Water Analysis Handbook 7°ed PDF

WATER ANALYSIS
HANDBOOK
7th Edition
Photometric Procedures
Titration Procedures
Ion-Selective Electrode Procedures
Microbiological Procedures
Chemical Procedures Explained
Digesting Liquids, Oils and Solids
HACH COMPANY
(970) 669-3050 or (800) 227-4224
Loveland, Colorado, U.S.A.
©Hach Company, 2008, 2012.

All rights reserved. Printed in the U.S.A.
Table of Contents
Applications Guide ........................................................................................................................... 11

Section 1 Abbreviations and Conversions .................................................................................. 15
1.1 Procedure abbreviations ............................................................................................................. 15
1.2 Conversions ................................................................................................................................ 16
Section 2 Laboratory Practices ...................................................................................................... 19
2.1 Temperature ............................................................................................................................... 19
2.2 Mixing ......................................................................................................................................... 19
2.3 Digestion ..................................................................................................................................... 20
2.4 Distillation ................................................................................................................................... 20
2.5 Filtration ...................................................................................................................................... 21
2.6 Reagents .................................................................................................................................... 24
2.7 Sample dilution ........................................................................................................................... 24
2.8 AccuVac® Ampuls ...................................................................................................................... 26
2.9 PermaChem® pillows ................................................................................................................. 27
2.10 Sample cells ............................................................................................................................. 28
2.11 Other apparatus ........................................................................................................................ 29
2.12 Achieve accuracy in measurement ........................................................................................... 29
Section 3 Chemical Analysis........................................................................................................... 31
3.1 Sample collection, preservation and storage .............................................................................. 31
3.2 Interferences ............................................................................................................................... 39
3.3 Method performance................................................................................................................... 40
3.4 Prepare a calibration curve ......................................................................................................... 43
3.5 Adapt procedures to other spectrophotometers ......................................................................... 43
Section 4 Sample Pretreatment by Digestion ............................................................................. 47
4.1 USEPA-approved digestions ...................................................................................................... 47
4.2 General Digesdahl digestion....................................................................................................... 48
Section 5 Water Management and Safety .................................................................................... 55
5.1 Waste minimization..................................................................................................................... 55
5.2 Regulatory overview ................................................................................................................... 55
5.3 Hazardous waste ........................................................................................................................ 55
5.4 Management of specific wastes.................................................................................................. 58
5.5 Resources................................................................................................................................... 58
5.6 Safety.......................................................................................................................................... 59
5.7 Material safety data sheets ......................................................................................................... 60
Section 6 International Guideline Comparison ........................................................................... 63
Section 7 Definitions of USEPA Approved and Accepted........................................................ 65
7.1 USEPA approved........................................................................................................................ 65
7.2 USEPA accepted ........................................................................................................................ 65
Sample cells and apparatus
Procedures for Analysis................................................................................................................... 69
Acid-Base, Acid Determination and Base Determination ....................................................................... 71
Acid-Base, Sodium Hydroxide for meq/L of Acid Sulfuric Acid for meq/L of Base ................................. 77
Acidity, Methyl Orange, Sodium Hydroxide with a Buret ........................................................................ 83
Acidity, Phenolphthalein, Sodium Hydroxide with a Buret ..................................................................... 87
Acidity, Methyl Orange and Phenolphthalein (Total) Acidity ................................................................... 91
Alachlor, Immunoassay Method.............................................................................................................. 97
Alkalinity, Phenolphthalein and Total Alkalinity..................................................................................... 105
Table of Contents
Page 3 of 10
Table of Contents
Alkalinity, USEPA Buret Titration Method ............................................................................................. 111

Aluminum, Aluminon Method.............................................................................................................................................. 117
Aluminum, Eriochrome Cyanine R Method........................................................................................... 125
Arsenic, Silver Diethyldithiocarbamate Method .................................................................................... 133
Atrazine, Immunoassay ........................................................................................................................ 141
Barium, Turbidimetric Method............................................................................................................... 151
Benzotriazole/Tolyltriazole, UV Photolysis Method............................................................................... 157
Boron, Carmine Method........................................................................................................................ 163
Boron, Azomethine-H Method .............................................................................................................. 169
Scope and Application: Test preparation ........................................................................................ 169
Bromine, DPD Method.......................................................................................................................... 177
Cadmium, Dithizone Method ................................................................................................................ 185
Carbon Dioxide, Digital Titrator Method Using Sodium Hydroxide ....................................................... 193
Carbon Dioxide, Buret Titration Method ............................................................................................... 197
Chelant, Free, Digital Titrator Using Magnesium Chloride ................................................................... 201
Chelant, Total, Bismuth Nitrate Method ................................................................................................ 205
Chloramine (Mono);
Nitrogen, Free Ammonia, Indophenol Method...................................................................................... 209
Chloramine (Mono), Indophenol Method .............................................................................................. 219
Chloramine (Mono), Indophenol Method .............................................................................................. 225
Chloride, Mercuric Thiocyanate Method ............................................................................................... 231
Chloride, Mercuric Nitrate Method ........................................................................................................ 237
Chloride, Silver Nitrate Method............................................................................................................. 243
Chloride, USEPA Silver Nitrate Buret Titration Method ........................................................................ 249
Chlorine Dioxide, DPD Method............................................................................................................. 255
Chlorine Dioxide, Direct Reading Method............................................................................................. 263
Chlorine Dioxide, Chlorophenol Red Method ....................................................................................... 267
Chlorine Dioxide, Amaranth Method..................................................................................................... 273
Chlorine Dioxide, Direct Reading Method............................................................................................. 277
Chlorine Demand/Requirement, .......................................................................................................... 281
Chlorine, Free, DPD Rapid Liquid Method ........................................................................................... 289
Chlorine, Free, DPD Method ................................................................................................................ 295
Chlorine, Free, USEPA DPD Method ................................................................................................... 301
Chlorine, Free, USEPA Amperometric Buret Titration Method............................................................. 309
Chlorine, Free, DPD Method ................................................................................................................ 313
Chlorine, Free, Indophenol ................................................................................................................... 319
Chlorine, Total, USEPA DPD Method ................................................................................................... 327
Chlorine, Total, DPD Rapid Liquid Method ........................................................................................... 335
Chlorine, Total, DPD Method ................................................................................................................ 341
Table of Contents
Page 4 of 10
Table of Contents

Chlorine, Free and Total, DPD-FEAS Method ...................................................................................... 373
Chlorine, Free, Amperometric Forward Titration using 0.00564 N PAO............................................... 379
Chlorine, Hypochlorite, Iodometric Method........................................................................................... 385
Chlorine, Total, Amperometric Back Titration ....................................................................................... 389
Chlorine, Total, Amperometric Forward Titration .................................................................................. 399
Chlorine, Total, Iodometric Method using Sodium Thiosulfate.............................................................. 405
Chlorine, Total, USEPA Amperometric Buret Titration Method............................................................. 409
Chlorine, Total, Iodometric Method Using Sodium Thiosulfate ............................................................. 413
Chromate, Titration Method using Sodium Thiosulfate......................................................................... 421
Chromium, Hexavalent, USEPA 1,5-Diphenylcarbohydrazide Method ................................................ 427
Chromium, Total, Alkaline Hypobromite Oxidation Method, ................................................................. 433
Cobalt, 1-(2-Pyridylazo)-2-Naphthol (PAN) Method ............................................................................. 439
Color, True and Apparent, Platinum-Cobalt Standard Method, , ......................................................... 445
Color, ADMI, ADMI Weighted Ordinate Method ................................................................................... 449
Copper, USEPA Bicinchoninate Method............................................................................................... 455
Copper, Porphyrin Method.................................................................................................................... 463
Cyanide, Pyridine-Pyrazalone Method ................................................................................................. 469
Cyanuric Acid, Turbidimetric Method.................................................................................................... 479
Fluoride, USEPA SPADNS Method...................................................................................................... 483
Fluoride, USEPA SPADNS 2................................................................................................................ 491
Formaldehyde, MBTH Method.............................................................................................................. 499
Hardness, Calcium and Magnesium;
Calmagite Colorimetric Method....................................................................................................... 505
Hardness, Calcium and Magnesium; Chlorophosphonazo Colorimetric Method ................................. 511
Hardness, Total, Calcium and Magnesium; Chlorophosphonazo Rapid Liquid Method ....................... 517
Hardness, Calcium, Titration Method using EDTA ............................................................................... 523
Hardness, Calcium, USEPA Buret Titration Method............................................................................. 529
Hardness, Total, Sequential, Titration Method using EDTA.................................................................. 537
Hardness, Total, Titration Method using EDTA..................................................................................... 545
Hardness, Total, USEPA ManVer 2 Buret Titration Method ................................................................. 553
Hardness, Total, Sequential, Buret Titration Method ............................................................................ 561
Hydrazine, p-Dimethylaminobenzaldehyde Method ............................................................................. 569
Iodine, DPD Method ............................................................................................................................ 575
Iron, Ferrozine® Method ....................................................................................................................... 583
Iron, Ferrous, 1-10 Phenanthroline Method.......................................................................................... 589
Iron, Total, USEPA FerroVer® Method.................................................................................................. 595
Iron, Total, TPTZ Method ...................................................................................................................... 603
Iron, FerroZine® Rapid Liquid Method .................................................................................................. 611
Iron, Total, FerroMo Method ................................................................................................................. 619
Table of Contents
Page 5 of 10
Table of Contents
Iron, TitraVer Titration Method.............................................................................................................. 625

Lead, USEPA Dithizone Method........................................................................................................... 629
Lead, LeadTrak™ Fast Column Extraction Method .............................................................................. 637
Manganese, USEPA Periodate Oxidation Method ............................................................................... 645
Manganese, 1-(2-Pyridylazo)-2-Naphthol PAN Method ....................................................................... 651
Mercury, Cold Vapor Mercury Concentration Method........................................................................... 657
Molybdenum, Mercaptoacetic Acid Method.......................................................................................... 669
Molybdenum, Ternary Complex Method............................................................................................... 675
Nickel, USEPA Heptoxime Method....................................................................................................... 681
Nickel, 1-(2 Pyridylazo)-2-Napthol (PAN) Method ................................................................................ 687
Nitrate, Chromotropic Acid Method....................................................................................................... 693
Nitrate, Cadmium Reduction Method.................................................................................................... 699
Nitrate, UV Screening Method .............................................................................................................. 725
Nitrite, Ferrous Sulfate Method............................................................................................................. 729
Nitrite, Diazotization Method................................................................................................................. 733
Nitrite, USEPA Diazotization................................................................................................................. 737
Nitrite, Ceric Acid Titration Method ....................................................................................................... 743
Nitrogen, Ammonia, USEPA Nessler Method....................................................................................... 749
Nitrogen, Ammonia, Salicylate Method................................................................................................. 755
Nitrogen, Free Ammonia, Indophenol Method...................................................................................... 773
Nitrogen, Total, Persulfate Digestion Method ....................................................................................... 781
Nitrogen, Total Inorganic, Titanium Trichloride Reduction Method ....................................................... 789
Nitrogen, Total Kjeldahl, Nessler Method (Digestion Required)............................................................ 797
Nitrogen, Total, Persulfate Digestion Method ....................................................................................... 805
Oil and Grease, USEPA Hexane Extractable Gravimetric Method....................................................... 813
Oil and Grease, USEPA Solid Phase Extraction Method ..................................................................... 829
Organic Carbon, Total, Direct Method .................................................................................................. 841
Organic Constituents
UV Absorbing (UV-254), Direct Reading Method ................................................................................. 865
Oxygen Demand, Biochemical, Dilution Method .................................................................................. 871
Oxygen Demand, Chemical, Manganese III Reactor Digestion Method (with optional chloride removal)..
885
Oxygen Demand, Chemical, (Manganese III Reactor Digestion Method) ........................................... 895
Manganese III Reactor Digestion Method (without chloride removal) .................................................. 895
Oxygen Demand, Chemical, USEPA Reactor Digestion Method ......................................................... 901
Table of Contents
Page 6 of 10
Table of Contents
Oxygen, Dissolved, HRDO Method ...................................................................................................... 913

Oxygen, Dissolved, Indigo Carmine Method ........................................................................................ 917
Oxygen, Dissolved, Ultra High Range Method ..................................................................................... 921
Oxygen, Dissolved, Azide Modification of Winkler Method................................................................... 925
Oxygen, Dissolved, USEPA Azide Modification of Winkler Method ..................................................... 933
Oxygen Scavengers, Iron Reduction Method for Oxygen Scavengers ................................................ 939
Ozone, Indigo Method .......................................................................................................................... 945
Polychlorinated Biphenyls (PCB) in Soil, Immunoassay Method.......................................................... 949
Phenols, USEPA 4-Aminoantipyrine Method........................................................................................ 959
Phosphonates, Persulfate UV Oxidation Method ................................................................................. 967
Phosphorus, Acid Hydrolyzable Digestion, USEPA Acid Digestion Method......................................... 975
Phosphorus, Acid Hydrolyzable, PhosVer™ 3 with Acid Hydrolysis Method ....................................... 979
Phosphorus, Reactive (Orthophosphate), Molybdovanadate Method .................................................. 987
Phosphorus, Reactive (Orthophosphate), Amino Acid Method ............................................................ 995
Phosphorus, Reactive, Molybdovanadate Rapid Liquid Method ........................................................ 1001
Phosphorus, Reactive, Ascorbic Acid Rapid Liquid Method............................................................... 1009
Phosphorus, Reactive (Orthophosphate), USEPA PhosVer 3 (Ascorbic Acid) Method ..................... 1017
Phosphorus, Reactive (Orthophosphate), USEPA PhosVer® 3 Method ............................................ 1025
Phosphorus, Reactive
(Orthophosphate), Molybdovanadate Method .................................................................................... 1031
Phosphorus, Total, USEPA
PhosVer® 3 with Acid Persulfate Digestion Method ..................................................................... 1039
Phosphorus, Total, Digestion, USEPA Acid Persulfate Digestion Method.......................................... 1047
Phosphorus, Total, Molybdovanadate Method with Acid Persulfate Digestion ................................... 1051
Potassium, Tetraphenylborate Method............................................................................................... 1059
Quaternary Ammonium Compounds, Direct Binary Complex Method ............................................... 1065
Salinity, Mercuric Nitrate Method ........................................................................................................ 1071
Selenium, Diaminobenzidine Method ................................................................................................. 1075
Silica, Silicomolybdate Method ........................................................................................................... 1085
Silica, Heteropoly Blue Method........................................................................................................... 1091
Silica, Heteropoly Blue Rapid Liquid Method...................................................................................... 1097
Silica, Heteropoly Blue Method........................................................................................................... 1105
Silver, Colorimetric Method................................................................................................................. 1113
Solids, Settleable Matter, Direct Measurement................................................................................... 1121
Solids, Nonfilterable Suspended Solids; Total and Volatile, USEPA Gravimetric Method .................. 1123
Solids, Total Filterable
(Total Dissolved Solids), USEPA Gravimetric Method ........................................................................ 1129
Solids, Volatile Dissolved and Fixed Dissolved, Gravimetric Method ................................................. 1133
Solids, Total Volatile and Fixed, Gravimetric Method.......................................................................... 1139
Solids, Total, USEPA Gravimetric Method .......................................................................................... 1143
Suspended Solids, Photometric Method............................................................................................. 1147
Table of Contents
Page 7 of 10
Table of Contents
Sulfate, USEPA SulfaVer 4 Method.................................................................................................... 1151

Sulfide, USEPA Methylene Blue Method............................................................................................ 1159
Sulfite, Iodate-Iodide Buret Titration Method ...................................................................................... 1163
Sulfite, Iodate-Iodide Method.............................................................................................................. 1167
Sulfite, Colorimetric Method................................................................................................................ 1171
Surfactants, Anionic (Detergents), Crystal Violet Method................................................................... 1175
Tannin and Lignin, Tyrosine Method................................................................................................... 1181
Toxicity, ToxTrak™ Method , ............................................................................................................................................ 1185
TPH (Total Petroleum Hydrocarbons), Immunoassay ........................................................................ 1191
Trihalomethanes, THM Plus™ Method............................................................................................... 1203
Trihalomethane Formation Potential (THMFP), ................................................................................. 1213
Volatile Acids, Esterification Method................................................................................................... 1221
Volatile Acids, Sodium Hydroxide Method .......................................................................................... 1227
Volatile Acids, Buret Titration Method................................................................................................. 1231
Zinc, USEPA Zincon Method .............................................................................................................. 1235
Microbiology .................................................................................................................................. 1241
Bacteria Test Guidelines, ................................................................................................................... 1243
Membrane Filtration Guidelines, ........................................................................................................ 1247
Bacteria, Membrane Filtration Method................................................................................................ 1255
Coliforms—Total, Fecal and E. coli, USEPA Membrane Filtration Method......................................... 1261
Coliforms—Fecal, USEPA Membrane Filtration Method .................................................................... 1275
Coliforms—E. coli, USEPA Membrane Filtration Method ................................................................... 1283
Coliforms—E. coli, USEPA Membrane Filtration Method ................................................................... 1289
Coliforms—Total and E. coli, USEPA Membrane Filtration Method ................................................... 1295
Enterococci, Membrane Filtration Method .......................................................................................... 1303
Heterotrophic Bacteria, Membrane Filtration Method ......................................................................... 1309
Pseudomonas, Membrane Filtration Method...................................................................................... 1333
MPN Dilution Guidelines, Coliforms—Fecal, USEPA A-1 Medium..................................................... 1343
Coliforms—Total and E. Coli, Lauryl Tryptose with MUG Broth.......................................................... 1349
Coliforms—Total, Fecal and E. Coli, USEPA Lauryl Tryptose Broth presumptive test with BGB, EC Medi-
um and EC/MUG confirmation ...................................................................................................... 1359
Coliforms—Total, Fecal and E. Coli, USEPA Lauryl Tryptose Broth presumptive test with BGB, EC Medi-
um and EC/MUG confirmation ...................................................................................................... 1369
Bacteria, Hydrogen Sulfide Producing, Most Probable Number (MPN) Method ................................ 1377
Coliforms, Presence/Absence ............................................................................................................ 1381
Bacteria, Hydrogen Sulfide Producing, Presence/Absence (P/A) Method ........................................ 1387
Heterotrophic Bacteria, Pour Plate Method ........................................................................................ 1391
Table of Contents
Page 8 of 10
Table of Contents

Total Aerobic Bacteria, Yeasts and Molds, Total Aerobic Bacteria (Amber)/Yeast and Mold (Red)
Total Aerobic Bacteria (Amber)/Total Coliform (Red)
Total Aerobic Bacteria (Amber)/Disinfection Control (Purple)....................................................... 1425
Electrochemistry ........................................................................................................................... 1431
Conductivity, USEPA Direct Measurement Method ............................................................................ 1433
Fluoride in Acid Solutions, Direct Measurement ISE Method ............................................................. 1439
Fluoride in Drinking Water, USEPA Direct Measurement ISE Method ............................................... 1447
Nitrate, Direct Measurement ISE Method ........................................................................................... 1457
Nitrate, Direct Measurement ISE Method ........................................................................................... 1469
Nitrogen, Ammonia, USEPA Direct Measurement ISE Method.......................................................... 1479
Nitrogen, Ammonia, USEPA Known Addition ISE Method ................................................................. 1489
Sodium, Direct Measurement ISE Method ......................................................................................... 1497
Oxygen, Dissolved, Direct Measurement Method .............................................................................. 1507
Oxygen Demand, Biochemical, Dilution Method ................................................................................ 1515
Oxygen, Dissolved, Direct Measurement Method .............................................................................. 1529
Oxidation Reduction Potential
(ORP), Direct Measurement Method .................................................................................................. 1533
pH, USEPA Electrode Method............................................................................................................ 1539
Chemical Procedures Explained ................................................................................................ 1545
Acidity, For water, wastewater and seawater ..................................................................................... 1547
Alkalinity, For water, wastewater and seawater .................................................................................. 1549
Aluminum, For water........................................................................................................................... 1552
Barium, For water, wastewater, oil-field water and seawater ............................................................. 1553
Boron, For water and wastewater....................................................................................................... 1554
Benzotriazole and Tolyltriazole, For water .......................................................................................... 1556
Carbon Dioxide, For water and seawater ........................................................................................... 1557
Chloramine (Mono), For water and wastewater.................................................................................. 1558
Chloramine (Mono);
Nitrogen, Free Ammonia, For determining free ammonia and monochloramine simultaneously in finished
chloraminated water ..................................................................................................................... 1559
Chloride, For water and wastewater ................................................................................................... 1561
Chlorine Dioxide, For water and wastewater ...................................................................................... 1563
Chlorine, Free and Total, For water, wastewater and seawater ......................................................... 1565
Chromium, For water and wastewater................................................................................................ 1567
Cobalt, For water ................................................................................................................................ 1568
Copper, For water, wastewater and seawater .................................................................................... 1570
Cyanide, For water, wastewater and seawater................................................................................... 1573
Fluoride, For water and seawater ....................................................................................................... 1575
Table of Contents
Page 9 of 10
Table of Contents
Formaldehyde, For water.................................................................................................................... 1577

Hardness, For water, wastewater and seawater ................................................................................ 1579
Hydrazine, For water and boiler water................................................................................................ 1583
Iron, For water and seawater.............................................................................................................. 1584
Langelier and Aggressive Indices, Method 8073................................................................................ 1588
Lead, For water and wastewater ........................................................................................................ 1593
Manganese, For water and wastewater.............................................................................................. 1594
Mercury, Cold Vapor, Molybdenum, Molybdate, For water ................................................................. 1597
Nickel, For water................................................................................................................................. 1599
Nitrogen, Ammonia, For water, wastewater and seawater ................................................................. 1600
Nitrogen, Kjeldahl, For water and wastewater .................................................................................... 1601
Nitrogen, Nitrate, For water and wastewater ...................................................................................... 1602
Nitrogen, Nitrite, For water and wastewater ....................................................................................... 1604
Nitrogen, Total, For water, wastewater and seawater......................................................................... 1606
Organic Carbon, Total, For water and wastewater ............................................................................. 1607
Oxygen Demand, Chemical, Mn III, For water and wastewater ......................................................... 1608
Oxygen Demand, Chemical, For wastewater ..................................................................................... 1610
Oxygen, Dissolved, For water, wastewater, and seawater................................................................. 1612
Oxygen Scavengers, For water .......................................................................................................... 1615
Ozone, For water ................................................................................................................................ 1616
pH, pH Indicators, For water and wastewater..................................................................................... 1621
Phenols, For water, wastewater and seawater................................................................................... 1626
Phosphonates, For water.................................................................................................................... 1627
Phosphorous, For water, wastewater and seawater........................................................................... 1628
Potassium, For water and wastewater................................................................................................ 1630
Selenium, For water and wastewater ................................................................................................. 1631
Silica, For water and seawater ........................................................................................................... 1632
Sulfate, For water, seawater and oil-field water.................................................................................. 1633
Sulfide, For water, wastewater and seawater..................................................................................... 1634
Sulfite, For water, wastewater and seawater...................................................................................... 1635
Turbidity, Zinc, For water and wastewater .......................................................................................... 1639
Technical Support ......................................................................................................................... 1641
Table of Contents
Page 10 of 10
Applications Guide
Metals/Mining, Mfg & Finishing
Pharmaceutical Manufacture
Semiconductor Manufacture
Beverages/Bottled Water
Commercial Laundries
Environmental Testing
Chemical Manufacture
Wastewater, Municipal
Wastewater, Industrial
Boiler/Cooling Water
Power Plant Utilities

Chlorine Production
Food/Feed Industry
Solid Waste/Sludge
Water Conditioning
Petroleum Industry
Pulp & Paper Mills

Aquarium Testing
Ultrapure Water
Textile Industry
Drinking Water
Pools & Spas

Aquaculture
Agriculture
Education
Acid/Base • • • • •
Acidity • • • • • • • • • •
Alkalinity • • • • • • • • • • • • • • • •
Aluminum • • • • • • • • •
Arsenic • • • • • • • •
Ascorbic Acid •
Bacteria • • • • • • • • • • • • • • • • • •
Barium • • • • •
BOD • • • • • • • • • • • •
Boron • • • • • • • • • •
Bromine • • • • • • •
Cadmium • • • • • • •
Calcium • • • • • • •
Carbon Dioxide • • • • • • •
Chelants • • • • •
Chloride • • • • • • • • • • • • • • • • • • • •
Chlorine • • • • • • • • • • • • • • • • • • • • •
Chlorine Dioxide • • • • • • •
Chromate • •
Chromium
(Hexavalent)
• • • • • • • • •
Chromium (Total) • • • • • • • • • • •
Cobalt • • • • • • •
COD • • • • • • • • • • • • • • • •
Color • • • • • • • • •
Conductivity • • • • • • • • • • • • •
Copper • • • • • • • • • • • • • • • • • •
Cyanide • • • • • •
Cyanuric Acid • • • •
Detergents • • • • •
Applications Guide
Page 11
Applications Guide

Chlorine Production
Food/Feed Industry
Solid Waste/Sludge
Water Conditioning
Petroleum Industry
Pulp & Paper Mills

Aquarium Testing
Ultrapure Water
Textile Industry
Drinking Water
Pools & Spas

Aquaculture
Agriculture
Education
Dissolved Oxygen • • • • • • • • • • • • • • •
Erythorbic Acid • • • • •
Fluoride • • • • • •
Formaldehyde • • • •
Gluteraldehyde • • • •
Glycols • • • • • •
Hardness • • • • • • • • • • • • • • • • • • • • •
Hydrazine • •
Hydrogen
Peroxide
• • • • • • • • •
Hydrogen Sulfide • • • • •
Iodide •
Iodine • • • • • • • •
Iron (Ferrous) • • • • •
Iron (Total) • • • • • • • • • • • • • • • • •
Lead • • • • • • • • • • •
Manganese • • • • •
Mercury • • • • • •
Molybdenum • • • • • • • • • • •
Nickel • • • • • • • •
Nitrogen
Ammonia
• • • • • • • • • • • • • • •
Nitrogen
(Inorganic)
• • • •
Nitrogen (Total) • • • • • • •
Nitrogen (Nitrate) • • • • • • • • • • • • • •
Nitrogen
(Monochloramine)
• • •
Nitrogen (TKN) • • • • • • • • •
Nitrogen (Nitrite) • • • • • • •
Oil and Grease • • • • • •
Oxygen Scavenger • • • • •
Applications Guide
Page 12
Applications Guide

Chlorine Production
Food/Feed Industry
Solid Waste/Sludge
Water Conditioning
Petroleum Industry
Pulp & Paper Mills

Aquarium Testing
Ultrapure Water
Textile Industry
Drinking Water
Pools & Spas

Aquaculture
Agriculture
Education
Ozone • • • • • • • • • •
PCB • • • • • •
Permanganate • •
pH • • • • • • • • • • • • • • • • • • • • • • • •
Phenols • • • • • • • •
Phosphate • • • • • • • • • • • • • • • • • •
Phosphonates • • • • • • •
Phosphorus • • • • • • • • • • • • •
Potassium • • • • •
QAC • • • • •
Salinity • • • • • • • • •
Selenium • • • • • • •
Silica • • • • • • • • • • • • • • • •
Silver • • • • • • • • • • • • • • •
Sodium • • • • • • • • • •
Sodium Chromate • • • •
Sodium
Hydroxide
• •
Sulfate • • • • • • • • • •
Sulfide • • • • • • •
Sulfite • • • • • • • • • •
Tannin • • • •
TDS • • • • • • •
Toxicity • • • • • •
TPH • • • • •
Triazole • • •
Turbidity • • • • • • • • • • • • • • • • •
Volatile Acids • • • • •
Water in Oil • • •
Zinc • • • • • • • • • • • •
Applications Guide
Page 13
Applications Guide
Page 14
Section 1 Abbreviations and Conversions
1.1 Procedure abbreviations

The abbreviations in Abbreviations table are common in written chemical procedures:
Table 1 Abbreviations
Abbreviation Definition Abbreviation Definition
liter—volume equal to one cubic decimeter
°C degree(s) Celsius (Centigrade) L
(dm3)
°F degree(s) Fahrenheit LR low range
American Chemical Society reagent grade
ACS MDL method detection limit
purity
Standard Methods for the Examination of MDB marked dropping bottle
Water and Wastewater, published jointly by mg/L milligrams per liter (ppm)
the American Public Health Association
(APHA), the American Water Works µg/L micrograms per liter (ppb)
Association (AWWA) and the Water milliliter—1/1000 of a liter. It is approximately
APHA
Environment Federation (WEF), is the mL the same as a cubic centimeter (and is
Standard
standard reference work for water analysis. sometimes called a “cc”).
Methods
Order from Hach Company, requesting
MR medium range
Catalog No. 2270800 or from the Publication
Office of the APHA. Many procedures
National Interim Primary Drinking Water
contained in this manual are based on NIPDWR
Regulations
Standard Methods.
National Pollutant Discharge Elimination
AV AccuVac® NPDES
System
Bicn bicinchoninate P phosphorus
conc concentrated PCB poly chlorinated biphenyl
DB dropping bottle ppb parts per billion
DBP disinfection by-products ppm parts per million
CFR Code of Federal Regulations RL Rapid Liquid™
EDL Estimated detection limit SCDB self-contained dropping bottle
EPA Environmental Protection Agency THM trihalomethane
F&T free and total TNT Test ‘N Tube™
FM FerroMo® TOC total organic carbon
FV FerroVer® TPH total petroleum hydrocarbons
FZ FerroZine® TPTZ 2,4,6-Tri-(2-Pyridyl)-1,3,5-Triazine

United States Environmental
g grams USEPA
Protection Agency
gr/gal grains per gallon (1 gr/gal = 17.12 mg/L) ULR ultra low range
HR high range
Page 15
Abbreviations and Conversions
1.2 Conversions
1.2.1 Chemical species
Species conversion factors for many commonly used chemicals are listed in the
Conversion factors table.
Table 2 Conversion factors
To Convert From... To... Multiply By...
mg/L Al mg/L Al2O3 1.8895
mg/L B mg/L H3BO3 5.7
mg/L Ca-CaCO3 mg/L Ca2+ 0.4004
mg/L CaCO3 mg/L Ca2+ 0.4004
mg/L CaCO3 mg/L Mg2+ 0.2428
µg/L Carbo. µg/L Hydro. 1.92
µg/L Carbo. µg/L ISA 2.69
µg/L Carbo. µg/L MEKO 3.15
mg/L Cr6+ mg/L CrO42– 2.231
mg/L Cr6+ mg/L Na2CrO4 3.115
mg/L Cr6+ mg/L Cr2O72– 2.077
mg/L Mg–CaCO3 mg/L Mg2+ 0.2428
mg/L Mn mg/L KMnO4 2.876
mg/L Mn mg/L MnO4– 2.165
mg/L Mo6+ mg/L MoO42– 1.667
mg/L Mo6+ mg/L Na2MoO4 2.146
mg/L N mg/L NH3 1.216
mg/L N mg/L NO3– 4.427
mg/L Cl2 mg/L NH2Cl 0.726
mg/L Cl2 mg/L N 0.197
mg/L NH3–N mg/L NH3 1.216
mg/L NH3–N mg/L NH4+ 1.288
mg/L NO2– mg/L NaNO2 1.5
mg/L NO2– mg/L NO2––N 0.3045
mg/L NO2––N mg/L NaNO2 4.926
µg/L NO2––N µg/L NaNO2 4.926
mg/L NO2––N mg/L NO2– 3.284
µg/L NO2––N µg/L NO2– 3.284
mg/L NO3––N mg/L NO3– 4.427
mg/L PO43– mg/L P 0.3261
µg/L PO43– µg/L P 0.3261
mg/L PO43– mg/L P2O5 0.7473
µg/L PO43– µg/L P2O5 0.7473
mg/L SiO2 mg/L Si 0.4674
µg/L SiO2 µg/L Si 0.4674
Page 16
1.2.2 Hardness conversion

See the Hardness conversion factors table for the factors to convert hardness from one
unit of measure to another. For example, to convert mg/L CaCO3 to German
parts/100,000 CaO, multiply the value in mg/L x 0.056.
Table 3 Hardness conversion factors
British
American French German
Units of mg/L gr/gal lb/cu ft
gr/gal (US) Parts/ Parts/ meq/L1 g/L CaO
Measure CaCO3 (Imperial) CaCO3
CaCO3 100,000 CaCO3 100,000 CaO
CaCO3
mg/L
1.0 0.07 0.058 0.1 0.056 0.02 5.6x10–4 6.23x10–5
CaCO3
English
gr/gal 14.3 1.0 0.83 1.43 0.83 0.286 8.0x10–3 8.9x10–4
CaCO3
US gr/gal
17.1 1.2 1.0 1.72 0.96 0.343 9.66x10–3 1.07x10–3
CaCO3
Fr. p/
100,000 10.0 0.7 0.58 1.0 0.56 0.2 5.6x10–3 6.23x10–4
CaCO3
Ger.
p/100,000 17.9 1.25 1.04 1.79 1.0 0.358 1x10–2 1.12x10–3
CaO
meq/L 50.0 3.5 2.9 5.0 2.8 1.0 2.8x10–2 3.11x10–2
g/L CaO 1790.0 125.0 104.2 179.0 100.0 35.8 1.0 0.112
lb/cu ft
16,100.0 1,123.0 935.0 1,610.0 900.0 321.0 9.0 1.0
CaCO3
1 epm/L or mval/L
meq
= N • 1000
Note: -----------
L
Page 17
Page 18
Section 2 Laboratory Practices
2.1 Temperature
Most methods perform accurately when the sample temperature is between 20 and 25 °C
(68 to 77 °F). A note in the individual procedure will state any special temperature
requirements.
2.2 Mixing
Swirling is recommended when mixing samples in a graduated cylinder or a titration
flask. Swirling is the gentlest method of mixing and offers the least chance for
atmospheric contamination when testing for carbon dioxide and other gases.
1. Grip the cylinder (or flask) firmly with the tips of the thumb and first two fingers (see
the Swirl a cylinder and invert a sample cell figure).
2. Hold the cylinder at a 45-degree angle and make a circling motion from the wrist.
3. Move the cylinder in an approximately 12-inch circle, creating enough rotation to
complete the mixing in a few turns.
Mixing sample in a square sample cell:

1. Grasp the neck of the cell with the thumb and index finger of one hand. Rest the
concave bottom of the cell on the tip of the index finger of the other hand.
2. Mix by rotating the cell quickly one way and then in the reverse direction.
Figure 1 Rotate a sample cell
Page 19
Laboratory Practices
Inverting allows for thorough mixing in a capped sample cell or a mixing cylinder.
1. Hold the cell or cylinder, in a vertical position with the cap on top.
2. Invert so that the cap is on the bottom. Return the cell to its original position (see
Swirl a cylinder and invert a sample cell figure.) Repeat as needed.
Swirl a cylinder and invert a sample cell
Figure 2 Swirl a cylinder and invert a sample cell
2.3 Digestion
Several procedures require sample digestion. Digestion uses chemicals and heat to
break down a substance into components that can be analyzed. This section briefly
describes three different digestion procedures.
The Digesdahl system is a process that yields a digest suitable for the determination of
metals, total phosphorus and total Kjeldahl nitrogen (TKN). It is rapid, convenient and is
very effective at destroying interfering organic materials.
For USEPA reporting purposes, USEPA-approved digestions are required. USEPA
presents two digestions (mild and vigorous) for metals analysis. Other digestion
procedures are required for mercury, arsenic, phosphorus and TKN.
See Sample Pretreatment by Digestion for more information on sample digestion.
2.4 Distillation
Distillation is an effective, easy and safe method of separating some chemical
components for analysis. The following equipment is recommended for distillation:
• General Purpose Distillation Apparatus (Catalog No. 2265300), shown in the General
purpose distillation apparatus figure
• Arsenic Distillation Apparatus Set (Catalog No. 2265400)
• Cyanide Distillation Apparatus Set (Catalog No. 2265800)
• General Purpose Heater and Support Apparatus (Catalog No. 2274400, 115 VAC,
60 Hz)
• General Purpose Heater and Support Apparatus (Catalog No. 2274402, 230 VAC,
50 Hz)
Page 20
Note: When ordering the Cyanide or Arsenic Distillation Apparatus, always order in conjunction with
the General Purpose Distillation Apparatus and the General Heater and Support Apparatus.
The Distillation Apparatus is suitable for water and wastewater that requires sample
pretreatment by distillation. Applications for the General Purpose Apparatus include:
fluoride, albuminoid nitrogen, ammonia nitrogen, phenols, selenium and volatile acids.
The General Purpose Heater and Support Apparatus provides efficient heating and
anchoring of the glassware.
General purpose distillation apparatus
Figure 3 General purpose distillation apparatus
2.5 Filtration
Filtration separates particulates from an aqueous sample. It uses a porous medium that
retains particulates but allows liquids to pass through. It is useful for removing turbidity
(which may interfere in colorimetric analyses) from water samples.
The two methods most frequently used filtration methods are vacuum and gravity
filtration.
Page 21
2.5.1 Vacuum filtration

Vacuum filtration uses both suction and gravity to draw the liquid through the filter. An
aspirator or vacuum pump is used to create suction (see the Vacuum filtration figure).
Vacuum filtration is faster than gravity filtration alone.
To filter using a vacuum:

1. Use tweezers to place a filter paper into the filter holder.
2. Place the filter holder assembly in the filtering flask. Dampen the filter with deionized
water to make sure adhesion to the holder.
3. Position the funnel housing on the filter holder assembly.
4. While applying a vacuum to the filtering flask, transfer the sample to the filtering
apparatus.
5. When the filtration is complete, slowly release the vacuum from the filtering flask and
transfer the solution from the filter flask to another container.
Vacuum filtration
Figure 4 Vacuum filtration
2.5.2 Required apparatus for vacuum filtration

Table 4 Required apparatus for vacuum filtration
Description Unit Catalog. No.
Filter Discs, glass fiber, 47-mm 100/pkg 253000
Filter Holder, membrane, 47-mm each 1352900
Flask, filtering, 500-mL each 54649
Select one of the following: — —
Pump, vacuum, hand operated each 2824800
Pump, vacuum, portable, 115 VAC each 1469700
Pump, vacuum, portable, 230 VAC each 2824801
Tubing, vacuum — 2074145
Tweezers each 1428200
Page 22
2.5.3 Gravity filtration

Many chemical procedures use gravity filtration. This requires filter paper, a conical funnel
and a receiving flask (see the Gravity filtration figure). Gravity filtration is better for
retaining fine particles. The rate of filtration increases as the volume increases in the filter
cone, but never fill the cone more than three-quarters full.
Note: Pretreatment using acid and heat is often necessary for metals testing. Filter paper will not
withstand such an environment; therefore, vacuum filtration with glass fiber filter discs is
recommended. Also, glass filter discs do not retain colored species like the paper filters do.
To filter using gravity:

1. Place a folded filter paper into the funnel.
2. Dampen the filter paper with deionized water so it adheres to the funnel.
3. Place the funnel into an Erlenmeyer flask or graduated cylinder.
4. Pour the sample into the funnel.
Gravity filtration
Figure 5 Gravity filtration
Table 5 Required apparatus for gravity filtration

Cylinder, graduated, 100-mL each 50842
Funnel, poly, 65-mm each 108367
Filter Paper, 12.5-cm, pleated 100/pkg 189457
Flask, Erlenmeyer, 125-mL each 50543
Page 23
2.6 Reagents
2.6.1 Reagent and standard stability
In general, reagents and standards have the maximum shelf life when stored in a location
that is cool, dark, and dry. The product label will specify any special storage needs.
It is always good laboratory practice to date chemicals upon receipt and to rotate supplies
so the older supplies are used first. When the reagent shelf life is unknown or in doubt,
run a standard to check reagent effectiveness.
Absorption of moisture, carbon dioxide or other gases from the atmosphere, bacterial
action, high temperatures or light (with photosensitive compounds) may affect the reagent
shelf life. In some cases, reaction with the storage container or interaction of reagent
components may occur.
2.6.2 Reagent blank

In several tests, the contribution of the reagent(s) to the final reading is of such a
magnitude that it must be compensated for whenever the test is performed. Reagent
blank refers to that portion of the test result contributed solely by the reagent. This
produces a positive error in the test results.
Every effort is made to produce reagents with the lowest possible blank; and, for most
reagents, it is less than 0.009 absorbance units. However, it is sometimes impossible or
impractical to produce reagents with such a low blank. When using such reagents, it is
best to determine the reagent blank by performing the procedure using high-quality water
(deionized, distilled, etc.) in place of sample to “zero” the instrument. The resulting value
is then expressed in the concentration units of the test and is subtracted from each
sample determination that uses the same reagent lot. Spectrophotometer software allows
the reagent blank value to be stored and subtracted automatically from each sample
value. The reagent blank needs to be determined only at first use, when a new lot of
reagent has been opened or if contamination is suspected.
In most tests, the reagent blank is so small the instrument may be zeroed on either an
untreated portion of the original water sample or on deionized water. This will not result in
a significant loss of accuracy unless the test is for very low levels of the species of
interest. It is then best to use a reagent blank prepared as above.
2.7 Sample dilution

Most colorimetric tests use volumes of 10 and 25 mL. However, in some tests, the color
developed in the sample may be too intense to be measured due to high levels of
analyteor or unexpected colors may develop due to an interference. In either case, dilute
the sample to produce a measurable endpoint or to determine if interfering substances
are present.
To dilute the sample:

1. Pipet the chosen sample portion into a clean graduated cylinder (or volumetric flask
for more accurate work).
2. Fill the cylinder (or flask) to the desired volume with deionized water.
3. Mix well. Use the diluted sample when running the test.
The Sample dilution volumes table shows relative quantities and the multiplication factors
to use with a 25 mL graduated cylinder. The concentration of the sample is equal to the
diluted sample result multiplied by the multiplication factor.
Page 24
Table 6 Sample dilution volumes

mL Deionized Water
Sample
used to bring the volume Multiplication factor
volume (mL)
to 25 mL
25.0 0.0 1
12.5 12.5 2
10.01 15.0 2.5
5.01 20.0 5
2.51 22.5 10
1.01 24.0 25
0.2501 24.75 100
1 For sample sizes of 10 mL or less, use a pipet to measure the sample into the graduated cylinder or volumetric flask.
More accurate dilutions can be done with a pipet and a 100-mL volumetric flask
(See table for Multiplication factors for diluting to 100 mL).
1. Pipet the sample and dilute to volume with deionized water.
2. Stopper and invert to mix.stopper and invert to mix.
Table 7 Multiplication factors for diluting to 100 mL
Sample volume (mL) Multiplication factor
1 100
2 50
5 20
10 10
25 4
50 2
2.7.1 Sample dilution with interfering substances

Sample dilution may influence the level at which a substance interferes. The effect of the
interferences decreases as the dilution increases. In other words, higher levels of an
interfering substance can be tolerated in the original sample if it is diluted before analysis.
Example:
Copper does not interfere at or below 100 mg/L for a 25 mL sample in a procedure. If the
sample volume is diluted with an equal volume of water, what is the level at which copper
will not interfere?
Total volume
----------------------------------------- = Dilution factor
Sample volume
25 -
---------- = 2
12.5
Interference Level × Dilution Factor = Interference level in sample

100 × 2 = 200
The level at which copper will not interfere in the diluted sample is at or below 200 mg/L.
Page 25
2.8 AccuVac® Ampuls

AccuVac Ampuls contain pre-measured powder or liquid vacuum-packed in optical-quality
glass ampules.
To use AccuVac Ampuls:

1. Collect the sample in a beaker or other open container.
2. Use one of the following methods to break the tip off the ampule:
• Use the optional AccuVac Snapper (Catalog. No. 2405200). See How to use the
AccuVac snapper figure for instructions or;
• Place the ampule tip well below the sample surface and break the tip off (see How
to use AccuVac Ampuls figure) against the beaker wall. The break must be far
enough below the surface to prevent air from being drawn in as the level of the
sample drops.
3. Secure an ampule cap over the tip of the ampule. Invert the ampule several times to
dissolve the reagent. The cap protects from broken glass and provides a grip for
inserting and removing the ampul from the cell holder. Wipe the ampule with a
lint-free cloth to remove fingerprints.
Note: Without the cap, the liquid will stay in the ampule when inverted. DO NOT place fingers
over broken glass!
4. Insert the ampule into the sample cell holder and read the results directly.
How to use AccuVac Ampuls
Figure 6 How to use AccuVac Ampuls
2.8.1 Using the AccuVac Snapper
To use the AccuVac Snapper:

1. Hold the Snapper with the open end up.
2. Gently slip the ampule into the Snapper, point first, until the tip touches the ramp at
the bottom of the Snapper.
3. Hold the Snapper between the index and middle finger (like a syringe). With the
ampule tip down, lower the Snapper into the sample until the ampule shoulder is wet.
4. Press on the flat end of ampule with the thumb (as if depressing the plunger on a
syringe) until the tip snaps. Allow the ampule to fill before removing from sample.
5. Rinse the wet end of the Snapper and ampule with clean water, if desired. Remove
the ampule from Snapper.
6. Dispose of the ampule tip (retained in the Snapper) in a suitable waste receptacle.
Page 26
How to use the AccuVac snapper
Figure 7 How to use the AccuVac snapper
2.9 PermaChem® pillows

Permachem pillows utilize powdered reagents to minimize deterioration and the risk of
reagent spills.
To use PermaChem pillows (see the How to open PermaChem pillows figure):
1. Tap the pillow on a hard surface to collect the powdered reagent in the bottom.
2. Locate the tear notch and tear (or cut) across, holding the pillow away from the face.
3. Using two hands, push both sides toward each other to form a spout.
4. Pour the pillow contents into the sample cell and continue the procedure according to
the instructions.
How to open PermaChem pillows
Figure 8 How to open PermaChem pillows
Page 27
2.10 Sample cells

A set of sample cells are shipped with each photometric instrument. The same solution in
both cells will give the same absorbance (within ± 0.002 Abs for properly matched cells).
For more information, see the section on Match sample cells.
For accurate results, use only the sample cells specified in each procedure. Due to
differences in cell pathlengths, sample cell substitution will introduce bias in test results.
For example, 1-inch square cells have a pathlength approximately 8% longer than 1-inch
round cells; substitution of round cells for square cells will introduce a bias in the reading.
2.10.1 Orientation of sample cells

To minimize measurement variability when using a particular cell, always orient the cell in
the same manner before placing it into the cell holder. The fill marks on the cells can be
used as orientation guides for positioning the cells.
2.10.2 Maintain sample cells

When not in use, store the sample cells in the supplied boxes to protect from scratches
and breakage. After use, empty and clean the cells; avoid leaving colored solutions in the
cells for extended periods of time.
2.10.3 Clean sample cells

Most laboratory detergents can be used at recommended concentrations. Neutral
detergents, such as Liquinox, are safer when regular cleaning is required. Cleaning times
are reduced by increasing the temperature or by using an ultrasonic bath. Finish by
rinsing a few times with deionized water and allow to air dry.
Cells may also be cleaned with acid, followed by rinsing thoroughly with deionized water.
Note: Always use acid to clean cells used for low-level metal tests.
Individual procedures may require special cleaning methods. When using a brush to
clean cells, take extra care to avoid scratching the inner surfaces of the cells.
2.10.4 Match sample cells

Although sample cells shipped with the spectrophotometer instrument are distortion-free,
nicks and scratches from handling may cause an optical mismatch between two sample
cells and introduce error into the test results. This type of error may be avoided by
optically matching the sample cells as follows:
Note: Refer to the spectrophotometer user manual for specific steps required to select wavelengths
and zero the instrument.
1. Power on the instrument. Make sure Display Lock is off or the Reading mode is set to
Continuous.
2. Select a wavelength of 510 nm or the wavelength to be used for the test.
3. Pour at least 10 mL (25 mL for 25-mL cells) of deionized water into each of two
sample cells.
4. Place one sample cell into the cell holder with the fill mark facing the user.
5. Zero the instrument.
6. Place the other sample cell into the cell holder with the fill line facing the user.
7. Wait for the value to stabilize and read the absorbance. Record the resulting
absorbance.
Page 28
8. Turn the cell 180° and repeat step 6. Try to achieve an absorbance value within
± 0.002 Abs of the first cell. Note the orientation of the cell.
If the sample cells cannot be matched within ± 0.002 Abs, they may still be used by
compensating for the difference. For example, if the second cell reads 0.003 absorbance
units higher than the first cell, correct future readings (when using these two cells) by
subtracting 0.003 absorbance units (or the equivalent concentration) from the reading.
Likewise, if the second cell reads –0.003 absorbance units, add 0.003 absorbance units
to the reading.
2.11 Other apparatus

2.11.1 Boiling aids
Boiling is required in some procedures. Under some conditions bumping may occur
causing sample loss or injury. Bumping is caused by the sudden, almost explosive,
conversion of water to steam as it is heated. Use of a boiling aid, such as Boiling Chips
(Catalog. No. 1483531), reduces bumping.
Make sure the boiling aids will not contaminate the sample. Do not use boiling aids
(except glass beads, Catalog. No. 259600) more than once. Use a large enough flask or
beaker to allow significant headspace above the solution. Loosely covering the sample
during boiling will prevent splashing, reduce the chances of contamination and minimize
sample loss.
Individual procedures will recommend the specific boiling aid to use.
2.12 Achieve accuracy in measurement

2.12.1 Pipets and graduated cylinders
When smaller sample quantities are used, the accuracy of measurements becomes
increasingly important. The Reading the meniscus figure illustrates the proper way to
read the sample level using the meniscus formed when the liquid wets the graduated
cylinder or pipet walls.
Reading the meniscus
50
45
40
35
Figure 9 Reading the meniscus
Before filling, rinse the pipet or cylinder two or three times with the sample to be tested.
Use a pipet filler or pipet bulb to draw the sample into the pipet.
CAUTION
Never pipet chemical reagent solutions or samples by mouth.
When filling a pipet, keep the tip of the pipet below the surface of the sample as the
sample is drawn into the pipet.
Page 29
Serological pipets have marks that indicate the volume of liquid delivered by the pipet.
The marks may extend to the tip of the pipet or may be only on the straight portion of the
tube. If the marks are only on the straight part of the tube, fill serological pipets to the
zero mark and discharge the sample by draining the sample until the meniscus is level
with the desired mark. If the serological pipet has marks extending to the tip of the pipet,
fill the pipet to the desired volume and drain all the sample from the pipet. Then use a
pipet filler to blow the sample out of the pipet tip with the pipet filler for accurate
measurements.
Volumetric (transfer) pipets have a bulb in the middle and a single ring above the bulb to
indicate the volume of liquid when it is filled to the mark. To discharge a volumetric pipet,
hold the tip of the pipet at a slight angle against the container wall and drain. Do not
discharge the solution remaining in the tip of the pipet after draining. Volumetric pipets
are designed to retain a small amount of sample in the pipet tip.
If droplets of sample cling to the walls of the pipet, the pipet is dirty and is not delivering
the correct amount of sample. Wash the pipet thoroughly with a laboratory detergent or
cleaning solution and rinse several times with deionized water.
2.12.2 The Pour-Thru™ cell

The Pour-Thru Cell is an optional accessory that improves accuracy and makes
measuring more convenient for rapid liquid methods. Methods that use 25-mL samples
and sample cells can use the Pour-Thru Cell if specified in the procedure. The Pour-Thru
Cell cannot be used with 10-mL sample sizes and reagents.The Cell cannot be used
directly with a method unless it is specified in the procedure. For more information, see
the photometer user manual.
See the photometer user manual for installation and operation instructions.
• Pour the solution into the funnel of the installed Pour-Thru Cell Module. Avoid spilling
solution onto the instrument.
• The funnel height and orientation may be adjusted. The funnel height determines the
speed of sample flow through the cell. The higher the funnel, the faster the flow.
• To minimize air bubbles, adjust the funnel so that it drains completely with the final
level of liquid in the tube about 5 cm (2 inches) below the tip of the funnel.
• Take instrument readings after the solution has stopped flowing through the cell.
• Always rinse the cell thoroughly with deionized water after each series of tests or as
often as specified in the procedure.
Occasionally, remove the Pour-Thru Cell to check for any accumulation of film on the
windows. If the windows appear hazy, soak the cell in a detergent bath and rinse
thoroughly with deionized water.
Page 30
Section 3 Chemical Analysis
3.1 Sample collection, preservation and storage

Correct sampling and storage are critical for accurate testing. Sampling devices and
containers must be thoroughly clean to prevent carryover from previous samples.
Preserve the sample according to test-specific information about sample preservation.
3.1.1 Collecting water samples

Use a clean container. Rinse the container several times with the water to be sampled
before taking the sample. Document the location and procedure used for each sample
taken. For example:
From a tap—Take samples as close as possible to the source of the supply. This
decreases the influence of the distribution system on the sample. Let the water run long
enough to flush the system. Fill sample containers slowly with a gentle stream to avoid
turbulence and air bubbles.
From a well—Let the pump run long enough to draw fresh groundwater into the system.
Collect a sample from a tap near the well.
From open waters—Sample as near the middle of the body of water as is practical; at
least several feet from the shore or edge of the tank. Take the sample under the surface
of the water. When using a capped container, submerge it before removin
3.1.1.1 Types of containers

• The Example on page 23 lists recommended containers for specific parameters.
• Polypropylene and Polyethylene
• Quartz or TFE (tetrafluoroethylene, Teflon®)— higher quality and price
• Glass—Glass provides a good general-purpose container. Avoid using soft-glass
containers to collect samples to be tested for metals in the microgram-per-liter range.
• When determining silver, store samples in dark containers such as amber or
brown glass.
Acid washing will thoroughly clean sample containers before use.
3.1.1.2 Acid washing

If a procedure suggests acid washing, follow these steps:
1. Clean the glassware or plasticware with laboratory detergent. Phosphate-free
detergent is best. (When determining phosphates, phosphate-free detergent must
be used.)
2. Rinse well with tap water.
3. Rinse with a 1:1 hydrochloric acid solution or a 1:1 nitric acid solution. (Nitric acid is
best when testing for lead or other metals.)
4. Rinse well with deionized water. For chromium, 12–15 rinses may be necessary.
When determining ammonia and Kjeldahl nitrogen, the rinse water must be
ammonia-free.
5. Air dry. Protect the glassware from fumes and other sources of contamination during
storage.
Use chromic acid or chromium-free substitutes to remove organic deposits from glass
containers. Afterward, rinse thoroughly with water to remove all traces of chromium.
Avoid introducing metal contaminants from containers, distilled water or membrane filters.
Page 31
Chemical Analysis
3.1.1.3 Sample splits

Samples must often be split or divided into separate containers for intra- or
inter-laboratory use in studies, confirmation, alternative techniques or maintaining
additional sample for reference and stability studies.
It is very important that sample splits be done correctly:
• Collect a large volume of sample in a single container and transfer to smaller
containers; do not fill the smaller containers individually from the water source.
• Thoroughly mix samples containing particulates or solids before splitting so that all the
splits are homogeneous.
• If the sample requires filtering before analysis or storage, filter the entire sample
before splitting.
• Use the same kind of container for all the splits.
• Analyze biologically active splits on the same day or as close to the same day as is
possible.
• Preserve all splits in the same way; if this is not done, the differing methods must be
fully documented.
• When testing for volatile contaminants, fill containers samples to overflowing and cap
carefully. Do not leave any headspace or air in the container.
3.1.2 Storage and preservation

Because chemical and biological processes continue after collection, analyze the sample
as soon as possible. This also reduces the chance for error and minimizes labor. When
immediate analysis is not possible, the sample must be preserved. Preservation methods
include pH control, chemical addition, refrigeration and freezing.
Comparison of international drinking water and FDA bottled water guidelines presents an
overview of preservation methods and holding times for specific procedures.
Preserve aluminum, cadmium, chromium, cobalt, copper, iron, lead, nickel, potassium,
silver and zinc samples for at least 24 hours as follows:
1. Add one Nitric Acid Solution Pillow 1:1 (Catalog No. 254098) per liter of sample.
2. Check the pH with pH indicator paper or a pH meter to assure the pH is 2 or less.
Add additional pillows if necessary.
3. Adjust the sample pH prior to analysis by raising the pH to 4.5 with Sodium
Hydroxide Standard Solution, 1 N or 5 N.
3.1.3 Correcting for volume additions

When using a large volume of preservative or neutralizer, account for dilution by the acid
added to preserve the sample and/or the base used to adjust the pH to the range of the
procedure. Make this correction as follows:
1. Determine the volume of initial sample, the volume of acid and base added and the
total final volume of the sample.
2. Divide the total volume by the initial volume.
3. Multiply the test result by this factor.
Page 32
Chemical Analysis
Example:
A one-liter sample was preserved with 2 mL of nitric acid. It was neutralized with 5 mL of
5 N sodium hydroxide. The result of the analysis procedure was 10.00 mg/L. What is the
volume correction factor and correct result?
1. Total Volume = 1000 mL + 2 mL + 5 mL = 1007 mL
2. 1007
------------- = 1.007 = volume correction factor
1000
10.0 mg/L × 1.007 = 10.07 mg/L = correct result
Table 8 Required containers, preservation techniques and holding times 1

Parameter name Container2 Preservation3,4 Maximum holding time5
Bacterial tests
P, G Cool, 4 °C, 0.008%
Coliform, fecal and total 6 hours
Na2S2O3
Cool, 4 °C, 0.008%
Fecal streptococci P, G 6 hours
Na2S2O3
Aquatic toxicity tests
Toxicity, acute and chronic P, G Cool, 4 °C 36 hours
Chemical tests
Acidity P, G Cool, 4 °C 14 days
Alkalinity P, G Cool, 4 °C 14 days
Ammonia P, G Cool, 4 °C, H2SO4 to pH<2 28 days
Biochemical oxygen demand
P, G Cool, 4 °C 48 hours
(BOD)
Boron P, PFTE or quartz HNO3 to pH<2 6 months
Bromide P, G None required 28 days
Biochemical oxygen demand,
P, G Cool, 4 °C 48 hours
carbonaceous
Chemical oxygen demand P, G Cool, 4 °C, H2SO4 to pH<2 28 days
Chloride P, G None required 28 days
Chlorine, total residual P, G None required Analyze immediately
Color P, G Cool, 4 °C 48 hours
Cyanide, total and amenable Cool, 4 °C, NaOH to pH>12,
P, G 14 days7
to chlorination 0.6 g ascorbic acid6
Fluoride P None required 28 days
HNO3 to pH<2, H2SO4 to
Hardness P, G 6 months
pH<2
Hydrogen ion (pH) P, G None required Analyze immediately
Kjeldahl and organic nitrogen P, G Cool 4 °C, H2SO4 to pH<2 28 days
Metals8
Chromium VI P, G Cool, 4 °C 24 hours
Mercury P, G HNO3 to pH<2 28 days
Metals, except boron,

P, G HNO3 to pH<2 6 months
chromium VI and mercury
Nitrate P, G Cool, 4 °C 48 hours
Nitrate-nitrite P, G Cool 4 °C, H2SO4 to pH<2 28 days
Nitrite P, G Cool, 4 °C 48 hours
Page 33
Chemical Analysis
Table 8 Required containers, preservation techniques and holding times (continued)1

Cool, 4 °C, HCl or H2SO4 to
Oil and grease G 28 days
pH<2
Cool, 4 °C, HCl or H2SO4 or
Organic Carbon P, G 28 days
H3PO4 to pH<2
Orthophosphate P, G Filter immediately; Cool, 4 °C 48 hours
Oxygen, dissolved probe G Bottle and top None required Analyze immediately
Winkler G Bottle and top Fix on site and store in dark 8 hours
48. Phenols G only Cool 4 °C, H2SO4 to pH<2 28 days
Phosphorus, elemental G Cool, 4 °C 48 hours
Phosphorus, total P, G Cool, 4 °C, H2SO4 to pH<2 28 days
Residue, Total P, G Cool, 4 °C 7 days
Residue, Filterable P, G Cool, 4 °C 7 days
Residue, Nonfilterable (TSS) P, G Cool, 4 °C 7 days
Residue, Settleable P, G Cool, 4 °C 48 hours
Residue, Volatile P, G Cool, 4 °C 7 days
Silica P, PFTE or quartz Cool, 4 °C 28 days
Specific Conductance P, G Cool, 4 °C 28 days
Sulfate P, G Cool, 4 °C 28 days
Cool 4 °C, add zinc acetate
Sulfide P, G plus sodium hydroxide to 7 days
pH>9
Sulfite P, G none required Analyze immediately
Surfactants P, G Cool, 4 °C 48 hours
Temperature P, G None required Analyze immediately
Turbidity P, G Cool, 4 °C 48 hours
1 This table was adapted from Table II in the Code of Federal Regulations, July 1, 2000, Title 40, Part 136.3 (40 CFR 136.3), pages 23–25. Most organic
tests are not included.
2 Polyethylene (P) or glass (G) or PTFE Teflon
3 Sample preservation should be performed immediately upon sample collection. For composite chemical samples each portion should be preserved at
the time of collection. When use of an automated sampler makes it impossible to preserve each portion, then chemical samples may be preserved by
maintaining at 4 °C until compositing and sample splitting is completed.
4 When any sample is to be shipped by common carrier or sent through United States Mails, it must comply with the Department of Transportation
Hazardous Material Regulations (49 CFR Part 172). The person offering such material for transportation is responsible for ensuring such compliance.
For the preservation requirements of Table II, the Office of Hazardous Materials, Materials Transportation Bureau, Department of Transportation has
determined that the Hazardous Materials Regulations do not apply to the following materials: Hydrochloric acid (HCl) in water solutions at
concentrations of 0.04% by weight or less (pH about 1.96 or greater); Nitric acid (HNO3) in water solutions at concentrations of 0.15% by weight or less
(pH about 1.62 or greater); Sulfuric acid (H2SO4) in water solutions at concentrations of 0.35% by weight or less (pH about 1.15 or greater); and
Sodium hydroxide (NaOH) in water solutions at concentrations of 0.080% by weight or less (pH about 12.30 or less).
5 Samples should be analyzed as soon as possible after collection. The times listed are the maximum times that samples may be held before analysis
and still be considered valid. Samples may be held for longer periods only if the permittee or monitoring laboratory has data on file to show that the
specific types of samples under study are stable for the longer time and has received a variance from the Regional Administrator under §136.3(e).
Some samples may not be stable for the maximum time period given in the table. A permittee or monitoring laboratory is obligated to hold the sample
for a shorter time if knowledge exists to show that this is necessary to maintain sample stability. See §136.3(e) for details. The term “analyze
immediately” usually means within 15 minutes or less after sample collection.
6 Should only be used in the presence of residual chlorine.
7 Maximum holding time is 24 hours when sulfide is present. Optionally all samples may be tested with lead acetate paper before pH adjustments in order
to determine if sulfide is present. If sulfide is present, it can be removed by the addition of cadmium nitrate powder until a negative spot test is obtained.
The sample is filtered and then NaOH is added to pH 12.
8 Samples should be filtered immediately on-site before adding preservative for dissolved metals.
Page 34
Chemical Analysis
3.1.4 Checking for accuracy and precision

Accuracy defines the closeness of a test result to the true value. Precision defines the
closeness of repeated measurements to each other. Although precise results suggest
accuracy, they can be inaccurate. Both the accuracy and the precision of test results can
be evaluated by using standard additions or standard solutions.
Not accurate, Accurate, Precise, Accurate and

not precise not precise not accurate precise
Figure 10 Precision and accuracy illustrated
3.1.5 Standard solutions

A standard solution may be ordered as a prepared reagent or it may be made in the
laboratory. It is a solution of a known composition and concentration. The accuracy of the
analysis system may be checked by using a standard solution in place of the sample
water in a procedure.
3.1.6 Standard additions

Standard Additions is a common technique for checking test results. Other names are
“spiking” and “known additions.” The technique can test for interferences, bad reagents,
faulty instruments and incorrect procedures.
Perform Standard Additions by adding a measured small amount of a standard solution to
the sample and repeating the test. Use the same reagents, equipment and technique.
The result should be about 100% recovery. If not, there is an identifiable problem.
If Standard Additions works for the test, a Standard Additions Method section will be in
the procedure under Accuracy Check. Follow the detailed instructions given.
If the result is about 100% recovery for each addition, everything is working properly and
the results are correct.
If the result is not approximately 100% recovery for each addition, a problem exists. To
determine if the cause is an interference, repeat the Standard Additions using deionized
water as the sample. If the result is approximately 100% recovery for each addition, an
interference exists.
If results did show good recoveries with the deionized water, use the following checklist to
find the problem:
1. Check that the procedure is followed exactly:
a. Are the correct reagents used in the correct order?
b. Is the correct time allowed for color to develop?
c. Is the correct glassware being used?
d. Is the glassware clean?
Page 35
Chemical Analysis
e. Does the test need a specific sample temperature?

f. Is the sample pH in the correct range?
Refer to the written procedure to answer these questions.
2. Check the performance of the instrument per instructions in the user manual.
3. Check the reagents. Repeat the Standard Additions using new, fresh reagents.
If results are good, the original reagents were faulty.
4. If nothing else is wrong, the standard is almost certainly defective. Repeat the
Standard Additions with a new standard.
If the problem is still uncertain, call Technical Customer Support at 800-227-4224 (U.S.A.)
or 303-669-3050 for assistance.
3.1.7 Troubleshoot a test when results are in doubt

If the results from any chemistry are in doubt, troubleshoot as follows:
1. Run a proof-of-accuracy check. Take a standard solution, which has a known
concentration, through the same steps as the original sample. Include sampling and
storage, digestion and colorimetric determination, if applicable. If the results of the
standard solution check are correct, skip to step 4 below. If there is a variation in the
expected results, go to step 2.
2. If the standard solutions check does not match the expected results, check the
instrument set-up and method procedure as follows:
a. Verify that the correct program number for the test being performed is selected.
b. Verify that the units of concentration of the standard match the displayed units.
(One of the alternative forms of the analyte may be in the display.) For example:
Molybdenum may be shown as Mo instead of MoO4.
c. Verify that the sample cells specified in the procedures are used.
d. Verify that the reagents are correct for the sample size being analyzed.
e. Verify that the reagent blank value stored is for the current procedure. It may be
from a previous lot of reagents and therefore not representative of the current
reagent lot.
f. Verify the calibration curve adjustment (Standard Adjust) currently in use. The
factory-stored default calibration should be used initially to check the standard.
g. Verify that the dilution factor option is correct.
If the instrument setup is correct and the method procedure specifics are being followed
correctly, go to step 3.
3. If the standard solution check does not match the expected results, check the
reagents used in the test and the analytical technique as follows:
a. Determine the age of the reagents used in the test. Many factors affect reagent
shelf life (i.e., storage temperature, storage conditions, microbial contamination).
Replace suspect reagents and run the standards check again.
b. Run a deionized or distilled water blank through the entire process; include
sampling and storage, digestion and colorimetric determination. Some chemicals
will add a small amount of color to a test; this is not considered unusual. However,
color development in excess of 10% of the range of the test may indicate a
problem with one of the reagents or the dilution water.
c. To troubleshoot the procedure, delete the parts one by one. First, using the
standard solution, omit preservation and storage, doing only digestion and
colorimetry. If this analysis is correct, examine the procedure used to store the
Page 36
Chemical Analysis
sample. Make sure that it is the procedure prescribed for the chosen parameter.
If the sample is acidified for storage, be sure the correct acid is used and the
sample is adjusted to the proper pH level before testing.
If the standards check is still incorrect, run the standard on just the colorimetry. If the
results are correct, examine the digestion procedure. Make sure that the amount of
reagents used and the pH after the digestion are correct for the procedure. (See the
procedure for the parameter in question.)
4. If the standard solution gives a correct value, but the results of the sample
measurement are questionable, there may be an interference in the sample. To
check for an interference:
a. Spike the sample. Use a standard addition test instead of a standard solution test
to include any possible interferences.
b. To two cells containing fresh sample water, add an amount of standard equal to
two times the concentration of the sample.
c. Process both samples using the same reagents, instruments and technique. The
spiked sample should show an increase equal to the amount of standard added.
d. Calculate percent recovery as shown below. Ideally, the results should be 100%,
with results from 90 to 110% considered acceptable. Refer to the procedure notes
for possible interferences and ways to eliminate them.
e. Run a series of dilutions on the sample. Make sure the sample is within the range
of the test. A sample out of range for the method may give erroneous results
because of under- or over-development of the color, excess turbidity or even
sample bleaching. Run a series of dilutions to check for this possibility.
f. Because it may not be feasible to determine the cause of the interference, diluting
the sample past the point of interference is often the most economical and
efficient means of getting the correct result. If it is not possible to dilute out an
interference without diluting out the parameter to be measured, use a different
method, such as a different chemistry or an ion-selective electrode to measure the
parameter.
Calculating percent recovery:

1. Measure the unknown sample concentration.
2. Calculate the theoretical concentration of the spiked sample using the following
formula:
( Cu × Vu ) + ( Cs × Vs )
Theoretical Concentration = -------------------------------------------------------
-
Vu + Vs
Where:
Cu = measured concentration of the unknown sample
Vu = volume of the unknown sample
Cs = concentration of the standard
Vs = volume of the standard
3. Measure the spiked sample concentration.
4. Divide the spiked sample concentration by the theoretical concentration and multiply
by 100.
Page 37
Chemical Analysis
For example:
A sample was tested for manganese and the result was 4.5 mg/L. A separate 97-mL
portion of the same sample was spiked with 3 mL of a 100 mg/L standard solution of
manganese. This spiked solution was tested again for manganese using the same
method. The result was 7.1 mg/L.
The theoretical concentration of the spiked sample is:
( 4.5 mg/L × 97 mL ) + ( 100 mg/L × 3 mL )
------------------------------------------------------------------------------------------------------------ = 7.4 mg/L
97 mL + 3 mL
The percent spike recovery is:
7.1 mg/L
----------------------- × 100 = 96%
7.4 mg/L
USEPA calculation
The USEPA requires a more stringent calculation for percent recovery. This formula
calculates the percent recovery only for the standard added to the spiked sample and
yields a lower value than the above calculation. A complete explanation for the USEPA
formula is in USEPA Publication SW-846. The USEPA percent recovery formula is:
100 ( X s – X u )
%R = ----------------------------------
-
K
Where:
Xs = measured value of the spiked sample
Xu = measured value for the unspiked sample, adjusted for the dilution of the spike volume
K = known value of the spike in the sample
Example:
A sample measures 10 mg/L. A separate 100-mL portion of the sample was spiked with
5 mL of a 100-mg/L standard solution. The spiked solution was measured by the same
method as the original sample. The result was 13.7 mg/L.
X s = 13.7 mg/L
10 mg/L × 100 mL
X u = ------------------------------------------------ = 9.5 mg/L
105 mL
5 mL × 100 mg/L
K = -------------------------------------------- = 4.8 mg/L
105 mL
100 × ( 13.7 mg/L – 9.5 mg/L )

%R = ----------------------------------------------------------------------------- = 88%
4.8 mg/L
Acceptable percent recovery values are 80–120%.
3.1.8 Adjusting the standard curve

Spectrophotometers typically have many programs permanently installed in memory.
Many programs include a pre-programmed calibration curve. Each curve is the result of
an extensive calibration performed under ideal conditions and is normally adequate for
most testing. Deviations from the curve can occur from using compromised testing
reagents, defective sample cells, incorrect test procedure, incorrect technique or other
correctable causes. Interfering substances or other causes may be beyond the analyst’s
control.
In some situations, using the pre-programmed curve may not be convenient:
• Running tests where the reagents are highly variable from lot to lot.
• Running tests where frequent calibration curve checks are required.
• Testing samples which give a consistent test interference.
• Consider the following before adjusting the calibration curve:
Page 38
Chemical Analysis
• Will future test results be improved by adjusting the curve?

• Are interfering substances consistent in all the samples tested?
• Estimated detection limit, sensitivity, precision and test range information provided
with the procedure may not apply to an adjusted curve calibration.
The calibration curves can be adjusted by following the steps found in the test procedure.
Generally, add test reagents to a blank and standard solution. Working carefully is
important. After the adjustment, it is wise to run standard solutions of several
concentrations to make sure the adjusted curve is satisfactory. Performing standard
additions on typical samples may also help determine if the adjusted curve is acceptable.
Adjusting a measurement is a two-step process. First, the instrument measures the
sample using the pre-programmed calibration. Second, it multiplies this measurement by
an adjustment factor. The factor is the same for all concentrations. The instrument will
remember the factor until the program is exited and will display the standard adjustment
icon when it is used. Return to the pre-programmed curve any time by selecting the
original stored program from the main menu.
3.2 Interferences
Interferences are contaminants in a sample that are capable of causing changes in color
development, turbidity or unusual colors and odors, thereby creating errors in the results.
A list of common interferences is included in each procedure. Reagents are formulated to
eliminate many interferences; remove others by pretreating the sample as instructed in
the procedure.
Test strips are available for many of the common interferences. These can be
conveniently used to screen samples for the presence of interferences.
When test results appear inaccurate, develop an unexpected color or develop an unusual
odor or turbidity, repeat the test on a sample diluted with deionized water. (See Sample
dilution.) Correct the results for the dilution and compare them with those from the original
test. If they differ significantly, make a second dilution and check it against the first.
Repeat the dilutions until the same result (after volume corrections) is achieved twice in
succession.
For more information on interferences, see Standard additions. The APHA Standard
Methods book, an excellent reference for water analysis, also covers interferences in the
“General Introduction.”
pH interference
Chemical reactions are often pH dependent. Reagents contain buffers to adjust the pH of
the sample to the correct range. However, the reagent buffer may not be strong enough
for samples that are highly buffered or have an extreme pH.
The Sampling and Storage section of each procedure gives the pH range for that test.
Before testing, adjust the sample to the proper pH as instructed in the procedure or by
following these steps:
1. Measure the pH of the analyzed sample with a pH meter.
Note: Use pH paper when testing for chloride, potassium or silver to avoid contamination.
2. Prepare a reagent blank using deionized water as the sample. Add all reagents
called for in the procedure. Timer sequences, etc., may be ignored. Mix well.
3. Measure the pH of the reagent blank with a pH meter.
4. Compare the pH values of the analyzed sample with the reagent blank.
Page 39
Chemical Analysis
5. If there is little difference in the values of the analyzed sample and the reagent blank,
then pH interference is not the problem. Follow the Accuracy Check for the specific
procedure to more clearly identify the problem.
6. If there is a large difference between the value of the analyzed sample and the
reagent blank, adjust the sample pH to the value of the reagent blank. Adjust the
sample pH to this same pH for all future samples before analysis. Use the
appropriate acid, usually nitric acid, to lower the pH. Use the appropriate base,
usually sodium hydroxide, to raise the pH. Adjust the final result for any dilution
caused by adding acid or base; see Correcting for volume additions.
7. Analyze the sample as before.
8. Some purchased standards may be very acidic and will not work directly with test
procedures. Adjust the pH of these standards as described above. Adjust the final
concentration of the standard for the dilution. The standard solutions suggested in the
procedures are formulated so that no pH adjustment is necessary.
3.3 Method performance

3.3.1 Estimated detection limit (EDL)
Ranges for chemical measurements have limits. The lower limit is important because it
determines whether a measurement is different from zero. Many experts disagree about
the definition of this detection limit and determining it can be difficult. The Code of Federal
Regulations (40 CFR, Part 136, Appendix B) provides a procedure to determine the
“Method Detection Limit” or MDL. The MDL is the lowest concentration that is different
from zero with a 99% level of confidence. A measurement below this MDL is highly
suspect.
The MDL is not fixed; it varies for each reagent lot, instrument, analyst, sample type, etc.
Therefore, a published MDL may be a useful guide, but is only accurate for a specific set
of circumstances. Each analyst should determine a more accurate MDL for each specific
sample matrix using the same equipment, reagents and standards that will routinely be
used for measurements.
A sensitivity value (concentration change equivalent to an absorbance change of 0.010
abs) is provided as an estimate of the lower detection limit of each test. The sensitivity
value may be treated as an EDL for the purposes of MDL determination. It can be
considered a good starting concentration when determining a MDL. Do not use the EDL
as the MDL. The conditions for MDL determination must be exactly the same as the
conditions used for analysis. The EDL may be useful to the analyst as a starting point in
determining a MDL or as a way to compare methods. Measurements below the EDL may
also be valuable because they can show a trend, indicate the presence of analyte and/or
provide statistical data. However, these values have a large uncertainty.
3.3.2 Method detection limit (MDL)

This method is in accordance with the USEPA definition in 40 CFR, Part 136, Appendix B
in the 7-1-94 edition. The USEPA defines the method detection limit (MDL) as the
minimum concentration that can be determined with a 99% level of confidence that the
true concentration is greater than zero. Since the MDL will vary from analyst to analyst, it
is important that the MDL be determined under actual operating conditions.
Page 40
Chemical Analysis
The procedure for determining MDL is based on replicate analyses at a concentration

1 to 5 times the estimated detection limit. The MDL value is calculated from the standard
deviation of the replicate study results multiplied by the appropriate t value for a 99%
confidence interval. For this definition, the MDL does not account for variation in sample
composition and can only be achieved under ideal conditions.
1. Estimate the detection limit. Use the sensitivity value stated in the Method
Performance section of the analysis procedure.
2. Prepare a laboratory standard of the analyte, 1 to 5 times the estimated detection
limit, in deionized water that is free of the analyte.
3. Analyze at least seven portions of the laboratory standard and record each result.
4. Calculate the average and the standard deviation (s) of the results.
5. Compute the MDL using the appropriate t value (see table below) and the standard
deviation value:
MDL = t x s
Number of test portions t Value

7 3.143
8 2.998
9 2.896
10 2.821
For example:
The EDL for measuring iron using an iron test is 0.003 mg/L. An analyst accurately
prepared 1 liter of a 0.010 mg/L (about 3x the EDL) laboratory standard by diluting a
10-mg/L iron standard in iron-free deionized water.
Eight portions of the standard were tested according to the FerroZine method with the
following results:
Sample # Result (mg/L)

1 0.009
2 0.010
3 0.009
4 0.010
5 0.008
6 0.011
7 0.010
8 0.009
Using a calculator program, the average concentration = 0.010 mg/L and the standard
deviation (s) = 0.0009 mg/L.
Based on the USEPA definition, calculate the MDL as follows:
MDL for iron test = 2.998 (t) x 0.0009 (s)
MDL = 0.003 mg/L (agrees with initial estimate)
Note: Occasionally, the calculated MDL may be very different from the estimate of the detection
limit. To test how reasonable the calculated MDL is, repeat the procedure using a standard near the
calculated MDL. The average result calculated for the second MDL derivation should agree with the
Page 41
Chemical Analysis
initial calculated MDL. Refer to 40 CFR, Part 136, Appendix B (7-1-94), pages 635–637 for detailed
procedures to verify the MDL determination.
Run a laboratory blank containing deionized water without analyte through the test
procedure to confirm that the blank measurement is less than the calculated MDL. If the
blank measurement is near the calculated MDL, repeat the MDL procedure using a
separate blank for analysis for each portion of standard solution analyzed. Subtract the
average blank measurement from each standard and use the corrected standard values
to calculate the average and standard deviation used in the MDL.
3.3.3 Precision
Every chemical measurement has some degree of uncertainty. The quality of the entire
calibration curve determines the precision.
Uncertainty in chemical measurements may be due to systematic errors and/or random
errors. A systematic error is a mistake that is always the same for every measurement
made. For example, a blank can add to each measurement for a specific compound,
giving consistently high results (a positive bias). Random errors are different for every
test and can add either a positive or negative variation in response. Random errors are
most often caused by variation in analytical technique. Even with reliable reagents
developed to eliminate systematic errors, response variation occurs in all chemical
measurements.
3.3.4 Estimating precision

The method performance section in each procedure provides an estimate of test
precision. Most procedures use a replicate analysis estimate, based on real data.
For precision determined in this manner, the 95% confidence interval of the distribution is
reported.
In replicate analysis, the chemist prepares a specific concentration of the analyte in a
deionized water matrix. The standard is analyzed seven individual times on a single
instrument. The standard deviation is calculated and the 95% confidence interval of the
distribution is reported in the method. The reported value provides an estimate of the
“scatter” of results at a particular point in the calibration curve.
Precision estimates are based on a deionized water matrix. Precision on real samples
with varying matrices can be quite different from these estimates.
If the concentration obtained from running a standard solution is not as expected, refer to
Troubleshoot a test when results are in doubt.
3.3.5 Sensitivity
The definition of the sensitivity of a method is a change in concentration (ΔConcentration)
for a 0.010 change in absorbance (ΔAbs).
Use sensitivity when comparing different methods. For example, between three methods
for determining iron:
Iron Analysis Method Portion of curve ΔAbs ΔConcentration

FerroVer Entire range 0.010 0.022 mg/L
FerroZine Entire range 0.010 0.009 mg/L
TPTZ Entire range 0.010 0.012 mg/L
Page 42
Chemical Analysis
Notice that the FerroZine method has the greatest sensitivity of the three methods
because it will measure the smallest change in concentration. The technical definition of
sensitivity comes from a calibration curve with Abs on the x-axis and concentration on the
y-axis.
1. If the calibration is a line, the sensitivity is the slope of the line multiplied by 0.010.
2. If the calibration is a curve, the sensitivity is the slope of the tangent line to the curve
at the concentration of interest multiplied by 0.010.
The sensitivity value is also used as an estimate of the lower limit of the test. The value
may be used as a starting point in determining MDL.
3.4 Prepare a calibration curve

Note: Calibration curves are recommended when performing tests on different instruments or where
required by a regulator.
1. Prepare five or more standards of known concentration that cover the expected
range of the test. Run tests as described in the procedure on each prepared
standard. Then pour the customary volume of each known solution into a separate
clean sample cell of the type specified for the instrument.
2. Select the proper wavelength. Standardize (zero) the instrument using an untreated
water sample or a reagent blank, per procedure instructions.
3. Measure and record the absorbance of the known solutions within the time
constraints detailed in the procedure. To use absorbance vs. concentration, see the
section on Absorbance versus concentration calibration.
3.4.1 Absorbance versus concentration calibration

If absorbance values are measured, plot the results on linear graph paper. Plot the
absorbance value on the vertical axis and the concentration on the horizontal axis.
Plot increasing absorbance values from bottom to top. Plot increasing concentration
values from left to right. Values of 0.000 absorbance units and 0 concentration will begin
at the bottom left corner of the graph. A calibration table can be extrapolated from the
curve or the concentration values can be read directly from the graph. Or, determine an
equation for the line using the slope and y-intercept.
Alternately, use the User Program software in the spectrophotometer or a curve-fitting
program such as Excel to calculate the calibration curve.
3.5 Adapt procedures to other spectrophotometers

Test procedures may be used with more than one spectrophotometer, if calibration curves
are made that convert absorbance to concentration. Regardless of the
spectrophotometer used, prepare the sample and calibration standards following the
procedure and use the optimum wavelength used in the procedure.
To calibrate for a given analyte, a series of standards are prepared and measured to
establish the calibration curve. The absorbance vs. concentration is plotted as described
in the section on Absorbance versus concentration calibration. Points on the graph are
connected with a smooth line (curved or straight). If necessary, use the curve to make a
calibration table.
Page 43
Chemical Analysis
3.5.1 Select the best wavelength

When developing a new procedure or using procedures that are sensitive to wavelength,
select the wavelength where the instrument gives the greatest absorbance (see the
Select the best wavelength figure). Because procedures are usually developed to use the
best wavelength for the test, selecting the wavelength is not necessary for most stored
procedures.
3.5.1.1 Select the best wavelength on a spectrophotometer:
Note: When available, use of the Scan function is the easiest way to find the optimum wavelength.
1. Refer to the user manual for specific instructions for wavelength adjustments.
2. Select single wavelength adjustment.
3. Enter a wavelength in the range of interest.
Note: Sample color provides a good indication of what wavelength region to use. A yellow solution
absorbs light in the 400–500 nm region. A red solution absorbs light between 500–600 nm. A blue
solution absorbs light in the 600–700 nm range.
4. Prepare the sample and blank for analysis. Fill the appropriate sample cells with the
blank and the reacted sample solution.
5. Place the blank in the cell holder. Zero the instrument.
6. Place the prepared sample into the cell holder. Read the absorbance level.
7. Increase the wavelength so it is at least 100 nm greater than the range of interest.
Re-zero as in step 5. Measure and record the absorbance of the sample.
8. Repeat, decreasing the wavelength by 50 nm. Re-zero, then measure and record the
absorbance at each increment. Continue this process throughout the wavelength
range of interest. Note the wavelength of greatest absorbance (see the Example
in Table 9).
Table 9 Example
Wavelength Absorbance
550 nm 0.477
500 nm 0.762
450 nm 0.355
400 nm 0.134
9. Adjust the wavelength to 50 nm more than the highest absorbance point on the initial
search (step 8). Re-zero as in step 5.
Measure and record the absorbance. Repeat, decreasing the absorbance in 5-nm steps.
Re-zero, then measure and record the absorbance at each increment. Continue until the
entire range of interest is measured (see the Example in Table 10).
Table 10 Example
Wavelength Absorbance
520 nm 0.748
515 nm 0.759
510 nm 0.780
505 nm 0.771
500 nm 0.771
495 nm 0.651
490 nm 0.590
Page 44
Chemical Analysis
Check to be sure there is enough difference in absorbance between samples with low
and high analyte concentrations by measuring two sample solutions that contain the
expected low and high concentrations of analyte at the optimum wavelength. The change
in absorbance caused by increases/decreases in concentration depends on the
sensitivity of the procedure and the chemistry. Chemistries with small absorbance
changes are less sensitive, but tend to have larger ranges. Chemistries with large
absorbance changes are more sensitive, but tend to have smaller ranges.
Select the best wavelength
1.0
500 505 510 515 520

Absorbance
400 450 500 550 600 650 700
0.0 Wavelength (nm)
Figure 11 Select the best wavelength
Page 45
Page 46
Section 4 Sample Pretreatment by Digestion
Several procedures require sample digestion before determining total metal content.
Digestion uses acid and heat to break organo-metallic bonds and free ions for analysis.
4.1 USEPA-approved digestions

For USEPA reporting, USEPA-approved digestions are required. There are two methods
for metals analysis: mild and vigorous.
4.1.1 USEPA mild digestion

1. Acidify the entire sample at the time of collection with concentrated nitric acid by
adding 5 mL of acid per liter (or quart) of sample.
2. Transfer 100 mL of well-mixed sample to a beaker or flask. Add 5 mL of distilled
1:1 hydrochloric acid (HCl).
3. Heat using a steam bath or hot plate until the volume has been reduced to 15–20 mL.
Do not boil.
4. Filter the sample to remove any insoluble material.
5. Adjust the pH of the digested sample to a pH of 4 by adding 5.0 N Sodium Hydroxide
Standard Solution a drop at a time. Mix thoroughly and check the pH after each
addition.
6. Quantitatively transfer the sample to a 100-mL volumetric flask and dilute to volume
with deionized water. Continue with the procedure.
7. Process a reagent blank to match the sample.
4.1.2 USEPA vigorous digestion

For some samples mild digestion will not be sufficient. Use a vigorous digestion to make
sure that all the organo-metallic bonds are broken.
1. Use redistilled 1:1 Nitric Acid Solution to acidify the entire sample to a pH of less than
two. Do not filter the sample before digestion.
2. Transfer an appropriate sample volume (see the table on Vigorous digestion
volumes) into a beaker and add 3 mL of concentrated redistilled nitric acid.
3. Place the beaker on a hot plate and evaporate to near dryness, making certain the
sample does not boil.
4. Cool the beaker and add another 3 mL of the concentrated re-distilled nitric acid.
5. Cover the beaker with a watch glass and return it to the hot plate. Increase the
temperature of the hot plate so that a gentle reflux occurs. Add additional acid, if
necessary, until the digestion is complete (generally indicated when the digestate is
light in color or does not change color or appearance with continued refluxing).
Page 47
Sample Pretreatment by Digestion
6. Again, evaporate to near dryness (do not bake) and cool the beaker. If any residue or
precipitate results from the evaporation, add redistilled 1:1 hydrochloric acid (5 mL
per 100 mL of final volume). See the table on Vigorous digestion volumes.
7. Warm the beaker. Adjust the sample to pH 4 by drop-wise addition of 5.0 N Sodium
Hydroxide Standard Solution; mix thoroughly and check the pH after each addition.
8. Quantitatively transfer the sample to a volumetric flask and dilute to volume with
deionized water. See the Vigorous digestion volumes table for the suggested final
volume.
9. Multiply the result by the correction factor in the Vigorous digestion volumes table.
10. Process a reagent blank to match the sample.
Table 11 Vigorous digestion volumes
Suggested final
Expected metal Suggested sample Suggested volume of
volume after Correction factor
concentration vol. for digestion 1:1 HCl
digestion
1 mg/L 50 mL 10 mL 200 mL 4
10 mg/L 5 mL 10 mL 200 mL 40
100 mg/L 1 mL 25 mL 500 mL 500
4.2 General Digesdahl digestion

Many samples may be digested using the Digesdahl Digestion Apparatus
(Catalog. No. 2313020). It is designed to digest samples such as oils, wastewater,
sludges, feeds, grains, plating baths, food and soils. In this procedure the sample is
oxidized by a mixture of sulfuric acid and hydrogen peroxide. Digestion of a dry sample
requires less than ten minutes, while liquid samples require about 1 minute/mL. The
digestion is done in a special flat-bottomed, 100-mL volumetric flask. Aliquots (sample
portions) are taken for analysis using colorimetric methods.
Procedures for digestion using the Digesdahl Digestion Apparatus are based on the type
and form of the sample and are found in the Digesdahl Digestion Apparatus Instruction
Manual, which is included with each Digesdahl Digestion Apparatus.
Digesdahl digestion is a process that yields a digest suitable for the determination of
metals, total phosphorus and total Kjeldahl nitrogen (TKN). It is faster than traditional
methods, but yields comparable accuracy and precision. The digest can be used with
colorimetric, turbidimetric or titrimetric tests.
Procedures for using the Digesdahl Digestion Apparatus vary with sample type. Sample
types include food products, feeds, grains, wastewater sludges, plating baths, plant
tissues, fertilizers, beverages and oils. Most procedures use a two-phase digestion
process involving concentrated sulfuric acid and 50% hydrogen peroxide. Sulfuric acid
dehydrates and chars the sample. Hydrogen peroxide is added through the capillary flow
funnel to complete decomposition. The analyst controls the digestion time (exposure to
the hydrogen peroxide) by varying the volume of hydrogen peroxide used.
Some samples are more difficult to digest completely. For example, resistant or refractory
materials such as nicotinic acid require several minutes of continued peroxide digestion
after clearing to obtain 100% nitrogen recovery. To make sure complete sample
digestion, consider variables such as sample size, solution temperature and sample
contamination. Refer to the Digesdahl Manual (Catalog No. 2313018) for complete
information.
Page 48
4.2.1 Frequently asked questions regarding digestion procedures

This section provides answers to common questions about digestion.
The reading on my instrument is over-range. What should I do?

The concentration range tables found in digestion procedures are only guidelines. Take a
smaller analysis volume and repeat the procedure. Record the new analysis volume and
use it in the calculation.
Do I have to prepare a reagent blank each time I use reagents with the same
lot number?
To decide, first determine the reading of the reagent blank by zeroing the instrument with
deionized or distilled water. If the reagent blank has an insignificant concentration reading
and the reagents have the same lot number, a reagent blank does not have to be
prepared every time. If the reagent blank shows a reading, either analyze it daily or
subtract the reading from the sample reading. If a reagent blank is not run daily, zero the
instrument with deionized water.
Do I have to follow the exact sample amount and the exact analysis volume given
for each procedure?
The sample amount and the analysis volume for each procedure are only suggested
guidelines. Digest any aqueous solution or suspension sample amount up to 40 mL. Solid
or organic liquid samples require less than 0.5 g of anhydrous material—as a routine
practice, 0.25 g of sample is used.
How can I refine the initial amount of sample required for digestion and the analysis
volume to be used?
The amount of sample to be digested is a critical aspect of the digestion. The aliquot size
of the digest to be used in the analysis is also very important. Tables are provided in each
method for determining amount of initial sample to be digested. In order to optimize the
specific test being performed, the following equations have been developed. Before using
these equations, please refer to the manual specifications for the sample type.
To use the equations, first determine the approximate concentration (in ppm or mg/L or
mg/kg). Next determine the range of the colorimetric test being used (e.g., 0–50 mg/L)
and the midpoint of the test range. (This midpoint range is optimum but can be lowered to
accommodate very low sample concentrations). The midpoint of the test range is
determined by subtracting the lower limit of the range from the higher limit and dividing
by 2.
After finishing these determinations, the following equation may be used:
B×C×D
A = -------------------------
E×F
Where:
A = approximate concentration of sample
B = midpoint of colorimetric test range
C = final volume of digest
D = final volume of analysis
E = sample amount to digest
F = analysis volume of digest
Using algebra, the following two equations are obtained:

B×C×D
equation (1) E = -------------------------
A×F
B×C×D
equation (2) F = -------------------------
A×E
Page 49
Both equations contain two unknown values, E and F and some trial and error may be
required to achieve the optimum values.
Using equation (1): If analyzing for copper using the CuVer™ method with an initial
sample containing approximately 150 ppm Cu, the amount of sample required for
digestion and the aliquot volume to be used can be determined as follows:
Determine the test range. In this example, the test range is assumed to be 0–5.0 ppm
and the midpoint is 2.5. When using the Digesdahl system, the final volume of digest is
100 mL and the procedure calls for a final analysis volume of 25 mL.
Therefore:
E and F are
A = 150 B = 2.5 C = 100 D = 25
unknown
Substituting values into equation (1) gives:

2.5 × 100 × 25 41.7
E = ------------------------------------- or E = -----------
150 × F F
Since CuVer Copper Reagent is pH sensitive, a small analysis volume (0.5 mL) is desired
so that pH adjustment would not be necessary.
With this in mind, a 0.5-mL analysis volume would give:
41.7
E = ----------- = 83.4 mL digestion sample amount
0.5
Because the maximum digestion sample amount is 40 mL for Digesdahl digestions, a
0.5-mL analysis volume would not be acceptable for the range. (This is where trial and
error is necessary). Next, try a 5.0-mL analysis volume and the equation gives:
41.7
E = ----------- = 8.0 mL digestion sample amount
5.0
(Round to the nearest whole number for ease of measure).
From the calculation, an 8.0 mL sample amount would be digested and a 5.0-mL analysis
volume would be taken. A pH adjustment would be necessary before analysis.
Using equation (2): Equation (2) may be used when a minimum sample size is desired
or when a sample has already been digested for one parameter (such as copper) and
another parameter (such as zinc) also needs to be measured. Continuing the example for
copper, above, a zinc test may also be performed. The undigested sample contains
approximately 3 ppm zinc and the Zincon method is being used. The analysis volume can
be determined as follows.
In this example, the Zincon method’s test range is assumed to be 0–2.5 ppm so the
midpoint of the range is 1.25. Therefore values are:
A=3
B = 1.25
C = 100
D = 50
E = 8 (as determined above)
substituting:
1.25 × 100 × 50
F = ---------------------------------------- = 260 mL
3×8
Page 50
This is an extreme example but it illustrates the need to compare the values of D and F to
assure that the analysis volume (F) does not exceed the final analysis volume (D). If F
exceeds D, the analysis cannot be done. A test with a more suitable range is necessary
or a larger sample may be digested for this test. Care must also be taken to assure that
the volume of digest taken for analysis (F) is greater than 0.1 mL because accurately
pipetting less than 0.1 mL is difficult.
As a comparison, assume that the concentration of zinc is 75 ppm (A = 75 instead of 3)
and substituting again will give:
1.25 × 100 × 50
F = ---------------------------------------- = 10.5 mL
75 × 8
In this case, the aliquot volume is less than the final analysis volume and analysis may be
done as stated in the procedure.
Why is the factor in the calculation step either 75, 2500 or 5000 depending on the
method used and where does the factor come from?
In all cases, the factor is a correction for sample dilution. For example, in some tests the
factor is 2500. The Digesdahl digestion total volume is 100 mL, the analysis total volume
is 25 mL and 100 x 25 = 2500. The mL units are not included with the factor because
they cancel out in the formula.
How do I report my total concentration on a dry basis when I am analyzing a slurry?

The sample must be analyzed for moisture content. See the figure for Determining dry
basis weight.
Determining dry basis weight
Figure 12 Determining dry basis weight

1 Weigh an aluminum dish and 3 Place the dish in the oven 5 Weigh the aluminum dish with
record the weight as “A”. (103–105 °C) for two hours. oven-dried sample. Record as “C.”
Note: The oven-dried material
generally is unsuitable for
additional testing and should be
discarded.
2 Weigh out approximately 2 g of 4 Cool to room temperature by 6 Use the following formula to
solid sample into the dish. Record placing in a desiccator calculate the sample on a “dry
the exact weight added as “B.” basis.”
Test result (dry basis) =
C – A-
-------------
B–A
Note: Multiply the test result on an
“as is” basis, by the factor above, to
report as “dry basis.
Table 12 Required appartaus for dry basis weight

Balance, general laboratory, 50-g each 2610300
Page 51
Table 12 Required appartaus for dry basis weight (continued)

Desiccant, Drierite (without indicator) 454 g 2285901
Desiccator, vacuum (uses ceramic
each 2088800
plate)
Dish, moisture determination,
100/pkg 2164000
aluminum, 63 x 17.5 mm
Oven, laboratory
120 VAC each 1428900
or
240 VAC each 1428902
Tongs, crucible each 56900
Table 13 Optional apparatus

Description Unit Catalog No.
Desiccator, without stopcock each 1428500
4.2.2 Adjusting the pH
4.2.2.1 For a metals procedure

Note: If analyzing aliquots smaller than 0.5 mL, pH adjustment is unnecessary.
1. Determine the required volume of sample for analysis from the Sample and Analysis
Volume Tables following each digestion procedure. Determine the required volume of
sample for analysis from the Sample and Analysis Volume Tables following the
digestion procedure. Pipet this volume into a mixing graduated cylinder.
Note: Some methods require pipetting into a volumetric flask or a regular graduated cylinder.
2. Dilute to about 20 mL with deionized water.

3. Add one drop of 2,4 Dinitrophenol Indicator Solution (Cat. No. 186932).
4. Add one drop of 8 N Potassium Hydroxide (KOH) Standard Solution (Cat. No.
28232H), swirling between each addition, until the first flash of yellow appears (pH 3).
If analyzing for potassium, use 5 N sodium hydroxide (Cat. No. 245026) instead. Do
not use a pH meter if analyzing for potassium or silver.
5. Add one drop of 1 N KOH (Cat. No. 2314426). Stopper the cylinder and invert several
times to mix. If analyzing for potassium, use 1 N sodium hydroxide instead.
Note: Use pH paper to make sure that the pH is 3. If it is higher than 4, do not readjust with
acid; start over with a fresh aliquot.
6. Continue to add 1 N KOH in this manner until the first permanent yellow color
appears (pH 3.5–4.0).
7. View the cylinder from the top against a white background. Compare the cylinder to a
second cylinder filled to the same volume with deionized water.
Note: High iron content will cause precipitation (brown cloud) which will co-precipitate other
metals. Repeat this procedure with a smaller aliquot volume.
8. Add deionized water to the volume indicated in the colorimetric procedure for the
parameter under analysis.
9. Continue with the colorimetric procedure.
Page 52
4.2.2.2 For the total Kjeldahl Nitrogen colorimetric method

Consult the spectrophotometer or colorimeter procedure to complete the TKN analysis.
The following is only a guide to use if a procedure is not available.
1. Pipet an appropriate analysis volume into a mixing graduated cylinder.
2. Add one drop of TKN Indicator (Cat. No. 2251900).
3. Add one drop of 8 N KOH Standard Solution (Catalog No. 28232H), swirling between
each addition, until the first flash of pale blue appears (pH 3).
4. Add one drop of 1 N KOH (Cat. No. 2314426). Stopper the cylinder and invert several
times to mix.
Note: View the cylinder from the top against a white background. Compare the cylinder to a
second cylinder filled to the same volume with deionized water.
5. Continue to add 1 N KOH in this manner until the first permanent blue color appears.
6. Add deionized water to the volume indicated in the colorimetric procedure for the
parameter under analysis.
Continue with the colorimetric procedure.
Page 53
Page 54
Section 5 Water Management and Safety
This section provides guidelines for laboratory waste management. These guidelines are
only a summary of basic USEPA requirements and do not relieve the user from
complying with the complete regulations contained in the Code of Federal Regulations
(CFR). The regulations may change or additional state and local laws may apply; waste
generators are responsible for knowing and following all the laws and regulations that
apply to their operations.
5.1 Waste minimization

The most effective way to decrease waste management problems and expense is
through waste minimization. To do this:
• Use the smallest sample size that will produce accurate results.
• Where possible, choose methods that use reagents that pose fewer hazards.
• Eliminate the need to dispose of out-dated materials by purchasing in smaller
quantities.
• Use biodegradable detergents to clean glassware and apparatus unless solvents or
acids are specifically required.
5.2 Regulatory overview

The Resource Conservation and Recovery Act (RCRA) controls all solid waste disposal
with an emphasis on hazardous waste. Title 40 Code of Federal Regulations (CFR) part
260 contains the federal hazardous waste disposal regulations issued in accordance with
the RCRA. The regulations create a system to identify hazardous wastes and track waste
generation, transport and ultimate disposal from cradle to grave. Each facility involved in
hazardous waste management must be registered with the USEPA, with the exception of
conditionally exempt small quantity generators.
Federal regulations recognize three categories of generators with those generating larger
amounts of waste being under stricter control.
The categories are:
Conditionally Exempt Small Quantity Generator— less than 100 kg (220 lb) per month
Small Quantity Generator— between 100 kg (220 lb) and 1000 kg (2200 lb) per month
Large Quantity Generator— greater than 1000 kg (2200 lb) per month.
5.3 Hazardous waste

5.3.1 Definition
For regulatory purposes, a hazardous waste is a material that is subject to special
consideration by the USEPA under 40 CFR 261. State or local authorities may also
designate additional materials as hazardous waste in their areas.
Many toxic compounds are not regulated, but improper management or disposal may
lead to legal problems under CERCLA (Superfund) or common law tort.
Page 55
Water Management and Safety
The definition given by 40 CFR 261 defines a hazardous waste as a solid waste that is
not excluded from regulation and meets one or more of the following criteria:
• It is a discarded commercial chemical product, off-specification species, container
residue or spill residue of materials specifically listed in 40 CFR 261.33 (P- and
U-codes);
• It is a waste from a specific source listed in 40 CFR 261.32 (K-code);
• It is a waste from a non-specific source listed in 40 CFR 261.31 (F-code);
• and/or
• It displays any of the following characteristics of hazardous waste:
• ignitability
• corrosivity
• reactivity
• toxicity
There are exceptions to these regulations; review the regulations to determine applicable
exclusions.
5.3.2 Sample codes

Hazardous wastes are managed by specific codes assigned in 40 CFR 261.20–261.33.
These codes are provided to help identify hazardous waste. The generator is responsible
for making the actual waste code determination.
Selected characteristic waste codes for chemicals which may be generated using
methods for water analysis are given in the following table. A complete list of waste codes
is found in 40 CFR 261.20 through 40 CFR 261.33.
Table 14 Hazardous waste codes
Chemical abstract services Regulatory level
Characteristic USEPA code
(CAS) No. (mg/L)
Corrosivity D002 na na
Ignitability D001 na na
Reactivity D003 na na
Arsenic D004 6440-38-2 5.0
Barium D005 6440-39-3 100.0
Benzene D018 71-43-2 0.5
Cadmium D006 7440-43-9 1.0
Chloroform D022 67-66-3 6.0
Chromium D007 7440-47-3 5.0
Lead D008 7439-92-1 5.0
Mercury D009 7439-97-6 0.2
Selenium D010 7782-49-2 1.0
Silver D011 7440-22-4 5.0
Page 56
5.3.3 How to determine if waste is hazardous

Federal laws do not require testing of material to decide if it is a hazardous waste. If the
product is not specifically listed in the regulations, apply product or generator knowledge
to decide if it is hazardous. Often, there is enough information on a Material Safety Data
Sheet (MSDS) to decide. Look for characteristics of a hazardous waste:
• Flash point is below 60 °C (140 °F) or it is classified by DOT as an oxidizer (D001).
• The pH of the material is £2 orŠ³12.5 (D002).
• The material is unstable, reacts violently with water, may generate toxic gases when
mixed with water (D003).
• It is toxic (D004–D043).
Use the chemical composition data to decide if a material is toxic based on the
concentration of certain contaminants (Heavy metals and a number of organic
compounds). If the waste is a liquid, compare the concentration of contaminants to the
concentrations listed in 40 CFR 26. If the waste is a solid, analyze the sample by the
Toxicity Characteristic Leachability Procedure (TCLP) and then compare the results to the
concentrations in 40 CFR 261.24. Levels above the threshold amounts are considered
hazardous.
For more information on using the MSDS, see Material safety data sheets.
Some tests use or produce a number of chemicals that make the end product a
hazardous waste; for example, the COD tests and Nessler reagent. Hazardous waste
status may also result from substances present in the sample.
5.3.4 Disposal
Hazardous waste must be managed and disposed of according to federal, state and local
regulations. The waste generator is responsible for making hazardous waste
determinations. Analysts should check with their facility’s environmental compliance
department for specific instructions.
Most hazardous wastes should be handled by treatment, storage and disposal facilities
(TSDF) that have USEPA permits. In some cases, the generator may treat the hazardous
waste, but may need a permit from the USEPA and/or state agency. Laboratories are not
exempt from these regulations. If the facility is a “Conditionally Exempt Small Quantity
Generator,” special rules may apply. Check 40 CFR 261 to determine the laws and rules
that apply for a given generator.
The most common allowed treatment is elementary neutralization. This applies to wastes
that are hazardous only because they are corrosive or are listed only for that reason.
Neutralize acidic solutions by adding a base such as sodium hydroxide; neutralize basic
solutions by adding an acid such as hydrochloric acid. Slowly add the neutralizing agent
while stirring. Monitor the pH. When it is at or near 7, the material is neutralized and may
be flushed down the drain. Many generated wastes may be treated in this manner.
Other chemical or physical treatments such as cyanide destruction or evaporation may
require a permit. Check with the environmental department or local regulators to
determine which rules apply to each facility.
Laboratory chemicals may be mixed and disposed of with other hazardous wastes
generated at a facility. They may also be accumulated in accordance with 40 CFR 262.34
satellite accumulation rules. After collection they may be disposed of in a labpack. Many
environmental and hazardous waste companies offer labpacking services. They will
inventory, sort, pack and arrange for proper disposal of hazardous waste. Find
companies offering these services in the Yellow Pages under “Waste Disposal —
Hazardous” or contact state and local regulators for assistance.
Page 57
5.4 Management of specific wastes

Recyling programs for some forms of laboratory waste are available through Hach
Company. Obtain information on recycling services by calling 1-800-227-4224 or by
visiting www.hach.com."
Several documents are also available to assist in managing generated waste. Obtain
available documents by calling 1-800-227-4224 or 970-669-3050 and requesting the
literature codes given:
Table 15 Waste management literature
Literature code Title
9323 Mercury Waste Disposal Firms
9324 RCRA Waste Disposal Information
9325 COD Waste Disposal Information
9326 COD Heavy Metal Concentrations
5.4.1 Special considerations for Cyanide-containing materials

Several procedures in this manual use reagents that contain cyanide compounds. These
materials are regulated as reactive waste (D003) by the Federal RCRA. Instructions
provided with each procedure explain how to collect these materials for proper disposal.
It is imperative that these materials be handled safely to prevent the release of hydrogen
cyanide gas (an extremely toxic material with the smell of bitter almonds). Most cyanide
compounds are stable and can be safely stored for disposal, in highly alkaline solutions
(pH >11) such as 2 N sodium hydroxide. Never mix these wastes with other laboratory
wastes that may contain lower pH materials such as acids or even water.
If a cyanide-containing compound is spilled, avoid exposure to hydrogen cyanide gas.
Take the following steps to destroy the cyanide compounds in an emergency:
1. Use a fume hood, supplied air or self-contained breathing apparatus.
2. While stirring, add the waste to a beaker containing a strong solution of sodium
hydroxide and either calcium hypochlorite or sodium hypochlorite (household bleach).
3. Add an excess of hydroxide and hypochlorite. Let the solution stand for 24 hours.
4. Neutralize the solution and flush it down the drain with a large amount of water. If the
solution contains other regulated materials such as chloroform or heavy metals, it
may still need to be collected for hazardous waste disposal. Never flush untreated
hazardous wastes down the drain.
5.5 Resources
Many sources of information on proper waste management are available. The USEPA
has a hotline number for questions about the Resource Conservation and Recovery Act
(RCRA). The RCRA Hotline number is 1-800-424-9346. Copies of the appropriate
regulations are available. Federal hazardous waste regulations are found in 40 CFR
260-99. Obtain this book from the U.S. Government Printing Office or an alternate
vendor. Other documents that may be helpful to the hazardous waste manager in the
laboratory include:
• Task Force on Laboratory Waste Management. Laboratory Waste Management, A
Guidebook; American Chemical Society, Department of Government Relations and
Science Policy: Washington, DC 1994.
• Task Force on Laboratory Waste Management. Waste Management Manual for
Laboratory Personnel; American Chemical Society, Department of Government
Relations and Science Policy: Washington, DC 1990.
Page 58
• Task Force on Laboratory Waste Management. Less is Better;

2nd ed.; American Chemical Society, Department of Government Relations and
Science Policy: Washington, DC 1993.
• Committee on Chemical Safety. Safety in Academic Chemistry Laboratories, 5th ed.;
American Chemical Society: Washington, DC, 1990.
• Armour, Margaret-Ann. Hazardous Laboratory Chemicals Disposal Guide; CRC
Press: Boca Raton, FL, 1991.
• Environmental Health and Safety Manager’s Handbook; Government Institutes, Inc.:
Rockville, MD, 1988.
• Lunn, G.; Sansone, E.B. Destruction of Hazardous Chemicals in the Laboratory; John
Wiley and Sons: New York, 1990.
• National Research Council. Prudent Practices for Disposal of Chemicals from
Laboratories; National Academy Press: Washington, DC, 1983.
• National Research Council. Prudent Practices for Handling Hazardous Chemicals in
Laboratories; National Academy Press: Washington, DC, 1981.
• Environmental Protection Agency, Office of Solid Waste and Emergency Response.
The RCRA Orientation Manual; U.S. Government Printing Office: Washington, DC,
1991.
• Environmental Protection Agency, Office of Solid Waste and Emergency Response.
Understanding the Small Quantity Generator Hazardous Waste Rules: A Handbook
for Small Business; U.S. Government Printing Office: Washington, DC, 1986.
5.6 Safety
Safety is the responsibility of every analyst. Many chemical procedures use potentially
hazardous chemicals and equipment; it is important to prevent accidents by practicing
good laboratory techniques. The following guidelines apply to water analysis and are not
intended to cover every aspect of safety.
5.6.1 Read labels carefully

Read each reagent label carefully. Pay particular attention to the precautions given.
Never remove or cover the label on a container while it contains reagent. Do not put a
different reagent into a labeled container without changing the label. When preparing a
reagent or standard solution, label the container clearly. If a label is hard to read, relabel
promptly according to the hazard communication program.
Warning labels also appear on some of the apparatus used with the test procedures. The
protective shields with the Digesdahl Digestion Apparatus point out potential hazards. Be
sure these shields are in place during use and observe the precautions on the label.
5.6.2 Protective equipment

Use the right protective equipment for the chemicals and procedures. The MSDS
contains this information. Protective equipment may include:
• Eye protection, such as safety glasses or goggles, to protect from flying objects or
chemical splashes.
• Gloves to protect skin from toxic or corrosive materials, sharp objects, very hot or very
cold materials or broken glass. Use tongs or finger cots when transferring hot
apparatus.
• Laboratory coats or splash aprons to protect skin and clothing from splashes.
• Footwear to protect feet from spills. Open toed shoes should not be worn in chemistry
settings.
Page 59
• Respirators may be needed if adequate ventilation, such as fume hoods, are not
available. Use fume hoods when directed to do so by the procedure or as
recommended in the MSDS. For many procedures, adequate ventilation is enough if
there is plenty of fresh air and air exhaust to protect against unnecessary exposure to
chemicals.
5.6.3 First aid equipment and supplies

Most first aid instructions for chemical splashes in eyes or on skin call for thorough
flushing with water. Laboratories should have eyewash and shower stations. For field
work, carry a portable eyewash unit. Laboratories should also have appropriate fire
extinguishers and fume hoods.
5.6.4 General safety rules

Follow these rules when working with toxic and hazardous chemicals:
• Never pipet by mouth. Always use a mechanical pipet or pipet bulb to avoid ingesting
chemicals.
• Follow test procedures carefully and observe all precautionary measures. Read the
entire procedure before beginning.
• Wipe up all spills promptly. Get proper training and have the right response equipment
to clean up spills. See the safety director for more information.
• Do not smoke, eat or drink in an area where toxic or irritating chemicals are used.
• Use reagents and equipment only as directed in the test procedure.
• Do not use damaged labware and broken equipment.
• Minimize all chemical exposures. Do not breathe vapors or let chemicals touch the
skin. Wash hands after using chemicals.
• Keep work areas neat and clean.
• Do not block exits or access to emergency equipment.
5.7 Material safety data sheets

Material safety data sheets (MSDS) describe the hazards of chemical products. This
section explains the information found on the MSDS and tells how to locate important
information for safety and waste disposal. The information provided on the MSDS applies
to the product as sold by a specific manufacturer. The properties of any mixtures obtained
by using this product will be different.
5.7.1 How to obtain an MSDS

The MSDS is shipped to a customer with the first order of any chemical product. A new
MSDS may be sent when the information on the data sheet is updated. Please review all
new MSDS for new information. To obtain another copy of an MSDS, call 1-800-227-4224
or download directly from www.hach.com/msdsinfo.htm.
5.7.2 Sections of an MSDS

Each MSDS has ten sections. The sections and the information included are
described below.
Header information
The manufacturer order number, MSDS date, change number, company address and
telephone number and emergency telephone numbers are listed at the top of the MSDS.
Page 60
5.7.2.1 Product identification

This section contains:
• Product name
• Chemical Abstract Services (CAS) number
• Chemical name
• Chemical formula, if appropriate
• Chemical family to which the material belongs
5.7.2.2 Ingredients
This section lists each component in the product. It contains the following information for
each component:·
• PCT: Percent by weight of this component
• CAS NO.: Chemical Abstract Services (CAS) registry number for this component
• SARA: Superfund Amendments and Reauthorization Act, better known as the
“Community Right to Know Law”, informs if the component is listed in SARA 313. If the
component is listed and the facility uses more than the specified amount, report use to
the USEPA every year.
• TLV: Threshold Limit Value. The maximum airborne concentration for an 8-hour
exposure that is recommended by the American Conference of Governmental
Industrial Hygienists (ACGIH).
• PEL: Permissible Exposure Limit. The maximum airborne concentration for an 8-hour
exposure that is regulated by the Occupational Safety and Health Administration
(OSHA).
• HAZARD: Physical and health hazards of the component are explained.
5.7.2.3 Physical data

The physical properties of the product are given in this section. They include the physical
state, color, odor, solubility, boiling point, melting point, specific gravity, pH, vapor density,
evaporation rate, corrosivity, stability and storage precautions.
5.7.2.4 Fire, explosion hazard and reactivity data

This section contains the flash point and flammable limits of the material. It also includes
how to fight fires if the material catches on fire. Key terms in this section include:
• Flash point: The temperature at which a liquid will give off enough flammable vapor to
ignite.
• Flammability and ignitability are usually defined by the flash point.
• Lower Flammable Limit (LFL or LEL): The lowest concentration that will produce a fire
or flash when an ignition source is present.
• Upper Flammable Limit (UFL or UEL): The vapor concentration in air above which the
concentration is too rich to burn.
• NFPA Codes: The National Fire Protection Association (NFPA) has a system to rate
the degree of hazards presented by a chemical. These codes are usually placed in a
colored diamond. The codes range from 0 for minimal hazard to 4 for extreme hazard.
They are grouped into the following hazards: health (blue), flammability (red),
reactivity (yellow) and special hazards (white).
Page 61
5.7.2.5 Health hazard data

This section describes the pathways by which the chemical can enter the body (i.e.,
ingestion, inhalation, skin contact). It also gives acute (immediate) and chronic
(long-term) health effects. If the material causes cancer or genetic damage, it is stated in
this section.
5.7.2.6 Precautionary measures

This section contains special precautions for the material. These may include special
storage instructions, handling instructions, conditions to avoid and protective equipment
required to use this material safely.
5.7.2.7 First aid

First aid instructions for exposures to the chemical are given in this section. Be sure to
read this section before inducing vomiting in a victim. Some chemicals are better treated
by not inducing vomiting. Seek prompt medical attention for all chemical exposures.
5.7.2.8 Spill and disposal procedures

This section explains safe practices for cleaning up and disposing of spilled material. For
more information, see Hazardous waste. The waste generator is ultimately
responsible for meeting the federal, state and local laws that apply to each facility.
5.7.2.9 Transportation data

Domestic and International shipping information is provided in this section. It gives
shipping name, hazard class and ID number of the product.
5.7.2.10 References
This section lists the reference materials used to write the MSDS.
Following the Reference section, the product will be listed as having SARA 313
chemicals or California Proposition 65 List Chemicals, if applicable. Also found here is
any special information about the product.
5.7.3 OSHA chemical hygiene plan

The Occupational Safety and Health Administration (OSHA) enforces laws controlling
exposure to hazardous chemicals in laboratories. These regulations are in Title 29 CFR
1910.1450. The regulations apply to all employers who use hazardous chemicals and
require employers to develop and use a written Chemical Hygiene Plan and to appoint a
qualified person as the Chemical Hygiene Officer.
Page 62
Section 6 International Guideline Comparison
The following table shows a comparison between international drinking water and FDA
bottled water guidelines:
Table 16 Comparison of international drinking water and FDA bottled water guidelines1
Bottled Water
USEPA2 Canada3 EEC4 Japan5
U.S. Federal
maximum maximum maximum maximum WHO6
Parameter Drug
contaminant acceptable admissible admissible guideline
Administration
level (MCL) concentration concentration concentration
level
Aluminum 0.05–0.2 mg/L7 — 0.2 mg/L 0.2 mg/L 0.2 mg/L —
Ammonium — — 0.5 mg/L No standard 1.5 mg/L —
Antimony 0.006 mg/L — 0.01 mg/L 0.002 mg/L8 0.005 mg/L —
Arsenic 0.05 mg/L 0.025 mg/L 0.05 mg/L 0.01 mg/L 0.01 mg/L 0.05 mg/L
Barium 2.0 mg/L 1.0 mg/L No standard No standard 0.7 mg/L 2.0 mg/L
Boron — 5.0 mg/L 1.0 mg/L 0.2 mg/L8 0.3 mg/L —
Cadmium 0.005 mg/L 0.005 mg/L 0.005 mg/L 0.01 mg/L 0.003 mg/L 0.005 mg/L
Chloride 250 mg/L7 250 mg/L 250 mg/L 200 mg/L 250 mg/L
Chromium 0.1 mg/L 0.05 mg/L 0.05 mg/L 0.05 mg/L 0.05 mg/L 0.1 mg/L
Coliforms, total
Organisms/100 £% positive 0 0 or MPN £1 0 0 £1 MF
mL
Coliforms (E. coli)
Organisms/100 0 0 0 0 0 —
mL
Color 15 cu7 15 cu 20 mg Pt-Co/L 5 cu 15 cu <15 cu
Copper 1.3 mg/L7 1.0 mg/L 2.0 mg/L 1.0 mg/L 1–2 mg/L 1.0 mg/L
Cyanides 0.2 mg/L 0.2 mg/L 0.05 mg/L 0.01 mg/L 0.07 mg/L —
Fluoride 2.0-4.0 mg/L7 1.5 mg/L 0.7-1.5 mg/L 0.8 mg/L 1.5 mg/L —
Hardness — — 50 mg/L 300 mg/L — —
Iron 0.3 mg/L7 0.3 mg/L 0.2 mg/L 0.3 mg/L 0.3 mg/L —
Lead 0.015 mg/L 0.01 mg/L 0.01 mg/L 0.05 mg/L 0.01 mg/L 0.005 mg/L
Manganese 0.05 mg/L 0.05 mg/L 0.05 mg/L 0.01–0.05 mg/L 0.1–0.5 mg/L —
Mercury 0.002 mg/L 0.001 mg/L 0.001 mg/L 0.0005 mg/L 0.001 mg/L 0.002 mg/L
Molybdenum — — — 0.07 mg/L 0.07 mg/L —
Nickel 0.1 mg/L — 0.02 mg/L 0.01 mg/L8 0.02 mg/L —
Nitrate/Nitrite, total 10.0 mg/L as N — — 10.0 mg/L as N — 10 mg/L as N
50 mg/L as
Nitrates 10.0 mg/L as N 10.0 mg/L as N 50 mg/L 10 mg/L as N —
NO3-
3 mg/L as
Nitrites 1 mg/L as N 3.2 mg/L 0.1 mg/L 10 mg/L 1 mg/L as N
NO2-
2 dilution no.
@ 12 °C;
Odor 3 TON9 — 3 TON — —
3 dilution no.
@ 25 °C.
pH 6.5–8.5 6.5–8.5 6.5–9.5 5.8–8.6 6.5–8.5 —
Phosphorus — — 5 mg/L No Standard — —
0.5 µg/L
Phenols — 0.002 mg/L 0.005 mg/L — —
C6H5OH
Page 63
International Guideline Comparison
Table 16 Comparison of international drinking water and FDA bottled water guidelines1 (continued)
Bottled Water
USEPA2 Canada3 EEC4 Japan5
U.S. Federal
maximum maximum maximum maximum WHO6
Parameter Drug
contaminant acceptable admissible admissible guideline
Administration
level (MCL) concentration concentration concentration
level
Potassium — — 12 mg/L No Standard — —
Selenium 0.05 mg/L 0.01 mg/L 0.01 mg/L 0.01 mg/L 0.01 mg/L 0.05 mg/L
Silica Dioxide — — 10 mg/L No Standard — —
Silver 0.1 mg/L7 0.05 mg/L 0.01 mg/L No standard No standard —
Solids, total
500 mg/L7 500 mg/L No standard 500 mg/L 1000 mg/L —
dissolved
Sodium — — 75-150 mg/L 200 mg/L 200 mg/L —
Sulfate 250 mg/L7 500 mg/L 250 mg/L No Standard 250 mg/L —
Turbidity 0.5-5 NTU 1 NTU 4 JTU 1–2 units 5 NTU <5 NTU
Zinc 5 mg/L7 5.0 mg/L No Standard 1.0 mg/L 3.0 mg/L —
1 Contact the local regulatory agency for the most current information.
2 United States Environmental Protection Agency.
3 These limits are established by Health Canada.
4 In the EEC (European Economic Community), limits are set by the European Committee for Environmental Legislation.
5 In Japan, limits are established by the Ministry of Health and Welfare.
6 World Health Organization.
7 U.S. Secondary MCL.
8 Identified as a parameter to be regulated in the future.
9 Threshold Odor Number.
Page 64
Section 7 Definitions of USEPA Approved and Accepted
7.1 USEPA approved

The United States Environmental Protection Agency (USEPA) establishes limits for
maximum contamination levels of certain constituents in water. It also requires that
specific methodology be used to analyze for these constituents. Sometimes the USEPA
develops these methods; more often, the USEPA evaluates methods developed by
manufacturers, professional groups, and public agencies such as:
• American Public Health Association
• American Water Works Association
• Water Environmental Federation
• American Society for Testing and Materials
• United States Geological Survey
• Association of Official Analytical Chemists
When a method meets the USEPA criteria, it is approved. All USEPA approved methods
are cited in the Federal Register and compiled in the Code of Federal Regulations.
USEPA-approved methods may be used for reporting results to the USEPA and other
regulatory agencies.
7.2 USEPA accepted

Some procedures are equivalent to USEPA approved methods. Even though minor
modifications exist, the USEPA has reviewed and accepted certain procedures for
reporting purposes. These methods are not published in the Federal Register, but are
referenced to the equivalent USEPA method in the procedure.
Page 65
Definitions of USEPA Approved and Accepted
Page 66
Sample cells
2495402 2401906 1353702 1993500
2095000 2095100 2410212 2410222
2427606 4822800 2629250 5940506
2612602 4864302 2122800

Page 67 of 68
Instruments and adapters
DR 5000 DR 2800 and DR 2700 DR2500 DR2400
LZV583 LZV584 LZV585 LZV646
LZV479 5940400 5912200

Page 68 of 68
Procedures for Analysis
Page 69
Page 70
Acid-Base, DT, 8200 and 8233
Acid-Base DOC316.53.01165
Acid Determination and Base Determination Method 8200 and 8233

1 to 4000 meq/L Digital Titrator
Scope and Application: For water, wastewater and seawater.
Test preparation
Before starting the test:
Four drops of Phenolphthalein Indicator Solution1 can be substituted for the Phenolphthalein Indicator Powder Pillow.
A pH meter can be used in place of the indicators. The end point for the titration is pH 8.3.
To change the result from meq/L to normality (N), divide the result in meq/L by 1000.
For added convenience when stirring, use the TitraStir® stirring apparatus1.
1 See Optional reagents and apparatus.
Collect the following items:
Description Quantity
Phenolphthalein Indicator Powder Pillow 1 pillow
Ttitration cartridge (see Range-specific information) 1 cartridge
Digital titrator 1
Delivery tube for digital titrator 1
Graduated cylinder 1
Erlenmeyer flask, 250-mL 1
See Consumables and replacement items for reorder information.
Acid determination (Method 8200)
See
Table 1
1. Select a sample 2. Insert a clean delivery 3. Turn the delivery knob 4. Use a graduated
volume and sodium tube into the titration to eject air and a few drops cylinder or pipet to
hydroxide titration cartridge. Attach the of titrant. Reset the measure the sample
cartridge from the Range- cartridge to the titrator. counter to zero and wipe volume from the Range-
specific information table. the tip. specific information table.
Acid-Base
Page 71
Acid-Base
Acid determination (Method 8200)
5. Transfer the sample 6. Add the contents of 7. Place the delivery tube 8. Use the multiplier in
into a clean, 250-mL one Phenolphthalein into the solution and swirl the Range-specific
Erlenmeyer flask. If the Indicator Powder Pillow. the flask. Turn the knob on information table to
sample volume is less Swirl to mix. The solution the titrator to add titrant to calculate the
than 100 mL, dilute to should be colorless. the solution. Continue to concentration:
approximately 100 mL with swirl the flask and add digits x multiplier =
deionized water. titrant until a light pink meq/L acid
color forms and persists
for 30 seconds. Example: 100 mL of
sample was titrated with
Write down the number of the 8.00 N cartridge and
digits displayed on the 250 digits were used to
counter. reach the endpoint. The
concentration is 250 x 0.1
= 25 meq/L acid
Base determination (Method 8233)
See
Table 1
1. Select a sample 2. Use a graduated 3. Insert a clean delivery 4. Turn the delivery knob
volume and acid titration cylinder or pipet to tube into the titration to eject air and a few drops
cartridge from the Range- measure the sample cartridge. Attach the of titrant. Reset the
specific information table. volume from theRange- cartridge to the titrator. counter to zero and wipe
specific information table. the tip.
Acid-Base
Page 72
Acid-Base
Base determination (Method 8233)
into a clean, 250-mL one Phenolphthalein into the solution and swirl the Range-specific
sample volume is less Swirl to mix. The solution the titrator to add titrant to calculate the
than 100 mL, dilute to should be pink. the solution. Continue to concentration:
deionized water. titrant until the solution is meq/L base
colorless.
Example: 100 mL of
Write down the number of sample was titrated with
digits displayed on the the 8.00 N cartridge and
counter. 250 digits were used to
reach the endpoint. The
= 25 meq/L base
Table 17 Range-specific information

Range (meq/L) Sample volume (mL) Titration cartridge1 (N) Multiplier
1–4 100 1.600 0.02
4–10 50 1.600 0.04
10–40 100 8.00 0.1
20–80 50 8.00 0.2
50–200 20 8.00 0.5
100–400 10 8.00 1.0
200–800 5 8.00 2.0
500–2000 2 8.00 5.0
1000–4000 1 8.00 10.0
1 For acid determinations, use a sodium hydroxide titration cartridge. For base determinations, use a hydrochloric acid or sulfuric acid cartridge.
Interferences
Color or turbidity can mask the color change of the end point. Use a pH meter instead of the color
indicators and titrate to a pH of 8.3 to duplicate the phenolphthalein end point.
Acid-Base
Page 73
Acid-Base
Sample collection, preservation and storage

• Collect samples in clean plastic or glass bottles. Fill completely and cap tightly.
• Prevent excessive agitation or prolonged exposure to air.
• Complete the test procedure as soon as possible after collection for best accuracy. The
sample can be stored for at least 24 hours if cooled to 4 °C (39 °F) or below.
• Warm to room temperature before the test is started.
Accuracy check
The standard solution method can be used to confirm analytical technique and reagent
performance.
Standard solution method
Complete the following test to make sure the concentration of the titrant is accurate.
Required for accuracy check:
• For acid determination—0.500 N Sulfuric Acid Standard Solution
• For base determination—Alkalinity Standard Solution, 0.500 N Na2CO3
1. Add the standard solution to an Erlenmeyer flask:
• If using the 1.600 N Titration Cartridge, pipet 1.0 mL of the standard solution into the
Erlenmeyer flask and dilute to approximately 100 mL with deionized water.
• If using the 8.00 N Titration Cartridge, pipet 5.0 mL of the standard solution into the
Erlenmeyer flask and dilute to approximately 100 mL with deionized water.
2. Add one Phenolphthalein Powder Pillow and swirl to mix.
3. Follow the test procedure to titrate the standard to the end point. The expected digits to reach
the end point are 250.
Summary of method
A measured amount of sample is treated with a phenolphthalein colorimetric indicator and then
titrated with a strong acid or base to a pH of 8.3. The amount of titrant used is directly proportional
to the milliequivalents of acid or base in the sample. These titrations also can be performed using
a pH meter instead of a colorimetric indicator.
Acid-Base
Page 74
Acid-Base
Consumables and replacement items

Required reagents
Description Quantity/Test Unit Catalog number
Hydrochloric Acid Titration Cartridge, 8.00 N varies each 1439001
Phenolphthalein Indicator Powder Pillows 1 pillow 100/pkg 94299
Sodium Hydroxide Titration Cartridge, 1.600 N varies each 1437901
Sodium Hydroxide Titration Cartridge, 8.00 N varies each 1438101
Sulfuric Acid Titration Cartridge, 1.600 N varies each 1438901
Sulfuric Acid Titration Cartridge, 8.00 N varies each 1439101
Required apparatus
Digital Titrator each 1690001
Flask, Erlenmeyer, graduated, 250-mL 1 each 50546
Graduated cylinder—select one or more based on range:
Cylinder, graduated, 5-mL 1 each 50837
Recommended standards
Description Unit Catalog number
Alkalinity Standard Solution, 0.500 N Na2CO3, 10-mL ampules 16/pkg 1427810
Sulfuric Acid Standard Solution, 0.500 N 100 mL 212132
Optional reagents and apparatus

Stir bar, octagonal 28.6 mm x 7.9 mm each 2095352
TenSette Pipet, 0.1 to 1.0 mL each 1970001
Water, deionized 500 mL 27249
Pipet, volumetric, Class A, 1 mL each 1451535
Pipet Filler, safety bulb each 1465100
Bottle, sampling, poly, 500 mL each 2087079
pH meter each —
Delivery Tube, 180° hook 5/pkg 1720500
TitraStir stir plate, 115 Vac each 1940000
Stir Bar, Octagonal 28.6 mm x 7.9 mm each 2095352
Acid-Base
Page 75
FOR TECHNICAL ASSISTANCE, PRICE INFORMATION AND ORDERING: HACH COMPANY
In the U.S.A. – Call toll-free 800-227-4224 WORLD HEADQUARTERS
Outside the U.S.A. – Contact the HACH office or distributor serving you. Telephone: (970) 669-3050
On the Worldwide Web – www.hach.com; E-mail – techhelp@hach.com FAX: (970) 669-2932
© Hach Company, 2007, 2010, 2012. All rights reserved. Printed in the U.S.A. Edition 7
Acid-Base, BT, 8288 and 8289
Acid-Base DOC316.53.01148
Sodium Hydroxide for meq/L of Acid Method 8288

Sulfuric Acid for meq/L of Base Method 8289
(0 to 25,000 meq/L) Buret Titration
Scope and Application: For water, wastewater and seawater
Test preparation
Read the entire procedure before starting the test.
Four drops of Phenolphthalein Indicator Solution can be substituted for the Phenolphthalein Indicator
Powder Pillow.
To determine the normality of the sample, divide the milliequivalents per liter obtained by 1000.
Phenolphthalein indicator powder pillow 1
Sodium hydroxide standard solution (acid test) varies1
Sulfuric acid standard solution (base test) varies1
Buret clamp 1
Buret, Class A 1
Graduated cylinder varies1
Erlenmeyer flask 1
Support Stand 1
1 See Consumables and replacement items.
Acid-Base
Page 77
Acid-Base
Sodium hydroxide acid determination (Method 8288)
See
Table 1
1. Select the sample 2. Fill a 25-mL buret to 3. Measure the sample 4. Pour the sample into a
volume that corresponds the zero mark with the volume from the Sample 250-mL Erlenmeyer flask.
to the expected acid appropriate Sodium volume selection for Dilute to 50–100 mL with
concentration in Hydroxide Solution. expected acid deionized water, if
milliequivalents per liter concentration table using necessary.
(meq/L) or normality (N) a graduated cylinder or
from the Sample volume pipet.
selection for expected acid
concentration table.
5. Add the contents of 6. Swirl the flask while 7. Calculate:

one Phenolphthalein titrating until a light pink mL NaOH Titrant ×
Indicator Powder Pillow color forms and persists Multiplier used = meq/L of
and swirl to mix. The for 30 seconds. Acid
solution should be
colorless.
Table 18 Sample volume selection for expected acid concentration

Range Sample Volume Sodium Hydroxide Standard Catalog
Multiplier
(meq/L) (N) (mL) Solution Titrant (N) Number
0–10 0–0.010 50 0.020 19353 0.4

10–100 0.010–0.100 25 0.100 19153 4
100–500 0.100–0.500 50 1.000 104553 20
500–2500 0.500–2.50 10 1.000 104553 100
2500–25000 2.50–25.0 1 1.000 104553 1000
Acid-Base
Page 78
Acid-Base
Sulfuric acid base determination (Method 8289)
See
Table 2
1. Select the sample 2. Fill a 25-mL buret to 3. Measure the sample 4. Pour the sample into a
volume that corresponds the zero mark with the volume from Table 2 using 250-mL Erlenmeyer flask.
to the expected base appropriate Sulfuric Acid a graduated cylinder or Dilute to 50–100 mL with
concentration in Standard Solution. pipet. deionized water, if
milliequivalents per liter necessary.
(meq/L) or normality (N)
from the Sample volume
selection for expected
base concentration table.
5. Add the contents of 6. Swirl the flask while 7. Calculate:

one Phenolphthalein titrating just until the mL Sulfuric Acid Titrant X
Indicator Powder Pillow solution turns colorless. Multiplier used = meq/L of
and swirl to mix. The Base
solution should turn pink.
Table 19 Sample volume selection for expected base concentration

Range Sulfuric Acid Standard Solution
Sample Volume (mL) Catalog Number Multiplier
(meq/L) (N) Titrant (N)
0–10 0–0.010 50 0.020 20353 0.4

10–100 0.010–0.100 25 0.100 20253 4
100–500 0.100–0.500 50 1.000 127053 20
500–2500 0.500–2.50 10 1.000 127053 100
2500–25000 2.50–25.0 1 1.000 127053 1000
Acid-Base
Page 79
Acid-Base
Interferences
Highly colored or turbid samples may mask the color change at the end point. Use a pH meter for
these samples. Titrate to pH 8.3 to duplicate the colorimetric phenolphthalein end point.
Sampling and storage

Collect samples in clean plastic or glass bottles. Fill completely and cap tightly. Minimize agitation
or prolonged exposure to air. Samples may be stored at least 24 hours by cooling to 4 °C (39 °F)
or below if they cannot be analyzed immediately. Warm to room temperature before analyzing.
Accuracy check
For acid determinations, use a 20-mL volumetric pipet to measure Sulfuric Acid Standard Solution
of the same normality as the Sodium Hydroxide Standard Solution being used. Pipet the sulfuric
acid into an Erlenmeyer flask. Dilute to the 100-mL mark with deionized water. Perform the test; 20
mL of sodium hydroxide should be required.
For base determinations, use a 20-mL volumetric pipet to measure Sodium Hydroxide Standard
Solution of the same normality as the Sulfuric Acid Standard Solution being used. Pipet sodium
hydroxide into an Erlenmeyer flask. Dilute to the 100-mL mark with deionized water. Perform the
test; 20 mL of sulfuric acid should be required.
If incorrect results are obtained, refer to Method Performance in the Water Analysis Guide.
Summary of method
A measured amount of sample is treated with a phenolphthalein colorimetric indicator and then
titrated with a strong acid or base to pH 8.3. The amount of titrant used is directly proportional to
the milliequivalents of acid or base in the sample. These titrations also can be performed using a
pH meter instead of a colorimetric indicator.
Acid-Base
Page 80
Acid-Base
Required reagents
Description Quantity/test Unit Catalog number
Phenolphthalein Indicator Powder Pillow 1 pillow 100/pkg 94299
Water, deionized varies 4L 27256
For acid, pick one or more based on range:
Sodium Hydroxide Standard Solution, 0.020 N — 1L 19353
For base, pick one or more based on range:
Sulfuric Acid Standard Solution, 0.020 N — 1L 20353
Required apparatus
Buret clamp, double 1 each 32800
Buret, Class A, 25-mL 1 each 2636540
Pick one or more based on sample volume:
Cylinder, graduated, 5-mL — each 50837
Flask, Erlenmeyer, 250-mL 1 each 50546
Pipet, volumetric, Class A, 1-mL 1 each 1451535
Support stand 1 each 56300
Optional items
Pipet, 20-mL, volumetric each 1451520
Pipet filler, Safety bulb each 1465100
Phenolphthalein Indicator Solution, 5 g/L100 mL — 16232
Acid-Base
Page 81
Acid-Base, BT, 8219
Acidity, Methyl Orange DOC316.53.01150
Sodium Hydroxide with a Buret1 Method 8219

(0 to 10,000 mg/L as CaCO3) Buret Titration
1 Adapted from Standard Methods for the Examination of Water and Wastewater.
Test preparation
Avoid excessive agitation of the sample to prevent the loss of dissolved gases such as carbon dioxide, hydrogen sulfide
or ammonia.
Six drops of Phenolphthalein Indicator Solution may be substituted for the Phenolphthalein Powder Pillow.
When determining Methyl Orange Acidity, six drops of Bromphenol Blue Indicator Solution can be substituted for the
Bromphenol Blue Indicator Powder Pillow. Methyl Orange Indicator Solution can also be substituted for the indicator. When
using the Methyl Orange Indicator Solution, the end point color change then becomes red to orange.
The Methyl Orange and Phenolphthalein Acidity procedures can be run sequentially if both values are desired. First, titrate to
pH 3.7 and record the amount of sodium hydroxide used. Then titrate to pH 8.3.
Phenolphthalein acidity in mg/L CaCO3 x 0.0584 = grains per gallon
Bromphenol Blue indicator powder pillow 1
Sodium hydroxide standard solution, 0.020 N varies1
Buret clamp 1
Buret, Teflon plug, Class A 1
Erlenmeyer flask 1
Funnel, Micro 1
Support Stand 1
1 See Consumables and replacement items on page 86.
Acidity, Methyl Orange

Page 83
Buret titration (Method 8219)
See
Table 1
1. Select a sample 2. Use a graduated 3. Transfer the sample 4. Add the contents of
volume from the Sample cylinder or pipet to into a 250-mL Erlenmeyer one Bromphenol Blue
volume selection for measure the sample flask. Dilute to about Indicator Powder Pillow.
expected concentration volume. 50-mL with deionized Swirl to mix. (Omit this
table that corresponds to water if necessary. step when using a
the expected acidity pH meter.)
concentration in mg/L as
calcium carbonate
(CaCO3).
5. Fill a 50-mL buret to 6. While swirling the 7. Calculate:

the zero mark with 0.020 N flask, titrate the sample mL Titrant × multiplier
Sodium Hydroxide with 0.020 N Sodium used = mg/L methyl
Standard Solution. Hydroxide Standard orange acidity as CaCO3
Solution until the solution
color changes from yellow
to pure green (pH 3.7).
Table 20 Sample volume selection for expected concentration

Range
Sample Volume (mL) Sodium Hydroxide Multiplier
(mg/L as CaCO3)
1–1000 50 19353 20
800–2000 25 19353 40
2000–5000 10 19353 100
4000–10000 5 19353 200
Interferences
these samples. Titrate to pH 3.7.

Page 84
Add a drop of 0.1 N Sodium Thiosulfate Standard Solution to remove residual chlorine that may
interfere with the indicator.
Collect samples in clean plastic or glass bottles. Fill completely and cap tightly. Avoid excessive
agitation and prolonged exposure to air. Samples should be analyzed as soon as possible after
collection but can be stored at least 24 hours by cooling to 4 °C (39 °F) or below. Warm to room
temperature before analyzing.
Accuracy check
End point confirmation
To determine the correct end point, dissolve the contents of a Buffer Powder Pillow, pH 3.7, in 50
mL of deionized water in a 250-mL Erlenmeyer flask. Add the contents of one Bromphenol Blue
Indicator Powder Pillow and swirl to mix. Titrate the prepared water test samples to this same
color.
Standard additions method (Sample spike)
The standard additions method check can be performed as follows:
4. Use a TenSette Pipet to add 0.1 mL of Sulfuric acid standard solution 0.500 N to a prepared
sample titrated to the end point.
5. Swirl to mix and titrate again to the end point. Note the amount of additional titrant used.
6. Make 0.2-mL and 0.3-mL acid additions, titrating to the end point after each addition. The mL
of titrant required should increase by 2.5 mL for each 0.1-mL increment of acid added. If these
increases do not occur, refer to the Water Analysis Guide for the Standard Additions.

Sodium Hydroxide Standard Solution slowly absorbs carbon dioxide when exposed to air, causing
a partial loss of strength. Check the solution frequently (monthly) by titrating 50 mL of Potassium
Acid Phthalate Standard Solution as 100 mg/L CO2 and using a Bromphenol Blue Indicator
Powder Pillow. The titration should require 5.68 mL of Sodium Hydroxide Standard Solution. If the
volume required for the titration is greater than 5.88 mL, discard the solution and replace with a
fresh supply.
Summary of method
Acidity is classified by the pH value of the titration end point. Various pH indicators are used
depending on the pH end point selected. Acidity also can be determined by using a pH meter to
follow the solution pH to the correct end point value as the standard base is added.

Page 85
Required reagents
Bromphenol Blue Powder Pillows 1 pillow 100/pkg 1455099
Sodium Hydroxide Standard Solution, 0.020 N varies 1L 19353
Water, Deionized varies 500 mL 27249
Required apparatus
Buret, Teflon Plug, Class A, 25-mL 1 each 2636540
Buret Clamp, double 1 each 32800
Select one or more based on sample volume:
Pipet, volumetric, Class A, 5 mL — each 1451537
Support Stand 1 each 56300
Funnel, Micro 1 each 2584335
Required standards
Descripiton Unit Catalog number
Buffer Powder Pillows, pH 3.7 25 1455168
Potassium Acid Phthalate Standard Solution, 100-mg/L as CO2 100 mL 226142
Optional items
Description Catalog number
Meter, pH, SenION 1 with Platinum pH electrode 5170010
Pipet Filler 1465100
Sodium Thiosulfate Standard Solution, 0.1 N 32332

Acid-Base, BT, 8010
Acidity, Phenolphthalein DOC316.53.01149
Sodium Hydroxide with a Buret1 Method 8010

1 Adapted from Standard Methods for the Examination of Water and Wastewater, 23/0 B (4a).
Test preparation
Avoid excessive agitation of the sample to prevent the loss of dissolved gases such as carbon dioxide, hydrogen sulfide or
ammonia.
Six drops of Phenolphthalein Indicator Solution may be substituted for the Phenolphthalein Powder Pillow.
The Methyl Orange and Phenolphthalein Acidity procedures can be run sequentially if both values are desired. First, titrate to
pH 3.7 and record the amount of sodium hydroxide used. Then titrate to pH 8.3.
Phenolphthalein acidity in mg/L x 0.0584 = grains per gallon
Sodium hydroxide standard solution, 0.020 N varies1
Buret clamp 1
Buret, Teflon plug, Class A 1
Erlenmeyer flask 1
Funnel, Micro 1
Support Stand 1
Acidity, Phenolphthalein
Page 87
See
Table 1
volume from the Sample cylinder or pipet to into a 250-mL Erlenmeyer one Phenolphthalein
table that corresponds to water if necessary. step if you are using a
the expected acidity pH meter.)
calcium carbonate
(CaCO3).
5. Fill a 50-mL buret to 6. While swirling the 7. Calculate:

the zero mark with 0.020 N flask, titrate the sample mL Titrant × multiplier
Sodium Hydroxide with 0.020 N Sodium used = mg/L
Standard Solution. Hydroxide Standard phenolphthalein acidity
Solution until the solution as CaCO3
color changes from
colorless to a light pink
that persists for 30
seconds (pH 8.3).

Range
Sample Volume (mL) Sodium Hydroxide Multiplier
(mg/L as CaCO3)
1–1000 50 19353 20
800–2000 25 19353 40
2000–5000 10 19353 100
4000–10000 5 19353 200
Page 88
Interferences
these samples. Titrate to pH 8.3.
Add a drop of 0.1 N Sodium Thiosulfate Standard Solution to remove residual chlorine that may
interfere with the indicator.
Pretreat samples that contain significant amounts of hydrolyzable metals such as iron, aluminum
or manganese as follows:
1. Adjust the sample taken in step 1 of the procedure to pH 4.0 or less (if necessary) by adding
5.0-mL increments of 0.020 N Sulfuric Acid Standard Solution. Record the amount of acid
added. Use a pH meter or appropriate indicator to determine when pH 4.0 is reached. Remove
the pH electrodes after completing this step if a pH meter is used.
2. Using a 1-mL glass serological pipet and pipet filler, add five drops of 30% Hydrogen Peroxide
Solution.
3. Boil the solution for two to five minutes.
4. Cool to room temperature and titrate to pH 8.3 as described in the procedure beginning with
step 3 of the procedure.
5. Subtract the number of milliliters of 0.020 N Sulfuric Acid Standard Solution added from the
number of milliliters of 0.020 N Sodium Hydroxide Standard Solution used in the titration
before multiplying the volume of sodium hydroxide by the multiplier used in step 7 of the
procedure.

agitation and prolonged exposure to air. Samples should be analyzed as soon as possible after
collection but can be stored at least 24 hours by cooling to 4 °C (39 °F) or below. Warm to room
temperature before analyzing.
Accuracy check
To determine the correct end point, dissolve the contents of a Buffer Powder Pillow, pH 8.3, in 50
mL of deionized water in a 250-mL Erlenmeyer flask. Add the contents of one Phenolphthalein
Indicator Powder Pillow and swirl to mix. Titrate the prepared water test samples to this same
color.
The standard additions method check can be performed as follows:
1. Use a TenSette Pipet to add 0.1 mL of Sulfate standard solution 0.500 N to a prepared sample
titrated to the end point.
2. Swirl to mix and titrate again to the end point. Note the amount of additional titrant used.
3. Make 0.2-mL and 0.3-mL acid additions, titrating to the end point after each addition. The mL
of titrant required should increase by 2.5 mL for each 0.1-mL increment of acid added. If these
increases do not occur, refer to the Water Analysis Guide for Standard Additions.
Sodium Hydroxide Standard Solution slowly absorbs carbon dioxide when exposed to air, causing
a partial loss of strength. Check the solution frequently (monthly) by titrating 50 mL of Potassium
Acid Phthalate Standard Solution as 100 mg/L CO2and using a Phenolphthalein Indicator Powder
Pillow. The titration should require 5.68 mL of Sodium Hydroxide Standard Solution. If the volume
required for the titration is greater than 5.88 mL, discard the solution and replace with a fresh
supply.
Page 89
Summary of method
Acidity is classified by the pH value of the titration end point. Various pH indicators are used
depending on the pH end point selected. Acidity also can be determined by using a pH meter to
follow the solution pH to the correct end point value as the standard base is added.
Required reagents
Phenolphthalein Powder Pillows 1 pillow 100/pkg 94299
Water, Deionized varies 500 mL 27249
Required apparatus
Buret, Teflon Plug, Class A, 25-mL 1 each 2636540
Pipet, volumetric, Class A, 5 mL — each, 1451537
Pipet Filler, Safety bulb — each 1465100
Required standards
Sulfuric Acid Standard Solution, 0.500 N 100 mL MDB 212132
Buffer Powder Pillows, pH 8.3, 50 mL 25/pkg 89868
Optional items
Hydrogen Peroxide Solution. 30 % 473 mL 14411
Meter, pH, SensION 1 with Platinum pH electrode each 5170010
Phenolphthalein Indicator Solution 100 mL MDB 16232
Pipet, 1-mL glass serological pipet each 53235
Pipet Filler each 1465100
Sodium Thiosulfate Standard Solution, 0.1 N 100 mL MDB 32332

Acidity, DT, 8201 and 8202
Acidity DOC316.53.01164
Methyl Orange and Phenolphthalein (Total) Acidity Method 8201 and 8202
10 to 4000 mg/L CaCO3 Digital Titrator
Test preparation
Avoid excess agitation when collecting and swirling the sample to prevent the loss of gases such as carbon dioxide,
hydrogen sulfide and ammonia.
Six drops of Bromphenol Blue Indicator Solution1 can be substituted for the Bromphenol Blue Indicator Powder Pillow.
A pH meter can be used in place of the indicators. The end point for methyl orange acidity is pH 3.7. The end point for
phenolphthalein acidity is pH 8.3.
Bromphenol Blue Indicator Powder Pillow 1 pillow
Sodium Hydroxide Titration Cartridge (see Range-specific information) 1 cartridge
Digital Titrator 1
Delivery Tube for Digital Titrator 1
Graduated Cylinder 1
Erlenmeyer Flask, 250-mL 1
Acidity
Page 91
Acidity
Methyl orange acidity (Method 8201)
See
Table 1
volume and titration tube into the titration to eject air and a few drops cylinder or pipet to
cartridge from the Range- cartridge. Attach the of titrant. Reset the measure the sample
specific information. cartridge to the titrator. counter to zero and wipe volume from the Range-
the tip. specific information.
into a clean, 250-mL one Bromphenol Blue into the solution and swirl the Range-specific
sample volume is less Swirl to mix. the titrator to add titrant to calculate the
than 100 mL, dilute to the solution. Continue to concentration:
deionized water. titrant until the color mg/L as CaCO3 methyl
changes from yellow to orange acidity
blue-violet (pH 3.7).
Example: 100 mL of
Write down the number of sample was titrated with
= 25 mg/L as CaCO3
Acidity
Page 92
Acidity
Phenolphthalein (total) acidity (Method 8202)
1. Measure a second 2. Add the contents of 3. Place the delivery tube 4. Use the multiplier in
portion of the sample from one Phenolphthalein into the solution and swirl Range-specific information
the Range-specific Indicator Powder Pillow. the flask. Turn the knob on table to calculate the
information table into a Swirl to mix. the titrator to add titrant to concentration:
clean, 250-mL Erlenmeyer the solution. Continue to digits x multiplier =
flask. If the sample volume swirl the flask and add mg/L as CaCO3
is less than 100 mL, dilute titrant until the color phenolphthalein acidity
to approximately 100 mL changes from colorless to
with deionized water. a light pink color that Example: 100 mL of
persists for 30 seconds. sample was titrated with
the 1.600 N cartridge and
Write down the number of 250 digits were used to
digits displayed on the reach the endpoint. The
counter. concentration is 250 x 1.0
= 250 mg/L as CaCO3

Range (mg/L as CaCO3) Sample volume (mL) Titration cartridge (N NaOH) Multiplier
10–40 100 0.1600 0.1

40–160 25 0.1600 0.4
100–400 100 1.600 1.0
200–800 50 1.600 2.0
500–2000 20 1.600 5.0
1000–4000 10 1.600 10.0
Interferences
Interfering substances lists substances that can interfere with this test.
Table 23 Interfering substances
Interfering substance Interference level
Chlorine may interfere with the indicators. Add one drop of 0.1 N Sodium Thiosulfate to the
Chlorine
sample to remove chlorine before starting the test.
Color or turbidity can mask the color change of the end point. Use a pH meter instead of the
Color or turbidity color indicators and titrate to a pH of 3.7 for methyl orange acidity or pH 8.3 for
phenolphthalein acidity.
Acidity
Page 93
Acidity
Table 23 Interfering substances (continued)

Samples that contain hydrolyzable metals such as iron, manganese or aluminum should be
pretreated before the test for phenolphthalein acidity is started.
1. Measure the sample volume into the flask.
2. Use the Digital Titrator and a Sulfuric acid cartridge of the same normality as the one
being used for the Acidity test to adjust the sample pH to pH 4 or less. Write down the
number of digits of acid that was added to lower the pH.
Hydrolyzable metals 3. Add five drops of 30% hydrogen peroxide solution.
4. Boil the solution for 2–5 minutes.
5. Cool the solution to room temperature.
6. Add the phenolphthalein indicator and titrate the sample.
7. Subtract the number of digits of acid that were added in step 2 from the number of digits
that were used to reach the end point in step 6. Multiply this number by the multiplier in
Range-specific information to find the phenolphthalein acidity in the sample.

• Complete the test procedure as soon as possible after collection for best accuracy. The
sample can be stored for at least 24 hours if cooled to 4 °C (39 °F) or below.
Accuracy check
Use a buffer pillow with the same pH as the end point with the indicator to make sure the end point
color is accurate.
• Methyl orange acidity—Add 50 mL of deionized water to a flask. Add one pH 3.7 buffer
powder pillow and one Bromphenol Blue Indicator Powder Pillow and swirl to mix. Use this
solution for comparison during the titration with the sample.
• Phenolphthalein acidity—Add 50 mL of deionized water to a flask. Add one pH 8.3 buffer
powder pillow and one Phenolphthalein Indicator Powder Pillow and swirl to mix. Use this
solution for comparison during the titration with the sample.
Standard additions method (sample spike)
• Sulfuric Acid Standard Solution, 0.500 N
• Ampule breaker
• TenSette Pipet, 0.1–1.0 mL
1. Open the standard solution.
2. Use the TenSette Pipet to add 0.1 mL of the standard to the titrated sample. Swirl to mix.
3. Titrate the spiked sample to the end point. Write down the amount of titrant that was used to
reach the end point.
4. Use the TenSette Pipet to add 0.2 mL of standard to the titrated sample. Swirl to mix.
Acidity
Page 94
Acidity
8. Each 0.1 mL of standard that was added will use approximately 25 digits of the 1.600 N
titration cartridge or 250 digits of the 0.1600 N titration cartridge to reach the endpoint. If more
or less titrant was used, there can be an interference (see Interferences) or the concentration
of the titrant has changed.
Summary of method
Bromphenol blue or phenolphthalein indicator is used to titrate the sample with sodium hydroxide
to a colorimetric end point. Bromphenol blue gives a better end point than methyl orange indicator.
Titration to pH 3.7 determines strong mineral acidity (also referred to as methyl orange acidity),
whereas the pH 8.3 phenolphthalein end point includes weaker acid species as well and
represents the total acidity. The results are expressed in mg/L as calcium carbonate (CaCO3) at a
specified pH.
Required reagents
Acidity Reagent Set (approximately 100 tests) 2272800
(1) Bromphenol Blue Powder Pillows 1 pillow 100/pkg 1455099
(1) Phenolphthalein Indicator Powder Pillows 1 pillow 100/pkg 94299
(1) Sodium Hydroxide Titration Cartridge, 0.1600 N varies each 1437701
Required apparatus
Digital Titrator 1 each 1690001
SUlfuric Acid Standard Solution, 0.500 N H2SO4, 10-mL 100 mL 212132
Acidity
Page 95
Acidity

Buffer Powder Pillows, pH 3.7 25/pkg 1455168
Pipet tips 50/pkg 2185696
TitraStir Stir Plate, 115 VAC each 1940000
Sulfuric Acid DT cartridge, 0.1600 each 1438801
Sulfuric Acid DT cartridge, 1.600 each 1438901
Bottles, sampling, poly, 500 mL each 2087079
Bromphenol Blue indicator solution 100 mL MDB 1455232
Phenolphthalein Indicator solution, 5 g/L 100 mL MDB 16232
pH meter each —
Delivery tube, 180° hook 5/pkg 1720500

Alachlor
Alachlor DOC356.53.01001
Immunoassay Method1 Method 10202

Scope and Application: For water
1 This test is semi-quantitative. Results are expressed as greater or less than the threshold value used.
Test preparation
How to use instrument-specific information

The Instrument-specific information table displays requirements that may vary between
instruments. To use this table, select an instrument then read across to find the corresponding
information required to perform this test.
Table 24 Instrument-specific information

Instrument Adapter
DR 6000 —
DR 5000 A23618
DR 3900 LZV846(A)
DR3800, DR 2800, DR 2700 LZV583
This method analyzes for Alachlor in water. Sample calibrators and reagents are added to cuvettes coated with Alachlor-
specific antibodies. The color that develops is then measured and compared with the color measurements of the calibrators.
The test requires about 30 minutes for complete analysis. As many as 20 cuvettes (18 samples and 2 calibrators) can be run
simultaneously.
Read the entire procedure before starting. Identify and make ready all the necessary reagents, cuvettes, and other
apparatus before beginning the analysis.
Timing is critical; follow instructions carefully.
A consistent technique when mixing the cuvettes is critical to this test. The best results come from using the cuvette
rack and mixing as described in Using the 1-cm MicroCuvette Rack. Cuvettes can be mixed individually, but test results may
not be as consistent.
Handle the cuvettes carefully. Scratches on the inside or outside may cause erroneous results. Carefully clean the outside of
the cuvettes with a clean absorbent cloth or tissue before placing them into the instrument.
Antibody cuvettes and enzyme conjugate are made in matched lots. Do not mix reagent lots.
To avoid damaging the Color Developing Solution, do not expose it to direct sunlight.
The cuvette rack is designed to be inverted with the cuvettes in place. This is especially helpful when running many samples
at once; the cuvettes can remain in the rack and be processed together until they are read in the spectrophotometer.
Twenty Antibody Cuvettes are provided with each reagent set. One Antibody Cuvette will be used for each calibrator and
each sample. Cuvettes are not reusable.
Protective nitrile gloves are recommended for this procedure.
Alachlor
Page 97
Alachlor
Alachlor Reagent Set 1
Caps, flip spout 1
Marker, laboratory 1
Rack, for 1-cm Micro Cuvettes 1
Wipes, disposable 1
Pipet, TenSette®, 0.1–1.0 mL 1
Pipet tip for 19700-01, TenSette Pipet 1
Immunoassay for Water
Single Wavelength
4 5 0
OK
1. Press 2. Label an Antibody 3. Insert the cuvettes into 4. Pipet 0.5 mL of each
SINGLE WAVELENGTH Cuvette for each calibrator the rack snugly. calibrator into the
and each sample to be appropriately labeled
Press OPTIONS and the λ
tested. cuvette.
button.
Use a new pipette tip for
Enter 450 NM and press
each calibrator.
OK.
Insert an adapter if
required (Instrument-
specific information). Refer
to the user manual for
orientation.
Alachlor
Page 98
Alachlor
Immunoassay for Water (continued)
5. Pipet 0.5 mL of each 6. Immediately pipet 7. Set the instrument 8. After 10 minutes mix
sample to be tested into 0.5 mL of Alachlor timer for 20:00 minutes. the contents of the rack for
the appropriately labeled Enzyme Conjugate into Press OK to start a 20- 30 seconds using the
cuvette. each cuvette. minute reaction time. technique described
Use a new pipette tip for Use the same pipette tip Immediately mix the in Using the 1-cm
each sample. for each sample. contents of the cuvettes MicroCuvette Rack.
for 30 seconds using the
technique described
in Using the 1-cm
MicroCuvette Rack.
9. At the end of the 10. Wash each cuvette

20-minute period, discard forcefully and thoroughly
the contents of all the four times with deionized
cuvettes into an water. Empty the rinse
appropriate waste water into the waste
container. container.
Ensure that most of the
water is drained from the
cuvettes by turning the
cuvettes upside down and
tapping them lightly on a
paper towel.
Alachlor
Page 99
Alachlor
Color Development
Important Note: Timing is critical. Follow instructions carefully.
11. With the cuvettes still 12. Set the instrument 13. After 5 minutes, mix 14. At the end of the
held snugly in the rack, timer for 10:00 minutes. the contents of the rack a 10-minute reaction period,
pipet 0.5 mL of Color Press OK to start the second time for a period of pipette 0.5 mL of Stop
Developing Solution into reaction period. 30 seconds using the Solution into each cuvette
each Antibody Cuvette. Mix, using the instructions same technique as in the same order as the
Use the same pipette tip in Using the 1-cm step 12. Color Developing Solution
for each sample. MicroCuvette Rack. Solutions will turn blue in was added in step 11.
some or all of the cuvettes. Slide the rack for 20
seconds (Using the 1-cm
MicroCuvette Rack.)
Blue solutions will turn
yellow with the addition of
the Stop Solution.
Use the same pipette tip
repeatedly for this step.
Alachlor
Page 100
Alachlor
Measuring the Color
Zero
15. Label and fill a Zeroing 16. Insert the filled 17. ZERO the instrument. 18. Insert the first
Cuvette with deionized Zeroing Cuvette into the The display will show: calibrator into the cell
water. Wipe the outside of cell holder. 0.000 Abs holder.
all the cuvettes with a Orient the arrow in the READ the results in (ABS)
tissue to remove water, same direction for all for each calibrator
smudges, and fingerprints. cuvettes. and sample. Record the
results.
Repeat this step for all
remaining calibrators and
samples.
See Interpreting and
reporting results for help
with interpretation of
results.
Using the 1-cm MicroCuvette Rack
The MicroCuvette rack (Figure 1) has been designed specifically to aid in achieving precise and
accurate results when using the immunoassay technique to analyze several samples at the same
time.
Figure 1 The 1-cm MicroCuvette Rack
Alachlor
Page 101
Alachlor
Loading the rack—The cuvette rack is designed so that it may be inverted with the cuvettes in
place. Identify each cuvette with a sample or calibrator number and insert all the cuvettes in the
rack before beginning the procedure. Fit the cuvettes snugly into the rack, but do not force them or
they may be difficult to remove and their contents may spill. The cuvettes should remain in place
when the rack is inverted and tapped lightly.
Mixing—Set the rack on a hard, flat surface that is at least twice the length of the rack. Hold the
rack by one end and vigorously slide it back and forth along its long axis for 30 seconds. The rack
should move through a distance equal to its own length in each direction.
Interpreting and reporting results
There is an inverse relationship between the concentration of Alachlor and the absorbance
reading. In other words, the higher the reading, the lower the concentration of Alachlor (see
Relative Alachlor concentration)
Table 25 Relative Alachlor concentration

If the sample reading is... the sample Alachlor Concentration is...
…less than calibrator reading …greater than the calibrator concentration
…greater than calibrator reading …less than the calibrator concentration
Example
Readings:
0.1 ppb Alachlor Calibrator: 0.475 Abs
0.5 ppb Alachlor Calibrator: 0.245 Abs
Sample #1: 0.140 Abs
Interpretation
Sample #1—Sample reading is less than the readings for both calibrators. Therefore the sample
concentration of Alachlor is greater than both 0.1 ppb and 0.5 ppb Alachlor.
Sample #2—Sample reading is between the readings for the 0.1 ppb and 0.5 ppb Alachlor
calibrators. Therefore the sample concentration of Alachlor is between 0.1 ppb and 0.5 ppb.
Sample #3—Sample reading is greater than the readings for both calibrators. Therefore the
sample concentration of Alachlor is less than both 0.5 ppb and 0.1 ppb.
Storing and handling reagents
• Wear protective gloves and eyewear.
• When storing reagent sets for extended periods of time, keep them out of direct sunlight. Store
reagents at a temperature of 4 °C when not in use.
• Keep the foil pouch containing the Antibody Cuvettes sealed when not in use.
• If Stop Solution comes in contact with eyes, wash thoroughly for 15 minutes with cold water
and seek immediate medical help.
Sensitivity
The Alachlor immunoassay test cannot differentiate between certain herbicides and metabolites,
but it detects their presence to differing degrees. The Required concentration for selected
chemicals table shows the required concentration for selected chemicals.
Alachlor
Page 102
Alachlor
Table 26 Required concentration for selected chemicals

Concentration to give a positive Concentration to give a positive
Compound
response of 0.1 ppb Alachlor response of 0.5 ppb Alachlor
Acetochlor 0.45 ppb 4 ppb
Butachlor 0.09 ppm 1 ppm
2 Chloro-2',6'-Diethylacetaniline 0.030 ppm 2 ppm
Metolachlor 0.085 ppm 2 ppm
2,6-Diethylaniline 0.3 ppm 9 ppm
Propachlor 0.72 ppb 12 ppb
Table 27 Compounds tested and not detected up to 10,000 ppb

Atrazine 2, 4-D
Aldicarb Chlorpyrifos
Diazoton
Carbendazim
Carbofuran
Sample collection and storage

Collect samples in a clean glass bottle. Do not pre-rinse the bottle with the sample. If the
sample cannot be analyzed immediately, store the sample at 4 °C. Samples may be kept for as
long as 14 days. Warm the samples to room temperature before analysis.
Water sample dilution
Other levels of Alachlor can be tested by diluting the sample and comparing the results to the 0.1
ppb Calibrator. Select the appropriate sample volume from the Sample volume and concentration
table, place it in a graduated mixing cylinder, and dilute it to 50 mL with deionized water.
Table 28 Sample volume and concentration

mL sample Representative concentration using 0.1 ppb calibrator
0.5 10 ppb
1.0 5 ppb
2.5 2 ppb
5.0 1 ppb
Example:
Dilute 2.5 mL of sample to 50 mL with deionized water. Run the diluted sample in the procedure
along with the 0.1 ppb calibrator. If the absorbance of the diluted sample is less than the 0.1 ppb
calibrator, the concentration of the original sample is greater than 2 ppb.
Alachlor
Page 103
Alachlor
Summary of method
Immunoassay tests use antigen/antibody reactions to test for specific organic compounds in water
and soil. Alachlor-specific antibodies, attached to the walls of plastic cuvettes, selectively bind and
remove Alachlor from complex sample matrices. A prepared sample and a reagent containing
enzyme-conjugate molecules (analyte molecules attached to molecules of an enzyme) are added
to the Antibody Cuvettes. During incubation, enzyme-conjugate molecules and Alachlor compete
for binding sites on the antibodies. Samples with higher levels of analyte will have more antibody
sites occupied by Alachlor and fewer antibody sites occupied by the enzyme-conjugate molecules.
After incubation, the sample and unbound enzyme conjugate are washed from the cuvette and a
color-development reagent is added. The enzyme in the conjugate catalyzes the development of
color. Therefore, there is an inverse relationship between color intensity and the amount of
Alachlor in the sample. The resulting color is then compared with a calibrator to determine whether
the Alachlor concentration in the sample is greater or less than the threshold levels. Test results
are measured at 450 nm.
Required reagents
Description Unit Catalog Number
Alachlor Reagent Set1 20 cuvettes 2813000

1 Immunoassay components are manufactured by Beacon Analytical Systems, Inc.
Required apparatus
Caps, flip spout 2/pkg 2581802
Marker, laboratory each 2092000
Pipet, TenSette®, Pipet, 0.1–1.0 mL each 1970001
Pipet Tips, for TenSette Pipet 1970001 1000/pkg 2185628
Rack, for 1-cm Micro Cuvettes each 4879900
Wipes, disposable box 2097000

Deionized Water1 500 mL 27249

Glasses, Safety each 2756800
Gloves, Disposable, Nitrile, Medium1 each 2550502
Mixing Cylinder, graduated Class A, 50-mL1 each 2636341
Mixing Cylinder, graduated Class A, 25-mL1 each 2636340
Pipet Tips, for TenSette Pipet 19700-01 50/pkg 2185696
1 Other sizes available.

Alkalinity, DT, 8203
Alkalinity DOC316.53.01166
Phenolphthalein and Total Alkalinity Method 8203

10 to 4000 mg/L as CaCO3 Digital Titrator
Test preparation
Four drops of Bromcresol Green-Methyl Red Indicator Solution1 can be substituted for the Bromcresol Green-Methyl Red
Indicator Powder Pillow.
meq/L Alkalinity = mg/L as CaCO3 ÷ 50
Bromcresol Green-Methyl Red Indicator Powder Pillow 1 pillow
Sulfuric acid titration cartridge (see Range-specific information) 1 cartridge
Digital titrator 1
Alkalinity
See
Table 1
volume and titration tube into the titration to eject air and a few drops cylinder or pipet to
cartridge from the Range- cartridge. Attach the of titrant. Reset the measure the sample
specific information table. cartridge to the titrator. counter to zero and wipe volume from the Range-
the tip. specific information table.
Alkalinity
Page 105
Alkalinity
Alkalinity (continued)
into a clean, 250-mL one Phenolphthalein into the solution and swirl the Range-
Erlenmeyer flask. If the Indicator Powder Pillow. the flask. Turn the knob on specific information table
sample volume is less Swirl to mix. the titrator to add titrant to to calculate the
than 100 mL, dilute to If the solution turns pink, the solution. Continue to concentration:
approximately 100 mL with proceed to step 7. If the swirl the flask and add digits x multiplier =
deionized water. solution is colorless, the titrant until the color mg/L as CaCO3
Phenolphthalein (P) changes from pink to P alkalinity
alkalinity is zero. Proceed colorless.
Example: 100 mL of
to step 9. Write down the number of sample was titrated with
= 25 mg/L as CaCO3
9. Add the contents of 10. Continue the titration 11. Use the multiplier in
one Bromcresol Green- with sulfuric acid to a light the Range-
Methyl Red Indicator pink color. specific information table
Powder Pillow. Swirl to Write down the number of to calculate the
mix. digits displayed on the concentration:
counter. digits x multiplier =
Note: A pH meter may be mg/L as CaCO3
used to titrate to a specific total alkalinity
pH as required by sample Example: 100 mL of
composition. See the sample was titrated with
End point pH table. the 0.1600 N cartridge and
250 digits were used to
= 25 mg/L as CaCO3
Alkalinity
Page 106
Alkalinity

Range (mg/L as CaCO3) Sample volume (mL) Titration cartridge (N H2SO4) Multiplier
10–40 100 0.1600 0.1

40–160 25 0.1600 0.4
100–400 100 1.600 1.0
200–800 50 1.600 2.0
500–2000 20 1.600 5.0
1000–4000 10 1.600 10.0
Table 30 End point pH

Sample composition Total alkalinity Phenolphthalein alkalinity
Alkalinity about 30 mg/L pH 4.9 pH 8.3
Silicates or phosphates present pH 4.5 pH 8.3
Industrial wastes or complex system pH 4.5 pH 8.3
Routine or Automated Analyses pH 4.5 pH 8.3
Interferences
Chlorine at levels above 3.5 mg/L may cause a yellow-brown color when the Bromcresol
Chlorine Green-Methyl Red Powder Pillow is added. Add one drop of 0.1 N Sodium Thiosulfate to the
sample to remove chlorine before starting the test.
Color or turbidity color indicators and titrate to a pH of 8.3 for phenolphthalein acidity. For total alkalinity see
End point pH for the correct end point pH.

• Prevent excessive agitation or prolonged exposure to air. Complete the test procedure as
soon as possible after collection for best accuracy.
• The sample can be stored for at least 24 hours if cooled to 4 °C (39 °F) or below.
Alkalinity relationship table

Total alkalinity primarily includes hydroxide, carbonate and bicarbonate alkalinities. The
concentration of these alkalinities in a sample may be determined when the phenolphthalein and
total alkalinities are known (see Alkalinity relationships).
To use the table follow these steps:
g. Does the phenolphthalein alkalinity equal zero? If yes, use Row 1.
Alkalinity
Page 107
Alkalinity
h. Does the phenolphthalein alkalinity equal total alkalinity? If yes, use Row 2.
i. Divide the total alkalinity by 2 to give one-half the total alkalinity.
j. Select Row 3, 4 or 5 based on comparing the result of step c (one-half total alkalinity) with
the total alkalinity.
k. Perform the required calculations in the appropriate row, if any.
l. Check your results. The sum of the three alkalinity types will equal the phenolphthalein
alkalinity.
For example:
A sample has 170 mg/L as CaCO3 phenolphthalein alkalinity and 250 mg/L as CaCO3 total
alkalinity. What is the concentration of hydroxide, carbonate and bicarbonate alkalinities?
The phenolphthalein alkalinity does not equal 0 (it is 170 mg/L), see step g.
The phenolphthalein alkalinity does not equal total alkalinity (170 mg/L vs. 250 mg/L), see step h.
One-half of the total alkalinity (250 g/L) equals 125 mg/L. Because the phenolphthalein alkalinity
(170 mg/L) is greater than one-half the total alkalinity (125 mg/L), select row 5.
The hydroxide alkalinity is equal to:
2 x 170 = 340
340 – 250 = 90 mg/L hydroxide alkalinity
The carbonate alkalinity is equal to:
250 – 170 = 80
80 x 2 = 160 mg/L carbonate alkalinity
The bicarbonate alkalinity equals 0 mg/L.
Check: (See step l)
90 mg/L hydroxide alkalinity + 160 mg/L carbonate alkalinity + 0 mg/L bicarbonate alkalinity =
250 mg/L
The above answer is correct; the sum of each type equals the total alkalinity.
Table 32 Alkalinity relationships

Hydroxide alkalinity Carbonate alkalinity Bicarbonate alkalinity
Row Sample result
equals: equals: equals:
1 Phenolphthalein Alkalinity = 0 0 0 Total Alkalinity
2 Phenolphthalein Alkalinity equal Total Alkalinity 0 0
to Total Alkalinity
3 0 Phenolphthalein Alkalinity Total Alkalinity minus two
Phenolphthalein Alkalinity less
times 2 times Phenolphthalein
than one-half of Total Alkalinity
Alkalinity
4 Phenolphthalein Alkalinity equal 0 Total Alkalinity 0
to one-half of Total Alkalinity
5 Phenolphthalein Alkalinity 2 times Phenolphthalein 2 times the difference 0
greater than one-half of Total Alkalinity minus Total between Total and
Alkalinity Alkalinity Phenolphthalein Alkalinity
Alkalinity
Page 108
Alkalinity
Accuracy check
Use a buffer pillow with the same pH as the end point with the indicator to make sure the end point
color is accurate.
• Phenolphthalein alkalinity—Add 50 mL of deionized water to a flask. Add one pH 8.3
buffer powder pillow and one Phenolphthalein Indicator Powder Pillow and swirl to mix.
Use this solution for comparison during the titration with the sample.
• Total alkalinity—Add 50 mL of deionized water to a flask. Add one pH 4.5 buffer powder
pillow and one Bromcresol Green-Methyl Red Indicator Powder Pillow and swirl to mix.
Use this solution for comparison during the titration with the sample.
• Alkalinity Voluette® Ampule Standard Solution, 0.500 N
• Ampule breaker
• TenSette Pipet, 0.1–1.0 mL and Pipet Tips
1. Open the standard solution ampule.
4. Repeat steps 2 and 3, using two more additions of 0.1 mL. Titrate to the end point after each
addition.
titration cartridge or 250 digits of the 0.1600 N titration cartridge to reach the endpoint. If more
or less titrant was used, there may be an interference (see Interferences) or the concentration
of the titrant has changed.
Summary of method
The sample is titrated with sulfuric acid to a colorimetric end point corresponding to a specific pH.
Phenolphthalein alkalinity is determined by titration to a pH of 8.3, as evidenced by the color
change of phenolphthalein indicator and indicates the total hydroxide and one half the carbonate
present. M (methyl orange) or T (total) alkalinity is determined by titration to a pH between 4.3 and
4.9 and includes all carbonate, bicarbonate and hydroxide. Alternatively, total alkalinity end points
may be determined by using a pH meter and titrating to the specific pH required for the sample
composition.
Alkalinity
Page 109
Alkalinity

Required reagents
Alkalinity Reagent Set (approximately 100 tests) 2271900
(1) Bromcresol Green-Methyl Red Powder Pillows 1 pillow 100/pkg 94399
(1) Sulfuric Acid Titration Cartridge, 0.1600 N varies each 1438801
(1) Sulfuric Acid Titration Cartridge, 1.600 N varies each 1438901
Required apparatus
Alkalinity Standard Solution, Voluette® Ampule 0.500 N Na2CO3, 10-mL 16/pkg 1427810

Bromphenol Green-Methyl Red indicator solution 100 mL MDB 2329232
pH meter each —

Alkalinity, BT, 8221
Alkalinity DOC316.53.01151
USEPA1 Buret Titration Method2 Method 8221

1 USEPA Accepted
2 Adapted from Standard Methods for the Examination of Water and Wastewater, 2320 B
Test preparation
A pH meter is required for NPDES reporting and is recommended for best results.
Substitute six drops of Phenolphthalein Indicator Solution for the Phenolphthalein Indicator Powder Pillow if necessary
Substitute six drops of Bromcresol Green-Methyl Red Indicator Solution for the Bromcresol Green-Methyl Red Powder Pillow
if necessary.
Results in mg/L as CaCO3 ÷ 17.12 = grains per gallon
Bromcresol Green-Methyl Red indicator powder pillow 1
Sulfuric acid standard solution, 0.020 N varies1
Buret clamp, double 1
Buret, Class A, 25-mL 1
Funnel, Micro 1
Support Stand 1
1 See Consumables and replacement items on page 116.
Alkalinity
Page 111
Alkalinity
See
Table 1
volume from the Sample cylinder or pipet to into a 250-mL Erlenmeyer one Phenolphthalein
table that corresponds to water if necessary. step when using a
the expected alkalinity pH meter.)
calcium carbonate
(CaCO3).
5. Fill a 25-mL buret to 6. While swirling the 7. Calculate: 8. Add the contents of
the zero mark with 0.020 N flask, titrate the sample mL Titrant × multiplier one Bromcresol Green-
Sulfuric Acid standard until the solution color used = mg/L Methyl Red Indicator
solution. changes from pink to phenolphthalein alkalinity Powder Pillow to the
colorless (pH 8.3). as CaCO3. titrated sample. Swirl to
If the solution is colorless mix.
before titrating with sulfuric Do not add indicator if a
acid, the phenolphthalein pH meter is used.
alkalinity is zero. Specific sample
composition may require
titration to a specific pH
(see the
Alkalinity relationship
table).
Alkalinity
Page 112
Alkalinity
9. Continue the titration 10. Calculate:

until a light pink end point mL Titrant × multiplier
is reached. used = mg/L total alkalinity
as CaCO3.

Range
Sample Volume (mL) Sulfuric Acid Multiplier
(mg/L as CaCO3)
0–500 50 20353 20
400–1000 25 20353 40
1000–2500 10 20353 100
2000–5000 5 20353 200
The end points in the Alkalinity endpoints table are recommended for determining total alkalinity in
water samples of various compositions and alkalinity concentrations.
Table 34 Alkalinity endpoints

End point pH
Sample composition
Total Alkalinity Phenolphthalein Alkalinity
Silicates or phosphates present pH 4.5 pH 8.3
Industrial wastes or complex system pH 4.5 pH 8.3
Routine or Automated Analyses pH 4.5 pH 8.3
Alkalinity
Page 113
Alkalinity
Total alkalinity primarily includes hydroxide, carbonate, and bicarbonate alkalinities. The
concentration of these types in a sample may be determined when the phenolphthalein and total
alkalinities are known ( Alkalinity relationship table).
Table 35 Alkalinity relationship
Hydroxide Alkalinity Carbonate Alkalinity Bicarbonate
Row Result of Titration
Equals: Equals: Alkalinity Equals:
1 Phenolphthalein Alkalinity equal to 0 0 0 Total Alkalinity
2 Phenolphthalein Alkalinity equal to Total Total Alkalinity 0 0
Alkalinity
3 0 Phenolphthalein Alkalinity Total Alkalinity minus
Phenolphthalein Alkalinity less than times 2 two times
one-half of Total Alkalinity Phenolphthalein
Alkalinity
4 Phenolphthalein Alkalinity equal to one- 0 Total Alkalinity 0
half of Total Alkalinity
5 2 times 2 times the difference 0
Phenolphthalein Alkalinity greater than Phenolphthalein between Total and
one-half of Total Alkalinity Alkalinity minus Total Phenolphthalein Alkalinity
Alkalinity
Use the Alkalinity relationship table with the following procedure:
1. Does the phenolphthalein alkalinity equal zero? If yes, use Row 1.
2. Does the phenolphthalein alkalinity equal total alkalinity? If yes, use Row 2.
3. Divide the total alkalinity by 2 to calculate one-half the total alkalinity.
4. Select Row 3, 4 or 5 based on comparing the result of step c (one-half total alkalinity) with the
phenolphthalein alkalinity.
5. Perform the required calculations if any.
6. Check your results. The sum of the three alkalinity types will equal the total alkalinity.
Example:
A sample has 170 mg/L as CaCO3 phenolphthalein alkalinity and 250 mg/L as CaCO3 total
alkalinity. What is the concentration of hydroxide, carbonate, and bicarbonate alkalinities?
a. The phenolphthalein alkalinity does not equal zero but 170 mg/L.
b. The phenolphthalein alkalinity does not equal total alkalinity (170 mg/L vs. 250 mg/L).
c. One-half of the total alkalinity equals 125 mg/L.
d. Because the phenolphthalein alkalinity of 170 mg/L is greater than one-half the total
alkalinity of 125 mg/L, select Row 5.
The hydroxide alkalinity is equal to:
2 × 170 = 340
340 – 250 = 90 mg/L hydroxide alkalinity
The carbonate alkalinity is equal to:
250 – 170 = 80
80 × 2 = 160 mg/L carbonate alkalinity
Alkalinity
Page 114
Alkalinity
The bicarbonate alkalinity is equal to zero mg/L.

Check:
90 mg/L hydroxide alkalinity + 160 mg/L carbonate alkalinity + 0 mg/L bicarbonate alkalinity = 250 mg/L
The answer is correct.

The sum of each type equals the total alkalinity (250 mg/L).
Interferences
Chlorine at levels above 3.5 mg/L cause a yellow-brown color upon the addition of the Bromcresol
Green-Methyl Red Indicator Powder Pillow. Residual chlorine interference with the indicator may
be removed by adding a drop of 0.1 N Sodium Thiosulfate Standard Solution* before adding the
indicator.
these samples, titrating to pH 8.3 for phenolphthalein alkalinity and the appropriate pH (see the
Alkalinity endpoints table) for total alkalinity.
Collect samples in plastic or glass bottles. Fill completely and cap tightly. Avoid excessive agitation
and prolonged exposure to air. Samples should be analyzed as soon as possible after collection
but can be stored at least 24 hours by cooling to 4 °C (39 °F) or below. Warm to room temperature
before analyzing.
Accuracy check
• To accurately determine the phenolphthalein alkalinity end point, mix the contents of one
Phenolphthalein Indicator Powder Pillow and the contents of one pH 8.3 Buffer Powder Pillow
with 50 mL of deionized water in a 250-mL Erlenmeyer flask. The resulting color is the end
point.
• To accurately determine the total alkalinity end point, mix the contents of one pH 4.5 Buffer
Powder Pillow and the contents of one Bromcresol Green-Methyl Red Indicator Powder Pillow
with 50 mL of deionized water in a 250-mL Erlenmeyer flask. Titrate to a light pink color
change.
Standard additions method (Sample Spike)
Perform the standard additions method check as follows:
1. Break the top off an Alkalinity Voluette® Ampule Standard Solution, 0.500 N.
2. Use the TenSette Pipet* to add 0.1 mL of standard to the sample titrated in step 6 or step 9.
Resume titration back to the same end point. Record the volume of titrant needed.
3. Repeat, using two more additions of 0.1 mL. Titrate to the end point after each addition.
4. The mL of titrant required should increase by 2.5 mL for each 0.1 mL increment of standard
added.
Summary of method
Alkalinity is expressed as P (phenolphthalein) alkalinity or as T (total) alkalinity. Both types are
determined by titration with a Sulfuric Acid Standard Solution to an end point evidenced by the
color change of an indicator solution or determined with a pH meter. The P alkalinity is determined
by titration to a pH of 8.3 and registers the total hydroxide and one half the carbonate present. The
T alkalinity is determined by titration to a pH of 4.5. The total alkalinity includes all carbonate,
bicarbonate and hydroxide alkalinity. Alternatively, total alkalinity end points may be determined by
using a pH meter and titrating to the specific pH required for the sample composition.
* See Consumables and replacement items on page 116.
Alkalinity
Page 115
Alkalinity

Required reagents
Bromcresol Green-Methyl Red Indicator Powder Pillows 1 pillow 100/pkg 94399
Sulfuric Acid Standard Solution, 0.020 N varies 1L 20353
Required apparatus
Pipet, volumetric, Class A, 5-mL, — each 1451537
Pipet, volumetric, Class A, 10-mL — each 1451537
Pipet Filler, Safety Bulb — each 1465100
Ampule Breaker — each 2196800
Required standards
Alkalinity Standard Solution, Voluette® Ampules, 0.500 N, 10-mL 16/pkg 1427810
Water, deionized 4L 27256
Optional items
Sodium Thiosulfate Standard Solution, 0.1 N — 32332
TenSette Pipet, 0.1–1.0 mL — 1970001
Tips for Tensette Pipet 50/pkg 2185696

Aluminum, 8012
Aluminum DOC316.53.01002
Aluminon Method1 Method 8012

(0.008 to 0.800 mg/L) Powder Pillows
Scope and Application: For water and wastewater
Test preparation

Instrument Sample cell Cell orientation
DR 6000 2495402 Fill line faces right
DR 5000 2495402 Fill line faces user
DR 3800, DR 2800, DR 2700 2495402 Fill line faces right
Digestion is required for determining total aluminum. Refer to the Digestion section of the Water Analysis Guide.
Clean all glassware with 6.0 N HCl and deionized water before use to remove contaminants from the glass.
Check the sample temperature. It must be between 20–25 °C (68 –77 °F) for accurate results.
The Pour-Thru Cell can be used if rinsed well with deionized water between the blank and prepared samples.
For more accurate results, determine a reagent blank value for each new lot of reagent. Follow the procedure using
deionized water in place of the sample. Subtract the reagent blank value from the final results or perform a reagent blank
adjust.
AluVer® 3 Aluminum Reagent Powder Pillow 1
Ascorbic Acid Powder Pillow 1
Bleaching 3 Reagent Powder Pillow 1
50-mL graduated mixing cylinder with glass stopper 1
Sample cells (see Instrument-specific information) 2
Aluminum
Page 117
Aluminum
Aluminon method with powder pillows
Programs
10 Aluminum Alumin.
Start
1. Select the test. 2. Fill the cylinder to the 3. Add one AluVer 3 4. Start the instrument
Insert an adapter if 50-mL mark with sample. Aluminum Reagent timer.
required (Instrument- Add one Ascorbic Acid Powder Pillow. Insert the A one-minute reaction
specific information). Powder Pillow. stopper. period will begin.
Refer to the user manual Stopper. Invert several An orange to orange-red
for orientation. times to dissolve the color will develop if
powder. aluminum is present.
5. Invert repeatedly for 6. Blank Preparation: 7. Add one Bleaching 3 8. Start the instrument
one minute to dissolve the Pour 10 mL of the mixture Reagent Powder Pillow to timer.
powder. Undissolved into a square sample cell. the blank. A 30-second reaction time
powder will cause will begin.
inconsistent results.
9. Vigorously swirl the 10. Start the instrument 11. Prepared Sample: 12. Within five minutes
cell for 30 seconds. The timer. Pour 10 mL of solution after the timer expires,
solution should turn a light A 15-minute reaction from the cylinder into a wipe and dry the blank and
to medium orange. period will begin. second square sample place it into the cell holder.
cell.
Aluminum
Page 118
Aluminum
Aluminon method with powder pillows (continued)
Zero Read
13. ZERO the instrument. 14. Immediately wipe and 15. READ the results in 16. Clean the graduated
The display will show: dry the prepared sample mg/L Al3+. cylinder with soap and
and place it into the cell See the user manual for brush immediately after
0.000 mg/L Al3+ holder. the test. Rinse with
instructions on changing
the chemical form to deionized water.
Al2O3.
Interferences
Greater than 300 mg/L as CaCO3. Samples with greater than
300 mg/L acidity as CaCO3 must be treated as follows:
1. Add one drop of m-Nitrophenol Indicator Solution1 to the
sample taken in step 2., before adding ascorbic acid.
2. Add one drop of 5.0 N Sodium Hydroxide Standard
Acidity Solution1. Stopper the cylinder. Invert to mix. Repeat as
often as necessary until the color changes from colorless
to yellow.
3. Add one drop of 5.25 N Sulfuric Acid Standard Solution1
to change the solution from yellow back to colorless.
Continue with the test.
1000 mg/L as CaCO3. Interferences from higher alkalinity
concentrations can be eliminated by the following
pretreatment:
1. Add one drop of m-Nitrophenol Indicator Solution1 to
the sample taken in step 2, before adding ascorbic acid.
Alkalinity
A yellow color indicates excessive alkalinity.
2. Add one drop of 5.25 N Sulfuric Acid Standard Solution1.
Stopper the cylinder. Invert to mix. If the yellow color
persists, repeat until the sample becomes colorless.
Continue with the test.
Fluoride Interferes at all levels. See the Fluoride interference graph.
Iron Greater than 20 mg/L
Phosphate Greater than 50 mg/L
Polyphosphate interferes at all levels by causing negative
errors and must not be present. Before running the test,
Polyphosphate
polyphosphate must be converted to orthophosphate by acid
hydrolysis as described under the phosphorus procedures.
1 See Optional reagents and apparatus for reorder information.
Aluminum
Page 119
Aluminum
Fluoride interferes at all levels by complexing with aluminum. The actual aluminum
concentration can be determined using the Fluoride interference graph when the fluoride
concentration is known.
To use the fluoride interference graph:
1. Select the vertical grid line along the top of the graph that represents the aluminum reading
obtained in step 15.
2. Locate the point on the line where it intersects with the horizontal grid line that indicates how
much fluoride is present in the sample.
3. Extrapolate the true aluminum concentration by following the curved lines on either side of the
intersect point down to the true aluminum concentration.
For example, if the aluminum test result was 0.7 mg/L Al and the fluoride present in the sample
was 1 mg/L F–, the point where the 0.7 grid line intersects with the 1 mg/L F– grid line falls
between the 1.2 and 1.3 mg/L Al curves. In this case, the true aluminum content would be 1.27
mg/L.
mg/L Al3+ (Reading from instrument)

mg/L F–
True Aluminum Concentration
Figure 1 Fluoride interference graph

Collect samples in clean glass or plastic containers. Preserve the sample by adjusting the pH to 2
or less with nitric acid (about 1.5 mL per liter). Preserved samples can be stored up to six months
at room temperature. Before analysis, adjust the pH to 3.5–4.5 with 5.0 N Sodium Hydroxide.
Correct the test result for volume additions.
Aluminum
Page 120
Aluminum
Accuracy check
• Aluminum Voluette® Ampule Standard, 50-mg/L Al
• TenSette Pipet
• Mixing cylinders, (3)
1. After reading test results, leave the sample cell (unspiked sample) in the instrument. Verify the
chemical form.
2. Select Options>More>Standard additions from the instrument menu.
3. Press OK to accept the default values for standard concentration, sample volume, and spike
volumes. Press EDIT to change these values. After values are accepted, the unspiked sample
reading will appear in the top row.
4. Open an Aluminum Voluette® Ampule Standard, 50-mg/L Al.
5. Prepare three sample spikes. Fill three mixing cylinders* with 50 mL of sample. Use the
TenSette® Pipet to add 0.1 mL, 0.2 mL, and 0.3 mL of standard, respectively, to each sample
and mix thoroughly.
6. Analyze each sample spike as described in the procedure above, starting with the 0.1 mL
sample spike. Accept each standard additions reading by pressing READ. Each addition
should reflect approximately 100% recovery.
7. After completing the sequence, press GRAPH to view the best-fit line through the standard
additions data points, accounting for matrix interferences. Press IDEAL LINE to view
relationships between the sample spikes and the “Ideal Line” of 100% recovery.

1. Prepare a 0.4-mg/L aluminum standard solution as follows: Pipet 1.00 mL of Aluminum
Standard Solution, 100-mg/L as Al3+, into a 250-mL volumetric flask.
2. Dilute to the mark with deionized water. Prepare this solution daily. Follow the Aluminon
method with powder pillows test.
Or, use the following alternative procedure:
1. Using the TenSette Pipet, add 0.8 mL of solution from an Aluminum Voluette Ampule Standard
Solution (50-mg/L as Al) into a 100-mL volumetric flask.
2. Dilute to volume with deionized water. Follow the Aluminon method with powder pillows test.
3. To adjust the calibration curve using the reading obtained with the 0.4-mg/L aluminum
standard solution, select Options>More>Standard Adjust from the instrument menu.
4. Turn on the Standard Adjust feature and accept the shown concentration. If an alternate
concentration is used, enter the concentration and adjust the curve to that value.
* See Consumables and replacement items.
Aluminum
Page 121
Aluminum
Method performance
Precision Sensitivity
Program Standard 95% Confidence Limits of Concentration change
Distribution per 0.010 Abs change
10 0.40 mg/L Al3+ 0.385–0.415 mg/L Al3+ 0.008 mg/L Al3+
Summary of method
Aluminon indicator combines with aluminum in the sample to form a red-orange color. The
intensity of color is proportional to the aluminum concentration. Ascorbic acid is added before the
AluVer 3 reagent to remove iron interference. To establish a reagent blank, the sample is split after
the addition of the AluVer 3. Bleaching 3 Reagent is then added to one-half of the split sample to
bleach out the color of the aluminum aluminon complex. The AluVer 3 Aluminum reagent,
packaged in powder form, shows exceptional stability and is applicable for fresh water
applications. Test results are measured at 522 nm.
Aluminum
Page 122
Aluminum

Required reagents
Aluminum Reagent Set (100 Tests), includes: — — 2242000
(1) AluVer® 3 Aluminum Reagent Powder Pillow 1 100/pkg 1429099
(1) Ascorbic Acid Powder Pillow 1 100/pkg 1457799
(1) Bleaching 3 Reagent Powder Pillow 1 100/pkg 1429449
Hydrochloric Acid, 6.0 N varied 500 mL 88449
Water, deionized varied 4L 27256
Required apparatus
Description Quantity Unit Catalog number
Cylinder, graduated mixing, 50-mL, with glass stopper 1 each 189641
Aluminum Standard Solution, 100-mg/L as Al3+ 100 mL 1417442
Aluminum Standard Solution, 10-mL Voluette® Ampule, 50-mg/L as Al 16/pkg 1479210
Voluette Ampule Breaker each 2196800

Liqui-Nox Phosphate-free detergent 946 mL 2088153
m-Nitrophenol Indicator Solution 100 mL 247632
Nitric Acid Solution, 1:1 500 mL 254049
pH Paper, 0–14 pH range 100/pkg 2601300
Pipet, TenSette, 0.1–1.0 mL each 1970001
Pipet Tips for 1970001 50/pkg 2185696
Sodium Hydroxide Standard Solution, 5.0 N 50 mL 245026
Test Tube Brush each 69000
Volumetric Flask, Class A, 100-mL each 1457442
Aluminum
Page 123
Aluminum, 8326
Aluminum DOC316.53.01003
Eriochrome Cyanine R Method1 Method 8326

(0.006 to 0.250 mg/L Al3+) Powder Pillows
Test preparation

Clean all glassware with 6.0 N HCl and deionized water before use to remove contaminants from the glass.
deionized water in place of the sample. Subtract the reagent blank value from the final results or perform a reagent
blank adjust.
ECR Reagent Powder Pillow 1
ECR Masking Reagent Solution 1 drop
Hexamethylene-tetramine Buffer Reagent Powder Pillow 1
25-mL graduated mixing cylinder with glass stopper 1
Aluminum
Page 125
Aluminum
Eriochrome Cyanine R method with powder pillows
Programs
9 Aluminum ECR
Start
1. Select the test. 2. Fill the 25-mL mixing 3. Insert the stopper. 4. Start the instrument
Insert an adapter if cylinder to the 20-mL mark Invert several times to timer.
required (see Instrument- with sample. Add one ECR completely dissolve A 30-second reaction
specific information). Reagent Powder Pillow for powder. period will begin.
Refer to the user manual 20-mL sample size. Undissolved reagent will
for orientation. cause inconsistent results.
5. After the timer expires, 6. Insert the stopper. 7. Blank Preparation: 8. Pour 10 mL from the
add one Hexamethylene- Invert several times to Put one drop of ECR mixing cylinder into the
tetramine Buffer Reagent dissolve powder. Masking Reagent Solution blank cell. Swirl to mix.
powder pillow. A red-orange color will into a clean square sample The solution will begin to
develop if aluminum is cell. turn yellow.
present.
Zero
9. Prepared Sample: Fill 10. Start the instrument 11. Within five minutes 12. ZERO the instrument.
a second square sample timer. after the timer expires, The display will show:
cell to the 10-mL mark with A 5-minute reaction period wipe the blank then insert
the remaining solution in it into the cell holder. 0.000 mg/L Al3+
will begin.
the cylinder. This test uses a non-zero
intercept for the calibration
curve on some instrument
platforms.
Aluminum
Page 126
Aluminum
Eriochrome Cyanine R method with powder pillows (continued)
Read
13. Immediately wipe and 14. Read the results in

dry the prepared sample mg/L Al3+.
then insert it into the cell See the user manual for
holder. instructions to change to
alternate form Al2O3.
If fluoride is present,
measure the fluoride
concentration and refer to
the Fluoride Concentration
(mg/L) table.
Interferences

Interfering substance Interference level and treatments
Acidity Greater than 62 mg/L as CaCO3
Alkalinity Greater than 750 mg/L as CaCO3
Ca2+ Greater than 1000 mg/L as CaCO3
Cl– Greater than 1000 mg/L as Cl–

Cr6+ 0.2 mg/L (error is –5% of reading)
Cu2+ 2 mg/L (error is –5% of reading)
Fe2+ Greater than 4 mg/L (error is positive and equals mg/L Fe2+ x 0.0075)
Fe3+ Greater than 4 mg/L (error is positive and equals mg/L Fe2+ x 0.0075)
F– See the Fluoride Concentration (mg/L) table.
Hexameta phosphate 0.1 mg/L as PO43– (error is –5% of reading)
Mg2+ Greater than 1000 mg/L as CaCO3
Mn2+ Greater than 10 mg/L

NO2– Greater than 5 mg/L
NO3 – Greater than 20 mg/L
2.9–4.9 or 7.5–11.5. A sample pH between about 4.9 and 7.5 causes dissolved aluminum to
partially convert to colloidal and insoluble forms. This method measures much of that hard-
pH
to-detect aluminum without any pH adjusting pretreatment as is necessary in some other
methods.
PO43– (ortho) 4 mg/L (error is –5% of reading)
Aluminum
Page 127
Aluminum

Polyphosphate See procedure below.
SO42– Greater than 1000 mg/L
Zn2+ Greater than 10 mg/L
Polyphosphate interference can be reduced by converting polyphosphate to orthophosphate by

the following steps:
1. Rinse a 50-mL graduated mixing cylinder and a 125-mL Erlenmeyer flask containing a
magnetic stir bar with 6 N hydrochloric acid. Rinse again with deionized water. This will
remove any aluminum present.
Note: Rinse two Erlenmeyer flasks if a reagent blank is used; see step 2.
2. Measure 50 mL deionized water into the 125-mL Erlenmeyer flask using the graduated
cylinder. This is the reagent blank. Because of the test sensitivity, this step must be done only
when any of the reagents used in the following pretreatment are replaced—even if the new
reagent has a matching lot number. When the pretreated sample has been analyzed, correct
for the aluminum concentration of the reagent blank. Refer to the instrument user manual.
3. Measure 50 mL sample into the 125-mL Erlenmeyer flask using the graduated cylinder. Use a
small amount of deionized water to rinse the cylinder contents into the flask.
4. Add 4.0 mL of 5.25 N Sulfuric Acid Standard Solution*.
5. Use a combination hot plate/stirrer to boil and stir the sample for at least 30 minutes. Add
deionized water as needed to maintain a sample volume of 20-40 mL. Do not boil dry.
6. Cool the solution to near room temperature.
7. Add 2 drops of Bromphenol Blue Indicator Solution*.
8. Add 1.5 mL of 12.0 N Potassium Hydroxide Standard Solution* using the calibrated, plastic
dropper provided. Swirl to mix. The solution color should be yellow or green but not purple. If
the color is purple, begin with step 1 again using an additional 1 mL Sulfuric Acid Standard
Solution in step 4.
9. While swirling the flask, add 1.0 N Potassium Hydroxide Solution*, a drop at a time, until the
solution turns a dirty green color.
10. Pour the solution into the graduated cylinder. Rinse the flask contents into the graduated
cylinder with deionized water to bring the total volume to 50 mL.
11. Use this solution in step 2. of the ECR method.

Note: Fluoride interference can be corrected by using the Fluoride Concentration (mg/L) table.
Example:
If the fluoride concentration is known to be 1.00 mg/L F– and the ECR method gives a reading of
0.060 mg/L aluminum, what is the true mg/L aluminum concentration?
Intermediate values can be found by interpolation. Do not use correction graphs or charts found in
other publications.
Answer: 0.183 mg/L
* See Optional reagents and apparatus for reorder information
Aluminum
Page 128
Aluminum
Table 40 Fluoride Concentration (mg/L)

(mg/L) 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
0.010 0.010 0.019 0.030 0.040 0.052 0.068 0.081 0.094 0.105 0.117 0.131
0.020 0.020 0.032 0.046 0.061 0.077 0.099 0.117 0.137 0.152 0.173 0.193
0.030 0.030 0.045 0.061 0.077 0.098 0.124 0.146 0.166 0.188 0.214 0.243
0.040 0.040 0.058 0.076 0.093 0.120 0.147 0.174 0.192 0.222 — —
0.050 0.050 0.068 0.087 0.109 0.135 0.165 0.188 0.217 — — —
0.060 0.060 0.079 0.100 0.123 0.153 0.183 0.210 0.241 — — —
0.070 0.070 0.090 0.113 0.137 0.168 0.201 0.230 — — — —
0.080 0.080 0.102 0.125 0.152 0.184 0.219 — — — — —
0.090 0.090 0.113 0.138 0.166 0.200 0.237 — — — — —
0.100 0.100 0.124 0.150 0.180 0.215 — — — — — —
0.120 0.120 0.146 0.176 0.209 0.246 — — — — — —
0.140 0.140 0.169 0.201 0.238 — — — — — — —
0.160 0.160 0.191 0.226 — — — — — — — —
0.180 0.180 0.213 — — — — — — — — —
0.200 0.200 0.235 — — — — — — — — —
0.220 0.220 — — — — — — — — — —
0.240 0.240 True Aluminum Concentration (mg/L) Al

Collect samples in clean glass or plastic containers. Preserve samples by adjusting the pH to 2 or
less with concentrated nitric acid (about 1.5 mL per liter). Preserved samples can be stored up to
six months at room temperature. Before analysis, adjust the pH to 2.9–4.9 with 12.0 N Potassium
Hydroxide Standard Solution* and/or 1 N Potassium Hydroxide Solution*. Correct the test result for
volume additions.
Accuracy check
• Class A glassware (pipettes and volumetric flasks)
• Aluminum Standard Solution, 100 mg/L
OR
• Aluminum Voluette® Ampule Standard Solution, 50-mg/L
• Deionized water
Prepare a 0.100 mg/L aluminum standard solution as follows:
1. Using Class A glassware, pipet 1.00 mL of Aluminum Standard Solution 100 mg/L as Al3+, into
a 1000-mL volumetric flask.
2. Dilute to the mark with deionized water.
* See Optional reagents and apparatus for reorder information.
Aluminum
Page 129
Aluminum
3. Prepare this solution daily. Do the Eriochrome Cyanine R method with powder pillows. Go to
step 4.
OR
1. Add 2.0 mL of solution from an Aluminum Voluette Ampule Standard Solution (50-mg/L as Al)
into a 1000-mL volumetric flask.
2. Dilute to volume with deionized water. Prepare this solution daily.
3. Do the Eriochrome Cyanine R method with powder pillows test procedure.
5. Accept the shown concentration. If an alternate concentration is used, enter the actual
concentration.
Method performance
9 0.100 mg/L Al3+ 0.091–1.009 mg/L Al3+ 0.002 mg/L Al3+
Summary of method
Eriochrome Cyanine R combines with aluminum in a sample to produce an orange-red color. The
intensity of color is proportional to the aluminum concentration. Test results are measured at
535 nm.
Required reagents
Aluminum Reagent Set (100 Tests), includes: — — 2603700
ECR Reagent Powder Pillows 1 100/pkg 2603849
Hexamethylenetetramine Buffer Reagent Powder Pillows 1 100/pkg 2603999
ECR Masking Reagent Solution 1 drop 25 mL SCDB 2380123
Required apparatus
Cylinder, graduated mixing, 25-mL, with glass stopper 1 each 189640
Sample cell, 10 mL square, matched pair 2 2/pkg 2495402
Thermometer, –10 to 110 °C — each 187701
Aluminum
Page 130
Aluminum

Aluminum Standard Solution, 10-mL Voluette® Ampule, 50-mg/L as Al 16/pkg 1479210

Ampule breaker for Voluette Standards each 2196800
Bromphenol Blue Indicator Solution 100 mL 1455232
Cylinder, graduated mixing, 50-mL, Class A each 2636341
Deionized Water 500 mL 27249
Erlenmeyer Flask, 125-mL each 50543
Hot Plate with Stirrer, 7 x 7 inches, 115 VAC each 2881600
Hydrochloric Acid, 6.0 N 500 mL 88449
Nitric Acid 1:1 500 mL 254049
Pipet Tips for 1970001 50/pkg 2185696
Potassium Hydroxide Standard Solution, 12.0 N 100 mL 23032
Potassium Hydroxide Solution, 1.0 N 50 mL 2314426
Stir Bar, magnetic, octagonal, 22.2 x 7.9 mm each 2095350
Aluminum
Page 131
Arsenic, 8013
Arsenic DOC316.53.01005
Silver Diethyldithiocarbamate Method1 Method 8013

(0 to 0.200 mg/L As)
Scope and Application: For water, wastewater, and seawater; distillation is required; USEPA accepted2 for
reporting for drinking and wastewater analysis (distillation required)
2 Procedure is equivalent to Standard Method 3500-As for drinking water analysis.
Test preparation

instruments. To use this table, select an instrument then read across to find the corresponding DR
3800, DR 2800, DR 2700information required to perform this test.
Create a user-entered program for arsenic. See step 1 and User programming.
Prepare the arsenic absorber solution as directed in Reagent preparation.
Perform a user-entered calibration for each new lot of arsenic absorber solution. See the Calibration section. Some
variations of the calibration procedure are possible.
In bright light conditions (e.g. direct sunlight) it may be necessary to close the cell compartment with the protective cover
during measurements.
Do not use the Pour-Thru Cell with this test.
The arsenic absorber in this test is a silver solution in pyridine. Both silver (D011) and pyridine (D038) are regulated
by the Federal RCRA as hazardous waste. In addition, the cotton ball soaked in lead acetate (D008) solution is a
hazardous waste. These materials should not be poured down the drain. Refer to a current MSDS sheet for proper
disposal.
Arsenic
Page 133
Arsenic
Collect the following items :
Apparatus (see Required apparatus) —
Arsenic Standard Solution, 1000-mg/L As varies
Hydrochloric Acid, ACS 25 mL
Lead Acetate Solution, 10% 1 mL
Potassium Iodide Solution, 20% 3 mL
Pyridine, ACS 50 mL
Sample Cells (see Instrument-specific information) 2
Silver Diethyldithiocarbamate 1g
Stannous Chloride Solution 1 mL
Water, deionized varies
Zinc, 20-mesh, ACS 6g
Silver Diethyldithiocarbamate
User Programs
Arsenic
User Programs
Start
1. Perform the User 2. To run the test, press 3. Prepare the distillation 4. Dampen a cotton ball
programming procedure. USER PROGRAMS. apparatus for arsenic with 10% Lead Acetate
Make note of the program Select the test. recovery. See the Solution. Insert it in the
number. Distillation Manual for gas scrubber. Be certain
Insert an adapter if assembly instructions. Do that the cotton seals
required (Table 1). Refer to not connect to the against the glass.
the user manual for aspirator.
orientation.
Place the distillation
apparatus under a fume
hood to vent toxic fumes.
Arsenic
Page 134
Arsenic
5. Using a graduated 6. Using a graduated 7. Turn on the power 8. Using a graduated

cylinder, pour 25-mL of cylinder, pour 250 mL of switch. Set the stir control cylinder, add 25 mL of
prepared arsenic absorber sample into the distillation to 5. Set the heat control Hydrochloric Acid, ACS, to
solution (Reagent flask. to 0. the distillation flask.
preparation) into the
cylinder/gas bubbler
assembly.
Attach it to the distillation
apparatus.
9. Use a serological pipet 10. Use a serological pipet 11. Start the instrument 12. When the timer
to add 1 mL of Stannous to add 3 mL of Potassium timer. expires, weigh and add
Chloride Solution to the Iodide Solution to the A 15-minute reaction 6.0 g of 20-mesh zinc to
flask. flask. Cap. period will begin. the flask. Cap
immediately.
13. Set the heat control 14. Start the instrument 15. When the timer 16. Start the instrument
to 3. timer. expires, set the heat timer.
A second 15-minute control to 1. A third 15-minute reaction
reaction period will begin. period will begin.
Arsenic
Page 135
Arsenic
17. When the timer 18. Rinse the gas bubbler 19. Blank Preparation: 20. Wipe the blank and
expires, turn off the heater. by moving it up and down Fill a dry, 10-mL sample insert it into the cell holder.
Remove the cylinder/gas in the arsenic absorber cell with untreated arsenic
bubbler assembly as a solution. absorber solution.
unit. Stopper.
Zero
21. ZERO the instrument. 22. Prepared Sample: 23. Wipe the prepared
The display will show the Pour the reacted arsenic sample and insert it into
intercept as calculated absorber sample into a the cell holder.
from the user-entered sample cell. READ the results.
calibration curve. This will Close the sample cell.
probably be a non-zero
intercept.
Interferences

Antimony Salts May interfere with color development.

Collect samples in acid washed glass or plastic bottles. Adjust the pH to 2 or less with sulfuric acid
(about 2 mL per liter)*. Preserved samples may be stored up to six months at room temperature.
Correct the test result for volume additions.
* See Optional reagents and apparatus.
Arsenic
Page 136
Arsenic
Reagent preparation
Prepare the arsenic absorber solution as follows:
1. Weigh 1.00 g of silver diethyldithiocarbamate on an analytical balance.
2. Transfer the powder to a 200-mL volumetric flask. Dilute to volume with pyridine. Use pyridine
only in a fume hood and wear chemical resistant gloves. Read the MSDS before
using pyridine.
3. Mix well to dissolve. Store the reagent, tightly sealed, in an amber bottle. The reagent is stable
for one month if stored in this manner. Larger volumes of reagent can be prepared if the
reagent is used within one month.
Calibration
Standard preparation
Perform a new calibration for each lot of arsenic absorber solution.
1. Prepare a 10.0-mg/L arsenic working standard by pipetting 10.0 mL of Arsenic Standard
Solution, 1000 mg/L As into a 1000-mL volumetric flask.
2. Dilute to volume with deionized water.
3. Into three different 500-mL volumetric flasks, pipet 1.0, 2.0, and 10.0 mL of the 10.0 mg/L As
stock solution using Class A glassware.
4. Dilute to the mark with deionized water and mix thoroughly. These standards have
concentrations of 0.02, 0.04 and 0.20 mg/L As.
Note: Distill standards before making the calibration curve.
User programming
1. Press USER PROGRAMS on the main menu.
2. Press PROGRAM OPTIONS and NEW. Key any available program number (950–999) to use for
arsenic testing. Press OK.
3. Fill in the appropriate fields using the touch screen when the field is highlighted. Use the
alphanumeric keys to name the User Program ARSENIC. Press NEXT to move to the next
screen. Set up the rest of the parameters as follows:
•Program Type: Single Wavelength •Chemical Form: As

•Units: mg/L •Wavelength: 520 nm
•Concentration Resolution: 0.001 •Calibration: Read Standards
4. After entering Read Standards, press NEXT>EXIT. Fill in the appropriate fields for each of the
following. Use the touch screen to activate the parameter and press EDIT to enter the data
entry screen. Set up the rest of the parameters as follows:
•Timer1: 15 minutes •Upper Limit: 0.220 mg/L

•Timer2: 15 minutes •Lower Limit: –0.020 mg/L
•Timer3: 15 minutes
5. Press CALIBRATION: C = A + BA. Press EDIT.
Arsenic
Page 137
Arsenic
6. The Read Standards will be indicated. Enter the standard concentration values to be used to
perform the calibration: 0.00, 0.02, 0.04, and 0.20. To enter the concentration values press +
and enter the value followed by OK for each concentration value.
7. After the values are entered, press the UP arrow four times to move the cursor to the
0.00 concentration line.
8. Insert the 25-mL sample cell containing only unreacted arsenic absorber solution into the cell
holder. Press ZERO.
9. Press the DOWN arrow once to move to the next concentration. Insert the prepared sample in
the cell holder. Press READ to accept the absorbance value. Repeat steps for each standard.
Note: Standards must be distilled before absorbance values are measured.
10. Press GRAPH. Make sure FORCE ZERO is off.
11. If the graph is acceptable press DONE>EXIT.
12. “Store Program?” will appear on the display. Press YES.
The program is ready for use.

Some variations of the calibration procedure are possible. See the user manual for details.
Summary of method
Arsenic is reduced to arsine gas by a mixture of zinc, stannous chloride, potassium iodide, and
hydrochloric acid in a specially equipped distillation apparatus. The arsine is passed through a
scrubber containing cotton saturated with lead acetate for sulfide removal, and then into an
absorber tube containing silver diethyldithiocarbamate in pyridine. The arsenic reacts to form a red
complex which is read colorimetrically. This procedure requires a manual calibration. Test results
Required reagents
Arsenic Standard Solution, 1000-mg/L As varies 100 mL 1457142
Hydrochloric Acid, ACS 25 mL 500 mL 13449
Lead Acetate Solution, 10% 1 mL 100 mL 1458042
Potassium Iodide Solution, 20% 3 mL 100 mL 1456842
Pyridine, ACS 50 mL 500 mL 1446949
Silver Diethyldithiocarbamate 1g 25 g 1447624
Stannous Chloride Solution 1 mL 100 mL 1456942
Water, deionized varies 4 liters 27256
Zinc, 20-mesh, ACS 6g 454 g 79501
Required apparatus
Balance, analytical, Zeta series, 80-g capacity 1 each 2936701
Balls, cotton 1 100/pkg 257201
Boat, weighing, 8.9-cm square 2 500/pkg 2179000
Arsenic
Page 138
Arsenic
Required apparatus (continued)

Bottle, amber, 237-mL, glass 1 6/pkg 714441
Cap, polypropylene, for amber bottle 1 6/pkg 2166706
Distillation Apparatus, arsenic accessories 1 set 2265400
Distillation Apparatus, general purpose accessories 1 set 2265300
Flask, volumetric, Class A, 1000-mL, with glass stopper 1 each 1457453
Flask, volumetric, Class A, 200-mL 1 each 1457445
Flask, volumetric, Class A, 500-mL 6 each 1457449
Pipet Filler, safety bulb 1 each 1465100
Pipet, serological, 5-mL 2 each 53237
Pipet, volumetric, Class A, 1.00-mL 2 each 1451535
Select one based on available voltage:
Distillation Apparatus Heater, 115 VAC, 60 Hz 1 each 2274400
Distillation Apparatus Heater, 230 VAC, 50 Hz 1 each 2274402

Cylinder, mixing, 25-mL each 189640
Sulfuric Acid, 1.00 N 100 mL 127032
Gloves, chemical resistant, size 91 pair 2410104
Arsenic
Page 139
Atrazine, 10050
Atrazine DOC316.53.01006
Immunoassay1 Method 10050

Test preparation


Instrument Adapter
DR 6000 —
DR 5000 A23618
DR 3900 LZV846(A)
DR 3800, DR 2800, DR 2700 LZV583
This method analyzes for Atrazine in water. Sample calibrators and reagents are added to cuvettes coated with Atrazine-
specific antibodies. The color that develops is then measured and compared with the color measurements of the calibrators.
The test requires about 30 minutes for complete analysis. As many as 20 cuvettes (18 samples and 2 calibrators) can be run
simultaneously.
Read the entire procedure before starting. Identify and make ready all the necessary reagents, cuvettes, and other
Atrazine
Page 141
Atrazine
Atrazine Reagent Set 1
Caps, flip spout 1
Wipes, disposable 1
Pipet Tips, for TenSette Pipet 19700-01 1
Atrazine immunoassay method
Single Wavelength
4 5 0
OK
Single Wavelength Cuvette for each calibrator the rack snugly. calibrator into the
and each sample to be appropriately labeled
Press OPTIONS and the λ tested. cuvette.
button.
Use a new pipette tip for
Enter 450 nm and press each calibrator.
OK.
required (see Instrument-
specific information). Refer
to the user manual for
orientation.
Atrazine
Page 142
Atrazine
Atrazine immunoassay method (continued)
5. Pipet 0.5 mL of each 6. Immediately pipet 7. Press OPTIONS. Press 8. After 10 minutes mix
sample to be tested into 0.5 mL of Atrazine TIMER. Enter 20:00 the contents of the rack for
the appropriately labeled Enzyme Conjugate into minutes and press OK. 30 seconds (see Using the
cuvette. each cuvette. A 20-minute reaction time 1-cm MicroCuvette Rack.)
Use a new pipette tip for Use the same pipette tip will begin.
each sample. for each sample. Immediately mix the
contents of the cuvettes
for 30 seconds (see Using
the 1-cm MicroCuvette
Rack.)

Ensure that most of the
water is drained from the
cuvettes by turning the
cuvettes upside down and
paper towel.
Atrazine
Page 143
Atrazine

Color Development
11. With the cuvettes still 12. Set the instrument 13. After 5 minutes, mix 14. At the end of the
held snugly in the rack, timer for 10:00 minutes the contents of the rack a 10-minute reaction period,
pipet 0.5 mL of Color Press OK to start a 10- second time for a period of pipette 0.5 mL of Stop
Developing Solution into minute reaction time. 30 seconds using the Solution into each cuvette
each Antibody Cuvette. Mix, using the instructions same technique. in the same order as the
Use the same pipette tip is Using the 1-cm Solutions will turn blue in Color Developing Solution
for each sample. MicroCuvette Rack. some or all of the cuvettes. was added in step 11. Use
the same pipette tip
Slide the rack for 20
seconds (see Using the 1-
cm MicroCuvette Rack.)
the Stop Solution.
Atrazine
Page 144
Atrazine
Zero
water. Wipe the outside of cell holder. holder.
all the cuvettes with a 0.000 Abs
Orient the arrow in the READ the results. The
tissue to remove water, same direction for all display will give an
smudges, and fingerprints. cuvettes. absorbance reading.
Record the results for
each calibrator and
sample.
Repeat this step for all
remaining calibrators and
samples.
reporting results for help
with interpretation of
results.

The 1-cm MicroCuvette Rack has been designed specifically to aid in achieving precise and
accurate results when using the immunoassay technique to analyze several samples at the
same time.
Atrazine
Page 145
Atrazine
Loading the Rack—The cuvette rack is designed so that it may be inverted with the cuvettes in

There is an inverse relationship between the concentration of Atrazine and the absorbance
reading. In other words, the higher the reading, the lower the concentration of Atrazine. See the
Relative Atrazine concentration table.
Table 44 Relative Atrazine concentration

If the sample reading is... the sample Atrazine Concentration is...
Example
Readings:
0.5 ppb Atrazine Calibrator: 0.475 Abs
3.0 ppb Atrazine Calibrator: 0.245 Abs
Interpretation
concentration of Atrazine is greater than both 0.5 ppb and 3.0 ppb Atrazine.
Sample #2—Sample reading is between the readings for the 0.5 ppb and 3.0 ppb Atrazine
calibrators. Therefore the sample concentration of Atrazine is between 0.5 ppb and 3.0 ppb.
sample concentration of Atrazine is less than both 3.0 ppb and 0.5 ppb.
Storing and Handling Reagents

1. Wear protective gloves and eyewear.
2. When storing reagent sets for extended periods of time, keep them out of direct sunlight. Store
3. Keep the foil pouch containing the Antibody Cuvettes sealed when not in use.
4. If Stop Solution comes in contact with eyes, wash thoroughly for 15 minutes with cold water
Sensitivity
The Atrazine immunoassay test cannot differentiate between certain triazines and metabolites, but
it detects their presence to differing degrees. The Required concentrations for selected chemicals
Atrazine
Page 146
Atrazine
table shows the required concentration for selected chemicals. The Compounds tested but not
detectable up to 10,000 ppb table shows compounds not detectable at 10,000 ppb.
Table 45 Required concentrations for selected chemicals

Compound Concentration to give a positive result at 3 ppb (in ppb)
Ametryne 1
Atrazine 3
Atrazine, de-ethylated 115
Atrazine, de-isopropyl 714
Cyanazine 460
Cyromazine 1200
Prometon 8
Prometryne 0.7
Propazine 2.3
Simetryne 5.4
Simazine 37
Terbuthylazine 91
Terbutryne 8.3
Table 46 Compounds tested but not detectable up to 10,000 ppb

Alachlor 2, 4-D
Aldicarb Diaminoatrazine
Carbendazim Melamine
Carbofuran Metolachlor

• Collect samples in a clean glass bottle.
• Do not pre-rinse the bottle with the sample.
• If the sample cannot be analyzed immediately, store the sample at 4 °C.
• Samples may be kept for as long as 14 days.
• Warm stored samples to room temperature before analysis.
Atrazine
Page 147
Atrazine
Water sample dilution

Other levels of Atrazine can be tested by diluting the sample and comparing the results to the 0.1
ppb Calibrator. Select the appropriate sample volume from the Sample volume and concentration
table, place it in a graduated mixing cylinder, and dilute it to 50 mL with deionized water.
Table 47 Sample volume and concentration

mL sample Representative concentration using 0.1 ppb calibrator
0.5 10 ppb
1.0 5 ppb
2.5 2 ppb
5.0 1 ppb
Example:
Dilute 2.5 mL of sample to 50 mL with deionized water. Run the diluted sample in the procedure
along with the 0.1 ppb calibrator. If the absorbance of the diluted sample is less than the 0.1 ppb
calibrator, the concentration of the original sample is greater than 2 ppb.
Summary of method
and soil. Atrazine-specific antibodies, attached to the walls of plastic cuvettes, selectively bind and
remove Atrazine from complex sample matrices. A prepared sample and a reagent containing
enzyme-conjugate molecules (analyte molecules attached to molecules of an enzyme) are added
to the Antibody Cuvettes. During incubation, enzyme-conjugate molecules and Atrazine compete
for binding sites on the antibodies. Samples with higher levels of analyte will have more antibody
sites occupied by Atrazine and fewer antibody sites occupied by the enzyme-conjugate molecules.
color. Therefore, there is an inverse relationship between color intensity and the amount of
Atrazine in the sample. The resulting color is then compared with a calibrator to determine whether
the Atrazine concentration in the sample is greater or less than the threshold levels. Test results
Atrazine
Page 148
Atrazine
Required reagents
Description Unit Cat. No.
Atrazine Reagent Set1 20 cuvettes 2762700

Required apparatus
Pipet, TenSette®, 0.1–1.0 mL each 1970001

Atrazine Reagent Set 100 cuvettes 2762710
Glasses, safety each 2756800
Gloves, disposable, nitrile, medium1 100/pkg 2550502
Graduated Mixing Cylinder, 50-mL each 2636341
Graduated Mixing Cylinder, 25-mL each 2636340
1 Other sizes are available.
Atrazine
Page 149
Barium, 8014
Barium DOC316.53.01007
Turbidimetric Method1 Method 8014

(2 to 100 mg/L) Powder Pillows
Scope and Application: For water, wastewater, oil-field water, and seawater
1 Adapted from Snell and Snell, Colorimetric Methods of Analysis, Vol. II, 769 (1959).
Test preparation

Perform a standard curve adjustment or a new calibration for each new lot of reagent. See Standard solutions and
Calibration standard preparation.
blank adjust.
Filter highly colored or turbid water samples using a funnel1 and filter paper1. Large amounts of color or turbidity will interfere
and cause high readings.
If samples cannot be analyzed immediately, refer to Sample collection, preservation and storage. Adjust the pH of preserved
samples before analysis.
The Pour-Thru and Sipper Cell modules cannot be used with this procedure.
BariVer® 4 Barium Reagent Powder Pillows 1
Barium
Page 151
Barium
Turbidimetric method with powder pillows
Stored Programs
20 Barium
Start
1. Select the test. 2. Fill a square sample 3. Prepared Sample: 4. Start the instrument
Insert an adapter if cell with 10 mL of sample. Add the contents of one timer.
required (see Instrument- BariVer® 4 Barium A five-minute reaction
specific information). Reagent Powder Pillow to period will begin.
the cell. Swirl to mix.
Refer to the user manual Do not disturb the
for orientation. If barium is present, a sample during the
white turbidity will develop. reaction period.
If the reagent does not
dissolve readily in the
sample, mix the sample
and reagent in a 25-mL
graduated mixing cylinder
before pouring into the
sample cell.
5. Blank Preparation: 6. When the timer 7. Wipe and the 8. Clean the sample cell
Fill another square sample expires, wipe the blank prepared sample and immediately after each
cell with 10 mL of sample. and insert the blank into insert the prepared sample test. Use soap, water and
the cell holder. into the cell holder. a brush to prevent a film of
ZERO the instrument. The READ the results in mg/L barium sulfate from
display will show: Ba2+. forming inside the cell.
0 mg/L Ba2+
Interferences

Interfering substance Interference levels and treatments
Calcium 10,000 mg/L as CaCO3
Magnesium 100,000 mg/L as CaCO3
Barium
Page 152
Barium

Silica 500 mg/L
Sodium Chloride 130,000 mg/L as NaCl
Interferes at any level. If present, the total concentration between barium and strontium may
Strontium be expressed as a PS (Precipitated by Sulfate). While this does not distinguish between
barium and strontium, it gives an accurate indication of scaling tendency.
Highly buffered samples or
May exceed the buffering capacity of the reagents and require sample pretreatment.
extreme sample pH

Collect samples in an acid cleaned glass or plastic container. Adjust the pH to 2 or less with nitric
acid* (about 2 mL per liter). Preserved samples can be stored up to six months at room
temperature. Before analysis, adjust the pH to 5 with 5.0 N sodium hydroxide*. Correct the test
result for volume additions.
Standard solutions
Prepare a 90.0-mg/L barium standard solution as follows:
1. Pipet 9.00 mL of Barium Standard Solution, 1000-mg/L, into a 100-mL volumetric flask.
3. Prepare this solution daily. Follow the Turbidimetric method with powder pillows procedure.
4. To adjust the calibration curve using the reading obtained with the standard solution, select
Options>More>Standard Adjust from the instrument menu.
5. Turn on the Standard Adjust feature and accept the displayed concentration. If an alternate
Accuracy check
• Barium Standard Solution, 1000-mg/L Ba
• Pipet, TenSette®, 0.1–1.0 mL and tips
chemical form.
3. Default values for standard concentration, sample volume, and spike volumes can be
accepted or edited. After values are accepted, the unspiked sample reading will appear in the
top row. See the user manual for more information.
4. Open a Barium Standard Solution, 1000-mg/L Ba.
5. Prepare a 0.1 mL sample spike by adding 0.1 mL of standard to the unspiked sample. Touch
the timer icon. After the timer expires, read the result. Press READ to accept the reading.
Barium
Page 153
Barium
6. Prepare a 0.2 mL sample spike by adding 0.1 mL of standard to the 0.1 mL sample spike.
Touch the timer icon. After the timer expires, read the result. Press READ to accept
the reading.
Touch the timer icon. After the timer expires, read the result. Press READ to accept
the reading.
8. Each addition should reflect approximately 100% recovery.
additions data points, accounting for the matrix interferences. Press IDEAL LINE to view the
relationship between the sample spikes and the "Ideal Line" of 100% recovery.
Calibration standard preparation

Prepare calibration standards containing 10, 20, 30, 50, 80, 90, and 100 mg/L Ba as follows:
1. Into seven different 100-mL Class A volumetric flasks, pipet 1, 2, 3, 5, 8, 9, and 10 mL of the
1000-mg/L Barium Standard Solution using Class A glassware.
2. Dilute to the mark with deionized water. Mix thoroughly.
3. Using the turbidimetric method and the calibration procedure described in the user manual,
generate a calibration curve from the standards prepared above.
Method performance
Program Instrument Standard 95% Confidence Limits of Concentration change
20 DR 5000 30 mg/L Ba 25–35 mg/L Ba 1 mg/L Ba
Summary of method
The BariVer® 4 Barium Reagent Powder combines with barium to form a barium sulfate
precipitate, which is held in suspension by a protective colloid. The amount of turbidity present
caused by the fine white dispersion of particles is directly proportional to the amount of barium
present. Test results are measured at 450 nm.
Required reagents
BariVer® 4 Barium Reagent Powder Pillows 1 100/pkg 1206499
Required apparatus
Beaker, 50-mL 1 each 50041H
Barium
Page 154
Barium
Barium Standard Solution, 1000-mg/L Ba 100 mL 1461142

Filter Paper for Funnel, 12.5 cm 100/pkg 189457
Funnel, 65 mm each 108367
Liqui-Nox Phosphate-free Detergent 946 mL 2088153
Pipet Tips for TenSette Pipet 1970001 50/pkg 2185696
Sodium Hydroxide, 5.0 N 100 mL 245032
Test Tube Brush each 69000
Barium
Page 155
Benzotriazole/Tolyltriazole
Benzotriazole/Tolyltriazole DOC316.53.01008
UV Photolysis Method1 Method 8079

(Benzotriazole 1.0 to 16.0 mg/L)
Powder Pillows
(Tolyltriazole 1.0 to 20.0 mg/L)
Scope and Application: For cooling or boiler water.
1 Adapted from Harp, D., Proceedings 45th International Water Conference, 299 (October 22–24, 1984)
Test preparation


Avoid fingerprints on the quartz surface of the lamp. Rinse the lamp and wipe with a soft, clean tissue between tests.
If sample contains nitrite or borax (sodium borate), adjust the pH to 4–6 with 1 N sulfuric acid.
If the sample contains more than 500 mg/L hardness (as CaCO3), add 10 drops of Rochelle Salt Solution before
adding reagent.
Triazole Reagent powder pillows 1
Square bottle, 25-mL 1
Ultra-violet lamp with power supply 1
UV safety goggles 1
Page 157
UV Photolysis method for powder pillows
Stored Programs
30 Benzotriazole
730 Tolyltriazole
Start
1. Select the test. 2. Prepared Sample: 3. Add the contents of 4. Put on UV safety
Insert an adapter if Fill a marked mixing bottle one Triazole Reagent goggles.
required (see Instrument- to the 25 mL line with Powder Pillow.
specific information). sample. Swirl to dissolve
Refer to the user manual completely.
for orientation.
5. Insert the ultraviolet 6. Start the instrument 7. When the timer 8. Fill a square sample
lamp into the mixing bottle. timer. A 5-minute reaction expires, turn the lamp off. cell with 10 mL of reacted
Turn on the UV lamp. period will begin. Remove the lamp from the (prepared) sample.
Low results will occur if bottle. Swirl the bottle to
photolysis (lamp on) takes mix thoroughly.
place for more or less than
five minutes.
A yellow color will form if
triazole is present.
Page 158
UV Photolysis method for powder pillows (continued)
9. Blank Preparation: 10. Insert the blank into 11. Insert the prepared
Fill another square sample the cell holder. sample into the cell holder.
cell with 10 mL of sample. ZERO the instrument. READ the results in mg/L
The display will show: Benzotriazole or mg/L
Tolyltriazole.
0.0 mg/L Benzotriazole
or
0.0 mg/L Tolyltriazole
Interferences

Acrylates (as methyl acrylate) Greater than 50 mg/L
Alum Greater than 400 mg/L
Borate (as sodium tetraborate) Greater than 4000 mg/L. Adjust the pH to 4–6 with 1 N sulfuric acid1.
Chlorine (as Cl2) Greater than 20 mg/L
Chromium (as chromate) Greater than 12 mg/L
Copper Greater than 10 mg/L
Greater than 500 mg/L as CaCO3. Add 10 drops of Rochelle Salt Solution1
Hardness
before adding reagent.
Lignosulfonates Greater than 40 mg/L
Magnesium Greater than 300 mg/L as CaCO3
Molybdenum (as molybdate) Greater than 200 mg/L
Nitrite Greater than 4000 mg/L. Adjust the pH to 4–6 with 1 N sulfuric acid1.
Phosphonates (AMP or HEDP) Greater than 100 mg/L
Sulfate Greater than 200 mg/L
Zinc Greater than 80 mg/L
Strong oxidizing or reducing agents Interfere at all levels
Page 159

The most reliable results are obtained when samples are analyzed as soon as possible after
collection.
Accuracy check
• Benzotriazole Standard Solution, 500 mg/L
1. After reading test results, leave the sample cell (unspiked sample) in the instrument.
4. Open a 500-mg/L Benzotriazole Standard Solution*.
5. Prepare three sample spikes. Fill three mixing cylinders with 25 mL of sample. Use the
TenSette Pipet to add 0.1 mL, 0.2 mL, and 0.3 mL of standard, respectively, to each sample
and mix thoroughly.
6. Follow the UV Photolysis method for powder pillows test procedure for each of the spiked
samples, starting with the 0.1 mL sample spike. Accept each standard addition result by
pressing READ. Each addition should reflect approximately 100% recovery.
7. After completing the sequence, select GRAPH to view the best-fit line through the standard
additions data points, accounting for matrix interferences. Select IDEAL LINE to view
UV Lamp Check
To verify the ultraviolet lamp (normal life equals 5000 hours) is working properly, perform the
following test:
1. Prepare a 5.0 mg/L benzotriazole standard solution by pipetting 10.0 mL of Benzotriazole

Standard Solution, 500-mg/L benzotriazole, into a 1-liter volumetric flask. Dilute to volume.
2. Analyze according to the above procedure. If the result is significantly below 5 mg/L, replace
the lamp.
Method performance
30 DR 5000 10 mg/L benzotriazole 9.7–10.3 benzotriazole 0.2 mg/L benzotriazole
730 DR 5000 12 mg/L tolyltriazole 11.6–12.4 mg/L tolyltriazole 0.2 mg/L tolyltriazole
Page 160
Summary of method
Benzotriazole or tolyltriazole, used in many applications as corrosion inhibitors for copper and
copper alloys, are determined by a proprietary catalytic ultraviolet (UV) photolysis procedure
requiring less than 10 minutes to perform. Test results are measured at 425 nm.
Required reagents
Triazole Reagent Powder Pillows 1 100/pkg 2141299
Required apparatus
UV Safety Goggles 1 each 2113400
Bottle, Square, with 25 mL mark 1 each 1704200
Lamp Kit, UV, with power supply, 115 VAC, 60 Hz 1 each 2704500
Lamp Kit, UV, with power supply, 230 VAC, 50 Hz 1 each 2704502
Benzotriazole Standard Solution, 500-mg/L 100 mL 2141342

Flask, Volumetric, Class A, 1000 mL each 1457453
Pipet Bulb each 1465100
Sulfuric Acid, 1 N 100 mL 127032
Rochelle Salt Solution 29 mL 172533
Volumetric pipet, Class A, 10-mL each 1451538
Page 161
Boron, 8015
Boron DOC316.53.01009
Carmine Method1 Method 8015

Test preparation

All labware must be completely dry. Excess water will cause low results.
Use the BoroVer® 3 Reagent with adequate ventilation. See Reagent preparation.
Do not cap the sample cells or the Erlenmeyer flasks at any time during sample preparation or reaction time. Samples may
be capped immediately prior to placing in the instrument.
Sulfuric acid may contain residual moisture; this will cause low results. Make sure the sulfuric acid is suitable by running a
known boron standard before running any unknown samples.
Wear safety goggles and gloves during this test.
Boron
Page 163
Boron
BoroVer 3 Reagent Powder Pillow 1
Cylinders, graduated, 50-mL and 100 mL 1 of each
Flask, Erlenmeyer, 125-mL 2
Pipet Bulb 1
Pipet, volumetric, 2.0 mL 2
Sulfuric Acid, Concentrated 75 mL
Water, deionized 2.0 mL
Carmine method with powder pillows
Stored Programs
40 Boron
Start
1. Select the test. 2. Using a 100-mL 3. With good ventilation, 4. Blank Preparation:
Insert an adapter if graduated cylinder, add the contents of one Accurately pipet 2.0 mL of
required (see Instrument- measure 75 mL of BoroVer 3 Reagent deionized water into a
specific information). concentrated sulfuric acid. Powder Pillow to the flask. 125-mL Erlenmeyer flask.
Pour the acid into a Swirl to mix. Allow up to
Refer to the user manual 250-mL Erlenmeyer flask. five minutes for the
for orientation. powder to dissolve
completely.
Boron
Page 164
Boron
Carmine method with powder pillows (continued)
5. Prepared Sample: 6. Using a 50-mL 7. Start the instrument 8. When the timer
Accurately pipet 2.0 mL of graduated cylinder, add timer. expires, pour at least
sample into another 125- 35 mL of the solution A 25-minute reaction 10 mL from each flask into
mL Erlenmeyer flask. prepared in step 3 to each period will begin. separate square sample
Erlenmeyer flask. cells.
Swirl to mix completely.
Zero Read
9. Wipe the blank and 10. ZERO the instrument. 11. Wipe and insert the 12. READ the results in
insert the blank into the The display will show: prepared sample into the mg/L B.
cell holder. cell holder.
0.0 mg/L B

Collect samples in clean polyethylene or polypropylene bottles, or alkali-resistant,
boron-free glass.
Reagent preparation
To prepare additional BoroVer 3/Sulfuric Acid Solution, mix one BoroVer 3 Reagent Powder Pillow
per 75 mL of concentrated sulfuric acid, adding the powder pillows individually while stirring.
Preparation of this solution generates gaseous HCl when the indicator pillow is added to the
sulfuric acid. Use of a fume hood or other well-ventilated lab area is strongly advised. This solution
is stable for up to 48 hours if stored in plastic containers. Do not store in borosilicate glassware
(Pyrex® or Kimax®) for longer than one hour; the solution may leach boron from these
containers.
The BoroVer 3/Sulfuric Acid Solution is highly acidic. Refer to the current MSDS for safe
handling and disposal instructions.
Accuracy check
• Boron Standard Solution, 1000-mg/L as B
• Pipet Bulb
• Volumetric pipet, 5-mL
• Mixing cylinder, 25-mL
1. Prepare a 250 mg/L boron standard as follows:
Boron
Page 165
Boron
a. Pipet 5.00 mL of Boron Standard Solution, 1000 mg/L as B into a mixing cylinder.
b. Pipet 15.00 mL of demineralized water into the cylinder, stopper and mix thoroughly.
2. After reading test results, leave the unspiked sample in the instrument. Verify the chemical
form.
3. Select Options>More>Standard Additions from the instrument menu.
TenSette® Pipet to add 0.1 mL, 0.2 mL, and 0.3 mL of standard, respectively, to each sample
and mix thoroughly.
6. Follow the Carmine method with powder pillows test. Analyze 2.0 mL of each sample spike
starting with the 0.1 mL sample spike. Accept each standard additions reading by pressing
READ. Each addition should reflect approximately 100% recovery.
relationship between the sample spikes and the “Ideal Line” of 100% recovery.
Standard Solution Method

• Boron Standard Solution, 1000-mg/L B
• Volumetric flask, 200-mL
• Volumetric pipet, Class A and bulb
• Deionized water
1. Prepare a Boron Standard Solution, 10-mg/L as B. solution as follows:
a. Use Class A glassware to pipet 2.00 mL of the Boron Standard Solution, 1000-mg/L B,
into a 200-mL volumetric flask.
b. Dilute to volume with deionized water.
c. Swirl to mix. Follow the Boron test procedure.
Boron
Page 166
Boron
Method performance
Program Standard 95% Confidence Limits of Concentration change per 0.010 Abs
Distribution change
40 7.6 mg/L B 7.5–7.7 mg/L B 0.14 mg/L B (at 0.2 mg/L)
0.16 mg/L B (at 7.0 mg/L)
0.18 mg/L B (at 14.0 mg/L)
Summary of Method
Boron is determined by its reaction with carminic acid in the presence of sulfuric acid to produce a
reddish to bluish color. The amount of color is directly proportional to the boron concentration. Test
results are measured at 605 nm.
Boron
Page 167
Boron
Required reagents
BoroVer® 3 Boron Reagent Powder Pillows 1 100/pkg 1417099

Sulfuric Acid, ACS, concentrated 75 mL 2.5 L 97909
Water, deionized 2.0 mL 4L 27256
Required apparatus
Pipet, volumetric, 2.0-mL, Class A 2 each 1451536
Safety Bulb 1 each 1465100
Boron Standard Solution, 1000 mg/L as B 100 mL 191442

Description Unit Catalog number.
Cylinder, mixing, 25 mL each 2088640
Gloves, chemical resistant, size 9 to 9.5 pair 24101041
Pipet Tips, for 1970001 50/pkg 2185696
Pipet, volumetric, 5.00 mL, Class A each 1451537
Safety Goggles, vented each 2550700
1 Other sizes available

Boron, LR, 10061
Boron DOC316.53.01010
Azomethine-H Method1 Method Number

LR (0.02 to 1.50 mg/L as B) Powder Pillows
Scope and Application: For testing low levels of boron (boric acid or borates) in drinking water, cooling water,
industrial process waters or wastewaters.
1 Adapted from ISO Method 9390 Harp, D.L., Analytica Chimica Acta, 346(1997), pp. 373–379.
Test preparation

For best results, match two cells using the Cell matching procedure.
Sample temperature should be 22–24 °C (72–75 °F) for most accurate results.
If outside this range, measure and record the sample temperature. See Sample temperature compensation.
BoroTrace™ Reagent Set Includes: —
EDTA Solution, 1M 20 drops
BoroTrace™ #2 Reagent Powder Pillow 2
BoroTrace™ #3 Reagent Powder Pillow 2
Water, Ultra-pure, aldehyde-free 25 mL
Sample cell (see Instrument-specific information) 2
Boron
Page 169
Boron
Azomethine-H method
Stored Programs
45 Boron, LR
Start
1. Select the test. 2. Blank Preparation: 3. Prepared Sample: Fill 4. Add ten drops of
Insert an adapter if Fill a clean plastic sample a second clean plastic EDTA Solution, 1 M, to
required (see Instrument- cell to the 25-mL mark with sample cell to the 25-mL each cell. Cap and invert
specific information). ultra-pure water. mark with sample. each cell twice to mix.
Refer to the user manual If the sample is colored or

for orientation. turbid, sample
pretreatment may
be required See the
Interfering substances and
suggested treatments
table.
5. Open one 6. Cap the prepared 7. Start the instrument 8. Continue shaking
BoroTrace™ 2 Reagent sample and shake to timer. vigorously for 30 seconds,
powder pillow and add the dissolve the powder. A ten-minute reaction or until all powder is
contents to the Proceed immediately with period will begin. dissolved
prepared sample. steps 7 through 10. Let the cell sit capped for
the remaining reaction
period.
Boron
Page 170
Boron
Azomethine-H method (continued)
9. During the reaction 10. Cap the blank and 11. After the timer expires, 12. Wipe the blank and
period, add the contents of shake vigorously until the add the contents of one insert it into the cell holder.
a BoroTrace™ 2 Reagent powder is dissolved. BoroTrace™ 3 Reagent
Powder Pillow to the Powder Pillow to each cell.
blank. Cap and shake to
dissolve.
BoroTrace™ 3 Reagent
“stops” the reaction.
Zero Read
13. ZERO the instrument. 14. Wipe the prepared 15. READ the results in
The display will show: sample and insert it into mg/L B.
the cell holder. To correct for sample
0.00 mg/L B
temperature, refer to the
Sample temperature
compensation table. To
change to alternate form
H3BO3, refer to the
instrument user manual.
Cell matching procedure

1. Rinse and fill two cells with deionized water.
2. Wipe each cell with a soft cloth or tissue to remove liquid or fingerprints.
3. Using one of the cells, set the instrument absorbance at 410 nm to zero.
4. Read the absorbance of the other cell.

5. Cells that read within 0.002 absorbance are matched.
Boron
Page 171
Boron
Interferences
The substances listed in the Interfering substances table have been tested for interference and
found not to interfere up to the indicated levels (in mg/L). The Interfering substances and
suggested treatments table lists suggested treatments for interferences:

Aluminum (3+) 10
Benzotriazole 20
Biocides:
Carbamate-type 120
Isothiazolin-type 120
Quat-type 90
Thiocyanate-type 60
Calcium 1000 (as CaCO3)
Chloride 2500
Copper (2+) 20
Magnesium 1000 (as CaCO3)
Manganese (7+) 5
Molybdate (Mo6+) 60
Phosphonates, AMP 20
Phosphonates, HEDP 20
Polyacrylates 20 (as Acumer 1000, 1100)
Polymaleic Acid 40 (as Belclene 200)
Silica 120
Sulfate 1800
Sulfite 40
Tolyltriazole 20
Zinc (2+) 10
Table 55 Interfering substances and suggested treatments

1. Adjust sample pH to between 5–7 using 1.0 N Sulfuric Acid Solution1.

Alkalinity >500 mg/L (+ or –)
2. Continue with step 4 of the Azomethine-H method procedure.
1. Zero the instrument (0.00 mg/L B) using Ultra-Pure Water2. Measure and
record the apparent concentration, in mg/L B, due to the sample color.
Color (+)
2. Subtract the apparent concentration from the result in step 15 of the
Azomethine-H method procedure.
Halogen disinfectants in the sample can produce a red-color after the addition
of BoroTrace™ 2 Reagent. To eliminate this interference:
1. Add 1 pillow Dechlorinating Reagent1 to 25 mL each of Ultra-Pure Water2
Halogens (Bromine or Chlorine) all levels (+) and sample.
2. Cap and shake to dissolve.
3. Continue with step 4 of the test procedure.
Boron
Page 172
Boron
Table 55 Interfering substances and suggested treatments (continued)

High levels of iron in the sample can produce a red-color after the addition of
BoroTrace™ 2 Reagent. To compensate, increase the amount of EDTA2 from
Iron (Fe3+ or Fe2+), above 8 mg/L (+) 10 drops to 15 drops to be added to each cell (step 4). Alternatively, dilute the
sample with Ultra-Pure Water and continue with step 5 of the test procedure.
Correct the results (step 15) using the appropriate dilution factor.
1. Add 0.1 gram scoop Sulfamic Acid1 to 25 mL each Ultra-Pure Water and
sample in plastic cells.
2. Cap and shake to dissolve.
Nitrites, all levels (+) 3. Uncap and wait 5 minutes.
4. Add 5 N Sodium Hydroxide Reagent1 solution to each to adjust pH to 5–8
using pH paper.
5. Continue with step 4 of the test procedure.
Filter the sample through a 3 µm membrane1 prior to testing. Do not use a
Turbidity (+)
glass fiber filter.
2 See Required reagents.

Collect samples in clean polyethylene bottles. Do not use borate-based detergents or soaps to
clean sample containers or labware used for this method. After use, thoroughly rinse all plastic
containers with deionized water, allow to air dry, and keep covered.
Sample temperature compensation

The reaction chemistry is very dependent on the sample temperature. Calibrations are performed
at 23 °C (73 °F). If the sample temperature is outside the range of 22–24 °C (72–75 °F), multiply
the results, in mg/L, by the appropriate multiplier (see the Sample temperature multipliers table).
Table 56 Sample temperature multipliers
Sample Temp. Sample Temp.
Multiplier Multiplier
°C °F °C °F
5 41 0.70 20 68 0.94
7 45 0.73 25 77 1.04
10 10 0.78 26 79 1.06
12 54 0.81 27 81 1.08
14 57 0.84 28 82 1.10
16 61 0.87 29 84 1.12
18 64 0.91 30 86 1.15
Accuracy check
• 1000-mg/L Boron Standard Solution
• 100-mL plastic volumetric flask with stopper
• Deionized water
Boron
Page 173
Boron

• Pipet bulb
3. Accept the default values for standard concentration, sample volume, and spike volumes.
After the values are accepted, the unspiked sample reading will appear in the top row. See the
user manual for more information.
4. Prepare a 50.0-mg/L boron standard:
a. Pipet 5.0 mL of a 1000-mg/L Boron Standard Solution into a 100-mL plastic volumetric
flask.
b. Dilute with deionized water.
c. Insert a stopper and invert to mix.
TenSette® Pipet to add 0.1 mL, 0.2 mL, and 0.3 mL of the diluted standard, respectively, to
each sample and mix thoroughly.
6. Follow the Azomethine-H method test procedure for each of the spiked samples, starting with
the 0.1 mL sample spike. Measure each of the spiked samples in the instrument.
7. Select GRAPH to view the results. Select IDEAL LINE (or best-fit) to compare the standard
addition results to the theoretical 100% recovery.

• Boron Standard Solution, 1000 mg/L as B
• Deionized water
• Plastic volumetric flask, 1000-mL
• Plastic pipet
1. Prepare a 1.0-mg/L B standard:
a. Use a plastic pipet to transfer 1.0 mL of Boron Standard Solution, 1000 mg/L as B, into a
1000-mL plastic volumetric flask.
b. Dilute to volume with deionized water.
c. Insert a stopper and invert to mix.
* See Optional reagents and apparatus
Boron
Page 174
Boron
Method performance
45 0.92 mg/L B3+ 0.87–0.97 mg/L B 0.01 mg/L B
Summary of method
Azomethine-H, a Schiff base, is formed by the condensation of an aminonaphthol with an
aldehyde by the catalytic action of boron. Test results are measured at 410 nm.
Required reagents
BoroTrace™ Reagent Set, includes: — — 2666900
EDTA Solution, 1 M 20 drops 50 mL SCDB 2241926
BoroTrace™ #2 Reagent Powder Pillows 2 100/pkg 2666669
BoroTrace™ #3 Reagent Powder Pillows 2 100/pkg 2666799
Water, Ultra-pure, Aldehyde-free 25-mL 500 mL 2594649
Required apparatus (powder pillows)

Sample cell, 1-inch square plastic, with cap 2 12/pkg 2410212
Boron Standard Solution, 1000-mg/L as B 100 mL 191442

Dechlorinating Reagent, Powder Pillows 100/pkg 1436369
Filter Holder, 25 mm each 246800
Membrane, filter, 3 µm, 25 mm 25/pkg 2594025
pH Paper, 1.0–11.0 5 rolls/pkg 39133
Pipet, tips for 1970001 TenSette Pipet 50/pkg 2185696
Pipet, volumetric, Class A, 5.00-mL each 1451537
Pipet, volumetric, Class A, 1- mL each 1451537
Safety Bulb each 1465100
Sodium Hydroxide Reagent, 5.0 N, 50 mL each 245026
Boron
Page 175
Boron

Spoon, measuring, 0.1 g each 51100
Sulfamic Acid, 454 g each 234401
Sulfuric Acid Solution 1.0N, 100 mL each 127032
Syringe, 30 cc Luer-lock each 2225800
Thermometer, non-mercury, –10 to +225 °C each 1451535
Volumetric Flask, Class A 1000-mL each 1451535

Bromine, 8016
Bromine DOC316.53.01011
DPD Method1 Method 8016

(0.05 to 4.50 mg/L) Powder Pillows or AccuVac® Ampuls
Scope and Application: For testing bromine residuals (including hypobromite, hypobromous acid and
bromamines) used as disinfectants in process waters, treated water, estuary water, and seawater
1 Adapted from Standard Methods for the Examination of Water and Wastewater
Test preparation


Powder pillows AccuVac Ampuls
Instrument
Sample cell Cell orientation Sample cell Adapter
DR 6000 2495402 Fill line faces right 2427606 —
DR 5000 2495402 Fill line faces user 2427606 —
DR 3900 2495402 Fill line faces user 2427606 LZV846 (A)
DR 3800, DR 2800, DR 2700 2495402 Fill line faces right 2122800 LZV584 (C)
Analyze samples immediately. Do not preserve for later analysis.
blank adjust.
If the sample temporarily turns yellow after reagent addition, dilute a fresh sample and repeat the test. A slight loss of
bromine may occur because of the dilution. Multiply the result by the dilution factor.
Sample cell orientation may vary. Refer to the instrument user manual for correct cell orientation.
Wipe the outside of sample cells before each insertion into the instrument cell holder. Use a damp towel followed by a dry
one to remove fingerprints or other marks.
Bromine
Page 177
Bromine
Powder Pillow Test:
DPD Total Chlorine Reagent Powder Pillows 1
AccuVac Test:
DPD Total Chlorine Reagent AccuVac® Ampuls 1
Beaker, 50-mL 1
Stopper, 18 mm tube 1
See Consumables and replacement items on page 183 for reorder information.
DPD Powder Pillows
Stored Programs
50 Bromine
Start
1. Select the test. 2. Fill a sample cell with 3. Prepared Sample: 4. Start the instrument
Insert an adapter if 10 mL of sample. Add the contents of one timer.
required (see Instrument- DPD Total Chlorine A three-minute reaction
specific information). Powder Pillow to the period will begin.
sample cell.
Refer to the user manual Perform steps 5–6 during
for orientation. Swirl for 20 seconds to the reaction period.
mix.
A pink color will develop if
bromine is present.
Bromine
Page 178
Bromine
DPD Powder Pillows (continued)
5. Blank Preparation: 6. Wipe the blank and 7. Within three minutes

Fill a second sample cell insert it into the cell holder. after the timer expires,
with 10 mL of sample. ZERO the instrument. wipe the prepared sample
and insert it into the cell
The display will show: holder.
0.00 mg/L Br2 READ the results in mg/L
Br2.
DPD AccuVac® Ampuls
Stored Programs
50 Bromine
Start
1. Select the test. 2. Blank Preparation: 3. Prepared Sample: 4. Quickly invert the
Insert an adapter if Fill a round 1-inch sample Collect at least 40 mL of Ampul several times
required (see Instrument- cell with 10 mL of sample. sample in a 50-mL beaker. to mix.
specific information). Fill a DPD Total Chlorine A pink color will develop if
Refer to the user manual Reagent AccuVac Ampul bromine is present.
for orientation. with sample. Keep the tip
immersed while the Ampul
fills completely.
Bromine
Page 179
Bromine
DPD AccuVac® Ampuls (continued)
5. Start the instrument 6. Wipe the blank and 7. Within three minutes
timer. insert it into the cell holder. after the timer expires,
A three-minute reaction ZERO the instrument. wipe the Ampul and insert
period will begin. it into the cell holder.
The display will show:
Perform step 6 during the READ the results in
0.00 mg/L Br2 mg/L Br2.
reaction period.
Bromine
Page 180
Bromine
Interferences
Greater than 150 mg/L CaCO3. May not develop full color or color may fade instantly.
Acidity Neutralize to pH 6 –7 with 1 N Sodium Hydroxide1. Determine amount to be added on
separate sample aliquot, then add the same amount to the sample being tested. Correct for
volume addition.
Alkalinity Neutralize to pH 6 –7 with 1 N Sulfuric Acid1. Determine amount to be added on separate
sample aliquot, then add the same amount to the sample being tested. Correct for volume
addition.
Chlorine Interferes at all levels
Chlorine Dioxide Interferes at all levels
Chloramines, organic May interfere
Hardness No effect at less than 1000 mg/L as CaCO3
Iodine Interferes at all levels
1. Adjust sample pH to 6 –7.
2. Add 3 drops Potassium Iodide1 (30-g/L) to a 25-mL sample.
Manganese, Oxidized 3. Mix and wait one minute.
(Mn4+, Mn7+) or 4. Add 3 drops Sodium Arsenite1, 2 (5-g/L) and mix.
Chromium, Oxidized (Cr6+) 5. Analyze 10 mL of the treated sample as described in the procedure.
6. Subtract the result of this test from the original analysis to obtain the correct bromine
concentration.
Monochloramine Interferes at all levels
Ozone Interferes at all levels
Peroxides May interfere
Extreme sample pH or highly
Adjust to pH 6–7.
buffered samples
2 Samples treated with sodium arsenite for manganese or chromium interferences will be hazardous wastes as regulated by the Federal RCRA
for arsenic (D004).

Collect samples in clean, dry glass containers. If sampling from a tap, allow the water to flow at
least 5 minutes to ensure a representative sample. Avoid excessive agitation and exposure to
sunlight when sampling. Allow several volumes of water to overflow the container and cap the
container so there is no headspace above the sample. If sampling with a DR cell, rinse the cell
several times with the sample, then carefully fill to the 10-mL mark. Proceed with the analysis
immediately.
Method performance
50 2.80 mg/L Br2 2.75–2.85 mg/L Br2 0.05 mg/L Br2
55 2.80 mg/L Br2 2.71—2.89 mg/L Br2 0.05 mg/L Br2
Bromine
Page 181
Bromine
Summary of method
Bromine residuals react with iodide and DPD (N,N-diethyl-p-phenylenediamine) to form a pink
color which is proportional to the total bromine concentration. Test results are measured at 530
nm.
Bromine
Page 182
Bromine
Required reagents
DPD Total Chlorine Reagent Powder Pillows 1 100/pkg 2105669
OR
DPD Total Chlorine Reagent AccuVac® Ampuls 1 25/pkg 2503025
Required apparatus
AccuVac snapper 1 each 2405200
Sample cell, 10 mL round, 25 x 54 mm 1 each 2122800
Sample cell, 10 mL round, 25 x 60 mm 1 6/pkg 2427606
Chlorine Standard Solution, 2-mL PourRite® Ampule, 25–30 mg/L 20/pkg 2630020
Chlorine Standard Solution, 10-mL Voluette® Ampule, 50–75 mg/L 16/pkg 1426810
PourRite Ampule Breaker, 2-mL each 2484600
Voluette Ampule Breaker, 10-mL each 2196800

AccuVac Snapper each 2405200
Chlorine Demand-Free Water 500 mL 2641549
DPD Total Chlorine Reagent Powder Pillows 1000/pkg 2105628
DPD Total Chlorine Reagent Powder Pillows 300/pkg 2105603
pH Paper,0 –14 pH range 100/pkg 2601300
Potassium Iodide, 30 g/L 100 mL 34332
Sodium Arsenite, 5 g/L 100 mL 104732
Sodium Hydroxide, 1 N 100 mL 104532
Stopper for 18 mm tube 6/pkg 173106
Bromine
Page 183
Cadmium, 8017
Cadmium DOC316.53.01012
Dithizone Method1 Method 8017

(0.7 to 80.0 µg/L) Powder Pillows
Scope and Application: For water and wastewater; digestion is required to determine total cadmium.
1 Adapted from Standard Methods for the Examination of Water and Wastewater (method 3500 Cd D).
Test preparation

deionized water instead of the sample.
Clean all glassware with 6 N Hydrochloric Acid Solution and rinse with deionized water.
Sample cell orientation may vary. Refer to the instrument user manual for correct cell orientation.
Cloudy and turbid samples may require filtering before running the test. Report results as µg/L soluble cadmium. Use glass
membrane type filter to avoid loss of cadmium by adsorption onto the filter paper.
If samples cannot be analyzed immediately, see Sample collection, preservation and storage. Adjust the pH of preserved
The Flow Cell and Sipper Modules cannot be used with this procedure.
The DithiVer powder will not completely dissolve in the chloroform. For further notes see Dithiver solution preparation and
storage.
Read the MSDS before testing. Spilled reagent will affect test accuracy and is hazardous to skin and other materials.
Goggles and gloves are recommended.
Cadmium
Page 185
Cadmium
Citrate buffer powder pillows 1
Chloroform 30 mL
DithiVer Metals Reagent powder pillows 1
Potassium Cyanide 0.1 g
Sodium Hydroxide solution, 50% 20 mL
Cotton balls 1
Clippers 1
Cylinder, 25-mL graduated 1
Cylinder, 50-mL graduated mixing 1
Funnel, 500-mL separatory 1
Sample Cells 2
Spoon, measuring, 0.1-g 1
Support ring (4-inch) and stand (5 x 8-inch base) 1
Dithizone method with powder pillows

DANGER
Cyanide is a deadly poison. Use a fume hood. Maintain cyanide solutions at pH 11 or greater to prevent
formation of cyanide gas.
Stored Programs
60 Cadmium, Dithizone
Start
1. Select the test. 2. Fill a 250-mL 3. Add the contents of 4. DithiVer Solution
Insert an adapter if graduated cylinder to the one Buffer Powder Pillow Preparation:
required (see Instrument- 250-mL mark with sample. for heavy metals, citrate Add 30 mL of chloroform
specific information). Pour the sample into a type. Stopper the funnel to a 50-mL mixing
500-mL separatory funnel. and shake to dissolve. graduated cylinder. Add
Refer to the user manual the contents of one
for orientation. DithiVer Metals Reagent
Powder Pillow. Stopper the
cylinder. Invert several
times to mix.
Cadmium
Page 186
Cadmium
Dithizone method with powder pillows (continued)
5. Add 20 mL of 50% 6. Add a 0.1-g scoop of 7. Remove the stopper. 8. Add 30 mL of the
Sodium Hydroxide potassium cyanide to the Start the instrument timer. DithiVer solution to
Solution to the funnel. funnel. Stopper. Shake the 500-mL separatory
vigorously for 15 seconds. A 1-minute reaction period funnel. Stopper, invert, and
will begin. open stopcock to vent.
Close the stopcock and
shake funnel once or
twice; vent again.
9. Start the instrument 10. Start the instrument 11. Prepared Sample: 12. Blank Preparation:
timer. timer and allow the funnel Insert a cotton plug Fill a dry sample cell with
Close the stopcock and to stand undisturbed until the size of a pea into the at least 10 mL of
shake the funnel the timer expires. delivery tube of the funnel chloroform. Stopper.
vigorously during the The bottom (chloroform) and slowly drain the
1-minute time period. layer will be orange to pink bottom (chloroform) layer
if cadmium is present. into a dry 25-mL sample
cell (the prepared sample).
Stopper.
The cadmium-dithizone
complex is stable for more
than one hour if the
sample cell is kept tightly
capped and out of direct
sunlight.
Cadmium
Page 187
Cadmium
Dithizone method with powder pillows (continued)
Zero Read
13. Insert the blank into 14. ZERO the instrument. 15. Insert the prepared 16. READ the results in
the cell holder. The display will show: sample into the cell holder. µg/L cadmium.
0.0 µg/L Cd
Interferences

May exceed the buffering capacity of the reagents and
Highly buffered samples or extreme sample pH
require sample pretreatment.
Bismuth Greater than 80 mg/L. See treatment below.
Copper Greater than 2 mg/L. See treatment below.
Mercury All levels. See treatment below.
Silver Greater than 2 mg/L. See treatment below.
To eliminate interference from the metals listed in the Interfering substances table, insert the
following steps into the procedure after step 4.
1. Measure approximately 5 mL of the DithiVer solution into the separatory funnel. Stopper the
funnel, invert and open the stopcock to vent. Close the stopcock and shake the solution
vigorously for 15 seconds. Allow the funnel to stand undisturbed until the layers separate
(about 30 seconds). A yellow, red, or bronze color in the bottom (chloroform) layer confirms
the presence of interfering metals. Draw off and collect the bottom (chloroform) layer for
proper disposal.
2. Repeat the extraction with fresh 5-mL portions of the DithiVer solution (discarding the bottom
layer each time) until the bottom layer shows a pure dark green color for three successive
extracts. Extractions can be repeated several times without appreciably affecting the amount
of cadmium in the sample.
3. Extract the solution with several 2- or 3-mL portions of pure chloroform to remove any
remaining DithiVer, collecting the bottom layer each time for proper disposal.
4. Continue with step 5 of the procedure.
5. In step 8, substitute 28.5 mL of DithiVer solution for the 30 mL.
6. Continue with step 9 of the procedure.
Cadmium
Page 188
Cadmium
Table 61 Substances that do not interfere

Aluminum Lead
Antimony Magnesium
Arsenic Manganese
Calcium Nickel
Chromium Tin
Cobalt
Zinc
Iron

Collect samples in an acid-washed glass or plastic containers. Adjust the pH to 2 or less with nitric
acid (about 2 mL per liter). Store preserved samples up to six months at room temperature. Adjust
the pH to 2.5 with 5.0 N sodium hydroxide before analysis. Correct the test result for
volume additions.
Dithiver solution preparation and storage

Store DithiVer Powder Pillows away from light and heat. A convenient way to prepare this solution
is to add the contents of 16 DithiVer Metals Reagent Powder Pillows to a 500-mL bottle of
chloroform and invert several times until well mixed (carrier powder may not dissolve completely).
Store dithizone solution in an amber glass bottle. This solution is stable for 24 hours.
Accuracy check
• Cadmium Voluette Ampule Standard, 25-mg/L Cd
• TenSette® Pipet, 0.1–1.0 mL and tips
5. Use the TenSette Pipet to add 0.1 mL, 0.2 mL, and 0.3 mL of standard, respectively to three
250-mL samples and mix each thoroughly.
6. Follow the Dithizone method with powder pillows test procedure for each of the spiked
samples, starting with the 0.1 mL sample spike. Measure each of the spiked samples in the
instrument.

• Cadmium Standard Solution, 100-mg/L Cd.
• Deionized water
• 100-mL Volumetric flask
Cadmium
Page 189
Cadmium
• Class A volumetric pipet and bulb
1. Prepare a 5.0-mg/L cadmium standard solution:
a. Pipet 5.00 mL of Cadmium Standard Solution, 100-mg/L Cd, into a 100-mL volumetric
flask.
b. Dilute to the mark with deionized water. Prepare this solution daily.
2. Pipet 2.00 mL of the 5.0-mg/L Cadmium Standard Solution into 248 mL of deionized water in a
500-mL separatory funnel. This is a 40-µg/L cadmium solution. Perform Dithizone method with
powder pillows on this solution beginning with step 3 of the procedure.
3. To adjust the calibration curve using the reading obtained with the standard solution, elect
Method performance
60 40.0 µg/L Cd 39.3–40.7 µg/L Cd 0.73 µg/L
Summary of method
The dithizone method is designed for the determination of cadmium in water and wastewater. The
DithiVer Metals Reagent is a stable powder form of dithizone. Cadmium ions in basic solution react
with dithizone to form a pink to red cadmium-dithizonate complex, which is extracted with
chloroform. Test results are measured at 515 nm.
Pollution prevention and waste management

Both chloroform (D022) and cyanide (D003) solutions are regulated as hazardous wastes by the
Federal RCRA. Do not pour these solutions down the drain. Chloroform solutions and the cotton
plug used in the delivery tube of the separatory funnel should be collected for disposal with
laboratory solvent waste. Collect the cyanide solution as a reactive waste. Be sure that cyanide
solutions are stored in a caustic solution with a pH >11 to prevent potential release of hydrogen
cyanide gas. See the current MSDS for disposal information.
Required reagents
Cadmium Reagent Set — 100/pkg 2242200
Includes:(1) 14202-99, (1) 14458-17, (1) 12616-99, (1) 767-14, (4) 2180-49, (1) 2572-01
Buffer Powder Pillows, citrate 1 100/pkg 1420299
Chloroform, ACS 40 mL 4L 1445817
DithiVer Metals Reagent Powder Pillows 1 100/pkg 1261699
Potassium Cyanide 0.1 g 125 g 76714
Sodium Hydroxide Solution, 50% 20 mL 500 mL 218049
Cadmium
Page 190
Cadmium
Required reagents (continued)

Cotton Balls, absorbent 1 100/pkg 257201
Required apparatus
Clippers, for opening powder pillows 1 each 96800
Cylinder, graduated, mixing, 50-mL 1 each 189641
Funnel, separatory, 500-mL 1 each 52049
Sample cell, 25 mL square, matched pair with cap 2 2/pkg 2612602
Spoon, measuring, 0.1-g 1 each 51100
Support Ring, 4" 1 each 58001
Support Ring Stand, 5" x 8" base 1 each 56300
Cadmium Standard Solution, 100-mg/L Cd 100 mL 1402442
Chloroform, ACS 500 mL 1445849
Hydrochloric Acid Solution, 6.0 N 500 mL 88449
Sodium Hydroxide Standard Solution, 5.0 N 100 mL MDB 245032
Sodium Hydroxide Standard Solution, 5.0 N 59 mL SCDB 245026
Cadmium
Page 191
Cadmium

Filter Discs, glass, 47 mm 100/pkg 253000
Filter Holder, glass, for 47-mm filter each 234000
Flask, volumetric, Class A, 100-mL each 1457442
Gloves, chemical resistant, size 9 to 9.51 pair 2410104
Goggles, safety, vented each 2550700
Hot Plate, 3½-in. diameter, 120 VAC, 50/60 Hz each 1206701
pH Paper, pH 1.0 to 11.0 5 rolls/pkg 39133
Pipet, serological, 2-mL each 53236
Pipet, TenSette®, 0.1 to 1.0 mL each 1970001
Pipet, volumetric, 2.00-mL, Class A each 1451536
Tongs, crucible, 9-inch each 56900

Carbon Dioxide, DT, 8205
Carbon Dioxide DOC316.53.01167
Digital Titrator Method Using Sodium Hydroxide Method 8205

10 to 1000 mg/L CO2 Digital Titrator
Scope and Application: For water and seawater.
Test preparation
Avoid excess agitation when collecting and swirling the sample to prevent the loss of carbon dioxide from the sample. The
sample is measured directly in the Erlenmeyer flask to avoid agitation..
For more accurate results, check the calibration of the Erlenmeyer flask. Fill a graduated cylinder with the sample volume of
deionized water. Pour the water into the Erlenmeyer flask and make a mark at the correct level.
A pH meter can be used in place of the indicator. The end point pH is 8.3.
Sodium hydroxide titration cartridge (see Range-specific information) 1 cartridge
Digital titrator 1
Carbon dioxide
See
Table 1
1. Select a sample 2. Insert a clean delivery 3. Turn the delivery knob 4. Collect a water sample
volume and titration tube into the titration to eject air and a few drops directly into the titration
cartridge from the Range- cartridge. Attach the of titrant. Reset the flask by filling to the
specific information table. cartridge to the titrator. counter to zero and wipe appropriate mark.
the tip.
Carbon Dioxide
Page 193
Carbon Dioxide
Carbon dioxide (continued)
5. Add the contents of 6. Place the delivery tube 7. Use the multiplier in
one Phenolphthalein into the solution and swirl the Range-specific
Indicator Powder Pillow. the flask. Turn the knob on information table to
Swirl to mix. the titrator to add titrant to calculate the
If a pink color forms, no the solution. Continue to concentration:
carbon dioxide is present. swirl the flask and add digits x multiplier =
titrant until the color mg/L as CO2
changes from colorless to
a light pink color that Example: 100 mL of
persists for 30 seconds sample was titrated with
(pH 8.3). the 0.3636 N cartridge and
Write down the number of reach the endpoint. The
digits displayed on the concentration is 250 x 0.2
counter. = 50 mg/L CO2

Range (mg/L as CO2) Sample volume (mL) Titration cartridge (N NaOH) Multiplier
10–50 200 0.3636 0.1

20–100 100 0.3636 0.2
100–400 200 3.636 1.0
200–1000 100 3.636 2.0
Interferences
Other acids Other acid components in the sample will be titrated and interfere directly in this test.
Color or turbidity
color indicators and titrate to a pH of 8.3.
Carbon Dioxide
Page 194
Carbon Dioxide

• Complete the test procedure as soon as possible after collection for best accuracy.
• The sample can be stored for at least 24 hours if cooled to 4 °C (39 °F) or below.
Accuracy check
• Carbon Dioxide Voluette® Ampule Standard Solution, 10,000 mg/L CO2
• Ampule breaker
titration cartridge or 5 digits of the 3.636 N titration cartridge to reach the endpoint. If more or
less titrant was used, there may be an interference (see Interferences) or the concentration of
the titrant has changed.
Summary of method
Acidity due to carbon dioxide in a sample is titrated with sodium hydroxide to a phenolphthalein
end point. Strong acids are assumed to be absent or of insignificant concentration.
Carbon Dioxide
Page 195
Carbon Dioxide
Required reagents
Carbon Dioxide Reagent Set (approximately 100 tests) 2272700
Required apparatus
Carbon Dioxide Standard Solution, Voluette® Ampule, 10,000-mg/L as CO2, 10-mL 16/pkg 1427510

Phenolphthalein Indicator Solution, 5-g/L 100 mL MDB 16232
Tips for Tensette 50/pkg 2185696
Cylinder, graduated, 250 mL each 108146
Bottle, sampling, poly, 500 mL each 2087079
pH meter each —
Thermometer, -10 to 220 °C each 2635700
Ampule Breaker each 2196800
TitraStir Apparatus, 115 Vac each 1940000
Digital Titrator delivery tips 5/pkg 1720500

Carbon Dioxide, BT, 8223
Carbon Dioxide DOC316.53.01152
Buret Titration Method1 Method 8223

0 to 250 mg/L Buret Titration
Test preparation
Avoid excess agitation when collecting and swirling the sample to prevent the loss of carbon dioxide. Measure the sample
directly in the Erlynmeyer flask to avoid agitation.
For more accurate results, check the calibration of the Erlenmeyer flask. Fill a graduated cylinder with the sample volume of
deionized water. Pour the water into the Erlenmeyer flask and mark the correct level with a wax pencil or permanent marker.
The concentration of the sodium hydroxide standard solution will slowly decrease. To prevent deterioration, keep the bottle
tightly sealed after use. Fill the buret immediately before the test is started and discard the remaining solution after the test.
Follow the Accuracy check each month to make sure the solution will give accurate results.
A pH meter can be used in place of the indicators. The end point pH is 8.3.
For added convenience when stirring, use the TitraStir® stirring apparatus.
Phenolphthalein Indicator Powder Pillow 1
Sodium Hydroxide Standard Solution, 0.0227 N 1 bottle
Buret, Class A, 25-mL, with support stand 1
Erlenmeyer flask 1
Carbon Dioxide
Page 197
Carbon Dioxide
Buret titration
See
Table 1
1. Select a sample 2. Fill a 25-mL buret to 3. Fill a clean 4. Add the contents of
volume and flask from the the zero mark with Erlenmeyer flask to the one Phenolphthalein
Range-specific information 0.0227 N Sodium selected volume. If Indicator Powder Pillow
table. Hydroxide Standard collected from a faucet, and mix gently.
Solution. allow the sample to
overflow several times and
then pour off the excess
sample.
5. Titrate the sample 6. Calculate:

while gently swirling the mL titrant used x multiplier = mg/L as CO2
flask until a light pink color
forms and persists for 30 Example: 100 mL of sample was titrated and 15 mL of
seconds. titrant was used to reach the endpoint. The
concentration is 15 x 10 = 150 mg/L as CO2

Range (mg/L as CO2) Sample volume (mL) Flask size Multiplier
0–125 200 250 5

100–250 100 125 10
Interferences
Color or turbidity
phenolphthalein indicator and titrate to a pH of 8.3.
Acids Acids other than carbonic acid will be titrated and interfere directly.
Carbon Dioxide
Page 198
Carbon Dioxide

agitation or prolonged exposure to air. Complete the test procedure as soon as possible after
collection for best accuracy. The sample can be stored for at least 24 hours if cooled.
Accuracy check
The standard additions method can be used to find if the sample has an interference. The
standard solution method can be used to confirm analytical technique and reagent performance.
• Carbon Dioxide Voluette® Ampule Standard Solution, 10,000-mg/L as CO2
• Ampule breaker
• TenSette Pipet, 0.1–1.0 mL and Pipet Tips.
8. Each 0.1 mL of standard that was added will use 1.0 mL of titrant to reach the endpoint. If
more or less titrant was used, there may be an interference (see Interferences) or the
concentration of the titrant has changed (see Standard solution method).

A sodium hydroxide standard solution will slowly absorb carbon dioxide from the atmosphere,
which forms carbonic acid and neutralizes some of the hydroxide. When sealed in plastic bottles,
the sodium hydroxide concentration will decrease by approximately 1% per year. Complete the
following test to make sure the concentration is accurate.
• Potassium Acid Phthalate Standard Solution, 100-mg/L as CO2
1. Add 50.0-mL of Potassium Acid Phthalate Standard Solution, 100-mg/L as CO2 to an

Erlenmeyer flask.
2. Add the phenolphthalein indicator and swirl to mix.
3. Titrate the standard to the end point with the sodium hydroxide standard solution. The titration
should require 5.00 mL of sodium hydroxide solution. If the volume is greater than 5.25 mL,
discard the sodium hydroxide and replace it with a fresh supply.
Note: To correct for a reduction in strength, divide 5.00 by the number of mL required for this titration.
Multiply all sample results by this factor.
Carbon Dioxide
Page 199
Carbon Dioxide
Summary of method
The analysis for dissolved carbon dioxide in water is similar to that for acidity. A water sample is
titrated to a phenolphthalein end point at pH 8.3 with a sodium hydroxide standard solution. Strong
mineral acids are assumed to be absent or their effect to be negligible.
Required reagents
Required apparatus
Carbon Dioxide Standard Solution, Voluette® Ampule, 10,000-mg/L as CO2, 10-mL 16/pkg 1427510

Phenolphthalein Indicator Solution, 5-g/L 100 mL 16232
Sampling Bottle with cap, low density polyethylene, 500 mL 12/pkg 2087079
Thermometer, Non-Mercury, -10 to 225 °C each 2635700
Voluette Ampule breaker 10 mL each 2196800
1 Other sizes are available

Chelant, Free, DT, 8352
Chelant, Free DOC316.53.01168
Digital Titrator Using Magnesium Chloride Method 8352

0 to 20.0 mg/L as CaCO3 Digital Titrator
Scope and Application: For industrial waters.
Test preparation
All apparatus must be extremely clean and rinsed frequently with acid and deionized water to remove any hardness present
on the plastic or glass.
Filter turbid samples with filter paper1 and a funnel.
Four drops of ManVer Hardness Indicator Solution1 or a 0.1 g scoop of ManVer 2 Hardness Indicator Powder1 can be used
in place of the ManVer 2 Hardness Indicator Powder Pillow.
For best results, measure a reagent blank. Use 50 mL of deionized water in place of the sample. Subtract the number of
digits that were used for the reagent blank from the number of digits that were used for titrating the sample.
Results may be expressed as mg/L tetrasodium EDTA. (Digits required x 0.38 = mg/L as Na4 EDTA)
Hardness 1 Buffer Solution 1 bottle
ManVer 2 Hardness Indicator Powder Pillow 1 pillow
Magnesium Chloride Titration Cartridge, 0.0800 M 1 cartridge
Digital titrator 1
Chelant, Free
Page 201
Chelant, Free
Free chelant
7. Insert a clean delivery 8. Hold the Digital 9. Use a graduated 10. Add two full droppers
tube into the titration Titrator with the cartridge cylinder to measure (2 mL) of Hardness 1
cartridge. Attach the tip pointing up. Turn the 100 mL of sample into a Buffer Solution to the flask.
cartridge to the titrator. delivery knob to eject a 125-mL Erlenmeyer flask. Swirl to mix.
few drops of titrant. Reset
the counter to zero and
wipe the tip.
11. Add the contents of 12. Place the delivery tube 13. Calculate:
one ManVer 2 Hardness into the solution and swirl digits x 0.1 = mg/L Free Chelant as CaCO3
Indicator Powder Pillow. the flask. Turn the knob on
Swirl to mix. the titrator to add titrant to Example: 100 mL of sample was titrated with the
the solution. Continue to 0.0800 M cartridge and 120 digits were used to reach
If the solution turns blue, the endpoint. The concentration is 120 x 0.1 = 12 mg/L
free chelant is present. If swirl the flask and add
titrant until the color as CaCO3
the solution turns red, a
chelant deficiency exists. changes to red-violet.
Write down the number of
digits on the counter.
Chelant, Free
Page 202
Chelant, Free
Interferences
Orthophosphate Causes a slow end point.
Polyphosphate Must be absent for accurate results.
If chelant residual in boiler water is being analyzed, adjust the pH before adding the Buffer
Solution, Hardness 1 as follows:
1. Measure 100 mL of sample into a flask.
2. Add 2 drops of Phenolphthalein Indicator Solution.
pH
3. Add 5.25 N Sulfuric Acid Standard Solution by drops until the solution changes from pink
to colorless. Write down the number of drops that were added. Discard this sample.
4. To a fresh 100-mL portion of sample, add the same number of drops of 5.25 N Sulfuric
Acid Standard Solution before adding the buffer in step 10 of the test.

• Collect samples in clean plastic or glass bottles.
• Filter the sample if turbid with filter paper and a funnel.
Accuracy check
• EDTA Standard Solution, 0.035 N
6. Each 0.4 mL of standard that was added will use approximately 70 digits of the titration
cartridge to reach the endpoint. If more or less titrant was used, there can be an interference
(see Interferences) or the concentration of the titrant has changed.
Summary of method
Chelant residual is determined by titration with a standard solution of magnesium chloride at
pH 10. The end point is determined by a color change from blue to red-violet.
Chelant, Free
Page 203
Chelant, Free
Required reagents
Hardness 1 Buffer Solution 2 mL 100 mL 42432
ManVer® 2 Hardness Indicator Powder Pillows 1 pillow 100/pkg 85199
Magnesium Chloride Titration Cartridge, 0.0800 M varies each 2062501
Required apparatus
EDTA Standard Solution, 0.035 N 100 mL 2349932

Delivery tubes, with 180° hook 5/pkg 1720500
Delivery tubes, with 90° hook 5/pkg 4157800
Filter paper, folded, 12.5 cm 100/pkg 189457
Funnel, analytical, poly, 65 mm each 108367
ManVer 2 Hardness Indicator Powder 113 g 28014
ManVer Hardness Indicator Solution 100 mL 42532
Phenolphthalein indicator solution, 5 g/L 15 mL SCDB 16236
Sulfuric acid standard solutions, 5.25 N 100 mL MDB 244932

Chelant, Total, DT, 8350
Chelant, Total DOC316.53.01169
Bismuth Nitrate Method Method 8350

0 to 40.0 mg/L as Na4 EDTA Digital Titrator
Scope and Application: For industrial waters.
Test preparation
Filter turbid samples with filter paper and a funnel.
For best results, measure a reagent blank. Use 50 mL of deionized water in place of the sample. Subtract the number of
digits that were used for the reagent blank from the number of digits that were used for titrating the sample.
Methylthymol Blue Indicator Powder Pillow 1 pillow
Ascorbic Acid Powder Pillow 1 pillow
Bismuth Nitrate Titration Cartridge, 0.0200 M 1 cartridge
Sulfuric Acid Standard Solution, 5.25 N 1 bottle
Digital titrator 1
Graduated cylinder, 100-mL 1
Chelant, Total
Page 205
Chelant, Total
Total chelant
14. Insert a clean delivery 15. Hold the Digital 16. Use a graduated 17. Add the contents of
tube into the titration Titrator with the cartridge cylinder to measure 50 mL one Ascorbic Acid
cartridge. Attach the tip pointing up. Turn the of sample into a 125-mL Indicator Powder Pillow.
cartridge to the titrator. delivery knob to eject a Erlenmeyer flask. Swirl to mix.
few drops of titrant. Reset
the counter to zero and
wipe the tip.
18. Add the contents of 19. If the solution in the 20. Place the delivery tube 21. Calculate:
one Methylthymol Blue flask is yellow, add one into the solution and swirl digits x 0.188 = mg/L total
Indicator Powder Pillow. drop of 5.25 N Sulfuric the flask. Turn the knob on chelant as Na4 EDTA
Swirl to mix. Acid Standard Solution. the titrator to add titrant to
the solution. Continue to Example: 50 mL of sample
If the solution is blue, add was titrated and 120 digits
5.25 N Sulfuric Acid swirl the flask and add
titrant until the color were used to reach the
Standard solution by drops endpoint. The
until the color changes to changes from yellow to
blue-green. Titrate slowly concentration is
yellow. Add one additional 120 x 0.188 = 22.3 mg/L
drop. as the end point is
approached. total chelant as Na4 EDTA

digits displayed on the
counter.
Interferences
Interference from ferric iron (Fe3+) is minimized by the addition of ascorbic acid. Approach the end
point slowly in samples that contain ferric iron because the reduced iron decreases the sharpness
of the color change.
Chelant, Total
Page 206
Chelant, Total

• Filter the sample if turbid with filter paper and a funnel.
Summary of method
Total chelant is determined by titrating an acidic sample with bismuth nitrate in the presence of
methylthymol blue indicator. The end point is indicated by a color change from yellow to
blue-green.
Required reagents
Ascorbic Acid Powder Pillows 1 pillow 100/pkg 1457799
Bismuth Nitrate Titration Cartridge, 0.0200 M varies each 2434501
Methylthymol Blue Indicator Powder Pillows 1 pillow 100/pkg 2284799
Sulfuric Acid Standard Solution, 5.25 N varies 100 mL MDB 244932
Required apparatus
Cylinder, graduated, poly, 100-mL 1 each 108142
Delivery tubes, with 180° hook 1 5/pkg 1720500
Delivery tubes, with 90° hook 1 5/pkg 4157800

Filter paper, folded, 12.5 cm 100/pkg 189457
Sampling bottle 250 mL 2087076
Chelant, Total
Page 207
Chloramine (Mono) and Free Ammonia, 10200
Chloramine (Mono);
Nitrogen, Free Ammonia DOC316.53.01016
Indophenol Method1 Method 10200

(0.01–0.50 mg/L NH3–N; 0.04–4.50 mg/L Cl2) Powder Pillows
Scope and Application: For determining Free Ammonia and Monochloramine simultaneously in finished
chloraminated water.
1 U.S. Patent 6,315,950
Test preparation


Instrument Sample cell Cell orientation Adapter
DR 6000 4864302 Orientation key toward user A23618
DR 3900 4864302 Orientation key inserted in adapter slot LZV846 (A)
DR 3800, DR 2800, DR 2700 5940506 1-cm (flat) path aligned with the arrow on the adapter LZV585 (B)
For more accurate chloramine results, determine a reagent blank value for each new lot of reagent, using deionized water in
place of the sample. Subtract the reagent blank value from the final results or perform a reagent blank adjust.
Dispose of reacted solutions according to local, state and federal regulation. Use the guidance given on the Material Safety
Data Sheets to dispose of unreacted reagents. Consult local regulatory agencies for further disposal information.
Free Ammonia Reagent Solution 1 drop
Monochlor F Reagent Pillows 2
Chloramine (Mono); Nitrogen, Free Ammonia

Page 209
Indophenol method, powder pillows
Stored Programs
66 Monochloramine LR Zero
M
Start
FA M
1. Select the 2. Fill two 1-cm cells to 3. Wipe the 4. ZERO the instrument.
Monochloramine test. the 10-mL line with monochloramine cell and The display will show:
Insert an adapter if sample. insert it into the cell holder.
See Instrument-specific 0.00 mg/L Cl2
required (see Instrument- Label one cell “Free
specific information). Ammonia” and one cell information for cell
“Monochloramine”. orientation
M M FA
5. Remove the cell and 6. Cap and shake the 7. Add one drop of Free 8. Cap the reagent bottle
add the contents of one cell about 20 seconds to Ammonia Reagent to maintain reagent
Monochlor-F pillow to the dissolve. Solution to the cell for Free performance and stability.
sample for A green color will develop Ammonia measurement.
monochloramine if monochloramine is
measurement. present.

Page 210
Indophenol method, powder pillows (continued)
Read
M
FA
9. Cap and invert the 10. Start the instrument 11. When the timer 12. READ the results in
Free Ammonia cell to mix. timer. expires, wipe the mg/L Monochloramine
If the sample becomes A 5-minute reaction period Monochloramine cell and (as Cl2).
cloudy by the end of the will begin. insert it into the cell holder. Leave the cell in the
reaction period, pretreat Color development time instrument.
the sample and retest. See depends on sample
the Interferences section. temperature. For accurate
results allow the full
reaction period to occur.
See the Color
development table
Exit
Stored Programs
Zero
388 N, Ammonia Free
FA FA
Start
13. Select the Free 14. With the 15. Add the contents of 16. Cap and shake the
Ammonia test. monochloramine sample one Monochlor F pillow to cell about 20 seconds to
If Display Lock is on, the still in the cell holder, the cell for the Free dissolve the reagent.
display will show “Store ZERO the instrument. Ammonia measurement. A green color will form if
Data?”. Press YES or NO The display will show: The reaction period in step monochloramine or
and continue. 0.00 mg/L NH3–N f 10 must be complete ammonia is present.
before adding the
Remove the Monochlor F to the cell for
monochloramine cell. Free Ammonia
measurement.

Page 211
Indophenol method, powder pillows (continued)
Read
FA
17. Start the instrument 18. When the timer 19. READ the results in
timer. expires, insert the vial into mg/L NH3–N f.
A 5-minute reaction period the cell.
will begin.
Samples colder than 18 °C
will require additional time.
See the Color
development table.
Interferences
This method is intended for finished, chloraminated drinking water samples that have a
measurable combined (total) chlorine disinfectant residual. Samples where the disinfectant
residual has disappeared and samples which exhibit a chlorine demand may produce low
ammonia test results. Blanks and ammonia standards analyzed without a disinfectant residual
must be prepared using high quality, reagent grade water.
The substances listed in the Non-interfering substances table do not interfere in free ammonia
determination at or below the stated concentration:
Table 68 Non-interfering substances

Substance Maximum level tested
Aluminum 0.2 mg/L Al
Chloride 1200 mg/L Cl–
Copper 1 mg/L Cu
Iron 0.3 mg/L Fe
Manganese 0.05 mg/L Mn
Nitrate 10 mg/L NO3––N
Nitrite 1 mg/L NO2––N
Phosphate 2 mg/L o–PO4
Silica 100 mg/L SiO2
Sulfate 1600 ppm as SO42–

Zinc 5 ppm Zn

Page 212
Samples containing high levels of both Total Hardness and Alkalinity may become cloudy after the
addition of the Free Ammonia Reagent Solution. If this occurs by the end of the first reaction
period, the sample for Free Ammonia measurement must be pretreated as follows:
Note: The sample for Monochloramine measurement does not need pretreatment.
1. Measure 10 mL of sample into the cell for Free Ammonia measurement.
2. Add the contents of one Hardness Treatment Reagent Powder Pillow to the sample.
3. Cap the cell and invert until the reagent is dissolved.
4. Remove the cap.
5. Continue with the analysis at step 2 using the pretreated sample as the Free Ammonia cell.
Color development time

Test results are strongly influenced by sample temperature. Both reaction periods in the
procedure are the same and depend on the temperature of the sample. The reaction periods
indicated in the procedure are for a sample temperature of 18–20 °C (64–68 °F). Adjust both
reaction periods according to the Color development table. Samples can be read up to 15 minutes
after the listed development times.
Table 69 Color development

Sample temperature (°C) Sample temperature (°F) Development time (minutes)
5 41 10
7 45 9
9 47 8
10 50 8
12 54 7
14 57 7
16 61 6
18 64 5
20 68 5
23 73 2.5
25 77 2
greater than 25 greater than 77 2

Collect samples in clean glass bottles. Most reliable results are obtained when samples are
analyzed as soon as possible after collection.
Accuracy check (Monochloramine, Program 66)

• Buffer Powder Pillow, pH 8.3
• Nitrogen, Ammonia Standard Solution, 100-mg/L as NH3–N
• Chlorine Solution Ampules, 50–70 mg/L
• 100-mL Class A volumetric flask

Page 213
• Pipet, Volumetric 2 mL and pipet bulb

• Organic-free water
Important Note: Because of the strong buffer used in the preparation of this standard, it cannot be
used for accuracy verification of the Free Ammonia test.
To check test accuracy, prepare the following 4.5-mg/L (as Cl2) monochloramine standard
immediately before use.
1. Add the contents of one Buffer Powder Pillow, pH 8.3 to about 50-mL of organic-free water in a
clean 100-mL Class A volumetric flask. Swirl to dissolve the powder.
2. Using a Class A volumetric pipet, transfer 2.00 mL of Nitrogen, Ammonia Standard Solution,
100-mg/L as NH3–N into the flask.
3. Dilute to volume with organic-free water, cap and mix thoroughly. This is a 2.00-mg/L buffered
ammonia standard.
4. Pipet 50.00 mL of the buffered ammonia standard into a clean 100-mL beaker.
Add a stir bar.
5. Obtain a recent lot of Chlorine Solution Ampules, 50–70 mg/L, and note the actual free
chlorine concentration for this lot.
6. Calculate the amount of Chlorine Solution to be added to the ammonia standard using the
following equation:
455
mL chlorine solution required = ----------------------------------------------------------------------
free chlorine concentration
7. Open an ampule and use a glass Mohr pipet to add the calculated amount of Chlorine Solution
slowly to the ammonia standard, while mixing at medium speed on a stir-plate.
8. Allow the monochloramine solution to mix for 1 minute after all Chlorine Solution is added.
9. Quantitatively transfer the monochloramine solution to a clean 100-mL Class A volumetric

flask. Dilute to the mark with organic-free water, cap, and mix thoroughly. This is a nominal
4.5-mg/L (as Cl2) monochloramine standard.
10. Use this standard within 1 hour of preparation. Analyze according to the Low Range
Monochloramine procedure described above.
11. To adjust the calibration curve using the reading obtained with the 4.5-mg/L standard solution,
select Options>More>Standard Adjust from the instrument menu.
Accuracy check (Free Ammonia, Program 388)

• Ammonium Nitrogen Standard, 10 mg/L as NH3-N
• 100-mL plastic volumetric flask with stopper
• 50-mL mixing cylinders, three
• Deionized water

Page 214
Dilution water is required when testing a diluted sample and preparing standard solutions. Dilution
water must be free of ammonia, chlorine and chlorine demand. A convenient source is a
recirculating, deionizer system with carbon filtration which produces 18 megaohm-cm water.
Standard Additions Method
chemical form.
4. Prepare three spiked samples. Measure 50 mL of sample into three 50-mL mixing cylinders.
5. Use the TenSette Pipet to add 0.3, 0.6, and 1.0 mL of Ammonium Nitrogen Standard, 10 mg/L
as NH3-N to the three samples. Mix well.
6. Analyze each spiked sample starting with the 0.3 mL sample spike. Follow all steps in Method
10200. Accept each standard additions reading by pressing READ. Each addition should
reflect approximately 100% recovery.
additions data points, accounting for matrix interferences. Press IDEAL LINE to view the
1. Prepare a 0.20 mg/L ammonia nitrogen standard by diluting 2.00 mL of the Ammonia Nitrogen
Standard Solution, 10 mg/L, to 100 mL with dilution water. Or, using the TenSette Pipet,
prepare a 0.20 mg/L ammonia nitrogen standard by diluting 0.4 mL of a Ammonia Nitrogen
Voluette Standard Solution, 50 mg/L as NH3–N, to 100 mL with dilution water. Analyze the
Standard Solution, following all steps in Method 10200.
Method performance
66 2.60 mg/L Cl2 2.58–2.62 mg/L Cl2 0.04 mg/L Cl2
388 0.20 mg/L NH3–N 0.19–0.21 mg/L NH3–N 0.01 mg/L NH3–N

Page 215
Summary of method
Monochloramine (NH2Cl) and free ammonia (NH3 and NH4+) can exist in the same water sample.
Added hypochlorite combines with free ammonia to form more monochloramine. In the presence
of a cyanoferrate catalyst, monochloramine in the sample reacts with a substituted phenol to form
an intermediate monoimine compound. The intermediate couples with excess substituted phenol
to form a green-colored indophenol, which is proportional to the amount of monochloramine
present in the sample. Free ammonia is determined by comparing the color intensities, with and
without added hypochlorite. Test results are measured at 655 nm.

Page 216

Required reagents
Free Ammonia Reagent Set, includes: — 50/pkg 2879700
(1) 2802299, (1) 2877336
Free Ammonia Reagent Solution 1 drop 4 mL SCDB 2877336
Monochlor F Reagent Pillows 2 100/pkg 2802299
Recommended standards and reagents

Buffer, pH 8.3 Powder Pillows 25/pkg 89868
Hardness Treatment Reagent Pillows (1 per test) 50/pkg 2882346
Nitrogen Ammonia Standard Solution, 10 mg/L as NH3–N 500 mL 15349
Nitrogen Ammonia Standard Ampule, 50 mg/L as NH3–N, 10 mL 16/pkg 1479110
Nitrogen, Ammonia Standard Solution, 100-mg/L as NH3–N 500 mL 2406549
PourRite Ampule Breaker, for 2-mL ampules each 248460
Voluette Ampule Breaker, for 10-mL ampules each 2196800
Water, Organic-free 500-mL 2641549

Beaker, 100 mL, Polypropylene each 108042
Beaker, 100 mL, Glass each 50042H
Cylinder, 50 mL, mixing each 2088641
Flask, volumetric, Class A, 100 mL each 1457442
Free Ammonia Reagent Set 250/pkg 2879701
Monochloramine/Free Ammonia Spec Check Kit each 2507500
Pipet, Mohr, Glass, 10 mL each 2093438
Scissors each 2883100
Stir Bar, octagonal each 2095352
Stirrer, magnetic each 2881200
Thermometer, –10 to 110 °C each 187701
Wipers, Disposable, 30 x 30 cm, 280/box box 2097000

Page 217
Chloramine (Mono), HR, TNT, 10172
Chloramine (Mono) DOC316.53.01014

HR (0.1 to 10.0 mg/L Cl2) Test ‘N Tube
Scope and Application: Chlorinated wastewater.
1 Patent Pending
Test preparation

Instrument Light shield
DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
DR 3900, DR 3800, DR 2800 and DR 2700: Install the light shield into compartment #2 before performing this test. See the
blank adjust.
Dispose of reacted solutions according to local, state and federal regulations. Use the guidance given on the Material Safety
High-range Monochloramine Diluent Vials 1
Monochlor F Reagent Pillow 1
Funnel, micro 1
Light Shield (see Instrument-specific information) 1
Pipet, Mohr glass, 2.00 mL and pipet bulb 1
Test Tube Rack 1
Chloramine (Mono)
Page 219
Chloramine (Mono)
Indophenol method for monochloramine
Stored Programs
67 Monochlora. HR TNT Zero
Start
20. Select the test. 21. Remove the cap from 22. Wipe the vial and 23. ZERO the instrument.
Insert an adapter or light one HR Monochloramine insert it into the 16 mm cell The display will show:
shield if required Diluent vial. Use a glass holder.
pipet to add 2.0 mL 0.0 mg/L Cl2
(Instrument-specific
information). sample to the vial. Re-cap
and invert several times to
Refer to the user manual mix.
for orientation.
Read
24. Remove the vial from 25. Start the instrument 26. After the timer expires, 27. READ the results in
the holder. Using a micro- timer. re-insert the vial into the mg/L Cl2.
funnel, add the contents of A 5-minute reaction period cell holder.
one Monochlor F pillow to will begin.
the sample. Cap and
shake the cell about
20 seconds to dissolve.
Interferences
The substances in the Non-interfering substances table have been tested for interference and do
not interfere at or below the indicated levels. The Interfering substances table describes suggested
treatments for interfering substances.

Interfering substance and effects Interference level Recommended treatment
Ozone – Above 1 mg/L Usually does not coexist with monochloramine.
Sulfide + Turns a “rust” color if present. Usually does not coexist with monochloramine.
Thiocyanate – Above 0.5 mg/L This method will tolerate up to 2 mg/L.
Chloramine (Mono)
Page 220
Chloramine (Mono)

Substance Maximum level tested
Alanine 1 mg/L N
Aluminum 10 mg/L
Bromide 100 mg/L Br –
Bromine 15 mg/L Br2
Calcium 1000 mg/L as CaCO3
Chloride 18,000 mg/L Cl–
Chlorine Dioxide 5 mg/L ClO2
Chromium (III) 5 mg/L Cr
Copper 10 mg/L Cu
Cyanide 10 mg/L CN –
Dichloramine 10 mg/L as Cl2
Fluoride 5 mg/L F–
Free Chlorine 10 mg/L Cl2
Glycine 1 mg/L N
Iron (II) 10 mg/L Fe2+
Iron (III) 10 mg/L Fe3+
Magnesium 1000 mg/L as CaCO3
Manganese (VII) 10 mg/L
Lead 10 mg/L Pb
Nitrate 100 mg/L N
Nitrite 50 mg/L N
Phosphate 100 mg/L PO4
Sulfate 2600 mg/L SO42–

Sulfite 50 mg/L SO32–
Tyrosine 1 mg/L N
Urea 10 mg/L N
Zinc 5 mg/L Zn

Analyze samples for monochloramine immediately after collection. Rinse the sample container
several times with sample, letting the container overflow each time. If sampling from a tap, let the
water flow for at least 5 minutes, then cap the container so that there is no head space (air) above
the sample.
Chloramine (Mono)
Page 221
Chloramine (Mono)
Accuracy check
• Chlorine Solution Ampules, 50–70 mg/L Cl2
• Pipet, Volumetric, 2 mL and pipet bulb
ammonia standard.
4. Pipet 50.00 mL of the buffered ammonia standard into a clean 100-mL beaker.
Add a stir bar.
following equation:
455
slowly to the ammonia standard, while mixing at medium speed on a stir plate.

10. Use this standard within 1 hour of preparation. Follow the Indophenol method for
monochloramine test.
Chloramine (Mono)
Page 222
Chloramine (Mono)
Method performance
1 Use the LR Chloramine (Mono) test for concentrations below 4.5 mg/L Cl2.
Summary of Method
The sample is first diluted in a Test ‘N Tube. In the presence of a cyanoferrate catalyst,
monochloramine (NH2Cl) in the sample reacts with a substituted phenol to form an intermediate
monoimine compound. The intermediate couples with excess substituted phenol to form a green-
colored indophenol, which is proportional to the amount of monochloramine present in the sample.
Test results are measured at 655 nm.
Required reagents
HR Monochloramine Test ‘N Tubes, 50 tests, includes: — — 2805145
(50) HR Monochloramine Diluent Vials1 1 50/pkg —
Funnel, micro 1 each 2584335
1 Not sold separately
Required apparatus
Pipet, Mohr, glass 2.00 mL 1 each 2093636
Piper Filler, safety bulb — each 1465100
Test Tube Rack 1–2 each 1864100
Recommended standards and apparatus

Chlorine Solution Voluette® Ampule, 50–75 mg/L 16/pkg 1426810
Chlorine Solution 2 mL PourRite® Ampule, 50–75 mg/L 20/pkg 1426820
Chlorine Solution 2 mL PourRite® Ampule, 25–30 mg/L 20/pkg 2630020
Water, organic-free 500 mL 2641549
Chloramine (Mono)
Page 223
Chloramine (Mono)

Beaker, 100 mL each 50042H
Clippers each 96800
Pipet, Mohr, glass, 5-mL each 2093437
Pipet Tips for TenSette Pipet 50/pkg 2185696
Pipet, volumetric, Class A, 2-mL each 1451536
Stir bar, octagonal each 2095352
Shears each 2369400

Chloramine (Mono), LR, 10171
Chloramine (Mono) DOC316.53.01015

LR (0.04 to 4.50 mg/L Cl2) Powder Pillows
Scope and Application: Chloraminated drinking water and chlorinated wastewater
1 U.S. Patent 6,315,950
Test preparation

DR 3900 4864302 Orientation key away from user LZV846 (A)
adjust.
To determine chloramine (mono) and free ammonia on the same sample, use Method 10200 Nitrogen, Free Ammonia and
Chloramine (Mono)
Dispose of reacted solutions according to local, state and federal regulation. Use the guidance given on the Material Safety
Monochlor F Reagent Pillow 1
Sample Cell (see Instrument-specific information) 1
Chloramine (Mono)
Page 225
Chloramine (Mono)
Indophenol method, powder pillows
Stored Programs
66 Monochloramine LR Zero
Start
1. Select the test. 2. Fill the cell to the 3. Wipe the cell and 4. ZERO the instrument.
Insert an adapter if 10-mL line with sample. insert it into the cell holder. The display will show:
required (see Instrument- 0.00 mg/L Cl2
specific information).
Refer to the user manual
for orientation.
5. Remove the cell and 6. Cap and shake the 7. Start the instrument 8. After the timer expires,
add the contents of one cell about 20 seconds to timer. insert the vial into the cell.
Monochlor-F pillow to the dissolve. A 5-minute reaction period READ the results in mg/L
sample. will begin. Samples colder Cl2.
than 18 °C will require
additional time. See the
table.
Chloramine (Mono)
Page 226
Chloramine (Mono)
Interferences
The substances listed in the Non-interfering substances table have been tested for interference
and do not interfere at or below the indicated levels. The Interfering substances table suggests
treatments for interferences.

Alanine 1 mg/L N
Aluminum 10 mg/L Al
Bromide 100 mg/L Br –
Bromine 15 mg/L Br2
Chloride 18,000 mg/L Cl–
Chromium (III) 5 mg/L Cr3+
Copper 10 mg/L Cu
Cyanide 10 mg/L CN –
Fluoride 5 mg/L F–
Free Chlorine 10 mg/L Cl2
Glycine 1 mg/L N
Lead 10 mg/L Pb
Nitrate 100 mg/L N
Nitrite 50 mg/L N
Phosphate 100 mg/L PO4
Sulfate 2600 mg/L SO42+

Tyrosine 1 mg/L N
Urea 10 mg/L N
Zinc 5 mg/L Zn

Interfering substance and effects Interference level Recommended treatment
+ Add 5 drops Rochelle Salt Solution prior to testing.
Magnesium Above 400 mg/L CaCO3
OR: use the high range (HR) test.
Manganese (+7) – Above 3 mg/L Use the HR test; it will tolerate up to 10 mg/L.
Ozone – Above 1 mg/L Usually does not coexist with monochloramine.
Sulfide + Turns a “rust” color if present. Usually does not coexist with monochloramine.
Thiocyanate – Above 0.5 mg/L This method will tolerate up to 2 mg/L.
Chloramine (Mono)
Page 227
Chloramine (Mono)

Test results are strongly influenced by sample temperature. The reaction period indicated in the
procedure is for a sample temperature of 18–20 ºC (64–68 ºF). Adjust the reaction period
according to Table 76. Samples can be read up to 15 minutes after the listed development time.
Table 76 Color development time

Sample temperature
Development time (minutes)
°C °F
5 40 10
7 42 9
9 48 8
10 50 8
12 54 7
14 58 7
16 61 6
18 68 4
20 73 3
23 75 2.5
25 77 2

Analyze samples for monochloramine immediately after collection. Rinse the sample container
several times with sample, letting the container overflow each time. If sampling from a tap, let the
water flow for at least 5 minutes, then cap the container so that there is no head space (air) above
the sample.
Accuracy check
• Chlorine Solution Ampules, 50–70 mg/L
• Pipet, Volumetric, 2 mL and pipet bulb
Chloramine (Mono)
Page 228
Chloramine (Mono)
ammonia standard.
4. Pipet 50.00 mL of the buffered ammonia standard into a clean 100-mL beaker. Add a stir bar.
following equation:
455
slowly to the ammonia standard, while mixing at medium speed on a stir-plate.

10. Use this standard within 1 hour of preparation. Analyze according to the Low Range
Monochloramine procedure described above.
Method performance
Summary of method
In the presence of a cyanoferrate catalyst, monochloramine (NH2Cl) in the sample reacts with a
substituted phenol to form an intermediate monoimine compound. The intermediate couples with
excess substituted phenol to form a green-colored indophenol, which is proportional to the amount
of monochloramine present in the sample. Test results are measured at 655 nm.
Chloramine (Mono)
Page 229
Chloramine (Mono)
Required reagents
Description Quantity/Test Unit Cat. No.
Buffer Powder Pillows pH 8.3 25/pkg 89868
Chlorine Standard Solution,10-mL Voluette® Ampule 50–75 mg/L 16/pkg 1426810
Chlorine Standard Solution, 2-mL PourRite® Ampule 50–75 mg/L 20/pkg 1426820
Chlorine Standard Solution, 2-mL PourRite® Ampule 25–30 mg/L 20/pkg 2630020
Nitrogen, Ammonia Standard Solution, 1000-mg/L as NH3–N 1L 2354153
Organic-free Water 500-mL 2641549

Beaker, glass, 100 mL each 50042H
Monochlor F Reagent Powder Pillows 100/pkg 2802299
Monochloramine/Free Ammonia Spec Check Kit each 2507500
Pipet, Mohr, glass, 5-mL each 2093437
Rochelle Salt Solution 29 mL DB 172533
Stir Bar, octagonal each 2095352
Shears each 2369400
Thermometer, non-mercury, –10 to +225 °C each 2635700

Chloride, 8113
Chloride DOC316.53.01017
Mercuric Thiocyanate Method Method 8113

(0.1 to 25.0 mg/L Cl–)
Test preparation


Filter turbid samples with moderately rapid filter paper and a funnel before analysis.
Both the sample and the blank will contain mercury (D009) at a concentration regulated as a hazardous waste by the Federal
RCRA. Do not pour these solutions down the drain.
Refer to the MSDS sheet for safe handling and disposal of hazardous waste. Gloves are recommended.
Ferric Ion Solution 1 mL
Mercuric Thiocyanate Solution 2 mL
Deionized Water 10 mL
Pipet, TenSette®, 0.1 to 1.0 mL 1
Pipet tips for 0.1 to 1.0 mL TenSette pipet 2
Chloride
Page 231
Chloride
Mercuric Thiocyanate method for Chloride
Stored Programs
70 Chloride
Start
1. Select the test. 2. Prepared Sample: 3. Blank Preparation: 4. Pipet 0.8 mL of

Insert an adapter if Fill a sample cell with Fill another sample cell Mercuric Thiocyanate
required (Instrument- 10 mL of sample. with 10 mL of deionized Solution into each sample
specific information). water. cell. Note: Use 1.0 mL
with DR 5000.
for orientation.
5. Swirl to mix. 6. Pipet 0.4 mL of Ferric 7. Swirl to mix. An 8. Start the instrument
Ion Solution into each orange color will develop if timer.
sample cell. Note: Use 0.5 chloride is present. A two-minute reaction time
mL with DR 5000. will begin.
Zero Read
9. Within five minutes 10. ZERO the instrument. 11. Wipe the prepared 12. READ the results in
after the timer expires, The display will show: sample and insert it into mg/L Cl–.
wipe the blank and insert it the cell holder.
into the cell holder. 0.0 mg/L Cl–
Chloride
Page 232
Chloride
Interferences
Table 78 Interfering substances and levels

The pH should be about 2 after adding reagents.
If the sample is strongly acidic or alkaline, adjust a portion of sample before testing to a pH of
Extreme pH
about 7. Use either 5.0 N Sodium Hydroxide Standard Solution1 or a 1:5 dilution of perchloric acid.
Use pH paper; most pH electrodes will contaminate the sample with chloride.

Collect samples in glass or plastic containers. Samples can be stored for at least 28 days at room
temperature.
Accuracy check
• Chloride Standard Solution, 1000-mg/L
• 50 mL mixing cylinders (3)
• TenSette Pipet and tips
4. Prepare three sample spikes. Fill three 50 mL mixing cylinders with 50 mL of sample. Use the
TenSette® Pipet to add 0.1 mL, 0.2 mL and 0.3 mL of 1000-mg/L Chloride Standard Solution,
respectively, to the cylinders and mix each thoroughly.
5. Analyze a 10 mL portion of each sample spike as described in the Mercuric Thiocyanate

method for Chloride test, starting with the 0.1 mL sample spike. Each addition should reflect
approximately 100% recovery.

• Chloride Standard Solution, 1000-mg/L
• Volumetric Flask, 500-mL
• Pipet, Class A, 10 mL
1. Prepare a 20.0-mg/L chloride standard solution.
a. Pipet 10.00 mL of Chloride Standard Solution, 1000-mg/L, into a 500-mL volumetric flask.
b. Dilute to the mark with deionized water. Follow the Mercuric Thiocyanate method for
Chloride test.
Chloride
Page 233
Chloride
Method performance
Program Standard 95% Confidence Limits of Concentration change Portion of curve
70 20.0 mg/L Cl– 17.9–22.1 mg/L Cl– 0.1 mg/L Cl– 1.0 mg/L
0.3 mg/L Cl– 10.0 mg/L
0.6 mg/L Cl– 20.0 mg/L
Summary of method
Chloride in the sample reacts with mercuric thiocyanate to form mercuric chloride and liberate
thiocyanate ion. Thiocyanate ions react with the ferric ions to form an orange ferric thiocyanate
complex. The amount of this complex is proportional to the chloride concentration. Test results are
measured at 455 nm.
Required reagents
Chloride Reagent Set, includes: — 250 tests/pkg1 2319800
(1) Ferric Ion Solution 1 mL 100 mL 2212242
(1) Mercuric Thiocyanate Solution 2 mL 200 mL 2212129
Water, deionized 10 mL 4L 27256
1 This test requires a reagent blank. The number of tests shown refers to any combination of samples and blanks.
Required apparatus
Pipet, TenSette®, 0.1 to 1.0 mL 1 each 1970001

Pipet Tips, for TenSette Pipet 1970001 varies 50/pkg 2185696
Chloride Standard Solution, 1000-mg/L Cl– 500 mL 18349
Chloride
Page 234
Chloride

Chloride Standard Solution, 10-mL Voluette® Ampule, 12,500-mg/L Cl– 16/pkg 1425010
Chloride Standard Solution, 100-mg/L Cl– 1L 2370853
Filter Paper, funnel, 125 mm 100/pkg 69257
Funnel, poly, 75 mm each 108368
Gloves, chemical resistant, size 9–9.51 pair 2410104
Perchloric Acid, ACS 680 g 75765
pH Paper, 1.0–11.0 pH range, 15 foot roll 5/pkg 39133
1 Other sizes available
Chloride
Page 235
Chloride, DT, 8206
Mercuric Nitrate Method Method 8206

10 to 8000 mg/L as Cl– Digital Titrator
Test preparation
mg/L sodium chloride = mg/L chloride x 1.65
meq/L chloride = mg/L chloride / 35.45
Diphenylcarbazone Indicator Powder Pillow 1 pillow
Mercuric Nitrate titration cartridge (see Range-specific information) 1 cartridge
Digital titrator 1
Chloride
See
Table 1
1. Select a sample 2. Insert a clean delivery 3. Hold the Digital 4. Use a graduated
volume and titration tube into the titration Titrator with the cartridge cylinder or pipet to
cartridge from the Range- cartridge. Attach the tip pointing up. Turn the measure the sample
specific information table. cartridge to the titrator. delivery knob to eject a volume from the Range-
few drops of titrant. Reset specific information table
the counter to zero and in a 250 mL Erlenmeyer
wipe the tip. flask.
Chloride
Page 237
Chloride
Chloride (continued)
5. If the sample volume 6. Add the contents of 7. Place the delivery tube 8. Use the multiplier in
is less than 100 mL, dilute one Diphenylcarbazone into the solution and swirl the Range-specific
to approximately 100 mL Indicator Powder Pillow. the flask. Turn the knob on information table to
with deionized water. Swirl to mix. the titrator to add titrant to calculate the
Results will still be the solution. Continue to concentration:
accurate if a small amount swirl the flask and add digits x multiplier =
of powder does not titrant until the color mg/L Cl–
dissolve. changes from yellow to
light pink. Example: 100 mL of
= 25 mg/L Cl–

Range (mg/L as Cl–) Sample volume (mL) Titration cartridge (N Hg(NO3)2) Multiplier
10–40 100 0.2256 0.1

40–160 25 0.2256 0.4
100–400 100 2.256 1.0
200–800 50 2.256 2.0
500–2000 20 2.256 5.0
1000–4000 10 2.256 10.0
2000–8000 5 2.256 20.0

Collect samples in clean plastic or glass bottles. The sample can be stored for up to 7 days
before the analysis.
Chloride
Page 238
Chloride
Interferences
Bromide Interferes directly and is included in the test result.
Chromate Concentrations above 10 mg/L interfere with this method.
Ferric iron Concentrations above 10 mg/L interfere with this method.
Iodide Interferes directly and is included in the test result.
Neutralize strongly alkaline or acidic samples to a pH of 2 to 7 with 5.25 N sulfuric acid or
5.0 N sodium hydroxide. If a pH meter is used in the pH adjustment, use a separate sample
pH
to find the correct amount of acid or base to use. Then add the same amount of acid or base
to the sample to be tested. pH electrodes will contaminate the sample.
Complete the following steps to remove sulfide interference:
1. Add the contents of one Sulfide Inhibitor Reagent Powder Pillow to approximately
125 mL of sample.
Sulfide
2. Mix for one minute.
3. Filter through folded filter paper.
4. Use the filtered sample in the chloride test procedure.
Concentrations above 10 mg/L interfere with this method. Eliminate sulfite interference by
Sulfite
adding three drops of Hydrogen Peroxide, 30%, to the sample before the test is started.
Accuracy check
Use the standard additions method to find if the sample has an interference and to confirm
analytical technique.
• Chloride Voluette® Ampule Standard Solution, 12,500-mg/L Cl–
• Ampule breaker
8. Each 0.1 mL of standard that was added will use approximately 12.5 digits of the 2.256 N
titration cartridge or 125 digits of the 0.2256 N titration cartridge to reach the endpoint.
If more or less titrant was used, the problem can be due to user technique, an interference
(see Interferences) or a problem with reagents or apparatus.
Chloride
Page 239
Chloride
Summary of method
When using Mercuric Nitrate Standard Solution, the sample is titrated under acidic conditions in
the presence of diphenylcarbazone indicator. Upon addition of a slight excess of mercuric ion, a
pink-purple complex is formed with the indicator, signaling the end point.

Required reagents
Chloride Reagent Set (approximately 100 tests): 2272600
(2) Diphenylcarbazone Powder Pillows 1 pillow 100/pkg 83699
(1) Mercuric Nitrate Titration Cartridge, 0.2256 N varies each 1439301
(1) Mercuric Nitrate Titration Cartridge, 2.256 N varies each 92101
Required apparatus
Delivery tubes w/ 180° hook 1 each 1720500
Chloride Standard Solution, Voluette® Ampule, 12,500-mg/L Cl–, 10-mL 16/pkg 1425010
Voluette breaker 1 2196800
Chloride
Page 240
Chloride

Filter paper, 12.5 cm 100/pkg 69257
Hydrogen Peroxide, 30%, ACS 473 mL 14411
Sulfide Inhibitor Reagent Powder Pillow 100/pkg 241899
pH paper — 2601300
pH Test Strip , 0–14 pH 100/pkg 2601300
Chloride standard solution, 1000 mg/L 500 mL 18349
Clipper for Medium PWD PLWS each 96800
Chloride
Page 241
Chloride, DT, 8207
Silver Nitrate Method Method 8207

10 to 10,000 mg/L as Cl– Digital Titrator
Test preparation
mg/L sodium chloride = mg/L chloride x 1.65
meq/L chloride = mg/L chloride / 35.45
Chloride 2 Indicator Powder Pillow 1 pillow
Silver Nitrate titration cartridge (see Range-specific information) 1 cartridge
Digital titrator 1
Chloride
See
Table 1
the counter to zero and into a 250 mL Erlenmeyer
Chloride
Page 243
Chloride
Chloride (continued)
into a clean, 250-mL one Chloride 2 Indicator into the solution and swirl the Range-specific
Erlenmeyer flask. If the Powder Pillow. Swirl to the flask. Turn the knob on information table to
sample volume is less mix. the titrator to add titrant to calculate the
than 100 mL, dilute to Results will still be the solution. Continue to concentration:
approximately 100 mL with accurate if a small amount swirl the flask and add digits x multiplier =
deionized water. of powder does not titrant until the color mg/L Cl–
dissolve. changes from yellow to
red-brown. Example: 100 mL of
= 25 mg/L Cl–

Range (mg/L as Cl–) Sample volume (mL) Titration cartridge (N AgNO3) Multiplier
10–40 100 0.2256 0.1

25–100 40 0.2256 0.25
100–400 50 1.128 1.0
250–1000 20 1.128 2.5
1000–4000 5 1.128 10.0
2500–10,000 2 1.128 25.0

Collect samples in clean plastic or glass bottles. The sample can be stored for up to 7 days
before the analysis.
Interferences
Bromide Interferes directly and is included in the test result.
Cyanide Interferes directly and is included in the test result.
Iron Concentrations above 10 mg/L mask the end point.
Chloride
Page 244
Chloride

Iodide Interferes directly and is included in the test result.
Orthophosphate Concentrations above 25 mg/L will precipitate the silver.
Neutralize strongly alkaline or acidic samples to a pH of 2 to 7 with 5.25 N sulfuric acid or
5.0 N sodium hydroxide. If a pH meter is used in the pH adjustment, use a separate sample
pH
to find the correct amount of acid or base to use. Then add the same amount of acid or base
to the sample to be tested. pH electrodes will contaminate the sample.
Complete the following steps to remove sulfide interference:
1. Add the contents of one Sulfide Inhibitor Reagent Powder Pillow to approximately
125 mL of sample.
Sulfide
2. Mix for one minute.
3. Filter through folded filter paper.
4. Use the filtered sample in the chloride test procedure.
Concentrations above 10 mg/L interfere with this method. Eliminate sulfite interference by
Sulfite
adding three drops of Hydrogen Peroxide, 30%, to the sample before the test is started.
Accuracy check
Use the standard additions method to determine whether the sample has an interference and
confirm analytical technique.
• Chloride Voluette® Ampule Standard Solution, 12,500-mg/L Cl–
• Ampule breaker
8. Each 0.1 mL of standard that was added will use approximately 12.5 digits of the 2.256 N
titration cartridge or 25 digits of the 1.128 N titration cartridge to reach the endpoint.
Chloride
Page 245
Chloride
Summary of method
The sample is titrated with Silver Nitrate Standard Solution in the presence of potassium chromate
(from the Chloride 2 Indicator Powder). The silver nitrate reacts with the chloride present to
produce insoluble white silver chloride. After all the chloride has been precipitated, the silver ions
react with the excess chromate present to form a red-brown silver chromate precipitate, marking
the end point of the titration.

Required reagents
Chloride Reagent Set (approximately 100 tests): 2288000
(2) Chloride 2 Indicator Powder Pillows 1 pillow 50/pkg 105766
(1) Silver Nitrate Titration Cartridge, 0.2256 N varies each 1439601
(1) Silver Nitrate Titration Cartridge, 1.128 N varies each 1439701
Required apparatus
Chloride Standard Solution, Voluette® Ampule, 12,500-mg/L Cl–, 10-mL 16/pkg 1425010
Voluette breaker — 2196800
Chloride
Page 246
Chloride

Filter paper, 12.5 cm 100/pkg 69257
pH Test Strip, 0–14 pH 100/pkg 2601300
Chloride standard solution, 1000 mg/L 500 mL 18349
Dropper, glass 5/pkg 1419705
Chloride
Page 247
Chloride, BT, 8225
USEPA1 Silver Nitrate Buret Titration Method2 Method 8225

0 to 25,000 mg/L as Cl– Buret Titration
1 USEPA accepted for NPDES reporting when 0.0141 N silver nitrate standard solution is used.
2 Adapted from Standard Methods for the Examination of Water and Wastewater, (Standard Method 4500 CI- B).
Test preparation
Adjust highly acidic or alkaline samples to a pH between pH 7 and 9. Use pH paper to measure the pH. A pH meter will
contaminate the sample.
To calculate the result as mg/L sodium chloride (NaCl): mg/L chloride x 1.65 = mg/L sodium chloride
A small amount of silver nitrate is used to make the red-brown color in step 6. For most accurate results, follow the procedure
using 100 mL of deionized water in place of the sample. Titrate this solution and note the volume of titrant required. For all
samples, subtract this volume of titrant before calculating the mg/L chloride.
Chloride 2 Indicator Powder Pillow 1
Silver Nitrate Standard Solution (see Range-specific information) 1 bottle
Erlenmeyer flask, 250 mL 1
Buret titration
See
Table 1
1. Select the sample 2. Fill a 25-mL buret to 3. Use a graduated 4. Transfer the sample
volume and silver nitrate the zero mark with the cylinder or pipet to into a 250-mL Erlenmeyer
standard solution from Silver Nitrate Standard measure the sample flask. If the sample volume
Range-specific Solution. volume from Range- is less than 100 mL, dilute
information. specific information. to approximately 100 mL
with deionized water.
Chloride
Page 249
Chloride
Buret titration (continued)
5. Add the contents of 6. Titrate the sample 7. Calculate:

one Chloride 2 Indicator while swirling the flask mL titrant used x multiplier = mg/L chloride as Cl–
Powder Pillow. Swirl until the color changes
to mix. from yellow to red-brown. Example: 100 mL of sample was titrated with the
0.0141 N silver nitrate solution and 15 mL of titrant was
used to reach the endpoint.
The chloride concentration is: 15 x 5 = 75 mg/L as Cl–

Range (mg/L as Cl–) Sample volume (mL) Silver nitrate concentration Multiplier
0–125 100 0.0141 N 5
100–250 50 0.0141 N 10
200–500 25 0.0141 N 20
500–1250 100 0.141 N 50
1000–2500 50 0.141 N 100
2500–10,000 25 0.141 N 200
5000–25,000 10 0.141 N 500
Interferences
An interfering substance can mask the end point. A dilution can reduce the interference to a level
at which the substance does not interfere. If an interference is suspected, use a smaller amount of
fresh sample and repeat the test.
Bromide Cyanide, bromide and iodide interfere directly and are titrated as chloride.
Cyanide Cyanide, bromide and iodide interfere directly and are titrated as chloride.
Iodide Cyanide, bromide and iodide interfere directly and are titrated as chloride.
Iron Iron concentrations over 20 mg/L will mask the end point.
Orthophosphate Orthophosphate concentrations over 25 mg/L will cause a precipitate to form.
To remove interference from sulfide, add one Sulfide Inhibitor Reagent Powder Pillow to
Sulfide
approximately 125 mL of the sample, mix for one minute and filter through filter paper.
To remove interference from at least 10 mg/L sulfite, add 3 drops of 30% hydrogen peroxide
Sulfite
to 100 mL of sample before starting the test.
Chloride
Page 250
Chloride

Collect samples in clean plastic or glass bottles. Samples can be stored in sealed containers.
Accuracy check
Use the standard additions method to find if the sample has an interference. Use the standard
solution method to make sure that the user has followed the test correctly and that the reagents
are good.
• Chloride Voluette® Ampule Standard, 12,500-mg/L as Cl–
• Ampule breaker
Procedure for use with the 0.0141 N titrant:
8. Each 0.1 mL of standard that was added should use 2.5 mL of titrant to reach the endpoint. If
more or less titrant was used, there can be an interference (see Interferences) or the
Chloride
Page 251
Chloride

A silver nitrate standard solution will slowly decompose with exposure to light. Complete the
following test to make sure the concentration is accurate.
• Sodium Chloride Standard Solution, 1000-mg/L as Cl–
• 100-mL Class A volumetric flask (for use with 0.0141 N titrant only)
1. Add 10.0 mL of the sodium chloride standard solution, 1000-mg/L as Cl–, to a 100-mL Class A
volumetric flask. Dilute to 100 mL with deionized water and mix fully. This solution has a
concentration of 100 mg/L chloride.
1. Add 100.0 mL of the diluted sodium chloride standard solution, 100-mg/L as Cl–, to an
Erlenmeyer flask.
2. Add the Chloride 2 indicator and swirl to mix.
3. Titrate the standard to the end point with the 0.0141 N silver nitrate titrant and calculate the
result. If the result is more than 105 mg/L chloride, discard the silver nitrate titrant and replace
it with a fresh supply.
1. Add 100.0 mL of the sodium chloride standard solution, 1000-mg/L as Cl–, to an Erlenmeyer
flask.
2. Add the Chloride 2 indicator and swirl to mix.

3. Titrate the standard to the end point with the 0.141 N silver nitrate titrant and calculate the
result. If the result is more than 1050 mg/L chloride, discard the silver nitrate titrant and
replace it with a fresh supply.
Summary of method
Silver nitrate is used as the titrant and potassium chromate as the indicator. Silver nitrate first
reacts selectively with the chloride in the sample to produce insoluble white silver chloride. After all
the chloride has been precipitated, the silver nitrate reacts with the chromate to form an orange or
red-brown silver chromate precipitate.
Chloride
Page 252
Chloride
Required reagents
Chloride 2 Indicator Powder Pillows 1 pillow 50/pkg 105766
Titrant—select one or more based on range:
Silver Nitrate Standard Solution, 0.0141 N varies 1L 31653
Silver Nitrate Standard Solution, 0.141 N varies 500 mL 1255149
Required apparatus
Chloride Standard Solution, Voluette® Ampule, 12,500-mg/L as Cl–, 10-mL 16/pkg 1425010
Sodium Chloride Standard Solution, 1000-mg/L as Cl– 500 mL 18349
Voluette Ampule breaker, 10 mL each 2196800
Chloride
Page 253
Chloride

Filter Paper, 12.5 cm diameter 100/pkg 69257
Hydrogen Peroxide, 30% 473 mL 14411
Pipet, TenSette, Pipet, 0.1–1.0 mL each 1970001
Flask, Class A volumetric, 100 mL each 1457442
Dropper, glass 5/pkg 1419705
Clippers for powder pillows each 66800

Chlorine Dioxide, 10126
Chlorine Dioxide DOC316.53.01021

(0.04 to 5.00 mg/L) Powder Pillows and AccuVac® Ampuls
Scope and Application: For water and wastewater. USEPA accepted for reporting for drinking water analysis.2
2 Procedure is equivalent to Standard Methods, 18 ed., 4500 ClO2 D.
Test preparation


Instrument
Analyze samples immediately because chlorine dioxide is unstable and volatile. See Sample collection, preservation and
storage.
deionized water instead of the sample. Subtract the reagent blank value from the final results or perform a reagent blank
adjust.
After adding the DPD Free Chlorine Powder Pillow to the sample, a pink color will develop if chlorine dioxide is present.
If the chlorine dioxide concentration in the sample exceeds the upper limit of the test, the color may fade or the sample may
turn yellow. Dilute the sample with high quality water that is chlorine demand-free, and repeat the test. Some loss of chlorine
dioxide may occur. Multiply the result by the appropriate dilution factor.
Chlorine Dioxide
Page 255
Chlorine Dioxide
Powder Pillow Test:
DPD Free Chlorine powder pillow, 10-mL 1
Glycine Reagent 4 drops
Stopper for 18 mm tube 2
AccuVac Test:
DPD Free Chlorine Reagent AccuVac® Ampuls 1
Glycine Reagent 16 drops
Beaker, 50-mL 1
DPD method, powder pillows
Stored Programs
76 Chlro Diox DPD
Start
1. Select the test. 2. Blank Preparation: 3. Prepared Sample: 4. Wipe the blank and
Insert an adapter if Fill a sample cell with 10 Fill a second sample cell insert it into the cell holder.
required (see Instrument- mL of sample. Close the with 10 mL of sample.
specific information). sample cell. Close the sample cell.

for orientation.
Chlorine Dioxide
Page 256
Chlorine Dioxide
DPD method, powder pillows (continued)
Zero
5. ZERO the instrument. 6. Add four drops of 7. Add the contents of 8. Wait 30 seconds for
The display will show: Glycine Reagent to the one DPD Free Chlorine any undissolved powder to
0.00 mg/L ClO2 sample. Swirl to mix. Powder Pillow to the settle.
prepared sample cell. Immediately proceed to
Swirl the sample for step 9.
20 seconds to mix.
Read
9. Within one minute of 10. READ the results in

adding the DPD reagent, mg/L ClO2.
wipe the sample cell and
insert it into the cell holder.
DPD method, AccuVac® Ampuls
Stored Programs
77 Chlor Diox DPD AV
Start
1. Select the test. 2. Blank Preparation: 3. Wipe the blank and 4. Prepared Sample:
Insert an adapter if Fill a round sample cell insert it into the cell holder. Fill a 50-mL beaker with
required (see Instrument- with 10-mL of sample. ZERO the instrument. 40 mL of sample.
specific information). The display will show: Add 16 drops of Glycine
Refer to the user manual 0.00 mg/L ClO2 Reagent to the sample in
for orientation. the beaker. Swirl gently to
mix.
Chlorine Dioxide
Page 257
Chlorine Dioxide
DPD method, AccuVac® Ampuls (continued)
Read
5. Fill a DPD Free 6. Quickly invert the 7. Within one minute of 8. READ the results in
Chlorine Reagent Ampul several times to adding the sample, wipe mg/L ClO2.
AccuVac Ampul with mix. Wait 30 seconds for the Ampul and insert it into
sample. Keep the tip any undissolved powder to the cell holder.
immersed while the Ampul settle.
fills completely.
Interferences

Greater than 150 mg/L CaCO3. May not develop full color or color may fade instantly. Neutralize
Acidity to pH 6 –7 with 1 N Sodium Hydroxide1. Determine amount to be added on a separate sample
aliquot, then add the same amount to the sample being tested. Correct for volume addition.
Greater than 250 mg/L CaCO3. Color may not develop fully or may fade instantly. Neutralize to
Alkalinity pH 6 –7 with 1 N Sulfuric Acid1. Determine amount to be added on a separate sample aliquot, then
add the same amount to the sample being tested. Correct for the volume addition.
Bromine, Br2 Interferes at all levels.
May interfere at levels greater than 6 mg/L. Additional glycine may be able to compensate for
Chlorine, Cl2
this interference.
Chloramines, organic May interfere.
High levels of most flocculating agents can be tolerated. This tolerance is decreased if chlorine
Flocculating agents is present. See the information about metals in this table. In the presence of 0.6 mg/L Cl2,
Al(SO4)3 (< 500 mg/L) and FeCl2 (<200 mg/L) may be tolerated.
Hardness No effect at less than 1000 mg/L as CaCO3.
Iodine, I2 Interferes at all levels.
Oxidized manganese interferes at all levels. Oxidized chromium interferes at levels greater than
2 mg/L. To remove the interferences:
Manganese, oxidized 6. Add 3 drops Potassium Iodide1 (30 g/L) to a 25-mL sample.
(Mn4+, Mn7+) or 7. Mix and wait one minute.
Chromium, oxidized (Cr6+) 8. Add 3 drops Sodium Arsenite1, 2 (5 g/L) and mix.
9. Analyze 10 mL of the treated sample as described in the procedure.
10. Subtract the result of this test from the original analysis to obtain the correct chlorine
dioxide concentration.
Chlorine Dioxide
Page 258
Chlorine Dioxide

Various metals may interfere by combining with the glycine needed to remove the chlorine
interference. Metal interference is limited except when chlorine is present. In the presence of
Metals 0.6 mg/L Cl2, both copper (>10 mg/L) and nickel (>50 mg/L) interfere. Other metals may also
interfere, depending on their ability to prevent glycine from reacting with any Cl2 in the sample.
It may be necessary to add more glycine to overcome this interference.
Causes a gradual drift to higher readings. When read within 1 minute after reagent addition,
Monochloramine
3 mg/L monochloramine causes less than a 0.1 mg/L ClO2 increase in the reading.
Ozone Interferes at levels greater than 1.5 mg/L.
Peroxides May interfere.
Extreme sample pH Adjust to pH 6–7.
Highly buffered samples Adjust to pH 6–7.
2 Samples treated with sodium arsenite for interferences will be hazardous waste as regulated by Federal RCRA for arsenic (D004). Refer to a
current MSDS for proper disposal instructions.

Analyze samples for chlorine dioxide immediately after collection. Chlorine dioxide is a strong
oxidizing agent and is unstable in natural waters. It reacts rapidly with various inorganic
compounds, but oxidizes organic compounds more slowly. Many factors, including reactant
concentrations, sunlight, pH, temperature, and salinity influence decomposition of chlorine dioxide
in water.
Avoid plastic containers since these may have a large chlorine dioxide demand. Pretreat glass
sample containers to remove any chlorine or chlorine dioxide demand by soaking in a dilute bleach
solution (1 mL commercial bleach to 1 liter of deionized water) for at least one hour. Rinse
thoroughly with deionized or distilled water. If sample containers are rinsed thoroughly with
deionized or distilled water after use, only occasional pretreatment is necessary.
A common error in testing for chlorine dioxide is not obtaining a representative sample. If sampling
from a tap, let the water flow for at least 5 minutes to ensure a representative sample. Let the
container overflow with the sample several times, then cap the sample containers so there is no
headspace (air) above the sample. If sampling with a sample cell, rinse the cell several times with
the sample, then carefully fill to the 10-mL mark. Perform the chlorine dioxide analysis
immediately.
Accuracy check
Preparing chlorine dioxide standards is difficult and dangerous. In addition, these standards are
both explosive and volatile! Only a trained chemist should prepare the standards using appropriate
safety equipment and precautions. The manufacturer does not recommend preparation of chlorine
dioxide standards. If independent standard preparation is required, please see the instructions in
Standard Methods for the Examination of Water and Wastewater, Part 4500-ClO2 Chlorine
Dioxide, under the headings "Stock chlorine dioxide solution" and "Standard chlorine dioxide
solution". Prepare a chlorine dioxide standard.
Chlorine Dioxide
Page 259
Chlorine Dioxide
Method performance
76 DR 5000 3.00 mg/L ClO2 2.89–3.11 mg/L ClO2 0.04 mg/L ClO2
77 DR 5000 3.00 mg/L ClO2 2.91–3.09 mg/L ClO2 0.04 mg/L ClO2
Summary of method
Chlorine dioxide reacts with DPD (N, N-diethyl-p-phenylenediamine) to the extent of one-fifth of its
total available chlorine content, corresponding to reduction of chlorine dioxide to chlorite. The
resulting pink color intensity is proportional to the ClO2 in the sample. Chlorine interference is
eliminated by adding glycine, which converts free chlorine to chloroaminoacetic acid, but has no
effect on chlorine dioxide at the test pH. Test results are measured at 530 nm.
Required reagents
Chlorine Dioxide DPD/Glycine Reagent Set (100 tests), includes: 2770900
(1) DPD Free Chlorine Reagent Powder Pillows, 10-mL 1 100/pkg 2105569
(1) Glycine Reagent 4 drops 29 mL 2762133
OR
Chlorine Dioxide DPD/Glycine AccuVac® Ampul Reagent Set (25 tests), includes: 2771000
(1) DPD Free Chlorine Reagent AccuVac® Ampuls 1 25/pkg 2502025
(1) Glycine Reagent 16 drops 29 mL 2762133
Required apparatus
AccuVac Snapper 1 each 2405200
Stopper for 18 mm tube 1 6/pkg 173106
Chlorine Dioxide
Page 260
Chlorine Dioxide

AccuVac Vials, for sample blanks 25/pkg 2677925
DPD Free Chlorine Reagent Powder Pillows, 10-mL 1000/pkg 2105528
Standard Methods Book, most current edition each 2270800
Sulfuric Acid, 1 N, 100 mL each 127032
Chlorine Dioxide
Page 261
Chlorine Dioxide, HR, 8138
Direct Reading Method Method 8138

HR (5 to 1000 mg/L)
Test preparation


Chlorine dioxide is unstable and volatile. Analyze samples immediately.
Gloves and goggles are recommended.
Water, deionized 10 mL
Chlorine Dioxide
Page 263
Chlorine Dioxide
Direct Reading method
Stored Programs
75 Chlor Diox HR
Zero
Start
1. Select the test. 2. Blank Preparation: 3. Wipe the blank and 4. ZERO the instrument.
Insert an adapter if Fill a sample cell to the 10- insert it into the cell holder. The display will show:
required (see Instrument- mL mark with deionized
water. 0 mg/L ClO2
for orientation.
Read
5. Prepared Sample: 6. Wipe the prepared 7. READ the results in

Fill a second sample cell sample and insert it into mg/L ClO2.
to the 10-mL mark with the cell holder.
sample.
Interferences
Because this is a Direct Reading test, no known interferences exist.
Sample collection, storage and preservation

in water.
Chlorine Dioxide
Page 264
Chlorine Dioxide
immediately.
Accuracy check
solution". Prepare a 500-mg/L chlorine dioxide standard.
Method performance
75 469 mg/L ClO2 459–479 mg/L ClO2 5 mg/L ClO2
Summary of method
Chlorine dioxide, a yellow gas, can be measured directly in a water solution. Test results are
measured at 445 nm.
Required reagents and apparatus

Optional apparatus

Chlorine Dioxide
Page 265
Chlorine Dioxide, LR, 8065
Chlorophenol Red Method1 Method 8065

LR (0.01 to 1.00 mg/L)
1 Adapted from Harp, Klein and Schoonover, Jour. Amer. Water Works Assn., 73 387–388 (1981).
Test preparation

For most accurate results, analyze each portion of sample at the same temperature.
A TenSette® Pipet may be used to dispense Chlorine Dioxide Reagent 1 and Chlorine Dioxide Reagent 3
Chlorine Dioxide Reagent 1 2 mL
Dechlorinating Reagent Pillows 1
Cylinder, graduated mixing, 50 mL 2
Pipet, volumetric, Class A, 1 mL 3
Pipet Filler, with safety bulb 1
Chlorine Dioxide
Page 267
Chlorine Dioxide
Powder Pillows
Stored Programs
72 Chlor Diox CPR LR
Start
1. Select the test. 2. Fill two 50-mL mixing 3. Use a volumetric pipet 4. Invert several times to
Insert an adapter if cylinders to the 50-mL and pipet filler to add 1.0 mix.
required (see Instrument- mark with sample. mL of Chlorine Dioxide
specific information). Reagent 1 to each
cylinder. Stopper.
for orientation.
5. Blank Preparation: 6. Use a volumetric pipet 7. Invert several times to 8. Use a volumetric pipet
Add the contents of one to add exactly 1.00 mL of mix. and pipet filler to add
Dechlorinating Reagent Chlorine Dioxide Reagent 1.0 mL of Chlorine Dioxide
Powder Pillow to one 2 to each cylinder. Reagent 3 to each
cylinder. (This is Stopper. cylinder. Stopper.
the blank).
Stopper and invert several
times until dissolved.
The second cylinder,
which does not receive
dechlorinating reagent, is
the prepared sample.
Chlorine Dioxide
Page 268
Chlorine Dioxide
Powder Pillows
9. Invert several times to 10. Pour 10 mL from each 11. Wipe the blank and 12. Wipe the prepared
mix. cylinder into two insert it into the cell holder. sample and insert it into
sample cells. ZERO the instrument. the cell holder.
The display will show: READ the results in

0.00 mg/L ClO2 mg/L ClO2.
Interferences

May require 2.0 mL each of Chlorine Dioxide Reagent 1 and
Highly acidic or alkaline water
Chlorine Dioxide Reagent 3 instead of 1.0 mL
ClO– Greater than 5.5 mg/L
ClO2 – Greater than 6 mg/L
ClO3– Greater than 6 mg/L
CrO42– Greater than 3.6 mg/L
Fe3+ Greater than 5 mg/L
Hardness Greater than 1000 mg/L
Ozone Greater than 0.5 mg/L
Turbidity Greater than 1000 NTU

• Analyze samples for chlorine dioxide immediately after collection.
• Chlorine dioxide is a strong oxidizing agent and is unstable in natural waters. It reacts rapidly
with various inorganic compounds, but oxidizes organic compounds more slowly. Many
factors, including reactant concentrations, sunlight, pH, temperature and salinity influence
decomposition of chlorine dioxide in water.
• Avoid plastic containers since these may have a large chlorine dioxide demand.
• Pretreat glass sample containers to remove any chlorine or chlorine dioxide demand by
soaking in a dilute bleach solution (1 mL commercial bleach to 1 liter of deionized water) for at
least one hour. Rinse thoroughly with deionized or distilled water. If sample containers are
rinsed thoroughly with deionized or distilled water after use, only occasional pretreatment is
necessary.
Chlorine Dioxide
Page 269
Chlorine Dioxide
• A common error in testing for chlorine dioxide is not obtaining a representative sample. If
sampling from a tap, let the water flow for at least 5 minutes to ensure a representative
sample. Let the container overflow with the sample several times, then cap the sample
containers so there is no headspace (air) above the sample.
Accuracy check
solution". Prepare a 0.50-mg/L chlorine dioxide standard.
Method performance
72 0.53 mg/L ClO2 0.50–0.55 mg/L ClO2 0.01 mg/L ClO2
Summary of method
Chlorine Dioxide (ClO2) is determined by its combination with chlorophenol red at pH 5.2 to form a
colorless complex. The net effect is bleaching of the color in an amount proportional to the chlorine
dioxide concentration. The method is specific for ClO2 and is unreactive to other active chlorine or
moderate oxidizing compounds. Test results are measured at 575 nm.
Chlorine Dioxide
Page 270
Chlorine Dioxide
Required reagents
Chlorine Dioxide Reagent Set (100 Tests), includes: — each 2242300
(2) Chlorine Dioxide Reagent 1 2 mL 100 mL 2070042
(1) Dechlorinating Reagent Powder Pillows 1 100/pkg 1436369
Required apparatus
Cylinder, graduated mixing, 50-mL 2 each 189641

Standard Methods book, current edition each 2270800
Chlorine Dioxide
Page 271
Chlorine Dioxide, Amaranth, Europe only
Amaranth Method1
(20 to 500 µg/L)
Scope and Application: For water, drinking water
1 This method is under license of Elf Atofina. Reagent sets for this method are only available in Europe.
Test preparation

Chlorine dioxide is unstable and volatile. Analyze samples immediately. See Sample collection, preservation and storage.
For most accurate results, analyze each portion of sample at the same temperature.
For best precision, measurement of the reagent with a volumetric pipet or high precision pipettor is recommended.
A TenSette® Pipet may be used to dispense Chlorine Dioxide Reagent A.
Chlorine Dioxide Reagent Set 1
Volumetric Flask, 25-mL plastic 2
Syringe, 1-mL with needle 1
Chlorine Dioxide
Page 273
Chlorine Dioxide
Amaranth method
Stored Programs
78 Chlor Diox, Amaranth
Start
1. Select the test. 2. Blank Preparation: 3. Fill the volumetric flask 4. Pour 10 mL from the
Insert an adapter if Using the syringe and to the mark with deionized volumetric flask into a
required (see Instrument- needle provided, add water. Stopper. Invert at 10 mL sample cell.
specific information). 1.0 mL of Chlorine Dioxide least 7 times to mix.
Reagent A into a 25-mL
Refer to the user manual volumetric flask.
for orientation.
5. Prepared Sample: 6. Fill the second 7. Start the instrument 8. Prepared Sample:
Add 1.0 mL of Chlorine volumetric flask to the timer. A 1-minute reaction Pour 10 mL from the
Dioxide Reagent A into a mark with the sample. period will begin. second volumetric flask
second 25-mL volumetric Stopper. Invert at least 7 into a second sample cell.
flask. times to mix.
Use a volumetric pipet and
pipet filler or a TenSette
Pipet to add this reagent.
Zero Read
9. Wipe the blank and 10. ZERO the instrument. 11. When the timer 12. READ the results in
insert it into the cell holder. The display will show: expires, wipe the prepared µg/L ClO2.
sample and insert it into
0 µg/L ClO2 the cell holder.
Chlorine Dioxide
Page 274
Chlorine Dioxide
Interferences

ClO– Greater than 2.0 mg/L
ClO2– Greater than 2.0 mg/L
ClO3– Greater than 2.0 mg/L
CrO42– Greater than 0.2 mg/L
Fe3+ Greater than 0.5 mg/L
Hardness Greater than 1000 mg/L
Ozone Greater than 0.5 mg/L
Turbidity Greater than 1000 NTU

in water.
headspace (air) above the sample. Perform the analysis immediately.
Accuracy check
solution". Prepare a 0.25-mg/L (250-µg/L) chlorine dioxide standard.
Method performance
78 250 µg/L ClO2 192–308 µg/L ClO2 24 µg/L ClO2
Chlorine Dioxide
Page 275
Chlorine Dioxide
Summary of method
Chlorine dioxide (ClO2) is determined by its combination with Amaranth. Color intensity decreases
as the level of chlorine dioxide increases. Test results are measured at 521 nm.
Required reagents
Chlorine Dioxide Reagent Set (100 Tests)1 1 100/pkg LYW240
1 Available only in Europe.
Required apparatus
Chlorine Dioxide Tool Set1, includes: — each LZC140

(2) Flask, volumetric, 25-mL 2 each —
(1) Syringe, 1-mL, with needle 1 each —
1 Available only in Europe.


Standard Methods Book (most current edition) each 2270800

Chlorine Dioxide, MR, 8345
Direct Reading Method Method 8345

MR (1 to 50 mg/L)
Test preparation


DR 3800, DR 2800 2495402 Fill line faces right
Gloves and goggles are recommended.
Chlorine Dioxide
Page 277
Chlorine Dioxide
Direct Reading method
Stored Programs
73 Chlor Diox MR
Zero
Start
Insert an adapter if Fill a sample cell to the 10- insert it into the cell holder. The display will show:
required (see Instrument- mL mark with deionized
water. 0 mg/L ClO2
for orientation.
Read
5. Prepared Sample: 6. Wipe the prepared 7. READ the results in

Fill a second sample cell sample and insert it into mg/L ClO2.
to the 10-mL mark with the cell holder.
sample.

in water.
immediately.
Chlorine Dioxide
Page 278
Chlorine Dioxide
Accuracy check
dioxide standards. If independent standard preparation is required, see the instructions in
solution". Prepare a 25.0-mg/L chlorine dioxide standard.
Method performance
75 43 mg/L ClO2 41–45 mg/L ClO2 0.3 mg/L ClO2
Summary of method
Chlorine dioxide, a yellow gas, can be measured directly in a water solution. Test results are
measured at 360 nm.

Optional apparatus

Chlorine Dioxide
Page 279
Chlorine Demand/Requirement
Chlorine Demand/Requirement DOC316.53.01146
Method 10223
DPD Reagent 1
Scope and Application: For determining the chlorine demand and the chlorine requirement in drinking water
production. For establishing chlorine demand constants and establishing historical background data on raw water
quality. For determining chlorine demand on distributed waters.
1 Adapted from Standard Methods for the Examination of Water and Wastewater, Section 2350
Test preparation
Important Note: Read Getting started and all procedure steps before performing this test.
Develop a chlorine demand plan to detail the number of sample doses, the concentration of chlorine dose additions and
length of chlorine contact time. See Chlorine demand test plan and Getting started.
Precondition sample containers, test bottles and labware to be chlorine demand free. See Treatment of analysis labware.
Allow time for samples to equilibrate to the temperature indicated in the test plan before beginning the test.
DPD Free Chlorine Reagent PP, 10-mL or 25-mL Varies
Chlorine Dosing Solution Ampules Varies
Sample Bottles and Caps 6
Bottle Labels 6
pH Meter 1
Thermometer 1
Pipet, TenSette®, 0.1–1.0 mL and tips 1
Stir Plate 1
Stir Bar Magnets 6
Sample Cells, 10 mL 1-inch square or 1-cm/10 mL 2
Spectrophotometer or Colorimeter 1
Page 281
DPD Reagent
1. Complete a Chlorine 2. Prepare 6 chlorine 3. Use tweezers or tongs 4. Open a Chlorine

demand test plan. demand-free bottles. to insert a stir bar magnet Dosing Solution Ampule.
Measure and record the Rinse each bottle with into each bottle. Set Using a TenSette Pipet,
temperature and pH of the sample and fill each Bottle #1 on a stir plate add 0.1 mL of the chlorine
sample water to be tested. 118-mL bottle with and stir gently. A small solution to Bottle #1 while
approximately 100 mL of vortex should be visible on stirring. Immerse the end
the sample to be tested. the surface of the liquid. of the pipet tip under the
Label the bottles 1 to 6. Do not handle the stir water to dispense the
bar with fingers. Bare chlorine. Mixing while
fingers will add chlorine adding the chlorine is
demand to the sample. imperative to avoid highly
localized areas of chlorine
concentration.
Method 8021
Repeat steps 4–6 or
Method 10069
5. Turn off the stirrer and 6. Calculate the actual 7. Repeat Steps 4–6 for 8. After the prescribed
fill the bottle until it amount of chlorine added. bottles 2 through 6. contact is completed,
overflows with sample. See Chlorine addition Increase the amount of analyze the samples for
Cap and invert to mix. calculation for the formula chlorine added in Free Chlorine using DPD
Record the start time and and an example. increments of 0.1 mL. See Free Chlorine Reagent.
put the sample bottle in The amounts of Dosing Incremental addition of Use Method 8021 if the
the dark or wrap with foil. Solution added may be Cl2 dosing solution and desired chlorine residual is
Each 0.1 mL of chlorine increased or decreased Getting started. Stagger below 2.0 mg/L Cl2 or
dosing solution added will based on the expected the chlorine additions if the Method 10069 if residuals
add approximately 1.0 mg/ organic level of the sample contact time is expected to of greater than 2.0 mg/L
L Cl2 to the sample. water and chlorine be less than 30 minutes. Cl2 are required. Follow
contact time. This allows time to perform the procedure supplied
the chlorine analysis on with the
each sample bottle at the spectrophotometer or
specified contact time. colorimeter being used.
Page 282
DPD Reagent (continued)
9. Subtract the residual 10. Determine the chlorine

chlorine determined in requirement (chlorine
Step 8 from the original dosage) needed to meet
amount of chlorine added the operating goal:
to each bottle to determine Cl2 Requirement = Cl2
the chlorine demand: Demand + Cl2 Residual
Cl2 Demand = Cl2 added Required
concentration (mg/L) – Cl2 Report the chlorine
residual measured requirement according to
concentration (mg/L).1 the goals of the study.
Report the chlorine Example: The sample
demand according to the required a dose of
goals of the study. 3.0 mg/L chlorine to
Example: The sample achieve a free chlorine
dosed at 6.0 mg/L residual of 1.1 mg/L
consumed 4.1 mg/L chlorine after 2 hours at
chlorine after 2 hours at 20 °C and pH 8.1.
20 °C and pH 8.1.
1 Some bottles will have no residual chlorine if the chlorine demand exceeded the amount of chlorine added. Choose a bottle that has a chlorine
residual to determine the demand. See Chlorine demand results.
Chlorine addition calculation

Use the following formula to calculate the concentration of the chlorine added in step 6.
0.1 mL 〈 volume of standard added〉 × ampule certificate value 〈 mg/L Cl 2〉
mg/L Cl 2 = -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
-
125 mL
Example:
0.1 mL × certificate value of 1250 mg/L Cl
mg/L Cl 2 = ----------------------------------------------------------------------------------------------------------------2-
125 mL
mg/L Chlorine = 1.0
Page 283
Incremental reagent addition

Incremental addition of Cl2 dosing solution defines the incremental addition of a 1250 mg/L
chlorine dosing solution.
Table 93 Incremental addition of Cl2 dosing solution

Bottle # Cl2 dosing solution added (mL) Increases sample concentration (in mg/L Cl2) by
1 0.1 1.0
2 0.2 2.0
3 0.3 3.0
4 0.4 4.0
5 0.5 5.0
6 0.6 6.0
Chlorine demand results

Select the sample bottle that most closely fits the following criteria to calculate the
chlorine demand.
1. Residual chlorine measured is less than the chlorine dose added (0.03* mg/L).
2. Residual chlorine measured is greater than 0.03* mg/L.
3. Chlorine dose added is most similar to the dosage range expected in the field.
Criteria 1 and 2 ensure that the chlorine residual and demand are greater than the detection limit
of the DPD method used for determining the chlorine residual. If no sample portion satisfies all
criteria, repeat the test and adjust the chlorine doses accordingly.
Chlorine demand test plan

Chlorine demand test plans are developed for many purposes. The purpose should be well
defined and documented. This will develop the specific details of the plan, with the goal of
establishing reproducible test conditions to obtain reproducible data. This data is useful in
characterizing and optimizing a water treatment operation. Purposes for developing a chlorine
demand test plan might be:
Characterize the water system to establish a historical data baseline.
This baseline with chlorine demand date can be used to troubleshoot water quality problems,
provide background information for new employees and can provide additional support for
monitoring changes in water quality. The plan would include:
• The standard elements of temperature, water pH, chlorine dose rate and chlorine
contact time.
• Additional specific details to allow other analysts to reproduce the plan.
Characterize the chlorine demand of an influent raw water source.
Data obtained to establish chlorine demand data for use in understanding the effects of water
source changes, blending operations and seasonal weather variations. The plan would include:
• Source water description, sample location, time of year, specific or unusual
weather events
* The minimum detection limit for DPD Chlorine Method 8021 when calculating the concentrations by difference
(1.412 x 0.02 mg/L).
Page 284
• Additional complementary tests to be run, such as TOC, turbidity or UV-254, in addition to

the standard temperature, pH, chlorine dose rate and contact time.
Track the reduction in chlorine demand as water moves through the treatment process.
Data obtained would be used to establish a baseline for monitoring the effects of treatment
changes, seasonal water temperatures and overall changes in chlorine demand. The plan
would include:
• Specific sampling locations
• Treatment practices in use and flow rates
Getting started
Before starting this procedure, determine:
• The magnitude of the chlorine demand present in the water to be tested.
• Which chlorine method to use to determine chlorine residual.
First time users of this method or users evaluating a new water source should perform a screening
test to determine an approximate chlorine demand level before performing a full chlorine demand
test series.
1. Add 0.5 mL and 1.0 mL of Chlorine Dosing Solution to a 125-mL water sample.
2. Hold for the contact time indicated in the test plan and then analyze the chlorine residual.
3. Use the chlorine residual values to determine the specific dose requirements in the chlorine
demand test plan. As a rule, use the HR DPD Chlorine Method (Method 10069) for raw water
samples or where the desired chlorine residual will be greater than 2.0 mg/L chlorine and use
the LR DPD Chlorine Method (Method 8021) for low chlorine demand waters, such as treated
waters or samples where the desired chlorine residual is less than 2.0 mg/L chlorine.
Chlorine demand procedure modification

The chlorine demand procedure is an operationally defined procedure. The procedure is defined
by the user and may be modified to meet the specific requirements of the sample or the process
operation. Run chlorine demand studies under the range of conditions expected in the field. Use
the basic test protocol while changing contact time, temperature, sample pH and chlorine
concentrations. Total Chlorine or Monochloramine can be determined as required from the
residual measured at the end of the prescribed contact time.
Use the following guidelines if modifications are made:
• Make smaller chlorine concentration additions by using a larger sample size. A 237-mL bottle
(contains 250 mL when filled to overflowing) is available for low chlorine demand applications.
Each 0.1 mL of chlorine dosing solution added will increase the chlorine concentration by
approximately 0.5 mg/L. Substitute 250 mL for 125 mL in the above equation. A lower
concentration Chlorine Standard Solution, 50–75 mg/L as Cl2 is also available for testing low
chlorine demand waters.
• High chlorine demand waters require larger additions of chlorine. Use 0.2 mL, 0.4 mL, 0.6 mL,
etc., to spike the bottles in steps 4 and 7 in the procedure.
• Wrap sample bottles made of clear colorless glass in foil to protect from light or kept in the
dark during the contact time.
• Sample pH can be modified or standardized by adding a fixed amount of a pH buffer solution
to each bottle. Prepare a reagent blank bottle using organic free water. Add the same amount
of buffer to this blank, add a known amount of chlorine and carry the blank through the
procedure. Add only enough buffer to give the desired pH. This will check the chlorine demand
(if any) that was added by the buffer. Subtract the chlorine demand of the blank from the
sample chlorine demand values.
Page 285
• Chlorine demand tests that have an extended contact time will require temperature control if
the sample temperature is significantly different from the analysis environment. Use a
refrigerator, water bath or incubator as required. It is important to control and document these
variables in order to be able to duplicate the chlorine demand procedure on future samples.
Treatment of analysis labware

Glassware used in this test must be chlorine demand-free. Treat all glassware with a dilute
solution of chlorine bleach prepared by adding 0.5 mL of commercial bleach to 1 liter of water.
Alternatively, the sample bottles may be treated by adding 2.0 mL of the Chlorine Dosing Solution
to each 125-mL bottle and filling to overflowing with deionized water. Soak glassware in this
solution for at least one hour. After soaking, rinse the glassware with copious amounts of chlorine
demand-free water before filling with sample.

Most reliable results are obtained on fresh low solid samples that are analyzed immediately.
Samples may be stored up to 24 hours at 4 °C. Warm the samples to the required temperature
before running the chlorine demand test.
Summary of method
The chlorine demand of a water sample is defined as the difference between the concentration of
chlorine added to the sample and the concentration of the chlorine residual remaining at the end of
a predetermined contact time. The chlorine demand is a function of chlorine concentration, sample
temperature, contact time and sample pH.
The chlorine requirement is the amount of chlorine required to achieve a predetermined chlorine
residual at a prescribed contact time, pH and temperature.
Chlorine demand is caused by a complex set of reactions. Chlorine reacts with dissolved or
suspended organic materials in the water to form stable chlorinated organic compounds such as
trihalomethanes, haloacetic acids or other chlorinated organic compounds. Some of these
compounds (trihalomethanes) are referred to as disinfection by-product (DBPs) and are regulated
under the Disinfection/Disinfection By-Products Rule; other chlorinated organics contribute to taste
and odor problems. As a general rule, the lower the chlorine demand the lower the amounts of
DBPs formed and less taste and odor problems occur. Chlorine also is reduced by inorganic
reductants present such as ferrous, manganous, nitrite, sulfide and sulfite ions. Ammonia present
in the water also consumes chlorine to form chloramines.
Chlorine demand is significantly impacted by the physical and chemical characteristics of the
water sample. Chlorine demand studies ran at 10 °C will be considerably different than studies ran
at 20 °C. It is imperative that the sample temperature, pH and chlorine dose be accurately
measured and recorded. It is difficult to extrapolate chlorine demand data from one water source
to another. Demand studies need to be performed directly on the water source of interest. This
provides the information required to establish chlorine demand constants, to provide usable
historical data and to provide the test requirements for making repeatable and meaningful chlorine
demand measurements.
Page 286
Required reagents
DPD Free Chlorine Reagent Powder Pillows, 10 mL 2105569
OR —
Chlorine Dosing Solution Ampules, 1190–1310 mg/L as Cl2, 10-mL ampules, 16/pkg 2504810
Required Apparatus
Bottles, Amber Glass, 118-mL 6/pkg 714424
Caps, Black, PP Teflon liner, 12/pkg 2401812
Ampule Breaker, for Voluette Ampules 2196800
Bottles, Amber Glass, 237 mL, 6/pkg 714441
Buffer Powder Pillows, pH 6.86, 15/pkg 1409895
Buffer Solution, pH 7.0, demand-free, 500 mL 2155353
Caps, for 714441 Bottles, 6/pkg 2166706
Chlorine Standard Solution Ampules, 10 mL, 50–75 mg/L as Cl2, 16/pkg 1426810
DPD Free Chlorine AccuVacs, 25/pkg 2502025
DPD Free Chlorine SwifTest Dispenser with reagents 2802300
DPD Total Chlorine Reagent Powder Pillows, 25 mL 1406499
DPD Total Chlorine Reagent PP, 10 mL 2105969
DPD Total Chlorine SwifTest Dispenser with reagents 2802400
Graduated Cylinder, plastic, 100 mL 108142
Incubator, Model 205, 110 V, 0 to 40 °C 2616200
Labels, PolyPaper, 1.5 x 3 inches, 120/pkg 2091502
Monochlor F Reagent Powder Pillows, 10 mL 2802299
Page 287
Optional reagents and apparatus (continued)
Sension 2 Portable pH/ISE Meter, with electrode 5172510
Sodium Hydroxide Standard Solution, 0.100N, 500 mL 19153
Standard Methods Handbook 2270800
Stir Bar, Teflon-coated, 2.22 cm x 0.48 cm 4531500
Stir Plate, 120 V, 4.25 x 4.25 inches 2881200
Sulfuric Acid Std. Solution, 0.100 N, 500 mL 20253
TenSette Pipet, 0.1 - 1.0 mL 1970001
Tips for TenSette Pipet, 0.1-1.0 mL, 50/pkg 2185696
Thermometer, Double Scale, –20 to 110 °C (0–230 °F) 2095911
Tweezers 1428200
Water, Organic-Free 500 mL 2641549

Chlorine, Free, 10059
Chlorine, Free DOC316.53.01026
DPD Rapid Liquid Method1 Method 10059

(0.02 to 2.00 mg/L) Pour-Thru™ Cell
Scope and Application: For treated water.
Test preparation

Instrument Pour-thru Kit Cell orientation Adapter
DR 6000 LQV175.99.20002 Arrow faces right —
DR 5000 LZV479 — —
LQV157.99.10002 Align cell flow arrows with arrows on cell —

DR 3900
compartment
5940400 1-inch (round) path in line with the arrow on LZV585 (B)
DR 3800, DR 2800, DR 2700
the adapter
Refer to the instrument User Manual for Pour-Thru cell and module assembly and installation.
Protect the Pour-Thru Cell from contamination when not in use by inverting a small beaker over the top of the glass funnel.
The indicator reagent must be prepared in advance. See Reagent preparation.
Make sure the pour-thru cell is completely seated in the sample cell compartment.
DPD Indicator Powder varies
Free Chlorine Indicator Solution 1 mL
Free Chlorine Buffer Solution 1 mL
Cylinder, glass, mixing, 100-mL 1
Dispenser, Adjustable Volume 2
Pour-Thru Cell Module and Cell (see Instrument-specific information) 1
Chlorine, Free
Page 289
Chlorine, Free
Free Chlorine, rapid liquid method
Stored Programs
82 Chlorine, F&T RL
Zero
Start
1. Select the test. 2. Install the Pour-Thru 3. Pour approximately 4. When the flow stops,
(See Instrument-specific Cell module and cell. See 50 mL of sample into the press ZERO. The display
information). Instrument-specific Pour-Thru Cell. will show: 0.00 mg/L Cl2.
information for adapter
and cell orientation.
5. Add 1.0 mL of Free 6. Add 1.0 mL of 7. Carefully fill the mixing 8. Stopper the cylinder
Chlorine Buffer Solution to prepared Free Chlorine cylinder to the 80-mL mark and gently invert it twice to
a clean, dry 100-mL glass Indicator Solution to the with sample. mix. Proceed to step 8
mixing cylinder using the same mixing cylinder immediately.
Dispenser. using the dispenser. Swirl
to mix the reagents.
Proceed to step 7
immediately.
Chlorine, Free
Page 290
Chlorine, Free
Free Chlorine, rapid liquid method (continued)
Read
9. Fill the funnel of the 10. After the flow stops, 11. Flush the Pour-Thru
Pour-Thru Cell with the press READ. Results will Cell with at least 50-mL of
reacted sample from the appear in mg/L Cl2. deionized water
mixing cylinder. immediately after use.
(It is not necessary to pour
the entire sample into the
Pour-Thru Cell;
approximately half of the
sample may be
discarded.)
Reagent preparation
The Free Chlorine Indicator Solution must be prepared before use. Using a powder funnel, add the
contents of one 24 g bottle of DPD Powder* to one 473-mL bottle of Free Chlorine Indicator
Solution*. Invert several times and swirl until the powder is completely dissolved. A pale pink color
may develop, but should not affect results.
This solution will give accurate results for at least one month after mixing when stored at 20–25 °C
(68–77 °C). Write the date of preparation on the Indicator Solution Bottle. Discard any remaining
solution after one month. Use of this reagent after one month may result in high reagent blanks
and low values at high concentration. Do not combine fresh reagent with previously
mixed reagent.
Interferences

addition.
Hardness Levels below 1000 mg/L as CaCO3 will not interfere.
* See Required reagents.
Chlorine, Free
Page 291
Chlorine, Free

1. Adjust sample pH to 6 –7 with 1.000 N Sulfuric Acid1.

2. Add 9 drops Potassium Iodide (30 g/L)1 to an 80-mL sample.
Manganese, oxidized (Mn4+, 3. Mix and wait 1 minute.
Mn7+) or Chromium, oxidized 4. Add 9 drops Sodium Arsenite1, 2 (5 g/L) and mix.
(Cr6+) 5. Analyze the treated sample as described in the procedure above.
6. Subtract the result of this test from the original analysis to obtain the correct
concentration.
Samples containing monochloramine will cause a gradual drift to higher chlorine readings.
Monochloramine (NH2Cl) When read within one minute of reagent addition, 3.0 mg/L monochloramine will cause an
increase of less than 0.1 mg/L in the free chlorine reading.
Ozone Interferes at all levels.
1 See Optional standards and apparatus.
2 Samples treated with sodium arsenite for interferences will be hazardous waste as regulated by the Federal RCRA for arsenic (D004). Refer
to the current MSDS for safe handling and disposal instructions.

Samples must be analyzed immediately and cannot be preserved for later analysis.
A common testing error is introduced if the analyst does not obtain a representative sample. If
sampling from a tap, let the water flow for at least five minutes to ensure a representative sample.
Let the container overflow with the sample several times, then cap the sample container so there is
no headspace (air) above the sample. Perform the chlorine analysis immediately.
Avoid plastic containers since these may have a chlorine demand.
Pre-treat glass sample containers to remove any chlorine demand by soaking in a dilute bleach
solution (1 mL commercial bleach to 1 liter of deionized water) for at least 1 hour. Rinse thoroughly
If sample containers are rinsed thoroughly with deionized water after use, only occasional
pretreatment is necessary. A pre-treated glass BOD bottle with a ground-glass stopper makes an
ideal sample container for chlorine collection.
Treating analysis labware

Glassware used in this test must be chlorine demand-free. Fill the 100-mL mixing cylinder and
sample container with a dilute solution of chlorine bleach prepared by adding 1 mL of commercial
bleach to 1 liter of water. Soak in this solution at least one hour. After soaking, rinse thoroughly
with deionized water and allow to dry before use. If the mixing cylinder is thoroughly rinsed with
deionized water and allowed to dry after each use, only occasional pretreatment is necessary. Do
not use the same mixing cylinder for Free and Total Chlorine analysis.
Treat the Pour-Thru Cell similarly with dilute bleach and let stand for several minutes. Rinse
several times with deionized water.
Cleaning the Pour-Thru Cell

The Pour-Thru Cell may accumulate a buildup of colored reaction products, especially if the
reacted solutions are allowed to remain in the cell for long periods after measurement. Remove
the buildup by rinsing the cell with 5.25 N Sulfuric Acid followed by several rinsings with deionized
water.
Chlorine, Free
Page 292
Chlorine, Free
Accuracy check
• Chlorine Voluette® Ampule Standard Solution, 50 to 75-mg/L Cl2
3. Default values for standard concentration, sample volume and spike volumes can be accepted
or edited. Enter the chlorine concentration from the ampule package. After values are
accepted, the unspiked sample reading will appear in the top row. See the user manual for
more information.
4. Open a Chlorine Voluette® Ampule Standard Solution, 50 to 75-mg/L Cl2.
5. Prepare three sample spikes. Use the TenSette® Pipet to add 0.3, 0.6 and 0.9 mL of standard
to three 80-mL samples, respectively. Swirl gently to mix.
sample spike. Accept each standard additions value. Each addition should reflect
Method performance
Summary of method
Chlorine in the sample as hypochlorous acid or hypochlorite ion (free chlorine or free available
chlorine) immediately reacts with DPD (N,N-diethyl-p-phenylenediamine) indicator to form a pink
color which is proportional to the chlorine concentration. Test results are measured at 530 nm.
Required reagents
Free Chlorine Reagent Set, includes: — — 2556900
DPD Indicator Powder varies 24 g 2297255
Free Chlorine Indicator Solution 1 mL 473 mL 2314011
Free Chlorine Buffer Solution 1 mL 473 mL 2314111
Chlorine, Free
Page 293
Chlorine, Free
Required apparatus
Cylinder, mixing graduated, 100-mL, glass 1 each 2636342
Dispenser, adjustable-volume, 1.0 – 5.0 mL 2 each 2563137
Powder Funnel 1 each 2264467
Chlorine Standard Solution, Voluette® Ampule, 50-75 mg/L, 10-mL 16/pkg 1426810
OR
Chlorine Standard Solution, Pour-Rite® Ampule, 50-75 mg/L, 2-mL 20/pkg 1426820
PourRite Ampule breaker 2-mL each 2484600
Optional standards and apparatus

Pipet, TenSette® 0.1–1.0 mL each 1970001

pH Paper, 0 - 14 pH range 100/pkg 2601300

Chlorine, Free, TNT, 10102

(0.09 to 5.00 mg/L) Test ‘N Tube™ Vials
Scope and Application: For testing higher levels of free chlorine (hypochlorous acid and hypochlorite ion) in
drinking water, cooling water and industrial process water.
Test preparation

DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
DR 3900, DR 3800, DR 2700 and DR 2800: Install the light shield in Cell Compartment #2 before performing this test.
Analyze samples immediately. Do not preserve samples for later analysis.
blank adjust.
After adding sample to the Test ‘N Tube™, a pink color will develop if free chlorine is present.
Test ‘N Tube™ DPD Free Chlorine Reagent 1
Wipes, disposable varies
Chlorine, Free
Page 295
Chlorine, Free
DPD method for Test ‘N Tube vials
Stored Programs
89 CHlorine F&T TNT

Zero
Start
Insert an adapter if Fill an empty Test ‘N Tube insert it into the 16 mm cell The display will show:
required (see Instrument- vial to the top of the label holder.
with sample. 0.00 mg/L Cl2
Read
5. Remove the cap from 6. Prepared Sample: 7. Immediately wipe the 8. READ the results in
a Free Chlorine DPD Test Cap and invert slowly at sample and insert it in the mg/L Cl2.
‘N Tube. Add 10 mL of least 10 times to dissolve 16 mm cell holder.
sample to the tube. Fill the the powder. Invert by
vial to the top of the label. turning the vial upside
down, then returning it to
an upright position. Ten
inversions should take at
least 30 seconds for
complete recovery.
Interferences

Acidity Neutralize to pH 6 –7 with1 N sodium hydroxide1. Determine amount to be added on separate
addition.
Alkalinity Neutralize to pH 6–7 with 1 N sulfuric acid1. Determine amount to be added on separate sample
Bromine, Br2 Interferes at all levels
Chlorine Dioxide, ClO2 Interferes at all levels
Chlorine, Free
Page 296
Chlorine, Free
Table 97 Interfering substances and levels (continued)

Iodine, I2 Interferes at all levels
Adjust sample pH to 6 –7.
Add 3 drops potassium iodide1 (30-g/L) to a 25-mL sample.
Manganese, oxidized Mix and wait 1 minute.
(Mn4+, Mn7+) or Chromium, Add 3 drops sodium arsenite1,2 (5-g/L) and mix.
oxidized (Cr6+) Analyze 10 mL of the treated sample as described in the procedure.
Subtract the result from this test from the original analysis to obtain the correct chlorine
concentration in the sample.
For conventional free chlorine disinfection (beyond the breakpoint), typical monochloramine
concentrations are very low. If monochloramine is present in the sample, its interference in the
free chlorine test depends on the sample temperature, relative amount of monochloramine to
free chlorine and the time required to do the analysis. Typical interference levels of
monochloramine as mg/L Cl2 in the free chlorine test are listed below (1 minute test time).
NH2Cl Sample Temp. °C (°F)

Monochloramine
(as Cl2) 5 (41) 10 (50) 20 (68) 30 (83)
1.2 mg/L +0.15 0.19 0.30 0.29
2.5 mg/L +0.35 0.38 0.55 0.61
3.5 mg/L +0.38 0.56 0.69 0.73
Note: Determine Monochloramine levels using Hach method 10200.
Ozone, O3 Interferes at all levels
Adjust to pH 6–7 using acid (Sulfuric Acid1, 1.000 N) or base (Sodium Hydroxide1, 1.00 N).
buffered samples
2 Samples treated with sodium arsenite for interferences will be hazardous waste as regulated by Federal RCRA for arsenic (D004). Refer to
reagent MSDS for disposal instructions.

• Analyze samples for chlorine immediately after collection. Free chlorine is a strong oxidizing
agent and it is unstable in natural waters. It reacts rapidly with various inorganic compounds
and more slowly oxidizes organic compounds. Many factors, including reactant
concentrations, sunlight, pH, temperature and salinity influence decomposition of free chlorine
in water.
• Avoid plastic containers since these may have a large chlorine demand.
• Pretreat glass sample containers to remove any chlorine demand by soaking in a dilute bleach
solution (1 mL commercial bleach to 1 liter of deionized water) for at least 1 hour. Rinse
deionized or distilled water after use, only occasional pre-treatment is necessary.
• A common error in testing for chlorine is not obtaining a representative sample. If sampling
container overflow with the sample several times, then cap the sample containers so there is
Chlorine, Free
Page 297
Chlorine, Free
Accuracy check
• Chlorine PourRite® Ampule Standard*, 50–75 mg/L Cl2
• TenSette Pipet 0.1 – 1.0 mL and tips
• Ampule Breaker
1. After reading test results, leave the sample cell (unspiked sample) in the instrument. Verify
that the units displayed are in mg/L.
3. A keypad will appear. Enter the average chlorine concentration shown on the label of the
standard solution. Press OK. A summary of the Standard Additions procedure will appear.
Press OK.
4. Open a Chlorine PourRite® Ampule Standard*, 50–75 mg/L Cl2.
5. Use the TenSette® Pipet to add 0.1 mL to a 10-mL sample. Mix thoroughly.
6. Analyze the standard addition sample as described in the procedure above. Accept the
standard additions reading by pressing READ. The addition should reflect approximately 100%
recovery.
Method performance
Summary of method
Chlorine, Free
Page 298
Chlorine, Free
Required reagents
Test ‘N Tube™ DPD Free Chlorine Reagent 1 50/pkg 2105545


Wipes, disposable® 280/pkg 2097000
Thermometer, Non-Mercury, -10 to 225°C each 2635700
Test Tube Rack each 1864100
Potassium Iodide, 30-g/L 100mL 34332
Sodium Arsenite, 5-g/L 100mL 104732
Chlorine, Free
Page 299
Chlorine, Free, 8021
USEPA DPD Method1 Method 8021

Scope and Application: For testing free chlorine (hypochlorous acid and hypochlorite ion) in water, treated
waters, estuary and seawater. USEPA accepted for reporting for drinking water analyses.2
2 Procedure is equivalent to USEPA and Standard Method 4500-Cl G for drinking water.
Test preparation


Instrument
If the test over-ranges, dilute the sample with a known volume of high quality, chlorine demand-free water and repeat the
test. Some loss of chlorine may occur due to the dilution. Multiply the result by the dilution factor. Alternatively, samples with
high chlorine concentrations may be analyzed directly without dilution by using Method 10069, Chlorine, Free HR, or Method
10245, Chlorine Free MR .
The SwifTest Dispenser for Free Chlorine can be used in place of the powder pillow in step 4.
The sample cell shown is a generic representation. Refer to Instrument-specific information for the correct sample cell and
adapter configuration.
An empty AccuVac ampule can be used as a blank in place of the sample cell in Step 2.
Do not use the same sample cells for free and total chlorine. If trace iodide from the total chlorine reagent is carried over into
the free chlorine determination, monochloramine will interfere. It is best to use separate, dedicated sample cells for free and
total chlorine determinations.
Chlorine, Free
Page 301
Chlorine, Free
Powder Pillow Test:
DPD Free Chlorine Reagent Powder Pillows, 10-mL 1
AccuVac Test:
DPD Free Chlorine Reagent AccuVac® Ampuls 1
Beaker, 50-mL 1
Powder pillow procedure
Stored Programs
80 Chlorine, F&T PP
Start
Insert an adapter if Fill a sample cell with insert it into the cell holder. Fill a second cell with
required (see Instrument- 10 mL of sample. ZERO the instrument. 10 mL of sample.
specific information). The display will show: Add the contents of one
Refer to the user manual DPD Free Chlorine
0.00 mg/L Cl2 Powder Pillow to the
for orientation.
sample cell.
5. Swirl the sample cell 6. Within one minute of

for 20 seconds to mix. adding the reagent, insert
A pink color will develop if the prepared sample into
chlorine is present. the cell holder.
Proceed to step 6 Results are in mg/L Cl2.
immediately.
Chlorine, Free
Page 302
Chlorine, Free
AccuVac Ampuls procedure
Stored Programs
85 Chlorine, F&T AV
Start
Insert an adapter if Fill a sample cell with insert it into the cell holder. Collect at least 40 mL
required (see Instrument- 10-mL of sample. ZERO the instrument. The of sample in a 50-mL
specific information). display will show: beaker.
0.00 mg/L Cl2 Fill a DPD Free Chlorine
Reagent AccuVac Ampul
with sample. Keep the tip
fills completely.
5. Quickly invert the 6. Within one minute

Ampul several times to after sample addition, wipe
mix. Wipe off any liquid or the AccuVac Ampul and
fingerprints. insert it into the cell holder.
READ the results in
mg/L Cl2
Chlorine, Free
Page 303
Chlorine, Free
Interferences
Greater than 150 mg/L CaCO3. May not develop full color or
color may fade instantly. Neutralize to pH 6–7 with
Acidity 1 N Sodium Hydroxide. Determine amount to be added on
separate sample aliquot, then add the same amount to the
sample being tested. Correct for volume addition.
Greater than 250 mg/L CaCO3. May not develop full color or
color may fade instantly. Neutralize to pH 6 –7 with 1 N
Alkalinity Sulfuric Acid. Determine amount to be added on separate
sample aliquot, then add the same amount to the sample being
tested. Correct for volume addition.
2. Add 3 drops Potassium Iodide (30-g/L) to a 10-mL
sample.
3. Mix and wait one minute.
Manganese, Oxidized
(Mn4+, Mn7+) or Chromium, Oxidized (Cr6+) 4. Add 3 drops Sodium Arsenite 1 (5-g/L) and mix.
5. Analyze 10 mL of the treated sample as described in
the procedure.
6. Subtract the result from this test from the original
analysis to obtain the correct chlorine concentration.
Causes a gradual drift to higher readings. When read within
Monochloramine 1 minute after reagent addition, 3 mg/L monochloramine
causes less than a 0.1 mg/L increase in the reading.
Adjust to pH 6–7 using acid (Sulfuric Acid, 1.000 N) or base
Extreme sample pH or highly buffered samples
(Sodium Hydroxide, 1.00 N).
1 Samples treated with sodium arsenite for interferences will be hazardous waste as regulated by Federal RCRA for arsenic (D004). See the
current MSDS for proper disposal of hazardous material.

in water.
Chlorine, Free
Page 304
Chlorine, Free
no headspace (air) above the sample. If sampling with a sample cell, rinse the cell several
times with the sample, then carefully fill to the 10-mL mark. Perform the chlorine analysis
immediately.
Accuracy check
• Chlorine Standard Solution, 2-mL PourRite® Ampule, 25–30 mg/L
• Breaker, PourRite Ampules
3. Enter the average chlorine concentration shown on the label of the ampule container.
4. A summary of the standard additions procedure will be displayed. Press OK to accept the
default values for standard concentration, sample volume and spike volumes. After the values
are accepted, the unspiked sample reading will appear in the top row.
5. Open one Voluette ampule standard.

6. Prepare spiked samples: add 0.1 mL, 0.2 mL and 0.3 mL of standard to three 10-mL portions
of fresh sample.
Note: For AccuVac® Ampuls, add 0.4 mL, 0.8 mL and 1.2 mL of standard to three 50-mL portions of
fresh sample.
7. Follow the test procedure for each of the spiked samples using the powder pillows or AccuVac
ampules, starting with the smallest sample spike. Measure each of the spiked samples in the
instrument.
Note: If results are not within acceptable limits (± 10%), be sure that the sample volumes and sample spikes
are measured accurately. The sample volumes and sample spikes that are used should agree with the
selections in the standard additions menu. If all procedures are followed correctly but the standard
additions results are not within acceptable limits, the sample may contain an interference.
Method performance
Chlorine, Free
Page 305
Chlorine, Free
Summary of method
color, the intensity of which is proportional to the chlorine concentration. Test results are measured
at 530 nm.
Chlorine, Free
Page 306
Chlorine, Free

Required reagents
DPD Free Chlorine Reagent Powder Pillows, 10-mL 1 100/pkg 2105569
OR
DPD Free Chlorine Reagent AccuVac® Ampuls 1 1 2502025
Required apparatus
PourRite Ampule breaker, 2-mL each 2484600

Chlorine-demand Free Water 500 mL 2641549
Potassium Iodide, 30-g/L 100 mL 34332
Sodium Arsenite, 5-g/L 100 mL 104732
SwifTest Dispenser for Free Chlorine1 each 2802300
Pipet, TenSette®, Pipet, 0.1 - 1.0 mL each 1970001
AccuVac, vials for sample blanks 25/pkg 2677925
DPD Free Chlorine Reagent, 10 mL, SwifTest Dispenser refill vial 250 tests 2105560
SpecCheck Secondary Standard Kit, Chlorine DPD, 0–2.0 mg/L Set each 2635300
1 Includes one vial of 2105560 for 250 tests
Chlorine, Free
Page 307
Chlorine, free, BT, 8334
USEPA1 Amperometric Buret Titration Method2 Method 8334

0.5 mg/L and above Buret Titration
Scope and Application: For water and wastewater.
1 USEPA accepted; 40 CFR Part 141, Section 141.74.
2 Adapted from Standard Methods for the Examination of Water and Wastewater (4500 Cl-D).
Test preparation
Chlorine can be lost from the sample during sample collection. Review the precautions in Sample collection, preservation
and storage before the test is started.
Use only a 50-mm stir bar. The wrong size can cause the loss of chlorine, unstable readings and loss of method sensitivity,
especially when measuring low level chlorine concentrations.
For added convenience when stirring, use the TitraStir® apparatus.
When a new probe is placed in service or when the probe has not been used recently, prepare it according to the Probe
Stabilization instructions in the Amperometric Titrator Instruction Manual.
Phenylarsine Oxide Solution, 0.00564 N 1 bottle
Phosphate Buffer Solution, pH 7 1 mL
Amperometric Buret Titrator System 1
Beaker, 250-mL 1
Chlorine, Free
Page 309
Chlorine, Free
Buret titration
1. Fill the 5-mL automatic 2. Put a 50-mm stir bar 3. If the pH is less than 6 4. Place the beaker of
buret to the zero mark with into a 250-mL beaker. or greater than 7.5, add prepared sample on the
0.00564 N Phenylarsine Use a graduated cylinder 1.0 mL of pH 7 Phosphate TitraStir titration stand and
Oxide (PAO) Solution. to measure 200 mL of Buffer Solution to make turn on the stirring motor.
sample. Add the sample to the prepared sample. Put the tip of the probe
the beaker. fully into the prepared
sample. The platinum
wires must be submerged.
If a stir plate other than the
TitraStir® is used, set the
speed for moderate
mixing. Do not adjust the
speed after this point.
5. Turn the BIAS control 6. Dispense the titrant 7. Continue dispensing 8. Continue the titration
knob to adjust the value on into the beaker in small slowly. Near the end point by recording at least three
the display to increments while of the titration, write down points on the downward
approximately 1.00. monitoring the values on the value on the display sloping curve and at least
The BIAS adjustment the Amperometric Titrator. and the corresponding three points after the end
controls the slope of the The values will decrease. total volume of titrant that point has been reached.
titration curve. The actual was added. Read the The value on the display
value is not important. volume to the nearest 0.01 will not change
Only the relative value as mL. Add a small amount of significantly after the
the titration continues is titrant and wait several end point.
important. A precise seconds for a stable value.
adjustment is not Write down the value.
necessary.
Chlorine, Free
Page 310
Chlorine, Free
9. Make a graph of the 10. Draw the two best

titration. Plot the values intersecting lines through
from the amperometric the points as shown
titrator on the vertical axis above. Find the volume of
and the corresponding titrant to the nearest
volume of titrant on the 0.01 mL at the intersection
horizontal axis. of the two lines. This is the
mL titrant end point. This
volume is equivalent to the
in mg/L.
mL titrant =
mg/L free chlorine as Cl2
Interferences
Refer to the Amperometric Titrator Instruction Manual for a discussion of sources of errors and
interferences using the amperometric methods.

Start the chlorine analysis immediately after the samples are collected. Chlorine is a strong
oxidizing agent and is not stable in natural waters. Chlorine reacts quickly with various inorganic
compounds and slowly oxidizes organic compounds. Many factors such as sample composition,
sunlight, pH, temperature and salinity can cause the decomposition of chlorine in water.
Do not use plastic containers because plastic can react with and consume chlorine. Pretreat glass
sample containers to remove any chlorine demand by soaking in a dilute bleach solution (1 mL
commercial bleach to 1 liter of demineralized water) for at least 1 hour. Rinse thoroughly with
demineralized or distilled water. If sample containers are rinsed thoroughly with demineralized or
distilled water after use, only occasional pre-treatment is necessary.
Do not use the same sample containers for free and total chlorine. If trace iodide from the total
chlorine reagent is carried over into the free chlorine determination, monochloramine will interfere.
It is best to use separate, dedicated sample containers for free and total chlorine determinations.
A common error in testing for chlorine is introduced when a representative sample is not obtained.
If sampling from a tap, let the water flow for at least 5 minutes before sample collection. Let the
sample container overflow with the sample several times, then cap the container so that there is no
headspace (air) above the sample. Start the chlorine analysis immediately.
Chlorine, Free
Page 311
Chlorine, Free
Summary of method
Free chlorine is measured by a titration at pH 7 with PAO solution to the amperometric end point.
The amperometric titration method has greater sensitivity and accuracy when compared to
colorimetric methods. Refer to the Amperometric Titrator Instruction Manual for more information.
Required reagents
Phenylarsine Oxide Solution, 0.00564 N varies 1L 199953
Phosphate Buffer Solution, pH 7 1 mL 100 mL MDB 2155332
Required apparatus
Amperometric Buret Titrator System, 115 VAC each 1930010
Beaker, 250-mL each 50046H
Graduated Cylinder, 250-mL each 50846
Stir bar, 50 mm each 2095355
TitraStir® apparatus, 115 VAC each 1940000
TitraStir apparatus, 230 VAC each 1940010
pH Paper, 0–14 pH range 100/pkg) 2601300

Chlorine Standard Solution, 10-mL Voluette® Ampules, 50–75 mg/L 16/pkg 1426810
Chlorine Standard Solution, 2-mL PourRite Ampule, 25–30 mg/L 20/pkg 2630020
Voluette Ampule breaker 10-mL each 2196800

Chlorine, Free, HR, 10069

HR (0.1 to 10.0 mg/L as Cl2) Powder Pillows
Scope and Application: For testing higher levels of free chlorine (hypochlorous acid and hypochlorite ion) in
drinking water, cooling water and industrial process waters
Test preparation

DR 3900 4864302 Orientation key away from user A23618
DR 3800, DR 2800, DR 2700 5940506 1-cm (flat) path in line with the indicator arrow on the LZV585 (B)
adapter
If the chlorine concentration is less than 2 mg/L, use Method 8021, program number 80.
DPD Free Chlorine Reagent Powder Pillows 1
Chlorine, Free
Page 313
Chlorine, Free
Multi-path Cell
Stored Programs
88 Chlorine F&T HR
Zero
Start
1. Select the test. 2. Fill the sample cell to 3. Wipe the cell and 4. ZERO the instrument.
Insert an adapter if the 5-mL line with sample. insert it into the cell holder. The display will show:
required (see Instrument- 0.0 mg/L Cl2
for orientation.
5. Remove the cell and 6. Cap and shake the 7. Insert the prepared
add the contents of one cell about 20 seconds to sample into the cell holder.
DPD Free Chlorine dissolve reagent. READ the results in
powder pillow for 25-mL A pink color will develop if mg/L Cl2.
samples to the sample. chlorine is present.
Interferences

Acidity Neutralize to pH 6–7 with1 N Sodium Hydroxide1. Determine amount to be added on separate
addition.
addition.
Chlorine, Free
Page 314
Chlorine, Free


2. Add 2 drops Potassium Iodide1 (30 g/L) to a 5-mL sample.
concentration.
For conventional free chlorine disinfection (beyond the “breakpoint”), typical monochloramine
concentrations are very low. If monochloramine is present in the sample, its interference in
the free chlorine test is dependent on sample temperature, relative concentration of
monochloramine to free chlorine and the time required to perform the analysis.
Typical interference levels of NH2Cl (1 minute test time, interference as mg/L Cl2):
NH2Cl Level Sample Temperature ° C (° F)

Monochloramine (NH2Cl) (as Cl2) 5 (41) 10 (50) 20 (68) 30 (86)
1.2 mg/L +0.15 0.19 0.30 0.29
2.5 mg/L +0.35 0.38 0.55 0.61
3.5 mg/L +0.38 0.56 0.69 0.73
5.0 mg/L +0.68 0.75 0.93 1.05
Note: Determine Monochloramine levels using Hach method 10200.

Adjust to pH 6–7 using acid (Sulfuric Acid1) or base (Sodium Hydroxide1).
buffered samples

Analyze samples for chlorine immediately after collection. Free chlorine is a strong oxidizing agent
and reacts rapidly with various compounds. Many factors such as sunlight, pH, temperature and
sample composition will influence decomposition of free chlorine in water.
Avoid plastic containers since these may have a large chlorine demand. Pretreat glass sample
containers to remove any chlorine demand by soaking in a dilute bleach solution (1 mL
commercial bleach to 1 liter of deionized water) for at least 1 hour. Rinse thoroughly with deionized
or distilled water. If sample containers are rinsed thoroughly with deionized or distilled water after
use, only occasional pre-treatment is necessary.
Do not use the same sample cells for free and total chlorine. If trace iodide from the total chlorine
reagent is carried over to the free chlorine test, monochloramine could interfere. It is best to use
separate, dedicated sample cells for free and total chlorine determinations.
A common error in testing for chlorine is not obtaining a representative sample. If sampling from a
tap, let the water flow for at least 5 minutes to ensure a representative sample. Let the container
overflow with the sample several times, then cap the sample container so there is no headspace
(air) above the sample. If sampling with a sample cell, rinse the cell several times with the sample,
then carefully fill to the 5-mL mark. Proceed with the chlorine test immediately.
Chlorine, Free
Page 315
Chlorine, Free
Accuracy check
more information.
4. Open a Chlorine PourRite® Ampule Standard, 50–75 mg/L.
5. Prepare three sample spikes. Fill three mixing cylinders* with 5-mL of sample. Using the
TenSette® Pipet*, add 0.1 mL, 0.2 mL and 0.3 mL of standard, respectively, to each sample
and mix thoroughly.
6. Analyze each standard addition sample as described in the procedure above. Accept each
standard additions reading by pressing READ. Each addition should reflect approximately
100% recovery.
Method performance
Summary of method
The range of analysis using the DPD method for free chlorine can be extended by adding more
indicator in proportion to sample volume. Thus, a larger fill powder pillow of DPD Free Chlorine
Reagent is added to a 5-mL sample portion.
chlorine) immediately reacts with DPD (N, N-diethyl-p-phenylenediamine) indicator to form a pink
Chlorine, Free
Page 316
Chlorine, Free
Required reagents
DPD Free Chlorine Reagent Powder Pillows for 25-mL samples 1 100/pkg 1407099
Chlorine Standard Solution, 2-mL PourRite® Ampules, 50–75 mg/L 20/pkg 1426820
Spec √ Gel Secondary Standard Kit, Chlorine DPD, 0–10 mg/L 4/pkg 2893300

Cylinder, mixing, 25 mL tall form each 2088640
Sodium Hydroxide, 1N 100 mL 104532
Sulfuric Acid, 1N 100 mL 127032
Chlorine, Free
Page 317
Chlorine Free, Indophenol, 10241
Indophenol1 Method 10241

(0.04 to 4.50 mg/L Cl2) Powder Pillows
Scope and Application: For determining residual free chlorine levels in the presence of manganese, chloramines
and other oxidants which interfere with DPD colorimetric, DPD titrimetric, and amperometric methods for free
chlorine. For use in potable water, chlorinated drinking water, swimming pool water and treated wastewater
effluent.
1 Patent pending.
Test preparation

The Instrument-specific information table shows requirements that can vary between instruments.
To use this table, select an instrument, then read across to find the corresponding information
required to perform this test.
DR 5000 4864302 Orientation key toward user —
DR 3800, DR 2800, DR 2700 5940506 1-cm (flat) path aligned with arrow on adapter LZV585 (B)
Put the protective cover or lid over the cell compartment during measurements.
This method uses Program 66, Monochloramine LR.
The sample and reagent from one analysis can contaminate other analyses and interfere with the test results. Be sure to
rinse the cells and caps several times with deionized water or with the sample water to be tested before each test.
Do not switch the sample cell caps between the Blank and Sample during the analysis.
Tap sample cells lightly on a hard surface or slowly invert the cells to remove air bubbles from the cell walls.
Be sure to keep the cap on the sample cells when not used to avoid ammonia contamination.
Freechlor F Reagent Solution 5 drops
Chlorine, Free
Page 319
Chlorine, Free
Collect the following items: (continued)
Sample cell (refer to Instrument-specific information) 2
Indophenol Method
Stored Programs
66 Monochloramine LR
Start
1. Select the test. Insert 2. Fill 2 sample cells to 3. Add 5 drops of the 4. Cap and invert the cell
the correct adapter (refer the 10-mL line with Freechlor F Reagent to the to mix.
to Instrument-specific sample. Mark one cell as Sample cell.
information). the Blank. Mark the other
cell as the Sample.
Zero
5. Add the contents of 6. Press TIMER>OK. A 7. When the timer stops, 8. ZERO the instrument.
one Monochlor F Reagent 5-minute reaction will start. invert the Blank cell to The display will show:
Powder Pillow to the If the sample temperature mix. Insert the cell into the 0.00 mg/L Cl2
Blank cell and to the is less than 18 °C (64 °F), cell holder.
Sample cell. Cap and refer to Table 103 for the
shake both cells 20 correct reaction time.
seconds to dissolve.
Chlorine, Free
Page 320
Chlorine, Free
Indophenol Method (continued)
9. Invert the Sample cell

to mix. Insert the cell into
the cell holder. READ the
results in mg/L Cl2.
Color reaction time

Use Table 103 to find the reaction time when the sample temperature is less than 18 °C (64 °F).
The color is stable for 30 minutes after the reaction time is reached. If a temperature measurement
is not available, read the sample after the color is stable.
Table 103 Color development based on sample temperature

Sample Temperature
Reaction Time (minutes)
°C °F
5 41 10
7 45 9
9 47 8
10 50 8
12 54 7
14 57 7
16 61 6
18 64 4
20 68 3
23 73 2.5
25 77 2
Interferences
The substances listed in Table 104 have been tested for interference and do not interfere at or
below the indicated levels. Refer to Table 105 for substances that do interfere with the test.

Alanine 1 mg/L N
Aluminum 10 mg/L Al3+
Bromide 100 mg/L Br-
Chlorine, Free
Page 321
Chlorine, Free
Table 104 Non-interfering substances (continued)

Bromine 15 mg/L Br2

Chloride 18,000 mg/L Cl-
Chromium (III) 5 mg/L Cr3+
Copper 10 mg/L Cu
Cyanide 10 mg/L CN-
Fluoride 5 mg/L F-
Glycine 1 mg/L N
Iodine 4 mg/L I2
Lead 10 mg/L Pb
Manganese (7+) 3 mg/L MnO4–
Nitrate 100 mg/L NO3––N
Nitrite 50 mg/L NO2––N
Oxone®1 (potassium
30 mg/L
peroxomonopersulfate)
Phosphate 100 mg/L PO43-
Sulfate 2600 ppm SO42-

Tyrosine 1 mg/L N
Urea 10 mg/L N
Zinc 5 mg/L Zn2+
1 Oxone is a registered trademark of E.I. du Pont de Nemours & Co., Inc.

Ozone1 > 1 mg/L O3
Sulfide1 > 0.5 mg/L S2-

1 This compound does not usually exist with free chlorine.

concentrations, sunlight, pH, temperature and salinity influence the decomposition of free
chlorine in water.
• Avoid plastic containers because they can have a large chlorine demand.
Chlorine, Free
Page 322
Chlorine, Free
no headspace (air) above the sample. If sampling with a sample cell, rinse the cell several
times with the sample, then carefully fill to the 10-mL mark. Perform the chlorine analysis
immediately.
Chlorine, Free
Page 323
Chlorine, Free
Testing applications
Finished chlorinated drinking waters and distributions systems
Finished waters contain free chlorine and various levels of organic chloramines and inorganic
contaminants. It is generally assumed that the free chlorine has reacted with all easily oxidizable
species present and the remaining free chlorine is likely present in a steady-state equilibrium.
Replicate analyses for free chlorine on this type of water should give equivalent results. It is
especially important when testing water where free chlorine residual levels are low to observe all
precautions that refer to sample cell cleanliness, water temperature and sampling techniques.
At breakpoint
These waters may contain a mixture of free chlorine, chloramines and nuisance residuals
depending on water temperature, mixing efficiencies, sampling location and distance beyond the
theoretical breakpoint. It is important to note that the water can be in a state of "dynamic
equilibrium" and the chemical speciation can change rapidly, especially if one is at or near
breakpoint. The chemical speciation can change dynamically in both the Blank cell and the
Sample cell. The test must be conducted immediately on these types of samples. Test results may
be difficult to replicate on duplicate samples depending on the dynamics of the water. Test results
are best used to identify free chlorine trends and to monitor changes due to different mixing
efficiencies, sampling locations, temperature changes, increased chlorine feed rates, etc.
In chloramination kinetic studies
These waters will contain a mixture of free chlorine and chloramines depending on water
temperature, mixing efficiencies, sampling locations, feed rates for chlorine and ammonia and
contact time. It is important to note that the water is in a state of "dynamic equilibrium" and the
chemical speciation can change rapidly depending on water conditions. The chemical speciation
can change dynamically in both the Blank cell and the Sample cell. The test must be conducted
immediately on these types of samples. Test results may be difficult to replicate on duplicate
samples depending on the dynamics of the water. Test results are best used to identify free
chlorine trends and to monitor changes based on changes in mixing efficiencies, sampling
locations, water temperature changes, increased chlorine feed rates, etc.
With other oxidants such as Oxone, permanganate, chlorine dioxide, bromine and iodine
It is assumed that the free chlorine residual has stabilized in the presence of the other oxidants.
Replicate analyses for free chlorine on this type of water should give equivalent results. The levels
of alternate oxidants that can be present without interference have been tested only in laboratory
bench studies (refer to Table 104). Field data for free chlorine in the presence of these oxidants is
not available.
Accuracy check
Important Note: This procedure is only valid for stabilized or equilibrated free chlorine samples.

• Chlorine Standard Solution, 2-mL PourRite® Ampule, 25–30 mg/L
• PourRite Ampule Breaker
• Pipet, TenSette®, 0.1–1.0 mL
• Pipet tips
2. Select standard additions from the instrument menu (if available). Refer to the instrument user
manual for specific instructions. Refer to step 9 if a standard additions menu is not available.
3. Enter the average chlorine concentration from the label or certificate that is enclosed with the
standard solution ampules.
Chlorine, Free
Page 324
Chlorine, Free
5. Open one standard solution ampule.
6. Use the TenSette Pipet to prepare 3 spiked samples: add 0.1 mL, 0.2 mL, and 0.3 mL of
standard to three 10-mL portions of fresh sample.
7. Follow the Indophenol Method test procedure for each of the spiked samples, starting with the
0.1 mL sample spike. Measure each of the spiked samples in the instrument.
9. If a standard additions menu is not available, calculate the percent recovery:
c. Put the spiked sample in the cell holder and press READ.
d. Calculate the concentration of chlorine that was added to the sample:
0.1 mL × concentration of chlorine standard (mg/L Cl2 )
mg/L chlorine added = -----------------------------------------------------------------------------------------------------------------------------------------------
-
10.1 mL
e. Find the expected result of the spiked sample from the sum of the unspiked sample result
plus the concentration of chlorine that was added (step d).
f. Calculate the percent recovery from the actual (step c) and the expected (step e) results.
Note: If results are not within acceptable limits (± 10%), be sure that the sample volumes and sample spikes
are measured accurately. The sample volumes and sample spikes that are used should agree with the
selections in the standard additions menu. If all procedures are followed correctly but the standard
additions results are not within acceptable limits, the sample may contain an interference.
Method performance
In a single laboratory with a standard solution of 3.51 mg/L chlorine (as Cl2) and a single lot of
reagent with a single instrument (DR 5000), a single operator obtained a standard deviation of
± 0.04 mg/L Cl2.
Summary of method
An ammonia solution at a pH of 8.3 is added to a sample containing free chlorine. The free
chlorine is immediately converted into monochloramine (NH2Cl). The monochloramine is then
determined by the indophenol method using Monochlor F Reagent. In this method, the
monochloramine reacts with a substituted phenol in the presence of a cyanoferrate catalyst to form
an intermediate monoimine compound. The intermediate compound couples with excess
substituted phenol to form a green-colored indophenol compound, which is proportional to the
amount of free chlorine present in the sample. A sample blank containing Monochlor F Reagent
compensates for background color from the reagent and sample. Test results are measured with a
spectrophotometer at 655 nm or with a colorimeter at 610 nm.
Chlorine, Free
Page 325
Chlorine, Free
Required reagents
Freechlor F Reagent Solution 5 drops 50-mL SCDB 2964926

Ampule Breaker, 2-mL PourRite Ampules each 2484600
Ampule Breaker Kit, 10-mL Volulette Ampules each 2196800
Clippers (shears) each 2369400
Thermometer, non-mercury, –10 to 225 °C each 2635700
Wipers, disposable, 28 x 37 cm 188/pkg 2932800

Chlorine, Total, 8167
Chlorine, Total DOC316.53.01027
USEPA1 DPD Method2 Method 8167

Scope and Application: For testing residual chlorine and chloramines in water, wastewater, estuary water and
seawater; USEPA-accepted1 for reporting for drinking and wastewater analyses.
1 Procedure is equivalent to USEPA method and Standard Method 4500-Cl G for drinking water and wastewater analyses.
Test preparation


Instrument
Samples must be analyzed immediately and cannot be preserved for later analysis
If the test overranges, dilute the sample with a known volume of high quality, chlorine demand-free water and repeat the test.
Some loss of chlorine may occur due to the dilution. Multiply the result by the dilution factor. Or, analyze samples with high
chlorine concentrations directly without dilution by using Method 10070, Chlorine, Total HR, or Method 10250, Chlorine Total,
MR.
For chloramination disinfection control, use Method 10172, Chloramine (Mono), Low Range (program number 66) or High
Range (program number 67).
After adding reagent a pink color will develop.
The SwifTest Dispenser1 for Total Chlorine can be used in place of the powder pillow in step 3.
An AccuVac ampule for Blanks can be used as a blank in place of the sample cell in step 2.
1 Optional reagents and apparatus.
Chlorine, Total
Page 327
Chlorine, Total
Powder Pillow Test:
DPD Total Chlorine Reagent powder pillow, 10-mL 1
AccuVac Test:
Collect at least 40 mL of sample in a 50-mL beaker 40 mL
DPD Total Chlorine Reagent AccuVac® Ampul 1
Beaker, 50-mL 1
DPD method for powder pillows
Stored Programs
80 Chlorine, F&T PP
Start
required (see Instrument- DPD Total Chlorine Powder A three-minute reaction
specific information). Pillow to the sample cell. period will begin. Perform
Swirl the sample cell for 20 steps 5 and 6 during this
seconds to mix. time period.
5. Blank Preparation: 6. Wipe the blank sample 7. Within three minutes

Fill a second sample cell cell and insert it into the cell after the timer expires,
with 10-mL of sample. holder. wipe the prepared sample
ZERO the instrument. The and insert it into the cell
display will show: holder.
0.00 mg/L Cl2 READ the results in mg/L
Cl2.
Chlorine, Total
Page 328
Chlorine, Total
DPD method for AccuVac Ampuls
Stored Programs
85 Chlorine, F&T AV
Start
Insert an adapter if Fill a sample cell with Fill a DPD Total Chlorine Ampul several times to
required (see Instrument- 10-mL of sample. Reagent AccuVac® Ampul mix. Wipe off any liquid or
specific information). with sample. Keep the tip fingerprints.
Refer to the user manual immersed while the Ampul

for orientation. fills completely.
5. Start the instrument 6. Wipe the blank sample 7. Within three minutes
timer. cell and insert it into the after the timer expires,
A three-minute reaction cell holder. wipe the AccuVac Ampul
period will begin. Perform ZERO the instrument. The and insert it into the cell
steps 6 and 7 during this display will show: holder.
time period. 0.00 mg/L Cl2. READ the results in mg/L
Cl2.
Interferences
Interfering Substance Interference Levels and Treatments
Greater than 150 mg/L CaCO3. May not develop full color or color may fade instantly. Neutralize to
Acidity pH 6 –7 with 1 N sodium hydroxide1. Determine amount to be added on separate sample aliquot, then
add the same amount to the sample being tested. Correct for volume addition.
Greater than 300 mg/L CaCO3. May not develop full color or color may fade instantly. Neutralize to
Alkalinity pH 6–7 with 1 N sulfuric acid1. Determine amount to be added on separate sample aliquot, then add
the same amount to the sample being tested. Correct for volume addition.
Chlorine, Total
Page 329
Chlorine, Total

2. Add 3 drops potassium iodide1 (30 g/L) to a 25-mL sample.
Manganese, Oxidized
3. Mix and wait one minute.
(Mn4+, Mn7+) or
Chromium, Oxidized 4. Add 3 drops sodium arsenite1, 2 (5 g/L) and mix.
(Cr6+) 5. Analyze 10 mL of the treated sample as described in the procedure.
6. Subtract the result from this test from the original analysis to obtain the correct
chlorine concentration.
Extreme sample pH or
Adjust to pH 6 –7 using acid (Sulfuric Acid1, 1.000 N) or base (Sodium Hydroxide1, 1.00 N).
Highly buffered samples
for arsenic (D004). Reference the current MSDS for more information on proper disposal of these materials.

• Analyze samples for chlorine immediately after collection. Chlorine is a strong oxidizing agent
and it is unstable in natural waters. It reacts rapidly with various inorganic compounds and
more slowly oxidizes organic compounds. Many factors, including reactant concentrations,
sunlight, pH, temperature and salinity influence decomposition of chlorine in water.
• Do not use the same sample cells for free and total chlorine. If trace iodide from the total
chlorine reagent is carried over into the free chlorine determination, monochloramine will
interfere. It is best to use separate, dedicated sample cells for free and total chlorine
determinations.
container overflow with the sample several times, cap the sample containers so there is no
headspace (air) above the sample. If sampling with a sample cell, rinse the cell several times
with the sample, then carefully fill to the 10-mL mark.
• Perform the chlorine analysis immediately.
Accuracy check
• Chlorine Voluette® Ampule Standard, 25–30 mg/L Cl2.
• Ampule Breaker
Chlorine, Total
Page 330
Chlorine, Total

more information.
4. Open a LR Chlorine Voluette Ampule Standard, 25–30 mg/L Cl2.
TenSette® Pipet to add 0.1 mL, 0.2 mL and 0.3 mL of standard, respectively to three 10-mL
samples and mix each thoroughly.
Note: For AccuVac® Ampuls, fill three mixing cylinders with 50-mL of sample and spike with 0.4 mL, 0.8
mL and 1.2 mL of standard. Transfer 40 mL from each of the three mixing cylinders to three 50-mL
beakers*. Analyze each standard addition sample as described in the procedure above. Accept
each standard additions reading by pressing Read. Each addition should reflect approximately
100% recovery.
6. Analyze each sample spike as described in the procedure above, starting with the smallest
sample spike. Accept each standard additions reading by pressing Read. Each addition
Method performance
Summary of method
Chlorine can be present in water as free chlorine and as combined chlorine. Both forms can exist
in the same water and be determined together as the total chlorine. Free chlorine is present as
hypochlorous acid and/or hypochlorite ion. Combined chlorine exists as monochloramine,
dichloramine, nitrogen trichloride and other chloro derivatives. The combined chlorine oxidizes
iodide in the reagent to iodine. The iodine and free chlorine reacts with DPD (N,N-diethyl-p-
phenylenediamine) to form a pink color which is proportional to the total chlorine concentration. To
determine the concentration of combined chlorine, run a free chlorine test and a total chlorine test.
Subtract the results of the free chlorine test from the total chlorine test to obtain the combined
chlorine concentration. Test results are measured at 530 nm.
Chlorine, Total
Page 331
Chlorine, Total

Required reagents
DPD Total Chlorine Reagent Powder Pillows, 10-mL 1 100/pkg 2105669
OR
Required apparatus
Chlorine Standard Solution, 2-mL Pour-Rite® Ampule, 25–30 mg/L 20/pkg 2630020

Beakers, 50 mL each 50041H
Chlorine Demand-Free Water 500 mL 2641549
Deionized Water 4L 27256
SwifTest Dispenser for Total Chlorine each 2802400
DPD Total Chlorine Reagent Powder Pillows, 10-mL 1000/pkg 2105628
DPD Total Chlorine Reagent, 10 mL, SwifTest Dispenser refill vial 250 tests 2105660
SpecCheck Secondary Standard Kit, Chlorine DPD, 0 - 2.0 mg/L Set each 2635300
Chlorine, Total
Page 332
Chlorine, Total
Chlorine, Total
Page 333
DPD Rapid Liquid Method1 Method 10060

(0.02 to 2.00 mg/L) Pour-Thru Cell
Scope and Application: For treated water.
Test preparation

DR 5000 LZV479 — —
DR 3900 LQV157.99.10002 Align cell flow arrows with arrows on cell compartment —
DR 3800, DR 2800, DR 2700 5940400 1-inch (round) path aligned with arrow on the adapter LZV585 (B)
Refer to Reagent preparation
Make sure the Pour-Thru cell is completely seated in the sample cell compartment.
DPD Indicator Powder varies
Total Chlorine Indicator Solution 1 mL
Total Chlorine Buffer Solution 1 mL
Cylinder, glass, mixing, 100-mL 1
Pour-Thru Module and Cell (See instrument-Specific Information) 1
Chlorine, Total
Page 335
Chlorine, Total
DPD rapid liquid method for Pour-Thru Cell
Stored Programs
82 Chlorine F&T RL
Zero
Start
1. Select the test. 2. Pour approximately 3. When the flow stops, 4. Add 1.0 mL of Total
Insert an adapter if 50 mL of sample into the ZERO the instrument. The Chlorine Buffer Solution to
required (Instrument- Pour-Thru Cell. display will show: a clean, dry 100-mL glass
specific information). 0.00 mg/L Cl2. mixing cylinder using the
Repipet Jr. Dispenser.
Refer to Instrument-
specific information for
Pour-thru cell orientation.
5. Add 1.0 mL of 6. Carefully fill the mixing 7. Start the instrument 8. When the timer
prepared Total Chlorine cylinder to the 80-mL mark timer. expires, fill the funnel of
Indicator Solution to the with sample. Stopper the A two-minute reaction the Pour-Thru Cell with the
same mixing cylinder cylinder and gently invert it period will begin. reacted sample from the
using the Repipet Jr. twice to mix. Proceed to mixing cylinder.
Dispenser. Swirl to mix the step 7 immediately. Complete steps 8 and 9
within two minutes after It is not necessary to pour
reagents. Proceed to the entire sample into the
step 6 immediately. the timer expires.
Pour-Thru Cell;
approximately half of the
sample may be discarded.
Chlorine, Total
Page 336
Chlorine, Total
DPD rapid liquid method for Pour-Thru Cell (continued)
Read
9. After the flow stops, 10. Flush the Pour-Thru

READ the results in Cell with at least 50-mL of
mg/L Cl2. deionized water
immediately after use.
Reagent preparation
The Total Chlorine Indicator Solution must be prepared before use. Using a powder funnel, add the
contents of one 24 g bottle of DPD Powder to one 473-mL bottle of Total Chlorine Indicator
Solution*. Invert several times and swirl until the powder is completely dissolved. A pale pink color
may develop, but should not affect results.
This solution will give accurate results for at least one month after mixing when stored at 20–25 °C
(68–77 °F). Write the date of preparation on the Indicator Solution Bottle. Discard any remaining
solution after one month. Use of this reagent after one month may result in high reagent blanks
and low values at high concentration. Do not combine fresh reagent with previously
mixed reagent.
Interferences
Greater than 700 mg/L CaCO3. May not develop full color or color may fade instantly. Neutralize
Alkalinity to pH 6 –7 with 1 N Sulfuric Acid1. Determine amount to be added on separate sample aliquot, then
add the same amount to the sample being tested. Correct for volume addition.
Hardness Levels below 1000 mg/L as CaCO3 will not interfere.
Hexavalent Chromium Levels greater than 1 mg/L will cause a positive interference.
Manganese, oxidized 2. Add 9 drops Potassium Iodide (30 g/L)1 to an 80-mL sample.
(Mn4+, Mn7+) or 3. Mix and wait 1 minute.
Chromium, oxidized 4. Add 9 drops Sodium Arsenite1, 2 (5 g/L) and mix.
6. Subtract the result of this test from the original analysis to obtain the correct concentration.

Chlorine, Total
Page 337
Chlorine, Total

• Samples must be analyzed immediately and cannot be preserved for later analysis.
• A common testing error is introduced if the analyst does not obtain a representative sample. If
sampling from a tap, let the water flow for at least five minutes to make sure that it is a
representative sample. Let the container overflow with the sample several times, then cap the
sample container so there is no headspace (air) above the sample. Perform the chlorine
analysis immediately.
• Avoid plastic containers since these may have a chlorine demand.
• Pre-treat glass sample containers to remove any chlorine demand by soaking in a dilute
bleach solution (1 mL commercial bleach to 1 liter of deionized water) for at least 1 hour. Rinse
thoroughly with deionized water. If sample containers are rinsed thoroughly with deionized
water after use, only occasional pretreatment is necessary. A pre-treated BOD bottle with a
ground-glass stopper is an ideal sample container for chlorine collection.

with deionized water and allow to dry before use. If the mixing cylinder is thoroughly rinsed with
deionized water and allowed to dry after each use, only occasional pretreatment is necessary. Do
not use the same mixing cylinder for Free and Total Chlorine analysis.

water.
Accuracy check
Required for accuracy check
• Chlorine Voluette® Ampule Standard Solution, 50 to 75-mg/L Cl2
• TenSette® Pipet and tips
• Ampule Breaker
more information.Open a Chlorine Voluette Ampule Standard Solution, 50 to 75-mg/L Cl2.
4. Prepare three sample spikes. Use the TenSette Pipet to add 0.3, 0.6 and 0.9 mL of standard to
three 80-mL samples, respectively. Swirl gently to mix.
Chlorine, Total
Page 338
Chlorine, Total
Method performance
Sensitivity
Precision Concentration change
Program Standard 95% Confidence Limits of per 0.010 Abs change
Distribution
Point of curve Concentration
82 1.18 mg/L Cl2 1.17–1.19 mg/L Cl2 Entire range 0.02 mg/L Cl2
Summary of method
Chlorine can be present in water as free available chlorine and as combined available chlorine.
Both forms can exist in the same water and can be determined together as the total available
chlorine. Free chlorine is available as hypochlorous acid and/or hypochlorite ion. Combined
chlorine exists as monochloramine, dichloramine, nitrogen trichloride and other chloro derivatives.
The combined chlorine oxidizes iodide in the reagent to iodine. The iodine reacts with DPD (N,N-
diethyl-p-phenylenediamine) indicator along with free chlorine present in the sample to form a pink
color which is proportional to the total chlorine concentration. To determine the concentration of
combined chlorine, run a free chlorine test and a total chlorine test. Subtract the free chlorine
results from the results of the total chlorine test to obtain combined chlorine. Test results are
measured at 530 nm.

Required reagents
Rapid Liquid Total Chlorine Reagent Set, includes: — — 2557000
DPD Indicator Powder, 24 g varies each 2297255
Total Chlorine Indicator Solution 1 mL 473 mL 2263411
Total Chlorine Buffer Solution 1 mL 473 mL 2263511
Required apparatus
Cylinder, mixing graduated, 100-mL, glass 1 each 2636342
Dispenser, Adjustable volume, 1.0-mL – 5.0 mL 2 each 2563137
Funnel, powder 1 each 2264467
Chlorine Standard Solution, Voluette® Ampule, 50–75 mg/L, 10-mL 16/pkg 1426810
OR
Chlorine Standard Solution, Pour-Rite® Ampule, 50–75 mg/L, 2-mL 20/pkg 1426820
Chlorine, Total
Page 339
Chlorine, Total

Pipet Tips, for TenSette Pipet 1970001 50 pkg 2185696
Voluette Ampule Breaker each 2196800

Chlorine, Total, TNT, 10101

(0.09 to 5.00 mg/L) Test ‘N Tube™ Vials
Scope and Application: For testing higher levels of total (free plus combined) chlorine in drinking water, treated
wastewater, cooling water or industrial process water
Test preparation

DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
For DR 3900, DR 3800, DR 2800 and DR 2700: Install the light shield before performing this test.
Analyze samples immediately. Do not preserve samples for later analysis.
adjust.
For chloramination disinfection control, use Method 10172, Chloramine (Mono), High Range.
After adding sample to the Test ‘N Tube™, a pink color will develop if free chlorine is present.
Test ‘N Tube™ DPD Total Chlorine Reagent 1
Chlorine, Total
Page 341
Chlorine, Total
DPD method for Test ‘N’Tubes
Stored Programs
89 Chlorine F&T TNT
Start
1. Select the test. 2. Blank Preparation: 3. Wipe the outside of 4. Insert the blank into
Insert an adapter if Fill an empty Test ‘N Tube the vial to remove the cell holder.
required (see Instrument- vial to the top of the label fingerprints and other
specific information). with sample. marks.
Zero
5. ZERO the instrument. 6. Prepared Sample: 7. Cap and slowly invert 8. Start the instrument
The display will show: Remove the cap from a at least 10 times to timer.
Total Chlorine DPD Test ‘N dissolve the powder. (Ten A three-minute reaction
0.00 mg/L Cl2 Tube™. Add 10 mL of inversions should take at period will begin.
sample to the tube. (Fill least 30 seconds. One
the vial to the top of the inversion equals turning
label.) the vial upside down, then
returning it to an upright
position.)
Read
9. When the timer 10. Insert the sample in 11. READ the results in
expires, wipe the outside the cell holder. mg/L Cl2.
of the vial that contains the
prepared sample.
Chlorine, Total
Page 342
Chlorine, Total
Interferences
Table 111 Interfering Substances and Levels
Interfering Substance Interference Levels and Treatment
sample aliquot, then add the same amount to the sample being tested. Correct for volume addition.
Alkalinity Neutralize to pH 6–7 with 1 N Sulfuric Acid1. Determine amount to be added on separate sample
Manganese, oxidized
3. Mix and wait 1 minute.
(Mn4+, Mn7+)
or 4. Add 3 drops Sodium Arsenite1, 2 (5 g/L) and mix.
Chromium, oxidized (Cr6+) 5. Analyze 10 mL of the treated sample as described in the procedure.
Ozone, O3 Interferes at all levels
Extreme sample pH or
Adjust to pH 6 –7 using acid (Sulfuric Acid1, 1.000 N) or base (Sodium Hydroxide1, 1.00 N).
highly buffered samples
for arsenic (D004). See the current reagent MSDS for safe disposal instructions.

• Analyze samples for chlorine immediately after collection. Free chlorine and combined
chlorine are strong oxidizing agents and are unstable in natural waters. Many factors,
including reactant concentrations, sunlight, pH, temperature and salinity influence
decomposition of free chlorine in water.
• A common error in testing for chlorine is obtaining an unrepresentative sample. If sampling
Chlorine, Total
Page 343
Chlorine, Total
Accuracy check
• Chlorine PourRite® Ampule Standard, 50-75 mg/L Cl2
• Ampule Breaker
more information.
4. Open a Chlorine PourRite Ampule Standard, 50-75 mg/L Cl2.
5. Use the TenSette® Pipet to add 0.1 mL of standard to a 10-mL sample and mix thoroughly.
6. Analyze the standard addition sample as described in the procedure above. Accept the
standard additions reading. The addition should reflect approximately 100% recovery.
Method performance
Summary of method
Chlorine can be present in water as free chlorine and as combined chlorine. Both forms can exist
in the same water and be determined together as the total chlorine. Free chlorine is present as
hypochlorous acid and/or hypochlorite ion. Combined chlorine exists as monochloramine,
dichloramine, nitrogen trichloride and other chloro derivatives.
Free or combined chlorine oxidizes iodide in the reagent to iodine. The iodine and chlorine react
with DPD (N,N-diethyl-p-phenylenediamine) to form a pink color, which is proportional to the total
chlorine concentration. To determine the concentration of combined chlorine, run a free chlorine
test and a total chlorine test. Subtract the results of the free chlorine test from the total chlorine test
to obtain the combined chlorine concentration. Test results are measured at 530 nm.
Chlorine, Total
Page 344
Chlorine, Total
Required reagents
Test ‘N Tube™ DPD Total Chlorine Reagent 1 50/pkg 2105645

Test Tube Rack each 1864100
Sodium Arsenite 5 g/L 100 mL 104732
Chlorine, Total
Page 345
Chlorine, Total, HR, 10070

HR (0.1 to 10.0 mg/L as Cl2) Powder Pillows
Scope and Application: For testing higher levels of total chlorine (free and combined) in drinking water, cooling
water and industrial process waters
1 USEPA accepted for reporting drinking water analyses. Procedure is equivalent to USEPA, Standard Method 4500-Cl-G for Drinking Water
and Wastewater.
Test preparation

DR 6000 4864302 Orientation key toward user
DR 5000 4864302 Orientation key toward user —
If the chlorine concentration is less than 2 mg/L, use Method 8167, program number 80.
After adding the reagent, a pink color will develop if chlorine is present.
DPD Total Chlorine Reagent Powder Pillows 1
Sample Cell (See Instrument Specific Information) 1
Chlorine, Total
Page 347
Chlorine, Total
DPD method for powder pillows
Stored Programs
88 Chlorine F&T HR
Zero
Start
1. Select the test. 2. Fill the sample cell to 3. Wipe the cell and 4. ZERO the instrument.
Insert an adapter if the 5-mL line with sample. insert it into the cell holder The display will show:
required (Instrument- (Instrument-specific
information. 0.0 mg/L Cl2
for orientation.
5. Remove the cell and 6. Cap and shake the 7. Start the instrument 8. Insert the prepared
add the contents of one cell about 20 seconds to timer. sample into the
DPD Total Chlorine dissolve reagent. A 3-minute reaction period Instrument-specific
powder pillow for 25-mL will begin. information
samples to the sample. READ the results in
mg/L Cl2.
Interferences
addition.
Alkalinity Neutralize to pH 6–7 with 1 N Sulfuric Acid1. Determine amount to be added on separate sample
Chlorine, Total
Page 348
Chlorine, Total

1. Adjust sample pH to 6–7 with 1.000 N Sulfuric Acid1.
Manganese, oxidized (Mn4+,
3. Mix and wait 1 minute.
Mn7+) or Chromium, oxidized
(Cr6+) 4. Add 2 drops Sodium Arsenite1, 2 (5 g/L) and mix.
5. Analyze the treated sample as described in the procedure above.
6. Subtract the result of this test from the original analysis to obtain the correct concentration.
Adjust to pH 6 –7 using acid (Sulfuric Acid1) or base (Sodium Hydroxide1).
buffered samples


• Analyze samples for chlorine immediately after collection. Free and combined chlorine are
strong oxidizing agents and react rapidly with various compounds. Many factors such as
sunlight, pH, temperature and sample composition will influence decomposition of chlorine in
water.
• Do not use the same sample cells for free and total chlorine. If trace iodide from the total
chlorine reagent is carried over to the free chlorine test, monochloramine could interfere. It is
best to use separate, dedicated sample cells for free and total chlorine determinations.
• A common error in testing for chlorine is obtaining a representative sample. If sampling from a
tap, let the water flow for at least 5 minutes to ensure a representative sample. Let the
container overflow with the sample several times, then cap the sample container so there is no
headspace (air) above the sample. If sampling with a sample cell, rinse the cell several times
with the sample, then carefully fill to the 5-mL mark. Proceed with the chlorine test
immediately.
Accuracy check
• Chlorine PourRite® Ampule Standard, 50–75 mg/L
Chlorine, Total
Page 349
Chlorine, Total
more information.
4. Open a Chlorine PourRite® Ampule Standard, 50–75 mg/L*.
5. Prepare three sample spikes. Fill three mixing cylinders with 5-mL of sample. Using the
TenSette® Pipet*, add 0.1 mL, 0.2 mL and 0.3 mL of standard, respectively, to each sample
and mix thoroughly.
6. Analyze each standard addition sample as described in the procedure above. Accept each
standard additions reading by pressing Read. Each addition should reflect approximately
100% recovery.
Method performance
Sensitivity
Distribution
Point of Curve Concentration
88 5.4 mg/L Cl2 5.3–5.5 mg/L Entire range 0.1 mg/L Cl2
Use Method 8167 to test chlorine concentrations at levels typically less than 2 mg/L or Method
8370 to test chlorine concentrations at levels typically less than 500 µg/L.
Summary of method
The range of analysis using the DPD method for total chlorine can be extended by adding more
indicator in proportion to sample volume. Thus, a larger fill powder pillow of DPD Total Chlorine
Reagent is added to a 5-mL sample portion.
The combined chlorine oxidizes iodide in the reagent to iodine. The iodine reacts with DPD (N, N-
diethyl-p-phenylenediamine) along with free chlorine present in the sample to form a pink color
which is proportional to the total chlorine concentration. Test results are measured at 530 nm.
Required Reagents
DPD Total Chlorine Reagent Powder Pillows for 25-mL samples 1 100/pkg 1406499
Recommended Standards
Chlorine Standard Solution, 10-mL Voluette ampules, 50–75 mg/L 16/pkg 1426810
Chlorine, Total
Page 350
Chlorine, Total

Ampule breaker, Voluette, 10 mL each 2196800
Ampule breaker, PourRite, 2-mL each 2484600
Chlorine, Total
Page 351

ULR (2 to 500 µg/L as Cl2) Pour-Thru Cell and OriFlo™ Filtration
Scope and Application: For testing trace levels of chlorine and chloramines in treated domestic and industrial
wastewater; USEPA accepted for reporting for wastewater analysis2
2 U.S. Patent 5,362,650 covers the procedure. U.S. Patent 5,549,816 covers the OriFlo™ Filtration System.
Test preparation

Instrument Pour-thru Kit Cell orientation1 Adapter
DR 5000 LZV479 — —
1 Align for long path
Analyze samples immediately. Samples containing chlorine cannot be preserved for later analysis.
A reagent blank value for a combined lot of indicator/buffer reagent solutions should be determined at least once a day. If
sample color or turbidity fluctuates frequently during the day, determine a reagent blank for each sample. Refer to Treating
Analysis Labware on page 7.
The reagent blank value is normally less than 5 µg/L. If the value is greater than 5 µg/L, an interfering substance may be
present in the blanking water or the DPD Indicator may be degrading. If there is doubt about the reagents, repeat the reagent
blank determination using chlorine-demand-free water for the sample. Blanks up to 5 µg/L may be used.
Use a new filter for each test. Using an unspecified filter may give low analysis results or inability to filter the required volume.
Ampules contain more than 1.0 mL of solution for ease of transfer. Discard excess reagent in the ampule.
Use forceps to handle filters.
Chlorine, Total
Page 353
Chlorine, Total
ULR Chlorine Buffer Solution, 1.5-mL ampules 1 mL
DPD Indicator Solution for ULR Chlorine, 1.5-mL ampules 1 mL
Blanking Reagent for ULR Chlorine 1 mL
Membrane Filters, 3-micron, 25-mm 1
OriFlo Assembly 1
Beaker, 250 mL 1
Cylinder, graduated mixing, 50-mL. 1
Pipet Tips 2
Deionized water Varies
Ampule Breaker 1
Pour-Thru Module and cell 1
DPD method for Pour-Thru Cell
Stored Programs
86 Chlorine Total, ULR
Start
1. Select the test. 2. Flush the Pour-Thru 3. Unscrew the cap from 4. Install a new, 3-micron
Insert an adapter if cell with 50 mL of the OriFlo™ plunger filter (white) into the cap
required (Instrument- deionized water. assembly. Be sure that the well. Wet the filter with a
specific information). O-ring is properly seated few drops of deionized
in the cap. water. Reassemble and
hand-tighten the cap onto
the plunger.
Chlorine, Total
Page 354
Chlorine, Total
DPD method for Pour-Thru Cell (continued)
5. Open one ULR 6. Using a TenSette® 7. Open one ampule of 8. Use a TenSette Pipet
Chlorine Buffer Solution Pipet and a clean tip, DPD Indicator Solution for and a clean tip to transfer
Ampule. transfer 1.0 mL of buffer Ultra Low Range Chlorine. 1.0 mL of indicator from
from the ampule to a the ampule to the
clean, treated 50-mL graduated mixing cylinder.
graduated mixing cylinder. Swirl to mix.
Proceed to step 9 within
one minute.
9. Prepared Sample: 10. Press TIMER>OK. 11. Push the valve button 12. Insert the plunger into
Avoiding extra agitation, A three-minute reaction on the OriFlo™ barrel the barrel and slowly push
carefully fill the cylinder to time will begin. Perform assembly in (“closed” the plunger down with
the 50-mL mark with steps 11–16 during this position). Place the barrel even pressure, until the
sample. Stopper the period. assembly into its stand. plunger is fully seated.
cylinder. Gently invert it Pour approximately 50 mL
twice to mix. Measure the reacted of the original sample into
sample 3–6 minutes after the barrel.
mixing the sample and
reagents. If less than three The lower ring on the
minutes elapses, the barrel assembly
reaction with chloramines represents about a 50-mL
may be incomplete. A volume.
reading after six minutes
may result in higher
reagent blank values.
Chlorine, Total
Page 355
Chlorine, Total
Zero
13. Pour the filtered 14. When the flow stops, 15. Pull the barrel valve 16. Push the barrel valve
sample from the plunger ZERO the instrument. button out to the “open” button to the “closed”
reservoir into the The display will show: position. Pull the plunger position. Place the barrel
Pour-Thru Cell. 0 µg/L Cl2. up to separate it from the assembly into its stand.
barrel assembly. Discard
the remaining unfiltered
sample.
A new membrane may be
required for very turbid
samples. Alternatively, use
a second Quick Filter unit
with a new membrane filter
installed.
17. When the timer 18. Insert the plunger into 19. Pour the filtered, 20. Flush the Pour-Thru
expires, pour the contents the barrel and slowly push reacted sample from the Cell with at least 50-mL of
of the mixing cylinder into the plunger down with plunger reservoir into the deionized water
the barrel. even pressure, until the Pour-Thru Cell. immediately after use.
plunger is fully seated. READ the results in µg/L Subtract the reagent blank
chlorine. value (See Determining
If a dechlorinating agent the reagent blank value)
(e.g., sulfite or sulfur from the sample value
dioxide) is present, the obtained in step 19.
sample result, corrected
for the reagent blank, will
read “0” or a slightly
negative value.
Chlorine, Total
Page 356
Chlorine, Total
Determining the reagent blank value
Stored Programs
86 Chlorine Total ULR
Start
1. Select the test. 2. Install the Pour-Thru 3. Collect about 100 mL 4. Use a TenSette® Pipet
Make sure that the reagent module. of deionized or tap water to add 1.0 mL of Blanking
blank setting is off. Flush the Pour-Thru cell in a clean, 250-mL beaker. Reagent to the beaker.
with 50 mL of deionized Swirl several times to mix.
See the user manual for
information. water. The Blanking Reagent
removes chlorine and
chloramines from the
water.
Note: The solution from step
4 will be used in Step 10.
5. Access the general 6. After the timer expires, 7. Use a TenSette Pipet 8. Open one ampule of
timer and set it for five open one ampule of ULR and a clean tip to transfer DPD Indicator Solution for
minutes. Start the timer. Chlorine Buffer Solution. 1.0 mL of buffer from the Ultra Low Range Chlorine.
ampule a clean 50 mL
mixing graduated cylinder.
Chlorine, Total
Page 357
Chlorine, Total
Determining the reagent blank value (continued)
9. Use a TenSette Pipet 10. Fill the cylinder to the 11. Start the instrument 12. During the reaction
and a clean tip to transfer 50-mL mark with timer. period, flush the Pour-Thru
1.0 mL of indicator from dechlorinated water from A three-minute reaction Cell with the remainder of
the ampule to the cylinder. step 4. Cap and invert time will begin. original dechlorinated
Swirl to mix the reagents. twice to mix. Save the water from step 10.
Proceed to step 10 within remaining water for
one minute. step 12.
Zero
13. When the flow stops, 14. When the timer 15. Use this value to 16. Flush the Pour-Thru
ZERO the instrument. expires, pour the contents correct the sample result Cell with at least 50-mL of
The display will show: of the cylinder into the obtained in this procedure. deionized water
0 µg/L Cl2. Pour-Thru Cell. READ the See the user manual for immediately after use.
results in µg/L chlorine. details on saving the
reagent blank value.
Chlorine, Total
Page 358
Chlorine, Total
Interferences

Copper, Cu2+ Greater than 1000 µg/L
concentration.
mg/L nitrite Apparent µg/L chlorine

2.0 mg/L 3 µg/L
Nitrite, NO2– (uncommon in
5.0 mg/L 5 µg/L
clean waters)
10.0 mg/L 7 µg/L
15.0 mg/L 16 µg/L
20.0 mg/L 18 µg/L

Adjust to pH 6–7
buffered samples


• Analyze samples for chlorine immediately after collection. Chlorine is a strong oxidizing agent
and it is unstable in natural waters. It reacts rapidly with various inorganic compounds and
more slowly oxidizes organic compounds. Many factors, including reactant concentrations,
sunlight, pH, temperature and salinity influence decomposition of chlorine in water.
• Avoid plastic containers. These may have a large chlorine demand.
solution (1 mL commercial bleach to 1 liter of deionized water) for at least 1 hour.
• Rinse thoroughly with deionized or distilled water. If sample containers are rinsed thoroughly
with deionized or distilled water after use, only occasional pre-treatment is necessary.
no head space (air) above the sample.
Chlorine, Total
Page 359
Chlorine, Total

Glassware used in this test must be chlorine demand-free. Fill the mixing cylinder and sample
container with a dilute solution of chlorine bleach prepared by adding 1 mL of commercial bleach
to 1 liter of water. Soak in this solution at least one hour. After soaking, rinse thoroughly with
deionized water and allow to dry before use.
Cleaning the Pour-Thru cell

the buildup by rinsing the cell with 5.25 N Sulfuric Acid* followed by several rinsings with
deionized water.
Accuracy check
• Low Range Chlorine Voluette® Ampule Standard Solution, 25 to 30-mg/L
(25,000 to 30,000 µg/L) Cl2
• TenSette® Pipet and Pipet Tips
• Ampule Breaker
Standard additions method (samples spike)
accepted or edited. Enter the chlorine concentration from the ampule package. After values
are accepted, the unspiked sample reading will appear in the top row. See the user manual for
more information.
4. Snap the top off a Low Range Chlorine Voluette® Ampule Standard Solution, 25 to 30-mg/L
(25,000 to 30,000 µg/L).
5. Prepare three sample spikes. Use the TenSette® Pipet to add 0.1, 0.2, and 0.3 mL of standard
to three 50-mL samples, respectively. Swirl gently to mix.
Chlorine, Total
Page 360
Chlorine, Total
Method performance
Sensitivity
Distribution
Portion of Curve Concentration
86 295 µg/L Cl2 290–300 µg/L Cl2 Entire range 17 µg/L Cl2
Summary of method
Several modifications to the normal DPD chlorine method are necessary to measure trace levels
of chlorine. The Pour-Thru Cell must be used in the spectrophotometer. Liquid reagents are also
required. The reproducible optics of the Pour-Thru Cell give more stable readings than is possible
with movable sample cells, resulting in more stable measurements.
It is essential that interfering sample turbidity is removed using a 3-micron membrane filter. To
avoid chlorine loss, the filtration is done after reacting the DPD with the chlorine in the sample. The
filter used has been specifically selected to avoid retention of the colored product. Sample color is
compensated by zeroing the spectrophotometer on a filtered sample.
The reagents are packaged in ampules and sealed under argon gas to ensure stability. Use of
liquid reagents eliminates any slight turbidity that might be caused by using powdered reagents.
Due to the possible oxidation of the reagents (which could give a positive chlorine reading in the
blank), a reagent blank must be determined at least once a day for each lot of reagent used. This
reagent blank value is subtracted from the sample result and the corrected value is the actual
Chlorine, Total
Page 361
Chlorine, Total

Required reagents
ULR Chlorine Reagent Set (approximately 20 tests), includes: — — 2563000
ULR Chlorine Buffer Solution, 1.5-mL ampules 1 mL 20/pkg 2493120
DPD Indicator Solution for ULR Chlorine, 1.5-mL ampules 1 mL 20/pkg 2493220
Blanking Reagent for ULR Chlorine 1 mL 29 mL 2493023
Water deionized varies 4L 27256
Required apparatus
ULR Chlorine Apparatus Set, includes: — — 2595600
Membrane Filters, 3-micron, 25-mm 1 25/pkg 2594025
OriFlo™ Assembly 1 each 4966000
Breaker, ampule 1 each 2484600
Pipet Tips, for TenSette Pipet 1970001 2 50/pkg 2185696
Chlorine Standard Solution, Voluette® Ampule, 25–30 mg/L, 2-mL 20/pkg 2630020

Forceps, flat square tips, 115 mm each 1453700
Potassium Iodide, 30 g/L 100 mL MDB 34332
Sulfuric Acid, 1 N 100 mL 100 mL MDB 127032

USEPA1 DPD Method2 Method 8370

ULR (2 to 500 µg/L as Cl2) Pour-Thru™ Cell
Scope and Application: For detecting trace levels of chlorine and chloramines in clean waters relatively free of
color and turbidity; USEPA accepted for reporting for drinking water analysis.
1 USEPA accepted
2 U.S. Patent 5,362,650
Test preparation

DR 5000 LZV479 — —
Analyze samples immediately. Samples containing chlorine cannot be preserved for later analysis.
A reagent blank value for a combined lot of indicator/buffer reagent solutions should be determined at least once a day. If
sample color or turbidity fluctuates frequently during the day, determine a reagent blank for each sample.
Ampules contain more than 1.0 mL of solution for ease of transfer. Discard excess reagent in the ampule.
Refer to Treating analysis labware.
ULR Chlorine Buffer Solution, 1.5-mL ampules 1 mL
DPD Indicator Solution for ULR Chlorine, 1.5-mL ampules 1 mL
Blanking Reagent for ULR Chlorine 1 mL
Beaker, 250 mL 1
Cylinder, graduated mixing, 50-mL. 1
Pipet Tips for TenSette Pipet 2
Chlorine, Total
Page 363
Chlorine, Total
Pour-Thru Module and cell (See Instrument Specific Information) 1
Ampule Breaker 1
See Consumables and Replacement Items for reorder information.
DPD method for Pour-Thru Cell
Stored Programs

Zero
Start
1. Select the test. 2. Pour at least 50 mL of 3. When the flow stops, 4. When the timer
Insert an adapter if sample into the Pour-Thru start the instrument timer. expires, ZERO the
required (Instrument- Cell. A three-minute reaction instrument.
specific information). period will begin. This time The display will show:
allows turbidity or solids to 0 µg/L
settle and ensures a stable
reading.
5. Open one ULR 6. Using a TenSette® 7. Open one ampule of 8. Use a TenSette Pipet
Chlorine Buffer Solution Pipet and a clean tip, DPD Indicator Solution for and a clean tip to transfer
Ampule. transfer 1.0 mL of buffer Ultra Low Range Chlorine. 1.0 mL of indicator from
from the ampule to a the ampule to the
clean, treated 50-mL graduated mixing cylinder.
graduated mixing cylinder. Swirl to mix.
Proceed to step 9 within
one minute.
Chlorine, Total
Page 364
Chlorine, Total
Read
9. Prepared Sample: 10. Start the instrument 11. Pour the contents of 12. When the timer
Avoiding extra agitation, timer. the graduated mixing expires, READ the results
carefully fill the cylinder to A three-minute reaction cylinder into the Pour-Thru in µg/L chlorine.
the 50-mL mark with time will begin. cell. If a dechlorinating agent
sample. Stopper the (e.g. sulfite or sulfur
cylinder. Gently invert it Measure the reacted
sample 3–4 minutes after dioxide) is present, the
twice to mix. sample result (corrected
mixing the sample and
reagents. If less than three for the reagent blank) will
minutes elapses, the read “0” or a slightly
reaction with chloramines negative value.
may be incomplete. A
reading after four minutes
may result in higher
reagent blank values.
13. Flush the Pour-Thru

Cell with at least 50-mL of
deionized water
Chlorine, Total
Page 365
Chlorine, Total
Determining the reagent blank value
Stored Programs
Start
1. Select the test. 2. Install the Pour-Thru 3. Collect about 100 mL 4. Use a TenSette® Pipet
Make sure that the reagent module. of deionized or tap water to add 1.0 mL of Blanking
blank setting is off. Flush the Pour-Thru cell in a clean, 250-mL beaker. Reagent to the beaker.
with 50 mL of deionized Swirl several times to mix.
See the user manual for
information. water. The Blanking Reagent
removes chlorine and
chloramines from the
water. Note: This
solution will be used in
Step 10.
5. Access the general 6. After the timer expires, 7. Use a TenSette Pipet 8. Open one ampule of
timer and set it for five open one ampule of ULR and a clean tip to transfer DPD Indicator Solution for
minutes. Start the timer. Chlorine Buffer Solution. 1.0 mL buffer from the Ultra Low Range Chlorine.
ampule to a clean 50 mL
mixing graduated cylinder.
Chlorine, Total
Page 366
Chlorine, Total
Determining the reagent blank value (continued)
9. Use a TenSette Pipet 10. Fill the cylinder to the 11. Start the instrument 12. During the reaction
and a clean tip to transfer 50-mL mark with timer. period, flush the Pour-Thru
1.0 mL of indicator from dechlorinated water from A three-minute reaction Cell with the remainder of
the ampule to the cylinder. step 4. Cap and invert time will begin. original dechlorinated
Swirl to mix the reagents. twice to mix. Save the water from step 10.
Proceed to step 10 within remaining water for
one minute. step 12.
Zero Read
13. When the flow stops, 14. When the timer 15. Use this value to 16. Flush the Pour-Thru
ZERO the instrument. expires, pour the contents correct the sample result Cell with at least 50-mL of
The display will show: of the cylinder into the obtained in this procedure. deionized water
0 µg/L Cl2. Pour-Thru Cell. READ the See the user manual for immediately after use.
results in µg/L chlorine. details on saving the
reagent blank value.
Interferences
Copper, Cu2+ Greater than 1000 µg/L
Iron (Fe3+) Greater than 1000 µg/L
Chlorine, Total
Page 367
Chlorine, Total


concentration.
mg/L nitrite Apparent µg/L chlorine

2.0 mg/L 3 µg/L
Nitrite, NO2– (uncommon in 5.0 mg/L 5 µg/L
clean waters) 10.0 mg/L 7 µg/L
15.0 mg/L 16 µg/L
20.0 mg/L 18 µg/L

Adjust to pH 6–7
buffered samples

• Analyze samples for chlorine immediately after collection. Many factors, including reactant
concentrations, sunlight, pH, temperature and salinity influence decomposition of chlorine
in water.
• A common error in testing for chlorine is failure to obtain a representative sample. If sampling

with deionized water and allow to dry before use.
Chlorine, Total
Page 368
Chlorine, Total
Cleaning the Pour-Thru cell

water.
Accuracy check
• Low Range Chlorine PourRite® Ampule Standard Solution, 25 to 30-mg/L (25,000 to 30,000
µg/L) Cl2
• Ampule Breaker
more information.
4. Open a Low Range Chlorine Voluette Ampule Standard Solution, 25 to 30-mg/L (25,000 to
30,000 µg/L) Cl2.
5. Prepare three sample spikes. Use the TenSette Pipet to add 0.1, 0.2 and 0.3 mL of standard to
three 50-mL samples, respectively. Swirl gently to mix.
Chlorine, Total
Page 369
Chlorine, Total
Method performance
Program Standard 95% Confidence Concentration change
Limits of Distribution per 0.010 Abs change

86 295 µg/L Cl2 290–300 µg/L Cl2 Entire range 17 µg/L Cl2
Summary of method
This method is designed for clean water, low in color and turbidity. The main applications include
monitoring for trace chlorine break-through of activated carbon beds and feedwater to reverse
osmosis membranes or ion-exchange resins.
Several modifications to the normal DPD chlorine method are necessary to measure trace levels
of chlorine. The Pour-Thru Cell must be used in the spectrophotometer. Liquid reagents are also
required. The reproducible optics of the Pour-Thru Cell give more stable readings than is possible
with movable sample cells, resulting in more stable measurements.
The reagents are packaged in ampules and sealed under argon gas to ensure stability. Use of
liquid reagents eliminates any slight turbidity that might be caused by using powdered reagents.
Due to the possible oxidation of the reagents (which could give a positive chlorine reading in the
blank), a reagent blank must be determined at least once a day for each lot of reagent used. This
reagent blank value is subtracted from the sample result and the corrected value is the actual
Chlorine, Total
Page 370
Chlorine, Total
Consumables and Replacement Items

Required Reagents
ULR Chlorine Reagent Set (approximately 20 tests), includes: 2563000
ULR Chlorine Buffer Solution, 1.5-mL ampules 1 mL 20/pkg 2493120
DPD Indicator Solution for ULR Chlorine, 1.5-mL ampules 1 mL 20/pkg 2493220
Blanking Reagent for ULR Chlorine 1 mL 29 mL 2493023
Required Apparatus
PourRite® Ampule Breaker 1 each 2484600

Recommended Standards
Chlorine Standard Solution, PourRite® Ampule, 25–30 mg/L, 2-mL 20/pkg 2630020

Chlorine Standard Solution, 10-mL Voluette® ampules, 50–75 mg/L 16/pkg 1426810
1 other sizes are available
Chlorine, Total
Page 371
Chlorine, Free and Total, DT, 8210
Chlorine, Free and Total DOC316.53.01172
DPD-FEAS Method Method 8210

0 to 3.00 mg/L Cl2 Digital Titrator
Test preparation
It is best to use separate, dedicated flasks for free and total chlorine determinations.
mg/L combined chlorine = mg/L total chlorine – mg/L free chlorine
DPD Free Chlorine Powder Pillow 1 pillow
DPD Total Chlorine Powder Pillow 1 pillow
Ferrous Ethylenediammonium Sulfate (FEAS) Titration Cartridge 1 cartridge
Digital titrator 1
Pipet, volumetric, 25-mL, Class A and pipet filler 1
Chlorine, Free and Total

Page 373
17. Insert a clean delivery 18. Hold the Digital 19. Use a pipet to 20. Add the contents of
tube into the titration Titrator with the cartridge measure 25.0 mL of the one DPD Free Chlorine
cartridge. Attach the tip pointing up. Turn the sample into a 50-mL Powder Pillow. Swirl to
cartridge to the titrator. delivery knob to eject a Erlenmeyer flask. mix.
few drops of titrant. Reset Results will still be
the counter to zero and accurate if a small amount
wipe the tip. of powder does not
dissolve.
21. Place the delivery tube 22. Calculate: To measure total chlorine,
into the solution and swirl digits x 0.01 = follow the procedure with
the flask. Turn the knob on mg/L Free Chlorine as Cl2 the following modification:
the titrator to add titrant to Substitute a DPD Total
the solution. Continue to Example: 25.0 mL of a
sample was titrated and Chlorine Powder Pillow in
swirl the flask and add step 20. Wait three
titrant until the solution is 250 digits were used to
reach the endpoint. The minutes before proceeding
colorless. to step 21. The result in
Write down the number of = 2.50 mg/L Cl2 step 22 will be expressed
digits displayed on the as mg/L total chlorine.
counter.
Accuracy check
Use the standard additions method to find if the sample has an interference and to confirm the
• Chlorine Standard Solution, PourRite™ Ampule, 50–75 mg/L Cl2 (the exact concentration will
be shown on a certificate enclosed with the ampule)
• Ampule breaker

Page 374

2. Use the TenSette Pipet to add 0.1 mL, 0.2 mL and 0.3 mL of standard, respectively, to three
25-mL samples. Swirl to mix.
3. Follow the test procedure and titrate each of the spiked samples and a sample with no
standard added.
4. Each 0.1 mL of standard that was added will use 20–30 digits to reach the endpoint. To find
the exact number of digits that should be used for each 0.1-mL addition, multiply the exact
concentration by 4 and by the spike volume.
(Example: 50 mg/L x 0.1 mL x 4 = 20 digits)
Interferences
Greater than 150 mg/L CaCO3. May not develop full color or color may fade
instantly. Neutralize to pH 6–7 with 1 N Sodium Hydroxide. Determine amount to be
Acidity
added on separate sample aliquot, then add the same amount to the sample being
tested.
Greater than 250 mg/L CaCO3. May not develop full color or color may fade
Alkalinity instantly. Neutralize to pH 6 –7 with 1 N Sulfuric Acid. Determine amount to be added
on separate sample aliquot, then add the same amount to the sample being tested.
2. Add 3 drops Potassium Iodide (30-g/L) to a 10-mL sample.
Manganese, Oxidized 3. Mix and wait one minute.
(Mn4+, Mn7+) or Chromium, Oxidized 4. Add 3 drops Sodium Arsenite 1 (5-g/L) and mix.
(Cr6+) 5. Analyze 10 mL of the treated sample as described in the procedure.
6. Subtract the result from this test from the original analysis to obtain the correct
Extreme sample pH or Highly buffered Adjust to pH 6–7 using acid (Sulfuric Acid, 1.000 N) or base (Sodium Hydroxide,
samples 1.00 N).
Higher room temperatures tend to give higher free chlorine values due to reaction of
Temperature
chloramines. Higher room temperatures also result in increased color fading.
1 Samples treated with sodium arsenite for interferences will be hazardous waste as regulated by Federal RCRA for arsenic (D004). See the
current MSDS for proper disposal of hazardous material.

Page 375

in water.
container overflow with the sample several times,
• Cap the sample containers so there is no headspace (air) above the sample.
Summary of method
The DPD-FEAS method provides a titrimetric procedure for determining free available chlorine
and for estimating free and combined chlorine fractions that are present together. The magenta
species, resulting from the oxidation of DPD by chlorine, is destroyed quantitatively by titration with
ferrous ethylenediammonium sulfate. The volume of titrant required to reach a colorless end point
is proportional to the chlorine concentration. Total residual chlorine may also be determined by
this test.

Required reagents
Free and Total Chlorine Reagent Set (approximately 100 tests): 2445300
(1) DPD Free Chlorine Powder Pillows 1 pillow 100/pkg 1407099
(1) DPD Total Chlorine Powder Pillows 1 pillow 100/pkg 1406499
(1) FEAS Titration Cartridge, 0.00564 N varies each 2292301
Required apparatus
Chlorine Standard Solution, PourRite™ Ampule, 50–75 mg/L Cl2, 2-mL 20/pkg 1426820
PourRite Breaker each 2484600

Page 376

Potassium Iodide Solution, 30-g/L 100 mL MDB 34332
Sodium Arsenite Solution, 5-g/L 100 mL MDB 104732
Chlorine Standard, 50–75 mg/L, 10 mL 16/pkg 1426810
Chlorine Standard, 25–30 mg/L, 2 mL 16/pkg 2630020
Voluette breaker each 2196800

Page 377
Chlorine, Free, AFT, DT, 10024
P
Amperometric Forward Titration using

Method 10024
0.00564 N PAO1
(0 to 1000 µg/L Chlorine as Cl2) Digital Titrator
Scope and Application: For drinking water; USEPA Accepted for reporting
1 Procedure is equivalent to Standard Method (18th ed.) 4500 Cl D for drinking water.
Test preparation
Make sure that the proper stir bar is used. The wrong size can cause the loss of chlorine, unstable readings and loss of
method sensitivity, especially when measuring low level chlorine concentrations.
Amperometric Titrator assembly each
Digital Titrator each
Beaker, low-form, 250-mL each
Stir bar, octagonal, Teflon-coated, 50.8 x 7.9 mm each
Cylinder, graduated, 250-mL each
TitraStir® mixer/stand assembly, 115 VAC each
Probe Assembly, Amperometric Titrator each
Delivery Tubes, 90° with hook 5/pkg
Phenylarsine Oxide Solution, 0.00564 N Digital Titrator Cartridge each
Phosphate Buffer Solution, pH 7 1 bottle
Chlorine, Free
Page 379
Chlorine, Free
Amperometric forward titration using 0.00564 N PAO
1. Install the 0.00564 N 2. Assemble the 3. Without excessive 4. If the pH is less

Phenylarsine Oxide (PAO) Amperometric Digital agitation, measure 200 mL than 6 or greater than
cartridge. Flush the Digital Titrator System according of sample with a clean 7.5, add 1.0 mL of pH 7
Titrator delivery tube by to the instructions in the graduated cylinder. Phosphate Buffer Solution
turning the delivery knob Amperometric Titrator Transfer the sample to a to make the prepared
to eject a few drops of Instruction Manual. clean, 250-mL beaker sample.
titrant. Reset the counter containing the 50-mm
to zero and wipe the tip. stirring bar supplied with
the system.
5. Place the beaker on 6. Note the LED reading 7. Using the Digital 8. As the end point of the
the TitraStir® stand and on the Amperometric Titrator delivery knob, titration is approached,
immerse the tips of both Titrator. Unlock the BIAS dispense the PAO Titrant record the LED readings
the probe and the delivery control and adjust the Solution in 5 to 10 digit along with the
tube in the solution. The BIAS control knob until a increments while noting corresponding digits
probe’s platinum wires reading between 0.50 and the downward reading. displayed on the Digital
must be submerged. Turn 0.06 is obtained. Lock the If the chlorine content of Titrator counter. Near the
on the stirring motor. bias control. the sample is high, add titration end point, add 2 to
The BIAS adjustment titrant at a faster rate. 5 digits of titrant; wait a
controls the slope of the few seconds for a stable
titration curve. The actual reading and record.
instrument reading is not
important; the relative
readings as the titration
proceeds are important. A
precise adjustment is not
required.
Chlorine, Free
Page 380
Chlorine, Free
Amperometric forward titration using 0.00564 N PAO
9. Continue the titration, 10. Use linear graph 11. Draw the two best 12. Determine the mg/L
recording at least three paper to plot the recorded intersecting lines through free chlorine
points on the downward readings from the the points plotted as concentration:
sloping curve and at least Amperometric Titrator on shown above. Determine Digits at end point x 1.25 =
three points after the end the vertical axis and the the number of digits at the µg/L free chlorine as Cl2
point is reached. The latter corresponding Digital intersection of the two
points will cause little Titrator digits on the lines. This is the end point.
change in the LED horizontal axis.
readings.
Interferences

Interfering substance Interference
Silver ions Silver ions poison the electrode.
Copper ions Interfere by plating on the electrode
High turbid water Turbid water containing surface active agents
Oxidized manganese and
Positive interference
other oxidizing reagents
Samples containing organic
Some uncertainty in the endpoint may be observed on samples with high organic content.
content
Excess reducing agents, such as sulfur dioxide, sulfite and bisulfite, will cause either static,
Samples containing reducing
or increasing LED readings because no free chlorine is present; these samples cannot be
agents
titrated under the conditions of the test.
Buffered samples or extreme Highly buffered samples or extreme sample pH may exceed the buffering capacity of the
sample pH buffer reagent. If necessary, add additional buffer and check pH of sample prior to titration.
Chlorine, Free
Page 381
Chlorine, Free

Chlorine is rapidly lost from water.
• Avoid exposure to sunlight or other strong light.
• Avoid excessive agitation.
• Analyze samples immediately.
Accuracy check
• Chlorine Standard Solution Ampule
• Fresh sample
1. Snap the top off a Chlorine Standard Solution Ampule. Note the certificate concentration of the
standard in mg/L.
2. Split a fresh sample into two 200-mL portions.
3. Use a TenSette® Pipet to add 0.1 to 0.5 mL of the standard to one portion and swirl to mix. This
is the spiked sample.
4. Analyze both the sample and spiked sample and record the chlorine concentration of each.
5. Calculate the theoretical concentration of the spiked sample:
( Cu × Vu ) + ( Cs × Vs )
Theoretical concentration = -------------------------------------------------------
-
Vu + Vs
Where:
Cu = measured concentration of sample, in mg/L (µg/L divided by 1000)
Vu = volume of sample
Cs = concentration of chlorine standard (mg/L, certificate value)
Vs = volume of standard added
6. Calculate the percent spiked recovery:
Spiked sample result, in mg/L

% Spike recovery = -----------------------------------------------------------------------------------------------------------------------
Theoretical concentration calculated, in mg/L
Chlorine, Free
Page 382
Chlorine, Free
Example:
Sample result (Cu) = 120 µg/L or 0.120 mg/L
Spiked sample result = 185 µg/L or 0.185 mg/L
Volume Sample (Vu) = 200 mL
Volume Standard (Vs) = 0.2 mL
Chlorine Standard (Cs) = 68.1 mg/L
( 0.120 × 200 ) + ( 68.1 × 0.2 )

Theoretical concentration = ------------------------------------------------------------------------- = 0.188 mg/L
200 + 0.2
0.185 mg/L
% Spike recovery = ------------------------------ × 100 = 98%
0.188 mg/L
Ideally, the percent recovery should be 100%. Generally, results from 80–120% recovery are
considered acceptable.
Method performance
Precision
In a single laboratory, a single operator used a standard solution of 338 µg/L chlorine to obtain a
standard deviation of ±5.2 µg/L chlorine.
Detection Limit
With good operator technique, the estimated detectable concentration is approximately 15 µg/L
chlorine using 0.00564 N PAO.
Summary of method
In the amperometric forward titration procedure for free chlorine, a small electrical current is
applied across two identical platinum electrodes. No current can flow between the electrodes
unless a substance that can be oxidized at the anode and a substance that can be reduced at the
cathode are both present. In the case of the free chlorine titration with phenylarsine oxide (PAO),
chlorine is reduced to chloride at the cathode due to the addition of PAO, and PAO is oxidized from
the +3 oxidation state to the +5 oxidation state at the anode. Prior to the end point of the titration,
both free chlorine and chloride are present in solution; allowing current to flow, even with a very
small applied potential. At the end point, no free chlorine remains and the solution cannot conduct
even if excess PAO titrant is added. The end point is defined when no change in current occurs,
signaling all free chlorine has been reacted.
Chlorine, Free
Page 383
Chlorine, Free
Required reagents
Phenylarsine Oxide Solution, 0.00564 N Digital Titrator Cartridge each 199901
Phosphate Buffer Solution, pH 7 100 mL MDB 2155332
Required apparatus
Amperometric Titrator Assembly each 1929900
Beaker, low-form, 250-mL each 50046H
Delivery Tubes, 90° with hook 5/pkg 4157800
Probe Assembly, Amperometric Titrator each 1939000
Stir Bar, octagonal, Teflon-coated, 50.8 x 7.9 mm each 2095355
TitraStir® Mixer/Stand Assembly, 115 VAC each 1940000
OR
TitraStir® Mixer/Stand Assembly, 230 VAC 1940010
Chlorine Standard Solution Ampule, 50–75 mg/L 20/pkg 1426820
Water, demineralized, each 4L 27256

Chlorine, Hypochlorite, 10100
Chlorine, Hypochlorite DOC316.53.01219
Iodometric Method1 Method 10100

HR (50 to 150 g/L) as Cl2 (5 to 15%) Digital Titrator
Scope and Application: For testing concentrated liquid bleach (sodium hypochlorite, soda bleach) used as a
disinfectant in drinking water or wastewater treatment
1 Adapted from ASTM method D2022.
Test preparation
Digital Titrator Reagent Set, HR Hypochlorite (Bleach), 5–15% as Cl2 1
Potassium Iodide-Iodate Standard Solution, 0.0125 N 1L
Clippers, large 1
Delivery Tubes, 180° 1
Digital Titrator 1
TenSette Pipet, 0.1–1.0 mL 1
Tips, for TenSette® Pipet 1970001 1
Iodometric method
13. Insert a clean delivery 14. Flush the delivery tube 15. Fill the Erlenmeyer 16. Add the contents of
tube into the 2.26 N by turning the delivery flask to about the 75-mL one Potassium Iodide
Sodium Thiosulfate Titrant knob to eject a few drops mark with deionized water Powder Pillow to the flask
Solution cartridge. Attach of titrant. Reset the or tap water. and swirl to mix.
the cartridge to the titrator counter to zero and wipe The level of residual
body. off the tip. chlorine found in tap water
will not interfere with the
test.
Chlorine, Hypochlorite
Page 385
Iodometric method
17. Add the contents of 18. Attach a clean tip to 19. Swirl to mix. The 20. Place the delivery tube
one Acid Reagent Powder the TenSette Pipet. solution will turn dark tip into the solution and
Pillow to the flask and swirl Use the pipet to dispense brown. swirl the flask while
to mix. 0.2 mL of bleach sample titrating with the sodium
below the solution level in thiosulfate titrant until the
the flask. solution is pale yellow.
21. Add one dropperful of 22. Continue the titration 23. Calculate the g/L
Starch Indicator Solution until the solution becomes chlorine:
to the flask and swirl to colorless. Record the Digits required x 0.5 =
mix. A dark blue or green number of digits required. g/L chlorine
color will develop.
g/L chlorine x 0.10 =
% chlorine by volume
(“trade percent”)
Interferences
The iodometric method is relatively free of interferences. The test will determine chlorite ion
(ClO2 –) in addition to the hypochlorite ion (ClO–). However, the amount of chlorite in commercial
bleach is insignificant (typically less than 0.2%).

A large excess of caustic may cause low results.
1. After adding the Acid Reagent Powder Pillow (step 17), check the pH of the solution with
Caustic agent pH Paper. The pH should be less than 3.
2. If the pH is not less than 3, add additional Acid Reagent, one pillow at a time, until the
pH drops below 3.
For most accurate results, the temperature of the dilution water should be less than 20 °C
Temperature
(68 °F).
Page 386

Soda bleach solutions are relatively unstable.
• Avoid exposing the sample to heat or light.
• Collect samples in glass bottles and store in a cool, dark place until analyzed.
• Analyze as soon as practical.
Accuracy check
For optimum test results, the manufacturer strongly recommends that reagent accuracy be
checked with each new lot of reagents. The strength of the Sodium Thiosulfate Standard Solution
can be checked using Potassium Iodide-Iodate Standard Solution:
• Class A pipet, 50-mL
• Pipet filler
• 125-mL Erlenmeyer flask.
• Potassium Iodide Powder Pillow
• 50.00 mL of 0.0125 N Potassium Iodide-Iodate Standard Solution
7. Use a Class A pipet to transfer 50.00 mL of 0.0125 N Potassium Iodide-Iodate Standard
Solution to a clean 125-mL Erlenmeyer flask.
8. Add the contents of one Potassium Iodide Powder Pillow to the flask and swirl to mix.
9. Add the contents of three Acid Reagent Powder Pillows to the flask and swirl to mix. Swirl until
all powder is dissolved.
10. Continue the titration starting at step 20 of the procedure. It should take 217–227 digits of
2.26 N Thiosulfate Standard Solution to reach the end point.
Precision
In a single laboratory, using a commercial bleach sample of 91.2 g/L (9.12%) Cl2, a single operator
obtained a standard deviation of ±1.5 g/L (± 0.15%) Cl2.
Summary of method
Under acidic conditions, hypochlorite reacts with iodide to produce an equivalent amount of
triiodide (I3–). The released I3– is titrated with standard sodium thiosulfate solution to a colorless
end point. The number of digits of sodium thiosulfate required is proportional to the hypochlorite
concentration in the original bleach sample.
Page 387
Required reagents
Digital Titrator Reagent Set, HR Hypochlorite (Bleach), 5–15% as Cl2. Includes:
Acid Reagent Powder Pillows 1 pillow 100/pkg 104299
Potassium Iodide Powder Pillows 1 pillow 50/pkg 2059996
Sodium Thiosulfate Standard Solution Cartridge, 2.26 N varies each 2686901
Starch Indicator Solution 1 100 mL MDB 34932
Required apparatus
Clippers, large 1 each 96800
Delivery Tubes, 180° 1 5/pkg 1720500
TenSette Pipet, 0.1–1.0 mL 1 each 1970001
Tips, for TenSette® Pipet 1970001 1 50/pkg 2185696
Potassium Iodide-Iodate Standard Solution, 0.0125 N 1L 1400153

pH Test Strips, 0–14 pH 100/pkg 2601300
Bottle, sample, glass, 4 oz, with cap 3/pkg 2161303
Pipet, volumetric Class A, 50-mL each 1451541
Pipet Filler, Safety Bulb each 1465100

Chlorine, Total, ABT, DT, 10025
Amperometric Back Titration1 Method 10025

Scope and Application: For drinking water and wastewater; USEPA accepted for reporting
1 Procedure is equivalent to Standard Method (18th ed.) 4500-Cl C for wastewater.
Test preparation
Before starting the test
When a new probe is used or the probe has not been used recently, prepare it according to the Probe Stabilization
instructions in the Amperometric Titrator Instruction Manual.
Use the proper stir bar (Catalog number 2095355). The wrong size can cause the loss of chlorine, unstable readings, and
loss of method sensitivity, especially when measuring low level chlorine concentrations.
To preserve the strength of the iodine titrant solution, always remove the delivery tube from the Digital Titrator cartridge and
replace the cap when not in use. Protect the iodine titrant solution from direct sunlight.
The sample may be fixed at the sample site for brief transportation delays— but not for sample storage. (This fixing
technique is not acceptable for USEPA compliance monitoring.). See Sample collection, preservation and storage for
more information.
Standard Iodine Titrant Solution, Cartridge, 0.028 N each
Amperometric Titrator Assembly each
Stir Bar, octagonal, Teflon-coated, 50.8 x 7.9 mm each
Acetate Buffer Solution, pH 4.0 100 mL MDB
Potassium Iodide Powder Pillows 100/pkg
TitraStir® Mixer/Stand Assembly, 115 or 230 VAC each
Pipet, Volumetric, Class A, 1-mL each
Pipet Filler each
Sodium Thiosulfate Standard Solution, 0.00564 N 100 mL
Chlorine, Total
Page 389
Chlorine, Total
Part 1—Adjusting the electrode response slope
1. Install the Standard 2. Assemble the 3. Use a graduated 4. Add 1 mL of pH 4

Iodine Titrant Cartridge, Amperometric Digital cylinder to measure 200 Acetate Buffer and the
0.028 N. Flush the Digital Titrator System according mL of deionized water into contents of one Potassium
Titrator delivery tube by to the instructions in the a clean 250-mL beaker. Iodide Pillow.
turning the delivery knob Amperometric Titrator Place the 50-mm stirring
to eject a few drops of Instruction Manual. bar into the beaker.
titrant. Reset the counter
to zero and wipe the tip.
5. Place the beaker on 6. Use the Digital Titrator 7. Note the LED reading 8. Remove the probe
the TitraStir stand and delivery knob to add on the Amperometric arm from the beaker and
immerse the tips of both 50 digits of Standard Titrator. Unlock the BIAS rinse the platinum wires
the probe and the delivery Iodine Titrant Solution. control knob until a stable with deionized water.
tube in the solution. The reading between 0.50 and Discard the sample.
probe’s platinum wires 0.60 is obtained. Lock the The adjustment of the
must be submerged. Turn bias control. electrode response slope
on the stirring motor. is complete.
Chlorine, Total
Page 390
Chlorine, Total
Part 2—Standardization of the Iodine Titrant
1. Flush the Digital 2. Assemble the 3. Use a graduated 4. Use a Class A pipet to
Titrator delivery tube by Amperometric Digital cylinder to measure transfer 1.00 mL of
turning the delivery knob Titrator System according 200 mL of deionized water 0.00564 N Sodium
to eject a few drops of to the instructions in the into a clean 250-mL Thiosulfate Solution to the
titrant. Reset the counter Amperometric Titrator beaker. Place the 50-mm beaker. Swirl to mix.
to zero and wipe the tip. Instruction Manual. stirring bar into the beaker. Alternatively, use
0.00564 N Phenylarsine
Oxide (PAO) (Catalog
No. 199942) instead of
sodium thiosulfate.
5. Add 1 mL of pH 4 6. Place the beaker on 7. Note the LED reading 8. Using the Digital
Acetate Buffer Solution the TitraStir stand and on the Amperometric Titrator delivery knob,
and the contents of one immerse the tips of both Titrator. It should read 0.00 dispense 100 digits of
Potassium Iodide Powder the probe and the delivery ±0.05. DO NOT adjust the Standard Iodine Titrant
Pillow. tube in the solution. The bias control. Solution and note the
probe’s platinum wires reading.
must be submerged. Turn
on the stirring motor.
Chlorine, Total
Page 391
Chlorine, Total
Part 2—Standardization of the Iodine Titrant (continued)
9. Continue dispensing 10. Record at least three 11. After the end point of 12. Add five to ten digits of
titrant in five to ten digit points (the null current the titration (nominal 160 titrant and wait a few
increments while noting values) before the end digits), record the seconds for a stable
the reading. point is reached. increasing LED readings reading. Record it.
along with the Stop adding titrant when
corresponding digits the LED readings exceed
displayed on the Digital 0.60. LED readings above
Titrator counter. 0.60 will be excessively
noisy.
13. Use a linear graph 14. Draw the two best 15. Determine the number 16. Record the standard
paper to plot the recorded intersecting lines through of digits at the intersection end point digits value. Find
readings from the the plotted points as of the lines. That is the the multiplier from the Digit
Amperometric Titrator on shown above. standard end point. multipliers table. This
the vertical axis and the multiplier will be used to
corresponding Digital calculate the sample
Titrator digits on the chlorine concentration.
horizontal axis. Discard the sample.
Table 121 Digit multipliers

Digits (standard end point) Multiplier
160 6.25
165 6.06
170 5.88
175 5.71
180 5.56
185 5.40
190 5.26
195 5.13
200 5.00
Chlorine, Total
Page 392
Chlorine, Total
Part 3—Titration of sample for total residual Chlorine
1. Flush the Digital 2. Assemble the 3. Place a clean, 50-mm 4. Add 1 mL of pH 4

Titrator delivery tube by Amperometric Digital stir bar into a clean Acetate Buffer Solution to
turning the delivery knob Titrator System according 250-mL beaker. Use a the beaker. Then add
to eject a few drops of to the instructions in the Class A pipet to transfer 200 mL of the sample to
titrant. Reset the counter Amperometric Titrator 1.00 mL of 0.00564 N the beaker.
to zero and wipe the tip. Instruction Manual. Sodium Thiosulfate Minimize agitation when
Solution to the beaker. adding the sample.
Alternatively, use Swirl to mix.
0.00564 N Phenylarsine
Oxide (PAO) (Catalog No.
199942) instead of sodium
thiosulfate.
5. Place the beaker on 6. Add the contents of 7. Note the LED reading 8. Using the Digital
the TitraStir stand and one Potassium Iodide on the Amperometric Titrator delivery knob,
immerse the tips of both Reagent Power Pillow to Titrator. It should read 0.00 dispense Standard Iodine
the probe and the delivery the beaker and allow the ±0.05. DO NOT adjust the Titrant Solution in five to
tube in the solution. The powder to dissolve. bias control. ten digit increments while
probe’s platinum wires noting the reading.
must be submerged. Turn
on the stirring motor.
Chlorine, Total
Page 393
Chlorine, Total
Part 3—Titration of sample for total residual Chlorine (continued)
9. Record at least three 10. After the end point of 11. Add five to ten digits of 12. Using linear graph
points (the null current the titration is reached, titrant and wait a few paper, plot the recorded
values) before the end record the increasing LED seconds for a stable readings from the
point is reached. readings along with the reading. Record it. Amperometric Titrator on
corresponding digits Stop adding titrant when the vertical axis and the
displayed on the Digital the LED readings exceed corresponding Digital
Titrator counter. 0.60. LED readings above Titrator digits on the
0.60 will be excessively horizontal axis.
noisy.
13. Draw the two best 14. Determine the number 15. Calculate the µg/L total chlorine:
intersecting lines through of digits at the intersection [Digits (Standard End Point) – Digits (Sample End
the plotted points as of the lines. That is the Point)] x Multiplier = µg/L Cl2
shown above. sample end point.
Use the multiplier from Part 2—Standardization of the
Iodine Titrant, step 16.
Interpolation between values in the table is not
necessary.
Example:
Standard EP = 160 digits
Sample EP = 150 digits
Multiplier = 6.25, so:
µg/L total chlorine [160 – 150] x 6.25 = 62.5, or 63 (round up)
Chlorine, Total
Page 394
Chlorine, Total

Silver ions Silver ions poison the electrode
Copper ions Interfere by plating on the electrode.
Interferences are sometimes found in highly turbid water and those containing surface active
Turbid water
agents
Oxidized manganese Oxidized manganese and other oxidizing reagents give positive interferences.
Samples containing high Some uncertainty in the end point may be observed with samples containing high organic
organic content. content.
Iron and nitrite interference are minimized by buffering to pH 4 before adding potassium
Iron and nitrite
iodide.
Buffered samples or sample Highly buffered samples or extreme sample pH may exceed the buffering capacity of the
pH buffer reagent. If necessary, add additional buffer and check pH of sample prior to titration.
In samples that contain excess dechlorinating agents, such as sulfur dioxide, sulfite or
bisulfite, the titration end point (number of digits) will be greater than the number of digits
Dechlorinating agents obtained during the standardization. It is not necessary to continue the titrant addition if the
number of digits used in the sample titration exceeds that calculated for the standardization
end point. This indicates that no free or combined chlorine is present in the sample.

Chlorine is rapidly lost from water. Avoid exposure to sunlight or other strong light. Avoid excessive
agitation. Analyze samples immediately or fix the sample by pre-addition of standard thiosulfate
and buffer. To fix the sample:
1. Pipet 1.00 mL of 0.00564 N Sodium Thiosulfate and add 1.0 mL of Acetate Buffer into a clean,
dry glass sampling bottle (e.g. BOD bottle).
2. At the sample site, measure 200 mL of sample with a graduated cylinder and transfer to the
sampling bottle. Swirl to mix.
3. Before analysis, quantitatively transfer the entire contents of the sampling bottle to the 250-mL
beaker. Minimize delay between sampling and analysis (1 hour maximum) to prevent
decomposition of thiosulfate in the sample. (This fixing technique is not acceptable for USEPA
compliance monitoring and should be used for brief transportation delays—not for sample
storage.)
4. Start the analysis in Part 3—Titration of sample for total residual Chlorine at step 5.
Accuracy check
Use the bias control prior to performing the analysis to adjust the electrode sensitivity. Set the bias
adjustment by adding a known amount of standard iodine titrant to deionized water and adjusting
the bias control to a given value on the display. The electrode sensitivity will vary depending on the
probe conditioning. Adjustment should be made at least daily or before each series of samples.
The iodine titrant concentration is approximately 0.0282 N, which relates to 160 digits needed to
titrate 1.00 mL of 0.00564 N Thiosulfate. If the calculated end point is greater than 160 digits, this
indicates that the Standard Iodine Titrant is weaker than when packaged. Discard the Standard
Iodine Titrant cartridge if the calculated standard end point in Part 2—Standardization of the Iodine
Titrant is greater than 200 digits.
To preserve the strength of the iodine titrant solution, always remove the delivery tube from the
Digital Titrator cartridge and replace the cap when not in use. Protect the iodine titrant solution
from direct sunlight.
Chlorine, Total
Page 395
Chlorine, Total

Note: Standard additions is not applicable for samples containing excess reducing agents such as sulfur
dioxide, sulfite, or bisulfite.

• Chlorine Standard Solution Ampule
1. Snap the top off a Chlorine Standard Solution Ampule. Note the certificate concentration of the
standard in mg/L.
3. Using a TenSette Pipet (Catalog number 1970001), add 0.1 to 0.5 mL of the standard to one
portion and swirl to mix. This is the spiked sample.
( Cu × Vu ) + ( Cs × Vs )
-
Vu + Vs
Where:
6. Calculate the percent spiked recovery:

% Spike recovery = -----------------------------------------------------------------------------------------------------------------------
Example:
( 0.120 × 200 ) + ( 68.1 × 0.2 )

200 + 0.2
0.185 mg/L
% Spike recovery = ------------------------------ × 100 = 98%
0.188 mg/L
Chlorine, Total
Page 396
Chlorine, Total
Method performance
Precision
In a single laboratory, using a standard solution of 120 µg/L chlorine, a single operator obtained a
standard deviation of ±19 µg/L chlorine.
Detection limit
The estimated detectable concentration is equivalent to one digit of 0.0282 N Standard Iodine
Titrant Solution, or approximately 6 µg/L chlorine.
Summary of method
The back titration procedure minimizes errors caused by liberating the full concentration of iodine
in the sample and is the preferred method for amperometric measurement for total chlorine in
wastewaters. In this procedure, the end point signal is reversed because the remaining thiosulfate
(or phenylarsine oxide) added to the sample is titrated with standard iodine. The end point of the
back titration is reached just when free iodine exists in the sample resulting in a measurable
polarization current. The end point is estimated by continued addition of titrant, recording of the
current at each titrant addition, and graphing the data points. Where the best line between the data
points intersects the null current, the number of digits (from the Digital Titrator) at the end point can
be determined and the chlorine concentration calculated.

Required reagents
Acetate buffer solution, pH 4.0 100 mL MDB 1490932
Potassium Iodide powder pillows 100/pkg 107799
Standard Iodine titrant solution, cartridge, 0.028 N each 2333301
Sodium Thiosulfate standard solution, 0.00564 N 100 mL 2408842
Required apparatus
each 1929900
Amperometric titrator assembly

Delivery tubes, 90° with hook 5/pkg 4157800
Digital titrator each 1690001
Probe assembly, Amperometric titrator each 1939000
Stir bar, octagonal, Teflon-coated, 50.8 x 7.9 mm each 2095355
TitraStir® mixer/stand assembly, 115 VAC each 1940000
OR
TitraStir® mixer/stand assembly, 230 VAC 1940010
Chlorine standard solution Ampule, 50–75 mg/L 20/pkg 1426820
Water, demineralized, each 4L 27256
Chlorine, Total
Page 397
Chlorine, Total


Chlorine, Total, AFT, DT, 10026
Amperometric Forward Titration1 Method 10026

Scope and Application: For drinking water and wastewater; USEPA accepted for reporting
1 Procedure is equivalent to Standard Method (18th ed.) 4500-Cl D for drinking water and Standard Method (17th ed.) 4500-Cl D for wastewater
Test preparation
When a new probe is used or the probe has not been used recently, prepare it according to the Probe Stabilization
instructions in the Amperometric Titrator Instruction Manual.
Use the proper stir bar. The wrong size can cause the loss of chlorine, unstable readings and loss of method sensitivity,
Phenylarsine Oxide Solution, 0.00564 N Digital Titrator Cartridge each
Amperometric Titrator Assembly each
Stir Bar, octagonal, Teflon®-coated, 50.8 x 7.9 mm each
Potassium Iodide Powder Pillows 100/pkg
Acetate Buffer Solution, pH 4 100 mL MBD
TitraStir® Mixer/Stand Assembly, 115 or 230 VAC each
Graph paper varies
Chlorine, Total
Page 399
Chlorine, Total
Amperometric forward titration
1. Install the 2. Assemble the 3. Without excessive 4. Add the contents of

Phenylarsine Oxide (PAO) Amperometric Digital agitation, measure 200 mL one Potassium Iodide
Cartridge, 0.00564 N. Titrator System according of sample with a clean Powder Pillow and swirl to
Flush the Digital Titrator to the instructions in the graduated cylinder. dissolve.
delivery tube by turning Amperometric Titrator Transfer the sample to a
the delivery knob to eject a Instruction Manual. clean, 250-mL beaker
few drops of titrant. Reset containing the 50-mm
the counter to zero and stirring bar supplied with
wipe the tip. the system.
5. Add 1 mL of pH 4 6. Place the beaker on 7. Note the LED reading 8. Using the Digital
Acetate Buffer solution. the TitraStir stand and on the Amperometric Titrator delivery knob,
immerse the tips of both Titrator. Unlock the BIAS dispense the PAO Titrant
the probe and the delivery control and adjust the Solution in 5 to 10 digit
tube in the solution. The BIAS control knob until a increments while noting
probe’s platinum wires stable reading between the downward reading.
must be submerged. Turn 0.50 and 0.60 is obtained. If the chlorine content of
on the stirring motor. Lock the BIAS control. the sample is high, add
The BIAS adjustment titrant at a faster rate.
controls the slope of the
titration curve. The actual
instrument reading is not
important; the relative
readings as the titration
proceeds are important. A
precise adjustment is not
required.
Chlorine, Total
Page 400
Chlorine, Total
Amperometric forward titration (continued)
9. As the end point of the 10. Continue the titration, 11. Using linear graph 12. Draw the two best
titration is approached, recording at least three paper, plot the recorded intersecting lines through
record the LED readings points on the downward readings from the the points plotted as
along with the sloping curve and at least Amperometric Titrator on shown above. Determine
corresponding digits three points after the end the vertical axis and the the number of digits at the
displayed on the Digital point is reached. The latter corresponding Digital intersection of the two
Titrator counter. Near the points will cause little Titrator digits on the lines. This is the end point.
titration end point, add 2 to change in the LED horizontal axis.
5 digits of titrant; wait a readings.
few seconds for a stable
reading and record.
13. Determine the µg/L

total chlorine
concentration:
Digits at end point x 1.25 =
µg/L total chlorine as Cl2
Chlorine, Total
Page 401
Chlorine, Total
Interferences

Silver ions Silver ions poison the electrode.
Copper ions Interfere by plating on the electrode.
Turbid water and water
Interferences are sometimes found in highly turbid water and those containing surface active
containing surface active
agents.
agents
Oxidized manganese and
Oxidized manganese and other oxidizing reagents give positive interferences.
other oxidizing reagents
Samples containing organic
Some uncertainty in the endpoint may be observed on samples with high organic content.
content.
Samples containing excess reducing agents, such as sulfur dioxide, sulfite and bisulfite do
Samples containing excess
not contain free chlorine or chloramines and can not be titrated under the conditions of the
reducing agents
test.
Buffered samples or sample Highly buffered samples or extreme sample pH may exceed the buffering capacity of the
pH buffer reagent. If necessary, add additional buffer and check pH of sample prior to titration.

Chlorine is rapidly lost from water.
• Avoid exposure to sunlight or other strong light.
• Avoid excessive agitation.
Accuracy check
Standard additions method* (sample spike)
• Chlorine Standard Solution Ampule, 50–75 mg/L Cl2
1. Snap the top off a Chlorine Standard Solution Ampule, 50–75 mg/L Cl2. Note the specific
certificate concentration of the standard in mg/L.
3. Use the TenSette® Pipet (Catalog. Number. 1970001) to add 0.1 to 0.5 mL of the standard to
one portion and swirl to mix. This is the spiked sample.
( Cu × Vu ) + ( Cs × Vs )
-
Vu + Vs
* The Standard Additions technique is not applicable to samples that contain excess reducing agents such as sulfur dioxide,
sulfite, or bisulfite.
Chlorine, Total
Page 402
Chlorine, Total
Where:
Calculate the percent spiked recovery:
% Spike recovery = -----------------------------------------------------------------------------------------------------------------------
Example:
( 0.120 × 200 ) + ( 68.1 × 0.2 )

200 + 0.2
0.185 mg/L
% Spike recovery = ------------------------------ × 100 = 98%
0.188 mg/L
Method performance
Precision
In a single laboratory, using a standard solution of 347 µg/L chlorine, a single operator obtained a
standard deviation of ±3.2 µg/L chlorine.
Detection limit
The estimated detectable concentration is approximately 15 µg/L chlorine using 0.00564 N PAO.
Summary of method
In the amperometric forward titration procedure for total chlorine, a small electrical current is
applied across two identical platinum electrodes. No current can flow between the electrodes
unless a substance that can be oxidized at the anode and a substance that can be reduced at the
cathode are both present. In the case of the total chlorine, an equivalent amount of iodine forms
from the reaction of excess iodide with chlorine and combined chlorine at pH 4. During the titration
with phenylarsine oxide (PAO), the free iodine is reduced to iodide at the cathode and PAO is
oxidized from the +3 oxidation state to the +5 oxidation state at the anode. Prior to the end point of
the titration, both iodine and iodide are present in solution; therefore current can flow, even with a
very small applied potential. At the end point, no free iodine remains and the solution cannot
conduct even if excess PAO titrant is added. The end point is defined when no change in current
occurs, signaling all total chlorine has been reacted.
Chlorine, Total
Page 403
Chlorine, Total
Required reagents
Phenylarsine Oxide Solution, 0.00564 N Digital Titrator cartridge each 199901
Acetate Buffer Solution, pH 4 100 mL MBD 1490932
Potassium Iodide Powder Pillows 100/pkg 107799
Required apparatus
Amperometric Titrator Assembly each 1929900
Delivery Tubes, 90° with hook 5/pkg 4157800
Probe Assembly, Amperometric Titrator each 1939000
Stir Bar, octagonal, Teflon®-coated, 50.8 x 7.9 mm each 2095355
TitraStir® Mixer/Stand Assembly, 115 VAC each 1940000
OR
TitraStir® Mixer/Stand Assembly, 230 VAC 1940010
Chlorine Standard Solution Ampule, 50–75 mg/L 20/pkg 1426820
Water, deionized, each 4L 27256


Chlorine, total, BT, 8161
Iodometric Method using Sodium Thiosulfate1 Method 8161

0 to 40,000 mg/L Buret Titration
1 Adapted from Standard Methods for the Examination of Water and Wastewater (4500 Cl- D).
Test preparation
Dissolved Oxygen 3 Powder Pillow 1
Potassium Iodide Powder Pillow 1
Starch Indicator Solution 1 bottle
Sodium Thiosulfate Standard Solution (see the Range-specific information table) 1 bottle
Buret titration
See
Table 1
volume and corresponding the zero mark with the cylinder or pipet to into a 250-mL Erlenmeyer
sodium thiosulfate solution Sodium Thiosulfate measure the sample flask. If the sample volume
from the Range-specific Standard Solution. volume from the Range- is less than 50 mL, dilute
information table. specific information table. to approximately 50 mL
Chlorine, Total
Page 405
Chlorine, Total
5. Add the contents of 6. Add the contents of 7. Titrate the sample 8. Add one full dropper of
one Dissolved Oxygen 3 one Potassium Iodide while swirling the flask Starch Indicator Solution.
Powder Pillow. Swirl Powder Pillow. Swirl until the color changes to a Swirl to mix. The sample
to mix. to mix. very light yellow. will change to a dark blue
The addition of the powder color.
pillow will lower the pH to 4
or less. If the sample is
highly alkaline, make sure
that the solution pH is 4 or
less with a pH meter or pH
paper before proceeding.
9. Titrate the sample 10. Calculate:

while swirling the flask mL titrant used x multiplier = mg/L total chlorine as Cl2
until the color changes
from blue to colorless. Example: 100 mL of sample was titrated with the 0.10 N
sodium thiosulfate solution and 15 mL of titrant was
used to reach the endpoint.
The chlorine concentration is: 15 x 35.5 = 532 mg/L Cl2

Range (mg/L as Cl2) Sample volume (mL) Sodium thiosulfate concentration Multiplier
0–200 100 0.025 N 8.87

100–400 50 0.025 N 17.7
200–800 100 0.10 N 35.5
400–1,600 50 0.10 N 70.9
1,000–4,000 20 0.10 N 177
2,000–8,000 10 0.10 N 355
5,000–20,000 4 0.10 N 887
10,000–40,000 2 0.10 N 1773
Chlorine, Total
Page 406
Chlorine, Total
Interferences
Oxidized forms of manganese, oxidizing agents and reducing agents such as organic sulfides can
interfere.

sunlight, pH, temperature and salinity can cause the decomposition of chlorine in water.
Summary of method
When potassium iodide is added to a sample containing chlorine at a pH less than 4, free iodine is
liberated in direct proportion to the amount of total chlorine present. The iodine is then titrated with
sodium thiosulfate. Starch indicator is added to enhance the end point. This method measures
both free chlorine and combined chlorine.
Chlorine, Total
Page 407
Chlorine, Total
Required reagents
Dissolved Oxygen 3 Powder Pillows 1 pillow 100/pkg 98799
Starch Indicator Solution 1 mL 100 mL MDB 34932
Titrant—select one or more based on range:
Sodium Thiosulfate Standard Solution, 0.025 N varies 1L 35253
Sodium Thiosulfate Standard Solution, 0.10 N varies 1L 32353
Required apparatus
pH Paper, 0–14 pH range — 100/pkg 2601300

Chlorine, total, BT, 8168
USEPA1 Amperometric Buret Titration Method2 Method 8168

(0.5 mg/L and above) Buret Titration
1 USEPA accepted (4500 Cl- D).
Test preparation
Use only a 50-mm stir bar. The wrong size can cause the loss of chlorine, unstable readings and loss of method sensitivity,
For added convenience when stirring, use the TitraStir® apparatus.
Phenylarsine Oxide Solution, 0.00564 N 1 bottle
Acetate Buffer Solution 1 mL
Potassium Iodide Powder Pillow 1
Beaker, 250-mL 1
Chlorine, Total
Page 409
Chlorine, Total
Buret titration
1. Fill the 5-mL automatic 2. Put a 50-mm stir bar 3. Add the contents of 4. Add 1.0 mL of pH 4
buret to the zero mark with into a 250-mL beaker. one Potassium Iodide Acetate Buffer Solution to
0.00564 N Phenylarsine Use a graduated cylinder Powder Pillow. Swirl to make the prepared
Oxide (PAO) Solution. to measure 200 mL of mix. sample.
sample. Add the sample to
the beaker.
5. Place the beaker of 6. Turn the BIAS control 7. Dispense the titrant 8. Continue dispensing
prepared sample on the knob to adjust the value on into the beaker in small slowly. Near the end point
TitraStir titration stand and the display to increments while of the titration, write down
turn on the stirring motor. approximately 1.00. monitoring the values on the value on the display
Put the tip of the probe The BIAS adjustment the Amperometric Titrator. and the corresponding
fully into the prepared controls the slope of the The values will decrease. total volume of titrant that
sample. The platinum titration curve. The actual was added. Read the
wires must be submerged. value is not important. volume to the nearest
If a stir plate other than the Only the relative value as 0.01 mL. Add a small
TitraStir® is used, set the the titration continues is amount of titrant and wait
speed for moderate important. A precise several seconds for a
mixing. Do not adjust the adjustment is not stable value. Write down
speed after this point. necessary. the value.
Chlorine, Total
Page 410
Chlorine, Total
9. Continue the titration 10. Make a graph of the 11. Draw the two best
by recording at least three titration. Plot the values intersecting lines through
points on the downward from the amperometric the points as shown
sloping curve and at least titrator on the vertical axis above. Find the volume of
three points after the end and the corresponding titrant to the nearest
point has been reached. volume of titrant on the 0.01 mL at the intersection
The value on the display horizontal axis. of the two lines. This is the
will not change mL titrant end point. This
significantly after the volume is equivalent to the
end point. total chlorine
concentration in mg/L.
mL titrant =
mg/L total chlorine as Cl2
Interferences
Refer to the Amperometric Titrator Instruction Manual for a discussion of sources of errors and
interferences using the amperometric methods.

sunlight, pH, temperature, and salinity can cause the decomposition of chlorine in water.
A common error in testing for chlorine is introduced when a representative sample is not obtained.
If sampling from a tap, let the water flow for at least 5 minutes before sample collection. Let the
sample container overflow with the sample several times, then cap the container so that there is no
headspace (air) above the sample. Start the chlorine analysis immediately.
Summary of method
Total chlorine is measured after the addition of potassium iodide and acetate buffer by a titration at
pH 4 with PAO solution to the amperometric end point. The amperometric titration method has
greater sensitivity and accuracy when compared to colorimetric methods. Refer to the
Amperometric Titrator Instruction Manual for more information.
Chlorine, Total
Page 411
Chlorine, Total
Required reagents
Total Chlorine Reagent Set (approximately 200 tests), includes: 2460700
(2) Acetate Buffer Solution, pH 4 1 mL 100 mL MDB 1490932
(1) Phenylarsine Oxide Solution, 0.00564 N varies 1L 199953
(2) Potassium Iodide Powder Pillows 1 100/pkg 107799
Required apparatus
Stir bar, 50 mm each 2095355
pH paper, 0–14 pH range 100/pkg 2601300

Chlorine Standard Solution, 10 mL Voluette® Ampules, 50–75 mg/L 16/pkg 1426810
Chlorine Standard Solution, 2 mL PourRite® Ampule, 50–75 mg/L 20/pkg 1426820
PourRite Ampule breaker, 2 mL (each) 2484600

Chlorine, Total, DT, 8209
Iodometric Method Using Sodium Thiosulfate Method 8209

1 to 400 mg/L or 20 to 70,000 mg/L Cl2 Digital Titrator
Test preparation
To convert mg/L chlorine to percent chlorine, divide the result in mg/L by 10,000.
These procedures can be used to measure iodine or bromine concentration if chlorine is not present. Multiply the test result
(in mg/L chlorine) by 3.58 or 2.25, respectively, to accurately express the iodine or bromine content of the sample.
For higher concentrations, see the hypochlorite procedure Method 10100.
Acetate Buffer Solution, pH 4 (for 1 to 400 mg/L Cl2 range) 1 bottle
Dissolved Oxygen 3 Powder Pillow (for 20 to 70,000 mg/L Cl2 range) 1 pillow
Potassium Iodide Powder Pillow 1 pillow
Sodium Thiosulfate Ttitration cartridge (see Range-specific information—20 to 70,000 mg/L) 1 cartridge
Digital titrator 1
Chlorine, Total
Page 413
Chlorine, Total
1 to 400 mg/L chlorine
See
Table 1
1. Select a sample 2. Insert a clean delivery 3. Turn the delivery knob 4. Use a clean graduated
volume and titration tube into the titration to eject a few drops of cylinder or pipet to
cartridge from the Range- cartridge. Attach the titrant. Reset the counter measure the sample
specific information—1 to cartridge to the titrator. to zero and wipe the tip. volume from the Range-
400 mg/L table. specific information—1 to
400 mg/L table in a
125 mL Erlenmeyer flask.
5. Transfer the sample 6. Add two full droppers 7. Add the contents of 8. Place the delivery tube
into a clean, 125-mL (2 mL) of Acetate Buffer one Potassium Iodide into the solution and swirl
Erlenmeyer flask. If the Solution, pH 4. Swirl to Powder Pillow. Swirl to the flask. Turn the knob on
sample volume is less mix. mix. the titrator to add titrant to
than 100 mL, dilute to the solution. Continue to
approximately 100 mL with swirl the flask and add
deionized water. titrant until the solution is a
pale yellow.
Chlorine, Total
Page 414
Chlorine, Total
1 to 400 mg/L chlorine
9. Add one full dropper of 10. Continue the titration 11. Use the multiplier in
Starch Indicator Solution. until the solution changes the Range-specific
Swirl to mix. from dark blue to information—1 to 400 mg/
A dark blue color will colorless. L table to calculate the
develop. Write down the number of concentration:
digits displayed on the digits x multiplier =
counter. mg/L Total Chlorine (Cl2)
Example: 100 mL of
sample was titrated and
= 2.5 mg/L chlorine
Table 125 Range-specific information—1 to 400 mg/L

Range (mg/L as Cl2) Sample volume (mL) Titration cartridge (N) Multiplier
1–4 100 0.02256 0.01

2–8 50 0.02256 0.02
5–20 20 0.02256 0.05
100–400 1 0.02256 1.0
Chlorine, Total
Page 415
Chlorine, Total
20 to 70,000 mg/L chlorine
See
Table 2
1. Select a sample 2. Insert a clean delivery 3. Turn the delivery knob 4. Use a clean graduated
volume and titration tube into the titration to eject a few drops of cylinder or pipet to
cartridge from the Range- cartridge. Attach the titrant. Reset the counter measure the sample
specific information—20 to cartridge to the titrator. to zero and wipe the tip. volume from the Range-
70,000 mg/L table. specific information—20 to
70,000 mg/L table in a
125 mL Erlenmeyer flask.
5. Dilute to 6. Add the contents of 7. Add the contents of 8. Place the delivery tube
approximately 50 mL with one Dissolved Oxygen 3 one Potassium Iodide into the solution and swirl
deionized water. Powder Pillow. Swirl to Powder Pillow. Swirl to the flask. Turn the knob on
mix. mix. the titrator to add titrant to
Normally, the addition of the solution. Continue to
the powder pillow will swirl the flask and add
lower the pH to 4 or less. If titrant until the solution is a
the sample is large or pale yellow.
highly alkaline, make sure
that the solution is pH 4 or
less before proceeding.
Chlorine, Total
Page 416
Chlorine, Total
20 to 70,000 mg/L chlorine (continued)
9. Add one full dropper of 10. Continue the titration 11. Use the multiplier in
Starch Indicator Solution. until the solution changes the Range-specific
Swirl to mix. from dark blue to information—20 to 70,000
A dark blue color will colorless. mg/L table to calculate the
develop. Write down the number of concentration:
digits displayed on the digits x multiplier =
counter. mg/L Total Chlorine (Cl2)
Example: 10 mL of sample
was titrated with the
0.113 N titration cartridge
and 250 digits were used
to reach the endpoint. The
= 125 mg/L chlorine
Table 126 Range-specific information—20 to 70,000 mg/L

Range (mg/L as Cl2) Sample volume (mL) Titration cartridge (N) Multiplier
20–80 25 0.113 0.2

50–200 10 0.113 0.5
100–400 5 0.113 1.0
250–1000 2 0.113 2.5
500–2000 1 0.113 5
2000–9000 (0.2–0.9%) 4 2.00 22.2
5000–18,000 (0.5–1.8%) 2 2.00 44.3
10,000–35,000 (1.0–3.5%) 1 2.00 88.7
20,000–70,000 (2.0–7.0%) 0.5 2.00 177

• Analyze samples for chlorine immediately after collection.
no headspace (air) above the sample.
Chlorine, Total
Page 417
Chlorine, Total
Accuracy check
Use the standard additions method to find if the sample has an interference and to confirm
• Chlorine Standard Voluette® Ampule, 50–75 mg/L Cl2 (the exact concentration will be shown
on a certificate enclosed with the ampule)
• Ampule breaker
samples of the same volume as used in the procedure. Swirl to mix.
standard added.
4. Each 0.2 mL of standard that was added will use approximately 10 digits to reach the
endpoint. To find the exact number of digits that should be used for each 0.2-mL addition,
multiply the exact concentration by the spike volume.
(Example: 50 mg/L x 0.2 mL = 10 digits)
If more or less titrant was used, the problem can be due to user technique, an interference or
a problem with reagents or apparatus.

samples of the same volume as used in the procedure. Swirl to mix.
standard added.
4. Each 1.0 mL of standard that was added will use approximately 10–15 digits to reach the
endpoint. To find the exact number of digits that should be used for each 1.0-mL addition,
multiply the exact concentration by the spike volume and divide by 5.
(Example: 50 mg/L x 0.2 mL / 5 = 10 digits)
Summary of method
Total chlorine concentration equals the concentration of the free and the combined forms of
chlorine. Free chlorine reacts readily with ammonia to form combined chlorine such as
monochloramines. When potassium iodide is added to a sample containing chlorine at a pH less
than 8, free iodine is liberated in direct proportion to the amount of total chlorine present. The
iodine is then titrated with sodium thiosulfate.
Chlorine, Total
Page 418
Chlorine, Total

Required reagents
1–400 mg/L range:
Acetate Buffer Solution, pH 4 2 mL 100 mL MDB 1490932
Sodium Thiosulfate Titration Cartridge, 0.02256 N varies each 2409101
20–2000 mg/L range—Reagent set (approximately 100 tests): 2272500
(1) Dissolved Oxygen 3 Powder Pillows 1 pillow 100/pkg 98799
(1) Potassium Iodide Powder Pillows 1 pillow 100/pkg 107799
(1) Sodium Thiosulfate Titration Cartridge, 0.113 N varies each 2267301
(1) Starch Indicator Solution 1 mL 100 mL MDB 34932
2000–70,000 mg/L range—Reagent set (approximately 100 tests): 2444800
(1) Dissolved Oxygen 3 Powder Pillows 1 pillow 100/pkg 98799
(1) Starch Indicator Solution 1 mL 100 mL MDB 34932
Required apparatus
Chlorine Standard Voluette® Ampule, 50–75 mg/L, 10-mL 16/pkg 1426810

Voluette Breaker each 2196800
Chlorine, Total
Page 419
Chlorine, Total

PourRite breaker each 2484600

Chromate, DT, 8211
Chromate DOC316.53.01174
Titration Method using Sodium Thiosulfate Method 8211

20 to > 400 mg/L as CrO42– Digital Titrator
Scope and Application: Closed system cooling towers.
Test preparation
mg/L chromium = mg/L chromate x 0.448
mg/L sodium chromate = mg/L chromate x 1.4
Potassium Iodide Powder Pillow 1 pillow
Dissolved Oxygen 3 Reagent Powder Pillow 1 pillow
Sodium Thiosulfate titration cartridge (see Range-specific information) 1 cartridge
Starch Indicator Solution 1 mL
Digital titrator 1
Chromate
See
Table 1
Chromate
Page 421
Chromate
Chromate
5. Transfer the sample 6. Add the contents of 7. Add the contents of 8. Wait 3 minutes.
into a clean, 125-mL one Potassium Iodide one Dissolved Oxygen 3 Do not wait more than
Erlenmeyer flask. If the Powder Pillow. Swirl to Powder Pillow. Swirl to 10 minutes.
sample volume is less mix. mix.
than 50 mL, dilute to
approximately 50 mL with
deionized water.
9. Place the delivery tube 10. Add one full dropper of 11. Continue the titration 12. Use the multiplier in
into the solution and swirl Starch Indicator Solution. until the solution changes the Range-specific
the flask. Turn the knob on Swirl to mix. from dark blue to information table to
the titrator to add titrant to A dark blue color will colorless. calculate the
the solution. Continue to develop. Write down the number of concentration:
swirl the flask and add digits displayed on the digits x multiplier =
titrant until the solution is a counter. mg/L chromate (CrO42–)
pale yellow.
was titrated and 250 digits
were used to reach the
endpoint. The
= 50 mg/L CrO42–

Range (mg/L as CrO42–) Sample volume (mL) Titration cartridge (N Na2S2O3) Multiplier
20–80 50 0.2068 0.2

50–200 20 0.2068 0.5
100–400 10 0.2068 1.0
> 400 5 0.2068 2.0
Chromate
Page 422
Chromate
Interferences
Copper Interfere to give high results. The effects of iron and copper can be masked by adding a
Magnesium CDTA Powder Pillow, followed by two 1.0-gram measuring spoons of Sodium
Iron, ferric (Fe3+) Acetate to the sample in step 7.
Substances capable of oxidizing iodide to iodine under acidic conditions (such as ferric iron
Other oxidants
and copper) will interfere to give high results.

• Collect samples in an acid-washed plastic or glass bottles.
• If sample can not be analyzed immediately, add 1 mL of concentrated sulfuric acid and swirl to
mix.
Accuracy check
Use the standard additions method to determine whether the sample has an interference and to
confirm the analytical technique.
• Hexavalent Chromium Standard Solution, 1000-mg/L Cr6+
5. Use the TenSette Pipet to add 0.1 mL, 0.2 mL and 0.3 mL of the standard to three samples.
Use the same sample volume that was used for the analysis. Swirl to mix.
6. Follow the test procedure and titrate the spiked samples to the end point. Write down the
amount of titrant that was used to reach the end point.
7. Each 0.1 mL of standard that was added will use approximately 22 digits of the titration
cartridge to reach the endpoint.

Complete the following test to make sure that the reagents and user technique are accurate.
• Hexavalent Chromium Standard Solution, 1000-mg/L Cr6+
• 100-mL volumetric flask, Class A
1. Use a pipet to add 3.0 mL of the standard solution, 1000-mg/L as Cr6+, to a volumetric flask.
Dilute to 100 mL with deionized water and mix fully. The diluted standard is equivalent to
67 mg/L chromate.
2. Use 20 mL or 50 mL of the standard as the sample volume and follow the test procedure.
3. Titrate the standard to the end point and calculate the result.
Chromate
Page 423
Chromate
Summary of method
Chromate in the sample reacts with iodide under acidic conditions to form iodine as triiodide. The
addition of starch indicator produces a blue color complex with the iodine. This complex is titrated
with sodium thiosulfate to a colorless end point. The volume of titrant used is proportional to the
chromate concentration.

Required reagents
Chromate Reagent Set (approximately 100 tests): 2272400
(1) Dissolved Oxygen 3 Reagent Powder Pillows 1 pillow 100/pkg 98799
(1) Starch Indicator Solution 1 mL each 34932
Required apparatus
Chromium, Hexavalent, Standard Solution, 1000-mg/L as Cr6+ 100 mL 1466442
Chromate
Page 424
Chromate

Magnesium CDTA Powder Pillows 100/pkg 1408099
Sodium Acetate, trihydrate, ACS 454 g 17801H
Bottle, sampling 250 mL 2087076
Sulfuric Acid, ACS 500 mL 97949
Measuring spoon 1g 51000
Chromium standard, 50 mg/L 100 mL 81042H
Volumetric flask 100 mL 1457442
Volumetric Pipet 3 mL 1457442
Safety bulb each 1465100
Tensette Pipet 1–10 mL 1970010
Clippers each 96800
Chromate
Page 425
Chromium, Hexavalent, 8023
Chromium, Hexavalent DOC316.53.01033
USEPA1 1,5-Diphenylcarbohydrazide
Method 8023
Method2
(0.010 to 0.700 mg/L Cr6+) Powder Pillows or AccuVac® Ampuls
Scope and Application: For water and wastewater; USEPA accepted for reporting for wastewater analysis.3
1 Accepted USEPA and Standard Method 3500 Cr B.
3 Procedure is equivalent to USGS method 1-1230-85 for wastewater.
Test preparation


Instrument
At high chromium levels, a precipitate will form. Sample dilution may be necessary.
The final samples are highly acidic. Refer to reagent MSDS sheets for disposal information.
Powder Pillow Test:
ChromaVer® 3 Chromium Reagent Powder Pillows 1
Sample cells, 10-mL 2
AccuVac Test:
ChromaVer® 3 AccuVac® Ampuls 1
Beaker, 50-mL 1
Chromium, Hexavalent
Page 427
Sample cell, 10-mL round, with cap 1
1,5-Diphenylcarbohydrazide for powder pillows
Stored Programs
90 Chromium, Hex.
Start
required (Instrument- ChromaVer® 3 Reagent A five-minute reaction
specific information). Powder Pillow to the period will begin.
sample cell. Swirl to mix.
for orientation. A purple color will form if
hexavalent chromium is
present.
5. Blank Preparation: 6. When the timer 7. Insert the prepared

Fill a sample cell with 10 expires, insert the blank sample into the cell holder.
mL of sample. into the cell holder. READ the results in
ZERO the instrument. The mg/L Cr6+.
display will show:
0.000 mg/L Cr6+
Page 428
1,5-Diphenylcarbohydrazide for AccuVac® Ampuls
Stored Programs
95 Chromium, Hex. AV
Start
Insert an adapter if Fill a round sample cell Fill a ChromaVer 3 Ampul several times
required (Instrument- with 10 mL of sample. Reagent AccuVac® Ampul to mix.
specific information). with sample from the
beaker. Keep the tip
Refer to the user manual immersed while the Ampul
for orientation. fills completely.
5. Start the instrument 6. When the timer 7. Wipe the Ampul and
timer. expires, wipe the blank insert it into the cell holder.
A five-minute reaction and insert it into the cell READ the results in
period will begin. holder. mg/L Cr6+.
ZERO the instrument.
0.000 mg/L Cr6+
Interferences
Iron May interfere above 1 mg/L
Mercurous & Mercuric Ions Interfere slightly
Highly buffered samples or extreme sample pH may exceed the buffering capacity of the
pH
reagents and require sample pretreatment.
Vanadium May interfere above 1 mg/L. Allow 10 minutes for the reaction period before reading.
For turbid samples, treat the blank with the contents of one Acid Reagent Powder Pillow1.
Turbidity This will ensure that any turbidity dissolved by the acid in the ChromaVer 3 Chromium
Reagent will also be dissolved in the blank.
Page 429

Collect samples in a cleaned glass or plastic container. Store at 4 °C (39 °F) up to 24 hours.
Samples must be analyzed within 24 hours.
Accuracy check
• Chromium Voluette® Ampule Standard, 12.5 mg/L Cr6+
• TenSette Pipet and Pipet Tips
• Ampule Breaker
or edited. After values are accepted, the unspiked sample reading will appear in the top row.
See the user manual for more information.
4. Open a Chromium Voluette® Ampule Standard, 12.5 mg/L Cr6+.
5. For analysis using powder pillows, use the TenSette® Pipet to add 0.1 mL, 0.2 mL and 0.3 mL
of standard, respectively to three 25-mL samples and mix thoroughly. Transfer 10 mL of each
solution into a 10-mL sample cell and analyze as described above.
Note: For AccuVac Ampuls, fill three mixing cylinders* with 50-mL of sample and spike with 0.2 mL, 0.4 mL
and 0.6 mL of standard. Transfer 40 mL from each of the three mixing cylinders to three 50-mL beakers.
Analyze each standard addition sample as described in the procedure above.
6. Accept each standard additions reading. Each addition should reflect approximately 100%
recovery.

Prepare a 0.50-mg/L Cr6+ standard solution daily, as follows:
1. Using a 5.00 mL pipet transfer 5.00 mL of Hexavalent Chromium Standard Solution, 50 mg/L,
into a Class A 500-mL volumetric flask.
2. Dilute to the mark with deionized water. Perform the test procedure as described above.
3. To adjust the calibration curve using the reading obtained with the 0.50-mg/L Standard
Solution, select Options>More>Standard Adjust from the instrument menu.
Note: Refer to the instrument user manual for specific software navigation instructions.
Page 430
Method performance
90 0.500 mg/L Cr6+ 0.497–0.503 mg/L Cr6+ 0.005 mg/L Cr6+
95 0.500 mg/L Cr6+ 0.496–0.504 mg/L Cr6+ 0.006 mg/L Cr6+
Summary of method
Hexavalent chromium is determined by the 1,5-Diphenylcarbohydrazide method using a single dry
powder formulation called ChromaVer 3 Chromium Reagent. This reagent contains an acidic
buffer combined with 1,5-Diphenylcarbohydrazide, which reacts to give a purple color when
hexavalent chromium is present. Test results are measured at 540 nm.
Page 431
Required reagents
ChromaVer® 3 Chromium Reagent Powder Pillows 1 100/pkg 1271099

OR
ChromaVer® 3 AccuVac® Ampuls 1 25/pkg 2505025
Deionized Water varies 4L 27256
Required apparatus
Stopper for 18 mm Tube 1 6/pkg 173106
Chromium, Hexavalent Standard Solution, 10-mL Voluette® Ampules, 12.5-mg/L Cr6+ 16/pkg 1425610
Chromium, Hexavalent Standard Solution, 50.0-mg/L Cr6+ 100 mL 81042H

Acid Reagent Powder Pillow 100/pkg 212699
Pipet, Volumetric 5.00 mL each 1451537
Pipet FIller, safety bulb each 1465100

Chromium, Total, 8024
Chromium, Total DOC316.53.01034
Alkaline Hypobromite Oxidation Method1, 2 Method 8024

0.01 to 0.70 mg/L Cr Powder Pillows
2 This procedure is equivalent to Standard Method 3500-Cr D for wastewater.
Test preparation


Instrument Sample cell Orientation
DR 6000 2495402 (2) and 2401906 (1) Fill line faces right
DR 5000 2495402 (2) and 2401906 (1) Fill line faces user
DR 3900 2495402 (2) and 2401906 (1) Fill line faces user
DR 3800, DR 2800, DR 2700 2495402 (2) and 2401906 (1) Fill line faces right
deionized water instead of the sample. Subtract the reagent blank value from the final results or perform a reagent
blank adjust.
Prepare a boiling water bath for step 4. Use finger cots to handle hot sample cells.
Acid Reagent Powder Pillows 1
ChromaVer® 3 Chromium Reagent Powder Pillows 1
Chromium 1 Reagent Powder Pillows 1
Chromium 2 Reagent Powder Pillows 1
Hot Plate 1
Water Bath and Rack 1
Finger Cots 1 pair
Sample Cells (Instrument-specific information) varies
Chromium, Total
Page 433
Chromium, Total
Alkaline Hypobromite Oxidation method for powder pillows
Stored Programs
100 Chromium, Total
Start
1. Select the test. 2. Fill a 25-mL sample 3. Prepared Sample: 4. Remove the cap.
Insert an adapter if cell with 25 mL of sample. Add the contents of one Insert the prepared
required (Instrument- Chromium 1 Reagent sample into a boiling water
specific information). Powder Pillow. bath.
Refer to the user manual Swirl to mix.
for orientation.
5. Start the instrument 6. When the timer 7. Remove cap and add 8. Add the contents of
timer. expires, remove the the contents of one one Acid Reagent Powder
A five-minute reaction prepared sample. Cap the Chromium 2 Reagent Pillow. Swirl to mix.
period will begin. cell and use running water Powder Pillow. Cap and
to cool the cell to 25 °C. invert to mix.
9. Add the contents of 10. Start the instrument 11. While the sample is 12. Blank Preparation:
one ChromaVer 3 timer. reacting, pour 10 mL from When the timer expires, fill
Chromium Reagent A five-minute reaction the mixing bottle into a another sample cell with
Powder Pillow. Swirl to period will begin. second sample cell 10 mL of original sample.
mix. (Instrument-specific
information). This is the
prepared sample.
Chromium, Total
Page 434
Chromium, Total
Alkaline Hypobromite Oxidation method for powder pillows (continued)
Zero Read
13. Wipe the blank and 14. ZERO the instrument. 15. Wipe the prepared 16. READ the results in
insert it into the cell holder. The display will show: sample and insert it into mg/L Cr.
the cell holder.
0.00 mg/L Cr
Interferences
extreme sample pH
May inhibit complete oxidation of trivalent chromium. If high levels of organic material are
Organic material present, digestion may be required. Perform the analysis as described in this procedure on the
digested sample.
For turbid samples, treat a 25-mL blank and the sample the same during steps 3–8. Use this
Turbidity
solution as the blank.

• Collect samples in acid-washed glass or plastic containers.
• To preserve samples, adjust the pH to 2 or less with nitric acid. This requires approximately 2
mL per liter of the acid.
• Store preserved samples at room temperature up to six months.
• Adjust the pH to about 4 with 5.0 N Sodium Hydroxide before analysis.
• Correct the test result for volume additions.
Accuracy check
• Trivalent Chromium Voluette® Ampule Standard, 50-mg/L as Cr3+.
• Ampule Breaker
• Mixing cylinders
5. Prepare a 12.5 mg/L standard by pipetting 5.00 mL of Trivalent Chromium Standard Solution,
50 mg/L as Cr3+ into a mixing cylinder. Pipet 15.00 mL of demineralized water into the
cylinder. Stopper the cylinder and mix thoroughly.
Chromium, Total
Page 435
Chromium, Total
TenSette® Pipet to add 0.1 mL, 0.2 mL and 0.3 mL of standard, respectively, to each sample
and mix thoroughly.
10. Analyze each sample spike as described in the procedure above, starting with the smallest
sample spike. Accept each standard additions reading. Each addition should reflect

Prepare a 0.50-mg/L trivalent chromium standard as follows:
1. Dilute 5.00 mL of Trivalent Chromium Standard Solution, 50-mg/L as Cr3+, to 500 mL with
deionized water. Prepare this solution daily.
Method performance
90 0.500 mg/L Cr 0.47–0.53 mg/L Cr 0.005 mg/L Cr
Summary of method
Trivalent chromium in the sample is oxidized to the hexavalent form by hypobromite ion under
alkaline conditions. The sample is acidified. The total chromium content is determined by the 1,5-
Diphenylcarbohydrazide method. Determine trivalent chromium by subtracting the results of a
separate hexavalent chromium test from the results of the total chromium test. Test results are
measured at 540 nm.
Chromium, Total
Page 436
Chromium, Total
Required reagents
Total Chromium Reagent Set (100 tests), includes: — — 2242500
Acid Reagent Powder Pillows 1 100/pkg 212699
ChromaVer® 3 Chromium Reagent Powder Pillows 1 100/pkg 1206699
Chromium 1 Reagent Powder Pillows 1 100/pkg 204399
Chromium 2 Reagent Powder Pillows 1 100/pkg 204499
Required apparatus
Hot plate, 3½-inch diameter, 120 VAC, 50/60 Hz 1 each 1206701
OR
Hot Plate, stirrer, 220 – 240 VAC 1 each 2881602
Water Bath and Rack 1 each 195555
Chromium, Trivalent, Standard Solution, 50-mg/L Cr3+ 100 mL 1415142

Finger Cots 2/pkg 1464702
Pipette, volumetric, Class A, 5.00 mL each 1451537
Tensette Pipet 0.1–1.0 mL each 1970001
Pipet Tips, Tensette 50/pkg 2185696
Pipet, Volumetric Class A, 15 mL each 1451539
Cylinder, Mixing, 25 mL each 189640
Chromium, Total
Page 437
Chromium, Total

Cobalt, 8078
Cobalt DOC316.53.01036
1-(2-Pyridylazo)-2-Naphthol (PAN) Method1 Method 8078

0.01 to 2.00 mg/L Powder Pillows
Scope and Application: For water and wastewater; digestion is required for determining total recoverable cobalt;
if EDTA is present, use the vigorous digestion.
1 Adapted from Watanbe, H., Talanta, 21 295 (1974).
Test preparation


adjust.
If the sample is less than 10 °C (50 °F), warm to room temperature prior to analysis.
Nickel can be determined with the same sample prepared with this method. Use program Number 340. A reagent blank is
necessary for the nickel procedure.
The Pour-Thru Cell can be used with 25-mL reagents only.
Total recoverable cobalt requires a prior digestion. See the Digestion section in the Water Analysis Guide.
For samples containing iron (Fe3+), all of the powder must dissolve before mixing the sample in step 6.
Cobalt
Page 439
Cobalt
EDTA reagent powder pillows 2
Phthalate-Phosphate reagent powder pillows 2
PAN indicator solution 1 mL
Cylinder, graduated mixing, 25-mL 2
Sample cells (Instrument-specific information) 2
Stopper for sample cells 2
PAN method for powder pillows
Stored Programs
110 Cobalt
Start
1. Select the test. 2. Prepared Sample: 3. Blank Preparation: 4. Add the contents of
Insert an adapter if Fill a sample cell to the Fill a second sample cell one Phthalate-Phosphate
required (Instrument- 10-mL mark with room- to the 10-mL mark with Reagent powder pillow to
specific information). temperature sample. room-temperature each cell. Swirl to
deionized water. completely dissolve.
for orientation.
Cobalt
Page 440
Cobalt
PAN method for powder pillows (continued)
5. Use the plastic 6. Use a stopper to close 7. Start the instrument 8. When the timer
dropper provided to add each cell. timer. A three-minute expires, add the contents
0.5 mL of 0.3% PAN Invert several times to mix. reaction time will begin. of one EDTA Reagent
Indicator Solution to each During the reaction period, powder pillow to each cell.
cell. the sample solution color Use a stopper to close
may vary from green to each cell.
dark red, depending on Shake each cell to
the chemical makeup of dissolve.
the sample. The deionized
water blank should be
yellow.
Zero Read
insert it into the cell holder. The display will show: sample and insert it into mg/L Co.
the cell holder.
0.00 mg/L Co
Interferences

Al3+ 32 mg/L
Ca2+ 1000 mg/L as CaCO3
Cd2+ 20 mg/L
Cl– 8000 mg/L
Cr3+ 20 mg/L
Cr6+ 40 mg/L
Cu2+ 15 mg/L
Cobalt
Page 441
Cobalt

F– 20 mg/L
Fe2+ Interferes directly and must not be present
Fe3+ 10 mg/L
K+ 500 mg/L
Mg2+ 400 mg/L
Mn2+ 25 mg/L
Mo6+ 60 mg/L
Na+ 5000 mg/L
Pb2+ 20 mg/L
Zn2+ 30 mg/L
May exceed the buffering capacity of the reagents and
require sample pretreatment.

• Collect samples in acid-washed plastic bottles.
• Adjust the sample pH to 2 or less with nitric acid* (about 5 mL per liter).
• Preserved samples can be stored up to six months at room temperature.
• Adjust the sample pH between 3 and 8 with 5.0 N Sodium Hydroxide Standard Solution* just
before analysis. Do not exceed pH 8 as this may cause some loss of cobalt as a precipitate.
• Correct test results for volume additions.
Accuracy check
Prepare a 1.00 mg/L cobalt standard solution as follows:

1. Dilute 10.0 mL of a 10-mg/L working stock solution to 100 mL in a volumetric flask. Prepare
the 10-mg/L working stock solution daily by diluting 10.00 mL of Cobalt Standard Solution,
1000-mg/L as Co, to 1000 mL with deionized water.
Method performance
110 1.00 mg/L Co 0.99–1.01 mg/L Co 0.01 mg/L Co
Cobalt
Page 442
Cobalt
Summary of method
After buffering the sample and masking any Fe3+ with pyrophosphate, the cobalt is reacted with 1-
(2-Pyridylazo)-2-Naphthol indicator. The indicator forms complexes with most metals present.
After color development, EDTA is added to destroy all metal-PAN complexes except nickel and
cobalt, which can both be determined using the same sample.
Required reagents
Cobalt Reagent Set, 10-mL (100 tests), includes: — — 2651600
(2) EDTA Reagent Powder Pillows 2 100/pkg 700599
(2) Phthalate-Phosphate Reagent Powder Pillows 2 100/pkg 2615199
(1) PAN Indicator Solution, 0.3% 1 mL 100 mL 2150232
Required apparatus
Stopper, rubber 2 6/pkg 173106
Cobalt Standard Solution, 1000-mg/L Co 100 mL 2150342

Nitric Acid, 1:1 500 mL 254049
Pipet Filter, Safety bulb each 1465100
pH paper 100/pkg 2601300
Cobalt
Page 443
Color, True and Apparent, 8025
Color, True and Apparent DOC316.53.01037
Platinum-Cobalt Standard Method1, 2, 3 Method 8025

15 to 500 units
Scope and Application: For water, wastewater and seawater; equivalent to NCASI method 253 and NCASI
Method Color 71.01 for pulp and paper effluent using 465 nm (requires pH adjustment).
1 Adapted from Standard Methods for the Examination of Water and Wastewater and National Council for Air and Stream Improvement
(NCASI) Methods Manual.
2 Adapted from Wat. Res. Vol. 30, No. 11, pp. 2771–2775, 1996.
3 NCASI Method 253 approved at 40 CFR part 136.
Test preparation


The NCASI procedure requires pH adjustment. Adjust the pH to 7.6 with 1.0 N HCl or 1.0 N NaOH. When adjusting the pH, if
the overall volume change is greater than 1%, start over and use a stronger acid or base. Use program 125 when
performing the NCASI procedure.
One powder pillow of pH 8 Buffer (sodium phosphate/potassium phosphate) may be added to 50 ml of sample prior to final
pH adjustment to reduce sample dilution effects. Mix thoroughly to dissolve before making final pH adjustments.
To test for apparent color, omit steps 3–5 and step 7. Use unfiltered deionized water in step 6 and unfiltered sample in step 8.
For low level samples, use of a Pour-Thru Cell is recommended.
Buffer, pH 8.0 (Program 125) 1
Hydrochloric Acid Solution, 1.0 N (Program 125) varies
Sodium Hydroxide, 1.00 N (Program 125) varies
FIlter Apparatus: membrane filter, filter holder, filter flask and aspirator 1
Sample Cells (Instrument-specific information) 2
Color, True and Apparent

Page 445
Stopper, rubber, one hole, No. 7 1
Tubing, rubber 1
Plantinum-Cobalt method
Stored Programs
120 Color, 455 nm

OR
125 Color, 465 nm
Start
1. Select the test. 2. Collect 200 mL of 3. Assemble the filter 4. Filter about 50 mL of
NCASI: Use Program 125 sample in a 400-mL apparatus (0.45 micron deionized water to rinse
for the NCASI test. beaker. membrane filter, filter the filter. Discard the rinse
NCASI: Adjust the pH as holder, filter flask and water.
Insert an adapter if aspirator).
required (Instrument- described in Test
specific information). Preparation. NCASI: The NCASI test
prescribes a 0.8-micron
Refer to the user manual filter. A 1.0 micron prefilter
for orientation. can be first used for
difficult to filter samples.
5. Filter another 50 mL of 6. Blank Preparation: 7. Filter about 50 mL of 8. Prepared Sample: Fill

deionized water through Fill a sample cell with sample through the filter. a second sample cell with
the filter. 10-mL of filtered deionized 10 mL of filtered sample.
water from step 5.
Discard the excess water
in the flask.

Page 446
Plantinum-Cobalt method (continued)
Zero Read
insert it into the cell holder. The display will show: sample and insert it into mg/L Pt-Co.
the cell holder.
0 units Pt-Co

• Collect samples in clean plastic or glass bottles. Most reliable results are obtained when
samples are analyzed as soon as possible after collection. If prompt analysis is impossible, fill
bottles completely and cap tightly.
• Avoid excessive agitation or prolonged contact with air.
• Samples can be stored for 24 hours by cooling to 4 °C (39 °F).
• Warm to room temperature before analysis.
Accuracy check
Prepare a 250 platinum-cobalt units standard as follows:

1. Using Class A glassware, pipet 50.00 mL of a 500 Platinum-Cobalt Units Standard Solution
into a 100-mL volumetric flask. Dilute to the 100 mL mark with deionized water.
2. To adjust the calibration curve using the reading obtained with the 250 unit Standard Solution,
Select Options>More>Standard Adjust from the instrument menu.
Method performance
120 250 units Pt-Co 245–255 units Pt-Co 16 units Pt-Co
125 250 units Pt-Co 245–255 units Pt-Co 16 units Pt-Co

Page 447
Summary of method
Color may be expressed as “apparent” or “true” color. The apparent color includes that from
dissolved materials plus that from suspended matter. By filtering or centrifuging out the suspended
materials, the true color can be determined. The procedure describes true color analysis. If
apparent color is desired, it can be determined by measuring an unfiltered water sample. The
same stored program is used for both forms of color.
The stored program is calibrated in color units based on the APHA-recommended standard of 1
color unit being equal to 1 mg/L platinum as chloroplatinate ion. Test results for Programs 120 and
125 are measured at 455 and 465 nm, respectively.
Required reagents
Buffer, pH 8.0 1 15/pK 1407995
Hydrochloric Acid Solution, 1.0 N varies 1L 2321353
Sodium Hydroxide, 1.00 N varies 1L 104553
Required apparatus
Aspirator, Nalgene vacuum pump 1 each 213100
Filter, membrane, 47-mm, 0.8-microns, Program 125 1 100/pkg 2640800
Filter, membrane, 47-mm, 0.45-microns, Program 120 1 100/pkg 1353000
Flask, filtering, 500-mL 1 each 54649
Stopper, rubber, one hole, No. 7 1 6/pkg 211907
Tubing, rubber, 7.9 x 2.4 mm varies 12 ft 56019
Filter Holder, 47 mm, 300 mL graduated 1 each 1352900
Beaker, 400 mL 1 each 50048

Color Standard Solution, 500 platinum-cobalt units 1L 141453
Color Standard Solution, 500 platinum-cobalt units, 10-mL Voluette® Ampules 16/pkg 141410
Filter, Glass Microfiber, 47-mm, 1.0 micron 100/pk 2551400
Pipet, volumetric, Class A, 50.00 mL each 1451541

Color, ADMI, 10048
Color, ADMI DOC316.53.01122
ADMI Weighted Ordinate Method1 Method 10048

(5 to 250 units Pt-Co)
Scope and Application: For water and wastewater where color characteristics are significantly different from
platinum-cobalt standards, or for water and wastewater with color characteristics similar in hue to the standards.
Turbid samples must be filtered prior to analysis.
1 Adapted from Allen, et. al., 1973. Determination of color of water and wastewater by means of ADMI Color Values. Proc. 28th Ind. Waste
Conf., Purdue Univ., Eng. Ext. Ser. No. 142:661
Test preparation
The ADMI color method is available on the DR 5000 and DR 6000 spectrophotometer. One of
three sample cell sizes can be used (refer to Sample cell options). Select the test that corresponds
to the sample cells used (refer to step 1 of the procedure).
Table 136 Sample cell options
Program Sample cell Cell orientation
97 2495402 Fill line faces user
96 2095100 Clear side toward user
98 2629250 Clear side toward user
If the sample cannot be analyzed immediately, see Sample collection, preservation and storage.
Make pH adjustments with a minimum volume of acid or base. Make major adjustments with strong acid or base, then fine-
tune with the 0.1 N solution.
The sample cells shown in the procedure are a generic representation. Each of the three stored programs uses a cell of a
different shape.
Sodium Hydroxide or Sulfuric Acid Solution, 10 N varies
Sodium Hydroxide or Sulfuric Acid Solution, 0.100 N varies
Beaker, 250 mL polypropylene 2
Cylinder, graduated, 100 mL polypropylene 1
Filter Apparatus: membrane filter, filter holder, filter flasks (2), tubing, stopper and aspirator 1
pH meter with electrode 1
Sample Cells, 1-inch square, 1 cm (10 mm) or 5 cm (50 mm) 2
Color, ADMI
Page 449
Color, ADMI
ADMI weighted ordinate method
Stored Programs
97 Color ADMI 1 inch

96 Color ADMI 10 mm
98 Color ADMI 50 mm
Start
1. Select the test. 2. If the sample is not 3. Assemble the filtering 4. Rinse the filter by
Insert the multi-cell turbid, omit steps 3–6. apparatus (0.45 micron pouring about 50 mL of the
adapter. Refer to Sample Pour two 100-mL aliquots membrane filter, filter original sample aliquot
cell options for cell of sample into separate holder, filter flask, and through the filter. Discard
orientation. beakers. Adjust the pH of aspirator). the rinse water.
one of the aliquots to 7.6;
leave the other aliquot
as is.
Use 10 N sodium
hydroxide or concentrated
sulfuric acid to adjust the
pH. Use 0.1 N acid or base
near the end point.
5. Pour another 50 mL of 6. Repeat steps 3–5 for 7. Prepared Sample: 8. Blank Preparation:
the original sample aliquot the pH adjusted sample Fill a sample cell with the Fill a second sample cell
through the filter. and label it “pH adjusted”. pH-adjusted sample. with deionized water.
Label the flask “original”. Discard the excess.
Discard the excess water
in the flask.
Color, ADMI
Page 450
Color, ADMI
ADMI weighted ordinate method (continued)
Zero Read
9. Wipe the blank and 10. ZERO the instrument. 11. Wipe the prepared 12. READ the results.
insert it into the cell holder. The instrument will read sample (pH adjusted) and The instrument will read
Close the light shield. the percent transmittance insert it into the cell holder. the percent transmittance
(%T) at 10-nm intervals Close the light shield. (%T) at 10-nm intervals
from 700 nm to 400 nm. from 700 nm to 400 nm.
When finished, the
instrument will display the
ADMI color value of the
pH-adjusted sample.
Repeat steps 7–12 for the
original sample.
For USEPA reporting,
report both results.
Interferences
Turbidity interferes directly and must be removed using filtration.

samples are analyzed as soon as possible after collection.
• If prompt analysis is impossible, fill bottles completely and cap tightly.
• Avoid excessive agitation or prolonged contact with air.
• Samples can be stored for 24 hours by cooling to 4 °C (39 °F).
Color, ADMI
Page 451
Color, ADMI
Accuracy check
• Color Standard Solution, 500 platinum-cobalt units
• Deionized water
• 20-mL Class A volumetric pipet and pipet bulb
1. Prepare a 100 platinum-cobalt units standard solution as follows:
a. Pipet 20 mL of Color standard, 500 platinum-cobalt units, into a 100-mL volumetric flask.
b. Dilute to the mark with deionized water. Mix well.
2. Use this solution in place of the sample. Follow the ADMI weighted ordinate method test
procedure. Omit filtration steps 3–6.
3. To adjust the calibration curve using the reading obtained with the standard solution, press
OPTIONS>MORE>STANDARD ADJUST.
Summary of method
Three properties describe color: hue, chroma and value. Hue is “color”, whether it be blue, red,
green, yellow, etc. Chroma is color intensity (bright or dull). Value is the amount of color (light or
dark). This method measures only the amount of color, or color value. It is independent of the hue
and chroma.
This method determines the color value in a sample. Transmittance is measured from 400 to 700
nm and converted to a set of abstract numbers. These numbers describe the color as seen by an
average human eye. They are converted to a single number that indicates the color value. This
number is expressed on a scale used by the American Dye Manufacturers Institute (ADMI) to
measure color value. The ADMI has adopted the Platinum-Cobalt standard of the American Public
Health Association (APHA) as the standard for color value. Although this standard is yellow, the
ADMI method works for all hues. Three calibrations are stored in the instrument. Each calibration
corresponds to one of the three sample cell pathlengths.
Color, ADMI
Page 452
Color, ADMI
Required reagents
Sodium Hydroxide Solution, 10 N varies 500 mL 2545049
Sodium Hydroxide Standard Solution, 0.100 N varies 1000 mL 19153
Sulfuric Acid, 10 N varies 1000 mL 93153
Sulfuric Acid Standard Solution, 0.100 N varies 100 mL MDB 20232H
Water, deionized 10 mL 4 liters 27256
Required apparatus
Beaker, 250-mL polypropylene 2 each 108046
Cylinder, graduated, 100-mL, polypropylene 1 each 108142
pH meter, with electrode 1 each HQ11d
Sample cells, 10 mL square, matched pair 2 2/pkg 2495402

Aspirator, Nalgene vacuum pump each 213100
Filter Holder, 47-mm, 300-mL graduated each 1352900
Filter, membrane, 47-mm, 0.45-microns 100/pkg 1353000
Stopper, rubber, one hole, No. 7 6/pkg 211907
Tubing, rubber 12 ft 56019
Color, ADMI
Page 453
Copper, Bicinchoninate, 8506 and 8026
Copper DOC316.53.01039
Method 8506
USEPA1 Bicinchoninate Method2
Method 8026
Scope and Application: For water, wastewater and seawater3; Method 8506 USEPA approved for reporting
wastewater analysis (digestion required).4
1 Approved, USEPA and Standard Method 3500 Cu C or E
2 Adapted from Nakano, S., Yakugaku Zasshi, 82 486-491 (1962) [Chemical Abstracts, 58 3390e (1963)].
3 Pretreatment required; see Interferences (Using Powder Pillows).
4 Federal Register, 45 (105) 36166 (May 29, 1980).
Test preparation


Instrument
Digestion is required for determining total copper.
Adjust the pH of acid-preserved samples to 4–6 with 8 N KOH before analysis.
adjust.
If copper is present, the sample will turn purple when it mixes with the reagent powder.
Accuracy is not affected by undissolved reagent.
Copper
Page 455
Copper
Powder Pillow Test:
CuVer® 1 Copper Reagent powder pillow 1
Sample Cells, powder pillow test (Instrument-specific information) 2
AccuVac Test:
CuVer® 2 Copper Reagent AccuVac® Ampul 1
Beaker, 50-mL 1
Sample Cell, AccuVac test (Instrument-specific information) 1
Bicinchoninate method for powder pillows (Method 8506)
Stored Programs
135 Copper, Bicin.
Start
1. Select the test. 2. Prepared Sample: Fill 3. Add the contents of 4. Start the instrument
Insert an adapter if a cell with 10 mL of one CuVer® 1 Copper timer. A two-minute
required (Instrument- sample. Reagent powder pillow to reaction period will begin.
specific information). the prepared sample cell.
Swirl sample cell to mix.
for orientation. Use a CuVer 2 Copper
Reagent powder pillow for
samples containing high
levels of aluminum, iron
and hardness. A 25-mL
sample cell is required
(Interfering substances
and suggested treatments
for powder pillows).
Copper
Page 456
Copper
Bicinchoninate method for powder pillows (Method 8506) (continued)
Zero
5. Blank Preparation: 6. Insert the blank into 7. ZERO the instrument. 8. Within 30 minutes
When the timer expires, fill the cell holder. The display will show: after the timer expires,
a second sample cell with insert the prepared sample
10 mL of sample. 0.00 mg/L Cu into the cell holder.
READ the results in mg/L
Cu.
Bicinchoninate method for AccuVac® Ampuls (Method 8026)
Stored Programs
140 Copper, Bicin. AV
Start
1. Select the test. 2. Blank Preparation: 3. Prepared Sample: 4. Place the stopper and
Insert an adapter if Fill a round sample cell Collect at least 40 mL of quickly invert the Ampul
required (Instrument- with 10 mL of sample. sample in a 50-mL beaker. several times to mix. Wipe
specific information). Fill a CuVer 2 AccuVac off all liquid and
Ampul with sample from fingerprints with a cloth or
Refer to the user manual soft paper towel.
for orientation. the beaker. Keep the tip
fills completely.
Copper
Page 457
Copper
Bicinchoninate method for AccuVac® Ampuls (Method 8026) (continued)
Zero
5. Start the instrument 6. When the timer 7. ZERO the instrument. 8. Within 30 minutes
timer. expires, wipe the blank The display will show: after the timer expires,
A two-minute reaction and insert it into the cell wipe the Ampul and insert
holder. 0.00 mg/L Cu it into the cell holder.
period will begin.
READ the results in
mg/L Cu.
Interferences
The Interfering substances and suggested treatments for powder pillows table suggests
treatments for powder pillows. The Interfering substances and suggested treatments for AccuVac
Ampuls tables suggests treatments for AccuVac Ampuls. To differentiate free copper from that
complexed to EDTA or other complexing agents, use a 25-mL sample cell and Free Copper
Reagent Powder Pillow instead of the CuVer 1 Powder Pillow in step 3. Add a Hydrosulfite
Reagent Powder Pillow to the same sample and re-read the result. This result will include the total
dissolved copper (free and complexed). Unlike CuVer 1 Reagent, CuVer 2 Reagent Powder
Pillows and AccuVac Ampuls react directly with copper, which is complexed by chelants such as
EDTA.
Table 138 Interfering substances and suggested treatments for powder pillows
If the sample is extremely acidic (pH 2 or less) a precipitate may form. Add 8 N Potassium
Acidity
Hydroxide Standard Solution drop-wise until sample pH is above 4. Continue with step 3.
Follow the powder pillow procedure above, but substitute a CuVer 2 Copper Reagent Powder
Aluminum, Al3+ Pillow for the CuVer 1 Pillow used in step 3. Results obtained will include total dissolved
copper (free and complexed). Requires a 25-mL sample volume.
Prevents full color development. Before adding the CuVer 1 Powder Pillow Reagent, add 0.2
Cyanide, CN– mL of formaldehyde to the 10-mL sample. Wait 4 minutes before taking the reading. Multiply
the test results by 1.02 to correct for sample dilution by the formaldehyde.
Hardness Pillow for the CuVer 1 Pillow used in step 3. Results obtained will include total dissolved
Iron, Fe3+ Pillow for the CuVer 1 Pillow used in step 3. Results obtained will include total dissolved
If a turbidity remains and turns black, silver interference is likely. Add 10 drops of saturated
Silver, Ag+ Potassium Chloride Solution to 75 mL of sample, followed by filtering through a fine or highly
retentive filter. Use the filtered sample in the procedure.
Copper
Page 458
Copper
Table 139 Interfering substances and suggested treatments for AccuVac Ampuls
If the sample is extremely acidic (pH 2 or less) a precipitate may form. Add 8 N Potassium
Acidity
Hydroxide Standard Solution drop-wise until sample pH is above 4. Continue with step 3.
Aluminum, Al3+ Reagents accommodate high levels.
Prevents full color development. Add 0.5 mL of formaldehyde per 25-mL of sample before
Cyanide, CN– using CuVer 2 Reagent AccuVac Ampul. Wait 4 minutes before taking the reading. Multiply the
test results by 1.02 to correct for sample dilution by the formaldehyde.
Hardness Reagents accommodate high levels.
Iron, Fe3+ Reagents accommodate high levels.
If a turbidity remains and turns black, silver interference is likely. Add 10 drops of saturated
Silver, Ag+ Potassium Chloride Solution to 75 mL of sample, followed by filtering through a fine or highly
retentive filter. Use the filtered sample in the procedure.

• Collect samples in acid-cleaned glass or plastic containers.
• Adjust the pH to 2 or less with concentrated nitric acid (about 2 mL per liter).
• Store preserved samples up to six months at room temperature.
• Before analysis, adjust the pH to 4–6 with 8 N Potassium Hydroxide. Do not exceed pH 6, as
copper may precipitate.
• If only dissolved copper is to be determined, filter the sample before acid addition.
Accuracy check
• Copper Standard Solution, 100 mg/L Cu
• Mixing cylinders (3)
• Beakers (3)
1. Prepare a 12.5 mg/L Copper standard by pipetting 5.0 mL of Copper standard solution,
100mg/L into a 50 mL mixing cylinder. Dilute the solution to 40 mL, stopper and mix.
5. Prepare a 0.1 mL sample spike by adding 0.1 mL of standard to the unspiked sample. Press
the timer icon. After the timer expires, read the result.
Press the timer icon. After the timer expires, read the result.
Copper
Page 459
Copper
Press the timer icon. After the timer expires, read the result. Each addition should reflect
Note: For AccuVac Ampuls, fill three mixing cylinders with 50-mL of sample and spike with 0.2 mL, 0.4 mL
and 0.6 mL of Copper Voluette Ampule Standard, 75-mg/L Cu. Transfer 40 mL from each of the three
mixing cylinders to three 50-mL beakers. Analyze each standard addition sample as described in the
procedure above. Accept each standard additions reading by pressing READ. Each addition should

Prepare a 4.00-mg/L Standard as follows:

1. Using Class A glassware, pipet 4.00 mL of Copper Standard Solution, 100-mg/L as Cu, into a
100-mL volumetric flask. Dilute to volume with deionized water, stopper and invert to mix.
Perform the procedure as described above.
Method performance
135 1.00 mg/L Cu 0.97–1.03 mg/L Cu 0.04 mg/L Cu
140 1.00 mg/L Cu 0.97–1.03 mg/L Cu 0.03 mg/L Cu
Summary of method
Copper in the sample reacts with a salt of bicinchoninic acid contained in CuVer 1 or CuVer 2
Copper Reagent to form a purple colored complex in proportion to the copper concentration. Test
Required reagents
CuVer® 1 Copper Reagent Powder Pillows 1 100/pkg 2105869

OR
CuVer® 2 Copper Reagent AccuVac® Ampuls 1 25/pkg 2504025
Copper
Page 460
Copper
Required apparatus
Copper Standard Solution, 100-mg/L as Cu 100 mL 12842
Copper Voluette® Ampule Standard, 75-mg/L as Cu, 10-mL 16/pkg 1424710
Metals Drinking Water Standard, LR for Cu, Fe, Mn 500 mL 2833749
Metals Drinking Water Standard, HR for Cu, Fe, Mn 500 mL 2833649

Beaker, 50-mL each 500-41H
CuVer 2 Copper Reagent Powder Pillow 100/pkg 2188299
Cylinder, mixing,. 50 mL each 189641
Formaldehyde, ACS 100 mL MDB 205932
Nitric Acid, concentrated 500 mL 15249
Potassium Chloride Solution 100 mL 76542
Potassium Hydroxide Standard Solution, 8 N 100 mL MDB 28232H
Reagent Set for Free and Total Copper, includes: each 2439200
Hydrosulfite Reagent Powder Pillows 100/pkg 2118869
Free Copper Reagent Powder Pillows 100/pkg 2182369
Sample Cells, 25 mLmatched, 1” square 2/pkg 2612602
Sample Cells, 25 mm round 6/pkg 2401906
Copper
Page 461
Copper

Copper, LR, 8143
Copper DOC316.53.01038
Porphyrin Method1 Method 8143

LR (1 to 210 µg/L) Powder Pillows
Scope and Application: For water, wastewater and sea water
1 Adapted from Ishii and Koh, Bunseki Kagaku, 28 (473), 1979
Test preparation


Digestion is required for determining total copper.
adjust.
Wash all glassware with detergent. Rinse with tap water. Rinse again with 1:1 Nitric Acid Solution. Rinse a third time with
copper-free, deionized water.
If samples contain high levels of metals, a slight metallic deposit or yellow buildup may form in the sample cell. Wash the cell
as described above.
Copper Masking Reagent Powder Pillows 1
Porphyrin 1 Reagent Powder Pillows 2
Porphyrin 2 Reagent Powder Pillows 2
Nitric Acid Solution, 1:1 varies
Copper
Page 463
Copper
Porphyrin method
Stored Programs
145 Copper, Porphyrin
Start
1. Select the test. 2. FIll two sample cells 3. Blank Preparation: 4. Add the contents of
Insert an adapter if with 10 mL of sample. Add the contents of one one Porphyrin 1 Reagent
required (Instrument- Copper Masking Reagent powder pillow to each
specific information). powder pillow to one of the sample cell.
sample cells to create the Swirl to dissolve.
Refer to the user manual blank. Swirl to dissolve.
for orientation.
5. Add the contents of 6. Swirl to dissolve. 7. Start the instrument 8. When the timer
one Porphyrin 2 Reagent If copper is present the timer. A three-minute expires insert the blank
powder pillow to each sample will briefly turn reaction period will begin. into the cell holder.
sample cell. blue, then return to yellow.
Zero Read
9. ZERO the instrument. 10. Insert the prepared 11. READ the results in
The display will show: sample into the cell holder. µg/L Cu.
0 µg/L Cu
Copper
Page 464
Copper
Interferences

Aluminum, Al3+ 60 mg/L
Cadmium, Cd2+ 10 mg/L
Calcium, Ca2+ 1500 mg/L
Interfere at all levels unless either the Digesdahl or vigorous digestion is
Chelating agents
performed
Chloride, Cl– 90,000 mg/L
Chromium, Cr6+ 110 mg/L
Cobalt, Co2+ 100 mg/L
Fluoride, F – 30,000 mg/L
Iron, Fe2+ 6 mg/L
Lead, Pb2+ 3 mg/L
Magnesium 10,000 mg/L
Manganese 140 mg/L
Mercury, Hg2+ 3 mg/L
Molybdenum 11 mg/L
Nickel, Ni2+ 60 mg/L
Potassium, K+ 60,000 mg/L
Sodium, Na+ 90,000 mg/L
Zinc, Zn2+ 9 mg/L
May exceed the buffering capacity of the reagents and require sample
pretreatment.

• To preserve, adjust the pH to 2 or less with nitric acid (about 5 mL per liter).
• Before testing, adjust the pH of the preserved sample to between 2 and 6. If the sample is too
acidic, adjust the pH with 5.0 N Sodium Hydroxide Standard Solution*.
Accuracy check
• Copper Standard Solution, 4 mg/L Cu Pour-Rite Ampules
Copper
Page 465
Copper
7. Fill eight sample cells with 10 mL of sample. Use the TenSette® Pipet to add 0.1 mL from a
4-mg/L Pour-Rite Ampule, to two of the sample cells. Then pipet 0.2 mL of the standard
solution into two more cells. Finally, pipet 0.3 mL of the standard solution into two more cells.
8. Analyze each standard addition sample as described in the procedure above, using one of the
two spiked samples in each set as the blank. Accept each standard additions reading by
pressing READ. The copper concentration reading should reflect approximately 100%
recovery.
relationship between the sample spikes and the "Ideal Line" of 100% recovery.

1. To assure the accuracy of the test, prepare a 150-µg/L copper standard by pipetting 15.00 mL
of Copper Standard Solution, 10.0-mg/L Cu, into a 1000-mL volumetric flask.
2. Dilute to the mark with copper-free, reagent-grade water. Prepare this solution daily. Perform
the copper procedure as described above.
3. To adjust the calibration curve using the reading obtained with the 150-µg/L Standard Solution,
Method performance
145 50 µg/L Cu 47–53 µg/L Cu 1 µg/L Cu
Summary of method
The porphyrin method is very sensitive to trace amounts of free copper. The method is free from
most interferences and does not require any sample extraction or concentration before analysis.
Interferences from other metals are eliminated by the copper masking reagent. The porphyrin
indicator forms an intense, yellow-colored complex with any free copper present in sample. Test
Copper
Page 466
Copper

Copper Reagent Set (100 tests), includes: — — 2603300
(1) Copper Masking Reagent Powder Pillows 1 100/pkg 2603449
(2) Porphyin 1 Reagent Powder Pillows 2 100/pkg 2603549
(2) Porphyrin 2 Reagent Powder Pillows 2 100/pkg 2603649
Nitric Acid Solution, 1:1 varies 500 mL 254049
Copper Standard Solution, 4 mg/L, 2 mL Pour-Rite Ampules 20/pkg 2605720
Copper Standard Solution, 10-mg/L Cu 100 mL 12932

Sodium Hydroxide Standard Solution, 5.0 N MDB 100 mL 245032
Tensette Pipet, 0.1–1.0 each 1970001
Pipet, Volumetric, Class A, 15 mL each 1451539
Sample Cells, 1" square matched set 8/pkg 2495408
Copper
Page 467
Cyanide, 8027
Cyanide DOC316.53.01040
Pyridine-Pyrazalone Method1 Method 8027

–
(0.002 to 0.240 mg/L CN ) Powder Pillows
1 Adapted from Epstein, Joseph, Anal. Chem. 19(4), 272 (1947).
Test preparation


Before starting the test
The sample cell shown is a generic representation. Refer to Instrument-specific information for the correct sample cell and
adapter configuration.
Use a water bath to maintain the optimum temperature for the reaction in this test (25 °C). Samples at less than 23 °C require
longer reaction times and samples at greater than 25 °C yield low results.
blank adjust.
The timing for steps 3–10 is critical. You may find it useful to open the necessary reagents before starting this sequence.
All samples to be analyzed for cyanide should be treated by acid distillation except when experience has shown that there is
no difference in results obtained with or without distillation. See Acid distillation.
See Pollution prevention and waste management for proper disposal of solutions containing cyanide.
CyaniVer® Cyanide 3 Reagent Powder Pillow 1
Cylinder, graduated, 10-mL 1
Sample Cells, 1-inch square glass 2
Cyanide
Page 469
Cyanide
Pyridine-Pyrazalone
Stored Programs
160 Cyanide
Start
1. Select the test. 2. Using a graduated 3. Prepared Sample: 4. Shake the sample cell
Insert the adapter, if cylinder, fill a sample cell Add the contents of one for 30 seconds.
required (Instrument- with 10 mL of sample. CyaniVer 3 Cyanide
specific information). Reagent Powder Pillow.
Cap the cell.
5. Leave the sample cell 6. Add the contents of 7. Shake the sample for 8. Add the contents of
undisturbed for an one CyaniVer 4 Cyanide 10 seconds. Immediately one CyaniVer 5 Cyanide
additional 30 seconds. Reagent Powder Pillow. proceed to step 8. Reagent Powder Pillow.
Cap the sample cell. (Delaying the addition of Cap the sample cell.
the CyaniVer 5 for more
Undissolved reagent will than 30 seconds will
not affect accuracy. produce low test results.)
Cyanide
Page 470
Cyanide
9. Shake the cell 10. Set the timer. 11. Blank Preparation: 12. Wipe the blank and
vigorously. A 30-minute reaction When the timer expires, fill insert it into the cell holder.
If cyanide is present, a period will begin. a second sample cell with
pink color will develop. 10 mL of sample.
The solution will turn from
pink to blue.
Samples less than 25 °C
require a longer reaction
time. Samples greater
than 25 °C give low test
results.
Zero
13. ZERO the instrument. 14. Wipe the prepared

The display will show: sample and insert it into
the cell holder. Review the
0.000 mg/L CN –. user manual for cell
orientation.
Results are in mg/L CN–.
Cyanide
Page 471
Cyanide

Special considerations for materials containing cyanide
Samples analyzed by this procedure may contain cyanide, which is regulated as reactive (D003)
waste by the federal RCRA. It is imperative these materials be handled safely to prevent the
release of hydrogen cyanide gas (an extremely toxic material with the smell of almonds). Most
cyanide compounds are stable and can be safely stored for disposal in highly alkaline solutions
(pH >11) such as 2 N sodium hydroxide. Never mix these wastes with other laboratory wastes
which may contain lower pH materials such as acids or even water.
In the event of a spill or release, special precautions must be taken to prevent exposure to
hydrogen cyanide gas. The following steps may be taken to destroy the cyanide compounds in the
event of an emergency:
• Use a fume hood or supplied air or self contained breathing apparatus.
• While stirring, add the waste to a beaker containing a strong solution of sodium hydroxide and
calcium hypochlorite or sodium hypochlorite (household bleach).
• Maintain a strong excess of hydroxide and hypochlorite. Let the solution stand for 24 hours.
• Neutralize and flush the solution down the drain with a large excess of water.
Note: If the solution contains other regulated materials such as chloroform or heavy metals, it may still need
to be collected for hazardous waste disposal. Never flush hazardous wastes down the drain.
Interferences

Interfering
Interference levels and treatments
substance
Large amounts of chlorine in the sample will cause a milky white precipitate after the addition of the
Chlorine CyaniVer® 5 Reagent. If chlorine or other oxidizing agents are known to be present, pretreat the sample
before testing using the procedure in this table for oxidizing agents.
Nickel or cobalt in concentrations up to 1 mg/L do not interfere. Eliminate the interference from up to
20 mg/L copper and 5 mg/L iron by adding the contents of one HexaVer Chelating Reagent Powder
Metals
Pillow to the sample and then mixing before adding the CyaniVer 3 Cyanide Reagent Powder Pillow in
step 3. Prepare a reagent blank of deionized water and reagents to zero the instrument in step 12.
1. Adjust a 25-mL portion of the alkaline sample to pH 7–9 with 2.5 N Hydrochloric Acid Standard
Solution. Count the number of drops of acid added.
2. Add two drops of Potassium Iodide Solution and two drops of Starch Indicator Solution to the
sample. Swirl to mix. The sample will turn blue if oxidizing agents are present.
3. Add Sodium Arsenite Solution drop-wise until the sample turns colorless. Swirl the sample
Oxidizing agents
thoroughly after each drop. Count the number of drops.
4. Take another 25-mL sample and add the total number of drops of Hydrochloric Acid Standard
Solution counted in step 1.
5. Subtract one drop from the amount of Sodium Arsenite Solution added in step 3. Add this amount to
the sample and mix thoroughly. Continue with step 2 of the cyanide procedure.
1. Adjust a 25-mL portion of the alkaline sample to pH 7–9 with 2.5 N Hydrochloric Acid Standard
Solution. Count the number of drops added.
2. Add four drops of Potassium Iodide Solution and four drops of Starch Indicator Solution to the
sample. Swirl to mix. The sample should be colorless.
3. Add Bromine Water drop-wise until a blue color appears. Swirl the sample thoroughly after each
Reducing agents
addition. Count the number of drops.
4. Take another 25-mL sample and add the total number of drops of Hydrochloric Acid Standard
Solution counted in step 1.
5. Add the total number of drops of Bromine Water counted in step 3 to the sample and mix thoroughly.
6. Continue with step 2 of the cyanide procedure.
Cyanide
Page 472
Cyanide

Interfering
Interference levels and treatments
substance
Large amounts of turbidity will cause high readings. Use filter paper and a funnel to filter highly turbid
Turbidity
water samples before use in steps 1 and 11. The test results should then be recorded as soluble cyanide.

• Collect samples in glass or plastic bottles and analyze as quickly as possible.
• The presence of oxidizing agents, sulfides and fatty acids can cause the loss of cyanide during
sample storage. Samples containing these substances must be pretreated as described below
before preservation with sodium hydroxide. If the sample contains sulfide and is not
pretreated, it must be analyzed within 24 hours.
• Preserve the sample by adding 4.0 mL of 5.0 N Sodium Hydroxide Standard Solution to each
liter (or quart) of sample, using a glass serological pipet and pipet filler.
• Check the sample pH; 4-mL of sodium hydroxide is usually enough to raise the pH of most
water and wastewater samples to 12. Add more 5.0 N Sodium Hydroxide if necessary.
• Store the samples at 4 °C (39 °F) or less. Samples preserved in this manner can be stored for
14 days.
• Before testing, samples preserved with 5.0 N Sodium Hydroxide or samples that are highly
alkaline due to chlorination treatment processes or sample distillation procedures should be
adjusted to approximately pH 7 with 2.5 N Hydrochloric Acid Standard Solution.
• Correct the volume when significant amounts of preservative are used.
Oxidizing agents
Oxidizing agents such as chlorine decompose cyanides during storage. To test for their presence
and to eliminate their effect, pretreat the sample as follows:
1. Take a 25-mL portion of the sample and add one drop of 10-g/L m-Nitrophenol Indicator
Solution. Swirl to mix.
2. Add 2.5 N Hydrochloric Acid Standard Solution drop-wise until the color changes from yellow
to colorless. Swirl the sample thoroughly after the addition of each drop.
3. Add two drops of Potassium Iodide Solution, 30-g/L and two drops of Starch Indicator Solution,
to the sample. Swirl to mix. The solution will turn blue if oxidizing agents are present.
4. If step 3 suggests the presence of oxidizing agents, add two level, 1-g measuring spoonfuls of
Ascorbic Acid per liter of sample.
5. Withdraw a 25-mL portion of sample treated with ascorbic acid and repeat steps 1 to 3. If the
sample turns blue, repeat steps 4 and 5.
6. If the 25-mL sample remains colorless, preserve the remaining sample to pH 12 for storage
with 5 N Sodium Hydroxide Standard Solution.
7. Perform the procedure given under Interfering Substances and Levels, Reducing Agents, to
eliminate the effect of excess ascorbic acid, before following the cyanide procedure.
Cyanide
Page 473
Cyanide
Sulfides
–
Sulfides will quickly convert cyanide to thiocyanate (SCN ). To test for the presence of sulfide and
eliminate its effect, pretreat the sample as follows:
1. Place a drop of sample on a disc of Hydrogen Sulfide Test Paper that has been wetted with pH
4 Buffer Solution.
2. If the test paper darkens, add a 1-g measuring spoon of Lead Acetate to the sample. Repeat
step 1.
3. If the test paper continues to turn dark, keep adding Lead Acetate until the sample tests
negative for sulfide.
4. Filter the lead sulfide precipitate through Filter Paper and a Funnel. Preserve the sample for
storage with 5 N Sodium Hydroxide Standard Solution or neutralize to a pH of 7 for analysis.
Fatty acids
CAUTION
Perform this operation under a ventilation hood and complete as quickly as possible.
When distilled, fatty acids will pass over with cyanide and under the alkaline conditions of the
absorber, will form soaps. If the presence of fatty acid is suspected, use the following pretreatment
before preserving samples with sodium hydroxide.
1. Acidify 500 mL of sample to pH 6 or 7 with a 4:1 dilution of glacial Acetic Acid.

2. Pour the sample into a 1000-mL separation funnel and add 50 mL of Hexane.
3. Stopper the funnel and shake for one minute. Allow the layers to separate.
4. Drain off the lower, sample layer into a 600-mL beaker. If the sample is to be stored, add
enough 5 N Sodium Hydroxide Standard Solution to raise the pH above 12.
Acid distillation
All samples to be analyzed for cyanide should be treated by acid distillation except when
experience has shown that there is no difference in results obtained with or without distillation.
With most compounds, a one-hour reflux is adequate.
If thiocyanate is present in the original sample, a distillation step is absolutely necessary as
thiocyanate causes a positive interference. High concentrations of thiocyanate can yield a
substantial quantity of sulfide in the distillate. The “rotten egg” smell of hydrogen sulfide will
accompany the distillate when sulfide is present. The sulfide must be removed from the distillate
prior to testing.
If cyanide is not present, the amount of thiocyanate can be determined. The sample is not distilled
and the final reading is multiplied by 2.2. The result is mg/L SCN –.
The distillate can be tested and treated for sulfide after the last step of the distillation procedure by
using the following lead acetate treatment procedure.
1. Place a drop of the distillate (already diluted to 250 mL) on a disc of Hydrogen Sulfide Test
Paper that has been wetted with pH 4 Buffer Solution.
2. If the test paper darkens, add 2.5 N Hydrochloric Acid Standard Solution by drops to the
distillate until a neutral pH is obtained.
3. Add a 1-g measuring spoon of Lead Acetate to the distillate and mix. Repeat step 1.
4. If the test paper continues to turn dark, keep adding lead acetate until the distillate tests
negative for sulfide. Filter the black lead sulfide precipitate through filter paper and a funnel.
Neutralize the liquid filtrate to pH 7 and immediately analyze for cyanide.
Cyanide
Page 474
Cyanide
Distillation procedure
The following steps describe the distillation process using distillation apparatus and cyanide
glassware offered by the manufacturer:
1. Set up the distillation apparatus for cyanide recovery, leaving off the thistle tube. Refer to the
Distillation Apparatus Manual. Turn on the water and make certain it is flowing steadily through
the condenser.
2. Fill the distillation apparatus cylinder to the 50-mL mark with 0.25 N Sodium Hydroxide
Standard Solution.
3. Fill a clean 250-mL graduated cylinder to the 250-mL mark with sample and pour it into the
distillation flask. Place a stirring bar into the flask and attach the thistle tube.
4. Arrange the vacuum system as shown in the Distillation Apparatus Manual, but do not connect
the vacuum tubing to the gas bubbler. Turn on the water to the aspirator to full flow and adjust
the flow meter to 0.5 SCFH.
5. Connect the vacuum tubing to the gas bubbler, making certain that air flow is maintained
(check the flow meter) and that air is bubbling from the thistle tube and the gas bubbler.
6. Turn the power switch on and set the stir control to 5. Using a 50-mL graduated cylinder, pour
50 mL of 19.2 N Sulfuric Acid Standard Solution* through the thistle tube and into the
distillation flask.
7. Using a water bottle, rinse the thistle tube with a small amount of deionized water.
8. Allow the solution to mix for three minutes; then add 20 mL Magnesium Chloride Reagent*
through the thistle tube and rinse again. Allow the solution to mix for 3 more minutes.
9. Verify that there is a constant flow of water through the condenser.
10. Turn the heat control to 10.

11. Carefully monitor the distillation flask at this point in the procedure. Once the sample begins to
boil, slowly lower the air flow to 0.3 SCFH. If the contents of the distillation flask begin to back
up through the thistle tube, increase the air flow by adjusting the flow meter until the contents
do not back up through the thistle tube. Boil the sample for one hour.
12. After one hour, turn off the still, but maintain the air flow for 15 minutes more.
13. After 15 minutes, remove the rubber stopper on the 500-mL vacuum flask to break the vacuum
and turn off the water to the aspirator. Turn off the water to the condenser.
14. Remove the gas bubbler/cylinder assembly from the distillation apparatus. Separate the gas
bubbler from the cylinder and pour the contents of the cylinder into a 250-mL, Class A
volumetric flask. Rinse the gas bubbler, cylinder and J-tube connector with deionized water
and add the washings to the volumetric flask.
15. Fill the flask to the mark with deionized water and mix thoroughly. Neutralize the contents of
the flask and analyze for cyanide.
Cyanide
Page 475
Cyanide
Accuracy check
Standard solutions method
CAUTION
Cyanides and their solutions and the hydrogen cyanide liberated by acids, are very
poisonous. Both the solutions and the gas can be absorbed through the skin.

• Potassium Cyanide
• Deionized water
Prepare a 100-mg/L cyanide stock solution weekly as follows:
1. Dissolve 0.2503 grams of Potassium Cyanide in deionized water and dilute to 1000 mL.
2. Immediately before use, prepare a 0.200 mg/L cyanide working solution by diluting 2.00 mL of
the 100 mg/L stock solution to 1000 mL using deionized water.
3. To adjust the calibration curve using the reading obtained with the 0.200 mg/L standard
solution, select Options>More>Standard Adjust from the instrument menu.
Method performance
Sensitivity
Distribution
160 0.100 mg/L CN– 0.090–0.110 mg/L CN– Entire Range 0.002 mg/L CN–
Summary of method
The Pyridine-Pyrazalone method used for measuring cyanide gives an intense blue color with free
cyanide. A sample distillation is required to determine cyanide from transition and heavy metal
cyanide complexes. Test results are measured at 612 nm.
Cyanide
Page 476
Cyanide

Required reagents
Cyanide Reagent Set, includes: — — 2430200
(1) CyaniVer® 3 Cyanide Reagent Powder Pillow 1 100/pkg 2106869
Required apparatus
Stopper, poly, hollow — 6/pkg 1448000
Potassium Cyanide, ACS 125 g 76714

Acetic Acid, ACS 500 mL 10049
Ascorbic Acid 100 g 613826
Bromine Water, 30 g/L 29 mL 221120
Buffer Solution, pH 4 500 mL 1222349
Filter Paper, 12.5 cm 100/pkg 189457
Funnel, 65 mm each 108367
Hexane Solution, ACS 500 mL 1447849
HexaVer Chelating Reagent Powder Pillow 100/pkg 24399
Hydrochloric Acid Standard Solution, 2.5 N 100 mL MDB 141832
Hydrogen Sulfide Test Paper 100/pkg 2537733
m-Nitrophenol Indicator Solution, 10 g/L 100 mL MDB 247632
Magnesium Chloride Reagent 1L 1476253
Potassium Iodide Solution, 30 g/L 100 mL MDB 34332
Sodium Arsenite Solution, 5 g/L 100 mL 104732
Sodium Hydroxide Standard Solution, 5.0 N 1L 245053
Starch Indicator Solution 100 mL MDB 34932
Cyanide Glassware each 2265800
Distillation Apparatus, 115 VAC each 2274400
Cyanide
Page 477
Cyanide

Distillation Apparatus, 230 VAC each 2274402
Distillation Apparatus Set each 2265300
Pipet, seriological, 5 mL each 53237
Spoon, measuring, 1 g each 51000
Thermometer, -10–225 °C each 2635700

Cyanuric Acid, 8139
Cyanuric Acid DOC316.53.01183
Turbidimetric Method Method 8139

5 to 50 mg/L Powder Pillows
Scope and Application: For water.
Test preparation


Filter highly turbid samples with filter paper and a funnel.
Clean sample cells with soap, water, and a brush soon after each test to avoid a build-up of film on the sample cell.
Do not use the Pour-Thru Cell with this procedure.
Bottle, mixing, square glass 1
Cyanuric Acid 2 Reagent Powder Pillow 1
Sample cells (See Instrument-specific information) 2
Cyanuric Acid
Page 479
Cyanuric Acid
Turbidimetric method
Stored Programs
170 Cyanuric Acid
Start
1. Select the test. 2. Fill a mixing bottle with 3. Prepared Sample: 4. Start the instrument
required (see Instrument- Cyanuric Acid 2 Reagent A three-minute reaction
specific information). powder pillow. Swirl to mix. period will begin.
Refer to the user manual After adding the reagent a
for orientation. white turbidity will form if
cyanuric acid is present.
5. Blank Preparation: 6. Wipe and insert the 7. When the timer 8. Within seven minutes
Fill a sample cell with blank sample cell into the expires, fill a second after the timer expires,
10 mL of sample from the cell holder. sample cell with 10 mL of insert the prepared sample
mixing bottle. ZERO the instrument. prepared sample from the into the cell holder.
mixing bottle. READ the results in mg/L
Cyanuric Acid.
0 mg/L Cyan Acid

Collect samples in clean plastic or glass bottles. Samples must be analyzed within 24 hours.
Accuracy check
1. Dissolve 1.000 gram of cyanuric acid in 1 liter of deionized water to make a 1000-mg/L
solution. Cyanuric acid is difficult to dissolve; it may take several hours to completely dissolve.
This solution is stable for several weeks.
2. Dilute 3.00 mL of the 1000-mg/L solution to 100 mL with deionized water to make a 30-mg/L
solution. Prepare fresh daily. Testing the 30-mg/L solution should give test results of about
30 mg/L cyanuric acid.
Cyanuric Acid
Page 480
Cyanuric Acid
Method performance
Program Standard 95% Confidence Limits Concentration change per
of Distribution Portion of curve
0.010 Abs change
170 10 mg/L cyanuric acid 7–13 mg/L cyanuric acid 10 mg/L 0.3 mg/L cyanuric acid
30 mg/L 0.3 mg/L cyanuric acid
50 mg/L 0.4 mg/L cyanuric acid
Summary of method
The test for Cyanuric Acid uses the turbidimetric method. Cyanuric Acid 2 Reagent precipitates
any Cyanuric Acid present and holds it in suspension. The amount of turbidity caused by the
suspended particles is directly proportional to the amount of cyanuric acid present. Test results are
measured at 480 nm.
Cyanuric Acid
Page 481
Cyanuric Acid
Required reagents
Cyanuric Acid 2 Reagent Powder Pillow 1 50/pkg 246066
Required apparatus
Bottle, mixing, square, with 25 mL mark 1 each 1704200
Cyanuric Acid 25 g 712924

Balance, 600 g x 0.01 g, 100–240 VAC each 2937201
Brush, test tube each 69000
Filter, paper, for funnel filter 10/pkg 189457
Flask, volumetric, 100 mL each 2636642
Funnel, filter each 108367
Liqui-nox Solution 1L 2088153
Pipet, TenSette, 1.0 to 10.0 mL each 1970010
Tips for Pipet, TenSette 50/pkg 2199796

Fluoride, 8029
Fluoride DOC316.53.01041
USEPA SPADNS Method1 Method 8029

0.02 to 2.00 mg/L F– Reagent Solution or AccuVac® Ampuls
Scope and Application: For water, wastewater and seawater; USEPA accepted for reporting for drinking and
wastewater analyses (distillation required; see Distillation in this procedure).2
1 Adapted from Standard Methods for the Examination of Water and Wastewater, 4500-F B & D.
2 Procedure is equivalent to USEPA method 340.1 for drinking water and wastewater.
Test preparation

Reagent solution AccuVac Ampuls

Instrument
The sample and deionized water should be at the same temperature (±1 °C). Temperature adjustments may be made before
or after reagent addition.
SPADNS Reagent is toxic and corrosive. Use care while handling the reagent.
SPADNS Reagent contains sodium arsenite. Final solutions will contain arsenic (D004) in sufficient concentration to be
regulated as a hazardous waste for Federal RCRA. Refer to the MSDS for disposal instructions.
For best results, measure the volume of SPADNS Reagent as accurately as possible.
Do not use the Pour-Thru Cell with this test.
Solution Test:
SPADNS Reagent Solution 4 mL
Pipet, volumetric, 2-mL 1
Fluoride
Page 483
Fluoride
Pipet Filler Bulb 1
Sample cells (Instrument-specific information) 2
Thermometer, –10 to 110 °C 1
AccuVac Test:
SPADNS Fluoride Reagent AccuVac® Ampuls 2
Beaker, 50-mL 1
Stopper 2
SPADNS reagent solution method
Stored Programs
190 Fluoride
Start
1. Select the test. 2. Prepared Sample: 3. Blank Preparation: 4. Carefully pipet 2.0 mL
Insert an adapter if Pipet 10.0 mL of sample Pipet 10.0 mL of deionized of SPADNS Reagent into
required (Instrument- into a dry sample cell. water into a second dry each cell. Swirl to mix.
specific information). sample cell.

for orientation.
Zero
5. Start the instrument 6. When the timer 7. ZERO the instrument. 8. Insert the prepared
timer. expires, insert the blank The display will show: sample into the cell holder.
A one-minute reaction into the cell holder. READ the result in
0.00 mg/L F–
period will begin. mg/L F–.
Fluoride
Page 484
Fluoride
SPADNS AccuVac® Ampuls
Stored Programs
195 Fluoride AV
Start
1. Select the test. 2. Prepared Sample: 3. Blank Preparation: 4. Close with stopper
Insert an adapter if Collect at least 40 mL of Pour at least 40 mL of and quickly invert both
required (Instrument- sample in a 50-mL beaker. deionized water into a Ampuls several times
specific information). Fill one SPADNS Fluoride second beaker. to mix.
Refer to the user manual Reagent AccuVac Ampul Fill a second Ampul with
for orientation. with sample. Keep the tip deionized water. Keep the
immersed while the Ampul tip immersed while the
fills completely. Ampul fills completely.
5. Start the instrument 6. When the timer 7. Insert the Ampul that
timer. expires, wipe the blank contains the prepared
A one-minute reaction and insert it into the cell sample into the cell holder.
period will begin. holder. READ the results in
ZERO the instrument. mg/L F–.
0.00 mg/L F–
Fluoride
Page 485
Fluoride
Interferences
This test is sensitive to small amounts of interference. Glassware must be very clean (acid rinse
before each use). Repeat the test with the same glassware to ensure that results are accurate.

Alkalinity (as CaCO3) At 5000 mg/L it causes a −0.1 mg/L F– error.
At 0.1 mg/L it causes a −0.1 mg/L F– error.
To check for interferences from aluminum, read the
concentration one minute after reagent addition, then again
Aluminum after 15 minutes. An appreciable increase in concentration
suggests aluminum interference. Waiting 2 hours before
making the final reading will eliminate the effect of up to
3.0 mg/L aluminum.
Chloride At 7000 mg/L causes a +0.1 mg/L F– error.
SPADNS Reagent contains enough arsenite to eliminate
interference up to 5 mg/L chlorine. For higher chlorine levels,
Chlorine
add one drop of Sodium Arsenite Solution1 to 25 mL of
sample for each 2 mg/L of Chlorine.
Iron, ferric At 10 mg/L it causes a −0.1 mg/L F– error.
Phosphate, ortho At 16 mg/L it causes a +0.1 mg/L F– error.
Sodium Hexametaphosphate At 1.0 mg/L it causes a +0.1 mg/L F– error.
Sulfate At 200 mg/L it causes a +0.1 mg/L F– error.
Distillation
Distillation Solution Preparation:
1. Measure 60 mL of deionized water into a 250 mL glass Erlenmeyer flask.

2. With constant stirring, add 120 mL of concentrated Sulfuric Acid. Caution: The mixture will
become very hot. Allow the solution to cool before handling.
To eliminate most interferences, dilute the sample from the acid solution as described
below:
1. Set up the distillation apparatus for general purpose distillation. Refer to the Distillation
Apparatus manual for proper assembly. Use a 125-mL Erlenmeyer flask to collect the distillate.
2. Turn on the water and maintain a steady flow through the condenser.
3. Measure 100 mL of sample into the distillation flask using a 100-mL graduated cylinder. Add a
magnetic stir bar and 5 glass beads.
4. Turn the stirrer power switch on. Turn the stir control to 5.
5. Using a 250-mL graduated cylinder, carefully add 150 mL of Distillation Solution into the flask.
Note: When distilling samples with high amounts of chloride, add 5 mg of Silver Sulfate* to the sample for
every mg/L of chloride in the sample.
Fluoride
Page 486
Fluoride
6. With the thermometer in place, turn the heat control to 10. The yellow pilot lamp indicates the
heater is on.
7. When the temperature reaches 180 °C or when 100 mL of distillate has been collected, turn
the still off (requires about 1 hour).
8. Dilute the distillate to a volume of 100 mL, if necessary. The distillate may now be analyzed by
the SPADNS or the fluoride ion-selective electrode method.

• Samples may be stored in glass or plastic bottles for up to 28 days when cooled to 4 °C
(39 °F) or lower.
• Warm samples to room temperature before analysis.
Accuracy check
A variety of standard solutions covering the entire range of the test is available. Use these instead
of sample to verify technique.
Minor variations between lots of reagent become measurable above 1.5 mg/L. While results in this
region are usable for most purposes, better accuracy may be obtained by diluting a fresh sample
1:1 with deionized water and retesting. Multiply the result by 2.
Method performance
190 1.00 mg/L F– 0.97–1.03 mg/L F– 0.024 mg/L F– at 1 mg/L
Summary of method
The SPADNS Method for fluoride determination involves the reaction of fluoride with a red
zirconium-dye solution. The fluoride combines with part of the zirconium to form a colorless
complex, thus bleaching the red color in an amount proportional to the fluoride concentration. This
method is accepted by the EPA for NPDES and NPDWR reporting purposes when the samples
have been distilled. Seawater and wastewater samples require distillation. Test results are
measured at 580 nm.
Fluoride
Page 487
Fluoride

Required reagents
SPADNS Reagent Solution 4 mL 500 mL 44449
OR
SPADNS Fluoride Reagent AccuVac® Ampuls 2 25/pkg 2506025
Water, deionized 10–40 mL 4L 27256
Required apparatus (solution)

Pipet, volumetric, Class A, 10.00-mL. 1 each 1451538
Thermometer, –10 to 110 °C 1 each 187701
Required apparatus (AccuVac)

Fluoride Standard Solution, 0.2-mg/L F– 500 mL 40502

Fluoride Standard Solution, 100-mg/L F– 500 mL 23249
Standard, Drinking Water, Mixed Parameter, Inorganic for F–, NO3, PO4, SO4 500 mL 2833049
Distillation reagents and apparatus

Distillation Heater and Support Apparatus Set,115 VAC, 50/60 Hz 1 each 2274400
OR
Fluoride
Page 488
Fluoride

Distillation Heater and Support Apparatus Set, 230 VAC, 50/60 Hz 1 each 2274402
AND
Distillation Apparatus Set, General Purpose 1 each 2265300
Glass Beads 1 100/pkg 259600
Stir Bar, magnetic 1 each 1076416
Sulfuric Acid, concentrated, ACS 1 500 mL 97949

Silver Sulfate 113 g 33414
Sodium Arsenite Solution, 5.0 g/L 100 mL 104732
AccuVac Snapper, each 2405200
Wipes, disposable 280/pkg 2097000
Fluoride
Page 489
Fluoride SPADNS 2, 10225
Fluoride DOC316.53.01184
USEPA1 SPADNS 22 Method 10225

(0.02 to 2.00 mg/L F– ) Reagent Solution or AccuVac® Ampuls
Scope and Application: For water, wastewater and seawater; USEPA accepted for reporting for drinking and
wastewater analyses (distillation required; see Distillation in this procedure).
1 Procedure is equivalent to USEPA method 340.1 for drinking water and wastewater analysis
2 Adapted from Standard Methods for the Examination of Water and Wastewater, 4500-F B & D.
Test preparation


Instrument
The sample and deionized water should be at the same temperature (± 1 °C). Temperature adjustments may be made before
or after reagent addition.
SPADNS 2 Reagent is corrosive. Use care while handling the reagent.
For best results, measure the volume of SPADNS 2 Reagent as accurately as possible.
If the instrument displays Over Measure Range!, dilute a fresh sample with an equal volume of deionized water and repeat
the test, using this solution in step 2. Multiply the result by 2.
SPADNS 2 Reagent contains a non-toxic reducing agent to prevent chlorine interference. SPADNS 2 does not contain
sodium arsenite.
Fluoride
Page 491
Fluoride
Solution test
SPADNS 2 Reagent Solution 4 mL
Pipet Filler Bulb 1
Thermometer 1
AccuVac test
SPADNS 2 Fluoride Reagent AccuVac® Ampuls 2
Beaker, 50-mL 1
SPADNS 2 reagent solution
Stored Programs
190 Fluoride
Start
1. Select the test. 2. Prepared Sample: 3. Blank Preparation: 4. Carefully pipet 2.0 mL
Insert an adapter if Pipet 10.0 mL of sample Pipet 10.0 mL of deionized of SPADNS 2 Reagent into
required (see Instrument- into a dry sample cell. water into a second dry each cell. Swirl to mix.
specific information). sample cell.
Fluoride
Page 492
Fluoride
SPADNS 2 reagent solution (continued)
Zero
5. Start the instrument 6. When the timer 7. ZERO the instrument. 8. Insert the prepared
timer. expires, insert the blank. The display will show: sample cell.
A one-minute reaction 0.00 mg/L F– READ the results in
period will begin. mg/L F–.
SPADNS 2 AccuVac® Ampuls
Stored Programs
195 Fluoride AV
Start
1. Select the test. 2. Prepared Sample: 3. Blank Preparation: 4. Quickly invert both
Insert an adapter if Collect at least 40 mL of Pour at least 40 mL of Ampuls several times
required (see Instrument- sample in a 50-mL beaker. deionized water into a to mix.
specific information). Fill one SPADNS 2 second beaker.
Fluoride Reagent AccuVac Fill a second Ampul with
Ampul with sample. Keep deionized water. Keep the
the tip immersed while the tip immersed while the
Ampul fills completely. Ampul fills completely.
Fluoride
Page 493
Fluoride
SPADNS 2 AccuVac® Ampuls (continued)
5. Start the instrument 6. When the timer 7. Insert the prepared

timer. expires, insert the blank sample into the cell holder
A one-minute reaction into the cell holder. READ the results in
period will begin. ZERO the instrument. mg/L F–.
0.00 mg/L F–
Interferences
This test is sensitive to small amounts of interference. Glassware must be very clean (acid rinse
before each use). Repeat the test with the same glassware to make sure that the results are
accurate.

Alkalinity (as CaCO3) At 5000 mg/L causes a −0.1 mg/L F– error
At 0.1 mg/L causes a −0.1 mg/L F– error. To check for interferences from aluminum,
read the concentration one minute after reagent addition, then again after 15 minutes.
Aluminum An appreciable increase in concentration suggests aluminum interference. Waiting 2
hours before making the final reading will eliminate the effect of up to 3.0 mg/L
aluminum.
Chloride At 7000 mg/L causes a +0.1 mg/L F– error
SPADNS 2 Reagent contains enough non-toxic reductant to eliminate interference up
to 5 mg/L chlorine.
For higher chlorine levels:
1. Dilute sample with deionized water by a factor that will lower chlorine
Chlorine concentration to below 5 mg/L.
2. Perform the SPADNS 2 reagent solution or AccuVac procedure.
3. Multiply results by the dilution factor to obtain
mg/L Fluoride.
Iron, ferric At 10 mg/L causes a −0.1 mg/L F– error
Phosphate, ortho At 16 mg/L causes a +0.1 mg/L F– error
Sodium Hexametaphosphate At 1.0 mg/L causes a +0.1 mg/L F– error
Sulfate At 200 mg/L causes a +0.1 mg/L F– error
Fluoride
Page 494
Fluoride
Distillation
Distillation Solution Preparation:
1. Measure 60 mL of deionized water into a 250 mL glass Erlenmeyer flask.
2. With constant stirring, add 120 mL of concentrated Sulfuric Acid. Caution: The mixture will
become very hot. Allow the solution to cool before handling.
To eliminate most interferences, dilute the sample from the acid solution as described
below:
1. Set up the distillation apparatus for general purpose distillation. Refer to the Distillation
Apparatus manual for proper assembly. Use a 125-mL Erlenmeyer flask to collect the distillate.
2. Turn on the water and maintain a steady flow through the condenser.
3. Measure 100 mL of sample into the distillation flask using a 100-mL graduated cylinder. Add a
magnetic stir bar and 5 glass beads.
4. Turn the stirrer power switch on. Turn the stir control to 5.
5. Using a 250-mL graduated cylinder, carefully add 150 mL of Distillation Solution into the flask.
Note: When distilling samples with high amounts of chloride, add 5 mg of Silver Sulfate to the sample for
every mg/L of chloride in the sample.
6. With the thermometer in place, turn the heat control to 10. The yellow pilot lamp indicates the
heater is on.
7. When the temperature reaches 180 °C or when 100 mL of distillate has been collected, turn
the still off (requires about 1 hour).
8. Dilute the distillate to a volume of 100 mL, if necessary. The distillate may now be analyzed by
the SPADNS, SPADNS 2 or the fluoride ion-selective electrode method.

• Samples may be stored in glass or plastic bottles for at least seven days when cooled to 4 °C
(39 °F) or lower.
Accuracy check
A variety of standard solutions for the entire range of the test is available. Use standard solutions
instead of sample to verify the technique.
Minor variations between lots of reagent become measurable above 1.5 mg/L. While results in this
region are usable for most purposes, better accuracy may be obtained with steps 1–3.
1. Dilute a fresh sample 1:1 with deionized water.
2. Perform the test again
3. Multiply the result by 2.
4. To adjust the calibration curve with the reading obtained with the standard solution, select
Fluoride
Page 495
Fluoride
Method performance
Safety
Follow good safety habits and laboratory techniques throughout the procedure. Consult the
Material Safety Data Sheet for information specific to the reagents used.

SPADNS 2 Reagent does not contain sodium arsenite. Instead, it contains a non-toxic species to
prevent chlorine interference. Dispose of all waste safely in accordance with local and
federal guidelines.
Summary of method
The SPADNS 2 Method for fluoride determination involves the reaction of fluoride with a red
zirconium-dye solution. The fluoride combines with part of the zirconium to form a colorless
complex that bleaches the red color in an amount proportional to the fluoride concentration. This
method is equivalent to the EPA method for NPDES and NPDWR reporting purposes when the
samples have been distilled. Seawater and wastewater samples require distillation. Test results
Fluoride
Page 496
Fluoride
Required reagents
SPADNS 2 Reagent Solution 4 mL 500 mL 2947549
OR
SPADNS 2 Fluoride Reagent AccuVac® Ampuls 2 25/pkg 2527025
Required apparatus (solution)

Pipet, volumetric, Class A, 10.00-mL. 1 each 1451538
Thermometer 1 each 2635700


Fluoride Standard Solution, 100-mg/L F– 500 mL 23249
Fluoride
Page 497
Fluoride

AND
OR
Distillation Apparatus Set, General Purpose 1 each 2265300
Flask, Erlenmeyer, 250 mL, Glass 1 each 50546
Glass Beads 1 100/pkg 259600
Sulfuric Acid, ACS 1 500 mL 97949

Silver Sulfate 113 g 33414
Balance Analytical 80 g x 0.1 mg 100–240 V each 2936701
Weighing papers 500/pkg 1473800

Formaldehyde, 8110
Formaldehyde DOC316.53.01042
MBTH Method1 Method 8110

3 to 500 µg/L CH2O Powder Pillows
Scope and Application: For water.
1 Adapted from Matthews, T.G. and Howell, T.C., Journal of the Air Pollution Control Association, 31 (11) 1181-1184 (1981).
Test preparation

Wash glassware with Chromic Acid Cleaning Solution1 to remove trace contaminants.
Time and temperature are very important to this test. The sample should be 25 ± 1 °C and the times specified in the
procedure steps must be followed precisely. A temperature-controlled water bath is recommended for best accuracy.
Obtain formaldehyde-free water by distilling water from alkaline permanganate (4 g sodium hydroxide1, 2 g potassium
permanganate per 500 mL of water). Discard the first 50–100 mL of distillate.
The Pour-thru cell cannot be used for this test.

Developing Solution for LR Formaldehyde 5 mL
MBTH Powder Pillows 2
Pipet, serological, 5-mL 1
Pipet Filler 1
Formaldehyde
Page 499
Formaldehyde
MBTH method for powder pillows
Stored Programs
200 Formaldehyde
Start
Insert an adapter if Accurately measure 25 mL Accurately measure 25 mL one MBTH powder pillow
required (Instrument- of sample in a 50-mL of formaldehyde-free to the blank. Insert the
specific information). mixing cylinder. water in a second 50-mL cylinder stopper.
mixing cylinder.
for orientation.
5. Start the instrument 6. Immediately after the 7. Add the contents of 8. Shake vigorously for
timer. A 17-minute reaction period starts, one MBTH powder pillow 20 seconds.
reaction period will begin. shake the blank sample to the prepared sample
Proceed with step 6 vigorously for 20 seconds. when the timer shows
immediately after the timer Do not wait for the timer 15:00. Insert the cylinder
starts. to expire. stopper.
9. Add 2.5 mL of 10. Insert the cylinder 11. Add 2.5 mL of 12. Insert the cylinder
Developing Solution for stopper and invert to mix. Developing Solution for stopper and invert to mix.
Low Range Formaldehyde Low Range Formaldehyde
to the blank when the to the prepared sample
timer shows 12:00. when the timer shows
10:00.
Formaldehyde
Page 500
Formaldehyde
MBTH method for powder pillows (continued)
Zero
13. Just before the timer 14. Immediately wipe the 15. When the timer shows 16. Pour at least 10 mL of
shows 2:00, pour at least blank and insert it into the 2:00, ZERO the the prepared sample into a
10 mL of the blank into the cell holder. instrument. sample cell.
sample cell. Pour the The display will show:
solution slowly to avoid
bubble formation on the 0 µg/L CH2O
cell walls. If bubbles form,
swirl to dislodge them.
Read
17. Wipe the cell and 18. When the timer

insert it into the cell holder. expires, READ the results
in µg/L CH2O.
Interferences

Interfering substance Interference level Interfering substance Interference level
Acetate Greater than 1000 mg/L Iron (Fe3+) Greater than 12 mg/L
Aldehydes (other) Positive interference at all levels Lead Greater than 100 mg/L
Ammonium (as N) Greater than 10 mg/L Manganese Greater than 500 mg/L
Aniline Greater than 10 mg/L Mercury Greater than 70 mg/L
Bicarbonate Greater than 1000 mg/L Morpholine Greater than 0.36 mg/L
Calcium Greater than 3500 mg/L Nitrate Greater than 1000 mg/L
Carbonate Greater than 500 mg/L Nitrite Greater than 8 mg/L
Chloride Greater than 5000 mg/L Phenol Greater than 1050 mg/L
Copper Greater than 1.6 mg/L Phosphate Greater than 200 mg/L
Cyclohexylamine Greater than 250 mg/L Silica Greater than 40 mg/L
Formaldehyde
Page 501
Formaldehyde

Interfering substance Interference level Interfering substance Interference level
Ethanolamine Greater than 33 mg/L Sulfate Greater than 10,000 mg/L
Ethylenediamine Greater than 1.5 mg/L Urea Greater than 1000 mg/L
Glucose Greater than 1000 mg/L
Glycine Greater than 1000 mg/L
Accuracy check
• Formaldehyde Voluette® Ampule Standard, 4000-mg/L CH2O
• Flask, 100-mL volumetric Class A
• Mixing cylinders, 50-mL (3)
4. Open a Formaldehyde Voluette® Ampule Standard, 4000-mg/L CH2O.
5. Use a TenSette® Pipet to add 0.2 mL of the standard to a 100-mL volumetric Class A flask.
Dilute to volume with formaldehyde-free water and mix well. Prepare daily. This is an
8000-µg/L (8-mg/L) formaldehyde standard.
6. Prepare three sample spikes. Fill three 50-mL mixing cylinders* with 25 mL of sample. Use the
TenSette Pipet to add 0.1 mL, 0.2 mL and 0.3 mL of 8000-µg/L standard, respectively, to each
sample and mix thoroughly.
sample spike. Accept each standard addition. Each addition should reflect approximately
100% recovery.
relationship between the sample spikes and the “Ideal Line” of 100% recovery

Prepare a 320-µg/L Formaldehyde Standard Solution by pipetting 1.0 mL of the 8000-µg/L solution
into a 50-mL mixing cylinder. Dilute to 25.0 mL with formaldehyde-free water. Run the test directly
on this sample.
Formaldehyde
Page 502
Formaldehyde
Method performance
200 320 µg/L CH2O 312–328 µg/L CH2O 3 µg/L CH2O
Summary of method
Formaldehyde reacts with MBTH (3-methyl-2-benzothiazoline hydrazone) and a developing
solution to form a blue color in proportion to the formaldehyde concentration. Test results are
measured at 630 nm.

Required reagents
Formaldehyde Reagent Set (100 tests), includes: — — 2257700
Developing Solution for LR Formaldehyde 5 mL 500 mL 2257249
MBTH Powder Pillows 2 100/pkg 2257169
Required apparatus
Formaldehyde Standard Solution, 10-mL Voluette® Ampule, 4000-mg/L 16/pkg 2257310

Chromic Acid Cleaning Solution 500 mL 123349
Potassium Permanganate 454 g 16801H
Sodium Hydroxide ACS 500 g 18734
Pipet Tips, Tensette 50/pkg 2185696
Wipes, disposable 280/pkg 2097000
Formaldehyde
Page 503
Hardness, 8030
Hardness DOC316.53.01043
Calcium and Magnesium;

Method 8030
Calmagite Colorimetric Method
0.05 to 4.00 mg/L Ca and Mg as CaCO3
Test preparation

For the most accurate magnesium test results, keep the sample temperature between 21–29 °C (70–84 °F).
The test will detect any calcium or magnesium contamination in the mixing cylinder, measuring droppers or sample cells. To
test cleanliness, repeat the test until results are consistent.
Total hardness in mg/L equals mg/L Ca as CaCO3 plus mg/L Mg as CaCO3.
Remaining traces of EDTA or EGTA from previous tests will give erroneous results. Rinse sample cells thoroughly
before using.
Alkali Solution for Calcium and Magnesium test 1 mL
Calcium and Magnesium Indicator Solution 1 mL
EDTA Solution, 1 M 1 drop
EGTA Solution 1 drop
Cylinder, 100-mL, graduated mixing 1
Dropper, measuring, 0.5 and 1.0 mL 2
Hardness
Page 505
Hardness
Calmagite
Stored Programs
225 Hardness, Mg
Start
1. Select the test. 2. Pour 100 mL of 3. Use a 1.0 mL 4. Stopper the cylinder
Insert an adapter if sample into a 100-mL measuring dropper to add and invert it several times.
required (Instrument- graduated mixing cylinder. 1.0 mL of Calcium and
specific information). Magnesium Indicator
solution.
for orientation.
5. Add 1.0 mL of Alkali 6. Stopper the cylinder 7. Pour 10 mL of the 8. Blank Preparation:
Solution for Calcium and and invert it several times. solution into each of three Add one drop of 1 M EDTA
Magnesium Test using a sample cells. Solution to the first cell
1.0 mL measuring (blank).
dropper.
Hardness
Page 506
Hardness
Calmagite (continued)
9. Swirl to mix. 10. Magnesium Sample: 11. Swirl to mix. 12. Insert the blank (first
Add one drop of EGTA cell) into the cell holder.
Solution to the second
cell.
Zero Read
13. ZERO the instrument. 14. Insert the magnesium 15. READ the results in 16. Do not remove the cell
The display will show: sample (second cell) into mg/L magnesium as from the instrument.
the cell holder. calcium carbonate. Record or store the
0.00 mg/L Mg CaCO3 magnesium results before
This value is the amount of
magnesium in the sample proceeding with step 17.
expressed as CaCO3.
Exit
220 Hardness, Ca
Zero Read
Start
17. EXIT the magnesium 18. ZERO the instrument. 19. Calcium Sample: 20. READ the results in
program The display will show: Insert the third cell into mg/L calcium as calcium
Select the calcium test. the cell holder. carbonate.
0.00 mg/L Ca CaCO3
This value is the amount of
Remove the second cell. calcium in the sample
expressed as CaCO3.
Hardness
Page 507
Hardness
Interferences
Chromium (Cr 3+) Above 0.25 mg/L
Copper (Cu 2+) Above 0.75 mg/L
EDTA Above 0.2 mg/L as CaCO3
Traces remaining in sample cells from previous tests will give erroneous results. Rinse cells
EDTA or EGTA
thoroughly before using.
Iron (Fe 2+) Above 1.4 mg/L
Iron (Fe 3+) Above 2.0 mg/L
Manganese (Mn 2+) Above 0.20 mg/L
Zinc (Zn 2+) Above 0.050 mg/L
For the most accurate calcium test result, rerun the test on a diluted sample if the calcium is over
Ca >1.0 mg/L;
1.0 and the magnesium is over 0.25 mg/L as CaCO3. No retesting is needed if either is below
Mg >0.25 mg/L
those respective concentrations.

• Adjust the sample pH to 2 or less with Nitric Acid (about 5 mL per liter).
• Cool samples to 4 °C. Preserved samples can be stored up to six months.
• Before analysis, adjust the sample pH to between 3 and 8 with 5.0 N Sodium Hydroxide
Standard Solution*.
• Correct the test results for volume additions.
Method performance
220 2.00 mg/L Ca 1.90–2.10 mg/L Ca 0.05 mg/L Ca
225 2.00 mg/L Mg 1.92–2.08 mg/L Mg 0.02 mg/L Mg
Summary of method
The colorimetric method for measuring hardness supplements the conventional titrimetric method
because the colorimetric method can measure very low levels of calcium and magnesium. Also,
some metals (those listed in Table 152) that interfere in the titrimetric method may be
inconsequential when diluting the sample to bring it within the range of this test. The indicator dye
is calmagite, which forms a purplish-blue color in a strongly alkaline solution and changes to red
when it reacts with free calcium or magnesium. Calcium and magnesium determinations are made
by chelating calcium with EGTA to destroy any red color due to calcium and then chelating the
calcium and magnesium with EDTA to destroy the red color due to both calcium and magnesium.
* See Optional Reagents and Apparatus on page 4
Hardness
Page 508
Hardness
By measuring the red color in the different states, calcium and magnesium concentrations are
determined. Test results are measured at 522 nm.
Hardness
Page 509
Required reagents
Hardness Reagent Set (100 tests), includes: — — 2319900
Alkali Solution for Calcium and Magnesium test 1 mL 100 mL MDB 2241732
Calcium and Magnesium Indicator Solution 1 mL 100 mL MDB 2241832
EDTA Solution, 1 M 1 drop 50 mL SCDB 2241926
EGTA Solution 1 drop 50 mL SCDB 2229726
Required apparatus
Cylinder, 100 mL, graduated mixing 1 each 189642
Dropper, measuring, 0.5 and 1.0 mL 2 20/pkg 2124720

Nitric Acid, ACS 500 mL 15249
pH Paper, 0–14 100/pkg 2601300

Hardness, 8374
Hardness DOC316.53.01044

Method 8374
Chlorophosphonazo Colorimetric Method
(8 to 1000 µg/L Ca and Mg as CaCO3) Solution Pillow
Scope and Application: For boiler and ultra-pure water.
Test preparation


The test will detect any calcium or magnesium contamination in the measuring droppers or sample cells. To test cleanliness,
repeat the test until results are consistent.
If the sample concentration is greater than 750 µg/L, a 1:1 dilution of the sample is recommended for greatest accuracy. Use
ultra-pure (aldehyde-free) water for the dilution. Repeat the analysis on the diluted sample and multiply the resulting
concentration by two.Alternate forms should only be used when the sample is known to contain only Mg or Ca. This method
does not distinguish between the two forms.
Alternate forms should only be used when the sample is known to contain only Mg or Ca. This method does not distinguish
between the two forms.
One mL of Chlorphosphonazo Solution may be used instead of the solution pillow in step 4.
Use dedicated plasticware for this analysis.
Use a bottle-top dispenser to dispense the reagent
ULR Hardness Reagent Set 1
Hardness
Page 511
Hardness
Chlorophosphonazo Indicator Solution Pillow 1
CDTA Solution 1 drop
Shears for opening powder pillow 1
Sample Cells (2410212) 1
Chlorophosphonazo method
Stored Programs
228 Hardness Tot ULR
Start
1. Select the test. 2. Rinse a plastic sample 3. Fill the plastic sample 4. Add the contents of
cell and the cap three cell to the 25-mL mark with one Chlorophosphonazo
times with the water to be sample. Solution Pillow to the
tested. Do not allow the sample cell.
underside of the cap to A small amount of solution
come in contact with may remain in the pillow.
surfaces that may This will not affect results.
contaminate it.
Zero
5. Cap the cell and swirl 6. Insert the cell into the 7. ZERO the instrument. 8. Remove the cell from
to mix. cell holder. The display will show: the holder. Add one drop
of CDTA Reagent for Ultra
0 µg/L CaCO3 Low Range Hardness.
Complete steps 10-11
within 1-2 minutes.
Hardness
Page 512
Hardness
Chlorophosphonazo method (continued)
Read
9. Cap the cell and swirl 10. Insert the cell into the 11. READ the results in
to mix. holder. µg/L CaCO3 .
Interferences
Interference studies were conducted at various hardness levels between 0 and 500 µg/L as
CaCO3 (Interfering substances and levels). Various cations and anions were evaluated at levels in
the range appropriate for ultra pure water applications. An ion is said to interfere when the
resulting concentration is changed by ± 10%.
Aluminum Negative interference above 150 µg/L
Ammonium No interference at or below 1000 µg/L
Copper Positive interference above 250 µg/L
Formaldehyde No interference at or below 47,000 µg/L
Nitrate Positive interference above 250 µg/L
Potassium No interference at or below 1000 µg/L
Silicon Positive interference above 1000 µg/L
Sodium Negative interference above 79,000 µg/L

• Do not use glass containers.
• Collect samples in clean plastic containers, preferably with screw-type closures.
• Rinse containers several times with the water to be analyzed before collecting the final
sample.
• Seal to avoid contamination during transport.
• Analyze as soon as possible.
Accuracy check
• Calcium Chloride Standard Solution, 50 mg/L (50,000 µg/L) as CaCO3
Hardness
Page 513
Hardness
1. Prepare a 20,000 µg/L (20 mg/L) CaCO3 standard by pipetting 20 mL of 50 mg/L CaCO3
standard solution into a 50 mL plastic volumetric flask. Dilute the solution with 50 mL of
ultra-pure water.

4. Accept the default sample volume (25 mL).
5. Use the TenSette Pipet to add 0.1 mL, 0.2 mL and 0.3 mL of the prepared 20,000 µg/L
standard, respectively to three 25-mL samples and mix each thoroughly.
6. Follow the test procedure for each of the spiked samples.
1. Use a 0.50 mg/L CaCO3 (500-µg/L as CaCO3) Calcium Chloride Standard Solution in place of
the sample.
Method performance
278 500 µg/L 478–522 mg/L 8 µg/L Ca
Summary of method
Calcium and magnesium combine equivalently with the Chlorophosphonazo III indicator to form a
colored complex which absorbs light very strongly at 669 nm. One drop of the CDTA reagent
breaks up this complex and the resultant decrease in color is proportional to the amount of calcium
and magnesium in the sample (as CaCO3). Test results are measured at 669 nm.
Required reagents
ULR Hardness Reagent Set (100 tests), includes: — — 2603100
(1) Chlorophosphonazo Indicator Solution Pillows 1 100/pkg 2589599
(1) CDTA Solution 1 drop 10 mL SCDB 2589636
(1) Sample cell, 1-inch square, molded 1 each 2410201
(1) Sample cell cap 1 each 2410202
Hardness
Page 514
Hardness
Required reagents
OR
ULR Hardness Reagent Set (500 tests), includes: — — 2603101
(1) Chlorophosphonazo Indicator Solution 1 mL 500 mL 2589549
(2) CDTA Solution 1 drop 10 mL SCDB 2589636
(1) Sample cell, 1-inch square, molded 1 each 2410201
(1) Sample cell cap 1 each 2410202
Chlorophosphonazo Indicator Solution Pillows 1 100/pkg 2589599
CDTA Solution 1 drop 10 mL SCDB 2589636
Required apparatus
Shears 1 each 2369400

Calcium Chloride Standard Solution, 50 mg/L as CaCO3 946 mL 2127716
Calcium Chloride Standard Solution, 500 µg/L as CaCO3 946 mL 2058016
Chlorophosphonazo Indicator Solution 500 mL 2589549

Dispenser, 1.0-mL, Repipet Jr. each 2111302
Flask, volumetric, polypropylene, 50 mL each 1406041
Pipet filler, Safety Bulb each 1465100
Sample cell, 25-mL plastic with cap 2 12/pkg
Hardness
Page 515
Hardness

Hardness, Total, 8374
Hardness, Total DOC316.53.01045

Method 8374
Chlorophosphonazo Rapid Liquid Method
ULR (4 to 1000 µg/L Ca and Mg as CaCO3) Pour-Thru Cell
Scope and Application: For boiler and ultra pure water.
Test preparation

DR 5000 LZV479 — —
Pre-clean the Pour-Thru Cell and all labware as specified in Treating analysis labware.
The test will detect any calcium or magnesium contamination in the mixing cylinder, measuring droppers, or sample cells. To
test cleanliness, repeat the test until results are consistent.
If the sample concentration is greater than 750 µg/L, a 1:1 dilution of the sample is recommended for greatest accuracy. Use
ultra-pure (aldehyde-free) water for the dilution. Repeat the analysis on the diluted sample and multiply the resulting
concentration by two.
Alternate forms should only be used when the sample is known to contain only Mg or Ca. This method does not distinguish
between the two forms.
Use dedicated plasticware for this analysis.
In bright light conditions (e.g. direct sunlight) it may be necessary to close the DR 2800 or DR 2700 cell compartment with the
protective cover during measurements.
Ensure the pour-thru cell is completely seated in the sample cell compartment.
Hardness, Total
Page 517
Hardness, Total
Chlorophosphonazo Indicator Solution 2 mL
CDTA Reagent for Ultra Low Range Hardness 1 drop
Water, Ultra-pure Varies
Cylinder, graduated, 50-mL, poly 1
Flask, Erlenmeyer, PMP w/cap, 125-mL 1
Pour-Thru Cell Module (see Instrument-specific information table) 1
Chlorophosphonazo rapid liquid method
Stored Programs
227 Hardness Tot RL ULR
Start
1. Select the test. 2. Flush the Pour-Thru 3. Fill a clean, 125-mL 4. Rinse a clean, 50-mL
Insert an adapter if Cell with 50 mL of ultra- plastic Erlenmeyer flask to plastic graduated cylinder
required (Instrument- pure water. overflowing with sample. three times with sample.
specific information). Collect sample directly in
the flask if possible.
5. Fill the rinsed cylinder 6. Pour the contents of 7. Add 2.0 mL of 8. Pour approximately
to the 50-mL mark with the 50-mL cylinder back Chlorophosphonazo half (25 mL) of the sample
sample from the flask. into the flask. Reagent to the sample into the Pour-Thru Cell.
Discard remaining with the Dispenser. Swirl Use a clean, dry, plastic
contents of the flask. to mix. 25-mL graduated cylinder
to measure the sample.
Hardness, Total
Page 518
Hardness, Total
Chlorophosphonazo rapid liquid method (continued)
Zero Read
9. After the flow stops, 10. Add one drop of CDTA 11. Pour the remaining 12. READ the results in
ZERO the instrument. Reagent for Ultra Low sample into the µg/L CaCO3.
The display will show: Range Hardness to the Pour-Thru Cell.
remaining sample in the
0 µg/L CaCO3 flask. Swirl to mix.
Complete steps 11 and 12
within one to two minutes.
13. Using a wash bottle,

rinse the Pour-Thru Cell
with at least 75 mL of ultra-
pure water immediately
after use.
Rinse the flask with ultra-
pure water. Cap when
finished.
Hardness, Total
Page 519
Hardness, Total
Interferences
Aluminum Negative interference above 150 µg/L
Ammonium No interference at or below 1000 µg/L
Copper Positive interference above 250 µg/L
Formaldehyde No interference at or below 47,000 µg/L
Potassium No interference at or below 1000 µg/L
Silicon Positive interference above 1000 µg/L
Sodium Negative interference above 79,000 µg/L

Clean all containers used in this test thoroughly to remove any traces of calcium or magnesium. If
possible, use plastic containers for all analysis and storage. Clean containers by normal means,
then rinse with ultra-pure (aldehyde-free) water. Fill and soak for 10 minutes with a 1:25 dilution of
Chlorophosphonazo Reagent in ultra-pure water. Rinse well with ultra-pure water. Keep containers
tightly closed and dedicate them for ULR Hardness only. If containers are rinsed and capped after
each use, only occasional soaking is necessary. Fill the Pour-Thru cell with this same mixture of
chlorophosphonazo and water and let stand for several minutes. Rinse with ultra-pure water.
Avoid contamination of the Chlorophosphonazo Reagent bottle when placing the Repipet
dispenser on the bottle. Rinse the inlet tubing and inside of the dispenser cap with copious
amounts of ultra-pure water using a wash bottle. Place the inlet tubing into a beaker of ultra-pure
water and depress the plunger 10–15 times to rinse the inside of the dispenser. (For best results,
pour a small amount of reagent into the beaker of rinse water.) Remove the dispenser from the
water and depress the plunger until all of the water has been expelled.
Shake off any excess water on the dispenser, place the dispenser on the bottle and tighten.

• Do not use glass containers.
• Collect samples in clean plastic containers, preferably with screw-type closures.
• Rinse containers several times with the water to be analyzed before collecting the final
sample.
• Seal to avoid contamination during transport.
• Analyze as soon as possible.
Accuracy check
• Calcium Chloride Standard Solution, 50 mg/L (50,000 µg/L) as CaCO3
• TenSette® Pipet and Pipet Tips
1. Prepare a 20,000 µg/L (20 mg/L) CaCO3 standard by pipetting 20 mL of 50 mg/L CaCO3
standard solution into a 50 mL plastic volumetric flask. Dilute the solution with 50 mL of ultra-
pure water.
Hardness, Total
Page 520
Hardness, Total
or edited. Accept the default values then read the unspiked sample measurement. After
values are accepted, the unspiked sample reading will appear in the top row. See the user
manual for more information.
5. Use the TenSette Pipet to prepare spiked samples: add 0.2 mL, 0.4 mL, and 0.6 mL of the
prepared 20,000 µg/L standard to three 50 mL portions of fresh sample.
6. Follow the test procedure for each of the spiked samples, starting with the 0.2 mL sample
spike. Measure each of the spiked samples in the instrument.

1. Using a 0.50 mg/L (500 µg/L as CaCO3) Calcium Chloride Standard Solution, perform the
procedure using the standard in place of the sample.
Method performance
227 0.500 mg/L Cr6+ 495–505 µg/L 8µg/L Ca
Summary of method
Calcium and magnesium combine equivalently with the Chlorophosphonazo Indicator to form a
colored complex which absorbs light very strongly at 669 nm. One drop of the CDTA reagent
breaks up this complex, and the resultant decrease in color is proportional to the amount of
calcium and magnesium (as CaCO3) in the sample. Test results are measured at 669 nm.
Required reagents
Chlorophosphonazo Indicator Solution 2 mL 500 mL 2589549
CDTA Reagent for Ultra Low Range Hardness 1 drop 10 mL SCDB 2589636
Required apparatus
Cylinder, graduated, 50 mL, poly 1 each 108141
Dispenser, variable-volume 1 each 2563137
Flask, Erlenmeyer, PMP w/cap, 125 mL 1 each 2089843
Hardness, Total
Page 521
Hardness, Total
Calcium Standard Solution, 50 mg/L as CaCO3 946 mL 2127716
Calcium Standard Solution, 0.50 mg/L as CaCO3 946 mL 2058016
Water, ultra-pure (aldehyde-free) 500 mL 2594649



Hardness, Calcium, DT, 8204
Hardness, Calcium DOC316.53.01175
Titration Method using EDTA Method 8204

Test preparation
Magnesium is not included in the results but must be present for a sharp end point. If magnesium is not present, add one to
two drops of Magnesium Standard Solution, 10-g/L as CaCO3 to the sample before the test is started.
One German degree of hardness (G.d.h.) = 17.9 mg/L hardness as CaCO3
mg/L Ca = Ca hardness in mg/L as CaCO3 x 0.40
A 0.1-g scoop of CalVer® 2 Calcium Indicator Powder can be used in place of the CalVer 2 Calcium Indicator Powder Pillow.
CalVer 2 Calcium Indicator Powder Pillow 1 pillow
Potassium Hydroxide Standard Solution, 8 N 1 bottle
EDTA titration cartridge (see Range-specific information—mg/L (mg/L) or Range-specific 1 cartridge

information—G.d.h. (German degrees of hardness))
Digital titrator 1
Hardness, Calcium
Page 523
Hardness, Calcium
Hardness, Calcium
See
Table 1
or
Table 2
specific information—mg/L cartridge to the titrator. delivery knob to eject air volume from the Range-
table or the Range-specific and a few drops of titrant. specific information—mg/L
information—G.d.h. table. Reset the counter to zero or the Range-specific
and wipe the tip. information—G.d.h. table.
Transfer the sample into a
clean, 250-mL Erlenmeyer
flask. If the sample volume
is less than 100 mL, dilute
to approximately 100 mL
is 100 mL, add 2 mL of one CalVer 2 Calcium into the solution and swirl the Range-specific
8 N Potassium Hydroxide Indicator Powder Pillow. the flask. Turn the knob on information—mg/L table
Standard Solution. If the Swirl to mix. the titrator to add titrant to (or the Range-specific
sample volume is 50 mL or the solution. Continue to information—G.d.h. table)
less, add 1 mL of swirl the flask and add to calculate the
8 N Potassium Hydroxide titrant until the color concentration:
Standard Solution. Swirl to changes from red to pure digits x multiplier =
mix. blue. mg/L (or G.d.h.) Ca as
Write down the number of CaCO3
counter.
Example:
50 mL of sample titrated with the 0.800 M EDTA titration cartridge, and 250 digits to reach the
endpoint yields a calcium concentration of 250 x 2.0 = 500 mg/L as CaCO3 (or with the 0.714 M
EDTA titration cartridge, 250 x 0.1 = 25 mg/L G.d.h.)
Hardness, Calcium
Page 524
Hardness, Calcium
Table 157 Range-specific information—mg/L

Range (mg/L as CaCO3) Sample volume (mL) Titration cartridge (M EDTA) Multiplier
10–40 100 0.0800 0.1

40–160 25 0.0800 0.4
100–400 100 0.800 1.0
200–800 50 0.800 2.0
500–2000 20 0.800 5.0
1000–4000 10 0.800 10.0
Table 158 Range-specific information—G.d.h.

Range (G.d.h as CaCO3) Sample volume (mL) Titration cartridge (M EDTA) Multiplier
1–4 100 0.1428 0.01

4–16 25 0.1428 0.04
10–40 50 0.714 0.1
25–100 20 0.714 0.25
> 100 10 0.714 0.5
Interferences
WARNING
Chemical hazard. Potassium cyanide is toxic. Always add it after the potassium hydroxide.
Follow local hazardous waste regulations for disposal of all cyanide-containing waste.
An interfering substance can prevent the color change at the titration end point. A dilution can
often reduce the interference to a level at which the substance does not interfere. If an interference
is suspected, decrease the sample volume, dilute to 100 mL and repeat the test.
Acidity The test can tolerate 10,000 mg/L acidity.
Alkalinity The test can tolerate 10,000 mg/L alkalinity .
Interferes by causing a slow end point but up to 200 mg/L aluminum can be tolerated by
Aluminum
allowing sufficient time for the color change.
Barium is titrated along with calcium but is seldom found in natural waters in significant
Barium
amounts.
Chloride Saturated solutions do not give a distinct end point. The test can be run directly in sea water
Interferes at all levels. 0.5 grams of potassium cyanide can be added after the potassium
Cobalt
hydroxide solution to remove interference from up to 20 mg/L cobalt.
Interferes at 0.1 mg/L copper. 0.5 grams of potassium cyanide can be added after the
Copper
potassium hydroxide solution to remove interference from up to 100 mg/L copper.
Interferes above 8 mg/L by causing an orange-red to green end point. Accurate results can
Iron
still be obtained up to approximately 20 mg/L iron with this end point.
Interference from magnesium is prevented up to 200 mg/L by the formation of magnesium
Magnesium
hydroxide at the high test pH but higher levels prevent a distinct end point.
Manganese Interferes above 5 mg/L.
Hardness, Calcium
Page 525
Hardness, Calcium

Interferes at 0.5 mg/L nickel. 0.5 grams of potassium cyanide can be added after the
Nickel
potassium hydroxide solution to remove interference from up to 200 mg/L nickel.
Causes a slow end point but does not interfere if the calcium phosphate that forms is allowed
Orthophosphate
to redissolve during the titration.
Polyphosphates Interfere directly and must be absent.
Strontium is titrated along with calcium but is seldom found in natural waters in significant
Strontium
amounts.
Samples at about 20 °C (68 °F) or colder should be titrated slowly near the end point to allow
Temperature
sufficient time for the color change.
Interferes at 5 mg/L zinc. 0.5 grams of potassium cyanide can be added after the potassium
Zinc
hydroxide solution to remove interference from up to 100 mg/L zinc.
Highly buffered samples or May exceed the buffering capacity of the reagents. Adjust the pH before starting the test
extreme sample pH (see Sample collection, preservation and storage).

• Collect samples in plastic or glass bottles that have been washed with a detergent and rinsed
with tap water.
• Rinse the bottles in 1:1 nitric acid solution and deionized water.
• The following storage instructions are necessary only when immediate analysis is not
possible. To preserve the sample, add 1.5 mL of nitric acid per liter (or quart) of sample. Mix.
Measure the sample pH to make sure that the pH is 2 or less. Add more nitric acid in 0.5-mL
increments if necessary. Mix thoroughly and check the pH after each addition until the pH is 2
or less.
• Preserved samples can be stored at least six months at room temperature.
• Before running the test, adjust the sample to pH 7 by adding potassium hydroxide standard
solution and mixing thoroughly.
Accuracy check
Use the standard additions method to find if the sample has an interference and to make sure that
the user has followed the procedure correctly.
• Hardness Voluette Ampule Standard Solution, 10,000-mg/L as CaCO3
• Ampule breaker
4. Repeat steps 2 and 3.
Hardness, Calcium
Page 526
Hardness, Calcium
5. Each 0.1 mL of standard that was added will use approximately 10 digits of the 0.800 M
titration cartridge or 100 digits of the 0.0800 M titration cartridge to reach the endpoint
(11 digits of 0.714 M or 56 digits of 0.1428 M titrant).
Summary of method
The sample is made alkaline (pH 12 to 13) with potassium hydroxide to precipitate magnesium as
magnesium hydroxide. CalVer 2 Calcium Indicator is added as an indicator and combines with any
calcium to form a red color. As the EDTA is added, it will react with all the free calcium ions
present. At the end point of the titration, when no free calcium ions are present, the EDTA will
remove the calcium complexed with the indicator. The indicator will then change from red to blue.

Required reagents
(1) CalVer 2 Calcium Indicator Powder Pillows 1 pillow 100/pkg 85299
(1) Potassium Hydroxide Standard Solution, 8 N 1–2 mL 100 mL MDB 28232H
(1) EDTA Titration Cartridge, 0.0800 M varies each 1436401
1–16 G.d.h. range—Reagent set (approximately 100 tests): 2447300
10–100+ G.d.h. range—Reagent set (approximately 100 tests): 2447400
Required apparatus
Hardness, Calcium
Page 527
Hardness, Calcium
Hardness Standard Solution, Voluette ampule, 10,000-mg/L as CaCO3, 10-mL 16/pkg 218710

CalVer® 2 Calcium Indicator Powder 113 g 28114H
Magnesium Standard Solution, 10-g/L as CaCO3 29 mL 102233
Nitric Acid Solution, ACS 500 mL 15249
CDTA Magnesium Salt Powder Pillow 100/pkg 1408099
Hardness 2 Indicator Solution 100 mL 42532
HexaVer Hardness Titrant, 0.020 N 1L 74053
Sodium Hydroxide Standard Solution, 5 N 50 mL 245026
Potassium cyanide 125 g 76714
Tensette Pipet tips 50/pkg 218596
Potassium hydroxide, 8 N 500 mL 28249
Bromphenol Green-Methyl Red indicator solution 100 mL MDB 2329232
pH meter each —
Delivery Tube, 180° Hook 5/pkg 1720500

Hardness, Calcium, BT, 8222
Hardness, Calcium DOC316.53.01157
USEPA1 Buret Titration Method2 Method 8222

0 to 25,000 mg/L as CaCO3 Buret Titration
1 USEPA accepted when 0.020 N titrant is used.
Test preparation
Magnesium is not included in the results but must be present for a sharp end point. If magnesium is not present, add one to
two drops of Magnesium Standard Solution, 10-g/L as CaCO3 to the sample before the test is started.
CalVer 2 Calcium Indicator Powder Pillow 1
Potassium Hydroxide Standard Solution, 8 N 1 mL
TitraVer Hardness Titrant (see Range-specific information) 1 bottle
Hardness, Calcium
Page 529
Hardness, Calcium
Buret titration
See
Table 1
volume and titrant the zero mark with the cylinder or pipet to into a 250-mL Erlenmeyer
concentration from the TitraVer Hardness Titrant. measure the sample flask. If the sample volume
Range-specific information volume from the Range- is less than 50 mL, dilute
table. specific information table. to approximately 50 mL
with deionized water. If
magnesium is known to be
absent, add one or two
drops of Magnesium
Standard Solution,
10-g/L as CaCO3.
5. Add 1 mL of 6. Add the contents of 7. Titrate the sample 8. Use the multiplier in
8 N Potassium Hydroxide one CalVer 2 Calcium while swirling the flask Range-specific information
Standard Solution using Indicator Powder Pillow. until the color changes to calculate the
the 1-mL dropper. Swirl to Swirl to mix. from red to pure blue. concentration:
mix. Magnesium must be mL titrant used x multiplier
present (and usually is in = mg/L calcium as CaCO3
natural waters) for a sharp Example: 50 mL of sample
end point, although it is not was titrated with the
measured in the titration. 0.020 N titrant solution
and 15 mL of titrant was
used to reach the
endpoint.
The calcium concentration
is: 15 x 20 = 300 mg/L as
CaCO3
Hardness, Calcium
Page 530
Hardness, Calcium

Range (mg/L as CaCO3) Sample volume (mL) Hardness titrant concentration Multiplier
0–500 50 0.020 N 20
400–1000 25 0.020 N 40
1000–2500 10 0.020 N 100
2000–5000 5 0.020 N 200
1000–5000 50 0.200 N 200
4000–10,000 25 0.200 N 400
10,000–25,000 10 0.200 N 1000
Table 161 Hardness conversions

To convert from To Multiply by
mg/L as CaCO3 mg/L as Ca 0.400
grains per gallon (gpg) 0.058
German degrees hardness (Gdh) 0.056
Interferences
WARNING
Dispose of all cyanide containing wastes according to local regulations.
reduce the interference to a level at which the substance does not interfere. If an interference is
suspected, decrease the sample volume, dilute to 50 mL and repeat the test.
Alkalinity The test can tolerate 10,000 mg/L alkalinity.
Interferes by causing a slow end point but up to 200 mg/L aluminum can be tolerated by
Aluminum
allowing sufficient time for the color change.
Barium is titrated along with calcium but is seldom found in natural waters in significant
Barium
amounts.
Saturated solutions do not give a distinct end point, but the test can be run directly under sea
Chloride
water.
Cobalt
Copper
Iron
Interference from magnesium is prevented up to 200 mg/L by the formation of magnesium
Magnesium
hydroxide at the high test pH but higher levels prevent a distinct end point.
Nickel
Hardness, Calcium
Page 531
Hardness, Calcium

Orthophosphate
Strontium is titrated along with calcium but is seldom found in natural waters in significant
Strontium
amounts.
Samples at about 20 °C (68 °F) or colder should be titrated slowly near the end point to allow
Temperature
sufficient time for the color change.
Zinc

Collect samples in plastic or glass bottles that have been washed with a detergent and rinsed with
tap water. Then rinse the bottles in 1:1 nitric acid solution and deionized water.
The following storage instructions are necessary only when immediate analysis is not possible. To
preserve the sample, add 1.5 mL of nitric acid per liter (or quart) of sample. Mix. Measure the
sample pH to make sure that the pH is 2 or less. Add more nitric acid in 0.5-mL increments if
necessary. Mix thoroughly and check the pH after each addition until the pH is 2 or less. Preserved
samples can be stored at least six months at room temperature.
Before running the test, adjust the sample to pH 7 by adding potassium hydroxide standard
solution and mixing thoroughly. Correct the test result for volume additions.
Accuracy check
solution method to confirm anaytical technique and reagent performance.
• Ampule breaker
• TenSette Pipet, 0.1–1.0 mL or 1.0–10.0 mL and Pipet Tips.

Hardness, Calcium
Page 532
Hardness, Calcium
concentration of the titrant has changed (see Accuracy check).

Complete the following test to confirm analytical technique and reagent performance.
1. Add 25.0 mL of a calcium chloride standard solution, 1000-mg/L as CaCO3, to an Erlenmeyer
flask. Dilute to 50 mL with deionized water and mix fully.
2. Add the potassium hydroxide and CalVer 2 indicator. Swirl to mix.
3. Titrate the standard to the end point with the 0.020 N hardness titrant and calculate the result.
The titration should use 25 mL of titrant.
1. Add 10.0 mL of a Hardness Voluette Ampule Standard Solution, 10,000-mg/L as CaCO3, to an

Erlenmeyer flask. Dilute to 50 mL with deionized water and mix fully.
2. Add the potassium hydroxide and CalVer 2 indicator. Swirl to mix.

The titration should use 10 mL of titrant.
Summary of method
The sample is made alkaline (pH 12 to 13) with potassium hydroxide to precipitate magnesium as
magnesium hydroxide. CalVer 2 Calcium Indicator is added as an indicator and combines with any
calcium to form a red color. As the TitraVer (EDTA) is added, it will react with all the free calcium
ions present. At the end point of the titration, when no free calcium ions are present, the EDTA will
remove the calcium complexed with the indicator. The indicator will then change from red to blue.
Hardness, Calcium
Page 533
Hardness, Calcium
Required reagents
Hardness (Calcium) Reagent Set (approximately 100 tests), includes: 2447000
CalVer 2 Calcium Indicator Powder Pillows 1 100/pkg 85299
Potassium Hydroxide Standard Solution, 8 N 1 mL 100 mL MDB 28232H
TitraVer® Hardness Titrant, 0.020 N varies 1L 20553
TitraVer Hardness Titrant, 0.200 N varies 500 mL 102149
Required apparatus

Calcium Chloride Standard Solution, 1000-mg/L as CaCO3 1L 12153
Pipet, Class A volumetric, 10 mL each 1451538
Hardness, Calcium
Page 534
Hardness, Calcium

Potassium Hydroxide Standard Solution, 8 N 500 mL 28249
Hardness, Calcium
Page 535
Hardness, Total, Sequential 8329
Hardness, Total, Sequential DOC316.53.01230

Test preparation
The first titration gives calcium hardness and the second titration gives total hardness. The difference between the values is
magnesium hardness. All of these concentrations are in mg/L as CaCO3. See Hardness conversions for conversions to
other units.
Four drops of Hardness 2 Indicator Solution or a 0.1-g scoop of ManVer 2 Hardness Indicator Powder can be added in place
of the ManVer 2 Hardness Indicator Powder Pillow.
Hardness 1 Buffer Solution 1 mL
ManVer 2 Hardness Indicator Powder Pillow 1
Sulfuric Acid Standard Solution, 5.25 N 1 mL

Digital titrator 1
Hardness, Total, Sequential

Page 537
Hardness, total, sequential
See
Table 1
or
Table 2
cartridge from Range- cartridge. Attach the tip pointing up. Turn the measure the sample
specific information—mg/L cartridge to the titrator. delivery knob to eject a volume from Range-
or Range-specific few drops of titrant. Reset specific information—mg/L
information—G.d.h.. the counter to zero and or Range-specific
wipe the tip. information—G.d.h..
is 100 mL, add 2 mL of one CalVer 2 Calcium into the solution and swirl Range-specific
8 N Potassium Hydroxide Indicator Powder Pillow. the flask. Turn the knob on information—mg/L (or
Standard Solution. If the Swirl to mix. the titrator to add titrant to Range-specific
sample volume is 50 mL or the solution. Continue to information—G.d.h.) to
less, add 1 mL of swirl the flask and add calculate the
8 N Potassium Hydroxide titrant until the color concentration:
Standard Solution. Swirl to changes from red to pure Total digits from step 7 and
mix. blue. step 12 x multiplier =
Write down the number of mg/L (or G.d.h.) Calcium
digits on the counter. as CaCO3

Page 538
Hardness, total, sequential (continued)
9. Add 1 mL of 5.25 N 10. Add 2 mL of 11. Add the contents of 12. Place the delivery tube
Sulfuric Acid Standard Hardness 1 Buffer one ManVer 2 Hardness into the solution and swirl
Solution. Continue to add Solution. Swirl to mix. Indicator Powder Pillow. the flask. Turn the knob on
the acid by drops while Swirl to mix. the titrator to add titrant to
swirling until the solution the solution. Continue to
changes from pure blue to swirl the flask and add
purple and finally to red. titrant until the color
Swirl the flask to make changes from red to pure
sure that all the blue.
precipitated magnesium Write down the number of
hydroxide has dissolved. digits on the counter.
13. Use the multiplier in

Range-specific
information—mg/L (or
Range-specific
information—G.d.h.) to
calculate the
concentration:
digits x multiplier =
mg/L (or G.d.h.) total
hardness as CaCO3

Page 539

10–40 100 0.0800 0.1

40–160 25 0.0800 0.4
100–400 100 0.800 1.0
200–800 50 0.800 2.0
500–2000 20 0.800 5.0
1000–4000 10 0.800 10.0

1–4 100 0.1428 0.01

4–16 25 0.1428 0.04
10–40 50 0.714 0.1
25–100 20 0.714 0.25
> 100 10 0.714 0.5

mg/L as Mg 0.243
Hardness relationships
mg/L Mg Hardness as CaCO 3
= mg/L Total Hardness as CaCO 3 – mg/L Ca Hardness as CaCO 3
mg/L MgCO 3 = mg/L Mg Hardness as CaCO 3 × 0.842
mg/L Mg = mg/L MgCO × 0.29

3
Interferences
WARNING:
Do not use potassium cyanide to eliminate interferences because it will generate deadly
hydrogen cyanide gas when the sulfuric acid solution is added.
• Although less common than calcium and magnesium, other polyvalent metal ions cause the
same hardness effects and will be included in the results.
• Some transition and heavy metals complex the indicator and prevent the color change at the
end point.

Page 540
• Iron does not interfere up to 15 mg/L. Above this level it causes a red-orange to green end
point which is sharp and usable up to 30 mg/L iron. Substitute a 0.0800 M CDTA or 0.800 M
CDTA titration cartridge for the 0.0800 M EDTA or 0.800 M EDTA titration cartridges,
respectively, if iron interference is probable. For results in G.d.h., divide the mg/L result by
17.9.
• Manganese titrates directly up to 20 mg/L but masks the end point above this level. Adding a
0.1-gram scoop of hydroxylamine hydrochloride raises this level to 200 mg/L manganese.
• Copper interferes at levels of 0.10 and 0.20 mg/L. Cobalt and nickel interfere at all levels and
must be absent or masked.
• Orthophosphate causes a slow end point and polyphosphate must be absent for accurate
results.
• Acidity and alkalinity at 10,000 mg/L (as CaCO3) do not interfere.
• Saturated sodium chloride solutions do not give a distinct end point, but the titration can be run
directly on sea water.
• Adding the contents of one CDTA Magnesium Salt Powder Pillow removes metal interferences
at or below the levels shown in Metal interferences.
• If more than one metal is present at or above the concentrations shown in Metal interferences,
an additional CDTA Magnesium Salt Powder Pillow may be required.
• Results obtained by this procedure include the hardness contributed by polyvalent metal ions.
If the concentration of each metal is known, a correction can be applied to obtain the calcium
and magnesium hardness concentration. The hardness (in mg/L as CaCO3) contributed by
each mg/L of metal is listed in Hardness contributed by each mg/L of metal. Hardness
contributed by metals can be subtracted from the total hardness value obtained to determine
the calcium and magnesium hardness concentration.
• Barium, strontium and zinc titrate directly.
Table 166 Metal interferences

CDTA Removes Interference Below this
Metal
Level
Aluminum 50 mg/L
Cobalt 200 mg/L
Copper 100 mg/L
Iron 100 mg/L
Manganese 200 mg/L
Nickel 400 mg/L
Zinc 300 mg/L

Page 541
Table 167 Hardness contributed by each mg/L of metal

Hardness as CaCO3 Contributed
Metal
by Each mg/L of Metal
Aluminum 3.710
Barium 0.729
Cobalt 1.698
Copper 1.575
Iron 1.792
Manganese 1.822
Nickel 1.705
Strontium 1.142
Zinc 1.531

with tap water. Then rinse the bottles in 1:1 nitric acid solution and deionized water.
• Measure the sample pH to make sure that the pH is 2 or less. Add more nitric acid in 0.5-mL
increments if necessary.
• Mix thoroughly and check the pH after each addition until the pH is 2 or less.
• Store samples at 4 °C (39 °F) or below. Preserved samples can be stored for at least six
months.
• Before starting the test, warm the sample to room temperature and adjust the pH to
approximately pH 7 with potassium hydroxide solution. Mix thoroughly.
• If a significant amount of nitric acid was added, make a volume correction for the extra acid
and hydroxide.
• Divide the total volume (sample + acid + hydroxide) by the volume of the sample and multiply
the result from the test by this value.
Summary of method
This test procedure is a combination of the calcium and total hardness procedures. Refer to each
method for a detailed description of the methods.

Page 542

Required reagents
Calcium and Total Hardness Reagent Set (approximately 100 tests) 2272100
(1) Buffer Solution, Hardness 1 1 mL 100 mL MDB 42432
(1) CalVer 2 Calcium Indicator Powder Pillows 1 100/pkg 94799
(1) ManVer 2 Hardness Indicator Powder Pillows 1 100/pkg 85199
(2) Potassium Hydroxide Standard Solution, 8 N 1 mL 100 mL MDB 28232H
Sulfuric Acid Standard Solution, 5.25 N varies 100 mL MDB 244932
Required apparatus
Delivery tube for Digital Titrator, 180° hook 1 5/pkg 1720500
Delivery tube for Digital Titrator, 90° hook 1 5/pkg 4157800

Page 543


pH paper, 0–14 100/pkg 2601300
Spoon, measuring, 0.1 g capacity each 51100
Hydroxylamine Hydrochloride 113 g 24614
Magnesium CDTA powder pillows 100/pkg 1408099
CDTA cartridge for digital titrator, 0.08 M each 1440201
CDTA cartridge for digital titrator, 0.80 M each 1440301

Hardness, Total, DT, 8213

Test preparation
Hardness conversions: grains per gallon (gpg) as CaCO3 = mg/L x 0.058; German degrees hardness (Gdh) = mg/L x 0.056;
mg/L Total Hardness as Ca = mg/L Total Hardness as CaCO3 x 0.40
mg/L Total Hardness as CaCO3 = mg/L Ca as CaCO3 + mg/L Mg as CaCO3.

Digital titrator 1
Hardness, Total
Page 545
Hardness, Total
Hardness, Total
See
Table 1
or
Table 2
specific information—mg/L cartridge to the titrator. delivery knob to eject air volume from the Range-
table or the Range-specific and a few drops of titrant. specific information—mg/L
information—G.d.h. table. Reset the counter to zero table or the Range-specific
and wipe the tip. information—G.d.h. table.
Hardness, Total
Page 546
Hardness, Total
Hardness, Total
5. Add 2 mL of Hardness 6. Add the contents of 7. Place the delivery tube 8. Use the multiplier in
1 Buffer Solution and swirl one ManVer 2 Hardness into the solution and swirl the Range-specific
to mix. Indicator Powder Pillow. the flask. Turn the knob on information—mg/L table
Swirl to mix. the titrator to add titrant to (or the Range-specific
the solution. Continue to information—G.d.h. table)
swirl the flask and add to calculate the
titrant until the color concentration:
changes from red to pure digits x multiplier =
blue. mg/L (or G.d.h.) total
Write down the number of hardness as CaCO3
digits on the counter. Example: 50 mL of sample
0.800 M EDTA titration
cartridge and 250 digits
endpoint. The total
hardness concentration is:
250 x 2.0 = 500 mg/L as
CaCO3 (or with the
0.714 M EDTA titration
cartridge, 250 x 0.1 = 25
mg/L G.d.h.)

10–40 100 0.0800 0.1

40–160 25 0.0800 0.4
100–400 100 0.800 1.0
200–800 50 0.800 2.0
500–2000 20 0.800 5.0
1000–4000 10 0.800 10.0
Hardness, Total
Page 547
Hardness, Total

1–4 100 0.1428 0.01

4–16 25 0.1428 0.04
10–40 50 0.714 0.1
25–100 20 0.714 0.25
> 100 10 0.714 0.5
Interferences
WARNING
Follow local hazardous waste regulations for disposal of all cyanide-containing waste.
often reduce the interference to a level at which the substance does not interfere. If an interference
is suspected, decrease the sample volume, dilute to 100 mL and repeat the test.
Alkalinity The test can tolerate 10,000 mg/L alkalinity and can be run directly in sea water.
Aluminum interferes above 0.20 mg/L aluminum. 0.5 grams of potassium cyanide can be
Aluminum
added after the buffer solution to remove interference from up to 1 mg/L aluminum.
Barium Barium is included in the results but is seldom found in natural waters in significant amounts.
Cobalt
Copper
Iron
Nickel
Orthophosphate
Although less common than calcium and magnesium, other polyvalent metal ions cause the
Polyvalent metal ions
Sodium chloride Saturated solutions do not give a distinct end point.
Strontium is included in the results but is seldom found in natural waters in significant
Strontium
amounts.
Zinc
Hardness, Total
Page 548
Hardness, Total
The addition of one CDTA Magnesium Salt Powder Pillow will remove metals interferences at or
below the levels shown in Interference level with CDTA pillow. If more than one metal is present at
or above the concentrations in Interference level with CDTA pillow, an additional CDTA
Magnesium Salt Powder Pillow may be necessary.
Table 171 Interference level with CDTA pillow
Interfering substance Interference level, mg/L
Aluminum 50
Cobalt 200
Copper 100
Iron 100
Manganese 200
Nickel 400
Zinc 300
The results obtained with CDTA Magnesium Salt will include the hardness contributed by the
metals. If the concentration of each metal is known, a correction can be made to obtain the
hardness contributed by calcium and magnesium only. The hardness contributed per mg/L metal
ion is listed in Hardness equivalence factors. The mg/L of metal in the sample multiplied by its
calcium carbonate hardness equivalent factor should be subtracted from the total hardness
determined in step 3 of the procedure to obtain the hardness contributed by calcium and
magnesium only.
Table 172 Hardness equivalence factors
Interfering substance Hardness equivalence factors, mg/L as CaCO3
Aluminum 3.710
Barium 0.729
Cobalt 1.698
Copper 1.575
Iron 1.792
Manganese 1.822
Nickel 1.705
Strontium 1.142
Zinc 1.531
Hardness, Total
Page 549
Hardness, Total

with tap water.
• Rinse the bottles in 1:1 nitric acid solution and deionized water.
Measure the sample pH to make sure that the pH is 2 or less. Add more nitric acid in 0.5-mL
increments if necessary. Mix thoroughly and check the pH after each addition until the pH is 2
or less.
• Store samples at 4 °C (39 °F) or below. Preserved samples can be stored for at least seven
days.
approximately pH 7 with 5.0 N sodium hydroxide.
• Mix thoroughly. If a significant amount of nitric acid was added, make a volume correction for
the extra acid and hydroxide. Divide the total volume (sample + acid + hydroxide) by the
volume of the sample and multiply the result from the test by this value.
Accuracy check
• Ampule breaker
titration cartridge or 100 digits of the 0.0800 M titration cartridge to reach the endpoint
(11 digits of 0.714 M or 56 digits of 0.1428 M titrant).
Hardness, Total
Page 550
Hardness, Total

Complete the following test to make sure that the reagents are user technique are accurate.
• Calcium Chloride Standard Solution, 1000-mg/L as CaCO3
1. Add 20.0 mL of the standard solution to an Erlenmeyer flask. Dilute to about 100 mL with
deionized water and mix fully.
2. Add the Hardness 1 Buffer Solution and ManVer 2 indicator. Swirl to mix.
3. Titrate the standard to the end point with the titration cartridge and calculate the result. The
result should be close to 1000 mg/L or 55.9 G.d.h. as CaCO3.
Summary of method
In the total hardness test, the water sample is first buffered (using an organic amine and one of its
salts) to a pH of 10.1. An organic dye, calmagite, is added as the indicator for the test. This dye
reacts with calcium and magnesium ions to give a red-colored complex.
EDTA (ethylenediaminetetraacetic acid) is added as a titrant. The EDTA will react with all free
calcium and magnesium ions in the sample. At the end point of the titration, when free magnesium
ions are no longer available, EDTA will remove magnesium ions from the indicator, causing a color
change from red to blue.

Required reagents
1–16 G.d.h. range—Reagent set (approximately 100 tests): 2447800
10–100+ G.d.h. range—Reagent set (approximately 100 tests): 2447900
Required apparatus
Hardness, Total
Page 551
Hardness, Total


Pipet, Volumetric, 10 mL Class A each 1451538
Pipet, Volumetric, 20 mL Class A each 1451520
Pipet filler safety bulb each 1465100
Delivery Tube, 180° Hook 5/pkg 1720500

Hardness, Total, BT, 8226
USEPA1 ManVer 2 Buret Titration Method2 Method 8226

1 USEPA accepted when 0.020 N titrant is used.
Test preparation
Hardness conversions: grains per gallon (gpg) as CaCO3 = mg/L x 0.058; German degrees hardness (Gdh) = mg/L x 0.056
Buret titration
See
Table 1
volume and titrant the zero mark with the cylinder or pipet to into a 250-mL Erlenmeyer
concentration from Range- TitraVer Hardness Titrant. measure the sample flask. If the sample volume
specific information. volume from the Range- is less than 50 mL, dilute
specific information table. to approximately 50 mL
Hardness, Total
Page 553
Hardness, Total
Hardness 1 Buffer one ManVer 2 Hardness while swirling the flask the Range-
Solution using the 1-mL Indicator Powder Pillow. until the color changes specific information to
dropper. Swirl to mix. Swirl to mix. from red to pure blue. calculate the
concentration:
mL titrant used x multiplier
= mg/L total hardness as
CaCO3
0.020 N titrant and 15 mL
of titrant was used to
reach the endpoint.
The hardness
concentration is: 15 x 20 =
300 mg/L as CaCO3

0–500 50 0.020 N 20
400–1000 25 0.020 N 40
1000–2500 10 0.020 N 100
1000–5000 50 0.200 N 200
4000–10,000 25 0.200 N 400
10,000–25,000 10 0.200 N 1000
Interferences
WARNING
Chemical hazard. Potassium cyanide is toxic. Always add it after the buffer solution.
Dispose of all cyanide containing wastes according to local regulations
reduce the interference to a level at which the substance does not interfere. If an interference is
suspected, decrease the sample volume, dilute to 50 mL and repeat the test.
Hardness, Total
Page 554
Hardness, Total
Acidity Acidity at 10,000 mg/L as CaCO3 does not interfere.
Alkalinity Alkalinity at 10,000 mg/L as CaCO3 does not interfere.

Aluminum interferes above 0.20 mg/L aluminum. 0.5 grams of potassium cyanide can be added
Aluminum
after the buffer solution to remove interference from up to 1 mg/L aluminum.
Barium Barium is titrated directly but is seldom found in natural waters in significant amounts.
Chloride Saturated solutions do not give a distinct end point. The test can be run directly in sea water.
Interferes at all levels and must be absent or masked. 0.5 grams of potassium cyanide can be
Cobalt
added after the buffer solution to remove interference from up to 100 mg/L cobalt.
Copper interferes above 0.10 mg/L copper. 0.5 grams of potassium cyanide can be added after
Copper
the buffer solution to remove interference from up to 100 mg/L copper.
still be obtained up to approximately 30 mg/L iron with this end point. For slightly sharper end
Iron
points in solutions containing higher levels of iron, HexaVer Hardness Titrant (CDTA) can be
used in place of the TitraVer Hardness Titrant (EDTA).
Interferes above 20 mg/L. Adding a 0.10-gram scoop of Hydroxylamine Hydrochloride
Manganese
Monohydrate raises this level to 200 mg/L manganese.
Interferes at all levels and must be absent or masked. 0.5 grams of potassium cyanide can be
Nickel
added after the buffer solution to remove interference from up to 100 mg/L nickel.
Causes a slow end point but does not interfere if the calcium phosphate that forms is allowed to
Orthophosphate
redissolve during the titration.
Although less common than calcium and magnesium, other polyvalent metal ions cause the
Polyvalent metal ions
Strontium Strontium is titrated directly but is seldom found in natural waters in significant amounts.
Titrates directly. 0.5 grams of potassium cyanide can be added after the buffer solution to
Zinc
remove interference from up to 100 mg/L zinc.
The addition of one CDTA Magnesium Salt Powder Pillow will remove metals interferences at or
below the levels shown in the Interference level with CDTA pillow table. If more than one metal is
present at or above the concentrations in the Interference level with CDTA pillow table, an
additional CDTA Magnesium Salt Powder Pillow may be necessary.
Table 175 Interference level with CDTA pillow
Interfering substance Interference level, mg/L
Aluminum 50
Cobalt 200
Copper 100
Iron 100
Manganese 200
Nickel 400
Zinc 300
The results obtained with CDTA Magnesium Salt will include the hardness contributed by those
soluble metal ions present. If the concentration of soluble metal ion is known, a correction can be
made to obtain the hardness contributed by calcium and magnesium only. The hardness
Hardness, Total
Page 555
Hardness, Total
contributed per mg/L metal ion is listed in Hardness equivalence factors for the individual metals.
The mg/L of metal present multiplied by its calcium carbonate hardness equivalent factor should
be subtracted for each metal present from the total hardness determined in step 8 of the procedure
to obtain the hardness contributed by calcium and magnesium only.
Table 176 Hardness equivalence factors

Interfering substance Hardness equivalence factors, mg/L as CaCO3
Aluminum 3.710
Barium 0.729
Cobalt 1.698
Copper 1.575
Iron 1.792
Manganese 1.822
Nickel 1.705
Strontium 1.142
Zinc 1.531

Collect samples in plastic or glass bottles that have been washed with a detergent and rinsed with
tap water. Then rinse the bottles in 1:1 nitric acid solution and deionized water.
The following storage instructions are necessary only when immediate analysis is not possible. To
preserve the sample, add 1.5 mL of nitric acid per liter (or quart) of sample. Mix. Measure the
sample pH to make sure that the pH is 2 or less. Add more nitric acid in 0.5-mL increments if
necessary. Mix thoroughly and check the pH after each addition until the pH is 2 or less. Store
samples at 4 °C (39 °F) or below. Preserved samples can be stored for at least seven days.
Before starting the test, warm the sample to room temperature and adjust the pH to approximately
pH 7 with 5.0 N sodium hydroxide. Mix thoroughly. If a significant amount of nitric acid was added,
make a volume correction for the extra acid and hydroxide. Divide the total volume (sample + acid
+ hydroxide) by the volume of the sample and multiply the result from the test by this value.
Accuracy check
solution method to confirm analytical technique and reagent performance.
• Ampule breaker
• TenSette Pipet, 0.1–1.0 mL or 1.0–10.0 mL and Pipet Tips
Hardness, Total
Page 556
Hardness, Total

Complete the following test to confirm the analytical technique and reagent performance.

The titration should use 25 (± 0.3) mL of titrant.

Hardness, Total
Page 557
Hardness, Total
Summary of method
In the total hardness test, the water sample is first buffered (using an organic amine and one of its
salts) to a pH of 10.1. An organic dye, calmagite, is added as the indicator for the test. This dye
reacts with calcium and magnesium ions to give a red-colored complex.
EDTA (ethylenediaminetetraacetic acid) is added as a titrant. The EDTA will react with all free
calcium and magnesium ions present in the sample. At the end point of the titration, when free
magnesium ions are no longer available, EDTA will remove magnesium ions from the indicator,
causing a color change from red to blue.

Required reagents
Hardness (Total) Reagent Set (approximately 100 tests), includes; 2447600
(1) TitraVer® Hardness Titrant, 0.020 N varies 1L 20553
Hardness (Total) Reagent Set (approximately 100 tests), includes: 2447700
(1) TitraVer® Hardness Titrant, 0.200 N varies 500 mL 102149
Required apparatus

Hardness, Total
Page 558
Hardness, Total

Hardness, Total
Page 559
Hardness, Total, Sequential, BT, 8338
Hardness, Total, Sequential DOC316.53.01159

Test preparation
The first titration gives calcium hardness and the second titration gives total hardness. The difference between the values is
magnesium hardness. All of these concentrations are in mg/L as CaCO3. See Hardness conversions for conversions to
other units.
Two drops of Hardness 2 Indicator Solution can be added in place of the ManVer 2 Hardness Indicator Powder Pillow.
Sulfuric Acid Standard Solution, 5.25 N 1 mL

Page 561
Buret titration
See
Table 1
1. Select the sample 2. Fill a 25-mL buret to 3. Use a graduated 4. Add 1 mL of

volume and titrant the zero mark with the cylinder or pipet to 8 N Potassium Hydroxide
concentration from Range- TitraVer Hardness Titrant. measure the sample Standard Solution using
specific information. volume from Range- the 1-mL dropper. Swirl to
specific information. mix.
250-mL Erlenmeyer flask.
If the sample volume is
less than 50 mL, dilute to
approximately 50 mL with
deionized water.
5. Add the contents of 6. Titrate the sample 7. Use the multiplier in 8. Add 1 mL of 5.25 N
one CalVer 2 Calcium while swirling the flask Range-specific information Sulfuric Acid Standard
Indicator Powder Pillow. until the color changes to calculate the Solution. Continue to add
Swirl to mix. from red to pure blue. concentration: the acid by drops while
Magnesium must be mL titrant used x multiplier swirling until the solution
present (and usually is in = mg/L calcium as CaCO3 changes from pure blue to
natural waters) for a sharp purple and finally to red.
end point. Record the Swirl the flask to make
volume of titrant used. sure that all the
precipitated magnesium
hydroxide has dissolved.

Page 562
Hardness 1 Buffer one ManVer 2 Hardness while swirling the flask Range-specific information
Solution using the 1-mL Indicator Powder Pillow. until the color changes to calculate the
dropper. Swirl to mix. Swirl to mix. from red to pure blue. concentration:
Record the volume of Total mL titrant used in
titrant used. step 6 and step 11 x
multiplier = mg/L total
hardness as CaCO3
0.020 N titrant and 15 mL
of titrant was used to
reach the endpoint.
The hardness
concentration is: 15 x 20 =
300 mg/L as CaCO3

0–500 50 0.020 N 20
400–1000 25 0.020 N 40
1000–2500 10 0.020 N 100
1000–5000 50 0.200 N 200
4000–10,000 25 0.200 N 400
10,000–25,000 10 0.200 N 1000

mg/L as Mg 0.243

Page 563
Hardness relationships
mg/L Mg Hardness as CaCO 3
= mg/L Total Hardness as CaCO – mg/L Ca Hardness as CaCO

3 3
mg/L MgCO = mg/L Mg Hardness as CaCO × 0.842

3 3
mg/L Mg = mg/L MgCO × 0.29

3
Interferences
WARNING:
Do not use potassium cyanide to eliminate interferences because it will generate deadly
hydrogen cyanide gas when the sulfuric acid solution is added.
• Although less common than calcium and magnesium, other polyvalent metal ions cause the
• Some transition and heavy metals complex the indicator and prevent the color change at the
end point.
• Iron does not interfere up to 15 mg/L. Above this level it causes a red-orange to green end
point which is sharp and usable up to 30 mg/L iron. For results in G.d.h., divide the mg/L result
by 17.9.
• Manganese titrates directly up to 20 mg/L but masks the end point above this level. Adding a
0.1-gram scoop of hydroxylamine hydrochloride raises this level to 200 mg/L manganese.
• Copper interferes at levels of 0.10 and 0.20 mg/L. Cobalt and nickel interfere at all levels and
must be absent or masked.
• Orthophosphate causes a slow end point and polyphosphate must be absent for accurate
results.
• Acidity and alkalinity at 10,000 mg/L (as CaCO3) do not interfere.
• Saturated sodium chloride solutions do not give a distinct end point, but the titration can be run
directly on sea water.
• Adding the contents of one CDTA Magnesium Salt Powder Pillow removes metal interferences
at or below the levels shown in the Metal interferences table.
• If more than one metal is present at or above the concentrations shown in the Metal
interferences table, an additional CDTA Magnesium Salt Powder Pillow may be required.

Page 564
• Results obtained by this procedure include the hardness contributed by polyvalent metal ions.
If the concentration of each metal is known, a correction can be applied to obtain the calcium
and magnesium hardness concentration. The hardness (in mg/L as CaCO3) contributed by
each mg/L of metal is listed in the Hardness of each mg/L of metal table. Hardness contributed
by metals can be subtracted from the total hardness value to determine the calcium and
magnesium hardness concentration.
• Barium, strontium and zinc titrate directly.
Table 179 Metal interferences

CDTA removes interference below this
Metal
level
Aluminum 50 mg/L
Cobalt 200 mg/L
Copper 100 mg/L
Iron 100 mg/L
Manganese 200 mg/L
Nickel 400 mg/L
Zinc 300 mg/L
Table 180 Hardness of each mg/L of metal

Hardness as CaCO3 contributed
Metal
by each mg/L of Metal
Aluminum 3.710
Barium 0.729
Cobalt 1.698
Copper 1.575
Iron 1.792
Manganese 1.822
Nickel 1.705
Strontium 1.142
Zinc 1.531

with tap water. Then, rinse the bottles in 1:1 nitric acid solution and deionized water.
• The storage instructions are necessary only when immediate analysis is not possible. To
preserve the sample, add 1.5 mL of nitric acid per liter (or quart) of sample. Mix.
• Measure the sample pH to make sure that the pH is 2 or less. Add more nitric acid in 0.5-mL
increments if necessary.
• Mix thoroughly and check the pH after each addition until the pH is 2 or less.
• Store samples at 4 °C (39 °F) or below. Preserved samples can be stored for at least six
months.
approximately pH 7 with potassium hydroxide solution. Mix thoroughly.
• If a significant amount of nitric acid was added, make a volume correction for the extra acid
and hydroxide.

Page 565
• Divide the total volume (sample + acid + hydroxide) by the volume of the sample and multiply
the result from the test by this value.
Accuracy check
solution method to make sure that the user has followed the test correctly and that the reagents
are good.
• Ampule breaker
2. Use the TenSette Pipet to add 0.1 mL of the standard to the titrated sample from step 7 or
step 12. Swirl to mix.
2. Use the TenSette Pipet to add 1.0 mL of the standard to the titrated sample from step 7 or
step 12. Swirl to mix.

Page 566

Complete the following test to make sure that the reagents are user technique are accurate.

2. Add the reagents as shown in the test procedure. Swirl to mix.

2. Add the reagents as shown in the test procedure. Swirl to mix.
Summary of method
This test procedure is a combination of the calcium and total hardness procedures. Refer to each
method for a detailed description of the methods.

Required reagents
Hardness (Total) Reagent Set (approximately 100 tests), includes; 2448200
(1) CalVer 2 Calcium Indicator Powder Pillows 1 100/pkg 94799
(1) Potassium Hydroxide Standard Solution, 8 N 1 mL 100 mL MDB 28232H
(1) Sulfuric Acid Standard Solution, 5.25 N varies 100 mL MDB 244932
(1) TitraVer® Hardness Titrant, 0.020 N varies 1L 20553
TitraVer® Hardness Titrant, 0.200 N varies 500 mL 102149
Required apparatus

Page 567

pH paper, 0–14 100/pkg 2601300
Hydroxylamine Hydrochloride 113 g 24614
Magnesium CDTA powder pillows 100/pkg 1408099
Hardness 2 indicator solution 100 mL MDB 42532

Hydrazine, 8141
Hydrazine DOC316.53.01046
p-Dimethylaminobenzaldehyde Method1 Method 8141

4 to 600 µg/L N2H4 Reagent Solution or AccuVac® Ampuls
Scope and Application: For boiler water/feedwater.
1 Adapted from ASTM Manual of Industrial Water, D1385-78, 376 (1979).
Test preparation

Reagent solution AccuVac Ampuls

Instrument
Sample cell Cell orientation Adapter
DR 6000 2495402 Fill line faces right —
DR 5000 2495402 Fill line faces user —
DR 3900 2495402 Fill line faces user LZV846 (A)
DR 3800, DR 2800, DR 2700 2495402 Fill line faces right LZV584 (C)
Samples cannot be preserved and must be analyzed immediately
Sample temperature should be 21 ± 4 °C (70 ± 7 °F).
After adding the HydraVer® 2 Hydrazine Reagent, a yellow color will develop in the sample if hydrazine is present. The blank
may also have a faint yellow color.
Important Note: The final samples will have a pH less than 2, which is considered corrosive (0002) by the
Federal RCRA. Refer to the MSDS for disposal instructions.
Solution Test:
HydraVer 2 Reagent Solution 1 mL
Graduated Cylinder, 25 mL 1
AccuVac Test:
HydraVer 2 Reagent AccuVac® Ampuls 2
Hydrazine
Page 569
Hydrazine
Collect the following items (continued):
Beaker, 50 mL 1
Stopper for Ampules 2
Reagent solution method
Stored Programs
231 Hydrazine
Start
1. Select the test. 2. Blank Preparation: 3. Prepared Sample: 4. Add 0.5 mL of

Insert an adapter if Use a graduated cylinder Use a graduated cylinder HydraVer 2 Hydrazine
required (Instrument- to pour 10 mL of deionized to pour 10 mL of sample Reagent to each sample
specific information). water into a sample cell. into a second sample cell. cell. Swirl to mix.

for orientation.
Zero
5. Start the instrument 6. Insert the blank cell. 7. ZERO the instrument. 8. Insert the prepared
timer. The display will show: sample cell.
A 12-minute reaction 0 µg/L N2H4 Immediately after the
period will begin. timer expires READ the
Complete steps 6–8 results in µg/L N2H4.
during this period.
Hydrazine
Page 570
Hydrazine
AccuVac® Ampuls
Stored Programs
232 Hydrazine AV
Start
1. Select the test. 2. Prepared Sample: 3. Start the instrument 4. Blank Preparation:
Insert an adapter if Collect at least 40 mL of timer. Pour at least 40-mL of
required (Instrument- sample in a 50-mL beaker. A 12-minute reaction deionized water into a
specific information). Fill a HydraVer Hydrazine period will begin. second beaker.
Refer to the user manual AccuVac® Ampul with Complete steps 5–7 Fill a second Ampul with
for orientation. sample. Keep the tip during this period. deionized water. Keep the
immersed while the Ampul tip immersed while the
fills completely. Cap with a Ampul fills completely.
stopper and mix. Cap with a stopper and
mix.
Zero
5. Insert the blank into 6. ZERO the instrument. 7. Insert the prepared .
the cell holder. The display will show: sample in the cell holder.
0 µg/L N2H4 Immediately after the
timer expires READ the
results in µg/L N2H4.
Interferences

No interference up to 10 mg/L. May cause a positive interference of up to 20% at 20
Ammonia
mg/L.
Prepare a blank by oxidizing the hydrazine in a portion of the sample with a 1:1
mixture of deionized water and household bleach. Add one drop of the mixture to 25
Highly colored or turbid samples
mL of sample in a graduated mixing cylinder and invert to mix. Use this solution in step
2, instead of deionized water, to prepare the blank.
Morpholine No interference up to 10 mg/L.
Hydrazine
Page 571
Hydrazine

• Samples collected in glass or plastic bottles should be filled completely and capped tightly.
• Avoid excessive agitation or exposure to air.
• Samples must be analyzed immediately after collection and cannot be preserved for later
analysis.
Accuracy check
1. Prepare a 25 mg/L stock solution. Dissolve 0.1016 g of hydrazine sulfate in 1000 mL of

oxygen-free deionized water. Prepare this stock solution daily.
2. Using Class A glassware, prepare a 0.25 mg/L (250 µg/L) hydrazine working solution by
diluting 10.00 mL of the 25 mg/L stock solution to 1000 mL with deoxygenated
deionized water. Prepare just before analysis. Perform either hydrazine procedure.
Method performance
231 DR 5000 250 µg/L N2H4 247–253 µg/L N2H4 4 µg/L N2H4
232 DR 5000 250 µg/L N2H4 246–254 µg/L N2H4 4 µg/L N2H4
Summary of method
Hydrazine in the sample reacts with the p-dimethylaminobenzaldehyde from the HydraVer 2
Reagent to form a yellow color which is proportional to the hydrazine concentration. Test results
Required reagents
HydraVer® 2 Hydrazine Reagent 1 mL 100 mL MDB 179032

OR
HydraVer 2 Hydrazine Reagent AccuVac® Ampuls 2 25/pkg 2524025
Water, deionized 10–40 mL 4L 27256
Hydrazine
Page 572
Hydrazine
Required apparatus (Reagent solution)

Cylinder, graduated, 25-mL. 1 each 50840

Beaker, 50 mL 1 each 50041H
Stopper 2 6/pkg 173106
Hydrazine Sulfate, ACS 100 g 74226

Cylinder, graduated mixing each 189640
Hydrazine
Page 573
Iodine, 8031
Iodine DOC316.53.01047
DPD Method 1 Method 8031

0.07 to 7.00 mg/L Powder Pillows or AccuVac® Ampuls
Scope and Application: For testing dissolved iodine residual used as disinfectant in process water, treated water,
estuary water and seawater
1 Adapted from Palin, A.T., Inst. Water Eng., 21 (6), 537-547 (1967).
Test preparation


Instrument
Analyze samples immediately. Do not preserve for later analysis
blank adjust.
Use DPD Total Chlorine reagents. Do not use DPD Free Chlorine reagents for this test.
If the sample temporarily turns yellow after reagent addition, dilute a fresh sample. Repeat the test. A slight loss of iodine
may occur due to the dilution. Apply the appropriate dilution factor.
Iodine
Page 575
Iodine
Powder Pillow Test:
DPD Total Chlorine Reagent Powder Pillow 1
Sample cells, 1-inch square, 10-mL (see Instrument-specific information) 2
AccuVac Test:
DPD Total Chlorine Reagent AccuVac® Ampul 1
Beaker, 50-mL 1
Stopper for 18 mm Tube 2
DPD for powder pillows
Stored Programs
245 Iodine
Start
1. Select the test. 2. Prepared sample: Fill 3. Swirl for about 20 4. Start the instrument
Insert an adapter if a sample cell with 10 mL second to mix. timer.
required (see Instrument- of sample. A pink color will develop if A three-minute reaction
specific information). Add the contents of one iodine is present. time will begin.
Refer to the user manual DPD Total Chlorine
for orientation. Powder Pillow to the
sample cell.
Iodine
Page 576
Iodine
DPD for powder pillows (continued)
Zero
5. Blank preparation: 6. Wipe the blank and 7. ZERO the instrument. 8. Within three minutes
Fill a second sample cell insert it into the cell holder. The display will show: after the timer expires,
with 10 mL of sample. (See Instrument- wipe the prepared sample
specific information.) 0.00 mg/L I2 and insert it into the cell
Close the cover. holder.
(See Instrument-
specific information.)
Read
9. READ the results in

mg/L I2.
DPD for AccuVac® Ampuls
Stored Programs
246 Iodine AV
Start
1. Select the test. 2. Prepared Sample: 3. Quickly invert the 4. Start the instrument
Insert an adapter if Collect at least 40 mL of Ampul several times timer.
required (see Instrument- sample in a 50-mL beaker. to mix. A three-minute reaction
specific information). Fill a DPD Total Chlorine A pink color will develop if period will begin.
Reagent AccuVac® Ampul iodine is present.
Refer to the user manual with sample. Keep the tip
for orientation. immersed while the Ampul
fills completely.
Iodine
Page 577
Iodine
DPD for AccuVac® Ampuls (continued)
Zero
5. Blank Preparation: 6. Wipe the blank and 7. ZERO the instrument. 8. Wipe the prepared
Fill a round sample cell insert it into the cell holder. The display will show: sample and insert it into
with 10 mL of sample. the cell holder.
0.00 mg/L l2.
READ the results in
mg/L I2.
Interferences

Acidity Neutralize to pH 6–7 with 1 N Sodium Hydroxide1. Determine amount to be added on
separate sample aliquot, then add the same amount to the sample being tested. Correct
for volume addition.
Greater than 250 mg/L CaCO3: May not develop full color or color may fade instantly.
1. Neutralize to pH 6–7 with 1 N Sulfuric Acid1.
Alkalinity 2. Determine amount to be added on separate sample aliquot. Add the same amount to
the sample to be tested.
3. Correct for volume addition.
Bromine Interferes at all levels
Chlorine and chloramines Causes a positive interference at all levels
1. Adjust sample pH to 6–7.
2. Add 3 drops Potassium Iodide1 (30-g/L) to a 25-mL sample.
Manganese, Oxidized (Mn4+, 3. Mix and wait 1 minute.
Mn7+) or 4. Add 3 drops Sodium Arsenite1, 2 (5-g/L) and mix.
Chromium, Oxidized (Cr6+) 5. Analyze 10 mL of the treated sample as described in the procedure.
6. Subtract the result from this test from the original analysis to obtain the correct iodine
concentration.
Adjust to pH 6–7.
buffered samples
Iodine
Page 578
Iodine
for arsenic (D004). Refer to the current MSDS for disposal information.

• Collect samples in clean, dry glass containers.
• If sampling from a tap, allow the water to flow at least 5 minutes to ensure a representative
sample.
• Avoid excessive agitation and exposure to sunlight when sampling.
• Allow several volumes of water to overflow the container and cap the container so there is no
headspace above the sample.
• If sampling with a sample cell, rinse the cell several times with the sample, then carefully fill to
the 10-mL mark.
• Proceed with the analysis immediately.
Accuracy check
Standard additions method (sample spike) for powder pillows
• LR Chlorine PourRite® Ampule Standard, 25–30 mg/L
• Ampule breaker
1. Analyze the sample and record the result.
3. Use the TenSette Pipet to prepare a spiked sample: add 0.1 mL of standard to a 10-mL portion
of reacted sample. Swirl to mix.
4. Insert the spiked sample cell in the instrument.
6. Read the result.
7. Calculate the equivalent concentration of mg/L iodine added to the sample:

0.1 (vol. standard added) × mg/L Chlorine (certificate value) × 3.6
mg/L Iodine = ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------
10.1 (sample + standard volume)
8. The spiked sample result from step 6 should reflect the analyzed sample result in step 1 plus
the added, calculated mg/L iodine in step 7. If the result does not reflect the increase, refer to
Standard Additions in the Water Analysis Guide.
Standard additions method (sample spike) for AccuVac Ampuls
• LR Chlorine PourRite® Ampule Standard, 25–30 mg/L
• DPD Total Chlorine AccuVac Ampul (2)
• Graduated cylinder
• Beakers
• Ampule breaker
Iodine
Page 579
Iodine
• TenSette Pipet
2. Use a graduated cylinder to measure 25 mL of sample into a beaker. Use the TenSette Pipet
to add 0.2 mL of standard to the beaker. Swirl to mix. This is the spiked sample.
3. Use a graduated cylinder to measure 25 mL of sample into a second beaker.
4. Fill an AccuVac Ampul from the spiked sample. Fill the second Ampul from the second beaker.
5. Follow the DPD for AccuVac® Ampuls test procedure.
7. Read the result.
8. Calculate the equivalent concentration of mg/L iodine added to the sample:

0.2 (vol. standard added) × mg/L Chlorine (certificate value) × 3.6
mg/L Iodine = ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------
25.2 (sample + standard volume)
9. The spiked sample result should reflect the analyzed sample result plus the added, calculated
mg/L iodine in step 8. If the result does not reflect the increase, refer to Standard Additions in
the Water Analysis Guide.
Method performance
240 4.47 mg/L I2 4.40–4.54 mg/L I2 0.07 mg/L I2
242 4.47 mg/L I2 4.33–4.61 mg/L I2 0.07 mg/L I2
Summary of method
Iodine reacts with DPD (N, N-diethyl-p-phenylenediamine) to form a pink color, the intensity of
which is proportional to the total iodine concentration. Test results are measured at 530 nm.
Iodine
Page 580
Iodine

Required reagents
DPD Total Chlorine Reagent Powder Pillows 1 100/pkg 2105669
OR
Required apparatus

AccuVac Snapper Kit each 2405200
Ampule Breaker Kit each 2196800
Potassium Iodide, 30 g/L 100 mL MDB 34332
Sodium Hydroxide, 1 N 100 mL MDB 104532
Stopper for 18 mm tube 25 / pkg 173125
Sulfuric Acid, 1 N 100 mL MDB 127032
Chlorine Standard, 25-30 mg/L ampules 20/pkg 2630020
TenSette Pipet, 0.1–1.0 mL each 1970001
Pipet tips for TenSette Pipet 1970001 50 / pkg 2185696
Pipet tips for TenSette Pipet 1970001 1000 / pkg 2185628
Iodine
Page 581
Iron, FerroZine, 8147
Iron DOC316.53.01048
Ferrozine® Method1 Method 8147

0.009 to 1.400 mg/L FerroZine Reagent Solution Pillows
1 Adapted from Stookey, L.L., Anal. Chem., 42(7), 779 (1970)
Test preparation


Digestion is required for total iron determination.
Rinse glassware with a 1:1 hydrochloric acid solution. Rinse again with deionized water. These two steps will remove iron
deposits that can cause slightly high results.
blank adjust.
Instead of solution pillows, 0.5 mL of FerroZine® Iron Reagent Solution can be used.
If the sample contains rust, see Interferences.
Use clean clippers, free of rust and wipe with a dry towel. Do not allow clippers to contact contents of the pillow.
FerroZine Iron Reagent may crystallize or precipitate when exposed to cold temperatures during shipment. Reagent quality
is not affected. Place the reagent in warm water to redissolve.
Iron
Page 583
Iron
FerroZine Iron Reagent Solution Pillows 1
OR
FerroZine Iron Reagent Solution 0.5 mL
Cylinder, 25 mL graduated mixing, with stopper 1
Clippers for solution pillows 1
FerroZine solution pillows
Stored Programs
260 Iron, FerroZine
Start
1. Select the test. 2. Fill a clean 25 mL 3. Prepared sample: 4. Start the instrument
Insert an adapter if graduated mixing cylinder Add the contents of one timer.
required (see Instrument- to the 25 mL mark with FerroZine® Iron Reagent A five-minute reaction
specific information). sample. Solution Pillow to the period will begin. A purple
mixing cylinder. color will develop if iron is
for orientation. Stopper and invert to mix. present.
Zero
5. Blank preparation: 6. When the timer 7. Insert the blank into 8. ZERO the instrument.
Fill a sample cell with expires, pour 10 mL of the the cell holder. The display will show:
10 mL of sample. prepared sample into a
second clean sample cell. 0.000 mg/L Fe
Iron
Page 584
Iron
FerroZine solution pillows (continued)
Read
9. Insert the prepared 10. READ the results in

sample into the cell holder. mg/L Fe.
Interferences

Interfere at all levels. Use the FerroVer® or TPTZ methods for these samples. Use the
Strong chelants (EDTA)
TPTZ method for low iron concentrations.
Cobalt May give slightly high results
Copper May give slightly high results
Boil the sample, with the FerroZine® Iron Reagent added to it from step 3, for 1 minute in a
Hydroxides boiling water bath. Cool to 24 °C (75 °F) before proceeding with step 4. Return the sample
volume to 25 mL with deionized water.
1. Fill a 25 mL graduated cylinder with 25 mL of sample.
2. Transfer this sample into a 125-mL Erlenmeyer flask.
3. Add the contents of one FerroZine® Iron Reagent Solution Pillow and swirl to mix.
4. Place the flask on a hot plate or over a flame and bring to a boil.
5. Continue boiling gently for 20 to 30 minutes.
Magnetite (black iron oxide) or
Ferrites Note: Do not allow to boil dry. A purple color will develop if iron is present.
6. Return the boiled sample to the 25 mL graduated cylinder. Rinse the Erlenmeyer flask
with small amounts of deionized water and empty into the graduated cylinder.
7. Return the sample volume to the 25 mL mark with deionized water.
8. Pour this solution into a sample cell and swirl to mix.
9. Proceed with steps 5–10.
Boil the sample, with the FerroZine Iron Reagent added to it from step 3, for 1 minute in a
Rust boiling water bath. Cool to 24 °C (75 °F) before proceeding with step 4. Return the sample
Iron
Page 585
Iron

• Collect samples in acid-washed glass or plastic bottles.
• To preserve samples, adjust the sample pH to 2 or less with concentrated Nitric Acid, ACS*
(about 2 mL per liter). Samples preserved in this manner can be stored up to six months at
room temperature.
• If only reporting dissolved iron, filter the sample immediately after collection and before adding
nitric acid.
• Before testing, adjust the sample pH to 3–5 with Ammonium Hydroxide 10%.
• Do not exceed pH 5 or iron may precipitate.
Accuracy check
• Iron Voluette® Ampule Standard, 10 mg/L Fe
• Ampule breaker
3. Accept the default values for standard concentration, sample volume and spike volumes. After
the values are accepted, the unspiked sample reading will appear in the top row. See the user
5. Follow the FerroZine solution pillows test procedure for each of the spiked samples:
a. Prepare a 0.1 mL sample spike by adding 0.1 mL of standard to the unspiked sample.
Start the instrument timer. After the timer expires, read the result.
b. Prepare a 0.2 mL sample spike by adding 0.1 mL of standard to the 0.1 mL sample spike.
c. Prepare a 0.3 mL sample spike by adding 0.1 mL of standard to the 0.2 mL sample spike.
Start the instrument timer. After the timer expires, read the result. Each addition should


• Iron Standard Solution, 100-mg/L
• 500 mL Class A volumetric flask
• Class A volumetric pipet, 5 mL and Pipet Filler
Iron
Page 586
Iron

1. Prepare a 1.0 mg/L Fe standard solution:
a. Pipet 5.00 mL of Iron Standard, 100-mg/L into a 500-mL volumetric flask.

c. Prepare this solution daily.
2. Follow the FerroZine solution pillows test procedure.
3. To adjust the calibration curve using the reading obtained with the 1.00 mg/L Standard
Method performance
260 DR 5000 1.000 mg/L Fe 0.985–1.015 mg/L Fe 0.009 mg/L Fe
Summary of method
The FerroZine® Iron Reagent forms a purple-colored complex with trace amounts of iron in
samples that are buffered to a pH of 3.5. This method is applicable for determining trace levels of
iron in chemical reagents and glycols and with digestion can be used to analyze samples
containing magnetite (black iron oxide) or ferrites. Test results are measured at 562 nm.
Iron
Page 587
Iron
Required reagents
FerroZine® Iron Reagent Solution 0.5 mL 500 mL 230149

OR
FerroZine® Iron Reagent Solution Pillows 1 50/pkg 230166
Required apparatus
Clippers for solution pillows 1 each 96800
Cylinder, graduated mixing, 25 mL with stopper 1 each 2088640

Iron Standard Solution, 100 mg/L Fe 100 mL 1417542
Iron Standard Solution, 10 mL Voluette® ampule, 25 mg/L Fe 16/pkg 1425310

Ammonium Hydroxide, 10% 100 mL MDB 1473632
Hydrochloric Acid, 1:1, 6N 500 mL 88449
Nitric Acid, ACS, concentrated 500 mL 15249

Iron, Ferrous, 8146
Iron, Ferrous DOC316.53.01049
1-10 Phenanthroline Method1 Method 8146

1 Adapted from Standard Methods for the Examination of Water and Wastewater, 15th ed. 201 (1980)
Test preparation


Instrument
adjust.
Analyze samples as soon as possible to prevent air oxidation of ferrous iron to ferric iron, which is not determined.
If ferrous iron is present, an orange color will form after adding the reagent.
Powder Pillow Test:
Ferrous Iron Reagent Powder Pillows 1
AccuVac Test:
Ferrous Iron Reagent AccuVac® Ampuls 1
Beaker, 50 mL (AccuVac test) 1
Iron, Ferrous
Page 589
Iron, Ferrous
1-10 Phenanthroline method for powder pillows
Stored Programs
255 Iron, Ferrous
Start
1. Select the test. 2. Fill a clean graduated 3. Prepared Sample: 4. Insert a stopper and
Insert an adapter if mixing cylinder with 25 mL Add the contents of one invert to mix. Undissolved
required (see Instrument- of sample. Ferrous Iron Reagent powder does not affect
specific information). powder pillow to the accuracy.
cylinder.
for orientation.
5. Start the instrument 6. Blank Preparation: 7. Fill a second sample 8. When the timer
timer. Fill a sample cell with cell with the prepared expires, insert the blank
A three-minute reaction 10 mL of sample. sample from the mixing into the cell holder.
period will begin. cylinder in step 4.
Zero Read
The display will show: sample into the cell holder. mg/L Fe2+.
0.00 mg/L Fe2+
Iron, Ferrous
Page 590
Iron, Ferrous
1-10 Phenanthroline method for AccuVac® Ampuls
Stored Programs
257 Iron, Ferrous AV
Start
Insert an adapter if Fill a round sample cell Fill a Ferrous Iron Reagent Ampul several times
required (see Instrument- with 10 mL of sample. AccuVac® Ampul with to mix.
specific information). sample from the beaker.
Keep the tip immersed
Refer to the user manual while the Ampul fills
for orientation. completely.
5. Start the instrument 6. Wipe the blank and 7. Wipe the Ampul and
timer. insert it into the cell holder. insert it into the cell holder.
A three-minute reaction ZERO the instrument. READ the results in
period will begin. The display will show: mg/L Fe2+.
0.00 mg/L Fe2+

• Collect samples in plastic or glass bottles.
• Analyze samples as soon as possible after collection.
Accuracy check

• Ferrous Ammonium Sulfate, hexahydrate, 0.7022 g
• 1 L Class A volumetric flask
• Deionized water
Iron, Ferrous
Page 591
Iron, Ferrous
• Analytical balance
• 2 mL Class A volumetric pipet and pipet filler
1. Prepare a 100 mg/L Fe2+ ferrous iron stock solution as follows:
a. Dissolve 0.7022 grams of Ferrous Ammonium Sulfate, hexahydrate, in deionized water.

b. Dilute to one liter in a Class A volumetric flask.
c. In a 100 mL Class A volumetric flask, dilute 2.00 mL of this solution to 100 mL with
deionized water to make a 2.0 mg/L standard solution. Prepare this solution immediately
before use.
2. Follow the 1-10 Phenanthroline method for powder pillows or the 1-10 Phenanthroline method
for AccuVac® Ampuls test procedure.
Method performance
255 DR 5000 2.00 mg/L Fe2+ 1.99–2.01 mg/L Fe2+ 0.021 mg/L Fe2+
257 DR 2800 2.00 mg/L Fe2+ 1.98–2.02 mg/L Fe2+ 0.023 mg/L Fe2+
Summary of method
The 1-10 phenanthroline indicator in the Ferrous Iron Reagent reacts with ferrous iron (Fe2+) in the
sample to form an orange color in proportion to the iron concentration. Ferric iron (Fe3+)does not
react. The ferric iron concentration can be determined by subtracting the ferrous iron concentration
from the results of a total iron test. Test results are measured at 510 nm.
Iron, Ferrous
Page 592
Iron, Ferrous
Required reagents
Ferrous Iron Reagent Powder Pillows 1 100/pkg 103769
OR
Ferrous Iron Reagent AccuVac® Ampuls 1 25/pkg 2514025
Required apparatus

Balance, analytical, 80 g x 0.1 mg 100–240 VAC each 2936701
Ferrous Ammonium Sulfate, hexahydrate, ACS 113 g 1125614
Pipet filler, safety bulb each 1465100
Pipet, volumetric, 2.00 mL each 1451535
Wipers, disposable 280/pkg 2097000
Iron, Ferrous
Page 593
Iron Total FerroVer, 8008
Iron, Total DOC316.53.01053
USEPA1 FerroVer® Method2 Method 8008

Scope and Application: For water, wastewater and seawater; digestion is required for determining total iron
1 USEPA approved for reporting wastewater analysis, Federal Register, June 27, 1980; 45 (126:43459)
Test preparation


Instrument
Digestion is required for determining total iron for EPA reporting purposes. Use the mild or vigorous digestion. Refer to the
Water Analysis Guide for more information.
adjust. See the user manual for more information.
Adjust pH of stored samples before analysis.
For turbid samples, treat the blank with one 0.1-g scoop of RoVer Rust Remover. Swirl to mix.
Powder Pillow Test:
FerroVer® Iron Reagent Powder Pillow 1
Iron, Total
Page 595
Iron, Total
AccuVac® Ampul test:
FerroVer® Iron Reagent AccuVac® Ampul 1
Beaker, 50-mL 1
Stopper of 18 mm tubes 1
FerroVer method for powder pillows
Stored Programs
265 Iron, FerroVer
Start
1. Select the test. 2. Prepared sample: Fill 3. Add the contents of 4. Start the instrument
Insert an adapter if a clean sample cell with one FerroVer Iron Reagent timer.
required (see Instrument- 10 mL of sample Powder Pillow to the A three-minute reaction
specific information). sample cell. Swirl to mix. period will begin. An
Refer to the user manual Accuracy is not affected by orange color will form, if
for orientation. undissolved powder. iron is present.
(Allow samples that
contain rust to react for at
least 5 minutes.)
Zero
5. Blank preparation: 6. When the timer 7. ZERO the instrument. 8. Insert the prepared
Fill a second sample cell expires, insert the blank The display will show: sample into the cell holder.
with 10 mL of sample. into the cell holder. READ the results in
0.00 mg/L Fe
mg/L Fe.
Iron, Total
Page 596
Iron, Total
FerroVer method for AccuVac® Ampuls
Stored Programs
267 Iron, FerroVer AV
Start
Insert an adapter if Fill a round sample cell Collect at least 40 mL of Ampul several times
required (see Instrument- with 10 mL of sample. sample in a 50-mL beaker. to mix.
specific information). Fill a FerroVer Iron Accuracy is not affected by
AccuVac® Ampul with undissolved powder.
Refer to the user manual sample from the beaker.
for orientation. Keep the tip immersed
while the Ampul fills
completely.
Zero
5. Start the instrument 6. Wipe the blank and 7. ZERO the instrument. 8. Wipe the Ampul and
timer. insert it into the cell holder. The display will show: insert it into the cell holder.
A three-minute reaction 0.00 mg/L Fe READ the results in
period will begin. An mg/L Fe.
orange color will develop if
iron is present.
Interferences

Calcium, Ca2+ No effect at less than 10,000 mg/L as CaCO3.
Chloride, Cl– No effect at less than 185,000 mg/L.

Copper, Cu2+ No effect. Masking agent is contained in FerroVer Reagent.
High Iron Levels Inhibit color development. Dilute sample and re-test to verify results.
Requires mild, vigorous or Digesdahl digestion. After digestion, adjust sample to pH 3–5 with
Iron Oxide
sodium hydroxide, then analyze.
Magnesium No effect at 100,000 mg/L as calcium carbonate.
Iron, Total
Page 597
Iron, Total

Molybdate Molybdenum No effect at 50 mg/L as Mo.
1. Treat in fume hood or well-ventilated area. Add 5 mL hydrochloric acid1, ACS to 100 mL
sample in a 250 mL Erlenmeyer flask. Boil 20 minutes.
2. Cool. Adjust pH to 3–5 with Sodium Hydroxide1. Readjust volume to 100 mL with
High Sulfide Levels, S2–
deionized water.
3. Analyze using FerroVer method for powder pillows or FerroVer method for AccuVac®
Ampuls.
1. Add 0.1 g scoop of RoVer® Rust Remover to the blank. Swirl to mix.
2. Zero the instrument with this blank.
3. If sample remains turbid, add three 0.2 g scoops of RoVer to a 75 mL sample.
Turbidity
Let stand 5 minutes.
4. Filter through a Glass Membrane Filter and Filter Holder1.
5. Use the filtered sample as the prepared sample and the blank.
Extreme Sample pH Adjust pH to 3–5.
Highly Buffered Samples Adjust pH to 3–5.

• Collect samples in acid-cleaned glass or plastic containers. No acid addition is necessary if
analyzing the sample immediately.
• To preserve samples, adjust the pH to 2 or less with concentrated nitric acid (about 2 mL per
liter). Preserved samples may be stored up to six months at room temperature.
• Before analysis, adjust the pH to between 3 and 5 with 5.0 N Sodium Hydroxide Standard
Solution.
• If only dissolved iron is to be determined, filter the sample before acid addition.
Accuracy check
• Iron Voluette® Ampule Standard, 25 mg/L
• Ampule breaker
5. Prepare a 0.1 mL sample spike by adding 0.1 mL of standard to 10 mL of unspiked sample.

6. Prepare a 0.2 mL sample spike by adding 0.1 mL of standard to the 0.1 mL sample spike. Start
the instrument timer. After the timer expires, read the result.
Iron, Total
Page 598
Iron, Total
7. Prepare a 0.3 mL sample spike by adding 0.1 mL of standard to the 0.2 mL sample spike. Start
the instrument timer. After the timer expires, read the result.
Standard additions method for AccuVac Ampuls (sample spike)
1. Fill three mixing cylinders each with 50 mL of sample and spike with 0.2 mL, 0.4 mL and
0.6 mL of standard. Stopper and invert to mix.
2. Transfer 40 mL from each of the three mixing cylinders to three 50 mL beakers.
3. Analyze each standard addition sample as described in the FerroVer method for AccuVac®
Ampuls.
recovery.


• Iron Standard Solution, 100 mg/L
• 100-mL volumetric flask
• Class A volumetric pipet, 2 mL
• Deionized water
• Pipet filler
1. Prepare a 2.00-mg/L Fe standard solution as follows:

a. Pipet 2.00 mL of Iron Standard Solution, 100 mg/L, into a 100 mL volumetric flask.
b. Dilute to the mark with deionized water. Mix well. Prepare this solution daily.
2. Use the 2.00 mg/L Fe standard solution in place of the sample. Follow the FerroVer method for
powder pillows test procedure.
3. To adjust the calibration curve using the reading obtained with the Standard Solution, select
concentration is used, enter the concentration and adjust the curve to that value. Mixed-
parameter standards are also available to simulate various matrices.
Method performance
265 2.00 mg/L Fe 1.99–2.01 mg/L Fe 0.021 mg/L Fe
Iron, Total
Page 599
Iron, Total
Summary of method
FerroVer Iron Reagent converts all soluble iron and most insoluble forms of iron in the sample to
soluble ferrous iron. The ferrous iron reacts with the 1-10 phenanthroline indicator in the reagent to
form an orange color in proportion to the iron concentration. Test results are measured at 510 nm.
Required reagents
FerroVer® Iron Reagent Powder Pillows (for 10-mL sample) 1 100/pkg 2105769
OR
FerroVer® Iron Reagent AccuVac® Ampuls 1 25/pkg 2507025
Required apparatus
Iron Standard Solution, 100 mg/L 100 mL 1417542
Iron Standard Solution, 10 mL Voluette® Ampule, 25 mg/L as Fe 16/pkg 1425310

Hydrochloric Acid, concentrated 500 mL 13449
Iron, Total
Page 600
Iron, Total

Glass Membrane Filter, 47 mm 100/pkg 253000
Glass Membrane Filter Holder each 234000
RoVer Rust Remover 454 g 30001
Iron, Total
Page 601
Iron, Total

Iron Total, 8112
TPTZ Method1 Method 8112

1 Adapted from G. Frederic Smith Chemical Co., The Iron Reagents, 3rd ed. (1980)
Test preparation


Instrument
Digestion is required for determining total iron.
adjust.
Rinse all glassware with a 1:1 Hydrochloric Acid Solution1. Rinse again with deionized water. This process will remove iron
After adding reagent, a blue color will develop if iron is present.
Adjust the pH of stored samples to 3–4. Do not exceed pH 5 or iron may precipitate.
Powder Pillow Test:
TPTZ Iron Reagent Powder Pillow 2
Iron, Total
Page 603
Iron, Total
AccuVac Test:
TPTZ Low Range Iron Reagent AccuVac® 1
Beaker, 50 mL 1
TPTZ method for powder pillows
Stored Programs
270 Iron, TPTZ
Start
1. Select the test. 2. Prepared Sample: Fill 3. Start the instrument 4. Blank Preparation:
Insert an adapter if one clean sample cell with timer. Fill a second sample cell
required (see Instrument- 10 mL of sample. A three-minute reaction with 10 mL of deionized
specific information). Add the contents of one period will begin. water.
Refer to the user manual 10-mL TPTZ Iron Reagent Proceed with step 4 and 5
for orientation. Powder Pillow to the while the timer is running.
prepared sample. Swirl at
least 30 seconds to
dissolve.
Zero
5. Add the contents of 6. When the timer 7. ZERO the instrument. 8. Insert the prepared
one 10-mL TPTZ Iron expires, insert the blank The display will show: sample into the cell holder.
Reagent Powder Pillow to into the cell holder. READ the results in
the blank. Swirl at least 30 0.000 mg/L Fe
mg/L Fe.
seconds to dissolve. This
will be the reagent blank.
Iron, Total
Page 604
Iron, Total
TPTZ method for AccuVac® Ampuls
Stored Programs
272 Iron, TPTZ AV
Start
Insert an adapter if Fill a sample cell with Collect at least 40 mL of Ampul several times
required (see Instrument- 10 mL of sample. sample in a 50-mL beaker. to mix.
specific information). Fill a TPTZ Iron AccuVac
Ampul with sample. Keep
Refer to the user manual the tip immersed while the
for orientation. Ampul fills completely.
Zero
5. Start the instrument 6. Wipe the blank and 7. ZERO the instrument. 8. Wipe the Ampul and
timer. insert it into the cell holder. The display will show: insert it into the cell holder.
A three-minute reaction 0.000 mg/L Fe READ the results in
period will begin. mg/L Fe
Iron, Total
Page 605
Iron, Total
Interferences
Interferences are tested (see the Interfering substances table) with an iron concentration of
0.5 mg/L. The following do not interfere with the method when present up to the levels given.

Cadmium 4.0 mg/L
Chromium 3+ 0.25 mg/L
Chromium 6+ 1.2 mg/L
Cobalt 0.05 mg/L
Copper 0.6 mg/L
Cyanide 2.8 mg/L
Manganese 50.0 mg/L
Mercury 0.4 mg/L
Molybdenum 4.0 mg/L
Nickel 1.0 mg/L
Nitrite Ion 0.8 mg/L
In the powder pillow procedure, if the sample, without a TPTZ Iron Reagent Powder Pillow,
Color or turbidity has a color or turbidity greater than the blank (deionized water plus TPTZ Iron Reagent),
then use the sample as the blank.
A sample pH of less than 3 or greater than 4 after the addition of reagent may inhibit color
formation, cause the developed color to fade quickly or to result in turbidity. Adjust the
sample pH in the sample cell before the addition of reagent:
1. Measure the current pH by using a pH meter or pH paper.
pH 2. Adjust the sample pH to between 3 and 4 by adding an appropriate amount of iron-free
acid or base such as 1.0 N Sulfuric Acid Standard Solution1 or 1.0 N Sodium Hydroxide
Standard Solution1.
3. Make a volume correction if significant volumes of acid or base are used. Refer to the

• To preserve samples, adjust the sample pH to 2 or less with about 2 mL/L Nitric Acid, ACS*.
• If reporting only dissolved iron, filter sample immediately after collection and before adding
nitric acid.
• Before testing, adjust the pH of the stored sample to between 3–4 with 5.0 N Sodium
Hydroxide Standard Solution*. Do not exceed pH 5 as iron may precipitate.
Iron, Total
Page 606
Iron, Total
Accuracy check
Standard additions method for powder pillows (sample spike)
• Iron Standard Solution, 10 mg/L Fe
• Beakers, 50 mL (3)
• Graduated mixing cylinders, 50 mL (3)
• TenSette Pipet
4. Open a fresh bottle of standard solution.
5. Use the TenSette Pipet to prepare spiked samples: add 0.1 mL, 0.2 mL and 0.3 mL of
6. Follow the TPTZ method for powder pillows test procedure for each of the spiked samples
using the powder pillows or AccuVac ampules, starting with the 0.1 mL sample spike. Measure
each of the spiked samples in the instrument.

1. Fill three mixing cylinders each with 50 mL of sample and spike with 0.5 mL, 1.0 mL and 1.5
mL of standard. Stopper and invert to mix.
2. Transfer 40 mL from each of the three mixing cylinders to three 50 mL beakers.
3. Analyze each standard addition sample as described in the TPTZ method for AccuVac®
Ampuls.
4. Accept each standard additions reading. Each addition should reflect approximately
100% recovery.


• Iron Standard Solution, 1 mg/L or
• 5 mL Class A volumetric pipet and pipet filler
• Deionized water
1. Use a 1 mg/L Iron Standard Solution or prepare a 1.00 mg/L iron standard solution as follows:
Iron, Total
Page 607
Iron, Total
2. Use the 1.00 mg/L iron standard solution in place of the sample. Follow the TPTZ method for
powder pillows or Accuvac Ampuls.
3. To adjust the calibration curve using the reading obtained with the 1.00 mg/L Standard
parameter standards are also available to simulate various matrices.
Method performance
Summary of method
The TPTZ Iron Reagent forms a deep blue-purple color with ferrous iron (Fe2+). The indicator is
combined with a reducing agent that converts precipitated or suspended iron, such as rust, to the
ferrous state. The amount of ferric iron (Fe3+) present can be determined as the difference
between the results of a ferrous iron test and the concentration of total iron. Test results are
measured at 590 nm.
Iron, Total
Page 608
Iron, Total
Required reagents
TPTZ Iron Reagent Powder Pillows (for 10-mL sample) 1 100/pkg 2608799
OR
TPTZ Low Range Iron Reagent AccuVac® Ampuls 1 25/pkg 2510025
Required apparatus

Catalog
Description Unit
number
Nitric Acid, ACS concentrated 500 mL 15249
Sodium Hydroxide, 5.0 N 50 mL SCDB 245026
Sulfuric Acid, 1.0 N 100 mL MDB 104532
Tensette Pipet, 0.1–1.0 mL each 1970001
Pipet, volumetric, 5 mL Class A each 1451537
Pipet Filler, Safety bulb each 1465100
Iron, Total
Page 609
Iron, FerroZine, 8147
Iron DOC316.53.01050
FerroZine® Rapid Liquid Method1 Method 8147

0.009 to 1.400 mg/L Fe Pour-Thru Cell
Scope and Application: For boiler, cooling and natural waters
1 Adapted from Stookey, L.L., Anal.Chem., 42 (7) 779 1970.
Test preparation


DR 5000 LZV479 — —
If sample contains rust, see Interferences.
adjust.
If iron is present, a purple color will form after adding the reagent
Rinse glassware with a 1:1 Hydrochloric acid1 solution. Rinse again with deionized water. This will remove residual iron that
may interfere
Prepare the Pour-Thru cell by preparing a solution of 1 mL Ferrozine Reagent per 50 mL of deionized water. Pour this into
the cell and allow to stand for approximately 5 minutes to react with any trace iron in the cell and cell tubing. Flush with
deionized water.
FerroZine Iron Reagent may crystallize or precipitate when exposed to cold temperatures during shipment; reagent quality is
not affected. Place the reagent bottle in warm water to dissolve.
Iron
Page 611
Iron
FerroZine® Iron Reagent Solution 1.0 mL
Cylinder, graduated, 50 mL poly 1
Dispenser, fixed volume, 1.0 mL, with bottle 1
Flask, Erlenmeyer, PMP w/cap, 125 mL 2
Pour-Thru Cell Module (see Instrument-specific information) 1
FerroZine Rapid Liquid Method for Pour-Thru Cell
Stored Programs
261 Iron, FerroZine RL
Start
1. Select the test. 2. Flush the Pour-Thru 3. Rinse two clean 4. Rinse a clean 50-mL
Insert an adapter if Cell with 50 mL of 125 mL Erlenmeyer flasks plastic graduated cylinder
required (see Instrument- deionized water. with the sample three three times with the
specific information). times. sample.
for orientation.
5. Fill the rinsed cylinder 6. Pour the contents of 7. Prepared sample: 8. Start the instrument
to the 50 mL mark with the 50 mL cylinder into Add 1.0 mL of FerroZine timer.
sample. one of the flasks. Iron Reagent Solution to A five-minute reaction
flask using the Dispenser. period will begin.
Swirl to mix.
Iron
Page 612
Iron
FerroZine Rapid Liquid Method for Pour-Thru Cell (continued)
Zero
9. Blank preparation: 10. When the timer 11. When the flow stops, 12. Pour the contents of
Measure a second 50 mL expires the display will ZERO the instrument. the flask containing the
portion of sample into the show “mg/L Fe.” The display will show: prepared sample into the
graduated cylinder and Pour the contents of the Pour-Thru Cell.
pour the contents into the 0.000 mg/L Fe
flask containing the blank
second flask. into the Pour-Thru Cell
Read
13. When the flow stops, 14. Flush the Pour-Thru

READ the results in Cell with at least 50 mL of
mg/L Fe. deionized water
Iron
Page 613
Iron
Interferences

Interfere at all levels. Use the FerroVer® or TPTZ methods for these samples. Use the
Strong Chelants (EDTA)
TPTZ method for low iron concentrations.
Cobalt May give slightly high results
Copper May give slightly high results
Boil the sample with the FerroZine® Iron Reagent added to it from step 7 of the test procedure
Hydroxides for 1 minute in a boiling water bath. Cool to 24 °C (75 °F) before proceeding with step 9.
Return the sample volume to 50 mL with deionized water.
1. Fill a 50 mL graduated cylinder with 50 mL of sample.
2. Transfer the sample into a clean glass 125 mL Erlenmeyer flask.
3. Add 1.0 mL of FerroZine Iron Reagent Solution1 and swirl to mix.
4. Place the flask on a hot plate or over a flame and bring to a boil.
5. Continue boiling gently for 20 to 30 minutes. Do not allow to boil dry.
Magnetite (black iron oxide) or
Ferrites 6. Return the boiled sample to the graduated cylinder. Rinse the Erlenmeyer flask with
small amounts of deionized water and empty into the graduated cylinder. A purple color
will develop if iron is present.
7. Return the sample volume to the 50 mL mark with deionized water.
8. Pour the solution into a 125 mL Erlenmeyer flask and swirl to mix.
9. Proceed with steps 9–14 of the test procedure.
Boil the sample, with the FerroZine® Iron Reagent added to it from step 7, for 1 minute in a
Rust boiling water bath. Cool to 24 °C (75 °F) before proceeding with step 9. Return the sample

• To preserve samples, adjust the sample pH to 2 or less with concentrated Nitric Acid, ACS*
(about 2 mL per liter). Samples preserved in this manner can be stored up to six months at
room temperature.
• If only reporting dissolved iron, filter the sample immediately after collection and before adding
nitric acid.
• Before testing, adjust the sample pH to 3–5 with Ammonium Hydroxide, 10%. Do not exceed
pH 5 or iron may precipitate.
• Correct test results for volume additions. Refer to the Water Analysis Guide for more
information.
Iron
Page 614
Iron
Labware
• All containers used in this test must be cleaned thoroughly to remove any traces of iron.
• Rinse labware and the Pour-Thru Cell with a 1:1 HCl solution* or with a 1:50 dilution of
FerroZine® Reagent. Rinse several times with deionized water.
• Keep flasks tightly closed when not in use. Dedicate these containers for iron analysis only. If
containers are rinsed and capped after each use, only occasional treatment with HCl or
FerroZine® is necessary.

The Pour-Thru Cell may accumulate a buildup of color products, especially if the reacted solutions
are allowed to stand in the cell for long periods after measurement. Remove the color by rinsing
with a 1:5 dilution of Ammonium Hydroxide*, followed by several rinses with deionized water.
Cover the Pour-Thru Cell after use.
Accuracy check
• Ampule breaker
• Graduated mixing cylinder, 50 mL (3)
standard to three 50 mL portions of fresh sample.
6. Follow the FerroZine Rapid Liquid Method for Pour-Thru Cell test procedure for each of the
spiked samples starting with the 0.2 mL sample spike. Measure each of the spiked samples in
the instrument.
addition results to the theoretical 100% recovery. Each addition should reflect approximately
100% recovery.


• 1.0 mg/L Iron Standard Solution or
• 100 mg/L Iron Standard Solution
• Deionized water
Iron
Page 615
Iron
• 5 mL Class A volumetric pipet and filler
1. To check the accuracy, use a 1.0 mg/L Iron Standard Solution or prepare a 1.0 mg/L iron
working solution as follows:
a. Pipet 5.00 mL of iron standard solution, 100 mg/L Fe, into a 500 mL volumetric flask.
b. Dilute to volume with deionized water. Prepare this solution daily.
2. Follow the FerroZine Rapid Liquid Method for Pour-Thru Cell test procedure.
parameter standards are also available to simulate various test matrices.
Method performance
Summary of method
The FerroZine® Iron Reagent forms a purple colored complex with trace amounts of iron in
samples that are buffered to a pH of 3.5. This method is applicable for determining trace levels of
iron in chemical reagents and glycols and with digestion can be used to analyze samples
containing magnetite (black iron oxide) or ferrites. The test results are measured at 562 nm.
Iron
Page 616
Iron
Required reagents
FerroZine® Iron Reagent Solution 1 mL 500 mL 230149

Required apparatus
Cylinder, graduated, 50-mL, poly 1 each 108141
Dispenser, fixed volume, 1.0-mL 1 each 2111302
Flask, Erlenmeyer, PMP w/cap, 125-mL 2 each 2089843

Iron Standard Solution, 100-mg/L Fe 100 mL 1417542
Iron Standard Solution, Voluette® ampule, 25-mg/L Fe, 10-mL 16/pkg 1425310
Pipet, TenSette 0.1–1.0 mL each 1970001

Ammonium Hydroxide, ACS, 58% 500 mL 10649
Ammonium Hydroxide, ACS, 10% 100 mL MDB 1473632
Hydrochloric Acid, 1:1, 6N 500 mL 88449
Nitric Acid ACS, concentrated 500 mL 15249
Iron
Page 617
Iron Total FerroMo, 8365
FerroMo Method1 Method 8365

Scope and Application: For cooling water containing molybdate-based treatment
1 Adapted from G. Frederick Smith Chemical Co., The Iron Reagents, 3rd ed. (1980)
Test preparation


adjust.
Rinse glassware with a 1:1 hydrochloric acid solution. Rinse again with deionized water. These two steps will remove iron
After the addition of the reagent, the sample pH should be between 3–5.
If the sample contains high levels of molybdate (100 mg/L MoO42– or greater), read the sample immediately after zeroing
the blank.
FerroMo® Reagent 1 Powder Pillow 1
FerroMo® Reagent 2 Powder Pillow 1
Cylinder, graduated mixing, 25 mL, with stopper 1
Cylinder, graduated mixing, 50 mL, with stopper 1
Iron, Total
Page 619
Iron, Total
FerroMo method for powder pillows
Stored Programs
275 Iron, FerroMo
Start
1. Select the test. 2. Prepared sample: Fill 3. Add the contents of 4. Invert several times to
Insert an adapter if a 50-mL graduated mixing one FerroMo Iron dissolve the reagents.
required (see Instrument- cylinder with 50 mL of Reagent 1 Powder Pillow
specific information). sample. to the graduated mixing
cylinder. Insert the
Refer to the user manual stopper.
for orientation.
5. Fill a clean, 25-mL 6. Developed sample: 7. Insert the stopper and 8. Start the instrument
graduated cylinder to the Add the contents of one invert to dissolve the timer.
25-mL mark with prepared FerroMo Iron Reagent 2 reagents. A blue color will A three-minute reaction
sample. Save the Powder Pillow to the develop if iron is present. time will begin.
remaining prepared sample in the 25-mL A small amount of
sample for step 10. mixing cylinder. undissolved reagent will
not affect the results.
Iron, Total
Page 620
Iron, Total
FerroMo method for powder pillows (continued)
Zero
9. When the timer 10. Blank preparation: Fill 11. Insert the blank into 12. ZERO the instrument.
expires, pour 10 mL of the a second sample cell with the cell holder. The display will show:
developed sample from 10 mL of the remaining
step 7 into a sample cell. prepared sample from 0.00 mg/L Fe
step 5.
Read
13. Insert the developed 14. READ the results in

sample into the cell holder. mg/L Fe.
Interferences

A sample pH of less than 3 or greater than 4 after the addition of reagent may inhibit color
formation, cause the developed color to fade quickly or result in turbidity. Adjust the sample
pH to between 3 and 8 in the graduated cylinder before the addition of reagent:
pH 1. Add by drops an appropriate amount of iron-free acid or base such as 1.0 N Sulfuric
Acid Standard Solution1 or 1.0 N Sodium Hydroxide Standard Solution1.
2. Make a volume correction if significant volumes of acid or base are used. Refer to the

• To preserve samples, adjust the sample pH to 2 or less with hydrochloric acid (about 2 mL per
liter)*. Samples preserved in this manner can be stored up to six months at room temperature.
Iron, Total
Page 621
Iron, Total
• If only dissolved iron is to be reported, filter sample immediately after collection through a 0.45
micron filter or equivalent medium before adding hydrochloric acid.
• Before testing, adjust the sample pH to 3–5 with 5.0 N Sodium Hydroxide Standard Solution*.
Do not exceed pH 5 as iron may precipitate.
Accuracy check
• Mixing cylinders, 50 mL (3)
• Ampule breaker
6. Follow the FerroMo method for powder pillows test procedure for each of the spiked samples,
starting with the 0.1 mL sample spike. Measure each of the spiked samples in the instrument.
Accept each standard additions reading. Each addition should reflect approximately 100%
recovery.


• Iron Standard Solution, 1 mg/L or
• 1 mL Class A volumetric pipet
• Deionized water
1. Use a 1.0 mg/L standard or prepare a 1.00 mg/L iron standard solution as follows:
2. Use the 1.00 mg/L iron standard solution in place of the sample. Follow the FerroMo method
for powder pillows test procedure.
Iron, Total
Page 622
Iron, Total
Method performance
Summary of method
FerroMo Iron Reagent 1 contains a reducing agent combined with a masking agent. The masking
agent eliminates interference from high levels of molybdate. The reducing agent converts
precipitated or suspended iron, such as rust, to the ferrous state. FerroMo Iron Reagent 2 contains
the indicator combined with a buffering agent. The indicator reacts with ferrous iron in the sample,
buffered between pH 3 and 5, resulting in a deep blue-purple color. Test results are measured at
590 nm.
Required reagents
FerroMo® Iron Reagent Set (100 tests), includes: — — 2544800

(4) FerroMo® Reagent 1 Powder Pillows 1 25/pkg 2543768
(2) FerroMo® Reagent 2 Powder Pillows 1 50/pkg 2543866
Required apparatus
Cylinder, graduated mixing, 25 mL, with stopper 1 each 2088640
Cylinder, graduated mixing, 50 mL, with stopper 1 each 2088641
Iron Standard Solution, 10 mL Voluette® ampule, 50 mg/L Fe. 16/pkg 1425410
Iron, Total
Page 623
Hydrochloric Acid, 1:1 500 mL 88449

Iron, DT, 8214
Iron DOC316.53.01177
TitraVer Titration Method Method 8214

10 to 1000 mg/L as Fe Digital Titrator
Test preparation
Citrate Buffer Powder Pillow 1 pillow
Sodium Periodate Powder Pillow 1 pillow
Sulfosalicylic Acid Powder Pillow 1 pillow
TitraVer Standard Solution titration cartridge (see Range-specific information) 1 cartridge
Digital titrator 1
Iron
See
Table 1
Iron
Page 625
Iron
Iron
5. If the sample volume 6. Add the contents of 7. Add the contents of 8. Add the contents of
is less than 50 mL, dilute one Citrate Buffer Powder one Sodium Periodate one Sulfosalicylic Acid
to approximately 50 mL Pillow. Swirl to mix. Powder Pillow. Swirl to Powder Pillow. Swirl to
with deionized water. mix. mix.
A yellow color will develop A red color will develop if
if iron is present. iron is present.
9. Place the delivery tube 10. Use the multiplier in

into the solution and swirl the Range-specific
the flask. Turn the knob on information table to
the titrator to add titrant to calculate the
the solution. Continue to concentration:
swirl the flask and add digits x multiplier =
titrant until the color mg/L Fe
changes from red to the
original yellow. Example: 50 mL of sample
Write down the number of 0.0716 N cartridge and
= 25 mg/L Fe

Range (mg/L as Fe) Sample volume (mL) Titration cartridge (M TitraVer) Multiplier
10–40 50 0.0716 0.1
25–100 20 0.0716 0.25
100–400 50 0.716 1.0
250–1000 20 0.716 2.5
Iron
Page 626
Iron

Collect samples in clean plastic or glass bottles.
Accuracy check
• Iron Standard Solution, 1000-mg/L as Fe

titration cartridge or 100 digits of the 0.0716 M titration cartridge to reach the endpoint.
Summary of method
Ferrous iron (Fe2+) is oxidized by sodium periodate to ferric ion (Fe3+). The ferric ion forms a red
complex with sulfosalicylic acid. The red complex is destroyed by titration with EDTA. Citric acid is
used to buffer the solution and to stabilize the ferric ion in solution.

Required reagents
10–100 mg/L range—Reagent Set (approximately 100 tests): 2449200
(1) Citrate Buffer Powder Pillows 1 pillow 100/pkg 2081599
(1) Sodium Periodate Powder Pillows 1 pillow 100/pkg 98499
(1) Sulfosalicylic Acid Powder Pillows 1 pillow 100/pkg 2081669
(1) TitraVer Standard Solution Titration Cartridge, 0.0716 M varies each 2081701
100–1000 mg/L range—Reagent Set (approximately 100 tests): 2449300
(1) Citrate Buffer Powder Pillows 1 pillow 100/pkg 2081599
(1) Sodium Periodate Powder Pillows 1 pillow 100/pkg 98499
(1) Sulfosalicylic Acid Powder Pillows 1 pillow 100/pkg 2081669
(1) TitraVer Standard Solution Titration Cartridge, 0.716 M varies each 2081801
Iron
Page 627
Iron
Required apparatus
Iron Standard Solution, 1000-mg/L as Fe 100 mL 227142

Bottle, sampling 250 mL 2087076
Iron standard, 10 mg/L 500 mL 14049
Iron standard, 25 mg/L 10 mL/16 1425310
Iron standard, 50 mg/L 10 mL/16 1425410
Iron standard, 100 mg/L 100 mL 1417542

Lead, 8033
Lead DOC316.53.01055
USEPA1 Dithizone Method2 Method 8033

3 to 300 µg/L Powder Pillows
1 USEPA accepted for reporting for wastewater analysis (digestion is required)
2 Procedure is equivalent to Standard Method 3500-Pb D for wastewater analysis.
Test preparation


Clean all glassware with a 1:1 Nitric Acid Solution. Rinse with deionized water.
Cloudy and turbid samples may require filtering before running the test. Report results as µg/L soluble lead. Use glass
membrane type filter to avoid loss of lead by adsorption onto the filter paper.
If samples cannot be analyzed immediately, see Sample collection, preservation and storage. Adjust the pH of preserved
For more accurate results, adjust the sample to pH 11.0–11.5 using a pH meter in step 11. Omit the five additional drops of
Sodium Hydroxide Standard Solution in step 12
The DithiVer powder will not completely dissolve in the chloroform. For further notes see DithiVer solution preparation,
storage and reagent blank.
Read the MSDS before testing. Spilled reagent will affect test accuracy and is hazardous to skin and other materials.
Digestion is required to for determine the total lead for EPA reporting purposes. Use mild or vigorous digestion.
Lead
Page 629
Lead
Citrate Buffer Powder Pillows 1
Chloroform 50 mL
DithiVer Metals Reagent Powder Pillows 1
Potassium Cyanide 2g
Sodium Hydroxide Standard Solution, 5.0 N varies
Cotton Balls 1
Clippers 1
Cylinder, 50 mL graduated mixing 1
Cylinder, 5 mL graduated 1
Funnel, 500 mL separatory 1
Spoon, measuring, 1.0 g 1
Support Ring (4 inch) and Stand (5 x 8 inch base) 1
Dithizone method for powder pillows
Stored Programs
280 Lead Dithizone
Start
1. Select the test. 2. Fill a 250 mL 3. Transfer the sample 4. Add the contents of
Insert an adapter if graduated cylinder to the into 500 mL separatory one Buffer Powder Pillow,
required (see Instrument- 250 mL mark with sample. funnel. citrate type.
for orientation.
Lead
Page 630
Lead
Dithizone method for powder pillows (continued)
5. Insert the stopper into 6. DithiVer Solution 7. Stopper the cylinder. 8. Measure 30 mL of the
the funnel and shake to preparation: Invert several times to mix. prepared dithizone
dissolve. Add 50 mL of chloroform solution with a second
to a 50-mL mixing graduated cylinder and
graduated cylinder. Add add to the separatory
the contents of one funnel.
DithiVer Metals Reagent Insert the stopper and
Powder Pillow. invert to mix. Open
stopcock to vent. Close
the stopcock.
9. Add 5 mL of 5.0 N 10. Stopper. Invert. Open 11. Continue adding 5.0 N 12. Add 5 more drops of
Sodium Hydroxide stopcock to vent. Close Sodium Hydroxide 5.0 N Sodium Hydroxide
Standard Solution. the stopcock and shake Standard Solution Standard Solution.
the funnel once or twice dropwise and shaking the A pink color in the bottom
and vent again. funnel after every few (chloroform) layer at this
If the solution turns orange drops until the color of the point does not necessarily
after shaking, the pH is too solution being shaken indicate lead is present.
high. Add a few drops of changes from blue-green Only after adding the
5.25 N Sulfuric Acid to the to orange. potassium cyanide in the
solution to decrease the Large amounts of zinc next step will the presence
pH. cause the color transition of lead be confirmed by a
The blue-green color will at the end point to be pink color.
reappear (alternatively, to indistinct.
avoid higher blanks,
repeat on new sample and
use less sodium hydroxide
in step 9).
Lead
Page 631
Lead
Dithizone method for powder pillows (continued)
13. Add 2 heaping 1.0 g 14. Wait one minute for 15. Prepared sample: 16. Blank preparation:
scoops of potassium the layers to separate. The Insert a cotton plug Measure 10 mL of
cyanide to the funnel. bottom (chloroform) layer the size of a pea into the chloroform into another
Stopper. will be pink if lead is delivery tube of the funnel sample cell.
Shake vigorously until the present. and slowly drain the Insert the stopper.
potassium cyanide is all bottom (chloroform) layer
dissolved (about 15 into a dry sample cell.
seconds). Insert the stopper.
The lead-dithizone
complex is stable for at
least thirty minutes if the
sample cell is kept tightly
capped and out of direct
sunlight.
Zero Read
17. Insert the blank into 18. ZERO the instrument. 19. Insert the prepared 20. READ the results in
the cell holder. The display will show: sample into the cell holder µg/L Pb2+.
0 µg/L Pb2+
Lead
Page 632
Lead
Interferences
Table 198 Substances that do not interfere

Non-interfering substance Non-interfering substance
Aluminum Lead
Antimony Magnesium
Arsenic Manganese
Calcium Nickel
Chromium Tin
Cobalt
Zinc
Iron
Interference from the metals in the Interfering substances table can be eliminated by inserting the
Interference treatment for metals procedure after step 6 of the Dithizone method for powder
pillows procedure.
All levels. See Interference treatment for metals.
extreme sample pH
Bismuth All levels. See Interference treatment for metals.
Copper All levels. See Interference treatment for metals.
Mercury All levels. See Interference treatment for metals.
Silver All levels. See Interference treatment for metals.
Tin All levels. See Interference treatment for metals.
Interference treatment for metals
1. Measure about 5 mL of the DithiVer solution into the separatory funnel. Stopper the funnel,
invert and open the stopcock to vent. Close the stopcock and shake the solution vigorously for
15 seconds. Allow the funnel to stand undisturbed until the layers separate (about 30
seconds). A yellow, red or bronze color in the bottom (chloroform) layer confirms the presence
of interfering metals. Draw off and collect the bottom (chloroform) layer for proper disposal.
2. Repeat extraction with fresh 5 mL portions of prepared dithizone solution (collecting the
bottom layer each time in appropriate waste collection vessel) until the bottom layer shows a
pure dark green color for three successive extracts. Extractions can be repeated a number of
times without appreciably affecting the amount of lead in the sample.
3. Extract the solution with several 2 or 3 mL portions of pure chloroform to remove any
remaining dithizone, again collecting the bottom layer each time for proper disposal.
4. Continue the procedure, substituting 28.5 mL of prepared dithizone solution for the 30 mL in
step 8.
Lead
Page 633
Lead
DithiVer solution preparation, storage and reagent blank

• Store DithiVer Powder Pillows away from light and heat.
• A convenient way to prepare this solution is to add the contents of 10 DithiVer Metals Reagent
Powder Pillows to a 500 mL bottle of chloroform.
• Invert several times until well mixed (carrier powder may not dissolve).
• Store dithizone solution in an amber glass bottle. This solution is stable for 24 hours.
• Carry out a reagent blank using deionized water through the entire method to obtain the most
accurate results.

• Collect samples in an acid-washed glass or plastic containers.
• Adjust the pH to 2 or less with nitric acid (about 2 mL per liter).
• Adjust the pH to 2.5 with 5.0 N sodium hydroxide before analysis.
Accuracy check
• Lead Voluette Ampule Standard, 50 mg/L Pb
• Ampule breaker
1. After reading test results, leave the sample cell (unspiked sample) in the instrument.Verify that
units are in µg/L.

6. Follow the Dithizone method for powder pillows test procedure for each of the spiked samples


• Lead Standard Solution, 100 mg/L
• Deionized water
Lead
Page 634
Lead

• Pipet filler
1. Prepare a 10 mg/L lead standard solution as follows:
a. Pipet 10.00 mL of Lead Standard, 100 mg/L, into a 100 mL volumetric flask.
2. Prepare a 200 µg/L lead standard solution as follows:
Use a graduated cylinder to measure 245 mL of deionized water into the 500 mL separatory
funnel (step 3 of the Dithizone method for powder pillows test). Pipet 5.00 mL of the 10.0 mg/L
Lead standard into the funnel.
3. Follow the Dithizone method for powder pillows test procedure.
4. To adjust the calibration curve using the reading obtained with the 200 µg/L Standard Solution,
Method performance
280 150 µg/L Pb 140–160 µg/L Pb 2.3 µg/L
Summary of method
The dithizone method is designed for the determination of lead in water and wastewater. The
DithiVer Metals Reagent is a stable powder form of dithizone. Lead ions in basic solution react with
dithizone to form a pink to red lead-dithizonate complex, which is extracted with chloroform. Test

Required reagents
Lead Reagent Set (100 Tests) — — 2243100
Includes: (1) 1420299, (2) 1445817, (1) 1261699, (2) 76714, (1) 245053, (2) 245026
Buffer Powder Pillows, citrate 1 100/pkg 1420299
Chloroform, ACS 30 mL 4L 1445817
DithiVer Metals Reagent Powder Pillows 1 100/pkg 1261699
Potassium Cyanide 0.1 g 125 g 76714
Sodium Hydroxide Solution, 5.0 N 5 mL 1000 mL 245053
Sodium Hydroxide Standard Solution, 5.0 N varies 59 mL DB 245026
Lead
Page 635
Lead
Required apparatus
Cylinder, graduated, 5 mL 1 each 50837
Cylinder, graduated, mixing, 50 mL 1 each 189641
Funnel, separatory, 500 mL 1 each 52049
pH Meter, sension™1, portable, with electrode 1 each 5170010
Spoon, measuring,1 g 1 each 51000
Support Ring, 4" 1 each 58001
Support Ring Stand, 5" x 8" base 1 each 56300
Sample Cell, 1-inch square, w/stopper, matched pair 2 2/pkg 2612602
Lead Standard Solution, 100 mg/L Pb 100 mL 1261742
Lead Standard Solution, 10 mL Voluette Ampules, 50 mg/L Pb 16/pkg 1426210

Chloroform, ACS 500 mL 1445849
Filter Discs, glass, 47 mm 100/pkg 253000
Filter Holder, glass, for 47-mm filter each 234000
Flask, Erlenmeyer, 500 mL each 50549
Flask, filtering, 500 mL each 54649
Pipet, serological, 2 mL each 53236
Sulfuric Acid, 5.25 N 100 mL MDB 244932

Lead, LeadTrak, 8317
Lead DOC316.53.01054
LeadTrak™ Fast Column Extraction Method Method 8317

5 to 150 µg/L
Scope and Application: For drinking water
Test preparation


adjust.
The sampling requirements for “first-draw” analysis are detailed in Sample collection, preservation and storage.
Reagents will stain the sample cells, rinse the cells with 1:1 nitric acid, LeadTrak followed by deionized water.
LeadTrak™ Reagent Set 1
Beaker, polypropylene, 150 mL 2
Beaker, polypropylene, 250 mL 1
Clamp, 2-prong extension, with clamp holder 1
Cylinder, graduated polypropylene, 25 mL 1
Cylinder, graduated polypropylene, 100 mL 1
Dropper, 0.5 and 1.0 mL marks 1
Support for Ring Stand 1
Lead
Page 637
Lead
LeadTrak Fast Column Extraction
Stored Programs
283 Lead, LeadTrak
Start
1. Select the test. 2. Fill a 100 mL plastic 3. Using a plastic 1mL 4. Start the instrument
Insert an adapter if graduated cylinder with dropper, add 1.0 mL of timer.
required (see Instrument- 100 mL of the sample. pPb-1 Acid Preservative A two-minute reaction
specific information). Pour the measured Solution to the sample and period will begin.
sample into a 250 mL swirl to mix.
Refer to the user manual plastic beaker.
for orientation. If the sample has been
preserved previously with
pPb-1 Acid Preservative at
a ratio of 1.0 mL per 100
mL sample, omit steps 3
and 4.
Samples preserved with
Nitric Acid require steps 3
and 4.
5. When the timer 6. Mount a new Fast 7. Soak the cotton plug 8. Pour the prepared
expires, use a second Column Extractor in a ring with deionized water and sample slowly into the
1 mL plastic dropper to stand with a clamp. Place compress it with the center of the Column
add 2.0 mL of pPb-2 Fixer a 150-mL plastic beaker plunger. Remove the Extractor. Wait for the
Solution. Swirl to mix. under the Extractor. plunger. If the cotton plug sample to flow through.
Field samples that have A Fast Column Extractor is moves up the column, The sample solution
been preserved with nitric included in the LeadTrak® push it back to the bottom should flow relatively
acid or samples that have Reagent Set. A new with a clean, blunt rod. slowly (2 drops per
been digested may extractor is required for The cotton plug should fit second) through the
exceed the buffer capacity each test. snugly against the inner column.
of the Fixer Solution. After wall of the column. Keep the level of the
step 5, check the pH of sample solution just above
these samples and adjust the cotton plug.
with 5 N Sodium
Hydroxide to a pH of 6.7–
7.1 before proceeding with
step 6.
Lead
Page 638
Lead
LeadTrak Fast Column Extraction (continued)
9. After the flow has 10. Place a clean, dry 150 11. Allow the Eluant 12. Using a 1 mL plastic
stopped, fully compress mL beaker under the Solution to drip slowly from dropper, add 1.0 mL of
the absorbent pad in the Extractor. Using a 25 mL the Extractor. pPb-4 Neutralizer Solution
Extractor with the plunger. plastic graduated cylinder, After the flow has stopped, to the beaker. Swirl
Discard the contents of the add 25 mL of pPb-3 Eluant fully compress the thoroughly to mix and
beaker. Slowly withdraw Solution to the Extractor. absorbent pad. proceed immediately to
the plunger from the Keep the level of the step 13.
Extractor. eluent solution just above
The absorbent pad should the absorbent pad.
remain at the bottom of the
Extractor when the
plunger is removed. If the
cotton plug moves up the
column, push it back to the
bottom with a clean,
blunt rod.
13. Add the contents of 14. Pour 10 mL of solution 15. Start the instrument 16. When the timer
one pPb-5 Indicator into a sample cell. timer. expires, insert the sample
Powder Pillow to the A two-minute reaction cell.
beaker and swirl period will begin.
thoroughly to mix.
The solution will turn
brown.
Lead
Page 639
Lead
LeadTrak Fast Column Extraction (continued)
Zero Read
17. ZERO the instrument. 18. Remove the sample 19. Insert the sample cell 20. READ the results in
The display will show: cell and add 3 drops of into the cell holder. µg/L Pb.
pPb-6 Decolorizer Solution
0 µg/L Pb to the cell. Swirl vigorously
to mix
Interferences
Interference studies were conducted by preparing a known lead solution of 25 µg/L as well as the
potential interfering ion. The ion was said to interfere when the resulting lead concentration
changed by ± 10%. Samples containing levels exceeding these concentration values may be
diluted 1:1 and analyzed. Multiply the value obtained by a factor of 2 to determine the lead present
in the original sample.
To avoid contamination, do not use black rubber stoppers, black dropper bulbs and droppers with
inked graduations. Use the plastic droppers provided in the reagent set.
Acid-wash all glassware and plasticware to prevent sample contamination, especially if the
previous sample had a high lead level (see Apparatus and sample preparation).
The Extractor plunger may be reused for more than one test but should be rinsed with lead-free
water between uses.

Aluminum, Al3+ 0.5 mg/L

Ammonium, NH4+ 500 mg/L
Barium, Ba2+ 6 mg/L
Calcium, Ca2+ 500 mg/L
Chloride, Cl– 1000 mg/L
Copper, Cu2+ 2 mg/L
Fluoride, F– 10 mg/L
Iron, Fe2+ 2 mg/L
Magnesium, Mg2+ 500 mg/L
Manganese, Mn2+ 0.5 mg/L
Nitrate, NO3– 1000 mg/L
Sulfate, SO42– 1000 mg/L
Zinc, Zn2+ 1 mg/L
Lead
Page 640
Lead
Apparatus and sample preparation

Because lead is very common to our environment, care must be taken to prevent sample
contamination. Follow these steps for greatest test accuracy:
• Lead-free water is necessary to minimize sample contamination when rinsing apparatus or
diluting sample. The water may be either distilled or deionized. If the water is obtained from a
grocery store, verify the lead concentration is zero from the label. If the lead concentration is
uncertain, determine the lead concentration with the LeadTrak test.
• Plastic or glass sample containers and lids may be checked for contamination by rinsing with 1
mL of pPb-1 Acid Preservative Reagent*. Add 100 mL of lead-free water. After 24 hours,
analyze this solution using the LeadTrak® test to confirm the absence of lead.
• Rinse glassware used in this test with a small amount of dilute lead-free 0.1 N nitric acid or
pPb-1 Acid Preservative Reagent followed by rinsing with lead-free water.
• pPb-5 Indicator may be rinsed from the glass sample cells with a few drops of pPb-1 Acid
Preservative Reagent or a small amount of dilute lead-free nitric acid.
• Acidify solutions containing lead with Nitric Acid or pPb-1 to below pH 2 to prevent adsorption
of lead onto the container walls. See Sample collection, preservation and storage.

• Samples may be collected either from household pipes (point-of-use) or from water sources.
• Preserved samples may be stored up to six months.
• Each sample type typically requires different sampling procedures. Consult with the
appropriate regulatory agency for more information about specific sampling requirements.
Sampling for lead contamination in household pipes for point-of-use drinking water
• The sample should be collected after sitting in pipes with no flow for a minimum of six hours.
• Add 10 mL of pPb-1 Acid Preservative* to a one-liter bottle.
• Turn on tap and collect exactly the first liter of water in the bottle containing acid preservative.
• Cap and invert several times to mix.
• After two minutes the sample is ready for analysis. Steps 3 and 4 are skipped in the analysis
procedure. Use 100 mL of this preserved sample directly in step 5.
Sampling for lead contamination from drinking water sources such as well water or water from main
supply lines
• Add 10 mL of pPb-1 Acid Preservative* to a one-liter bottle.
• Turn on the tap for 3–5 minutes or until the water temperature has been stable for 3 minutes.
• Collect exactly one liter of water into the bottle containing the acid preservative.
• Cap and invert several times to mix.
• After two minutes the sample is ready for analysis. Steps 3 and 4 are skipped in the analysis
procedure. Use 100 mL of this preserved sample directly in step 5.
• At least one liter should be collected to obtain a representative sample. If less than one liter is
collected, use 1 mL of pPb-1 Acid Preservative per 100 mL of sample.
• If nitric acid is to be substituted for pPb-1 as a preservative or the sample is digested, the
buffering capacity of the pPb-2 Fixer Solution* may be exceeded. Adjust the sample pH to
6.7–7.1 pH with 5 N Sodium Hydroxide* after step 6.
Lead
Page 641
Lead
Accuracy check
• 10 mg/L (10,000 µg/L) Lead Standard Solution
• TenSette Pipet and pipet tips
6. Follow the LeadTrak Fast Column Extraction test procedure for each of the spiked samples


• Lead Standard Solution, 1000 mg/L or Lead Voluette® Ampule Standard Solution, 50-mg/L
as Pb
• Lead-free water or Deionized water
• 100 mL Class A volumetric flask or 100-mL plastic volumetric flask
• 1.0 mL Class A volumetric pipet
1. Prepare a 100 µg/L Lead standard solution as follows:
a. Pipet 1.0 mL of Lead Standard, 1000 mg/L, into a 100 mL volumetric flask.
b. Use a Tensette Pipet to add 0.2 mL of concentrated nitric acid to the flask
c. Dilute to the mark with lead-free deionized water.
d. Pipet 10.00 mL of this prepared solution into a 1 liter plastic volumetric flask.
e. Add 2.0 mL of nitric acid to the flask.
f. Dilute to the mark with lead-free water.
g. Prepare this solution immediately before use.
OR
1. Prepare a 100 µg/L Lead Standard Solution as follows:
a. Use a Tensette Pipet to add 0.2 mL from a Lead Voluette® Ampule Standard Solution, 50
mg/L as Pb into a 100 mL plastic volumetric flask.
b. DIlute to volume with deionized water. Prepare solution immediately before use
Lead
Page 642
Lead
2. Use the standard solution in place of the sample. Follow the LeadTrak Fast Column Extraction
test procedure.
3. To adjust the calibration curve using the reading obtained with the 100-mg/L Standard
Method performance
Sensitivity
Distribution
283 50 µg/L Pb2+ 45–55 µg/L Pb2+ Entire curve 4 µg/L Pb2+
Summary of method
Acid soluble lead, as Pb2+, in a potable water sample is first concentrated on a Fast Column
Extractor. The lead is then eluted from the Extractor and determined colorimetrically with an
indicator. Test results are measured at 477 nm.

Required reagents
LeadTrak™ Reagent Set 1 20/pkg 2375000
Required apparatus
Beaker, polypropylene, 150 mL 2 each 108044
Beaker, polypropylene, 250 mL 1 each 108046
Clamp, 2-prong extension 1 each 2114500
Clamp Holder 1 each 32600
Cylinder, graduated polypropylene, 25 mL 1 each 108140
Cylinder, graduated polypropylene, 100 mL 1 each 108142
Dropper, 0.5 and 1.0 mL marks 1 20/pkg 2124720
Support for Ring Stand 1 each 56300

Lead Standard Solution, 1000 mg/L as Pb 100 mL 1279642
Lead Standard Solution, 50-mg/L 10 mL Voluette® Ampules 16/pkg 1426210
Lead Standard Solution, 10 mg/L 25 mL 2374820
Lead
Page 643
Lead
Recommended standards and apparatus (continued)



pPb-1 Acid Preservative Reagent 236 mL 2368531
pPb-2 Fixer Solution 43 mL 2368655
Sodium Hydroxide, 5.0 N 1L 245053

Manganese, HR, 8034
Manganese DOC316.53.01058
USEPA1 Periodate Oxidation Method2 Method 8034

HR (0.1 to 20.0 mg/L) Powder Pillows
Scope and Application: For soluble manganese in water and wastewater
1 USEPA Approved for reporting wastewater analyses (digestion required). Federal Register, 44(116)34 193 (June 14, 1979)
Test preparation


Digestion is required for reporting wastewater analyses.
If only dissolved manganese is to be determined, filter the sample before acid addition.
blank adjust.
High Range Manganese Reagent Set 1
Manganese
Page 645
Manganese
Periodate Oxidation method for powder pillows
Stored Programs
295 Manganese, HR
Start
1. Select the test. 2. Prepared Sample: 3. Add the contents of 4. Stopper or cap and
Insert an adapter if Fill a sample cell with 10 one Buffer Powder Pillow, invert to mix.
required (see Instrument- mL of sample. Citrate Type for
specific information). Manganese.
5. Add the contents of 6. Stopper or cap and 7. Start the instrument 8. Blank Preparation:
one Sodium Periodate invert to mix. timer. Fill a second sample cell
Powder Pillow to the A violet color will develop if A two-minute reaction time with 10 mL of sample.
sample cell. manganese is present. will begin.
Zero Read
9. When the timer 10. ZERO the instrument. 11. Within eight minutes 12. READ the results in
expires, insert the blank The display will show: after the timer expires, mg/L Mn.
into the cell holder. insert the sample into the
0.0 mg/L Mn cell holder
Manganese
Page 646
Manganese
Interferences

Calcium 700 mg/L
Chloride 70,000 mg/L
Iron 5 mg/L
Magnesium 100,000 mg/L
pH

• Collect samples in acid-washed plastic bottles. Do not use glass containers due to possible
adsorption of Mn to glass.
• If samples are acidified, adjust the pH to 4–5 with 5.0 N Sodium Hydroxide before analysis.
• Do not exceed pH 5, as manganese may precipitate.
Accuracy check
• Manganese Voluette® Ampule Standard, 250 mg/L Mn
• Ampule breaker
• TenSette Pipet
standard to three 10-mL portions of fresh sample. Mix thoroughly.
6. Follow the Periodate Oxidation method for powder pillows test procedure for each of the
spiked samples using the powder pillows, starting with the 0.1 mL sample spike. Measure


• Manganese Standard Solution, 1000 mg/L
• Deionized water
Manganese
Page 647
Manganese

• Pipet filler, safety bulb
1. Prepare a 10.0 mg/L manganese standard solution as follows:
a. Pipet 10.0 mL of Manganese Standard, 1000 mg/L, into a 1000 mL (1 liter) volumetric
flask.
2. Use this solution in place of the sample. Follow the Periodate Oxidation method for powder
pillows test procedure.
Method performance
Sensitivity
Program Standard 95% Confidence per 0.010 Abs change
Limits of Distribution
Portion of Curve Concentration
295 10.0 mg/L Mn 9.6–10.4 mg/L Mn Entire curve 0.1 mg/L Mn
Summary of method
Manganese in the sample is oxidized to the purple permanganate state by sodium periodate, after
buffering the sample with citrate. The purple color is directly proportional to the manganese
concentration. Test results are measured at 525 nm.
Required reagents
Manganese Reagent Set, High Range (100 tests), includes: — — 2430000
Buffer Powder Pillows, citrate type for Manganese 1 100/pkg 2107669
Sodium Periodate Powder Pillows for Manganese 1 100/pkg 2107769
Required apparatus
Stopper, rubber 1 6/pkg 173106
Manganese
Page 648
Manganese
Manganese Standard Solution, 1000 mg/L Mn 100 mL 1279142
Manganese Standard Solution, 250 mg/L Mn, 10-mL Voluette® ampule 16/pkg 1425810
Voluette Ampule breaker each 2196800

Manganese Standard Solution, 2 mL PourRite® Ampule, 25 mg/L 20/pkg 2112820

Manganese Standard Solution, 2 mL PourRite® Ampule, 10 mg/L 20/pkg 2605820
pH paper, 0–14 100/pkg 2601300
Pipe filler, safety bulb each 1465100
PourRite® Ampule breaker each 2484600
Volumetric flask, Class A, 1000 mL each 1457453
Volumetric pipet, Class A, 10 mL each 1451538
Manganese
Page 649
Manganese, LR, 8149
Manganese DOC316.53.01057
1-(2-Pyridylazo)-2-Naphthol PAN Method1 Method 8149

LR (0.006 to 0.700 mg/L) Powder Pillows
Scope and Application: For water and wastewater; digestion is required for determining total manganese
1 Adapted from Goto, K., et al., Talanta, 24, 652-3 (1977)
Test preparation


Rinse all glassware with 1:1 Nitric Acid Solution. Rinse again with deionized water.
The alkaline cyanide solution contains cyanide. Cyanide solutions should be collected for disposal as a reactive (D001)
waste. Be sure cyanide solutions are stored in a caustic solution with pH >11 to prevent release of hydrogen cyanide gas.
Refer to the current MSDS for safe handling and disposal instructions.
Total manganese determination requires a prior digestion. Refer to the Water Analysis Guide for more information.
Alkaline Cyanide Reagent 12 drops
Ascorbic Acid Powder Pillows 2
PAN Indicator Solution, 0.1% 12 drops
Stoppers for 18 mm tube 2
Manganese
Page 651
Manganese
Stored Programs
290 Manganese, LR PAN
Start
1. Select the test. 2. Blank Preparation: 3. Prepared Sample: 4. Add the contents of
Insert an adapter if Pour 10.0 mL of deionized Pour 10.0 mL of sample one Ascorbic Acid Powder
required (see Instrument- water into a sample cell. into another sample cell. Pillow to each cell.
specific information). Total manganese
determination requires
prior digestion.
5. Stopper and invert to 6. Add 12 drops of 7. Add 12 drops of PAN 8. Start the instrument
dissolve the powder. Alkaline-Cyanide Reagent Indicator Solution, 0.1%, timer.
Solution to each cell. Swirl to each sample cell. Swirl A two-minute reaction
gently to mix. gently to mix. period will begin.
A cloudy solution may An orange color will
form. The turbidity should develop in the sample if
dissipate after step 7. manganese is present.
Zero Read
9. When the timer 10. ZERO the instrument. 11. Wipe the prepared cell 12. READ the results in
expires, wipe the blank The display will show: and place it in the holder. mg/L Mn.
and place it in the cell
holder. 0.000 mg/L Mn
Manganese
Page 652
Manganese
Interferences
For samples that contain hardness greater than 300 mg/L CaCO3, add 4 drops of Rochelle Salt
Solution to the sample after adding the Ascorbic Acid Powder Pillow in step 4.

Aluminum 20 mg/L
Cadmium 10 mg/L
Cobalt 20 mg/L
Copper 50 mg/L
25 mg/L (If sample contains more than 5 mg/L iron, allow a 10-minute reaction period in
Iron
step 8.)
Lead 0.5 mg/L
Nickel 40 mg/L
Zinc 15 mg/L

• Collect samples in a clean plastic container.
• Adjust the pH to 2 or less with Concentrated Nitric Acid* (about 2 mL per liter).
• Preserved samples can be stored up to six months at room temperature.
• Adjust the pH to between 4–5 with 5.0 N Sodium Hydroxide before analysis.
Accuracy check
• Manganese PourRite® Ampule Standard, 10-mg/L Mn
• Ampule breaker, PourRite
• TenSette Pipet
standard to three 10 mL portions of fresh sample. Mix thoroughly.
Manganese
Page 653
Manganese
6. Follow the PAN method for powder pillows test procedure for each of the spiked samples
using the powder pillows, starting with the 0.1 mL sample spike. Measure each of the spiked
samples in the instrument.


• Manganese Voluette Standard Solution, 250 mg/L Mn
• Deionized water
• Pipet filler, safety bulb
1. Prepare a 0.5 mg/L manganese standard solution as follows:
a. Pipet 2.0 mL of Manganese Standard, 250 mg/L as Mn, into a 1000 mL (1 liter)
volumetric flask.
2. Use this solution in place of the sample. Follow the PAN method for powder pillows test
procedure.
Method performance
290 0.500 mg/L Mn 0.491–0.509 mg/L Mn 0.006 mg/L Mn
Summary of method
The PAN method is a highly sensitive and rapid procedure for detecting low levels of manganese.
An ascorbic acid reagent is used initially to reduce all oxidized forms of manganese to Mn2+. An
alkaline-cyanide reagent is added to mask any potential interferences. PAN Indicator is then
added to combine with the Mn2+ to form an orange-colored complex. Test results are measured at
560 nm.
Manganese
Page 654
Manganese
Required reagents
Manganese Reagent Set, 10 mL (50 tests), includes: — — 2651700
Alkaline Cyanide Reagent 12 drops 50 mL SCDB 2122326
Ascorbic Acid Powder Pillows 2 pillows 100/pkg 1457799
PAN Indicator Solution, 0.1% 12 drops 50 mL SCDB 2122426
Required apparatus
Stoppers for 18 mm Tube 2 6/pkg 173106
Manganese Standard Solution, 10-mg/L Mn, 2 mL PourRite® ampule 20/pkg 2605820

Manganese Standard Solution, 250-mg/L Mn, 10-mL Voluette® ampule 16/pkg 1425810
PourRite® Ampule breaker 2 mL each 2484600

pH paper, 0-14 100/pkg 2601300
Rochelle Salt Solution 29 mL 172533
PourRite® Ampule breaker 2 mL each 2484600
Manganese Standard Solution, 2-mL PourRite® Ampule, 25 mg/L 20/pkg 2112820
Manganese
Page 655
Mercury, 10065
Mercury DOC316.53.01059
Cold Vapor Mercury Concentration Method1 Method 10065

0.1 to 2.5 µg/L Hg
1 Patent number 5,733,786
Test preparation

Perform phase 1 of the procedure in a fume hood. Toxic chlorine or other gases may be produced.
Use dedicated digestion glassware and sample cells for this procedure.
Determine a reagent blank for each new lot of reagent by running the entire procedure, including the digestion, using one liter
of deionized water instead of sample. Add the same amount of potassium permanganate as required by the sample. Subtract
the reagent blank value from the final results or perform a reagent blank adjust.
Cold Vapor Mercury Reagent Set (see Required apparatus for contents of reagent set) 1
Digestion Reagents and Apparatus (see Required digestion reagents and apparatus) varies
Cold Vapor Mercury Apparatus Set 1
Sample Cells, 1-inch square, 10-mL, matched pair (wherever applicable) 2
See Required apparatus for a complete list of required apparatus.
Mercury
Page 657
Mercury
Phase 1: Sample digestion

DANGER
Toxic gas hazard. Test must be performed under a fume hood.
1. Transfer one liter of 2. Add 50 mL of 3. Add 25 mL of 4. Add 4.0 g of

the sample to a 2000 mL concentrated sulfuric acid concentrated nitric acid to potassium persulfate to
Erlenmeyer flask. Add a to the sample. the sample. the sample. Stir until
50-mm magnetic stir bar to dissolved.
the sample. Set the flask Alternatively, add one
on a magnetic stirring hot 5 gram measuring scoop
plate and begin stirring. of potassium persulfate to
the sample.
5. Add 7.5 g of 6. Cover the flask with a 7. Continue to stir and 8. Cool the digested
potassium permanganate watch glass. Begin heating heat the sample at 90 °C sample to room
to the sample. Stir until the sample to a for two hours. temperature. Turn the hot
dissolved. temperature of 90 °C after The solution must remain plate off.
Alternatively, add a the reagents have dark purple throughout the A brown/black precipitate
10 gram measuring scoop dissolved. Do not boil. entire digestion. Some of manganese dioxide
of potassium For a mercury standard or samples, such as sea may settle during cooling.
permanganate to the reagent blank in distilled waters, industrial effluents If the digested sample
sample. water, the heating step is or other samples high in does not have a purple
not necessary. organic matter or chloride color, the digestion may be
concentration, require incomplete. Add more
additional permanganate. potassium permanganate.
It may be difficult to see a Return the sample to the
dark purple color if the magnetic stirring hot plate
sample contains black/ and continue the digestion
brown manganese dioxide until the purple color
precipitate. Add more persists.
potassium permanganate
if the solution is not dark
purple.
Mercury
Page 658
Mercury
Phase 1: Sample digestion (continued)

DANGER
9. Return the cool, 10. Using a 0.5 g 11. Remove the stir bar. 12. The digested sample
digested sample to the measuring spoon, add is now ready for
cool, magnetic stirring hot 0.5 g additions of processing by cold vapor
plate. Turn on the stirrer. hydroxylamine- separation and
hydrochloride until the preconcentration. Proceed
purple color disappears. to Phase 2.
Wait 30 seconds after
each addition to see if the
purple disappears. Add
hydroxylamine-
hydrochloride until all
manganese dioxide is
dissolved.
Phase 2: Cold vapor separation and preconcentration of mercury

DANGER
1. Transfer the digested 2. Set the Gas Washing 3. Connect the 100 mL 4. Pipet 8 mL of
sample to the Cold Vapor Bottle in the support ring. Erlenmeyer flask to the HgEx Reagent B into the
Gas Washing Bottle. (The Place the top on the Gas mercury absorber column. Mercury Absorber column.
volume of the digested Washing Bottle. Wait until
sample should contain step 9 to connect the
0.1 to 2.5 µg Hg.) mercury absorber column
to the Gas Washing Bottle.
Mercury
Page 659
Mercury
Phase 2: Cold vapor separation and preconcentration of mercury (continued)

DANGER
5. Connect the power to 6. Disconnect the 7. Remove the 100 mL 8. Pipet 2 mL of HgEx
the vacuum pump and vacuum using the quick Erlenmeyer flask from the Reagent C into the
apply vacuum to the disconnect when Mercury Absorber Mercury Absorber
Mercury Absorber HgEx Reagent B begins to Column. Replace it with Column.
Column. Draw most of the drip from the inner delivery the 10 mL Distilling
HgEx Reagent B into the tube on the Mercury Receiver.
Erlenmeyer flask. Absorber Column (about
10 seconds after starting
the vacuum). Do not draw
enough air through the
column to begin drying the
packing.
9. Connect the Mercury 10. Shake an ampule of 11. Stopper the side neck 12. Reconnect the
Absorber column to the HgEx Reagent A to on the Glass Washing vacuum to the Mercury
Gas Washing Bottle using suspend undissolved Bottle. Absorber Column using
the glass elbow. reagent. the quick disconnect. The
Open the ampule and vacuum will pull HgEx
gently shake the contents Reagent C through the
into the Gas Washing Mercury Absorber Column
Bottle through the side packing and into the
neck. 10 mL receiver. Air
bubbles should be
produced at the gas
dispersion tube in the Gas
Washing Bottle. Perform
steps 13–14 immediately.
Mercury
Page 660
Mercury

DANGER
Stored Programs
312 Mercury, Cold Vap
Start
13. Select the test. 14. Start the instrument 15. After the timer expires, 16. Pipet 8 mL of HgEx
timer. remove the glass elbow Reagent B into the
A five-minute reaction from the top of the Mercury Absorber Column
period will begin. Let the Mercury Absorber to elute the captured
solution bubble for this Column. Keep the vacuum mercury.
period. pump on. Continue to apply vacuum
Air flow rate through the to pull the HgEx Reagent
Gas Washing Bottle B into the Distilling
should be between Receiver.
1–5 L/min. Allow more
bubbling time for lower air
flow rates. For example, if
the air flow rate is 1 L/min.,
let the solution bubble for
10 minutes.
Mercury
Page 661
Mercury

DANGER
17. Turn off or disconnect 18. Remove the distilling 19. Pipet 3 mL of HgEx
power to the vacuum Receiver from the Mercury Reagent B into the
pump when the volume in Absorber Column. Mercury Absorber Column
the Distilling Receiver Reconnect the 100-mL without applying vacuum.
reaches the 10 mL mark. Erlenmeyer flask to the This keeps the absorber
If necessary, the volume in column. packing wet between
the Distilling Receiver may tests.
be brought up to 10 mL The Mercury Absorber
with HgEx Reagent B. To Column eluate in the
avoid low volumes in the Distilling Receiver is ready
future, disconnect the for analysis.
vacuum a little sooner in Proceed to Phase 3.
step 6. This leaves more
HgEx Reagent B in the
packing of the Mercury
Absorber Column.
Phase 3: Colorimetric analysis

DANGER
1. Using the funnel 2. Add the contents of 3. Add 8 drops of HgEx 4. Start the instrument
provided, add the contents one HgEx Reagent 4 foil Reagent 5 to the Distilling timer.
of one HgEx Reagent 3 foil pillow to the Distilling Receiver. Stopper the A two-minute reaction
pillow to the eluate in the Receiver using the funnel Receiver. Invert to mix. period will begin.
Distilling Receiver. provided. Stopper the
Stopper the receiver. receiver. Invert to dissolve
Invert to dissolve the the reagent.
reagent.
Mercury
Page 662
Mercury
Phase 3: Colorimetric analysis (continued)

DANGER
Zero
5. During the reaction 6. Wipe the sample cell. 7. ZERO the instrument. 8. Remove the cell from
period, transfer the After the timer expires, The display will show: the cell holder. Add the
solution to a sample cell. insert the sample into the contents of one HgEx
cell holder. 0.1 µg/L Hg (this Reagent 6 foil pillow to the
program uses a non-zero solution. Swirl the cell until
intercept) the reagent is completely
dissolved. Immediately go
to step 9.
Do not use the funnel to
add HgEx Reagent 6 to
the sample cell. Any HgEx
Reagent 6 in the funnel
will make mercury
undetectable in
subsequent tests.
Read
9. Return the sample cell 10. READ the results in

to the cell holder. µg/L Hg. This is the
concentration in the
original sample.
Interferences
Standards were used to prepare a single test solution with substances at the concentrations listed
in the Interfering substances and levels table. A second test solution containing only mercury at
the same concentration was prepared as the control. The two solutions were digested, then
analyzed concurrently. There was no interference from the matrix of the test solution at the
concentrations listed.
In addition, no interference occurred with a test solution containing 1000 mg/L Na+, 1000 mg/L K+,
1000 mg/L Mg2+ and 400 mg/L Ca2+.
Mercury
Page 663
Mercury

Ag+ 7 mg/L Ag+

Al+3 10 mg/L Al+3
Au+3 500 µg/L Au+3
Cd+2 10 mg/ L Cd+2
Co+2 10 mg/L Co+2
Cr+6 10 mg/L Cr+6
Cu+2 10 mg/L Cu+2
F– 1.0 mg/L F–
Fe+2 100 mg/L Fe+2
Hg+2 1 µg/L Hg+2
Mo+6 10 mg/L Mo+6
Ni+2 10 mg/L Ni+2
NO3 ––N 50 mg/L NO3––N
Pb2+ 10 mg/L Pb2+
SiO2 100 mg/L SiO2
Zn+2 10 mg/L Zn+2

• Collect 1000 mL of sample in an analytically clean, glass or polyethylene terephthalate (PET)
container.
• Add 10 mL of concentrated hydrochloric acid to preserve the sample before sample collection.
Fill the container completely full to minimize air space when closed.
Note: Close a glass container with a ground glass stopper. Close a PET container with a PET cap or a
polypropylene cap (no liner).
• Store aqueous samples at 2–6 °C. Acid-preserved samples are stable for at least 6 months.
Accuracy check
Standard additions method
1. Prepare a 10.0 mg/L Mercury Standard Solution as described under Standard solution
method, step 3a.
2. Use a TenSette® Pipet to add 0.10 mL of the 10.0 mg/L Mercury Standard Solution to the
purged solution in the Gas Washing Bottle after an analysis has been performed. Immediately
stopper the Gas Washing Bottle.
3. Begin at step 3 of Phase 2. Follow the procedure steps.

4. Test the eluate as described in Phase 3. The displayed concentration should be 0.9–1.1 µg/L
Hg.
1. Transfer 800 mL of deionized water into the Gas Washing Bottle.
Mercury
Page 664
Mercury
2. Add 50 mL of concentrated sulfuric acid and 25 mL of concentrated nitric acid to the water.
Swirl to mix.
3. Prepare a 0.1 mg/L mercury standard solution by serially diluting a 1000 mg/L Mercury
Standard Solution:
a. To make a 10.0 mg/L standard, add 1.0 mL of concentrated nitric acid to a 500 mL
volumetric flask. Dilute 5.00 mL of a 1000 mg/L standard to 500 mL with deionized water.
Mix well.
b. To make a 1.0 mg/L standard solution, add 0.2 mL of concentrated nitric acid to a 100 mL
volumetric flask. Dilute 10.0 mL of the 10.0 mg/L standard to 100 mL with deionized water.
Mix well.
c. To make a 0.1 mg/L standard solution, add 0.2 mL of concentrated nitric acid to a 100 mL
volumetric flask. Dilute 10.00 mL of the 1.0 mg/L solution to 100 mL with deionized water.
Mix well.
4. Pipet 10.0 mL of the 0.1 mg/L mercury standard solution into the Gas Washing Bottle. Swirl to
mix.
Hg.
System start up
For more accurate results, perform a few analyses on mercury standards and blanks for system
equilibration before beginning sample testing. This allows the system to stabilize before
processing samples.
Startup standard
1. Test a mercury standard solution by following the procedure under Accuracy check using the
Standard solution method. Continue with step 2 of the Startup Standard procedure if the value
is not within specified limits.
2. Pipet 10.0 mL of the 0.1 mg/L mercury standard solution into the purged solution in the Gas
Washing Bottle. Immediately stopper the Gas Washing Bottle.

Hg. Repeat steps 1–3 if the value is not within these limits.
Startup blank
Run a system blank by using the purged solution in the Gas Washing Bottle after a satisfactory test
of the Startup Standard has been completed.
1. Leave the purged solution in the Gas Washing Bottle. Do not add an aliquot of
mercury standard.
3. Test the eluate as described in Phase 3. The displayed concentration should be

≤ 0.2 µg/L Hg. Repeat the Startup Blank procedure until a reproducible value is obtained.
Mercury
Page 665
Mercury
Method performance
312 1.0 µg/L Hg 0.9–1.1 µg/L Hg 0.03 µg/L Hg
Storage and maintenance of the cold vapor mercury apparatus

Storage
Store the apparatus as follows for fastest system stabilization and greatest sensitivity:
• Store the Gas Washing Bottle filled with deionized water containing 15 mL of concentrated
sulfuric acid. Seal the bottle with the Gas Washing Bottle stopper and top.
• Store the Mercury Absorber Column with the packing wetted with HgEx Reagent B. The
Erlenmeyer flask should be kept attached underneath the column. The top of the Mercury
Absorber column should be attached to the Gas Washing Bottle with the glass elbow as in the
procedure.
Glassware care
Use of dedicated glassware and sample cells is recommended because of the sensitivity of this
procedure. Thoroughly clean the glassware and sample cells between tests. After washing, rinse
with 1:1 hydrochloric acid solution, then rinse several times with deionized water.
Maintaining the system
• With proper care and storage, the Mercury Absorber Column may be used an unlimited
number of times.
• Replace the Mercury Scrubber in the air trap housing at least once for every reagent set used.
• Moisture build up on the Gas Washing Bottle side of the Acro 50 Vent Filter will reduce the
purging air flow rate. If this occurs replace the filter or dry it in an oven at 110 °C.
Summary of method
The sample is digested to convert all forms of mercury in the sample to mercuric (Hg2+) ions. The
mercuric ions in the digested sample are converted to mercury vapor in a semi-closed system. The
vapor is carried by ambient air into a chemically activated absorber column where the mercury
vapor is converted to mercuric chloride.
The mercuric chloride is eluted off the column and a sensitive indicator is added. The instrument is
zeroed using the absorbance peak of the unreacted indicator. A complexing agent is added to
break the mercury:indicator complex. The increase in unreacted indicator causes an increase in
absorbance proportional to the amount of mercury in the original sample. Test results are
measured at 412 nm.
Safety
Wear personal protective equipment such as safety glasses with side shields or a face shield to
protect your eyes. Use other protective equipment as necessary (such as a fume hood) to avoid
chemical exposure. Perform all steps exactly as prescribed in the procedure.

Proper management and disposal of waste is the responsibility of the waste generator. It is up to
the generator to arrange for proper disposal and comply with applicable local, state and federal
Mercury
Page 666
Mercury
regulations governing waste disposal. The manufacturer makes no guarantees or warranties,

express or implied, for the waste disposal information represented in this procedure.
1. Dispose of the solution in the Gas Washing Bottle by neutralizing the solution to a pH of 6–9
and flushing to the sanitary sewer with water for several minutes.
2. The mercury contained in one liter of sample is concentrated by a factor of 100 by the Mercury
Absorber Column. Mercury analysis within the range of the test may produce a solution in the
sample cell that is above the RCRA Toxicity Characteristic limit of 0.20 mg/L Hg or other
regulatory limits. The sample cell will contain 0.25 mg/L mercury if the original sample was at
2.5 µg/L mercury (the upper limit of the test range). Dispose of the solution in the sample cell
according to applicable regulations.
3. The mercury scrubber will capture mercury vapor if the Mercury Absorber Column is not
properly activated using HgEx Reagent B and HgEx Reagent C. In addition, mercury is also
captured if the capacity of the Absorber Column is exceeded. If the Mercury Scrubber has
captured mercury vapor, it must be disposed of according to applicable regulations.
Required reagents
Cold Vapor Mercury Reagent Set (25 tests), includes: 2658300
HgEx™ Reagent A, Stannous Sulfate Solution, 1 25/pkg 2658825
20 mL ampules
HgEx™ Reagent B, Sulfuric Acid Solution 19 mL 500 mL 2658949
HgEx™ Reagent C, Sodium Hypochlorite Solution 2 mL 55 mL 2659059
HgEx™ Reagent 3, Alkaline Reagent Powder Pillows 1 pillow 25/pkg 2658448
HgEx™ Reagent 4, Indicator Powder Pillows 1 pillow 25/pkg 2658548
HgEx™ Reagent 5, Sodium Hydroxide Solution 8 drops 10 mL SCDB 2658636
HgEx™ Reagent 6, Complexing Reagent Powder Pillows 1 pillow 25/pkg 2658748
Mercury Scrubber 2/reagent set 2/pkg 2655800
Required apparatus
Cold Vapor Mercury Apparatus Set, includes: 2674400
Acro 50 Vent Filter 1 18/pkg 2683318
Air Trap Holder Assembly 1 each 2663900
Ampule Breaker 1 each 2564000
Breaker/Capper Tool for Mercury Scrubber 1 each 2664000
C-flex Tubing, 0.25-inch ID, white 4 ft 25 ft 2327367
Clamp for Mercury Absorber Column 1 each 2656200
Clamp Holder 2 each 32600
Sample cell 10 mL Matched 2/pkg 2495402
Riser Cell, 1” Square DR2000/2010 1 each 4528200
Riser cell, 1”, Square DR3000 1 each 4840300
Distilling Receiver, 10-mL 1 each 2655438
Mercury
Page 667
Mercury

Gas Washing Bottle, 1200-mL 1 each 2662200
Glass Elbow, 90-degree, with hose adapter 1 each 2655200
Mercury Absorber Column 1 each 2655510
Support Ring for Gas Washing Bottle 1 each 2656300
Stopper, for Distilling Receiver 1 each 2655900
Stopper, for Gas Washing Bottle 1 each 2662300
Support, Base and Rod 1 each 32900
Tubing Quick Disconnect, HDPE 1 12/pkg 1481000
Pipet, TenSette, 1.0 to 10.0 mL 1 each 1970010
Vacuum Pump, 115 VAC w/ North American Plug 1 each 2824800
Vacuum Pump, 230 VAC w/ North American Plug 1 each 2824801
Vacuum Pump, 230 V w/ European Plug 1 each 2824802
Required digestion reagents and apparatus

Flask, Erlenmeyer, 2000 mL 1 each 2489454
Hot Plate/Stirrer, 7 x 7, Cimarec Digital, 115 VAC 1 each 2881600
Digital Hot Plate 7 x 7, 230 VAC 1 each 2881502
Hydroxylamine Hydrochloride, ACS varies 113 g 24614
Nitric Acid, ACS 25 mL 500 mL 15249
Potassium Permanganate, ACS varies 454 g 16801H
Potassium Persulfate, ACS 4.0 g 454 g 2617501
Sulfuric Acid, ACS, concentrated 50 mL 2.5 L 97909
Spoon, measuring, 0.5 g 1 each 90700
Stir Bar, Octagonal 50.8 x 7.9 mm 1 each 2095355
Thermometer, –20 to 110 °C 1 each 56601
Watch Glass, Pyrex, 65 mm 1 each 57867
Mercury Standard Solution, 1000 mg/L Hg (NIST) 100 mL 1419542

Molybdenum, HR, 8036
Molybdenum DOC316.53.01061
Mercaptoacetic Acid Method1 Method 8036

HR (0.2 to 40.0 mg/L Mo) Powder Pillows or AccuVac® Ampuls
Scope and Application: For water, wastewater, boiler and cooling waters
1 Adapted from Analytical Chemistry. 25(9) 1363 (1953)
Test preparation


Instrument
deionized water instead of the sample. Subtract the reagent blank from the result or adjust a reagent blank.
Filter turbid samples using filter paper and a funnel.
After all reagents have been added, the presence of molybdenum will cause a yellow color to form.
Adjust pH of acidified stored samples to pH 7 before analysis.
Powder Pillow Test:
MolyVer® 1 Molybdenum Reagent Powder Pillows 1
Sample Cell for blank (see Instrument-specific information) 2
Molybdenum
Page 669
Molybdenum
AccuVac Test:
CDTA Solution, 0.4 M 4 drops
MolyVer® 6 Reagent AccuVac® Ampuls 1
Beaker, 50 mL 1
Mercaptoacetic Acid for powder pillows
Stored Programs
320 Molybdenum HR
Start
1. Select the test. 2. Fill a sample cell with 3. Prepared Sample: 4. Add the contents of
Insert an adapter if 10-mL of sample. Add the contents of one one MolyVer 2 Reagent
required (see Instrument- MolyVer® 1 Reagent Powder Pillow. Swirl
specific information). Powder Pillow. Swirl to mix.
to mix.
5. Add the contents of 6. Start the instrument 7. Blank Preparation: 8. Insert the blank into
one MolyVer 3 Reagent timer. When the timer expires, fill the cell holder
Powder Pillow. Swirl to A five-minute reaction a second sample cell with
mix. period will begin. 10 mL of the original
sample.
Molybdenum
Page 670
Molybdenum
Mercaptoacetic Acid for powder pillows (continued)
Zero Read
The display will show: sample into the cell holder mg/L Mo6+.
0.0 mg/L Mo6+
Mercaptoacetic Acid for AccuVac® Ampuls
Stored Programs
322 Molybdenum HR
Start
1. Select the test. 2. Blank Preparation: 3. Prepared Sample: 4. Fill a MolyVer 6

Insert an adapter if Fill a sample cell with Collect 40 mL of sample in AccuVac® Ampul with the
required (see Instrument- 10 mL of sample. a 50-mL beaker. Add four treated sample. Make sure
specific information). drops of 0.4 M CDTA that the tip is immersed
Standard Solution to the when filling the Ampul.
sample in the beaker. Swirl
to mix
Zero Read
5. Quickly invert the 6. Start the instrument 7. When the timer 8. Insert the prepared
Ampul several times timer. expired insert the blank sample into the cell holder.
to mix. A five-minute reaction into the cell holder. READ the results in
period will begin. ZERO the instrument. mg/L Mo6+
0.0 mg/L Mo6+
Molybdenum
Page 671
Molybdenum
Interferences

Aluminum Greater than 50 mg/L
Chromium Greater than 1000 mg/L
Samples containing 10 mg/L copper or more will exhibit an increasing positive interference
Copper upon standing. Read these samples as soon as possible after the five minute reaction period
is complete.
Nickel Greater than 50 mg/L
Interference from up to 2000 mg/L as NO2– can be eliminated by adding one Sulfamic Acid
Nitrite
Powder Pillow1 to the sample.
extreme sample pH

• Adjust the pH to 2 or less with nitric acid (about 2 mL/L).
• Preserved samples can be stored up to 6 months at room temperature.
• Adjust the pH to 7 with 5.0 N Sodium Hydroxide before analysis.
Accuracy check
Standard additions method (sample spike) for powder pillows
• Molybdenum Standard Solution, 1000-mg/L Mo6+
• TenSette Pipet, 0.1 to 1.0 mL and Pipet tips
6. Follow the Mercaptoacetic Acid for powder pillows test procedure for each of the spiked
samples using the powder pillows, starting with the 0.2 mL sample spike. Measure each of the
spiked samples in the instrument.
Molybdenum
Page 672
Molybdenum

1. Fill three 100 mL mixing cylinders each with 60-mL of sample and spike with 0.4 mL, 0.8 mL
and 1.2 mL of standard.
2. Transfer 40 mL from each of the three mixing cylinders to three 50-mL beakers.
3. Analyze each standard addition sample as described in the Mercaptoacetic Acid for
AccuVac® Ampuls.
recovery.


• Molybdenum Standard Solution,10.0-mg/L Mo6+
1. Use the molybdenum standard solution in place of the sample. Follow the Mercaptoacetic Acid
for powder pillows or the Mercaptoacetic Acid for AccuVac® Ampuls test procedure.
Method performance
320 10.0 mg/L Mo6+ 9.7–10.3 mg/L Mo6+ 0.2 mg/L Mo6+
Summary of method
MolyVer 1 and 2 Reagents are added to buffer and condition the sample. MolyVer 3 provides the
mercaptoacetic acid which reacts with molybdate molybdenum to form a yellow color proportional
to the molybdenum concentration. Test results are measured at 420 nm.
Required reagents
Molybdenum Reagent Set, for 10-mL samples (100 tests): — — 2604100
MolyVer® 1 Molybdenum Reagent Powder Pillows 1 100/pkg 2604299
OR
MolyVer® 6 Reagent AccuVac® Ampuls 1 25/pkg 2522025
CDTA Solution, 0.4 M 4 drops 15 mL SCDB 2615436
Molybdenum
Page 673
Molybdenum
Required apparatus
Molybdenum Standard Solution, 10-mg-L as Mo 100 mL 1418742
Molybdenum Standard Solution, 1000-mg-L as Mo 100 mL 1418642

Beakers, 50 mL each 50041H
Filter Paper, folded, 12.5 cm 100/pkg 189457
Sulfamic Acid Powder Pillows 100/pkg 105599

Molybdenum, LR, 8169
Molybdenum DOC316.53.01062
Ternary Complex Method Method 8169

LR (0.02 to 3.00 mg/L Mo) Powder Pillows
Scope and Application: For boiler and cooling tower waters
Test preparation


Analyze samples immediately after collection.
Filter turbid samples using filter paper1 and a funnel1.

Molybdenum Reagent Set for 20-mL sample
Molybdenum 1 Reagent (LR) Molybdate Powder Pillow 1
Molybdenum 2 Reagent Solution 0.5 mL
Molybdenum
Page 675
Molybdenum
Ternary Complex Method for powder pillows
Stored Programs
315 Molybdenum LR
Start
1. Select the test. 2. Fill a 25-mL graduated 3. Add the contents of 4. Prepared Sample:
Insert an adapter if mixing cylinder with 20 mL one Molybdenum 1 Stopper the cylinder and
required (see Instrument- of sample. Reagent Powder Pillow to shake to completely
specific information). the graduated cylinder. dissolve the reagent.
for orientation.
5. Pour 10-mL of the 6. Developed Sample: 7. Swirl to mix. 8. Start the instrument
prepared sample into a Add 0.5 mL of timer.
sample cell. Molybdenum 2 Reagent to A two-minute reaction time
the sample cell. will begin.
Zero
9. Blank Preparation: 10. Wipe the blank and 11. ZERO the instrument. 12. Wipe the developed
When the timer expires, fill insert it into the cell holder. The display will show: sample and insert it into
a sample cell with 10 mL the cell holder.
of the remaining prepared 0.00 mg/L Mo6+
READ the results in
sample. mg/L Mo6+.
Molybdenum
Page 676
Molybdenum
Interferences
Interference studies were conducted by preparing a molybdenum standard solution
(2 mg/L Mo6+) containing the potential interfering ion. When the standard solution concentration
changed by ± 5% with a given ion concentration, the ion was considered an interference.
Interference results are summarized in Substances that cause a negative interference,
Substances that cause a positive interference and Non-interfering substances.
Highly buffered samples or extreme sample pH may exceed the buffering capacity of the reagent
and require sample pretreatment. Adjust the sample pH to between 3–5 by adding, by drops, an
appropriate amount of acid or base such as 1.0 N Sulfuric Acid Standard Solution* or 1.0 N
Sodium Hydroxide Standard Solution*. If significant volumes of acid or base are used, a volume
correction should be made by dividing the total volume (sample + acid + base) by the original
volume and multiplying the test result by this factor.
After a number of samples have been analyzed, the sample cells may exhibit a slight bluish
discoloration. Rinse the cells with 1:1 Hydrochloric Acid Solution* to eliminate this build-up.
Table 211 Substances that cause a negative interference

Alum Greater than 7 mg/L
AMP (Phosphonate) Greater than 15 mg/L
Bicarbonate Greater than 5650 mg/L
Bisulfate Greater than 3300 mg/L
Borate Greater than 5250 mg/L
Chloride Greater than 1400 mg/L
Chromium Greater than 4.5 mg/L1
Diethanoldithiocarbamate Greater than 32 mg/L
EDTA Greater than 1500 mg/L
Ethylene Glycol Greater than 2% (by volume)
Lignin Sulfonate Greater than 105 mg/L
Nitrite Greater than 350 mg/L
Orthophosphate Greater than 4500 mg/L
Phosphonohydroxyacetic Acid Greater than 32 mg/L
Positive interference of about 10% up to 30 mg/L. As the concentration increases above
Phosphonate HEDP 30 mg/L, a decrease in the molybdenum concentration reading occurs (negative
interference).
Sulfite Greater than 6500 mg/L
1 Read the molybdenum concentration immediately after the 2-minute reaction period.
Table 212 Substances that cause a positive interference

Benzotriazole Greater than 210 mg/L
Carbonate Greater than 1325 mg/L
Morpholine Greater than 6 mg/L
Molybdenum
Page 677
Molybdenum
Table 212 Substances that cause a positive interference (continued)

The presence of the phosphonate HEDP at concentrations up to 30 mg/L will increase the
Phosphonate HEDP apparent molybdenum concentration reading by approximately 10% (positive interference).
Multiply the value obtained in step 12 by 0.9 to obtain the actual Mo6+ concentration.
Silica Greater than 600 mg/L

Bisulfite 9600 mg/L
Calcium 720 mg/L
Chlorine 7.5 mg/L
Magnesium 8000 mg/L
Manganese 1600 mg/L
Nickel 250 mg/L
PBTC (phosphonate) 500 mg/L
Sulfate 12,800 mg/L
Zinc 400 mg/L

• Collect samples in glass or plastic bottles.
Accuracy check
• Molybdenum Standard Solution, 1000-mg/L Mo6+
• Graduated cylinder, 250 mL
• Erlenmeyer flasks (3)
• TenSette Pipet and Pipet tips
chemical form.
4. Open the standard solution bottle.
6. Follow the Ternary Complex Method for powder pillows test procedure for each of the spiked
Molybdenum
Page 678
Molybdenum


• Molybdenum Standard Solution, 10.00-mg/L
• Deionized water
• 10 mL, Class A volumetric pipet
• Pipet filler
1. Prepare a 2.00-mg/L molybdenum standard solution as follows:
a. Pipet 10.00 mL of Molybdenum Standard Solution, 10.00-mg/L, into a 50-mL volumetric

flask.
2. Use this solution in place of the sample. Follow the Ternary Complex Method for powder
Method performance
Summary of method
The ternary complex method for molybdenum determination is a method in which molybdate
molybdenum reacts with an indicator and a sensitizing agent to give a stable blue complex. Test
Molybdenum
Page 679
Molybdenum

Required reagents
Molybdenum Reagent Set for 20-mL sample (100 tests), includes: — — 2449400
(1) Molybdenum 1 Reagent (LR) Molybdate Powder Pillows 1 100/pkg 2352449
(1) Molybdenum 2 Reagent Solution 0.5 mL 50 mL MDB 2352512
Required apparatus
Molybdenum Standard Solution, 10-mg/L Mo6+ 100 mL 1418742

Molybdenum Standard Solution, 1000-mg/L Mo6+ 100 mL 1418642

Cylinder graduated, 250 mL each 108146
Pipet, Volumetric Class A, 10 mL each 1451538
Hydrochloric Acid Solution 1:1 500 mL 88449

Nickel, 8037
Nickel DOC316.53.01064
USEPA1 Heptoxime Method2 Method 8037

0.02 to 1.8 mg/L Ni Powder Pillows
Scope and Application: For Water, Wastewater and Seawater
1 USEPA accepted for reporting wastewater analyses (digestion required). Procedure is equivalent to Standard Method 3500-Ni D for
wastewater
2 Adapted from Chemie Analytique, 36 43 (1954)
Test preparation


adjust.
Make the cotton plug pea-size. A larger plug will restrict the flow; a smaller plug may become dislodged from the delivery
tube of the funnel.
Chloroform (D022) solutions are regulated as hazardous waste. Do not pour these materials down the drain. Water saturated
with chloroform, chloroform solutions and the cotton plug used in the delivery tube of the separatory funnel should be
collected for proper disposal. Refer to a current MSDS for safe handling and disposal instructions.
In bright light conditions (e.g. direct sunlight) it may be necessary to close the DR 3900, DR 3800, DR 2800 and DR 2700 cell
compartment with the protective cover during measurements.
Chloroform, ACS 30 mL
Nickel 1 Reagent Powder Pillow 1
Nickel 2 Reagent Powder Pillow 1
Nickel
Page 681
Nickel
Clippers for Opening Pillows 1
Cotton Balls varies
Cylinder, graduated. 10-mL 1
Funnel, separatory with stand and stopper 1
Heptoxime method for powder pillows
Stored Programs
335 Nickel Heptoxime
Start
1. Select the test. 2. Measure 300 mL of 3. Add the contents of 4. Start the instrument
Insert an adapter if sample in a 500-mL one Nickel 1 Reagent timer.
required (see Instrument- graduated cylinder. Pour Powder Pillow to the A five-minute reaction time
specific information). into a 500-mL separatory funnel. Stopper and invert will begin.
funnel. to mix.
5. When the timer 6. Start the instrument 7. When the timer 8. Close the stopcock
expires, add the contents timer. expires, add 10 mL of and invert for 30 seconds.
of one Nickel 2 Reagent A second five-minute chloroform. Insert the
Powder Pillow to the reaction time will begin. stopper and invert gently.
funnel. Stopper and invert With the funnel inverted
to mix. and the tip pointed away
from people, open the
stopcock to vent.
Nickel
Page 682
Nickel
Heptoxime method for powder pillows (continued)
9. Start the instrument 10. Prepared Sample: 11. Repeat steps 7 12. Cap the sample cell
timer. When the timer expires, through 10 two additional and invert to mix the
A third five-minute reaction wait for the layers to times with 10-mL volumes extracts. The final volume
period will begin. separate. Insert a of chloroform. The will be about 25 mL due to
pea-sized cotton plug into five-minute reaction period the slight solubility of
Invert the funnel several the delivery tube of the is not necessary. chloroform in water.
times over the five minute funnel. Remove the
period. stopper and drain the
chloroform layer (bottom
layer) into a sample cell.
Insert the stopper into the
funnel.
Zero
13. Blank Preparation: 14. Wipe the blank and 15. ZERO the instrument. 16. Wipe the prepared
Fill a second sample cell insert it into the cell holder. The display will show: sample and insert it into
with 10 mL of chloroform. the cell holder.
0.00 mg/L Ni
Cap the cell. READ the results in mg/L
Ni.
Interferences
Cobalt, copper and iron interferences can be overcome by adding additional Nickel 1 Reagent
Powder Pillows in step 3 of the Heptoxime method for powder pillows. The tolerance limits of these
interferences are shown in the Interfering substances table.
A preliminary acid digestion is required to determine any suspended or precipitated nickel and to
eliminate interference by organic matter. To eliminate this interference or to determine total
recoverable nickel perform the USEPA approved digestion.
Nickel
Page 683
Nickel
Pillows of Nickel 1 Tolerance Limit (mg/L)

Reagent Cobalt Copper Iron
1 1 10 20
2 7 16 65
3 13 22 110
4 18 28 155
5 25 35 200

• Adjust the sample pH to 2 or less with Nitric Acid*, about 5 mL per liter. Preserved samples
can be stored up to six months at room temperature.
• Before analysis, adjust the sample pH to between 3–8 with 5.0 N Sodium Hydroxide Standard
Solution*. Do not exceed pH 8 as this may cause some loss of nickel as a precipitate.
Accuracy check
Prepare a 300 mg/L nickel standard by pipetting 15 mL of 1000 mg/L Nickel standard into a 50 ml
volumetric flask. Dilute to volume and mix well.
• Nickel Standard Solution, 1000-mg/L Ni
• Volumetric Pipet, 15 mL
• Volumetric Flask, 50 mL
• Pipet Filler
300 mg/L standard to three 300-mL portions of fresh sample.
5. Follow the Heptoxime method for powder pillows test procedure for each of the spiked
Nickel
Page 684
Nickel


• Nickel Standard Solution, 1000-mg/L
• Deionized water
• Volumetric flasks, 500 mL and 1000 mL
• Volumetric pipets, 10 mL and 50 mL
• Pipet filler
1. Prepare a 10.0 mg/L nickel working solution as follows:
a. Pipet 10.0 mL of a Nickel Standard Solution, 1000-mg/L, into a 1000-mL (1 liter)

volumetric flask.
2. Prepare a 1.0-mg/L nickel standard solution by diluting 50.0 mL of the 10-mg/L working
standard solution to 500 mL in a volumetric flask.
3. Use the standard solution in place of the sample. Follow the Heptoxime method for powder
Method performance
335 1.00 mg/L Ni 0.93–1.07 mg/L Ni 0.02 mg/L Ni
Summary of method
Nickel ion reacts with heptoxime to form a yellow-colored complex which is then extracted into
chloroform to concentrate the color and enable a more sensitive determination. Chelating agents
are added to the sample to overcome the interferences caused by cobalt, copper and iron.
Readings are taken at 430 nm.
Required reagents
Nickel Reagent Set (50 Tests), includes: — — 2243500
(3) Chloroform, ACS 30 mL 500 mL 1445849
(2) Nickel 1 Reagent Powder Pillows 1 25/pkg 212368
(2) Nickel 2 Reagent Powder Pillows 1 25/pkg 212468
Nickel
Page 685
Nickel
Required apparatus
Clippers 1 each 96800
Ring, support, 4-inch 1 each 58001
Sample Cell, 1-inch square, w/stopper, matched pair 2 2/pkg 2612602
Stand, support, 5 x 8-inch base 1 each 56300
Nickel Standard Solution, 1000-mg/L Ni (NIST) 100 mL 1417642

Pipet, volumetric, 10 mL each 1451538

Nickel, 8150
Nickel DOC316.53.01063
1-(2 Pyridylazo)-2-Napthol (PAN) Method1 Method 8150

0.006 to 1.000 mg/L Ni Powder Pillows
Scope and Application: For water and wastewater; digestion is required for determining total nickel
1 Adapted from Watanabe, H., Talanta, 21 295 (1974)
Test preparation


Cobalt concentration can be determined with the same sample by using Program Number 110.
If the sample is less than 10 °C (50 °F), warm to room temperature before analysis. Adjust the pH of acidified stored
samples.
EDTA Powder Pillow 2
Phthalate-Phosphate Reagent Powder Pillow 2
PAN Indicator Solution, 0.3% 1 mL
Stoppers 2
Nickel
Page 687
Nickel
Stored Programs
340 Nickel, PAN
Start
Insert an adapter if Fill a sample cell to the Fill a second sample cell one Phthalate-Phosphate
required (see Instrument- 10-mL mark with sample. to the 10-mL mark with Reagent Powder Pillow to
specific information). deionized water. each cell.
for orientation.
5. Stopper the cells. 6. Using the plastic 7. Insert stoppers into 8. Start the instrument
Immediately shake to dropper provided, add the cells. Invert several timer.
dissolve. 0.5 mL of 0.3% PAN times to mix. A 15-minute reaction
If the sample contains iron, Indicator Solution to period will begin.
make sure that all the each cell.
During color development,
powder is dissolved before the sample solution color
proceeding to step 6. may vary from yellowish-
orange to dark red,
depending on the
chemical makeup of the
sample. The blank should
be yellow.
Nickel
Page 688
Nickel
PAN method for powder pillows (continued)
Zero
9. When the timer 10. Stopper the cells and 11. Wipe the blank and 12. ZERO the instrument.
expires, add the contents shake to dissolve. insert it into the cell holder The display will show:
of one EDTA Reagent
Powder Pillow to each cell. 0.00 mg/L Ni
The instrument will zero at
560 and 620 nm.
Read
13. Wipe the sample cell 14. READ the results in

and insert it into the cell mg/L Ni and Co.
holder.
Interferences

Al3+ 32 mg/L
Ca2+ 1000 mg/L as (CaCO3)
Cd2+ 20 mg/L
Cl– 8000 mg/L
Interfere at all levels. Use either the Digesdahl or vigorous digestion to eliminate
Chelating agents (e.g., EDTA)
this interference.
Cr3+ 20 mg/L
Cr6+ 40 mg/L
Cu2+ 15 mg/L
F– 20 mg/L
Fe3+ 10 mg/L
Nickel
Page 689
Nickel

Fe2+ Interferes directly and must not be present.
K+ 500 mg/L
Mg2+ 400 mg/L
Mn2+ 25 mg/L
Mo6+ 60 mg/L
Na+ 5000 mg/L
Pb2+ 20 mg/L
Zn2+ 30 mg/L
extreme sample pH

• Adjust the sample pH to 2 or less with Nitric Acid*, about 5 mL per liter. Preserved samples
can be stored up to six months at room temperature.
• Before analysis, adjust the sample pH to between 3 and 8 with 5.0 N Sodium Hydroxide
Standard Solution*. Do not exceed pH 8 as this may cause some loss of nickel as a
precipitate.
• If the sample is less than 10 °C, warm it to room temperature.
Accuracy check
• Nickel Standard solution, 1000-mg/L Ni
• 5-mL Volumetric Pipet, Class A
• 100 mL Volumetric Flask
• Pipet Filler
• Deionized Water
1. Prepare a 50 mg/L Nickel standard by pipetting 5.00 mL of 1000 mg/L Ni standard solution into
a 100 mL volumetric flask. Dilute the solution to the required volume and mix well.
Nickel
Page 690
Nickel
standard to three 25-mL portions of fresh sample. Mix well.
6. Transfer 10-mL of each solution into sample cells. Follow the PAN method for powder pillows
test procedure for each of the spiked samples using the powder pillows, starting with the


• Nickel Standard Solution, 1000-mg/L as Ni
• Deionized water
• 1-L volumetric flask, Class A
• 100-mL Volumetric flask
• Volumetric pipets, 5 mL and 10 mL
• Pipet filler
1. Prepare a 5.00 mg/L nickel stock solution as follows:
a. Pipet 5.00 mL of Nickel Standard Solution, 1000-mg/L as Ni, into a 1000-mL (1 liter)
volumetric flask.
2. Prepare a 0.5 mg/L nickel working solution as follows:
a. 10.0 mL of the 5.00-mg/L nickel stock solution into a 100-mL volumetric flask.
3. Use the working solution in place of the sample. Follow the PAN method for powder pillows
test procedure.
Method performance
340 0.500 mg/L Ni 0.492–0.508 mg.L Ni 0.006 mg/L Ni
Summary of method
After buffering the sample and masking any Fe3+ with pyrophosphate, the nickel is reacted with 1-
(2-Pyridylazo)-2-Naphthol indicator. The indicator forms complexes with most metals present.
After color development, EDTA is added to destroy all metal-PAN complexes except nickel and
cobalt. The instrument automatically adjusts for cobalt interference by measuring the absorbance
Nickel
Page 691
Nickel
of the sample at both 560 nm and 620 nm. This method is unique because both nickel and cobalt
can be determined on the same sample when using a spectrophotometer.
Required reagents
Nickel Reagent Set (100 Tests), includes: — — 2651600
(2) EDTA Reagent Powder Pillows 2 100/pkg 700599
(2) Phthalate-Phosphate Reagent Powder Pillows 2 100/pkg 2615199
(1) PAN Indicator Solution, 0.3% 1 mL 100 mL MDB 2150232
Required apparatus
Stoppers 2 6/pkg 173106
Nickel Standard Solution, 1000-mg/L Ni (NIST) 100 mL 1417642


Nitrate HR, TNT, 10020
Nitrate DOC316.53.01068
Chromotropic Acid Method Method 10020

HR (0.2 to 30.0 mg/L NO3––N) Test ‘N Tube™ Vials
Test preparation


DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
For DR 3900, DR 3800, DR 2800 and DR 2700: Install the light shield before performing this test.
deionized water (nitrate-free) in place of the sample. Subtract the reagent blank value from the final results or perform a
reagent blank adjust.
This test is technique-sensitive. Invert the vials as described here to avoid low results: Hold the vial in a vertical position with
the cap pointing up. Turn the vial upside-down. Wait for all of the solution to flow down to the cap. Pause. Return the vial to
an upright position. Wait for all the solution to flow to the bottom of the vial. This process equals one inversion.
Test ‘N Tube™ NitraVer® X Reagent Set 1
Funnel, micro, poly 1
Pipet, TenSette®, 0.01 to 1.0 mL, plus tips 1
Test Tube Rack 1
Nitrate
Page 693
Nitrate
Chromotropic Acid method for TNT
Stored Programs
344 N, Nitrate HR, TNT
Start
1. Select the test. 2. Blank Preparation: 3. Cap the tube and 4. Wipe the blank and
Insert an adapter if Remove the cap from a invert ten times to mix. insert it into the 16-mm cell
required (see Instrument- NitraVer X Reagent A Test holder.
specific information). ‘N Tube vial and add
1.00 mL of sample.
for orientation.
Zero
5. ZERO the instrument. 6. Prepared Sample: 7. Cap and invert ten 8. Start the instrument
The display will show: Remove the vial from the times to mix. timer.
instrument. Using a funnel, Some solid matter will A five-minute reaction time
0.0 mg/L NO3––N add the contents of one not dissolve. will begin. Do not invert
NitraVer X Reagent B the vial again.
Powder Pillow to the vial.
A yellow color will develop
if nitrate is present.
Read
9. Within five minutes 10. READ the results in

after the timer expires, mg/L NO3––N.
wipe the prepared sample To display other chemical
and insert it into the cell forms, refer to the
holder. instrument user manual.
Nitrate
Page 694
Nitrate
Interferences

Barium A negative interference at concentrations greater than 1 mg/L.
Chloride Does not interfere below 1000 mg/L.
A positive interference at concentrations greater than 12 mg/L. (Remove nitrite interference
Nitrite up to 100 mg/L by adding 400 mg (one full 0.5 g Hach measuring spoon) of Urea to 10 mL of
sample. Swirl to dissolve. Proceed with the nitrate test as usual.)
Copper Positive interference at all levels.

• Store the samples at 4 °C (39 °F) or lower if the sample will be analyzed within 24 to 48 hours.
For longer storage periods (up to 14 days), adjust sample pH to 2 or less with concentrated
Sulfuric Acid, ACS* (about 2 mL per liter). Sample refrigeration is still required.
• Before testing, warm the stored sample to room temperature and neutralize with 5.0 N Sodium
Hydroxide Standard Solution*. Do not use mercury compounds as preservatives.
• Correct the test result for volume additions:
( acid volume + base volume + sample volume )
Corrected result = ---------------------------------------------------------------------------------------------------------------------------- × test result
original sample volume
Accuracy check
• High Range Nitrate Nitrogen Voluette® Ampule Standard, 500 mg/L NO3––N
• Ampule breaker
standard to three mixing cylinders with 25-mL portions of fresh sample.
6. Add 1 mL of the 0.1 mL spiked sample to a TNT vial and follow the Chromotropic Acid method
for TNT test procedure. Repeat for each spiked sample. Measure each of the spiked samples
in the instrument.
Nitrate
Page 695
Nitrate


• 10.0-mg/L Nitrate Nitrogen Standard Solution
1. Use the 10.0-mg/L Nitrate Nitrogen Standard Solution solution in place of the sample. Follow
the Chromotropic Acid method for TNT test procedure.
Method performance
344 10.0 mg/L NO3––N 9.5–10.5 mg/L NO3––N 0.2 mg/L NO3––N
Summary of method
Nitrate in the sample reacts with chromotropic acid under strongly acidic conditions to yield a
yellow product with a maximum absorbance at 410 nm.
Required reagents
Test ‘N Tube™ NitraVer® X Nitrate Reagent Set 1 50/pkg 2605345

Funnel, micro, poly 1 each 2584335
Test Tube Rack 1 each 1864100
Nitrate Nitrogen Standard Solution, 10-mg/L: N03–N 500 mL 30749
Nitrate Nitrogen Standard Solution, Voluette® Ampule, 500-mg/L N03–N 16/pkg 1426010
Wastewater Influent Inorganics Standard for NH3–N, NO3–N, PO4, COD, SO4, TOC 500 mL 2833149
Nitrate
Page 696
Nitrate

Ampule breaker each 2196800
Pipet Tips, for TenSette Pipet 1970001 each 1000/pkg
Sulfuric Acid ACS, Concentrated 500 mL 97949
Urea, ACS grade 100 g 1123726
Nitrate
Page 697
Nitrate, HR, 8039
Cadmium Reduction Method Method 8039

HR (0.3 to 30.0 mg/L NO3––N) Powder Pillow or AccuVac® Ampuls
Test preparation


Instrument
adjust.
A deposit of unoxidized metal will remain at the bottom of the cell after the NitraVer® 5 dissolves. The deposit will not affect
results.
This method is technique-sensitive. Shaking time and technique influence color development. For most accurate results,
make successive tests on a 10-mg/L Nitrate Nitrogen Standard solution. Adjust shaking time and technique to obtain the
correct result. Use this technique for all the samples.
Rinse the sample cell immediately after use to remove all cadmium particles. Prepared samples will contain cadmium and
must be disposed of according to federal, state and local hazardous waste regulations. Refer to the current MSDS for safe
handling and disposal instructions.
Nitrate
Page 699
Nitrate
Powder Pillow Test:
NitraVer® 5 Nitrate Reagent Powder Pillow 1
Sample Cells, 1-inch, 10-mL, with stopper 2
AccuVac Test:
NitraVer® 5 Nitrate Reagent AccuVac® Ampul 1
Beaker, 50-mL 1
Sample Cell, for blank 1
Cadmium Reduction Method for powder pillows

CAUTION
Hazardous waste exposure. Prepared samples contain cadmium. Refer to the current MSDS for safe
handling and disposal instructions. Follow all federal, state and local hazardous waste
disposal regulations.
Stored Programs
355 N, Nitrate HR PP
Start
Insert an adapter if 10 mL of sample. Add the contents of one timer. A one-minute
required (see Instrument- NitraVer 5 Nitrate Reagent reaction time will begin.
specific information). Powder Pillow. Stopper.

for orientation.
Nitrate
Page 700
Nitrate
Cadmium Reduction Method for powder pillows (continued)

CAUTION
5. Shake the cell 6. When the timer 7. Blank Preparation: 8. Wipe the blank and
vigorously until the timer expires, start the timer When the timer expires, fill insert it into the cell holder.
expires. again. A five-minute a second sample cell with
Note: Some solid materials reaction period will begin. 10 mL of sample.
may not dissolve.
An amber color will
develop if nitrate is
present.
Zero Read
9. ZERO the instrument. 10. Within one minute 11. READ the results in
The display will show: after the timer expires, mg/L NO3––N.
wipe the prepared sample To display other chemical
0.0 mg/L NO3––N and insert it into the cell forms, refer to the user
holder. manual.
Nitrate
Page 701
Nitrate
Cadmium Reduction Method for AccuVac® Ampuls

CAUTION
Stored Programs
361 N, Nitrate HR AV
Start
1. Select the test. 2. Prepared Sample: 3. Tap the bottom of a 4. Start the instrument
Insert an adapter if Collect at least 40 mL of NitraVer 5 Nitrate timer.
required (see Instrument- sample in a 50-mL beaker. AccuVac® Ampul on a A one-minute reaction
specific information). hard surface to dislodge period will begin.
powder. Fill the Ampul with
Refer to the user manual sample. Keep the tip
for orientation. immersed while the Ampul
fills completely. Insert a
cap over the Ampul tip.
5. Invert the Ampul 6. When the timer 7. Blank Preparation: 8. Wipe the blank and
48–52 times as the timer expires, start the timer When the timer expires, fill insert it into the cell holder.
counts down. again. A five-minute a round sample cell with
reaction period will begin. 10 mL of sample.
Do not agitate or disturb
the sample during this
time.
An amber color will
present.
Nitrate
Page 702
Nitrate
Cadmium Reduction Method for AccuVac® Ampuls (continued)

CAUTION
Zero Read
9. ZERO the instrument. 10. Within one minute 11. READ the results in
wipe the Ampul and insert
0.0 mg/L NO3––N into the cell holder.
Interferences

Chloride concentrations above 100 mg/L will cause low results. The test may be used at high
Chloride chloride concentrations (seawater), but a calibration must be done using standards spiked to
the same chloride concentration. Refer to Seawater calibration.
Ferric iron Interferes at all levels
Interferes at all levels
Compensate for nitrite interference as follows:
Before performing step 3, add 30-g/L Bromine Water1 dropwise to the sample until a yellow
Nitrite
color remains.
Add one drop of 30-g/L Phenol Solution1 to destroy the color.
Proceed with step 3. Report the results as total nitrate and nitrite.
pH
Strong oxidizing and reducing
Interfere at all levels
substances
Nitrate
Page 703
Nitrate
Seawater calibration
Chloride concentrations above 100 mg/L will cause low results. To perform this test in water with
high interference level, calibrate the water using standards spiked to the same chloride
concentrations as the required samples. To prepare calibration standards containing 5.0, 10.0,
20.0 and 30.0 mg/L nitrate as NO3––N:
1. Prepare a 1 L volume of chloride water that matches the concentration of the samples, using
the following equation:
c. Add necessary Chloride concentration (g/L) x (1.6485) = g of ACS grade NaCl to 1 L of

deionized water. 18.8 g/L is a typical seawater chloride concentration.
d. Mix this solution thoroughly to get a homogeneous solution. Use this water as the dilution
water instead of the deionized water when preparing the nitrate standards.
2. Use Class A glassware or a Tensette Pipet to pipet 0.5, 1.0, 2.0, and 3.0 mL of the 1000 mg/L
Nitrogen-Nitrate as NO3––N (NIST) Standard Solution (Catalog Number 1279249) into four
different 100 mL Class A volumetric flasks.
3. Dilute to the mark with the prepared chloride water. Mix thoroughly.
4. Use the prepared chloride water for the 0-mg/L nitrate as NO3––N standard.

• More reliable results are obtained when samples are analyzed as soon as possible after
collection. If prompt analysis is impossible, store samples in clean plastic or glass bottles for
up to 24 hours at 4 °C. To preserve samples for longer periods, add 2 mL of Concentrated
Sulfuric Acid (H2SO4)* per liter and store at 4 °C. The results are reported as total nitrate plus
nitrite.
• Before analysis, warm the sample to room temperature and adjust the pH to 7 with 5.0 N
Sodium Hydroxide Standard Solution*. Do not use mercury compounds as preservatives.
• Correct the test result for volume additions by dividing the total volume (acid + base + sample)
by the original sample volume and multiplying the test result by this factor.
Accuracy check
Standard additions method for powder pillows (sample spike)
• Nitrate Nitrogen Standard solution, 1000-mg/L NO3––N
• Mixing cylinders
• 25-mL Volumetric pipet
• Pipet filler
• 100 mL Volumetric Flask
1. Prepare a 250-mL nitrate nitrogen standard solution by pipetting 25 mL of a 1000 mg/L Nitrate
Nitrogen standard solution into a 100 mL volumetric flask. Dilute the solution to the required
volume with deionized water and mix thoroughly.
Nitrate
Page 704
Nitrate
6. Follow the Cadmium Reduction Method for powder pillows test procedure for each of the
1. Fill three mixing cylinders each with 50-mL of sample and spike with 0.4 mL, 0.8 mL and
1.2 mL of 250-mg/L standard.
3. Analyze each standard addition sample as described in the Cadmium Reduction Method for
AccuVac® Ampuls.
recovery.


1. Use the 10.0-mg/L Nitrate Nitrogen Standard Solution in place of the sample. Follow the
Cadmium Reduction Method for powder pillows and Cadmium Reduction Method for
AccuVac® Ampuls test procedures.
Method performance
Sensitivity
Distribution
355 10 mg/L NO3––N 9.3–10.7 mg/L NO3––N 0 ppm 0.3 mg/L NO3––N
10 ppm 0.5 mg/L NO3––N
361 10 mg/L NO3––N 9.3–10.7 mg/L NO3 ––N 0 ppm 0.5 mg/L NO3––N
Nitrate
Page 705
Nitrate
Summary of method
Cadmium metal reduces nitrates in the sample to nitrite. The nitrite ion reacts in an acidic medium
with sulfanilic acid to form an intermediate diazonium salt. The salt couples with gentisic acid to
form an amber colored solution. Test results are measured at 500 nm.
Required reagents
NitraVer® 5 Nitrate Reagent Powder Pillows (for 10-mL sample) 1 100/pkg 2106169
OR
NitraVer® 5 Nitrate Reagent AccuVac® Ampul 1 25/pkg 2511025

Stopper, Neoprene, solid, size #1 2 12/pkg 1480801
or

Nitrate Nitrogen Standard Solution, 10.0-mg/L NO3––N 500 mL 30749
Nitrate Nitrogen Standard Solution 1000 mg/L NO3––N 500 mL 1279249

Wastewater Influent Standard, Mixed Parameter, for NH3–N, NO3–N, PO4, COD, SO4, 500 mL 2833149
TOC

Pipet, Volumetric, Class A, 0.5 mL each 1451534
Nitrate
Page 706
Nitrate

Phenol Solution, 30 g/L 29 mL 211220
Sulfuric Acid, concentrated, ACS 500 mL 97949
Nitrate
Page 707
Nitrate, LR, 8192

LR (0.01 to 0.50 mg/L NO3––N) Powder Pillows
Test preparation


adjust.
results.
Shaking time and technique influence color development. Analyze a standard solution several times and adjust the shaking
time to obtain the correct result. Use this technique for analyzing samples. See the Standard solution method.
Rinse the sample cell and mixing cylinder immediately after use to remove all cadmium particles.
Properly dispose of the used sample. Prepared samples contain cadmium and must be disposed of according to Federal,
State and local hazardous waste regulations. Refer to the current MSDS for safe handling and disposal information.
NitraVer® 6 Nitrate Reagent powder pillow 1
NitriVer® 3 Nitrite Reagent powder pillow 1
Cylinder, graduated, mixing, 25-mL 1
Nitrate
Page 709
Nitrate
Cadmium Reduction Method for powder pillows

CAUTION
Stored Programs
351 N, Nitrate LR
Start
1. Select the test. 2. Fill a 25-mL graduated 3. Add the contents of 4. Start the instrument
Insert an adapter if mixing cylinder with 15 mL one NitraVer 6 Reagent timer.
required (see Instrument- of sample. Powder Pillow to the A three-minute reaction
specific information). cylinder. Insert the time will begin.
stopper.
for orientation.
5. Shake the cylinder 6. When the timer 7. When the timer 8. Prepared Sample:
vigorously during the expires, start the expires, carefully pour Add the contents of one
three-minute timer. instrument timer again. 10 mL of the sample into a NitriVer 3 Nitrite Reagent
Note: Some solid materials
A two-minute reaction clean sample cell. Do not Powder Pillow to the
may not dissolve. transfer any cadmium sample cell.
period will begin.
particles to the sample
cell.
Nitrate
Page 710
Nitrate
Cadmium Reduction Method for powder pillows (continued)

CAUTION
9. Start the instrument 10. Cap and shake the 11. Start the instrument 12. Blank Preparation:
timer. A 30-second sample cell gently during timer. When the timer expires, fill
reaction time will begin. the 30-second timer. A 15-minute reaction a second square sample
A pink color will develop if period will begin. cell with 10 mL of original
nitrate is present. sample.
Zero Read
13. Wipe and insert the 14. ZERO the instrument. 15. Wipe and insert the 16. READ the results in
blank into the cell holder. The display will show: prepared sample into the mg/L NO3––N.
cell holder.
0.00 mg/L NO3––N
Nitrate
Page 711
Nitrate
Interferences

Calcium 100 mg/L
Chloride chloride concentrations (seawater) but a calibration must be done using standards spiked to
the same chloride concentration. (Refer to Sea Water Calibration.)
Ferric iron All levels
All levels: This method measures both the nitrate and nitrite in the sample. If nitrite is
present, the nitrite nitrogen test (Program #371) should be done on the sample. Pretreat the
nitrate nitrogen sample with the following pretreatment. Then subtract the amount of nitrite
found from the results of the LR nitrate nitrogen test.
Nitrite 1. Add 30-g/L Bromine Water1 dropwise to the sample in step 3 until a yellow color
remains. Mix after each drop.
2. Add one drop of 30-g/L Phenol Solution1 to destroy the color.
3. Proceed with the Cadmium Reduction Method for powder pillows.
pH
substances
high-interference level, calibrate the water using standards spiked to the same chloride
concentrations as the required samples. To prepare calibration standards containing 0.06, 0.1, 0.3
and 0.4 mg/L nitrate as NO3–N:
e. Add necessary Chloride concentration (g/L) x (1.6485) = g of ACS grade NaCl to 1 L of

deionized water. (
Note: 18.8 g/L is a typical seawater chloride concentration.
f. Mix this solution thoroughly to make sure that it is a homogeneous solution. Use this water
as the dilution water instead of the deionized water when preparing the nitrate standards.
2. Use Class A glassware or a Tensette Pipet to pipet 0.6, 1, 3 and 4 mL of the 10 mg/L
Nitrogen-Nitrate as NO3-N (NIST) Standard Solution (Catalog Number 30749) into four
4. Use the prepared chloride water for the 0-mg/L nitrate as NO3–N standard.

up to 48 hours at 4 °C. The results will be in total nitrate and plus nitrate.
• To preserve samples for longer periods, add 2 mL of Concentrated Sulfuric Acid* per liter and
store at 4 °C.
Nitrate
Page 712
Nitrate
Accuracy check
• Nitrate Nitrogen Standard, Solution, 100-mg/L NO3––N
• TenSette Pipet 1–10 mL and tips
• 50-mL Volumetric Flask
• Pipet filler
1. Prepare a 12-mL nitrate nitrogen standard solution by pipetting 6.0 mL of a 100 mg/L Nitrate
Nitrogen standard solution into a 50 mL volumetric flask. Dilute the solution to required
volume with deionized water and mix thoroughly.
5. Use the TenSette Pipet to prepare spiked samples in three mixing cylinders: add 0.1 mL, 0.2
mL and 0.3 mL of 12 mg/L standard to three 15-mL portions of fresh sample.
6. Follow the Cadmium Reduction Method for powder pillows test procedure for each of the


• Nitrate Nitrogen Standard Solution, 10-mg/L
• Deionized water
• Volumetric flask, 100 mL
• Volumetric pipet, 4 mL
1. Prepare a 0.40-mg/L NO3––N standard solution as follows:
a. Pipet 4.00 mL of Nitrate Nitrogen Standard Solution, 10 mg/L, into a 100 mL volumetric
flask.
Nitrate
Page 713
Nitrate
2. Use this solution in place of the sample. Follow the Cadmium Reduction Method for powder
Method performance
Summary of method
with sulfanilic acid to form an intermediate diazonium salt. The salt couples with chromotropic acid
to form a pink-colored product. Test results are measured at 507 nm.
Required reagents
Low Range Nitrate Reagent Set (100 tests), includes: — — 2429800
NitraVer® 6 Nitrate Reagent Powder Pillows 1 100/pkg 2107249
NitriVer® 3 Nitrite Reagent Powder Pillows 1 100/pkg 2107169
Required apparatus
Nitrate Nitrogen Standard Solution, 10.0 mg/L NO3––N 500 mL 30749
Nitrate Nitrogen Standard Solution, 100 mg/L NO3 ––N 500 mL 194749
Nitrate
Page 714
Nitrate

Flask, Volumetric, 50 mL each 1457441
Phenol Solution, 30-g/L 29 mL 211220
Sodium Hydroxide Standard Solution, 5.0 N 1L 245053
Sulfuric Acid, concentrated 500 mL 97949
Sodium chloride, ACS 454 g 18201H
Pipet, TenSette 1–10 mL each 1970010

Nitrate
Nitrate
Page 716
Nitrate, MR, 8171

MR (0.1 to 10.0 mg/L NO3 ––N) Powder Pillows or AccuVac® Ampuls
Test preparation


Instrument
results.
This method is technique-sensitive. Shaking time and technique influence color development. For most accurate results,
make successive tests on a 10.0-mg/L Nitrate Nitrogen Standard solution. Adjust shaking times to obtain the correct result.
Rinse the sample cell immediately after use to remove all cadmium particles. Retain the used sample for proper hazardous
waste disposal for cadmium.
Prepared samples will contain cadmium and must be disposed of according to Federal, State and local hazardous waste
regulations. Refer to the current MSDS for safe handling and disposal instructions.
Nitrate
Page 717
Nitrate
Powder Pillow Test:
NitraVer® 5 Nitrate Reagent Powder Pillow 1
Stopper, Neoprene, #1, solid 2
AccuVac Test:
NitraVer® 5 Nitrate Reagent AccuVac® Ampul 1
Beaker, 50-mL 1
Sample Cell for blank (see Instrument-specific information) 1
Cadmium reduction method for powder pillows
Stored Programs
353 N, Nitrate MR PP
Start
required (see Instrument- NitraVer 5 Nitrate Reagent A one-minute reaction
specific information). Powder Pillow. Insert a period will begin.
stopper into the cell.
Refer to the user manual Shake the cell vigorously
for orientation. until the timer expires.
Note: Some solid material
will not dissolve.
Nitrate
Page 718
Nitrate
Cadmium reduction method for powder pillows (continued)
Zero
5. When the timer 6. Blank Preparation: 7. Wipe and insert the 8. ZERO the instrument.
expires, start the timer When the timer expires, fill blank into the cell holder. The display will show:
again. a second sample cell with
10 mL of sample. 0.0 mg/L NO3––N
A five-minute reaction
period will begin.
An amber color will
present.
Read
9. Within two minutes 10. READ the results in

after the timer expires, mg/L NO3––N.
wipe and insert the Refer to the user manual
prepared sample into the to display other chemical
cell holder. forms.
Nitrate
Page 719
Nitrate
Cadmium reduction method for AccuVac® Ampuls
Stored Programs
359 N, Nitrate MR AV
Start
1. Select the test. 2. Prepared Sample: 3. Fill a NitraVer 5 Nitrate 4. Start the instrument
Insert an adapter if Collect at least 40 mL of AccuVac® Ampul with timer.
required (see Instrument- sample in a 50-mL beaker. sample. Keep the tip A one-minute reaction
specific information). immersed while the Ampul period will begin.
fills completely. Place a
Refer to the user manual stopper over the Ampul tip.
for orientation.
5. Invert the Ampul 48– 6. When the timer 7. Blank Preparation: 8. Wipe the blank and
52 times as the timer expires, start the timer When the timer expires, fill insert it into the cell holder.
counts down. again. a round sample cell with
A five-minute reaction 10 mL of sample.
period will begin. An
amber color will develop if
nitrate is present.
Zero Read
9. ZERO the instrument. 10. Within two minutes 11. READ the results in
wipe the Ampul and insert
0.0 mg/L NO3––N it into the cell holder.
Nitrate
Page 720
Nitrate
Interferences
Chloride chloride concentrations (seawater) but a calibration must be done using standards spiked to
the same chloride concentration (see Seawater calibration).
Ferric iron Interferes at all levels
Compensate for nitrite interference as follows:
Nitrite 1. Add 30-g/L Bromine Water1 drop-wise to the sample until a yellow color remains.
2. Add one drop of 30-g/L Phenol Solution1 to destroy the color.
3. Proceed with Step 2 of the test. Report the results as total nitrate and nitrite.
pH
substances
high interference level, calibrate the water using standards spiked to the same chloride
concentrations as the required samples. To prepare calibration standards containing 1.0, 3.0, 5.0
and 10.0 mg/L nitrate as NO3–N:
c. Add necessary Chloride concentration (g/L) x (1.6485) = g of ACS grade NaCl to 1 L of

deionized water.
Note: 18.8 g/L is a typical seawater chloride concentration.
d. Mix this solution thoroughly to make sure that it is a homogeneous solution. Use this water
as the dilution water instead of the deionized water when preparing the nitrate standards.
2. Use Class A glassware or a Tensette Pipet to pipet 1.0, 3.0, 5.0, and 10.0 mL of the 100 mg/L
Nitrogen-Nitrate as NO3––N (NIST) Standard Solution (Catalog Number 194749) into four
4. Use the prepared chloride water for the 0-mg/L nitrate as NO3–N standard.

• Most reliable results are obtained when samples are analyzed as soon as possible after
up to 24 hours at 4 °C. To preserve samples for longer periods, add 2 mL of Concentrated
Sulfuric Acid (H2SO4)* per liter and store at 4 °C. The results are reported as total nitrate and
nitrite.
Nitrate
Page 721
Nitrate
Accuracy check
• Nitrate Nitrogen Standard,100-mg/L NO3––N
4. Open the standard solution bottle.
6. Follow the Cadmium reduction method for powder pillows test procedure for each of the
spiked samples, starting with the 0.1 mL sample spike. Measure each of the spiked samples in
the instrument.

• 500 mg/L Nitrate Nitrogen Ampule Standard Solution
• Ampule breaker
• Mixing cylinder, 50-mL (3)
1. Fill three mixing cylinders each with 50-mL of sample and spike with 0.1 mL, 0.2 mL and 0.3
mL of 500 mg/L Nitrate Nitrogen Ampule Standard Solution.
3. Analyze each standard addition sample as described in the Cadmium reduction method for
AccuVac® Ampuls.
recovery. Standard solution method

• 5.0-mg/L Nitrate Nitrogen Standard Solution (prepared)
• 100-mg/L Nitrate Nitrogen Standard
• Deionized water
Nitrate
Page 722
Nitrate
• 5-mL Volumetric pipet and pipet filler
OR

1. Prepare a 5.0-mg/L nitrate nitrogen standard solution as follows:
a. Pipet 5.0 mL of 100-mg/L Nitrate Nitrogen Standard, into a 100-mL volumetric flask.
2. Use this 5.0 mg/L nitrate nitrogen standard solution in place of the sample. Follow the
Cadmium reduction method for powder pillows test procedure.
Method performance
359 5.0 mg/L NO3––N 4.6–5.4 mg/L NO3 ––N 0.05 mg/L NO3––N
Summary of method
with sulfanilic acid to form an intermediate diazonium salt. The salt couples with gentisic acid to
form an amber colored solution. Test results are measured at 400 nm.

Required reagents
NitraVer® 5 Nitrate Reagent Powder Pillows (for 10 mL sample) 1 100/pkg 2106169

OR
NitraVer® 5 Nitrate Reagent AccuVac® Ampul 1 25/pkg 2511025

Stopper, Neoprene, solid, size #1 2 12/pkg 1480801
Nitrate
Page 723
Nitrate

Mixed Parameter Drinking Water Standard, for F, NO3–N, PO4, SO4 500 mL 2833049
Nitrate Nitrogen Standard Solution, 100-mg/L NO3––N 500 mL 194749
Nitrate Nitrogen Standard Solution, 500 mg/L NO3–N, 10-mL ampules 16/pkg 1426010

Ampule breaker for 10 mL ampules each 2196800
Bromine Water, 30-mg/L 29 mL 221120
Flask, volumetric, 100-mL each 1457442
5.0 N Sodium Hydroxide Standard Solution 1L 245053

Nitrate Nitrogen, UV, 10049
UV Screening Method1 Method 10049

0.1 to 10.0 mg/L NO3 ––N
Scope and Application: For the screening of uncontaminated natural and potable water supplies containing low
concentrations of organic matter
1 Adapted from Standard Methods for the Examination of Water and Wastewater, part 4500-NO3–B.
Test preparation

The instrument specific information table displays requirements that may vary between

Instrument Sample cell Adapter
DR 6000 4822800 A23618
DR 5000 4822800 A23618
Turbid samples must be filtered prior to analysis.
This method is very sensitive to small differences in the measurement wavelength. For best results, use the Standard Adjust
feature (as described in Accuracy check, Standard solution method) to optimize results on each instrument.
Hydrochloric Acid Standard Solution, 1.0 M 1 mL
Beaker, 100-mL 1
Sample Cells, 1-cm quartz 2
Nitrate
Page 725
Nitrate
UV screening method
Stored Programs
357 N Nitrate UV
Start
1. Select the test. 2. Prepared Sample: 3. Add 1 mL of 1.0 N 4. Rinse and fill a 1-cm
Insert an adapter if Collect 50 mL of clear Hydrochloric Acid quartz sample cell with
required. sample in a 100-mL Standard Solution to the sample. Discard the
beaker. beaker and swirl to mix. excess.
for orientation.
Zero
5. Blank Preparation: 6. Insert the blank into 7. ZERO the instrument. 8. Insert the prepared
Fill another 1-cm quartz the cell holder and close The display will show: sample into the cell holder.
sample cell with deionized the lid. For the DR 5000, The instrument will read
water. the transparent face of the 0.0 mg/L NO3––N
the sample at 220 and 275
cell should face the user. nm. When finished, the
For the DR 6000, the display will show the
transparent face of the cell sample nitrate nitrogen
should face right. concentration.
Interferences

Chlorate May interfere
Chromium All levels
Dissolved organic matter All levels
Nitrite All levels
Surfactants All levels
Suspended particulate matter Remove using filtration.
Nitrate
Page 726
Nitrate

collection.
• If prompt analysis is impossible, store samples in clean plastic or glass bottles for up to 24
hours at 4 °C.
• To preserve samples for longer periods, add 2 mL of Concentrated Sulfuric Acid (H2SO4)* per
liter and store at 4 °C.
Accuracy check

• Deionized water
• 100 mL volumetric flask
• 5-mL Volumetric pipet and pipet filler
• 100 mg/L Nitrate Nitrogen Standard
OR
• 5 mg/L Nitrate Nitrogen Voluette Ampul Standard
1. Prepare a 5.0 mg/L Nitrate Nitrogen standard solution as follows:

a. Pipet 5.0 mL of 100 mg/L Nitrate Nitrogen Standard, into a 100-mL volumetric flask.
b. Dilute to volume with deionized water. Insert a stopper and invert to mix. Alternatively,
open a 5 mg/L Nitrate Nitrogen Voluette Ampul Standard.
2. Use the 5.0 mg/L nitrate nitrogen standard solution in place of the sample. Follow the UV
screening method test procedure.
Method performance
Summary of method
The UV nitrate direct screening method offers rapid determination of nitrate. Because both nitrate
and organic constituents absorb at 220 nm and nitrate does not absorb at 275 nm, the second
reading at 275 nm is used to correct for the absorbance attributed to organic matter. Although this
Nitrate
Page 727
Nitrate
method is useful for monitoring nitrate, it is not recommended for samples containing high
concentrations of organics. Adding hydrochloric acid prevents interference from hydroxide or
carbonate concentrations up to 1000 mg/L CaCO3.

Required reagents
Hydrochloric Acid Standard Solution, 1.0 M 1 mL 1L 2321353
Required apparatus
Sample cell with cap, 1-cm quartz, matched pair 2 2/pkg 4822800
Nitrate Nitrogen Standard Solution, 100-mg/L NO3–N 500 mL 194749
Nitrate Nitrogen Voluette Standard, 5 mg/L NO3-N, 10 mL ampuls 16/pkg 2557810

Ampule Breaker - Voluette each 2196800

Aspirator, Nalgene vacuum pump each 213100
Dropper, 0.5 and 1.0 mL 20/pkg 2124720
FIlter holder, 47-mm each 1352900
Filter membrane, 47-mm, 0.45 micron 100/pkg 1353000
Flask, volumetric, 100-mL each 1457442
Stopper, No. 7, one hole 6/pkg 211907
Sulfuric Acid, concentrated, ACS 500 mL 97949
Tubing, rubber latex 12 ft 56019

Nitrite, HR, 8153
Nitrite DOC316.53.01075
Ferrous Sulfate Method1 Method 8153

HR (2 to 250 mg/L NO2 –) Powder Pillows
Scope and Application: For cooling systems
1 Adapted from McAlpine, R. and Soule, B., Qualitative Chemical Analysis, New York, 476, 575 (1933)
Test preparation


blank adjust.
After adding the reagent, a greenish-brown color will develop if nitrite is present.
NitriVer® 2 Nitrite Reagent Powder Pillows 1
Deionized water varies
Stopper, Neoprene, solid # 1 2
Nitrite
Page 729
Nitrite
Ferrous sulfate method for powder pillows
Stored Programs
373 N, Nitrite HR PP
Start
1. Select the test. 2. Fill a sample cell with 3. Prepared Sample: 4. Insert the stopper and
Insert an adapter if 10 mL of sample. Add the contents of one shake to dissolve.
required (see Instrument- NitriVer® 2 Nitrite Reagent
specific information). Powder Pillow.
for orientation.
Zero
5. Start the instrument 6. Blank Preparation: 7. Wipe the blank and 8. ZERO the instrument.
timer. Fill a second sample cell insert it into the cell holder. The display will show:
A ten-minute reaction with 10 mL of sample. 0 mg/L NO2–
period will begin. To
prevent low results, leave
the sample on a flat
surface and do not
disturb it during the
reaction period.
Read
9. After the timer expires, 10. Wipe the prepared 11. READ the results in
cap and gently invert the sample and insert it into mg/L NO2–.
prepared sample twice. the cell holder.
Avoid excessive mixing or
low results may occur.
Nitrite
Page 730
Nitrite
Interferences
This test does not measure nitrates nor is it applicable to glycol-based samples. Dilute glycol-
based samples and follow the Low Range Nitrite procedure, Method 8507.

The following storage instructions are necessary when prompt analysis is impossible. Store at 4 °C
(39 °F) or lower if the sample is to be analyzed within 24 to 48 hours. Warm to room temperature
before running the test. Do not use acid preservatives.
Accuracy check
1. Preparing nitrite standards is difficult. Use the standard preparation instructions in Standard
Methods for the Examination of Water and Wastewater. Prepare a 200-mg/L standard using
Sodium Nitrite, ACS*, reagent grade.
2. Use the 200-mg/L solution in place of the sample. Follow the Ferrous sulfate method for
3. To adjust the calibration curve using the reading obtained with the standard solution, navigate
to Standard Adjust in the software OPTIONS>MORE>STANDARD ADJUST.
Method performance
373 Standard: 200 mg/L NO2– 191–209 mg/L NO2– 1.4 mg/L NO2–
Summary of method
The method uses ferrous sulfate in an acidic medium to reduce nitrite to nitrous oxide. Ferrous
ions combine with the nitrous oxide to form a greenish-brown complex in direct proportion to the
nitrite present. Test results are measured at 585 nm.
Required reagents
NitriVer® 2 Nitrite Reagent Powder Pillows 1 100/ pkg 2107569
Nitrite
Page 731
Nitrite

Stopper, Neoprene, solid #1 2 12/pkg 1480801

Balance, Analytical, 80 g capacity each 2936701
Handbook, Standard Methods for the Examination of Water and Wastewater each 2270800
Sodium Nitrite, ACS 454 g 245201

Nitrite, LR, 10019
Diazotization Method Method 10019

LR (0.003 to 0.500 mg/L NO2 ––N) Test ‘N Tube™ Vials
Test preparation


DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
DR 3900, DR 3800, DR 2800 and DR 2700: Install the light shield in cell compartment #2 before performing this test.
blank adjust.
Test ‘N Tube™ NitriVer® 3 Nitrite Reagent Set 1
Pipet, TenSette®, 1.0 to 10.0 mL, plus tips 1
Nitrite
Page 733
Nitrite
Diazotization method, TNT
Stored Programs
345 N, Nitrite LR TNT
Start
1. Select the test. 2. Fill a Test ‘N Tube 3. Prepared Sample: 4. Start the instrument
Insert an adapter if NitriVer® 3 Nitrite vial with Cap and shake to dissolve timer.
required (see Instrument- 5 mL of sample. the powder. A 20-minute reaction
specific information). A pink color will develop if period will begin.
Refer to the user manual nitrite-nitrogen is present.
for orientation.
Zero
5. Blank Preparation: 6. Wipe the blank and 7. ZERO the instrument. 8. Wipe and insert the
When the timer expires, fill insert it into the 16-mm The display will show: prepared sample cell into
an empty Test ‘N Tube™ round cell holder. 0.000 mg/L NO2––N the 16-mm round cell
vial with 5 mL of sample. holder.
READ the results in
mg/L NO2––N.
Interferences

Antimonous ions Interfere by causing precipitation
Auric ions Interfere by causing precipitation
Bismuth ions Interfere by causing precipitation
Chloroplatinate ions Interfere by causing precipitation
Cupric ions Cause low results
Ferric ions Interfere by causing precipitation
Ferrous ions Cause low results
Lead ions Interfere by causing precipitation
Nitrite
Page 734
Nitrite

Mercurous ions Interfere by causing precipitation
Metavanadate ions Interfere by causing precipitation
Very high levels of nitrate (>100 mg/L nitrate as N) appear to undergo a slight amount of
Nitrate reduction to nitrite, either spontaneously or during the course of the test. A small amount of
nitrite will be found at these levels.
Silver ions Interfere by causing precipitation
substances

• Store the samples at 4 °C (39 °F) or lower if the sample is to be analyzed within 24 to 48
hours.
• Warm the sample to room temperature before running the test.
Accuracy check
Methods for the Examination of Water and Wastewater, Method 4500-NO2– B. Prepare a
0.300-mg/L standard. Use this solution in place of the sample. Follow the Diazotization
method, TNT test procedure.
Method performance
95% Confidence Limits of Sensitivity—ΔConcentration

Program Standard
Distribution per 0.010 ΔAbs
Summary of method
Nitrite in the sample reacts with sulfanilic acid to form an intermediate diazonium salt. This couples
with chromotropic acid to produce a pink colored complex directly proportional to the amount of
Nitrite
Page 735
Nitrite
Required reagents
Test ‘N Tube™ NitriVer® 3 Nitrite Reagent Set 1 50/pkg 2608345
Required apparatus

Recommended standards, reagents and apparatus


Nitrite, LR, 8507
USEPA1 Diazotization Method 8507

LR (0.002 to 0.300 mg/L NO2––N) Powder Pillows or AccuVac® Ampuls
1 USEPA approved for wastewater analysis, Federal Register, 44(85), 25505 (May 1, 1979)
Test preparation


Instrument
Powder Pillow Test:
NitriVer® 3 Nitrite Reagent Powder Pillows 1
AccuVac Test:
NitriVer® 3 Nitrite Reagent AccuVac® Ampul. 1
Beaker, 50-mL 1
Nitrite
Page 737
Nitrite
Diazotization method for powder pillows
Stored Programs
371 N, Nitrite LR PP
Start
1. Select the test. 2. Fill a sample cell with 3. Prepared Sample: 4. Swirl to dissolve.
Insert an adapter if 10 mL of sample. Add the contents of one A pink color will develop if
required (see Instrument- NitriVer 3 Nitrite Reagent nitrite is present.
specific information). Powder Pillow.
for orientation.
Zero
5. Start the instrument 6. Blank Preparation: 7. Wipe the blank and 8. ZERO the instrument.
timer. When the timer expires, fill insert it into the cell holder. The display will show:
A 20-minute reaction a second sample cell with
10 mL of sample. 0.000 mg/L NO2––N
period will begin.
Read
9. Wipe the prepared 10. READ the results in

sample and insert it into mg/L NO2––N.
the cell holder.
Nitrite
Page 738
Nitrite
Diazotization method for AccuVac® Ampuls
Stored Programs
375 N, Nitrite LR AV
Start
1. Select the test. 2. Prepared Sample: 3. Invert the Ampul 4. Start the instrument
Insert an adapter if Collect at least 40 mL of several times to mix. A timer.
required (see Instrument- sample into a 50-mL pink color will develop if A 20-minute reaction
specific information). beaker. nitrite is present. period will begin.
Refer to the user manual Fill a NitriVer 3 Nitrite
for orientation. AccuVac® Ampul with
sample. Keep the tip
fills.
5. Blank Preparation: 6. Wipe the blank and 7. Wipe the Ampul and
When the timer expires, fill insert it into the cell holder. insert it into the cell holder.
a sample cell with 10 mL ZERO the instrument. READ the results in
of sample. mg/L NO2––N.
0.000 mg/L NO2––N.
Nitrite
Page 739
Nitrite
Interferences

Antimonous ions Interfere by causing precipitation
Auric ions Interfere by causing precipitation
Bismuth ions Interfere by causing precipitation
Chloroplatinate ions Interfere by causing precipitation
Cupric ions Cause low results
Ferric ions Interfere by causing precipitation
Ferrous ions Cause low results
Lead ions Interfere by causing precipitation
Mercurous ions Interfere by causing precipitation
Metavanadate ions Interfere by causing precipitation
Very high levels of nitrate (>100 mg/L nitrate as N) appear to undergo a slight amount of
Nitrate reduction to nitrite, either spontaneously or during the course of the test. A small amount of
nitrite will be found at these levels.
Silver ions Interfere by causing precipitation
substances

• Store at 4 °C (39 °F) or lower if the sample is to be analyzed within 24 to 48 hours.
• Warm to room temperature before running the test.
• Do not use acid preservatives.
Accuracy check
Methods for the Examination of Water and Wastewater, Method 4500—NO2-B. Prepare a
0.150-mg/L standard.
2. Use the 0.150 mg/L solution in place of the sample. Follow the Diazotization method for
Nitrite
Page 740
Nitrite
Method performance
Summary of method
Nitrite in the sample reacts with sulfanilic acid to form an intermediate diazonium salt. This couples
with chromotropic acid to produce a pink colored complex directly proportional to the amount of
Required reagents
NitriVer® 3 Nitrite Reagent Powder Pillows 1 100/pkg 2107169
OR
NitriVer® 3 Nitrite Reagent AccuVac® Ampul 1 25/pkg 2512025
Required apparatus
Recommended standards, reagents and apparatus

AccuVac ampules, for blanks 25/pkg 2677925
AccuVac Drainer each 4103600
Nitrite
Page 741
Nitrite

Nitrite, DT, 8351
Ceric Acid Titration Method Method 8351

100 to 2500 mg/L as NaNO2 Digital Titrator
Scope and Application: For cooling tower waters.
Test preparation
A pipet is recommended for measuring the sample volume when the volume is less than 10 mL.
Ferroin Indicator Solution 1 bottle
Sulfuric Acid Standard Solution, 5.25 N 1 bottle
Ceric Standard Solution Titration Cartridge, 0.5 N 1 cartridge
Digital titrator 1
Nitrite
Page 743
Nitrite
Nitrite
See
Table 1
volume from the Range- tube into the titration Titrator with the cartridge cylinder or pipet to
specific information table. cartridge. Attach the tip pointing up. Turn the measure the sample
cartridge to the titrator. delivery knob to eject a volume from the Range-
5. Dilute to 6. Add five drops of 7. Add one drop of 8. Place the delivery tube
approximately 75 mL with 5.25 N Sulfuric Acid Ferroin Indicator Solution into the solution and swirl
deionized water. Standard Solution to the to the flask. Swirl to mix. the flask. Turn the knob on
flask. Swirl to mix. the titrator to add titrant to
the solution. Continue to
swirl the flask and add
titrant until the color
changes from orange to
pale blue.
counter.
Nitrite
Page 744
Nitrite
Nitrite (continued)
9. Use the multiplier in

the Range-specific
information table to
calculate the
concentration:
digits x multiplier =
mg/L sodium nitrite
(NaNO2)
was titrated and 250 digits
endpoint. The
= 215 mg/L as NaNO2
Nitrite
Page 745
Nitrite

Range (mg/L as NaNO2) Sample volume (mL) Multiplier
100–400 25 0.86
400–800 10 2.15
800–1500 5 4.31
1500–2500 2 10.78

Collect samples in clean plastic or glass bottles. Prompt analysis is recommended.
If prompt analysis is not possible, store samples for 24 to 48 hours at 4 °C (39 °F) or lower. Warm
to room temperature before analysis. Do not use acid preservatives.
Accuracy check
• Sodium Nitrite, ACS
1. Prepare a 1000-mg/L sodium nitrite standard solution as follows. Add 1.000 gram of sodium
nitrite to the volumetric flask. Add deionized water to the mark and mix fully.
2. Add 5.0 mL of the solution to an Erlenmeyer flask. Dilute to approximately 75 mL with

deionized water and mix fully.
3. Add the sulfuric acid and Ferroin indicator. Swirl to mix.
4. Titrate the standard to the end point with the titration cartridge and calculate the result. The
result should be close to 1000 mg/L as NaNO2.
Standardization of ceric standard solution

The normality of the ceric standard solution will sometimes decrease over time. Before use, verify
the normality with the following procedure. This standardization should be done monthly.
1. Use a graduated cylinder or pipet to measure 50 mL of deionized water into a 125-mL

Erlenmeyer flask.
2. Add 5 mL of 19.2 N Sulfuric Acid Standard Solution. Swirl to mix.
3. Insert a clean delivery tube into a Ceric Standard Titration Cartridge.
4. Hold the Digital Titrator with the cartridge tip pointing up. Turn the delivery knob until a few
drops of titrant are expelled. Reset the counter to zero and wipe the tip.
5. Place the delivery tube tip into the solution. While swirling the flask, add 200 digits of Ceric
Standard.
6. Insert a clean delivery tube into a 0.200 N Sodium Thiosulfate Titration Cartridge.
7. Hold the Digital Titrator with the cartridge tip pointing up. Turn the delivery knob until a few
drops of titrant are expelled. Reset the counter to zero and wipe the tip.
8. Place the delivery tube tip into the solution. While swirling the flask, titrate with the sodium
thiosulfate from an intense yellow color to a faint yellow color. Record the number of digits
required. This step should require about 400–450 digits of titrant.
Nitrite
Page 746
Nitrite
9. Add one drop of Ferroin Indicator Solution. Swirl to mix. The solution will turn a faint blue.
10. Continue titrating with the Sodium Thiosulfate Standard Solution from a faint blue to orange
color. Record the number of digits required.
11. Divide the number of digits by 500 to calculate the correction factor (number of digits ¸ 500 =
correction factor).
12. Multiply the mg/L sodium nitrite from the titration procedure by the correction factor to obtain
the correct sodium nitrite concentration.
Summary of method
Sodium nitrite is titrated with tetravalent cerium ion, a strong oxidant, in the presence of ferroin
indicator. After the cerium oxidizes the nitrite, the indicator is oxidized and causes a color change
from orange to pale blue. The concentration of sodium nitrite is proportional to the amount
of titrant.
Required reagents
Ceric Standard Solution Titration Cartridge, 0.5 N 1 pillow each 2270701
Ferroin Indicator Solution 1 pillow 29 mL DB 181233
Sulfuric Acid Standard Solution, 5.25 N 1 pillow 100 mL MDB 244932
Required apparatus
Sodium Thiosulfate Titration Cartridge, 0.200 N each 2267501
Nitrite
Page 747
Nitrite

Thermometer -10 to 225 °C 405 mm 2635700
Volumetric flask, 100 mL each 1457453
Volumetric pipet, 5 mL each 1451537
Sampling bottle, 250 mL each 2087076
Analytical balance each 2936701

Nitrogen, Ammonia, 8038
Nitrogen, Ammonia DOC316.53.01078
USEPA1 Nessler Method2 Method 8038

0.02 to 2.50 mg/L NH3–N
Scope and Application: For water, wastewater and seawater; distillation is required for wastewater and
seawater; USEPA accepted for wastewater analysis (distillation required); see Distillation in this procedure.
1 USEPA accepted for wastewater analysis (distillation required)
2 Adapted from Standard Methods for the Examination of Water and Wastewater 4500-NH3 B & C.
Test preparation


Nessler Reagent contains mercuric iodide. Both the sample and the blank will contain mercury (D009) at a concentration
regulated as a hazardous waste. Do not pour these solutions down the drain. Refer to a current MSDS for safe disposal and
handling instructions.
When dispensing reagent from a dropper bottle, hold the bottle vertically. Do not hold the bottle at an angle.
If the Flow Cell is used, periodically clean the cells by pouring a few sodium thiosulfate pentahydrate crystals in to the cell
funnel. Flush with enough deionized water to dissolve. Rinse the cell thoroughly.
Nitrogen, Ammonia
Page 749
Nitrogen, Ammonia
Ammonia Nitrogen Reagent set 1
Graduated Mixing Cylinders 2
Sample Cells, 1-inch, 10-mL 2
Serological Pipet, 1-mL 1
Nessler method
Stored Programs
380 N, Ammonia, Ness
Start
1. Select the test. 2. Prepared Sample: 3. Blank Preparation: 4. Add three drops
Insert an adapter if Fill a 25-mL mixing Fill a 25-mL mixing of Mineral Stabilizer to
required (see Instrument- graduated cylinder to the graduated cylinder to the each cylinder. Stopper and
specific information). 25-mL mark with sample. 25-mL mark with invert several times to mix.
deionized water.
for orientation.
5. Add three drops of 6. Pipet 1.0 mL of 7. Start the instrument 8. Pour 10 mL of each
Polyvinyl Alcohol Nessler Reagent into each timer. solution into a sample cell.
Dispersing Agent to each cylinder. Stopper and A one-minute reaction
cylinder. Stopper and invert several times to mix. period will begin.
invert several times to mix.
Nitrogen, Ammonia
Page 750
Nitrogen, Ammonia
Nessler method (continued)
Zero Read
9. When the timer 10. ZERO the instrument. 11. Wipe the prepared 12. READ the results in
expires, insert the blank The display will show: sample and insert it into mg/L NH3 –N.
into the cell holder. the cell holder.
0.00 mg/L NH3 –N
Interferences

Remove residual chlorine from a 250 mL sample by adding 1 drop of sodium thiosulfate for
Chlorine each mg/L chlorine (Cl2). Sodium arsenite can be used instead of sodium thiosulfate. See
Sample collection, preservation and storage.
A solution containing a mixture of 500 mg/L CaCO3 and 500 mg/L Mg as CaCO3 does not
Hardness interfere. If the hardness concentration exceeds these concentrations, add extra
Mineral Stabilizer.
Iron Interferes at all levels by causing turbidity with Nessler Reagent.
May be analyzed by adding of 1.0 mL (27 drops) of Mineral Stabilizer to the sample before
analysis. This complexes the high magnesium concentrations found in sea water, but the
Seawater sensitivity of the test is reduced by 30 percent due to the high chloride concentration. For best
results, perform a calibration, using standards spiked to the equivalent chloride concentration
or distill the sample as described below.
Sulfide Interferes at all levels by causing turbidity with Nessler Reagent.
Glycine, various aliphatic and
aromatic amines, organic May cause greenish or other off colors or turbidity. Distill the sample if these compounds
chloramines, acetone, are present.
aldehydes and alcohols

• Collect samples in clean glass or plastic bottles.
• If chlorine is present, add one drop of 0.1 N Sodium Thiosulfate* for each 0.3 mg/L Cl2 in a
1-liter sample.
• Preserve the sample by reducing the pH to 2 or less with sulfuric acid (at least 2 mL/L). Store
at 4 °C (39 °F) or less.
• Preserved samples may be stored up to 28 days.
• Warm samples to room temperature and neutralize with 5 N Sodium Hydroxide* before
analysis.
Nitrogen, Ammonia
Page 751
Nitrogen, Ammonia
Accuracy check
• Nitrogen Ammonia Voluette® Ampule Standard, 50-mg/L NH3–N
• Ampule breaker
• 25-mL Mixing cylinders (3)
2. Select standard additions from the instrument menu: OPTIONS>MORE>STANDARD

ADDITIONS.
standard to three 25-mL portions of fresh sample in three mixing cylinders.
6. Follow the Nessler method test procedure for each of the spiked samples, starting with the 0.1
mL sample spike. Measure each of the spiked samples in the instrument.


• Nitrogen Ammonia Standard solution 1-mg/L NH3–N
1. Use a 1-mg/L Nitrogen Ammonia Standard solution in place of the sample. Follow the Nessler
method test procedure.
to Standard Adjust in the software: OPTIONS>MORE>STANDARD ADJUST.
Distillation
1. Measure 250 mL of sample into a 250-mL graduated cylinder and pour into a 400-mL beaker.
Destroy chlorine, if necessary, by adding 1 drop of Sodium thiosulfate Solution 0.1 N per mg/L
Cl2.
2. Add 25 mL of Borate Buffer Solution and mix. Adjust the pH to about 9.5 with 1 N sodium
hydroxide solution. Use a pH meter.
3. Set up the General Purpose Distillation Apparatus as shown in the Distillation Apparatus
Manual. Pour the solution into the distillation flask. Add a stir bar.
Nitrogen, Ammonia
Page 752
Nitrogen, Ammonia
4. Use a graduated cylinder to measure 25 mL of deionized water into a 250-mL Erlenmeyer

flask. Add the contents of one Boric Acid Powder Pillow. Mix thoroughly. Set the flask under
the distillation apparatus drip tube. Elevate the flask so the end of the tube is immersed in the
solution.
5. Turn on the heater power switch. Set the stir control to 5 and the heat control to 10. Turn on
the water and adjust to maintain a constant flow through the condenser.
6. Turn off the heater after collecting 150 mL of distillate. Immediately remove the collection flask
to avoid sucking solution into the still. Measure the distillate to ensure 150 mL was collected
(total volume = 175 mL).
7. Adjust the pH of the distillate to about 7 with 1 N sodium hydroxide. Use a pH meter.
8. Pour the distillate into a 250-mL volumetric flask; rinse the Erlenmeyer with deionized water.
Add the rinsings to the volumetric flask. Dilute to the mark. Stopper. Mix thoroughly. Analyze
as described above.
Method performance
380 DR 5000 1.00 mg/L NH3–N 0.99–1.01 mg/L NH3 –N 0.02 mg/L NH3 –N2
Summary of method
The Mineral Stabilizer complexes hardness in the sample. The Polyvinyl Alcohol Dispersing Agent
aids the color formation in the reaction of Nessler Reagent with ammonia and certain other
amines. A yellow color is formed proportional to the ammonia concentration. Test results are
measured at 425 nm.
Required reagents
Ammonia Nitrogen Reagent Set, includes: — — 2458200
Nessler Reagent 2 mL 500 mL 2119449
Mineral Stabilizer 6 drops 50 mL SCDB 2376626
Polyvinyl Alcohol Dispersing Agent 6 drops 50 mL SCDB 2376526
Required apparatus
Nitrogen, Ammonia
Page 753
Nitrogen, Ammonia

Nitrogen, Ammonia Standard Solution, 1-mg/L NH3–N 500 mL 189149
Nitrogen, Ammonia Standard Solution, 10-mL Voluette® Ampule, 50-mg/L NH3–N 16/pkg 1479110
Pipet, TenSette® 0.1 - 1.0 mL each 1970001

Wastewater, Effluent Inorganics, for NH3–N, NO3–N, PO4, COD, SO4, TOC 500 mL 2833249

Distillation Apparatus, General each 2265300
Heater and Support Apparatus, 115 VAC, 60 Hz each 2274400
Pour-Thru Cell Kit for DR 2800 each 5940400
Pour-Thru Cell Module for DR 5000 each LZV479
Ampule breaker, Voluette each 2196800
Sodium Thiosulfate, 0.1 N 100 mL 32332

Nitrogen, Ammonia, 8155
Salicylate Method1 Method 8155

0.01 to 0.50 mg/L NH3–N Powder Pillows
1 Adapted from Clin. Chim. Acta., 14, 403 (1966)
Test preparation


A green color will develop if ammonia nitrogen is present.
Ammonia Cyanurate Reagent pillows 2
Ammonia Salicylate Reagent pillows 2
Stopper for sample cells 2
Nitrogen, Ammonia
Page 755
Nitrogen, Ammonia
Salicylate method for powder pillows
Stored Programs
385 N, Ammonia, Salic
Start
Insert an adapter if Fill a sample cell to the 10- Fill a second sample cell one Ammonia Salicylate
required (see Instrument- mL mark with sample. to the 10-mL mark with Powder Pillow to each cell.
specific information). deionized water.
for orientation.
5. Insert the stopper or 6. Start the instrument 7. When the timer 8. Stopper or cap and
cap the cell and shake to timer. expires, add the contents shake to dissolve.
dissolve. A three-minute reaction of one Ammonia
period will begin. Cyanurate Reagent
Powder Pillow to each cell.
Zero
9. Start the instrument 10. When the timer 11. ZERO the instrument. 12. Wipe the sample and
timer. expires, wipe the blank The display will show: insert it into the cell holder.
A 15-minute reaction and insert it into the cell 0.00 mg/L NH3–N. READ the results in mg/L
period will begin. holder NH3–N.
A green color will develop
if ammonia-nitrogen
is present.
Nitrogen, Ammonia
Page 756
Nitrogen, Ammonia
Interferences

Calcium Greater than 1000 mg/L as CaCO3
All levels. Correct for iron interference as follows:
1. Determine the amount of iron present in the sample by following one of the Iron, Total,
Iron procedures.
2. Add the same iron concentration to the ammonia-free water in step 2. The interference
will be successfully blanked out.
Magnesium Greater than 6000 mg/L as CaCO3
Monochloramine present in chloraminated drinking water interferes directly at all levels,
Monochloramine giving high results. Use Method 10200, Free Ammonia and Monochloramine, to determine
free ammonia in these sample matrices.
Nitrate Greater than 100 mg/L as NO3––N
Nitrite Greater than 12 mg/L as NO2––N
Phosphate Greater than 100 mg/L as PO43––P
Sulfate Greater than 300 mg/L as SO42–
Sulfide will intensify the color. Eliminate sulfide interference as follows:
1. Measure about 350 mL of sample in a 500-mL Erlenmeyer flask1.
Sulfide 2. Add the contents of one Sulfide Inhibitor Reagent1 Powder Pillow. Swirl to mix.
3. Filter the sample through a Folded Filter Paper1 and Filter Funnel1.
4. Use the filtered solution in step 3.
Less common interferences such as hydrazine and glycine will cause intensified colors in
the prepared sample. Turbidity and color will give erroneous high values. Samples with
Other Substances
severe interferences require distillation. Use the distillation procedure with the General
Purpose Distillation Set.

samples are analyzed as soon as possible after collection.
• If chlorine is known to be present, add one drop of 0.1 N Sodium Thiosulfate* for each 0.3
mg/L Cl2 in a one-liter sample.
• Adjust the pH to 2 or less with concentrated (about 2 mL per liter) Sulfuric Acid.
• Store samples at 4 °C or less. Samples preserved in this manner can be stored up to 28 days.
• Just before testing the stored sample, warm to room temperature and neutralize with 5.0 N
Sodium Hydroxide Standard Solution.
Nitrogen, Ammonia
Page 757
Nitrogen, Ammonia
Accuracy check
• Ammonia Nitrogen Standard Solution, 10-mg/L as NH3–N
• 25-mL Mixing cylinders (3)
2. Select standard additions from the instrument menu OPTIONS>MORE>STANDARD

ADDITIONS.
6. Follow the Salicylate method for powder pillows test procedure for each of the spiked samples,


OR
• Ammonia Nitrogen Voluette® Standard Solution, 50-mg/L as NH3–N

• Deionized water
• 4-mL Class A volumetric pipet
1. Prepare a 0.40 mg/L ammonia nitrogen standard solution as follows:

• Dilute 4.00 mL of Ammonia Nitrogen Standard Solution, 10-mg/L to 100 mL with
deionized water.
OR
• Use the TenSette® Pipet to dilute 0.8 mL of an Ammonia Nitrogen Voluette Standard
Solution, 50-mg/L as NH3–N, to 100 mL with deionized water
2. Use the 0.40-mg/L solution in place of the sample. Follow the Salicylate method for powder
Nitrogen, Ammonia
Page 758
Nitrogen, Ammonia
Method performance
Precision—95% Confidence Sensitivity—ΔConcentration

Program Standard
Limits of Distribution per 0.010 ΔAbs
Summary of method
Ammonia compounds combine with chlorine to form monochloramine. Monochloramine reacts
with salicylate to form 5-aminosalicylate. The 5-aminosalicylate is oxidized in the presence of a
sodium nitroprusside catalyst to form a blue-colored compound. The blue color is masked by the
yellow color from the excess reagent present to give a final green-colored solution. Test results are
measured at 655 nm.

Required reagents
Ammonia Nitrogen Reagent Set for 10-mL samples (100 tests), — — 2668000
includes:
Includes:
(2) Ammonia Cyanurate Reagent Powder Pillows 2 100/pkg 2653199
(2) Ammonia Salicylate Reagent Powder Pillows 2 100/pkg 2653299

Nitrogen, Ammonia Standard Solution, 10-mg/L NH3–N 500 mL 15349
Nitrogen, Ammonia Standard Solution, 10-mL Voluette® Ampule, 50-mg/L NH3–N 16/pkg 147910
Wastewater, Effluent Inorganics, for NH3–N, NO3–N, PO4, COD, SO4, TOC 500 mL 2833249
Pipet, TenSette® 0.1 - 1.0 mL each 1970001
Stopper, 18 mm each 6/pkg
Sample cell, 10 mL square, matched pair 2/pkg 2495402

Nitrogen, Ammonia
Page 759
Nitrogen, Ammonia

Distillation Set, general purpose each 2265300
Erlenmeyer Flask, 500 mL each 50549
Filter Funnel, Analytical PP, 65 mm each 108367
Heater and support apparatus; 115 Vac, 60 Hz each 2274400
Heater and support apparatus, 230 Vac, 50 Hz each 2274402
Pipet, 2 mL Serological each 53236
Ampule breaker, Voluette each 2196800
Sulfuric Acid, Conc 500 mL 97949

Nitrogen, Ammonia, HR, 10031
Salicylate Method Method 10031

HR (0.4 to 50.0 mg/L NH3–N) Test ‘N Tube™ Vials
Test preparation

DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
Small sample sizes (such as 0.1 mL) may not be representative of the entire sample. Mix the sample well before testing or
repeat the test, sampling from different portions of the sample.
Good safety habits and laboratory techniques should be used throughout the procedure. Consult the Material Safety Data
Sheet (MSDS) for information specific to the reagent used.
In some lab environments, airborne cross-contamination of the blank is possible. To avoid the transfer of ammonia, complete
the preparation of the blank before opening or handling samples and standards. If the sample or standard containers have
already been opened, move to a separate area of the lab to prepare the blank.
High Range Test ‘N Tube AmVer™ Nitrogen Ammonia Reagent 2
Funnel, micro (for adding reagent) 1
Pipet Tips, for TenSette Pipet varies
Nitrogen, Ammonia
Page 761
Nitrogen, Ammonia
Salicylate method, TNT
Stored Programs
343 N, Ammonia HR TNT
Start
Insert an adapter or light Add 0.1 mL of sample to Add 0.1 mL of ammonia- one Ammonia Salicylate
shield if required (see one AmVer™ Diluent free water to one AmVer™ Reagent Powder Pillow for
Instrument-specific Reagent Test ‘N Tube for Diluent Reagent Test ‘N 5 mL sample to each vial.
information). High Range Ammonia Tube for High Range
Nitrogen. Ammonia Nitrogen.
for orientation.
5. Add the contents of 6. Cap the vials tightly 7. Start the instrument 8.
one Ammonia Cyanurate and shake thoroughly to timer.
Reagent Powder Pillow to dissolve the powder. A 20-minute reaction
each vial. period will begin.
Zero Read
9. ZERO the instrument. 10. 11. READ the results in

The display will show: mg/L NH3–N.
0.0 mg/L NH3–N
Nitrogen, Ammonia
Page 762
Nitrogen, Ammonia
Interferences

Calcium 50,000 mg/L as CaCO3
Glycine, hydrazine Will cause intensified colors in the prepared sample.
Monochloramine present in chloraminated drinking water interferes directly at all levels
Eliminate iron interference as follows:
1. Determine the amount of iron present in the sample using one of the total iron
Iron procedures.
2. Add the same iron concentration to the deionized water in step 3.
The interference will then be successfully blanked out.
Nitrite 600 mg/L as NO2––N
Nitrate 5000 mg/L as NO3––N
Orthophosphate 5000 mg/L as PO43––P

Acidic or basic samples should be adjusted to approximately pH 7. Use 1 N Sodium
pH Hydroxide Standard Solution1 for acidic samples and 1 N Hydrochloric Acid Standard
Solution1 for basic samples.
Sulfate 6000 mg/L as SO42–
Sulfide will intensify the color. Eliminate sulfide interference as follows:
1. Measure about 350 mL of sample in a 500-mL Erlenmeyer flask.
Sulfide
2. Add the contents of one Sulfide Inhibitor Reagent Powder Pillow1. Swirl to mix.
3. Filter the sample through folded filter paper1. Use the solution in step 2.
Other

• Collect samples in clean plastic or glass bottles. Best results are obtained with immediate
analysis.
• Preserve samples by reducing the pH to 2 or less with at least 2 mL of Hydrochloric Acid.
• Store at 4 °C (39 °F) or less.
• Neutralize preserved samples to a pH of 7.0 with 5.0 N Sodium Hydroxide before analysis.
Nitrogen, Ammonia
Page 763
Nitrogen, Ammonia
Accuracy check
• Nitrogen, Ammonia Ampule Standard, 150-mg/L NH3–N
• Ampule breaker
• Mixing cylinders, 25-mL, (3)

ADDITIONS.
5. Use the TenSette Pipet to prepare spiked samples: add 0.2 mL, 0.4 mL, and 0.6 mL of
6. Follow the Salicylate method, TNT test procedure for each of the spiked samples, starting with
7. Select GRAPH to view the results. Select IDEAL LINE (or best-fit) to compare the standard.


• 100-mg/L Ammonia Nitrogen standard
• Deionized water
• 20-mL Class A volumetric pipet and pipet filler
a. Pipet 20.00 mL of 100-mg/L Ammonia Nitrogen standard, into a 50-mL volumetric flask.
2. Use the 40.0 mg/L solution in place of the sample. Follow the Salicylate method, TNT test
procedure.
Nitrogen, Ammonia
Page 764
Nitrogen, Ammonia
Method performance
Precision—95% Sensitivity—
Program Standard Confidence Limits of ΔConcentration
Summary of method
sodium nitroprusside catalyst to form a blue colored compound. The blue color is masked by the
yellow color from the excess reagent present to give a green-colored solution. Test results are
measured at 655 nm.
Required reagents
Reagent Set, High Range Test ‘N Tube™ AmVer™ Nitrogen Ammonia 2 25 tests 2606945
Required apparatus
Funnel, micro (for adding reagent) 1 each 2584335
Pipet Tips, for TenSette® Pipet 1970001 varies 50/pkg 2185696
Tube Rack, 16 mm 1 each 1864100
Nitrogen Ammonia Standard Solution, 10-mg/L NH3–N 500 mL 15349
Nitrogen Ammonia Standard Solution, 100-mg/L NH3–N 500 mL 2406549
Nitrogen Ammonia Standard Solution, 150-mg/L NH3–N, 10-mL Voulette® Ampules 16/pkg 2128410
Nitrogen Ammonia Standard Solution, 50-mg/L NH3–N, 10-mL Voluette Ampules 16/pkg 1479110
Wastewater, Effluent Inorganics Standard, for NH3–N, NO3–N, PO4, COD, SO4, TOC 500 mL 2833249

Cylinders, mixing, 25 mL tall form each 2088640
Distillation Set, general purpose – 2265300
Nitrogen, Ammonia
Page 765
Nitrogen, Ammonia

Ampule Breaker, Voluette each 2196800
Heater and Support Apparatus, 230 VAC, 50 Hz; each 2274402
Filter Funnel, poly, 65 mm each 108367
Pipet, 2 mL serological each 50549
Hydrochloric Acid Standard Solution, 1 N 500 mL 13449
Sulfide Inhibitor Reagent Powder Pillows 100/pkg 241899
Hydrochloric Acid, conc 500 mL 13449

Nitrogen, Ammonia, LR, 10023
Salicylate Method1 Method 10023

LR (0.02 to 2.50 mg/L NH3–N) Test ‘N Tube™ Vials
1 Adapted from Clin. Chim. Acta, 14, 403 (1966)
Test preparation


DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
Good safety habits and laboratory techniques should be used throughout the procedure. Consult the Material Safety Data
Sheet (MSDS) for information specific to the reagent used.
Low Range Test ‘N Tube AmVer™ Nitrogen Ammonia Reagent 2
Funnel, micro (for adding reagent) 1
Pipet Tips, for TenSette Pipet varies
Nitrogen, Ammonia
Page 767
Nitrogen, Ammonia
Salicylate method, LR, TNT
Stored Programs
342, Ammonia LR TNT
Start
Insert an adapter or light Add 2.0 mL of sample to Add 2.0 mL of ammonia- one Ammonia Salicylate
shield if required (see one AmVer™ Diluent free water to one AmVer™ Reagent Powder Pillow to
Instrument-specific Reagent Test ‘N Tube for Diluent Reagent Test ‘N each vial.
information). Low Range Ammonia Tube for Low Range
Nitrogen. Ammonia Nitrogen.
for orientation.
5. Add the contents of 6. Cap the vials tightly 7. Start the instrument 8.
one Ammonia Cyanurate and shake thoroughly to timer.
Reagent Powder Pillow to dissolve the powder. A 20-minute reaction
each vial. period will begin.
Zero Read
9. ZERO the instrument. 10. 11. READ the results in

The display will show: mg/L NH3–N.
0.00 mg/L NH3–N
Nitrogen, Ammonia
Page 768
Nitrogen, Ammonia
Interferences

Determine the amount of iron present in the sample by using one of the Iron, Total,
procedures.
Iron
Add the same iron concentration to the ammonia-free water in step 3. The interference will
then be successfully blanked out.
Monochloramine present in chloraminated drinking water Interferes directly at all levels
Nitrate 250 mg/L as NO3––N
Nitrite 30 mg/L as NO2––N
Orthophosphate 250 mg/L as PO43––P
Acidic or basic samples should be adjusted to approximately pH 7. Use 1 N Sodium
pH Hydroxide Standard Solution1 for acidic samples and 1 N Hydrochloric Acid Standard
Solution1 for basic samples.
Sulfate 300 mg/L as SO42–
1. Measure about 350 mL of sample in a 500-mL Erlenmeyer flask.
Sulfide
3. Filter the sample through a folded filter paper1.
4. Use the filtered solution in step 3.
Other

analysis.
• Preserve samples by reducing the pH to 2 or less with at least 2 mL of Hydrochloric Acid.
• Store at 4 °C (39 °F) or less.
• Neutralize preserved samples to a pH of 7.0 with 5.0 N Sodium Hydroxide before analysis.
Nitrogen, Ammonia
Page 769
Nitrogen, Ammonia
Accuracy check
• Nitrogen, Ammonia Ampule Standard, 50-mg/L NH3–N
• Ampule breaker
• Mixing cylinders, 25-mL, (3)

ADDITIONS.
6. Follow the Salicylate method, LR, TNT test procedure for each of the spiked samples, starting
with the 0.2 mL sample spike. Measure each of the spiked samples in the instrument.
7. Select GRAPH to view the results. Select IDEAL LINE (or best-fit) to compare the standard.


• 1.0-mg/L Ammonia Nitrogen standard
1. Use a 1.0-mg/L Ammonia Nitrogen standard in place of the sample. Follow the Salicylate
method, LR, TNT test procedure.
to Standard Adjust in the software: OPTIONS>MORE>STANDARD ADJUST. .
Method performance
Program Standard Confidence Limits of DConcentration
Distribution per 0.010 DAbs
Nitrogen, Ammonia
Page 770
Nitrogen, Ammonia
Summary of method
sodium nitroprusside catalyst to form a blue colored compound. The blue color is masked by the
yellow color from the excess reagent present to give a green-colored solution. Test results are
measured at 655 nm.

Required reagents
Reagent Set, Low Range Test ‘N Tube™ AmVer™ Nitrogen Ammonia 2 25 tests 2604545
Required apparatus
Funnel, micro (for adding reagent) 1 each 2584335
Tube Rack, 16 mm 1–3 each 1864100
Nitrogen Ammonia Standard Solution, 1.0-mg/L NH3–N 500 mL 189149
Nitrogen Ammonia Standard Solution, 50-mg/L NH3–N, 10-mL Voluette® Ampules 16/pkg 1479110
Wastewater, Effluent Inorganics Standard, for NH3–N, NO3–N, PO4, COD, SO4, TOC 500 mL 2833249

Ampule Breaker, Voluette each 2196800
Cylinders, mixing, 25 mL each 2088640
Distillation Set, general purpose each 2265300
Filter Funnel, poly, 65 mm each 108367
Hydrochloric Acid Standard Solution, 1 N 1000 mL 2321353
Hydrochloric Acid, concentrated ACS 500 mL 13449
Pipet, 2 mL serological each 50549
Nitrogen, Ammonia
Page 771
Nitrogen, Ammonia


Nitrogen, Free Ammonia, 10201
Nitrogen, Free Ammonia DOC316.53.01084

(0.01 to 0.50 mg/L NH3–N) Powder Pillows
Scope and Application: For controlling free ammonia levels during the production of chloramines, at booster
stations and for monitoring free ammonia levels in potable distribution system waters.
1 U.S. Patent 6,315,950
Test preparation


Instrument Sample Cell Cell Orientation Adapter
DR 6000 4864302 Orientation key faces right —
DR 3800, DR 2800, DR 2700 5940506 1-cm (flat) path aligned with the arrow LZV585 (B)
on the adapter
Use Method 10200, Nitrogen, Free Ammonia and Chloramine (Mono) to determine free ammonia and monochloramine
simultaneously on the same sample.
Free Ammonia Reagent Set —
Free Ammonia Reagent Solution 1 drop
Nitrogen, Free Ammonia

Page 773
Indophenol method for powder pillows
Stored Programs
388 N, Ammonia Free
Start
1. Select the test. 2. Fill two cells to the 3. Prepared Sample: 4. Cap the reagent bottle
Insert an adapter if 10-mL line with sample. Add one drop of Free to maintain reagent
required (see Instrument- Label one cell ‘sample’ Ammonia Reagent performance and stability.
specific information). and one cell ‘blank’. Solution to the sample.
for orientation.
5. Cap and invert the 6. Start the instrument 7. When the timer 8. Cap and shake both
sample to mix. timer. expires, add the contents cells about 20 seconds to
If the sample becomes A 5-minute reaction period of one MonoChlor F dissolve the reagent.
cloudy by the end of the will begin. powder pillow to each cell. A green color will develop
reaction period, pretreat Color development time if monochloramine is
the sample and retest. See depends on sample present.
Interferences. temperature. For accurate
See Color development
based on sample
temperature.

Page 774
Indophenol method for powder pillows (continued)
Zero
9. Start the instrument 10. When the timer 11. ZERO the instrument. 12. Insert the sample into
timer. expires, insert the blank The display will show: 0.00 the cell
A 5-minute reaction period vial into the cell mg/L NH3–N f
will begin. Remove the blank.
depends on sample
temperature. For accurate
See Color development
based on sample
temperature.
Read
13. READ the results in

mg/L NH3–N f.
Interferences
This method is intended for finished, chloraminated drinking water samples that have a
measurable combined (total) chlorine disinfectant residual. Samples where the disinfectant
residual has disappeared and samples which exhibit a chlorine demand may produce low
ammonia test results. Blanks and ammonia standards analyzed without a disinfectant residual
must be prepared using high quality, reagent grade water.
The following do not interfere in the free ammonia determination at or below the stated
concentration.

Page 775

Aluminum 0.2 mg/L
Chloride 1200 mg/L Cl-
Copper 1 mg/L Cu
Iron 0.3 mg/L Fe
Manganese 0.05 mg/L Mn
Nitrate 10 mg/L NO3–N
Nitrite 1 mg/L NO2–N
Phosphate 2 mg/L o–PO4
Sulfate 1600 ppm as CaCO3
Zinc 5 ppm Zn
Samples containing high levels of both Total Hardness and Alkalinity may become cloudy after the
addition of the Free Ammonia Reagent Solution. If this occurs by the end of the first reaction
period, the sample for Free Ammonia measurement must be pretreated as follows:
Note: The blank does not need pretreatment.
1. Measure 10 mL of sample into the cell labeled for the sample.

2. Add the contents of one Hardness Treatment Reagent Powder Pillow to the sample.
3. Cap the cell and invert until the reagent is dissolved.
4. Remove the cap.

5. Continue with the analysis at step 2 using the pretreated sample as the sample.

Test results are strongly influenced by sample temperature. Both reaction periods in the
procedure are the same and depend on the temperature of the sample. The reaction periods
indicated in the procedure are for a sample temperature of 18–20 °C (64–68 °F). Adjust both
reaction periods (see Color development based on sample temperature). The samples can be
read up to 15 minutes after the development times listed in Table 244 as the developed color is
stable for an extended period of time.
Sample Temperature
Development Time (minutes)
°C °F
5 41 10
7 45 9
9 47 8
10 50 8
12 54 7
14 57 7
16 61 6
18 64 5

Page 776

Sample Temperature
Development Time (minutes)
°C °F
20 68 5
23 73 2.5
25 77 2

Collect samples in clean glass bottles. Most reliable results are obtained when samples are
analyzed as soon as possible after collection.
Accuracy check
Dilution water is required when testing a diluted sample and preparing standard solutions. Dilution
water must be free of ammonia, chlorine and chlorine demand. A convenient source is a
recirculating, deionizer system with carbon filtration which produces 18 megohm-cm water.
• Ammonia Nitrogen Standard, 10 mg/L as NH3-N
• Ampule breaker
• Mixing cylinders, 50 mL (3)

ADDITIONS.
6. Follow the Indophenol method for powder pillows test procedure for each of the spiked
instrument.

Page 777


• Ammonia Nitrogen Standard Solution, 10 mg/L NH3–N
• Deionized water
• Volumetric Pipet, 2 mL or Tensette Pipet and Pipet tips
• Volumetric flask, 100 mL
• Dilute 2.00 mL of Ammonia Nitrogen Standard Solution, 10 mg/L to 100 mL with deionized
water.
• Or, use the TenSette Pipet to dilute 0.4 mL of a Ammonia Nitrogen Voluette Standard
Solution, 50 mg/L as NH3–N, to 100 mL with dilution water.
2. Use this solution in place of the sample. Follow the Indophenol method for powder pillows test
procedure.
Method performance
388 0.20mg/L NH3–N 0.19–0.21 mg/L NH3–N 0.01 NH3–N
Summary of method
Monochloramine (NH2Cl) and free ammonia (NH3 and NH4+) can exist in the same water sample.
Added hypochlorite combines with free ammonia to form more monochloramine. In the presence
of a cyanoferrate catalyst, monochloramine in the sample reacts with a substituted phenol to form
an intermediate monoimine compound. The intermediate couples with excess substituted phenol
to form a green-colored indophenol, which is proportional to the amount of monochloramine
present in the sample. Free ammonia is determined by comparing the color intensities, with and
without added hypochlorite. Test results are measured at 655 nm.

Page 778
Required reagents
Free Ammonia Reagent Set (50 tests), includes: — — 2879700
(1) 2802299, (1) 2877336
Free Ammonia Reagent Solution 1 drop 4 mL SCDB 2877336
Hardness Treatment Reagent Pillows (1 per test) 50/pkg 2882346
Nitrogen Ammonia Standard Solution, 10 mg/L as NH3–N 500 mL 15349
Nitrogen Ammonia Standard Ampule, 50 mg/L as NH3–N, 10 mL 16/pkg 1479110
Water, organic-free water 500-mL 2641549

Thermometer, –10 to 110 °C each 187701
Wipers, Disposable, 30 x 30 cm, 280/box box 2097000
Cylinder, graduated mixing, 50 mL each 189641

Page 779
Nitrogen, Total, 10072
Nitrogen, Total DOC316.53.01085
Persulfate Digestion Method Method 10072

HR (2 to 150 mg/L N) Test ‘N Tube Vials
Test preparation


DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
DR 3900, DR 3800, DR 2800, DR 2700: Install the light shield in Cell Compartment #2 before performing this test.
Digestion is required for determining total nitrogen.
If the test overranges, repeat the digestion and measurement with diluted sample. The digestion must be repeated for
accurate results.
Use the deionized water provided in the reagent set or Organic-free Water to prepare the standards and perform
the procedure.
Nitrogen, Total
Page 781
Nitrogen, Total
Test ‘N Tube™ HR Total Nitrogen Reagent Set 1
DRB200 Reactor 1
Funnel, micro 1
Light Shield or Adapter (see Instrument-specific information) 1
Pipet, TenSette®, 0.1 to 1.0 mL plus tips 1
Test Tube Cooling Rack 1
Persulfate digestion method
1. Turn on the DRB200 2. Using a funnel, add 3. Prepared Sample: 4. Cap both vials. Shake
Reactor and heat to the contents of one Total Add 0.5 mL of sample to a vigorously for at least
105 °C. Nitrogen Persulfate vial. 30 seconds to mix. The
Reagent Powder Pillow to Blank Preparation: Add persulfate reagent may not
each of two HR Total 0.5 mL of the deionized dissolve completely after
Nitrogen Hydroxide water included in the kit to shaking. This will not affect
Digestion Reagent vials. a second vial. Use only accuracy.
Wipe off any reagent that water that is free of all
may get on the lip or the nitrogen-containing
tube threads. species as a substitute for
the deionized water
provided.
Nitrogen, Total
Page 782
Nitrogen, Total
Persulfate digestion method (continued)
Stored Programs
394 N, Total HR TNT
Start
5. Insert the vials in the 6. Using finger cots, 7. Select the test. 8. Remove the caps from
reactor and close the lid. immediately remove the Insert an adapter if the digested vials and add
Heat for exactly 30 hot vials from the reactor. required (see Instrument- the contents of one Total
minutes. Cool the vials to room specific information). Nitrogen (TN) Reagent A
temperature. Powder Pillow to each vial.
9. Cap the tubes and 10. Start the instrument 11. After the timer expires, 12. Cap the tubes and
shake for 15 seconds. timer. remove the caps from the shake for 15 seconds. The
A three-minute reaction vials and add one TN reagent will not completely
period will begin. Reagent B Powder Pillow dissolve. This will not
to each vial. affect accuracy. The
solution will begin to turn
yellow.
13. Start the instrument 14. Prepared Sample: 15. Cap the vials and 16. Start the instrument
timer. After the timer expires, invert ten times to mix. timer.
A two-minute reaction pipet 2 mL of digested, Use slow, deliberate A five-minute reaction
period will begin. treated sample into one inversions for complete period will begin.
TN Reagent C vial. recovery.
The yellow color will
Blank: Add 2 mL of The tubes will be warm to intensify.
digested, treated reagent the touch.
blank to the second TN
Reagent C vial.
Nitrogen, Total
Page 783
Nitrogen, Total
Zero Read
17. 18. ZERO the instrument. 19. Wipe the reagent vial 20. READ the results in
The display will show: and insert it into the 16- mg/L N.
mm round cell holder.
0 mg/L N
Blanks for colorimetric measurement

The reagent blank may be used up to seven days for measurements using the same lots of
reagents. Store it in the dark at room temperature (18–25 °C). If a small amount of white floc
appears within a week, discard the reagent blank and prepare a new one.
Interferences
The substances in the Non-interfering substances table have been tested and found not to
interfere up to the indicated levels (in mg/L). Interfering substances that resulted in a concentration
change of ±10% appear in the Interfering substances table.

Barium 10.4 mg/L
Calcium 1200 mg/L
Chromium (3+) 2 mg/L
Iron 8 mg/L
Lead 26.4 µg/L
Magnesium 2000 mg/L
Organic Carbon 600 mg/L
Phosphorus 400 mg/L
Silica 600 mg/L
Silver 3.6 mg/L
Tin 6 mg/L

Bromide > 240 mg/L; positive interference
Chloride > 3000 mg/L; positive interference
Nitrogen, Total
Page 784
Nitrogen, Total
This test performed with standard nitrogen solutions prepared from the following compounds
obtained 95% recovery:
• Ammonium chloride • Urea

• Ammonium sulfate • Glycine
• Ammonium acetate
Ammonium chloride or nicotinic-PTSA spikes in domestic influent, effluent and the ASTM standard
specification for substitute wastewater (D 5905-96) also resulted in ≥ 95% recovery.
The large amounts of nitrogen-free organic compounds in some samples may decrease digestion
efficiency by consuming some of the persulfate reagent. Samples known to contain high levels of
organics should be diluted and re-run to verify digestion efficiency.

analysis.
• Preserve the sample by reducing the pH to 2 or less with concentrated (at least 2 mL/L)
Sulfuric Acid.
• Store samples at 4 °C (39 °F) or less. Preserved samples may be stored up to 28 days.
• Warm stored samples to room temperature and neutralize with 5 N Sodium Hydroxide before
analysis.
Accuracy check
This method generally yields 95–100% recovery on organic nitrogen standards. For proof of
accuracy use Primary Standards for Kjeldahl Nitrogen.
1. Prepare one or more of the following three solutions. Each preparation is for an equivalent
120-mg/L N standard. Use the deionized water included in the kit or water that is free of all
organic and nitrogen-containing species.
• Weigh 1.6208 g of Ammonium p-Toluenesulfonate (PTSA). Dissolve in a 1000-mL
volumetric flask with deionized water. Add deionized water to the 1000-mL mark.
• Weigh 2.1179 g of Glycine p-Toluenesulfonate (PTSA). Dissolve in a 1000-mL volumetric
flask with deionized water. Add deionized water to the 1000-mL mark.
• Weigh 2.5295 g of Nicotinic p-Toluenesulfonate (PTSA). Dissolve in a 1000-mL volumetric
2. Analyze each of these solutions using the test procedure above. Calculate the percent
recovery for each using this formula. Refer to the Percent recovery table for more information.
measured concentration
% recovery = ---------------------------------------------------------------- × 100
120
Nitrogen, Total
Page 785
Nitrogen, Total
Table 248 Percent recovery

Compound Lowest Expected % Recovery
Ammonia-PTSA 95%
Glycine-PTSA 95%
Nicotinic-PTSA 95%
Analysts have found Ammonia-PTSA to be the most difficult to digest. Other compounds may yield
different percent recoveries.

ADDITIONS.
5. Follow the Persulfate digestion method test procedure for each of the spiked samples, starting

• 100-mg/L ammonia nitrogen standard solution
1. Substitute 0.5 mL of a 100-mg/L ammonia nitrogen standard solution in place of the sample.
Follow the Persulfate digestion method test procedure.
to Standard Adjust in the software: OPTIONS>MORE>STANDARD ADJUST..
Method performance
394 100 mg/L NH3–N 98–102 mg/L N 0.5 mg/L N
Nitrogen, Total
Page 786
Nitrogen, Total
Summary of method
An alkaline persulfate digestion converts all forms of nitrogen to nitrate. Sodium metabisulfite is
added after the digestion to eliminate halogen oxide interferences. Nitrate then reacts with
chromotropic acid under strongly acidic conditions to form a yellow complex with an absorbance
maximum at 410 nm.

Required reagents
Test ’N Tube™ Total Nitrogen Reagent Set 50 vials 2714100

DRB200 Reactor, 110 V, 15 x 16 mm 1 each LTV082.53.40001
OR
Pipet Tips, for TenSette Pipet 19700-10 2 50/pkg 2199796
Test Tube Cooling Rack 1 each 1864100
Finger cots 2 2/pkg 1464702
Ammonia Nitrogen Standard Sol., 1000-mg/L NH3–N 1L 2354153
Ammonia Nitrogen Standard Sol., 100-mg/L NH3–N 500 mL 2406549
Analytical balance, 80 g capacity, 0.1 mg resolution each 2936701
Cylinder, mixing with stopper, 25 mL each 2088640
Pipet tips for 1970001 1000/pkg 2185628
Pipet tips for 1970010 250-pkg 2199725
Primary Standard Set, for Kjeldahl Nitrogen set of 3 2277800
Wastewater Mixed Inorganic Standard for NH3-H, NO3-N, PO4, COD, SO4,TOC 500 mL 2833149
Ammonia Nitrogen Standard Solution, 10-mL Voluette® Ampules, 50 mg/L 16/pkg 1479110
Ammonia Nitrogen Standard Solution, 10-mL Voluette® Ampules, 150 mg/L 16/pkg 2128410
Nitrogen, Total
Page 787
Nitrogen, Total
Ammonia Nitrogen Standard Solution, 2-mL PourRite® Ampules, 50 mg/L 20/pkg 1479120
Ammonia Nitrogen Standard Solution, 10-mg/L NH3–N 500 mL 15349

Nitrogen, Total Inorganic, 10021
Nitrogen, Total Inorganic DOC316.53.01090
Titanium Trichloride Reduction Method Method 10021

0.2 to 25.0 mg/L N Test ‘N Tube™ Vials
Test preparation


Instrument Sample cell
DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
For safety, wear gloves while breaking ampules.
The ammonia salicylate reagent contains sodium nitroferricyanide. Cyanide solutions are regulated as hazardous wastes by
the Federal RCRA. Collect cyanide solutions for disposal as reactive (D001) waste. Be sure cyanide solutions are stored in a
caustic solution with pH >11 to prevent release of hydrogen cyanide gas. Refer to the current MSDS for safe handling and
disposal information.
Total Inorganic Nitrogen Pretreatment Reagent Set (TiCl3 Reduction Method) 1
Test ‘N Tube™ AmVer™ Nitrogen-Ammonia Reagent Set 1
Centrifuge 1
Funnel, micro 1
Light Shield or adapter (see Instrument-specific information) 1
Pipet, TenSette®, 1.0–10.0 mL with tips 1
Pipette, volumetric, Class A, 1.00-mL 1
Test Tube Rack 1
Nitrogen, Total Inorganic

Page 789
Titanium Trichloride Reduction method, TNT
Stored Programs
346 N Inorganic TNT
Start
1. Select the test. 2. Pipet 1 mL of Total 3. Prepared Sample: 4. Cap the vials and
Insert an adapter or light Inorganic Nitrogen Pipet 1 mL of sample into shake for 30 seconds to
shield if required (see Pretreatment Base one vial. mix.
Instrument-specific concentrate into each of Blank Preparation:
information). two Total Inorganic Pipet 1 mL of deionized
Nitrogen Pretreatment water into the second vial.
Diluent Vials.
5. Pour the contents of 6. Immediately cap the 7. Insert the vials in a 8. Start the instrument
one Total Inorganic vials. Shake gently for 30 centrifuge. timer.
Nitrogen Reductant seconds to mix the (If a centrifuge is not A three-minute timer will
ampule into the vial reagents. Allow the vials to available, wait at least begin.
containing the sample. stand for at least one 30 minutes for the solids to
minute. Centrifuge the vials for
Pour the contents of settle at the bottom of the three minutes.
another Total Inorganic The precipitate should vial. Proceed to step 9.)
Nitrogen Reductant remain black after
ampule into the vial shaking. Excessive
containing the blank. shaking will cause the
A black precipitate will precipitate to turn white
form immediately. and cause low results.

Page 790
Titanium Trichloride Reduction method, TNT (continued)
9. Using a pipet, add 10. Using a funnel, add 11. Using a funnel, add 12. Cap the vials tightly
2 mL of centrifuged the contents of one the contents of one and shake thoroughly to
sample to an AmVer™ Ammonia Salicylate Ammonia Cyanurate dissolve the powder.
Diluent Reagent Test ‘N Reagent Powder Pillow Reagent Powder Pillow A green color will develop
Tube™ for Low Range (for 5-mL samples) to each (for 5-mL samples) to each if nitrogen is present.
Ammonia Nitrogen. vial. vial.
Add 2 mL of centrifuged
blank to another AmVer™
Diluent Reagent Test ‘N
Tube™ for Low Range
Ammonia Nitrogen.
Pipet carefully to avoid
disturbing the sediment.
Zero
13. Start the instrument 14. 15. ZERO the instrument. 16. Wipe the prepared
timer. The display will show: sample and insert it into
A 20-minute reaction the 16-mm round cell
0.0 mg/L N holder.
period will begin.
READ the results in
mg/L N.

Page 791
Interferences
The substances in the Interfering substances table may interfere when present. The substances in
the Non-interfering substances table do not interfere below the levels listed.

Calcium Causes a positive interference at 1000 mg/L as CaCO3.
Manganese (IV) Causes a negative interference at 3 mg/L.
Magnesium Causes a positive interference at 1000 mg/L as CaCO3.
Sulfide Causes a negative interference at 3 mg/L.
Sulfate Causes a negative interference at 250 mg/L.

Al3+ 8 mg/L
Ba2+ 40 mg/L
Cu2+ 40 mg/L
Fe3+ 8 mg/L
Zn2+ 80 mg/L
F– 40 mg/L
PO43––P 8 mg/L
SiO2 80 mg/L
EDTA 80 mg/L

analysis.
• Preserve samples by reducing the pH to 2 or less with concentrated (at least 2 mL)
Hydrochloric Acid*. Store at 4 °C (39 °F) or less.
• Before analysis, warm stored samples to room temperature and neutralize with 5 N Sodium
Hydroxide*.

Page 792
Accuracy check
• HR Nitrate Nitrogen PourRite Ampule Standard, 500-mg/L NO3––N
• Ampule breaker
• TenSette Pipet 0.1–1.0 mL and tips
• 25 mL Mixing cylinders (3)

ADDITIONS.
6. Follow the Titanium Trichloride Reduction method, TNT test procedure for each of the spiked
instrument.


1. Use this solution in place of the sample. Follow the Titanium Trichloride Reduction method,
TNT test procedure.
Method performance
The total inorganic nitrogen test is designed to provide an estimate of the total nitrite, nitrate and
ammonia nitrogen load present in a water or wastewater sample. This test is most applicable to
the monitoring of samples taken from an industrial process stream or a wastewater treatment
stream where it is important to track the inorganic nitrogen load as it passes through the treatment
process. The test does exhibit different recoveries of each of the three nitrogen species, as
summarized below. The test is not recommended for use when quantifying only one of the three
species. In that case, specific procedures for each particular analyte would be more appropriate.

Page 793
Species Recovery
Nitrogen Form Recovery

NH3–N 112%
NO3––N 100%
NO2 ––N 77%
Sensitivity—
95% Confidence Limits of
Program Standard DConcentration
Distribution
per 0.010 DAbs
Summary of method
Titanium (III) ions reduce nitrate and nitrite to ammonia in a basic environment. After centrifugation
to remove solids, the ammonia is combined with chlorine to form monochloramine.
Monochloramine reacts with salicylate to form 5-aminosalicylate. The 5-aminosalicylate is oxidized
in the presence of a sodium nitroprusside catalyst to form a blue colored compound. The blue
color is masked by the yellow color from the excess reagent present to give a final green colored
solution. Test results are measured at 655 nm.

Page 794
Required reagents
Total Inorganic Nitrogen Pretreatment Reagent Set (TiCl3 Reduction — 25 tests 2604945
Method)
Test ‘N Tube™ AmVer™ Nitrogen-Ammonia Reagent Set — 25 tests 2604545
Water, deionized 1 mL 100 mL 27242
Required apparatus
Centrifuge, 115 VAC, 6 x 15 mL 1 each 2676500
OR
Centrifuge, 220 VAC, 6 x 15 mL 1 each 2676502
Pipet, TenSette®, 1.0–10.0 mL 1 each 1970010
Pipet Tips, for TenSette Pipet 19700-10 varies 50/pkg 2199796
Gloves, Nitrile, Large1 1 pair 100/pkg 2550503
Flask, volumetric Class A, 50-mL each 1457441
Nitrate Nitrogen Standard Solution, 10-mg/L NO3––N 500 mL 30749
Nitrate Nitrogen Standard Solution, 2-mL PourRite® Ampule, 500 mg/L 20/pkg 1426020
Pipet Tips, for TenSette Pipet 19700-10 250-pkg 2199725
Water, deionized 4 liters 27256

Page 795

Cylinder, 25 mL mixing, tall form each 2088640
Hydrochloric Acid, concentrated 500 mL 13449
Sodium Thiosulfate, 0.1 N 100 mL 32332
Pipette, volumetric, Class A, 1.00-mL each 1451535
Nitrate Nitrogen Standard Solution, 1-mg/L NH3–N 500 mL 204649
Nitrate Nitrogen Standard Solution, MDB, 15-mg/L NH3–N 100 mL 2415132

Nitrogen, Total Kjeldahl, 8075
Nitrogen, Total Kjeldahl DOC316.53.01091
Nessler Method1 (Digestion Required) Method 8075

1 to 150 mg/L
Scope and Application: For water, wastewater and sludge; digestion is required.
1 Adapted from Hach, et. al., Journal of Association of Official Analytical Chemists, 70(5) 783-787 (1987); Hach, et. al., Journal of Agricultural
and Food Chemistry, 33(6) 1117-1123 (1985); Standard Methods for the Examination of Water and Wastewater
Test preparation


adjust.
If using a Pour-Thru system, periodically clean the cell by pouring a few sodium thiosulfate pentahydrate crystals into the cell
funnel or rinse with a solution of sodium thiosulfate. Flush it through the funnel and cell with enough deionized water to
dissolve. Rinse out the crystals.
Hold droppers and dropper bottles vertically, not at an angle, when dispensing reagent.
Nessler reagent contains mercuric iodide. Both the sample and blank will contain mercury (D009) at concentrations
regulated as a hazardous waste by the Federal RCRA. Do not pour these solutions down the drain. Refer to the current
MSDS for safe handling and disposal instructions.
Because of the method’s sensitivity, it is recommended to use the Standards and Calibration Adjust feature..
Nitrogen, Total Kjeldahl

Page 797
Boiling Chips, silicon carbide 2–3
Cots, finger 2
Cylinder, graduated mixing, 25 mL 2
Digesdahl Digestion Apparatus 1
Hydrogen Peroxide, 50% 20 mL
Mineral Stabilizer 6 drops
Nessler Reagent 2 mL
Polyvinyl Alcohol Dispersing Agent 6 drops
Potassium Hydroxide (KOH) Standard Solution, 1.0 N varies
Potassium Hydroxide (KOH) Standard Solution, 8.0 N varies
Sulfuric Acid, ACS, concentrated 6 mL
TKN Indicator Solution 2 drops
Pipet, TenSette®, 0.1–1.0 mL, plus tips 1
Safety Shield 1

Page 798
Nessler method
Stored Programs
399 Nitrogen, TKN
Start
1. Select the test. 2. Prepared Sample: 3. Blank Preparation: 4. Select the appropriate
Insert an adapter if Digest the sample amount Digest an equal amount of analysis volume of the
required (see Instrument- as described in the deionized water as the digested sample given in
specific information). Digesdahl® Digestion blank. the Aqueous samples
Apparatus Instruction (solutions or suspensions
Refer to the user manual Manual. in water—less than 1%
for orientation. solids), Dry samples or
Oils and fats tables
depending on the sample.
Pipet the analysis volume
from the sample and the
blank into separate 25-mL
mixing graduated
cylinders.
5. Add one drop of TKN 6. If the aliquot is less 7. Add 1.0 N KOH to 8. Fill both cylinders to
Indicator to each cylinder. than 1 mL, proceed to each cylinder, one drop at the 20-mL mark with
step 7. a time, mixing after each deionized water.
If it is greater than 1 mL, addition. Continue until the
add drops of 8.0 N KOH to first permanent blue color
each cylinder until the first appears.
flash of blue color
appears. Stopper and
invert the cylinder after
each addition. Proceed to
the next step.

Page 799
9. Add three drops of 10. Add three drops of 11. Fill both cylinders to 12. Pipet 1.00 mL of
Mineral Stabilizer to each Polyvinyl Alcohol the 25-mL mark with Nessler Reagent to each
cylinder. Stopper and Dispersing Agent to each deionized water. Stopper cylinder. Stopper and
invert several times to mix. cylinder. Stopper and and invert several times to invert repeatedly.
invert several times to mix. mix. The solution should not be
hazy. Any haze (turbidity)
will cause incorrect
results.
Zero
13. Start the instrument 14. When the timer 15. Wipe the blank and 16. ZERO the instrument.
timer. expires, pour the contents insert it into the cell holder. The display will show:
A two-minute reaction of the cylinders into
separate square sample 0 mg/L TKN
period will begin.
cells.

Page 800
Read
17. Wipe the prepared 18. READ the results in 19. Calculate sample TKN
sample and insert it into mg/L TKN. as follows:
the cell holder. 75 × A
ppm TKN = ----------------
B×C
Where:
A = mg/L read from the
display
B = g (or mL of water)
sample taken for digest
C = mL analysis volume of
digested sample
Interferences
Table 253 Aqueous samples (solutions or suspensions in water—less than 1% solids)

Expected nitrogen concentration (mg/L) Analysis volume (mL)
0.5–28 10.0
2–112 5.0
11–560 2.00
45–2250 1.00
425–22500 0.50
Table 254 Dry samples

42–2200 10.0
106–5600 5.00
350–18,000 2.00
1000–56,000 1.00
4200–220,000 0.50

Page 801
Table 255 Oils and fats

85–4500 10.0
210–11,000 5.00
2100–110,000 1.00

• Collect samples in clean glass or plastic containers.
• Adjust sample pH to 2 or less with Sulfuric Acid (about 2 mL per liter) and cool to 4 °C (39 °F).
• Preserved samples can be stored up to 28 days.
Accuracy check
Kjeldahl Nitrogen standard method
This procedure checks digestion efficiency and indicates the amount of bound nitrogen that is
released during digestion. The methods and standards available to check digestion technique are
found in the Accuracy Check section following the procedure in the Digesdahl® Digestion
Apparatus Instruction Manual. Using the digested Kjeldahl standard, perform the Nessler method
on a colorimeter. The TKN value should come within ± 3% of the value of the prepared
Kjeldahl standard.
Standard solution method (to check calibration accuracy only)

• 1.0-mg/L NH3–N solution
• TKN indicator
• Dropper
• Graduated cylinders (2)
• Deionized water
• Mineral Stabilizer
• Polyvinyl Alcohol Dispersing agent
1. Add one drop of TKN Indicator to each 25-mL graduated mixing cylinder.
2. Fill one cylinder to the 20-mL mark with deionized water. Fill the other cylinder to the 20-mL
mark with a 1.0-mg/L NH3–N solution.
3. Add 3 drops of Mineral Stabilizer to each cylinder. Invert several times to mix.
4. Add 3 drops of Polyvinyl Alcohol Dispersing agent to each cylinder. Invert several times to mix.
5. Perform the TKN procedure as described in step 11 to step 18. Accurate calibrations will show
26–27 mg/L TKN.

Page 802
Method performance
Program Standard Confidence Limits of DConcentration
Distribution per 0.010 DAbs
399 76 mg/L NH3–N 70–82 mg/L NH3–N 1 mg/L NH3–N
Summary of method
The term Total Kjeldahl Nitrogen refers to the combination of ammonia and organic nitrogen.
However, only the organic nitrogen compounds appearing as organically bound nitrogen in the
trinegative state are determined in this test. Nitrogen in this form is converted into ammonium salts
by the action of sulfuric acid and hydrogen peroxide. The ammonia is then analyzed by a modified
Nessler method test. Test results are measured at 460 nm.
Required reagents
Nitrogen Reagent Set, 0–150 mg/L, Nessler Method, includes: — 250 tests 2495300
Hydrogen Peroxide, 50% 20 mL 490 mL 2119649
Mineral Stabilizer 6 drops 50 mL SCDB 2376626
Nessler Reagent 2 mL 500 mL 2119449
Polyvinyl Alcohol Dispersing Agent 6 drops 50 mL SCDB 2376526
Potassium Hydroxide Standard Solution, 1.0 N varies 50 mL SCDB 2314426
Potassium Hydroxide Standard Solution, 8.0 N varies 100 mL MDB 28232H
Sulfuric Acid, ACS, concentrated 6 mL 500 mL 97949
TKN Indicator Solution 2 drops 50 mL SCDB 2251926
Required apparatus
Boiling Chips, silicon carbide 2–3 500 g 2055734
Cots, finger 2 2/pkg 1464702
Digesdahl® Digestion Apparatus, 115 VAC 1 each 2313020
OR
Digesdahl® Digestion Apparatus, 220 VAC 1 each 2313021
Safety Shield 1 each 5003000

Page 803
Nitrogen, Primary Standard Set for TKN each 2277800
Nitrogen Standard Solution, 1-mg/L NH3–N 500 mL 189149
Nitrogen Standard Solution, Voluette® Ampule, 150-mg/L NH3–N, 10-mL 16/pkg 2128410
Wastewater Influent Inorganics Standard for NH3–N, NO3–N, PO4, COD, SO4, TOC 500 mL 2833149

Sodium Thiosulfate, Pentahydrate 454 g 46001
Pour-Thru Cell Kit (DR 2700, DR 2800) each 5940400
Pour-Thru Cell Kit (DR 5000) each LZV479
Pipet Tips, for TenSette Pipet 19700–10 50/pkg 2199796
Ammonia Nitrogen Standard Solution, 1000-mg/L NH3–N 1L 2354153
Ammonia Nitrogen Standard Solution, 10-mL Voluette Ampules, 50 mg/L 16/pkg 1479110
Balance, analytical, 80 g capacity 115 VAC 2936701
Weighing Paper, 76 x 76 mm 500/pkg 1473800
Ammonia Nitrogen Standard Solution, 2-mL PourRite® Ampule, 50 mg/L 20/pkg 1479120

Nitrogen, Total, LR, 10071
Nitrogen, Total DOC316.53.001086
Persulfate Digestion Method Method 10071

LR (0.5 to 25.0 mg/L N) Test ‘N Tube™ Vials
Test preparation


DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
Digestion is required for determining total nitrogen.
If the test overranges, repeat the digestion and measurement with diluted sample. The digestion must be repeated for
accurate results.
Use the deionized water provided in the reagent set or Organic-free Water to prepare the standards and perform the
procedure.
Test ‘N Tube™ LR Total Nitrogen Reagent Set 1
DRB200 Reactor 1
Funnel, micro 1
Test Tube Cooling Rack 1–3
Finger Cots 2
Nitrogen, Total
Page 805
Nitrogen, Total
Persulfate digestion method
1. Turn on the DRB200 2. Using a funnel, add 3. Prepared Sample: 4. Cap both vials. Shake
Reactor and heat to the contents of one Total Add 2 mL of sample to one vigorously for at least
105 °C. Nitrogen Persulfate vial. 30 seconds to mix. The
Reagent Powder Pillow to Blank Preparation: Add persulfate reagent may not
each of two Total Nitrogen 2 mL of the deionized dissolve completely after
Hydroxide Digestion water included in the kit to shaking. This will not affect
Reagent vials. Wipe off a second vial. accuracy.
any reagent that may get
on the lip or the tube Use only water that is free
threads. of all nitrogen-containing
Note: One reagent blank is species as a substitute for
sufficient for each set of the provided deionized
samples. water.
Stored Programs
350 N, Total LR TNT
Start
5. Insert the vials in the 6. Using finger cots, 7. Select the test. 8. Remove the caps from
reactor and close the lid. immediately remove the Insert an adapter if the digested vials and add
Heat for exactly 30 hot vials from the reactor. required (see Instrument- the contents of one Total
minutes. Cool the vials to room specific information). Nitrogen (TN) Reagent A
temperature. Powder Pillow to each vial.
Nitrogen, Total
Page 806
Nitrogen, Total
9. Cap the tubes and 10. Start the instrument 11. After the timer expires, 12. Cap the tubes and
shake for 15 seconds. timer. remove the caps from the shake for 15 seconds. The
A three-minute reaction vials and add one TN reagent will not completely
period will begin. Reagent B Powder Pillow dissolve. This will not
to each vial. affect accuracy. The
solution will begin to turn
yellow.
13. Start the instrument 14. Prepared Sample: 15. Cap the vials and 16. Start the instrument
timer. After the timer expires, invert ten times to mix. timer.
A two-minute reaction remove the caps from two Use slow, deliberate A five-minute reaction
period will begin. TN Reagent C vials and inversions for complete period will begin.
add 2 mL of digested, recovery.
treated sample to one vial. The yellow color will
The tubes will be warm to intensify.
Blank: Add 2 mL of the touch.
digested, treated reagent
blank to the second TN
Reagent C vial.
Nitrogen, Total
Page 807
Nitrogen, Total
Zero Read
17. Wipe the reagent 18. ZERO the instrument. 19. Wipe the reagent vial 20. READ the results in
blank and insert it into the The display will show: and insert it into the 16- mg/L N.
16-mm round cell holder. mm round cell holder.
0.0 mg/L N Note: Multiple samples may
be read after zeroing on one
reagent blank.
Blanks for colorimetric measurement

The reagent blank may be used up to seven days for measurements using the same lots of
reagents. Store it in the dark at room temperature (18–25 °C). If a small amount of white floc
appears within a week, discard the reagent blank and prepare a new one.
Interferences
The Non-interfering substances table shows substances that have been tested and found not to
interfere up to the indicated levels (in mg/L). Interfering substances that resulted in a concentration
change of ±10% appear in the Interfering substances table.

Barium 2.6 mg/L
Calcium 300 mg/L
Chromium (3+) 0.5 mg/L
Iron 2 mg/L
Lead 6.6 µg/L
Magnesium 500 mg/L
Organic Carbon 150 mg/L
Phosphorus 100 mg/L
Silica 150 mg/L
Silver 0.9 mg/L
Tin 1.5 mg/L

Bromide > 60 mg/L; positive interference
Chloride > 1000 mg/L; positive interference
Nitrogen, Total
Page 808
Nitrogen, Total
This test performed with standard nitrogen solutions prepared from the following compounds
obtained 95% recovery:
• Ammonium chloride • Urea
• Ammonium sulfate • Glycine
• Ammonium acetate
Ammonium chloride or nicotinic-PTSA spikes in domestic influent, effluent and the ASTM standard
specification for substitute wastewater (D 5905-96) also resulted in ≥ 95% recovery.
The large amounts of nitrogen-free organic compounds in some samples may decrease digestion
efficiency by consuming some of the persulfate reagent. Samples known to contain high levels of
organics should be diluted and re-run to verify digestion efficiency.

analysis.
• Preserve the sample by reducing the pH to 2 or less with concentrated (at least 2 mL/L)
Sulfuric Acid.
• Store samples at 4 °C (39 °F) or less. Preserved samples may be stored up to 28 days.
• Warm stored samples to room temperature and neutralize with 5 N Sodium Hydroxide before
analysis.
Accuracy check
This method generally yields 95–100% recovery on organic nitrogen standards. For proof of
accuracy use Primary Standards for Kjeldahl Nitrogen.
1. Prepare one or more of the following three solutions. Each preparation is for an equivalent 25-
mg/L N standard. Use the deionized water included in the kit or water that is free of all organic
and nitrogen-containing species.
a. Weigh 0.3379 g of Ammonium p-Toluenesulfonate (PTSA). Dissolve in a 1000-mL
volumetric flask with deionized water. Add deionized water to the 1000-mL mark.
b. Weigh 0.4416 g of Glycine p-Toluenesulfonate (PTSA). Dissolve in a 1000-mL volumetric
c. Weigh 0.5274 g of Nicotinic p-Toluenesulfonate (PTSA). Dissolve in a 1000-mL volumetric
2. Analyze each of these solutions using the test procedure above. Calculate the percent
recovery for each using this formula. Refer to the Percent recovery table for more information.
measured concentration
% recovery = ---------------------------------------------------------------- × 100
25
Refer to the Percent recovery table.

Table 259 Percent recovery
Ammonia-PTSA 95%
Nitrogen, Total
Page 809
Nitrogen, Total
Table 259 Percent recovery (continued)

Glycine-PTSA 95%
Nicotinic-PTSA 95%
Analysts have found Ammonia-PTSA to be the most difficult to digest. Other compounds may yield
different percent recoveries.
• Ampule breaker

ADDITIONS.
5. Follow the Persulfate digestion method test procedure for each of the spiked samples, starting


• 10-mg/L ammonia nitrogen standard solution
1. Substitute 2 mL of a 10-mg/L ammonia nitrogen standard solution in place of the sample.

Follow the Persulfate digestion method test procedure.
Method performance
350 10 mg/L NH3–N 9.6–10.4 mg/L N 0.5 mg/L N
Nitrogen, Total
Page 810
Nitrogen, Total
Summary of method
An alkaline persulfate digestion converts all forms of nitrogen to nitrate. Sodium metabisulfite is
added after the digestion to eliminate halogen oxide interferences. Nitrate then reacts with
chromotropic acid under strongly acidic conditions to form a yellow complex with an absorbance
maximum at 410 nm.
Required reagents
Test ’N Tube™ Total Nitrogen Reagent Set, LR 50 vials 2672245

DRB200 Reactor, 110 V, 15x16 mm 1 each LTV082.53.40001
OR
DRB200 Reactor, 220 V, 15x16 mm 1 each LTV082.52.40001
Test Tube Cooling Rack 1–3 each 1864100
Finger Cots 2 2/pkg 1464702
Ammonia Nitrogen Standard Solution, 1000-mg/L NH3–N 1L 2354153
Primary Standard Set, for Kjeldahl Nitrogen set of 3 2277800
Wastewater Mixed Inorganic Standard for NH3-H, NO3-N, PO4, COD, SO4,TOC 500 mL 2833149

Balance, analytical, 80 g capacity, 115 VAC each 2936701
Cylinder, mixing with stopper, 50 mL each 2088641
Pipet tips for TenSette Pipet 1970001 1000/pkg 2185628
Pipet tips for TenSette Pipet 1970010 250-pkg 2199725
Nitrogen, Total
Page 811
Nitrogen, Total

PourRite® Ampule breaker, 2-mL each 2484600
Voluette® Ampule breaker 10 mL each 2196800
Ammonia Nitrogen Standard Solution, 2-mL PourRite Ampule, 50 mg/L 20/pkg 1479120

Oil and Grease, LLE, 10056
Oil and Grease DOC316.53.01187
USEPA1 Hexane Extractable Gravimetric Method Method 10056

15 to 3000 mg/L HEM and SGT-HEM
1 Equivalent to USEPA Method 1664. Adapted from Standard Methods for the Examination of Water and Wastewater, Section 5520B.
Test preparation
Determine a blank value (350 mL) of distilled or deionized water) with each new lot of reagents. If the blank result is greater
than 5 mg, resolve the source of error or remove interferences before performing this procedure. Use the equivalent amount
of acid to determine the blank and all samples from each sampling source.
The sample must be at room temperature before analyzing.
Do not use plastic tubing to transfer the solvent between containers.
Do not pre-rinse the collecting vessel with sample.
Anhydrous sodium sulfate is used to remove traces of water from the hexane extraction layer. Dry the sodium sulfate at 200
to 250 °C for 24 hours for best performance.
Spilled reagent will affect test accuracy and is hazardous to skin and other materials.
If determining both the HEM and the SGT-HEM, clean and dry two distillation flasks (one for each procedure) in advance.
• HEM = n-hexane extractable materials
• SGT-HEM = Silica Gel Treated n-hexane extractable materials
Oil and Grease

Page 813
Oil and Grease
Hexane Extractable Gravimetric Method
1. Collect 350 mL of 2. Using a pipette and 3. Using an analytical 4. Add 20 mL of

sample in a clean 500-mL pipette filler, add 4 mL of balance, weigh a n-hexane to the
separatory funnel. 1:1 Hydrochloric Acid previously dried and separatory funnel.
If the sample is not solution to the separatory cleaned 125-mL distillation If the sample was
collected in the separatory funnel. Mix well. The pH flask containing 3–5 collected in a separate
funnel, set the empty must be ≤ 2 to hydrolyze boiling chips to the nearest container or if repeating
container and lid aside for oils and grease and 0.1 mg. Record the weight this step, rinse the
use in step 4. prevent a sodium sulfate of the flask. collecting vessel/
interference. volumetric flask which
Check sample pH after contained the sample/
acid addition by dipping a water layer with 20 mL of
glass rod into the sample n-hexane, then add the
and allowing a few drops 20-mL n-hexane rinse to
to touch the pH paper. Do the separatory funnel.
not dip the pH paper into
the sample. Rinse the
glass rod with a small
portion of hexane back
into the separatory funnel
to remove any grease/oil
on the rod.
Oil and Grease

Page 814
Oil and Grease
Hexane Extractable Gravimetric Method (continued)
5. Stopper the funnel. 6. Let the funnel stand 7. Slowly drain the lower 8. Set up the filtering
Invert the funnel and undisturbed for at least 10 water layer from the funnel. Put the glass
release the gases through minutes to ensure separatory flask into the funnel in the neck of the
the stopcock. Then, separation of the lower original sample container distillation flask. Place a
vigorously shake the water layer and the upper or 500-mL volumetric folded 12.5 cm filter paper
funnel for two minutes. solvent layer. flask. This should take in the funnel. Add 10 g of
To release gases from the The solvent layer may be about 3–4 minutes. Save anhydrous sodium sulfate
separatory funnel, invert it brown if a colored oil is the water layer for step 10. to the filter paper. Rinse
and shake it once very present. To ensure that water is not the sodium sulfate with a
hard (support the stopper transferred in step 9, allow small amount of the
If you repeat this step the hexane. Discard the
with your hand). Under a third time and the water several drops of solvent
hood, point the delivery layer to drain into the hexane properly.
layer is cloudy, allow the
tube in a safe direction separatory funnel to stand water layer until the Use the same filter, funnel
and slowly open the undisturbed for 20 minutes solvent layer is visible on and sodium sulfate when
stopcock to release any for better separation of the top of the water. repeating this step for the
gas. Close the stopcock. water and solvent layers. If the water layer drains second and third
Repeat the venting too quickly, excess water extractions. Remove large,
procedure until you no will be present in the hard sodium sulfate
longer hear the release of solvent layer. This causes chunks between
gases. sodium sulfate and water extractions to reduce
interference. sodium sulfate
contamination.
Oil and Grease

Page 815
Oil and Grease
Repeat
Steps 4–10
9. Drip-drain the solvent 10. Return the water layer 11. Repeat step 4 through 12. Rinse the separatory
layer into the pre-weighed to the separatory funnel. step 10 two more times. funnel with three separate
boiling flask through a Use the same glass funnel After the third extraction, 5-mL aliquots of fresh
funnel containing filter for the second and third discard the water layer. n-hexane to remove any
paper and 10 g anhydrous extraction (referred to in There may be small oil film left on the funnel
sodium sulfate. Gently stir step 11). amounts of acetone and/or walls. Drain each aliquot
the sodium sulfate with a n-hexane in the water through the funnel
glass stirring rod while the To reduce spillage, a containing the sodium
second funnel can be layer.
solvent layer is draining. sulfate into the distillation
Be careful not to rip the used when pouring the Refer to your local waste flask.
filter paper. water layer into the disposal procedure for
separatory funnel. proper disposal
Any spillage will cause instructions.
inaccurate results. To
reduce spillage, use a
glass rod to guide the
sample solution into the
filter.
Oil and Grease

Page 816
Oil and Grease
See See
Go to
Figure 1 Figure 1
Step 15
13. Rinse the tip of the 14. If HEM is to be 15. Using the distillation 16. Disconnect the
glass funnel with 5 mL of determined, go to step 15. assembly shown in condenser/connector
n-hexane while removing it If only the SGT-HEM is to Figure 13, distill off the portion of the distillation
from the distillation flask. be determined and the n-hexane. Distillation is assembly at the pinch
Check for sodium sulfate HEM is known, go to complete when there are clamp and remove the
contamination. step 21. no boiling bubbles or the distillation flask from the
Sodium sulfate distillation flask appears heat source with an
If only the SGT-HEM is to dry. anti-lint cloth or tongs.
contamination will appear be analyzed, the HEM is
as cubic crystals at the needed in order to Use a steam bath or a hot The distilled n-hexane
bottom of the distillation determine the amount of plate to maintain a water may be re-used in future
flask. If present, re-filter silica gel needed for the bath at the proper HEM extractions, but is not
the solvent layer through SGT-HEM. For each group temperature for the recommended for
filter paper without sodium of samples from a distillation. Do not place SGT-HEM due to the
sulfate. You must re-clean, discharge, determine the the flask directly on a hot potential increased water
dry and weigh the boiling HEM before the plate. This will cause low content of the solvent.
flask and boiling chips; or SGT-HEM. results and is dangerous
have an extra flask ready because n-hexane is
in case this is necessary. volatile.
Evaporation will be faster if
the long vertical arm of the
connector is wrapped with
insulation (paper towel,
cloth or asbestos
insulating tape). The
distillation should take less
than 30 minutes.
Oil and Grease

Page 817
Oil and Grease
17. Remove the remaining 18. Place the flask in a 19. Using an analytical 20. Calculate the test
solvent vapors from the desiccator for 30 minutes balance, weigh the flask to results:
distillation flask by (or longer if necessary) the nearest 0.1 mg. A – B × 1000 = mg/L HEM
attaching the vacuum until it cools to room Record this weight. Do not
connector/gas inlet temperature. touch the flask after A= Weight (mg) of residue
adapter to the flask. Apply If the silica gel indicator weighing; fingerprints will B= Weight (mg) of flask
a vacuum for 1–2 minutes has turned red, replace the add weight. with boiling chips (step 3).
or until all n-hexane silica gel. Always use a tong or a
solvent vapors have been Example:
lint-free wipe when
removed. handling the flask. A = 92.4659 g
Crystals on the bottom of Precise weighing is B = 92.4206 g
the flask indicate that necessary for accurate Sample volume = 0.350 L
sodium sulfate may have results; multiple weight (350 mL)
been dissolved in the measurements are
extraction steps. Re- recommended. Re-wipe 92.4659 – 92.4206
dissolve the extract in n- the flask before each ------------------------------------------------- =
0.350
hexane, filter into another measurement to ensure all
pre-weighed flask and 129.4 mg/L
contaminants are
repeat steps 14–16. This removed. Record each If yield is less than
is not necessarily true for weight; use the lowest 15 mg/L and additional
the “standard” extraction repeatable value for precision is needed, use a
since stearic acid is calculations. 1-liter sample.
crystalline below 69 °C
(156 °F). If sodium sulfate
is present in the standard,
big cubical crystals (not
the flattened stearic acid
crystals) will be visible.
Also, an unusually high
yield compared to the
expected value will result.
Oil and Grease

Page 818
Oil and Grease
21. If only calculating the 22. For a 350-mL water 23. Put a magnetic stir bar 24. Stir solution on a
HEM, stop here. sample, dilution is and the correct amount of magnetic stirrer for five
If continuing with necessary if the HEM is silica gel (based on the minutes (minimum).
SGT-HEM, re-dissolve above 2850 mg/L (for a equation below) into the
residue with approximately 1-liter sample, dilute if the flask with the solvent/
85 mL of fresh n-hexane. HEM is greater than product from step 22.
Heat slightly to ensure 1000 mg/L). 3 × mg/L HEM
-------------------------------------- =
re-dissolving of all HEM To dilute to a 1000 mg/L 100
materials. sample, pour the silica gel (g ± 0.3)
re-dissolved HEM into a
100-mL volumetric flask.
Rinse the distillation flask
3–4 times with 2–3 mL of
n-hexane. Fill the
volumetric flask to volume
with n-hexane. Mix well.
Into a 100-mL beaker,
volumetrically pipet the
amount (Va) determined
by this equation:
10000
V a = ----------------
Wh
where:
Va = Volume of aliquot to
be withdrawn (mL) to get
1000 mg of HEM.
Wh = Weight of HEM (mg)
(A –B x 1000 in step 19
(mg)). Dilute to about
100 mL with n-hexane.
Oil and Grease

Page 819
Oil and Grease
Perform
Steps 15—20.
25. Place a funnel on a 26. Follow step 15–

clean, dry distillation flask step 20. Weigh the
with 3–5 boiling chips in it. product remaining in the
Place a 12.5-cm filter bottom of the flask and
paper in the funnel. calculate the results using
Pre-moisten the filter the equation below:
paper with fresh n-hexane. A–B
Filter the solution through ------------------------------------------ =
Sample Volume
filter paper. mg/L SGT – HEM
Rinse the beaker
containing remaining silica where:
gel 3 times with 5-mL A = Weight (mg) of residue
aliquots of fresh n-hexane
B = Weight (mg) of flask
and pour the aliquots into
with boiling chips.
the distillation flask.
Oil and Grease

Page 820
Oil and Grease
Figure 13 Distillation assembly

1. Pinch Clamp 8. Water Bath
2. J-Shaped Connector 9. Hot Plate
3. Clamp, 3-Prong 10. Receiving Flask
4. Condenser 11. Water In
5. Pinch Clamp 12. Clamp Holders
6. Clamp, 3-Prong 13. Water Out
7. Distillation Flask 14. Support Stand and Rod Assembly
Oil and Grease

Page 821
Oil and Grease
Interferences
Substances extracted from samples will vary from source to source, depending upon the diversity
of the site being sampled. Some samples may contain high amounts of detergents or particulates
that can interfere with the extraction procedure. For these samples, it may be necessary to use a
350-mL sample rather than 1-liter (which is an option). In this circumstance, the 350-mL sample is
EPA accepted for reporting. Wash all glassware in hot water with detergent, rinse with tap and
distilled water and rinse with n-hexane or acetone.
If an emulsion forms between the two phases (at step 6) and is greater than one-third the volume
of the solvent layer, filter the emulsion and solvent layer through a funnel with glass wool in it. If an
emulsion still exists, other possible solutions include: stirring the solvent and emulsion layer with a
stir bar, using solvent phase separation paper, centrifugation, using an ultrasonic bath with ice,
adding NaCl or other physical methods. (Solid phase or other extraction techniques would fall
under performance based modifications.)
A milky solvent/product layer in the distillation flask indicates water in the solvent layer. Let the
flask stand one hour to allow the water to settle. Re-filter the solvent layer through sodium sulfate
to remove remaining water.
Extremely low yields could mean a poor extraction (step 5 through step 8) and a high yield could
indicate a problem in the solvent drying (step 8). Follow step 5 through step 8 very carefully and
run your blank before you run samples in order to identify any possible interference due to these
steps. If your blank indicates a yield above 1 mg per test, you should identify the source of
contamination before continuing; likely sources are sodium sulfate contamination and improperly
rinsed glassware.

• Collect samples in wide-mouth glass bottles or directly in the separatory funnel for immediate
analysis.
• Collection of sample may be done directly into the separatory funnel. Measure 350 mL of
water with a graduated cylinder.
• Pour this into the separatory funnel. Use a laboratory pen to mark the 350-mL level. Fill with
sample to this mark.
• Do not pre-rinse the bottle or separatory funnel with the sample.
• If analysis is delayed for more than a few hours, add 6 mL of 1:1 Hydrochloric Acid Solution for
each liter or quart of sample.
• Check the pH to ensure it is 2 or less. Do not place the pH paper directly into the sample, but
dip a glass rod into the sample and touch the pH paper with a drop of sample.
• Rinse the rod with n-hexane directly back into sample container after the pH has been
determined to make sure that no product has adhered to the glass rod.
• If necessary, add more acid to bring pH below 2.
• Store the sample between 0–4 °C (32–39 °F). Preserved samples can be stored for 28 days.
Handling glassware
Before analysis, careful cleaning and drying of the glassware and boiling chips is necessary. Clean
the chips and distillation flask by washing with hot water and detergent, rinsing with distilled water
and then rinsing with acetone or n-hexane. Place the cleaned flask and boiling chips in a drying
oven at 105–115 °C (220–240 °F) for 2 hours. Cool to room temperature in a desiccator for at least
30 minutes. Store in the desiccator until needed.
To eliminate errors, always handle the flask with tongs or an anti-lint wipe. If the same flasks are
used repeatedly, record their weights after drying in the oven without boiling chips. The drying step
Oil and Grease

Page 822
Oil and Grease
may be skipped if the flasks weigh the same after the acetone or n-hexane rinse as it does after
drying. Boiling chips will vary in weight; their weight should be added to the flask weight.
Definition of HEM and SGT-HEM

Oil and grease is the conventional term used to define pollutants of this nature. The new term
n-Hexane Extractable Materials (HEM) indicates this method may be applied to materials other
than oils and greases.
Likewise, the term Total Petroleum Hydrocarbons (TPH) was traditionally used to classify aliphatic
hydrocarbon materials. The new term Silica Gel Treated n-Hexane Extractable Material
(SGT-HEM), indicates that this method may be applied to materials other than aliphatic petroleum
hydrocarbons that are not adsorbed by silica gel.
Detection limit
This method is not applicable to measurements of materials that volatilize at temperatures below
approximately 85 °C (185 °F). Petroleum fuels from gasoline through #2 fuel oil may be partially
lost in the solvent removal operation. Some crude oils and heavy fuel oils contain a significant
percentage of materials that are not soluble in n-hexane. Recoveries of these materials may
be low.
The method is capable of measuring HEM and SGT-HEM in the range of 15 to 3000 mg/L when
using a 350-mL sample and may be decreased to as low as 5 mg/L if a 1-liter sample is used.
When using the 1-liter sample volume, use the amount of reagents listed for the 1-liter size in EPA
monitoring, testing procedures and modifications.
1. Transfer 400 ±4 mg stearic acid and 400 ±4 mg hexadecane into a 100-mL volumetric flask.
2. Pour 75 mL of acetone into the flask. Cover with a small beaker and stir gently. Heat slightly
until all material is in solution. Over-heating with the lid on causes pressure build up.
3. Fill to volume with acetone. Cover. Allow to equilibrate to room temperature and continue to fill
to volume until solution is at stable volume.
4. Using a volumetric pipet, transfer 5 mL of the solution from step 3 into 350 mL of deionized
reagent water. This standard solution should be 114.3-mg/L HEM or 57.1-mg/L SGT-HEM. If
using a 1-liter water sample, 5 mL gives concentrations of 40 mg/L HEM and 20 mg/L
SGT-HEM. To verify the concentration, pipette 5 mL of the solution from step 3 in a
pre-weighed flask, place in hood and allow acetone to evaporate off. Weigh the flask. Verify
that the weight difference before and after solution addition is 40 (±1) mg.
EPA monitoring, testing procedures and modifications

If the Oil and Grease tests are used for compliance reporting to the USEPA, make the following
changes to the procedure:
1. Use a 1-liter sample in a 2000-mL separatory funnel rather than a 350-mL sample in a 500-mL
separatory funnel (step 1).
2. Use 6 mL (instead of 4 mL) of 1:1 hydrochloric acid to bring the pH below 2 (step 2) and 30 mL
of n-hexane instead of 20 mL of n-hexane for the extraction (step 4).
Before testing real samples for oil and grease, you must be able to obtain a MDL (Minimum
Detection Limit) less than or equal to the EPA reported MDL and to report an IPR (Initial Precision
and Recovery). Before attempting the MDL and IPR, it is highly recommended to run
laboratory reagent water blanks to eliminate all interference.
Oil and Grease

Page 823
Oil and Grease
MDL: The USEPA-documented MDL is extremely difficult to reproduce if the slightest sodium
sulfate contamination occurs. EPA Method 1664 requires the MDL to be ≤ 1.4 mg/L for HEM and
≤ 1.6 mg/L for SGT-HEM. This is calculated by determining the standard deviation of seven
standard samples for HEM or seven standards for SGT-HEM and multiplying their respective
standard deviations by 3.143 (Student’s t test).
The recommended standard concentration for determining the MDL is about 5 mg/L. To prepare
the standard for HEM follow steps 1–3 in Standard preparation, but change step 1 to transfer
100 (± 4) mg stearic acid and 100 (± 4) mg hexadecane to a 250-mL volumetric flask. To prepare
the SGT-HEM standard, transfer 200 (± 4) mg of decahexane only into a 250-mL volumetric flask.
Transfer 5 mL of either standard into 1-liter of reagent water. Analysis of the standard should give
5 mg/L for either HEM or SGT-HEM.
IPR: Follow the procedure for HEM and SGT-HEM, using deionized water (void of any oil and
grease) as the blank. Perform the procedure four separate times using 5 mL of the standard
(40 mg/L 1:1 stearic acid/hexadecane) diluted into 1 liter of demineralized water. Report the
average percent recovery (X) and the standard deviation of the percent recovery(s) for both HEM
and SGT-HEM. X and s should fall within these parameters:
• HEM: Precision(s) ≤ 10%; Recovery(X) 83–101%
• SGT-HEM: Precision(s) ≤ 13%; Recovery(X) 83–116%
If not within these ranges, correct the problem and repeat IPR.
Once the MDL and IPR have been established, keep records for EPA verification or for future
reference.
Oil and grease reporting to EPA

To report the HEM and/or SGT-HEM, include the following data for each set of up to ten samples.
1. BLANK: Must be less than 5.0 mg/L for HEM and SGT-HEM.
Note: For compliance monitoring, it may be necessary to use a standard that either matches the
regulatory concentration limit or is 1 to 5 times higher than the concentration of the sample (B),
whichever is greater. The concentration of the spike (T) needs to be divided by 2 for SGT-HEM if
using the standard (40 mg/L 1:1 stearic acid/hexadecane).
2. OPR (Ongoing Precision and Recovery): Determine the amount of analyte in a one liter
sample containing 5 mL of the standard (40 mg/L 1:1 stearic acid/hexadecane). Continue on if
the HEM recovery is 70–114% and the SGT-HEM recovery is 66–114%. If recovery is lower,
an interference may be present or analysis technique may be faulty. Identify the cause and
repeat OPR until within range.
3. MS and MSD (matrix spike and matrix spike duplicate): Determine the background
concentration (B) of your sample by running HEM and SGT-HEM for the discharge water.
Spike two 1-liter discharge water samples with 5 mL of standard and measure the
concentration of the analyte after spiking (A).
Determine the Percent Recovery (P) as follows:

100 × ( A – B )
P HEM(40 mg/L) = -----------------------------------
T
100 × ( A – B )
P SGT – HEM = -----------------------------------
T⁄2
If HEM Recovery is 79–114% and SGT-HEM Recovery is 66–114%, then calculate the
Relative Percent Difference (RPD). If recovery is lower, an interference may be present.
Identify and correct the interference, then repeat MS and MSD.
Conc MS – Conc MSD
RPD = ---------------------------------------------------- × 200
Conc MS + Conc MSD
Oil and Grease

Page 824
Oil and Grease
If RPD for HEM ≤ 18 and RPD for SGT-HEM ≤ 24, then continue with step 4.
If recovery is lower, an interference may be present. Identify and correct the interference, then
repeat MS and MSD.
After every five MS/MSD tests, compute the average percent recovery (Pa) and standard
deviation of the percent recovery (sp). Record these numbers as Pa ±2sp.
4. Calibrate your analytical balance at 2 mg and 1000 mg using class “S” weights to ensure
calibration within ±10%.
5. Analyze up to 10 samples from the source used in the MS and MSD before starting back at
step 1. For every 10 samples you must determine a new blank, OPR, RPD, MS and MSD.
Summary
Each laboratory must first verify the MDL and IPR and ensure they are within proper parameters
before reporting oil and grease test results to the EPA. Once this is established for a laboratory, it
does not need to be done again.
For every 10 samples per discharge source, one must calibrate the balance, report one blank, one
OPR, one MS and one MSD. Logs must be kept on percent recovery and relative percent
differences for MS/MSD tests. For every five MS/MSD test, calculate and record the average
percent recovery and standard deviation.
Summary of method
Oil and Grease & Total Petroleum Hydrocarbons (TPH) include any material that may be
recovered as a substance that is soluble in the n-hexane extractant. These include substances
such as relatively non-volatile hydrocarbons, vegetable oils, animal fats, waxes, soaps, greases
and related materials. When measuring oil and grease (HEM) gravimetrically, the substances are
extracted from the sample with n-hexane, then the n-hexane is evaporated. The residue left is
weighed to determine the concentration of oil and grease materials in mg/L.
When measuring Total Petroleum Hydrocarbons (SGT-HEM) gravimetrically, the substances are
extracted from the sample with n-hexane, then mixed with silica gel to absorb non-TPH
components. Then the n-hexane is evaporated. Like the HEM, the residue left is weighed to
determine the concentration of total petroleum hydrocarbons.
Note: Careful technique is required to obtain accurate and precise results.
Required reagents
Hydrochloric Acid Standard Solution, 1:1 (6N) 4 mL 500 mL 88449
Hexane, ACS grade 100–200 mL 500 mL 1447849
pH Paper, 0–14 pH units varies 100/pkg 2601300
Silica Gel with indicator (for desiccator) varies 454 g 1426901
Silica Gel, 100–200 mesh 1–30 g 500 g 2665034
Sodium sulfate, anhydrous 10 g 113 g 709914
Oil and Grease

Page 825
Oil and Grease
Required apparatus
Adapter, vacuum connector/gas inlet, 28/15 1 each 1433900
Aspirator, vacuum 1 each 213100
Balance, Analytical, 115 VAC 60 Hz. 1 each 2936801
Boiling Chips 3–10 500 g 2055734
Clamp, 3-prong 2 each 42200
Clamp, holder 2 each 32600
Clamp, pinch type, No. 28, F/Glass Joints 2 each 1433800
Condenser, reflux, w/ground glass joints, 28/15 1 each 1433700
Desiccator 1 each 2088800
Desiccator Plate 1 each 1428400
Filter Funnel, 65-mm, short stem 1 each 2664700
Filter Paper, 12.5-cm, folded, pore size 8 to 12 µm 1 100/pkg 69257
Flask, Erlenmeyer, 125-mL, w/ground glass joint 28/15 2 each 1434000
Marker, Laboratory 1 each 2092000
Oven, drying, 120 VAC, 50 Hz 1 each 1428900
Pipette filler, safety bulb 1 each 1465100
Pipette, serological, 5-mL 1 each 53237
Ring Support, 4-inch 1 each 580-01
Rod, glass 1 3/pkg 177001
Steam bath, 8-inch, 5-ring 1 each 2347900
Hot Plate/Stirrer, 7.25 x 7.25", 120 VAC 1 each 2881600
Stir Bar, 22.2 x 7.9 mm 1 each 2095350
Tongs, crucible, 9-inch 1 each 56900
Tube, connecting, J-shaped, w/ground glass joint, 28/15 1 each 1814300
Tubing, black rubber, 7.9 X 2.4 mm 1 3.6 m 56019
Hexadecane, 99%, 400 mg 100 mL 2664842
Stearic Acid, 400 mg 500 g 2664934
Oil and Grease

Page 826
Oil and Grease

Acetone, ACS 500 mL 1442949
Separatory funnel, 2-liters each 52054
Balance, Weight Set each 2617601
Hotplate/Stirrer 7.25 x 7.25 in., 220–240 VAC each 2881602
Ring Support, 4.5 in. each 2656300
Furnace, Muffle, 120 VAC each 1429600
Pipet, Volumetric 5.0 mL each 14515-37
Flask, Volumetric 100 mL 14574-452
Cylinder, graduated, 100 mL each 508-42
Oil and Grease

Page 827
Oil and Grease, SPE, 10300
Oil and Grease DOC316.53.01186
USEPA1 Solid Phase Extraction Method Method 10300

5 to 1000 mg/L HEM and SGT-HEM Hexane Extractable Gravimetry
1 Equivalent to USEPA Method 1664A, Solid Phase Extraction (SPE)
Test preparation
Follow the instructions for sample collection and acidification in Sample collection, preservation and storage.
Assemble the Xenosep® SPE apparatus as shown in Figure 14. Be sure to put the waste collection tube in the large flask
before installing the funnel assembly. Put the pattern side of the SPE filter down in the SPE filter support (Figure 15).
Wash all glassware in hot water with detergent, rinse with tap and distilled water and rinse with acetone or n-hexane.
Rinse all glassware thoroughly with hexane to make sure the analyte is not retained on the apparatus. Inadequate rinsing will
result in low recovery.
Determine a blank value (1 liter of distilled or deionized water) with each new lot of reagents. If the blank result is greater
than 5 mg, resolve the source of error or remove interferences before performing this procedure.
If analyzing for SGT-HEM, rinse the inner cavity of the aluminum dish with a few milliliters of acetone and then with a few
milliliters of hexane to remove any potential artifacts.
Label the aluminum dish and heat in an oven for one hour at 105 °C. Cool the dish in a dessicator for 30 minutes. Weigh the
dish on an analytical balance to the nearest 0.1 mg. Record this weight as "B" for HEM step 22 or SGT-HEM step 12.
Make sure that the aluminum dish is removed from the hot plate before the solution has completely evaporated. Heating the
solution too long will result in low recovery.
Use a vacuum pump that can generate a Free Air Flow of at least 1 CFM (3 CFM preferred). Insufficient SPE disk drying will
result in low recovery.
Hydrochloric acid, 1:1 6 mL
Hexane (n-hexane) in Teflon-FEP wash bottle 1 wash bottle
Methanol in PE wash bottle 1 wash bottle
Deionized water in PE wash bottle 1 wash bottle
SPE Starter Kit, EPA Method 1664A 1 kit
SPE Consumables Kit 1 kit
SPE Solvent Recovery Kit 1 kit
Aluminum weighing dish 1
Hot plate 1
Vacuum pump 1
Oil and Grease

Page 829
Oil and Grease
pH paper, 0–14 pH units 1
Lab stand 1
Clamp, swivel 2
Desiccator 1
For SGT-HEM:
Silica gel 1 bottle
125-mL Erlenmeyer flask 1
100-mL volumetric flask (for HEM results over 1000 mg/L) 1
Magnetic stir plate 1
Aluminum weighing dish 1
Additional sodium sulfate column 1
Figure 14 Solid phase extraction assembly

1. Hot plate 7. Eluter tube
2. Aluminum dish 8. Clamp, tubing
3. Glass dome 9. 3-way valve
Oil and Grease

Page 830
Oil and Grease
4. Solvent recovery assembly 10. 2-way valve

5. Funnel assembly (see Figure 15) 11. To vacuum
6. Sodium sulfate column 12. Waste collection tube with O-Ring
Figure 15 Funnel assembly

1. Aluminum clamp 4. SPE filter—pattern side down
2. Funnel 5. Stainless steel support
3. Coupler with O-Rings 6. SPE starter holder
Solid phase extraction for HEM
1. Add approximately 2. Turn the vacuum on 3. Add approximately 4. Remove the tube and
10 mL of hexane to the for 1 minute to dry the 10 mL of methanol to the discard the solvent waste.
funnel. Wait 5 seconds. filter. Turn off the vacuum. funnel. Wait 5 seconds. Refer to the MSDS and
Turn the vacuum on and Turn the vacuum on and local regulations for safe
off to pull the solvent into off to pull the solvent into disposal of the solvent.
the waste collection tube. the waste collection tube.
Repeat with a second Do not allow the filter to
10-mL portion of hexane. become dry.
Make sure the valves are
set to pull vacuum from
the funnel holder and the
tubing clamp is open.
Oil and Grease

Page 831
Oil and Grease
Solid phase extraction for HEM (continued)
5. Add approximately 6. Slowly pour the 7. Leave the vacuum on 8. Put the funnel
20 mL of deionized water acidified sample into the for 4 to 8 minutes to air dry assembly on the eluter
to the funnel. Wait 5 funnel and turn on the the filter. Turn off the tube.
seconds. Turn the vacuum vacuum. Use deionized vacuum.
on and off to pull the water water to rinse any debris Do not leave the vacuum
into the flask. from the walls of the on for more than
Do not allow the filter to funnel. 8 minutes.
become dry. See Sample collection,
If the filter becomes dry, preservation and storage
repeat steps 3–5. for sample acidification.
9. Turn the valve to apply 10. Add 10 mL of hexane 11. Use a transfer pipet to 12. Turn the vacuum on
vacuum to the eluter tube. to the empty sample collect the hexane from and off to pull the solvent
Close the tubing clamp on bottle. Shake the bottle in the shoulder of the sample into the flat-sided flask.
the funnel holder tube. a horizontal, circular bottle. Slowly rinse the Rinse the walls of the
motion for 10 seconds to walls of the funnel with the funnel with approximately
rinse the bottle. hexane. Go around the 10 mL of hexane. Wait 5
funnel at least 3 times. seconds. Turn the vacuum
Repeat steps 10–11 two on and off.
more times.
Oil and Grease

Page 832
Oil and Grease
Solid phase extraction for HEM (continued)
13. Remove the flat-sided 14. Pour the hexane into 15. Rinse the flat-sided 16. Put the dish on the hot
flask from the eluter tube. a pre-weighed aluminum flask with approximately plate and place the glass
dish. 5 mL of hexane. Add the cover over the dish.
hexane to the dish.
17. Close the 3-way valve 18. Turn on the vacuum. 19. Examine the dish for 20. When the dish is dry,
and open the 2-way valve Turn the hot plate on low dry spots. As soon as put the dish in a desiccator
to pull vacuum from the heat (35–85 ºC). Heat for there is a dry spot remove for 30 minutes.
solvent recovery approximately 2 minutes. the dish from the hot plate.
apparatus. Put the dish in a fume
hood until the rest of the
hexane has evaporated.
21. After 30 minutes, 22. Calculate the test Example:

weigh the dish to the results: A= 6.2394 g
nearest 0.1 mg. Repeat A–B
---------------------------------------- × 1000 B= 6.2318 g
steps 20 and 21 until the sample volume
sample volume = 0.950 L
weight loss is less than A= weight of dish with
6.2394 g – 6.2318 g
0.5 mg from the previous residue (g) (step 21) ----------------------------------------------------- × 1000 =
0.950 L
weight. Record the weight. B= weight of dish (g)
8.0 mg/L HEM
Oil and Grease

Page 833
Oil and Grease
Solid phase extraction for SGT-HEM (HEM < 1000 mg/L)
1. Add hexane to the 2. Heat slightly to 3. Pour the mixture into a 4. Add 3 (± 0.3) g of
residue in the aluminum dissolve all of the residue. 125-mL Erlenmeyer flask. silica gel for every 100 mg
dish from the HEM test Rinse the pan and funnel of HEM or fraction thereof:
until the dish is several times with hexane 3 × mg/L HEM
approximately half full and add to the flask. Add -------------------------------------- =
100
(approximately 60 mL). hexane to approximately silica gel (g)
the 100-mL mark.
Example: if the HEM value
was 735 mg, round 7.35 g up
to the next whole gram
increment (8 g or 800 mg)
and add 3 x 800/100= 24 g of
silica gel.
Do not add more than 30 g
silica gel.
5. Add a stir bar to the 6. Put a new sodium 7. Install a clean 8. Adjust the valves to
flask and stir for 5 minutes. sulfate column into the flat-sided flask. pull vacuum from the
eluter tube. Put the funnel eluter tube.
on the eluter tube.
Oil and Grease

Page 834
Oil and Grease
Solid phase extraction for SGT-HEM (HEM < 1000 mg/L) (continued)
Go to the HEM
procedure.
9. Pour the solution from 10. Rinse the Erlenmeyer 11. Complete steps 13–21 12. Calculate the test
the Erlenmeyer flask into flask with 5 mL hexane. under Solid phase results:
the funnel. Turn the Add the hexane to the extraction for HEM to A–B
---------------------------------------- × 1000
vacuum on and off to pull funnel. Turn the vacuum evaporate the solvent. sample volume
the solution into the flat- on and off. A= weight of dish with
sided flask. Rinse the walls of the residue (g) (step 21)
funnel with approximately B= weight of dish (g)
5 mL of hexane. Turn the Example:
vacuum on and off. A= 6.2360 g
B= 6.2320 g
sample volume = 0.950 L
6.2360 g – 6.2320 g
----------------------------------------------------- × 1000 =
0.950 L
4.2 mg/L SGT-HEM
Report result as ≤ 5 mg/L
SGT-HEM
Solid phase extraction for SGT-HEM (HEM > 1000 mg/L)
1. Add hexane to the 2. Pour the mixture into a 3. Dilute to the mark with 4. Calculate the volume
residue in the aluminum 100-mL volumetric flask. hexane and mix well. to remove for the silica gel
dish from the HEM test Rinse the pan and funnel treatment:
until the dish is several times with hexane 100,000
approximately half full and add to the flask. V 1000 = -----------------------------
HEM value
(approximately 60 mL).
V1000 = volume that
Heat slightly to dissolve all contains 1000 mg HEM
of the residue.
Oil and Grease

Page 835
Oil and Grease
Solid phase extraction for SGT-HEM (HEM > 1000 mg/L) (continued)
Go to the SGT-HEM
(HEM < 1000 mg/L)
procedure.
5. Remove the amount 6. Add 30 g of silica gel 7. Complete steps 5–12 8. Correct the test result
calculated in step 4 from to the flask. in the procedure for Solid for the reduced volume
the volumetric flask and If the volume added in phase extraction for SGT- that was treated with the
add it to a 125-mL step 5 contained less than HEM (HEM < 1000 mg/L). silica gel:
Erlenmeyer flask. Add 100
1000 mg HEM, calculate W d × -------------- = W c
hexane to approximately V 1000
the amount of silica gel to
the 100-mL mark. add based on the mg HEM
that was added to the where:
flask. Wd = result from step 12
3 × mg HEM in aliquot
---------------------------------------------------------- = V1000 = volume removed
100
for silica gel treatment
silica gel (g)
Wc = corrected SGT-HEM
result
Interferences
For samples that are known to contain extremely high levels of oil and grease, use a smaller
sample volume and correct for the volume difference to give the result as a 1-L sample.
High concentrations of particulates in the water sample can clog the SPE filter or retain high levels
of water, which can lower the extraction efficiency. Inorganic particulates are easier to filter than
organic particulates. The following techniques may help to filter samples containing high levels of
particulates:
• Decanting—allow the sample to settle and pour the top portion into the funnel first. Just before
dryness, add the rest of the sample. Remove any sediment from the bottle and add it to the
SPE filter. Use deionized water to rinse any sediment that remains in the bottle.
• Prefilters or prefilter fibers—place the prefilter or prefilter fibers into the coupler before the
funnel is attached.
• Drying agents—add magnesium sulfate to the SPE filter or to the prefilter to remove water that
may be trapped in the particulates.
• Filtration aids—add materials such as sodium chloride, sand, diatomaceous earth or glass
beads to help speed the complete filtration of samples that contain organic particulates.
This method is not applicable to materials that volatilize at temperatures below approximately
85 °C (185 °F). Petroleum fuels from gasoline through #2 fuel oil may be partially lost in the solvent
removal operation. Some crude oils and heavy fuel oils contain a significant percentage of
materials that are not soluble in n-hexane. Recoveries of these materials may be low.
Oil and Grease

Page 836
Oil and Grease

Collect 1 L (950–1050 mL) of sample in a wide-mouth glass bottle. Do not rinse the bottle with
sample before collection. The sample must be at room temperature before the test.
Sample volume
Complete the steps that follow to measure the sample volume.
6. Use a laboratory pen to mark on the sample bottle the liquid level of the sample.
7. When the test is complete, fill the bottle to this mark with tap water.
8. Pour the tap water into a 1-L graduated cylinder and write down the volume. Use this volume
for the sample volume in the final step of the test procedure for HEM or SGT-HEM.
Acidification
The sample must be acidified with 1:1 hydrochloric acid (HCl) solution to a pH of 2 or less before
the test is started. To find the volume of HCl to use, collect a separate aliquot, add HCl until the pH
is less than 2 and add this volume of acid to each sample bottle before collection. Do not dip pH
paper, a pH electrode, a glass rod or other materials into the sample because oil and grease in the
sample may adhere to these items.
If the test can not be started for more than a few hours after collection, cool and store the sample
at 0–4 °C (32–39 °F). Acidified samples can be stored for 28 days.
Accuracy check
For USEPA reporting, each laboratory must first measure an acceptable MDL (Minimum Detection
Limit) and IPR (Initial Precision and Recovery) before samples can be measured. Before
measuring the MDL or IPR, run laboratory reagent water blanks to eliminate interferences.
Standard solution preparation
Required items:
• Stearic acid, 98% minimum
• Hexadecane, 98% minimum
• Acetone for Organic Residue Analysis, residue less than 1 mg/L
• 10.0-mL Class A volumetric pipet
• 15.0-mL Class A volumetric pipet
1. Put 200 (± 2) mg stearic acid and 200 (± 2) mg hexadecane into a 100-mL volumetric flask.
2. Add 75–85 mL of acetone and shake vigorously until all material has dissolved.
3. Allow to equilibrate to room temperature and fill to volume with acetone. Mix well. The
concentration of this stock solution is 4000 mg/L HEM (2000 mg/L SGT-HEM).
4. Use a volumetric pipet to dilute the stock solution for use in MDL, IPR or OPR measurements:
MDL standard solution:

a. Add 15 mL of the stock solution into a clean 100-mL volumetric flask. Dilute to the mark
with acetone and mix well.
b. Pipet 10 mL for HEM (or 20 mL for SGT-HEM) into a 1-L volumetric flask. Dilute to the
mark with deionized water at pH < 2 and mix well. The concentration of this standard
solution is 6 mg/L HEM (or 6 mg/L SGT-HEM).
Oil and Grease

Page 837
Oil and Grease
IPR or OPR standard solution:

Add 10 mL of the stock solution into 1 liter of deionized water and mix well. The concentration
of this solution is 40 mg/L HEM (20 mg/L SGT-HEM).
Note: To verify the concentration of the IPR/OPR standard, use a pipet to add 10 mL of the standard
solution into a pre-weighed flask or dish. Allow the acetone to evaporate in a fume hood. When dry,
weigh the flask or dish. The weight of the residue should be 40 (±1) mg.
EPA requirements for MDL and IPR

MDL: measure seven replicates of a 6 mg/L standard solution, find the standard deviation and
multiply the standard deviation by 3.143 (Student’s t test). The acceptable limits are:
• HEM: ≤ 1.4 mg/L
• SGT-HEM: ≤ 1.6 mg/L
IPR: follow the procedure for HEM and SGT-HEM (if necessary) four separate times using a
40 mg/L HEM (20 mg/L SGT-HEM) standard solution. Report the average percent recovery (x)
and the standard deviation for both HEM and SGT-HEM. The acceptable limits are:
• HEM: Precision(s) ≤ 11%; Recovery (x) 83–101%
• SGT-HEM: Precision(s) ≤ 28%; Recovery (x) 83–116%
A low result can be an indication of loss during quantitative transfers or loss during heating. A high
result can be an indication of incomplete drying or contamination. Run a blank before running
samples. If the blank result is more than 5 mg, identify and remove the source of contamination.
After acceptable values are obtained for the MDL and IPR, keep records for USEPA verification.
Oil and grease reporting to USEPA

Include the following data with the HEM and/or SGT-HEM results for each set of up to 20 samples
per discharge source.
1. Blank value: must be less than 5.0 mg/L for HEM and SGT-HEM.
Note: It may be necessary to use a standard that matches the regulatory concentration limit, is 1 to 5
times higher than the concentration of the sample (B) or is at the concentration of the Ongoing
Precision and Recovery, whichever is highest. Divide the concentration of the spike (T) by 2 for
SGT-HEM if using the 40 mg/L HEM (20 mg/L SGT-HEM) standard.
2. OPR (Ongoing Precision and Recovery): add 10 mL of the 40 mg/L HEM (20 mg/L SGT-
HEM) standard to a 1-liter sample and run the test. The acceptable limits for recovery are:
• HEM: 78–114%
• SGT-HEM: 64–132%
3. MS and MSD (matrix spike and matrix spike duplicate): measure the HEM and SGT-HEM
concentration of the sample (B). Spike two 1-L samples with 10 mL of the 40 mg/L HEM
(20 mg/L SGT-HEM) standard and measure the concentration after spiking (A).
Calculate the Percent Recovery (P) as follows:

100 × ( A – B )
P HEM (40 mg/L) = -----------------------------------
T
100 × ( A – B )
P SGT – HEM = -----------------------------------
T⁄2
where:
A = concentration of the unspiked sample
B = concentration of the spiked sample
T = concentration of the spike solution
Oil and Grease

Page 838
Oil and Grease
If the recovery for HEM and SGT-HEM is within the acceptable limits for OPR (step 2), then
calculate the Relative Percent Difference (RPD).
Conc MS – Conc MSD
RPD = ---------------------------------------------------- × 200
Conc MS + Conc MSD
If the RPD for HEM is ≤ 18 and for SGT-HEM ≤ 34, then continue with step 4. If the recovery is
lower than the RPD, an interference may be present. Identify and correct the interference,
then repeat MS and MSD.
After every five MS/MSD tests, calculate the average percent recovery (Pa) and standard
deviation of the percent recovery (sp). Record these numbers as Pa ±2sp. Update the
accuracy assessment on a regular basis (e.g., after five to ten new accuracy measurments).
4. Balance calibration: measure a 2 mg and a 1000 mg class “S” weight on the analytical
balance before and after each analytical batch. If the values are not within 10% of the actual
weight, recalibrate the balance.
Summary of method
Oil and Grease & Total Petroleum Hydrocarbons (TPH) include any material that may be
recovered as a substance that is soluble in the n-hexane extractant. These include substances
such as relatively non-volatile hydrocarbons, vegetable oils, animal fats, waxes, soaps, greases
and related materials. When measuring oil and grease (HEM) gravimetrically, the substances are
extracted from the sample with n-hexane, then the n-hexane is evaporated. The residue left is
weighed to determine the concentration of oil and grease materials in mg/L.
When measuring Total Petroleum Hydrocarbons (SGT-HEM) gravimetrically, the substances are
extracted from the sample with n-hexane, then mixed with silica gel to absorb non-TPH
components. Then the n-hexane is evaporated. Like the HEM, the residue left is weighed to
determine the concentration of total petroleum hydrocarbons.
Definition of HEM and SGT-HEM
Oil and grease is the conventional term used to define pollutants of this nature. The newer term
n-Hexane Extractable Materials (HEM) indicates this method may be applied to materials other
than oils and greases.
Likewise, the term Total Petroleum Hydrocarbons (TPH) was traditionally used to classify aliphatic
hydrocarbon materials. The newer term Silica Gel Treated n-Hexane Extractable Material
(SGT-HEM), indicates that this method may be applied to materials other than aliphatic petroleum
hydrocarbons that are not adsorbed by silica gel.
Required reagents
Hydrochloric Acid Standard Solution, 1:1 (6 N) 6 mL 500 mL 88449
Hexane, for Organic Residue Analysis 100–200 mL 1L 2510253
Methanol, ACS grade 10 mL 500 mL 1446449
Silica Gel, 100–200 mesh (for SGT-HEM) 1–30 g 500 g 2665034
Oil and Grease

Page 839
Oil and Grease
Required apparatus
Balance, Analytical, 115 VAC 60 Hz. 1 each 2936801
Bottle, wash, 500-mL Teflon, FEP 1 each 2505100
Bottle, wash, 500-mL 1 3/pkg 2927204
Bottle, wide-mouth, 1-L 1 each 2951401
Clamp, swivel 2 each 2503400
Cylinder, graduated, 1-L 1 each 108153
Desiccator 1 each 2088800
Desiccator Plate 1 each 1428400
Flask, Erlenmeyer, 125-mL (for SGT-HEM) 1 each 50543
Hot Plate (Thermolyne), 120 VAC, 50 Hz 1 each 2344100
Marker, laboratory 1 each 2092000
Oven, drying, 120 VAC, 50 Hz 1 each 1428900
Pump, vacuum, 27 in. Hg, 1.3 CFM 1 each 2824800
SPE Consumables Kit 1 24/pkg 2943800
SPE Starter Kit, EPA Method 1664A 1 each 2943231
SPE Solvent Recovery Kit 1 each 2514300
Stirrer, magnetic, 120 VAC 1 each 2343600
Stir Bar, 22.2 x 7.9 mm 1 each 2095350
Tongs, crucible, 9-inch 1 each 56900
Hexadecane, 99% 100 mL 2664842
Stearic acid 500 g 2664934

Acetone, for Organic Residue Analysis 1L 2510153
Flask, volumetric, Class A, 1-L each 1457453
pH Paper, 0–14 pH units 100/pkg 2601300
Silica Gel with indicator (for desiccator) 454 g 1426901

Organic Carbon, Total HR, 10128
Organic Carbon, Total DOC316.53.01095
Direct Method1 Method 10128

HR (100 to 700 mg/L C)
Scope and Application: For wastewater and industrial water
1 U.S. Patent 6,368,870
Test preparation

DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
A reagent blank is required for each series of samples.
To test for TOC levels below 100 mg/L C, use Method 10173 or Method 10129.
Total Organic Carbon Direct Method High Range Test ’N Tube™ Reagent Set 1
DRB200 Reactor 1
pH Paper 1
Magnetic Stirrer 1
Pipet, TenSette, 1.0 to 10.0 mL plus tips 1
Stir Bar, magnetic 1
Test Tube Rack 1
Organic Carbon, Total

Page 841
Water, organic-free 0.3 mL
Wipes, disposable 1
Direct method
1. Turn on the DRB200 2. Use a graduated 3. Add 0.4 mL of Buffer 4. Place the flask on a
reactor. Select the TOC cylinder to add 10 mL of Solution, pH 2.0. Use pH stir plate and stir at a
program. sample to a 50-mL paper to make sure the moderate speed for
Erlenmeyer flask that sample pH is 2. 10 minutes.
contains a stir bar.
5. Label two High Range 6. Use a funnel to add 7. Use a TenSette® Pipet 8. Rinse two blue MR/HR
Acid Digestion vials the contents of one TOC to add 0.3 mL of Indicator Ampules with
sample and reagent blank. Persulfate Powder Pillow organic-free water to the deionized water and wipe
to each Acid Digestion vial reagent blank vial and them with a soft, lint-free
(colorless liquid). 0.3 mL of prepared wipe. Do not touch the
sample to the sample vial. ampules sides after
Swirl to mix. wiping. Pick them up by
the top.

Page 842
Direct method (continued)
Stored Programs
426 Organic Carbon HR
Start
9. Lower one unopened 10. Cap the vial 11. Carefully remove the 12. Select the test.
ampule into each Acid assemblies tightly, insert vial assemblies from the Insert a light shield or an
Digestion vial. When the them in the reactor and reactor. Place them in a adapter if required (see
score mark on the ampule close the lid for 2 hours at test tube rack. Instrument-specific
is level with the top of the 103–105 °C. Allow the vials to cool for information).
Acid Digestion vial, break one hour for accurate
the top off the ampule and results.
allow it to drop into the
Acid Digestion vial. The liquid in the reagent
blank vial should be dark
Do not invert or tilt the blue.
vial after inserting the
ampule.
Zero
13. Wipe the reagent 14. 15. ZERO the instrument. 16. Wipe the sample vial
blank with a damp towel, The display will show: assembly with a damp
followed by a dry one, to towel, followed by a dry
remove fingerprints or 0 mg/L C one, to remove fingerprints
other marks. or other marks.
Read
17. Insert the sample vial 18. READ the results in

assembly in the 16-mm mg/L C.
round cell.

Page 843
Interferences
The Interfering substances table lists substances that have been tested for interference and found
not to interfere up to the levels indicated.
If the sample contains greater than 1000 mg/L CaCO3 alkalinity, lower the sample pH to less than
7 before testing by adding Sulfuric Acid Solution.
Most sample turbidity is either dissolved during the digestion stage or settled during the cooling
period. Sample turbidities up to 50 NTU have been tested without interference.

Aluminum 10 mg/L Al
Ammonia Nitrogen 1000 mg/L as N
ASTM Wastewater No effect
Bromide 500 mg/L Br–
Bromine 25 mg/L Br2
Chlorine 10 mg/L Cl2
Copper 10 mg/L Cu
Cyanide 10 mg/L CN–
Iodide 50 mg/L I–
Manganese (VII) 1 mg/L Mn
Monochloramine 14 mg/L NH2Cl as Cl2
Nitrite 500 mg/L NO2–

Ozone 2 mg/L O3
Phosphate 3390 mg/L PO43–


Sulfide 20 mg/L S2–
Zinc 5 mg/L Zn

• Collect samples in clean glass bottles.
• Rinse the sample bottle several times with the sample to be collected.
• Fill the bottle completely full before capping.
• Test samples as soon as possible.

Page 844
• Acid preservation is not recommended.

• Homogenize samples containing solids to assure representative samples.
Accuracy check
• TOC Standard Ampule, 1000 mg/L C
• Organic-free Reagent Water
• 1-L Class A volumetric flask
• Acid Digestion Vials (3)
• TOC Persulfate Powder Pillow (1)
• 50 mL Class A Volumentric flask
• 15 mL Volumetric Pipet and pipet filler

ADDITIONS.
4. Prepare a 300 mg/L C standard solution as follows:
a. Transfer 15.00 mL of the 1000-mg/L total organic carbon standard solution to a 50-mL
volumetric flask.
b. Dilute to the mark with Organic-free Reagent water. Insert the stopper and mix thoroughly.
Prepare this solution daily.
5. Use the TenSette Pipet to prepare spiked samples: add 0.1 mL, 0.2 mL and 0.3 mL of the
prepared 300-mg/L standard to three Acid Digestion Vials.
6. Add the contents of one TOC Persulfate Powder Pillow to each vial.
7. Add 0.3 mL of sample to each vial. Swirl to mix.
8. Continue the test starting at step 8 of the Direct method test procedure for each of the spiked
instrument.



Page 845
• 15 mL Volumetric pipet and pipet filler
a. Transfer 15.00 mL of the prepared 1000-mg/L organic carbon stock standard solution to a
50-mL volumetric flask.
2. Use this solution in place of the sample. Follow the Direct method test procedure.
Method performance
To test for TOC levels below 100 mg/L C, use Method 10173 or Method 10129.
426 350 mg/L C 337–363 mg/L C 4 mg/L C
Summary of method
The total organic carbon (TOC) is determined by first sparging the sample under slightly acidic
conditions to remove the inorganic carbon. In the outside vial organic carbon in the sample is
digested by persulfate and acid to form carbon dioxide. During digestion, the carbon dioxide
diffuses into a pH indicator reagent in the inner ampule. The absorption of carbon dioxide into the
indicator forms carbonic acid. Carbonic acid changes the pH of the indicator solution which, in
turn, changes the color. The amount of color change is related to the original amount of carbon
present in the sample. Test results are measured at 598 and 430 nm.
Required reagents
Total Organic Carbon Direct Method — 50 vials 2760445
High Range Test ’N Tube™ Reagent Set, includes:
Acid Digestion Solution Vials, High Range TOC1 1 50/pkg —
Buffer Solution, Sulfate1,2 0.4 mL 25 mL —
Indicator Ampules, MR/HR TOC1 1 10/pkg —
TOC Persulfate Powder Pillows1 1 50/pkg —
pH Paper 1 5/pkg 39133
Water, Organic-free 0.3 mL 500 mL 2641549
1 Not sold separately.
2 See alternate size below
Required apparatus

Page 846
Required apparatus
OR
Magnetic Stirrer 1 each 2881200
Wipes, Disposable 1 280/pkg 2097000
TOC Standard Solution Ampule (KHP Standard, 1000-mg/L C) 5/pkg 2791505

Buffer Solution, Sulfate, pH 2.0 500 mL 45249
Flask, volumetric class A, 50 mL each 1457441
Pipet, Volumetric, 15 mL each 1451539
Potassium Acid Phthalate 500 g 31534
Sulfuric Acid Solution, 5.25 N 100 mL 244932

Page 847

Organic Carbon, Total LR, 10129

LR (0.3 to 20.0 mg/L C)
Scope and Application: For water, drinking water and wastewater
1 U.S. Patent 6,368,870
Test preparation


DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
To test for higher ranges of TOC use Method 100173 or method 10128.
Total Organic Carbon Direct Method Low Range Test ’N Tube™ Reagent Set 1
DRB200 Reactor 1
pH Paper 1
Magnetic Stirrer 1

Page 849
Test Tube Rack 1
Wipes, disposable, Kimwipes® 1
Direct method
5. Label two Low Range 6. Use a funnel to add 7. Use a TenSette® Pipet 8. Rinse two blue Low
Acid Digestion vials the contents of one TOC to add 3.0 mL of Range Indicator Ampules
sample and reagent blank. Persulfate Powder Pillow organic-free water to the with deionized water and
to each Acid Digestion vial reagent blank vial and wipe them with a soft, lint-
(colorless liquid). 3.0 mL of prepared free wipe.
sample to the sample vial. Do not touch the ampule
Swirl to mix. sides after wiping. Pick
Note: The organic free water
them up by the top.
must contain less than 0.05
mg/L carbon.

Page 850
Stored Programs
427 Organic Carbon LR
Start
ampule into each Acid assemblies tightly, insert vial assemblies from the Insert an adapter if
Digestion vial. When the them in the reactor and reactor. Place them in a required (see Instrument-
score mark on the ampule close the lid for 2 hours at test tube rack. specific information).
is level with the top of the 103–105 °C. Allow the vials to cool for
Acid Digestion vial, snap one hour for accurate
ampule.
Zero
13. Wipe the reagent 14. Insert the reagent 15. ZERO the instrument. 16. Wipe the sample vial
blank with a damp towel, blank vial assembly in the The display will show: assembly with a damp
followed by a dry one, to 16-mm round cell holder. towel, followed by a dry
remove fingerprints or 0.0 mg/L C one, to remove fingerprints
Read

round cell.

Page 851
Interferences
The Interfering substances table lists substances that have been tested for interference and found
If the sample contains greater than 600 mg/L CaCO3 alkalinity, lower the sample pH to less than 7
before testing by adding Sulfuric Acid Solution.

Aluminum 10 mg/L Al
Bromine 25 mg/L Br2
Copper 10 mg/L Cu
Iodide 50 mg/L I–

Ozone 2 mg/L O3


Zinc 5 mg/L Zn

Page 852

Accuracy check

ADDITIONS.
4. Prepare a 150-g/L C standard:
a. Transfer 15.00 mL of 1000-mg/L TOC standard solution to a 100-mL Class A volumetric

flask.
b. Dilute to volume with organic-free water. Mix.
prepared standard to three Acid Digestion Vials.
instrument.


Page 853
• TOC Standard Ampule 1000 mg/L C

1. Prepare a 10.0 mg/L C standard solution as follows:
(1 liter) volumetric flask.
Reagent blanks
Water used for the reagent blank must contain less than 0.05 mg/L carbon. If the organic-free
water container is left open for extended periods, the water may absorb carbon dioxide (CO2) from
the atmosphere. To remove the dissolved CO2 from the organic-free water, it is necessary to acid-
sparge it (see steps 2–4 of the procedure).
Generally, water stored in plastic containers is not suitable for low-range TOC blanks. Water stored
in plastic may leach organic compounds from the container walls. The leached organic
compounds usually cannot be removed by acid sparging.
Method performance
427 10.0 mg/L C 9.1–10.9 mg/L C 0.2 mg/L C
Summary of method
conditions to remove the inorganic carbon. In the outside vial, organic carbon in the sample is

Page 854
Required reagents
Reagent Set, Total Organic Carbon Direct Method — 50 vials 2760345
Low Range Test ‘N Tube™, includes:
Buffer Solution, Sulfate1,2 0.4 mL 25 mL 45233
Indicator Ampule, Low Range TOC1 1 10/pkg —
Required apparatus
OR
TOC Standard Solution Ampule (KHP Standard, 1000-mg/L C) 5/pkg 2791505

Buffer Solution, Sulfate pH 2.0 500 mL 45249

Page 855

Pipet filler each 1465100
Sulfuric Acid Solution, 5.25 N 100 mL 244932

Organic Carbon, Total MR, 10173

MR (15 to 150 mg/L C)
Scope and Application: For water, drinking water and wastewater
1 U.S. Patent 6,368,870
Test preparation

DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
To test for higher ranges of TOC use Method 10128. To test lower ranges of TOC use method 10129.
Total Organic Carbon Direct Method Mid Range Test ’N Tube™ Reagent Set 1
DRB200 Reactor 1
pH Paper 1
Magnetic Stirrer 1

Page 857
Test Tube Rack 1
Wipes, disposable 1
Direct method
5. Label two Mid Range 6. Use a funnel to add 7. Use a TenSette® Pipet 8. Rinse two blue MR/HR
Acid Digestion vials the contents of one TOC to add 1.0 mL of Indicator Ampules with
sample and reagent blank. Persulfate Powder Pillow organic-free water to the deionized water and wipe
to each Acid Digestion vial reagent blank vial and them with a soft, lint-free
(colorless liquid). 1.0 mL of prepared wipe.
sample to the sample vial. Do not touch the ampule
Swirl to mix. sides after wiping. Pick
them up by the top.

Page 858
Stored Programs
425 Organic Carbon MR
Start
ampule into each Acid assemblies tightly, insert vial assemblies from the Insert a light shield or an
Digestion vial. When the them in the reactor and reactor. Place them in a adapter if required (see
score mark on the ampule close the lid for 2 hours at test tube rack. Instrument-specific
is level with the top of the 103–105 °C. Allow the vials to cool for information).
Acid Digestion vial, snap one hour for accurate
ampule.
Zero
13. Wipe the reagent 14. 15. ZERO the instrument. 16. Wipe the sample vial
blank with a damp towel, The display will show assembly with a damp
followed by a dry one, to towel, followed by a dry
remove fingerprints or 0 mg/L C. one, to remove fingerprints
Read

round cell.

Page 859
Interferences
The Interfering substances table lists substance that have been tested for interference and found
If the sample contains greater than 1000 mg/L CaCO3 alkalinity, lower the sample pH to less than
7 before testing by adding Sulfuric Acid Solution.

Aluminum 10 mg/L Al
Bromine 25 mg/L Br2
Copper 10 mg/L Cu
Iodide 50 mg/L I–

Ozone 2 mg/L O3


Zinc 5 mg/L Zn


Page 860
Accuracy check
• 1000-mg/L C TOC Standard Ampule

ADDITIONS.
4. Prepare a 300-mg/L C standard:
a. Transfer 15.00 mL of 1000-mg/L TOC standard solution to a 50-mL Class A volumetric

flask.
b. Dilute to volume with organic-free water. Insert the stopper and mix thoroughly.
5. Use the TenSette Pipet to prepare spiked samples: add 0.1 mL, 0.2 mL and 0.3 mL of the 300-
mg/L standard to three Acid Digestion Vials.
instrument.

Page 861


• TOC standard Ampule, 1000 mg/L C

volumetric flask.
b. Dilute to volume with Organic-free Reagent water. Insert the stopper and mix thoroughly.
Method performance

Program Instrument Standard
425 DR 5000 70 mg/L C 68–72 mg/L C 1.2 mg/L C
Summary of method
conditions to remove the inorganic carbon. In the outside vial, organic carbon in the sample is
Required reagents
Reagent Set, Total Organic Carbon Direct Method — 50 vials 2815945
Mid Range Test ‘N Tube™, includes:
Buffer Solution, Sulfate1,2 0.4 mL 25 mL —
Indicator Ampules, MR/HR TOC1 1 10/pkg —

Page 862
Required reagents
Required apparatus
OR
TOC Standard Solution Ampule, 20 mL (KHP Standard, 1000-mg/L C) 5/pkg 2791505

Sulfuric Acid Solution, 5.25 N 100 mL-MDB 244932
Buffer solution, sulfate, pH 2.0 500 mL 45249

Page 863

Organic constituents, UV absorbing, 10054
UV Absorbing (UV-254) DOC316.53.01092
Direct Reading Method1 Method 10054

Scope and Application: To indicate the total concentration of UV-absorbing organic compounds in drinking water
and drinking water source waters.
1 Adapted from Standard Methods for the Examination of Water and Wastewater, Method 5910.
Test preparation

DR 6000 2624410 Align clear windows facing right/left —
DR 5000 2624410 Align clear windows facing the user Multi-cell Adapter, 10-mm faces user
The sample pH should be between 4 and 10. If not, see Interferences in this procedure. Samples used for SUVA calculations
must not be pH adjusted
Any non-plastic filter assembly can be used for this test. Use a 0.45-µm or glass fiber filter of nominal pore size (1–1.5 µm)
without organic binder. A 0.45-µm filter must be utilized if the results are to be used for SUVA calculations.
Handle the cell on the frosted sides only.
Use only Organic-Free Reagent Water to zero the instrument.
The Chromic Acid Cleaning Solution is regulated as a hazardous waste for chromium (D007) and corrosivity (D002) when
disposed per Federal RCRA. Refer to the current MSDS for safe handling and disposal instructions.
Organic-free Reagent Water varies
Filter Assembly 1
Stand, buret 1
Organic Constituents UV Absorbing (UV-254)

Page 865
Stored Programs
410 Organics, UV - 254
Start
1. Select the test. 2. Assemble the filter 3. Mount the apparatus 4. Prewash the filter
Insert an adapter if apparatus. Be sure to use into a support stand and assembly by pouring at
required (see Instrument- the white PTFE support place a clean glass beaker least 50 mL of Organic-
specific information). plate. Insert the filter with underneath. Free Reagent Water
the wrinkled surface through the filter. Discard
upward. the filtered water.
Pre-rinsing removes any
soluble impurities from the
filter.
Zero
5. Prepared Sample: 6. Blank Preparation: 7. Align the clear 8. ZERO the instrument.
Pour 50 mL of sample Rinse a clean 1-cm quartz windows with the light The display will show:
through the filter and cell several times with path. Insert the blank into
collect the filtered sample. Organic-Free Reagent the cell holder. 0.000 cm–1
Water. Fill the cell with 1-cm cell
Organic-Free Reagent Lamp Warm Up will be
Water. Wipe the cell walls indicated if the UV lamp
thoroughly. has not been previously
on. This may take 2–3
minutes.

Page 866
Organic Constituents (continued)
Read
9. Discard the contents 10. After rinsing, fill the 11. Align the clear 12. READ the results in
of the blank cell and rinse cell with filtered sample. windows with the light absorbance per
the cell several times with Wipe the cell walls to path. Insert the cell centimeter (cm–1).
filtered sample. remove fingerprints. containing the prepared
sample into the cell holder.
For optimum results, the cm–1 value should fall between 0.005 and 0.900. If the value is less than
0.005 absorbance using a 1-cm cell, use a 5-cm or 10-cm quartz cell.
To test with the 5-cm or 10-cm cell:
1. Press OPTIONS>MORE>CHEMICAL FORMS.
2. Press 5 CM or 10 CM. Press OK>RETURN.
3. The displayed results (in absorbance per centimeter) will be corrected for the 5-cm or 10-cm
cell pathlength selected. If cm–1 results are greater than 0.900, accurately dilute the sample
with Organic-Free Reagent Water. Correct the test result by the appropriate dilution factor.
Interferences

Add either 1 N Sodium Hydroxide or 1 N Sulfuric Acid to the sample to adjust sample
Sample pH outside 4–10
between pH 4-10.
UV-absorbing inorganics
(bromide, ferrous iron, nitrate, Follow the UV scanning procedure below.
nitrites)
UV-absorbing Oxidants and
reductants (chloramines,
Follow the UV scanning procedure below.
chlorates, chlorites, ozone,
thiosulfates)
To determine the presence of interferences, a scan of the filtered sample versus Organic-Free
Reagent Water is recommended on a regular basis:
1. From the main menu, press WAVELENGTH SCAN>OPTIONS>λ.
2. Press 200>OK.
3. Press 400>OK.
4. Press 1 NM>OK.

Page 867
5. Insert the cell containing the Organic-Free Reagent Water (the blank) into the cell
compartment.
6. Press ZERO. The baseline scan from 200 to 400 nm will begin.
Note: Lamp Warm Up will be indicated if the UV lamp has not been previously on. This may take
2–3 minutes.
7. After the baseline scan is recorded, insert the cell containing the filtered sample into the cell
compartment. Press READ to scan the sample.
If the sample scan shows relatively sharp peaks, interferences may be present. Generally, natural
organic matter will show a relatively featureless curve in the UV region with increasing absorption
as the wavelength decreases. If the sharp peaks are indicated, an alternate wavelength should be
selected and reported.

• Collect samples in cleaned glass containers. Do not use plastic containers.
Cell cleaning
New or dirty cells should be soaked with Chromic Acid Cleaning Solution to remove trace organic
contamination.
1. Allow to soak overnight or up to 12 hours.
2. After soaking, rinse with at least 10 volumes of Organic-Free Reagent Water.
Treatment of cells with chromic acid is required only occasionally if cells are rinsed with Organic-
Free Reagent Water after use.
Method performance
Standard: There is no primary standard or calibration for the UV-254 method. Using a Potassium
Acid Phthalate solution equivalent to 30-mg/L as carbon, the following reproducibility data was
obtained using one instrument. Refer to Standard Methods for the standard preparation.
Program Precision—95% Confidence Limits of Distribution

410 0.431 - 0.433 cm-1
Summary of method
Filtered sample is measured at 254 nm against organic-free water as a indicator of organic
constituents in the sample water. Results are automatically reported in absorbance per centimeter
(cm–1). The results can be used in calculating Specific Ultraviolet Absorbance (SUVA).
Estimated detection limit
Because this test is a non-specific measurement for organic constituents, there is no estimated
detection limit for program 410.

Page 868
Required reagents
Organic-Free Reagent Water varies 500 mL 2641549
Required apparatus
Buret Stand each 32900
Clamp Holder each 32600
Clamp, 3-Prong each 42200
Filter Funnel Assembly, 7-cm each 2164100
Filter Plate, PTFE, for 21641-00 each 2164200
Filter, glass fiber, 70-mm 100/pkg 253053
Sample Cell, quartz, 1-cm (10 mm) each 2624410
Sulfuric Acid, 1 N 100 mL MDB 127032
Optional reagents
Chromic Acid Cleaning Solution 500 mL 123349
Sodium Hydroxide Standard Solution, 1.00 N 100 mL MDB1 104532
1 Larger sizes are available.
Optional apparatus
Cell Holder for 10-cm (100 mm) sample cells (DR 5000 only) each LZY421
Filter, membrane, 47-mm; 0.45-microns, hydrophilic, polyethersulfone SUVA each 2894700
Filter Holder, glass for vacuum filtration (SUVA) each 234000
Flask, filtering, glass, 1000-mL (SUVA) each 54653
Tubing, Latex rubber (SUVA) 12 ft 56019
Sample Cell, quartz, 1-cm (10 mm), matched pair pair 4822800
Standard Methods Book, most current edition 1 2270800
Aspirator (SUVA) 1 213100

Page 869
Oxygen Demand, Biochemical, 8043
Oxygen Demand, Biochemical DOC316.53.01200
Dilution Method1 Method 8043

1 Adapted from Standard Methods for the Examination of Water and Wastewater and from Klein, R.L.; Gibbs, C. Journal of Water Pollution
Control Federation, 1979, 51(9), 2257.
Test preparation
The BOD test is a 5-day test. Follow all steps carefully to make sure that the test does not have to be repeated.
The dilution water for this test must not have an oxygen demand or any toxins. When incubated for 5 days at 20 °C, the
dissolved oxygen concentration in the dilution water must not change by more than 0.2 mg/L.
Carbonaceous BOD (CBOD) can be determined by the addition of nitrification inhibitor. A test for CBOD is recommended for
biologically treated effluents, samples seeded with biologically treated effluents and river water.
The Troubleshooting—Graphical calculation method provides an alternate system for calculating results and is a convenient
tool for troubleshooting problems in BOD measurements. The graphical calculation method is not approved for regulatory
reporting.
BOD bottles, 300-mL, glass, with glass stoppers and plastic caps 6
Dilution water containing nutrient buffer and seed (see Dilution water preparation) varies
Nitrification inhibitor (for CBOD only) 1 bottle
Pipet, serological 1
Incubator 1
Oxygen Demand, Biochemical

Page 871
Dilution method
1. Prepare the dilution 2. Select the sample 3. Stir the sample gently 4. Fill an additional BOD
water using a BOD volumes. See Sample size with the pipet. Use the bottle with dilution water
Nutrient Buffer Pillow. See selection. pipet to add the minimum only. This will be the
Dilution water preparation. Note: If the minimum sample sample volume to the first dilution water blank.
volume is 3 mL or more, BOD bottle.
determine the dissolved
oxygen in the undiluted Add the remaining four
sample; this determination sample volumes to four
can be omitted when more BOD bottles. Mark
analysing sewage and settled the bottles and record the
effluents known to have a contents of each bottle.
dissolved oxygen content
near 0 mg/L.
When analyzing
disinfected samples or
industrial effluents, refer to
Interferences.
5. If the test is for CBOD, 6. Fill each bottle to just 7. Stopper the bottles 8. Measure the initial
add two portions of below the lip with dilution carefully to prevent air dissolved oxygen
Nitrification Inhibitor water. Allow the dilution bubbles from becoming concentration in each
(approximately 0.16 g) to water to flow down the trapped. Tightly twist the bottle. Use a probe and
each bottle. sides of the bottle to stopper into place. Press meter or titration. If a
The oxidation of nitrogen prevent air bubbles from down on the stopper and titration is used, two sets
compounds will be becoming trapped in the invert the bottles several of BOD bottles must be
prevented. Report results bottle. times to mix. prepared.
as CBOD. Be sure to measure the
DO of the dilution water
blank.

Page 872
Dilution method
9. Stopper the bottles 10. Place a plastic cap 11. After five days, 12. Calculate the BOD
carefully to prevent air over the lip of each bottle. measure the remaining value (see Calculation
bubbles from becoming Put the bottles in an dissolved oxygen Methods—Standard
trapped. Add dilution water incubator at 20 (±1) °C. concentration in each Methods).
to the lip of each BOD Incubate for five days. bottle.
bottle to make a water At least 1.0 mg/L DO
seal. should be left in each BOD
bottle.
Dilution water preparation

The dilution water must be prepared very carefully to make sure that no source of oxygen demand
or toxins are added. The water that is used to prepare the dilution water must be of very high
quality. The water must not have any organic compounds or any toxic compounds such as
chlorine, copper and mercury.
Use the following guidelines to make sure the dilution water is of high quality.
Guidelines
• Use distilled water from an alkaline permanganate distillation for the best results.
• Do not use deionized water from ion exchange columns. The resins in the cartridges
(especially new cartridges) will occasionally release organic materials that have an oxygen
demand. In addition, bacteria can grow on the columns and contaminate the dilution water.
• Store the distilled water in clean jugs in an incubator at 20 °C. Fill containers till about ¾ full
and shake the jugs to saturate the water with air, or cap the jugs loosely and store for 24 hours
or more, to allow dissolution of oxygen.
• A small aquarium pump or air compressor can be used to saturate the water with air. Make
sure that the air is filtered and that the filter does not grow bacteria. Clean the apparatus
before and after use.
• Add the nutrients and seed (if necessary) to the distilled water immediately before the test.
• The dissolved oxygen concentration in the dilution water must not change by more than
0.2 mg/L when incubated for 5 days at 20 °C.
Procedure
1. Prepare and store the distilled water at 20 °C (see Guidelines).
2. Select a BOD nutrient buffer pillow from the BOD nutrient buffer pillows table.
3. Tap the pillow on a hard surface then shake the pillow to mix the contents.

Page 873
4. Add the contents of the pillow to the distilled water in a jug with ample headspace above the
water. Cap the jug and shake vigorously for one minute to dissolve the nutrients and to
saturate the water with air.
5. If the sample is known to be low in bacteria, for example industrial waste or sewage that has
been disinfected, add 3 mL of bacterial seed to each liter of the dilution water. Use raw
sewage for the bacterial seed. Allow the sewage to stand undisturbed at 20 °C for 24 to
36 hours before use. Pipet from the upper portion of the sewage. Make sure to measure the
BOD of the seed so that it can be subtracted from the BOD of the sample. A seed that has a
BOD of 200 mg/L (a typical range for domestic sewage) will typically deplete at least 0.6 mg/L
DO, when added at a rate of 3 mL/L of dilution water. If insufficient oxygen depletion occurs,
increase the quantity of the seed.
Table 268 BOD nutrient buffer pillows
Volume of dilution water to prepare BOD nutrient buffer pillow catalog no.
300 mL (add pillow to each BOD bottle) 1416066
3 liters 1486166
4 liters 2436466
6 liters 1486266
19 liters 1486398
Note: To prepare dilution water by the conventional method, pipet 1 mL of each of the following solutions per
liter of distilled water at 20 °C: Calcium Chloride Solution, Ferric Chloride Solution, Magnesium Sulfate
Solution, and Phosphate Buffer Solution. Cap the bottle and shake vigorously for one minute. The
Phosphate Buffer Solution should be refrigerated to decrease the rate of biological growth. Use care with
all solutions to avoid contamination.
Sample size selection

Make an estimation of the sample volumes that are necessary for the test. At least 2.0 mg/L of
dissolved oxygen (DO) should be consumed during the test and at least 1.0 mg/L DO should be
left in the BOD bottle.
Samples such as raw sewage will have a high BOD. Small sample volumes must be used
because large samples will consume all of the oxygen. Samples with a low BOD must use larger
sample volumes to make sure that enough oxygen is consumed to give accurate results.
The elevation of the laboratory changes the amount of oxygen that can dissolve in water (see
Oxygen saturation values at various altitudes (20 °C)). At higher elevations, the amount of oxygen
that can dissolve in water decreases, so less oxygen is available to microorganisms.
Procedure
1. Refer to the Minimum sample volume table to select the minimum sample volume. For
example, if a sewage sample is estimated to contain 300 mg/L BOD, the minimum sample
volume is 2 mL. For sewage effluent with an estimated BOD of 40 mg/L, the minimum sample
volume is 15 mL.
2. Refer to the Maximum sample volume table to select the maximum sample volume. At 1000
feet, with an estimated BOD of 300 mg/L, the largest sample volume is 8 mL. For a BOD of 40
mg/L the maximum volume is 60 mL (also at 1000 feet).
3. Select two or more other sample volumes between the minimum and maximum volumes so
that there are four or five sample volumes total.

Page 874
Table 269 Minimum sample volume

Sample type Estimated BOD (mg/L) Minimum sample volume (mL)
Strong trade waste 600 1
Raw and settled sewage 300 2
200 3
150 4
120 5
100 6
75 8
60 10
Oxidized effluents 50 12
40 15
30 20
20 30
10 60
Polluted river waters 6 100
4 200
2 300
Table 270 Maximum sample volume1

BOD at sea level BOD at 1000 ft BOD at 5000 ft Maximum sample volume (mL)
615 595 508 4
492 476 406 5
410 397 339 6
304 294 251 8
246 238 203 10
205 198 169 12
164 158 135 15
123 119 101 20
82 79 68 30
41 40 34 60
25 24 21 100
12 12 10 200
8 8 7 300
1 Samples with higher concentrations should be pre-diluted, per Standard Methods.

Page 875
Table 271 Oxygen saturation values at various altitudes (20 °C)

Average Pressure in mBar at this Oxygen value (mg/L) in water
Altitude (ft)
altitude saturated with air
Sea level (0) 1013 9.09
1000 977 8.76
2000 942 8.44
3000 908 8.13
4000 875 7.82
5000 843 7.53
6000 812 7.24
Calculation Methods—Standard Methods

Use the Standard Methods calculation when the results must be reported to a regulatory agency.
When dilution water is not seeded:
D1 – D2
BOD 5, mg/L = -------------------
-
P
When dilution water is seeded:
( D 1 – D 2 ) – ( B 1 – B 2 )f
BOD 5, mg/L = ---------------------------------------------------------
-
P
where:
BOD5 = BOD value from the 5-day test
D1 = DO of diluted sample immediately after preparation, in mg/L
D2 = DO of diluted sample after 5 day incubation at 20 °C, in mg/L
P = Decimal volumetric fraction of sample used
B1 = DO of seed control before incubation, in mg/L
B2 = DO of seed control after incubation, in mg/L
f = ratio of seed in diluted sample to seed in seed control =
(% seed in diluted sample)/(% seed in seed control) OR
If seed material is added directly to sample or to seed control bottles:
f = (volume of seed in diluted sample)/(volume of seed in seed control)
Report results as CBOD5 if nitrification inhibitor was added.
Averaged results are acceptable if more than one sample dilution meets all of the following criteria:
• The remaining DO is at least 1 mg/L
• The final DO value is at least 2 mg/L lower than the initial DO value
• There is no evidence of toxicity at higher sample concentrations
• There are no obvious anomalies

Page 876
Interferences
Many chlorinated and industrial effluents require special handling to ensure reliable BOD results.
Usually, careful experimentation with the particular sample will indicate what modifications should
be made to the test procedure.
Toxins in the sample will adversely affect any microorganisms present and result in lower BODs.
To eliminate small amounts of residual chlorine, allow the sample to stand for one to two hours
at room temperature. For larger quantities, determine the amount of sodium thiosulfate to add to
the sample as follows:
c. Measure 100 mL of sample into a 250-mL Erlenmeyer flask. Using a 10-mL serological
pipet and a pipet filler, add 10 mL of 0.020 N Sulfuric Acid Standard Solution and 10 mL of
Potassium Iodide Solution, 100-g/L, to the flask.
d. Add three full droppers of Starch Indicator Solution and swirl to mix.
e. Fill a 25-mL buret with 0.025 N Sodium Thiosulfate Standard Solution and titrate the
sample from dark blue to colorless.
f. Calculate the amount of 0.025 N Sodium Thiosulfate Standard Solution to add to the
sample:
mL titrant used x volume of remaining sample
mL 0.025 N sodium thiosulfate required = -------------------------------------------------------------------------------------------------------------------------
100
g. Add the required amount of 0.025 N Sodium Thiosulfate Standard Solution to the sample.
Mix thoroughly. Wait 10 to 20 minutes before running the BOD test.
To eliminate the effect of phenols, heavy metals or cyanide, dilute the sample with high quality
distilled water. Alternately, the seed used in the dilution water may be acclimatized to tolerate such
materials. Acclimatize seed as follows:
a. Fill a one-gallon stainless steel or plastic container with domestic sewage and aerate for
24 hours. Allow the heavier material to settle.
b. After settling for one hour, siphon off three quarts of material and discard.
c. Fill the container with a mixture of 90% sewage and 10% wastes containing the toxic
material.
d. Aerate for 24 hours. Repeat steps b and c with increasing amounts of waste until the
container holds 100% toxic waste material.
Optimum pH for the BOD test is between 6.5 and 7.5. Adjust samples to pH 7.2 with Phosphate
Buffer Solution or 1 N Sulfuric Acid or Sodium Hydroxide Standard Solution if the pH is not in
this range.
Cold samples may be supersaturated with oxygen and will have low BOD results. Fill a one-quart
bottle about halfway with cold sample and shake vigorously for two minutes. Allow sample to reach
20 °C. Then shake the bottle vigorously for two minutes.

Page 877
Accuracy check
ezGGA Method
• BOD Standard Solution, Voluette® Ampule, 300-mg/L, 10-mL (300-mg/L of glucose and 300-
mg/L of glutamic acid)
• Seeded dilution water
• 4 BOD bottles
• 1.0–4.0 mL Class A volumetric pipets and pipet filler or 1–10 mL TenSette Pipet and Pipet tips
• Dissolved oxygen measurement apparatus
DO measurement with the LBOD probe:
1. Add the necessary seed to a 300-mL BOD bottle.
2. Fill the BOD bottle with dilution water until the water level is approximately ¼ inch up the
ground glass portion of the neck. (See dimension “x” in illustration).
3. Put the 2-mL BOD standard ampule into the ampule breaker and rinse the assembly with
deionized water.
4. Hold the ampule and breaker over the rim of the BOD bottle.
5. Use the ampule breaker to open the ampule and allow it to fall into the BOD bottle. Leave
ampule in the BOD bottle during incubation period.
6. Follow the general procedure for the BOD test.
7. Calculate the BOD concentration of the standard solution. The 2 mL in the vial is equivalent to
6 mL as prepared by Standard Methods. Calculate the BOD concentration as though there
were 6 mL added to the bottle instead of 2 mL. The dilution factor for this standard is 50x.
DO measurement with the Clark Cell electrode:

1. Add the necessary seed to a 300-mL BOD bottle.
2. Use the ampule breaker to open the ampule.
3. Pour the contents of the ampule into the BOD bottle. Tap the ampule on the rim of the bottle to
dislodge the contents. Do not drop ampule into the bottle when using a Clark Cell.
4. Fill the ampule with buffered dilution water and add the water to the BOD bottle.
5. Repeat step 4.
6. Fill the BOD bottle with dilution water until the water level is approximately ½ inch up the
ground glass portion of the neck.
7. Follow the general procedure for the BOD test.
8. Calculate the BOD concentration of the standard solution. The 2 mL in the vial is equivalent to
6 mL as prepared by Standard Methods. Calculate the BOD concentration as though there
were 6 mL added to the bottle instead of 2 mL. The dilution factor for this standard is 50x.
Note: The ampules include precisely 2 mL of 450 mg/L GGA. Pouring the entire solution into the bottle is
the same as adding 6 mL of 150 mg/L solution as per the Standard Methods.

Page 878
Troubleshooting—Graphical calculation method

The Graphical Method helps troubleshoot problems in BOD measurements. This method cannot
be used when the results must be reported to a regulatory agency.
1. Plot the mg/L dissolved oxygen (DO) remaining in each diluted sample versus the mL sample
taken. Draw the best straight line through the plotted points. See Dissolved Oxygen per mL of
Sample.
Note: An erroneous point is visually evident at this time and can be disregarded. However, at least three
points should be on the line or very close to it. For unseeded dilution water, the line should cross the
mg/L DO Remaining scale near or below the oxygen saturation value for the altitude of the laboratory as
discussed in Dilution water preparation.
2. To calculate the BOD, use the following equation which is mathematically equivalent to the
BOD equation in Standard Methods.
mg/L BOD = (A x 300) – B + C
where:
A = the slope
The slope of the line is equal to the mg/L DO consumed per mL of sample taken. Take any
point on the line and subtract the mg/L DO Remaining at that point from the mg/L DO where
the line crosses the DO scale (Y intercept, mg/L DO Remaining). Divide the difference by the
mL of sample at the point chosen.
300 = the volume of the BOD bottle
B = the Y intercept
This is the DO value where the line crosses the “DO Remaining” scale. (This should be very
close to the actual dilution water blank value.)
C = the sample DO
This is the DO of the undiluted sample.
Another way to write this equation is:
mg/L BOD = (Slope x 300) – Y intercept + Sample DO
Note: If the best straight line is obtained by linear regression through use of a calculator, the sign (-) of
the slope must be changed (+) before multiplying by 300.
Example:
The mg/L DO remaining was determined for a series of four dilutions of domestic sewage after five
days of incubation. Results were as follows:
mL of sample taken mg/L DO remaining
2.0 7.50
3.0 6.75
6.0 4.50
9.0 2.25
The DO values were plotted versus the mL of sample taken and a straight line drawn as in
Dissolved Oxygen per mL of Sample. If a set of BOD dilutions is run correctly with a homogeneous
sample, a graph of the mg/L DO remaining versus the sample volume would result in a straight
line. The value where the line intersects the y-axis is equal to the DO content of the dilution water

Page 879
after incubation, although this is not actually measured. In this case, it was equal to 9.0 mg/L and
the DO of the domestic sewage sample was assumed to be zero. If another type of sample is
used, the DO of an undiluted sample should be measured either by the Winkler titration or with a
luminescent or electrochemical probe.
The Calculation Methods—Standard Methods formula for calculating BOD also can be written as
follows (not approved for reporting purposes):
mg/L DO remaining w/smaller sample volume – mg/L DO remaining w/larger sample volume
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- × 300 – DO D + S = mg/L BOD
mL of larger sample volume – mL of smaller sample volume
Using this information in the example:
mg/L DO remaining with smaller sample volume = 7.50
mg/L DO remaining with larger sample volume = 2.25
mL of larger sample volume = 9.0
mL of smaller sample volume = 2.0
300 = volume (mL) of BOD bottle
DOD = mg/L DO of dilution water = 9.0
S = mg/L DO of sample = assumed in this case to be zero
Therefore:
7.50 – 2.25-
---------------------------- × 300 – 9 + 0 = mg/L BOD = 216 mg/L BOD
9.0 – 2.0
Using the equation below:
(slope x 300) – Y-Intercept + sample DO = mg/L BOD
To determine slope, arbitrarily select point A in Figure 1. At this point the mg/L DO remaining is
equal to 3.0 mg/L. The mL of sample at this point is 8 mL.The difference between the y-
intercept of 9.0 mg/L and 3.0 mg/L equals 6 mg/L; 6 mg/L divided by 8 mL = 0.75 mg/L per mL.
slope = 0.75 mg/L per mL
Y intercept = 9.0 mg/L
sample DO = 0 (Because the sample is domestic sewage, this is assumed to be zero.)
Therefore:
(0.75 x 300) – 9.0 + 0 = mg/L BOD = 216 mg/L BOD

Page 880
y Intercept
mg/L DO Remaining
mL of Sample
Figure 16 Dissolved Oxygen per mL of Sample

Page 881
Summary of method
Biochemical Oxygen Demand (BOD) is a measurement of the oxygen requirements of municipal
and industrial wastewaters and sewage. The test results are used to calculate the effect of waste
discharges on the oxygen resources of the receiving waters. The BOD test is of limited value in
measuring the actual oxygen demand because temperature change, biological population, water
movement, sunlight, oxygen concentration, and other environmental factors cannot be reproduced
accurately in the laboratory. The BOD test is of greatest value after patterns of oxygen uptake for a
specific effluent and receiving water have been established.
The BOD test is performed by incubating a sealed wastewater sample (or a prepared dilution) for
the standard five-day period and then determining the change in dissolved oxygen content. The
BOD value is then calculated from the results of the dissolved oxygen tests.
Required reagents
BOD Nutrient Buffer Pillows, for 3 liters of dilution water 1 pillow 50/pkg 1486166
Required apparatus
BOD Bottle, glass-stoppered, 300-mL, unlabelled 6 6/pkg 62106
BOD Bottle Cap 6 6/pkg 241906
Bottle, wash, 500-mL 1 each 62011
Pipet, serological:
Pipet Filler 1 each 1218900
Dissolved Oxygen measurement apparatus — — —
BOD Standard Solution, Voluette® Ampule, 300-mg/L, 10-mL 16/pkg 1486510

ezGGA BOD Standard Ampules, 450 mg/L, 2 mL 20/pkg —

Page 882

BOD Nutrient Buffer Pillows
for 300 mL of dilution water 50/pkg 1486166
for 4 liters of dilution water 50/pkg 2436466
Buffer Solution, APHA, for BOD, pH 7.2, phosphate type 500 mL 43149
Calcium Chloride Solution, APHA, for BOD 500 mL 42849
Ferric Chloride Solution, APHA, for BOD 1L 42953
Magnesium Sulfate Solution, APHA, for BOD 500 L 43049
Nitrification Inhibitor 35 g 253335
Dispenser Cap, for Nitrification Inhibitor each 45901
Potassium Iodide Solution, 100-g/L 500 mL 1228949
Sodium Hydroxide, pellets, ACS 500 g 18734
Sodium Thiosulfate Standard Solution, 0.025 N 1L 35253
Sulfuric Acid Standard Solution, 0.020 N 1L 20353
Potassium Permanganate 454g 16801H
Bottle, BOD, Serialized: 1-241 24/pkg 1486610
Bottle, BOD, Disposable 117/case 2943100
Stopper for Disposable BOD Bottle 25/pkg 2943900
Bottle Rack, BOD, 12 bottle each 2094200
Brush, cylinder, 2-in. diameter each 68700
Incubator, BOD, Compact Model 205, 110 Vac each 2616200
Incubator, BOD, Compact Model 205, 220/240 Vac each 2616202
Leash, rubber, for stopper & bottle 6/pkg 2091606
ATU (1-1-allyl-2-thiourea) 50 g 2845425
BOD Seed Inoculum, Polyseed 50 capsules 2918700
1 Other numerical series are available

Page 883
Oxygen Demand, Chemical with chloride removal 10067
Oxygen Demand, Chemical DOC316.53.01101
Manganese III Reactor Digestion Method

Method 10067
(with optional chloride removal)1
30 to 1000 mg/L COD Mn
1 U.S. Patent 5,556,787
Test preparation


DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
To determine if the sample contains chloride, use Quantab® Titrator Strips for low range chloride.
If the sample COD is expected to exceed 1000 mg/L, dilute the sample as described in the Multiplication factors table.
Homogenize the sample for even distribution of solids and better accuracy and reliability.
Run one blank with each lot of reagent. Run all samples and blanks with the same lot of vials. The lot number appears on the
container label.
The stability of the reagent blank allows for reuse. Verify the reagent blank quality by measuring the absorbance of the blank
vs. a clean COD vial filled with deionized water. The absorbance range should be about 1.41–1.47.
If the sample boils during the digestion, the vial is not properly sealed. Test results will be invalid.
Spilled reagent will affect test accuracy and is hazardous. Do not run tests with spilled vials.
The maximum range of the VPD gauge is 40 inches of water; it will not indicate the full vacuum level obtained. Full vacuum is
20–25 inches of mercury; this can be measured at the vacuum pump with a gauge calibrated for inches of mercury.
Oxygen Demand, Chemical

Page 885
Blender 1
DRB200 Reactor 1
Forceps, extra fine 1
Manganese III COD Reagent Vials, 20–1000 mg/L COD 1
Pipet, TenSette® (0.1 to 1.0 mL and 1.0 to 10.0 mL) 1 each
Pipet Tips for TenSette Pipet 2 of each
Sulfuric Acid, concentrated, ACS 1 mL
Test Tube Rack 1
Vacuum pretreatment device 1
Vacuum Pump 1
Vial, glass, for sample and acid 2
Acidified sample preparation
13. Turn on the DRB200 14. Homogenize 100 mL 15. Blank Preparation: 16. Prepared Sample:
Reactor and heat to of sample for 30 seconds Pipet 9.0 mL of deionized Pipet 9.0 mL of
150 °C or set to COD in a blender. water into an empty glass homogenized sample into
program. If suspended solids are mixing cell. another empty glass
present, continue to mix mixing cell
the sample while pipetting.

Page 886
Acidified sample preparation (continued)
17. Using an automatic 18. Cap the cells tightly Proceed to Vacuum
dispenser or TenSette® and invert several times. pretreatment.
Pipet, add 1.0 mL of The solution will become
concentrated sulfuric acid hot. Cool to room
to both the sample and the temperature before
blank. proceeding.
Acidified samples are
stable for several months
when refrigerated at 4 °C.
Calculate the multiplication factor

All dilutions require that the ratio of sample to sulfuric acid remain at 9:1. For other dilutions that
are not listed in the Multiplication factors table, add the sample volume and the deionized water
and divide by the sample volume to obtain the multiplication factor.
Note: Mixing concentrated sulfuric acid and water is not additive. Adding 1.0 mL of concentrated sulfuric acid
to 9.0 mL of sample does not result in a final volume of 10.0 mL. This factor is built into the
calibration curve.
Table 273 Multiplication factors

Sample (mL) Deionized Water (mL) Range (mg/L COD) Multiplication Factor
6.0 3.0 30–1500 1.5
3.0 6.0 60–3000 3
1.0 8.0 180–9000 9
0.5 8.5 360–18,000 18
For best results, use 0.5 mL or more of sample for diluting. If sample values exceed 18,000 mg/L
COD, use a separate sample dilution before performing the sample chloride removal procedure.
Example: Dilute the sample to a range of 90–4500 mg/L COD.
Sample Volume (2.0 mL) + Deionized water (7.0 mL) = Total Volume (9.0 mL)
Total Volume 9.0 mL
Multiplication Factor = ------------------------------------------ = ------------------ = 4.5
Sample Volume 2.0 mL
Standard test range is 50 to 1000 mg/L COD.
Example test range = 4.5(50) to 4.5(1000) = 225 to 4500 mg/L COD

Page 887
Vacuum pretreatment
1. Attach the Vacuum 2. Label each Mn III 3. Place the VPD top on 4. Turn on the vacuum
Pretreatment Device COD vial and remove the the base. Insert a fresh pump and adjust the
(VPD) to a vacuum pump cap. Insert the vials in the Chloride Removal vacuum regulator valve on
(not an aspirator-type numbered holes in the Cartridge (CRC) directly top of the VPD until the
vacuum) that can create a VPD base. above each Mn III COD internal gauge reads 20
vacuum of 20–25 inches of Reagent Vial. Plug any inches of water.
mercury. open holes in the VPD top
using the stoppers
provided.
5. Pipet 0.60 mL of 6. Close the vacuum 7. Open the VPD Proceed to Sample
acidified sample (see regulator valve completely regulator valve to release preparation and
Acidified sample to achieve full vacuum. the vacuum. Turn the measurement.
preparation) into the CRC. After one minute of full pump off. Remove the
Pipet 0.60 mL of acidified vacuum, slide the VPD VPD top and set it beside
blank into another CRC. It back and forth several the base.
should take 30–45 times to dislodge any Dispose of the used
seconds to draw the liquid drops clinging to the Chloride Removal
through the CRC into each cartridge. Cartridge. Do not reuse it.
vial.
If the sample does not flow
through the Chloride
Removal Cartridge (CRC),
increase the vacuum until
flow starts, then reduce
the vacuum down to 20
inches of water. Proceed
as usual.

Page 888
Sample preparation and measurement
1. Use forceps to remove 2. Insert each filter in the 3. Remove the Mn III 4. Invert the vials several
the filter from the top of corresponding Mn III COD COD vial from the vacuum times to mix.
each CRC. Vial. (Use numbers on the chamber and replace the
If the sample does not VPD as a guide.) original cap. Screw the
contain suspended solids, To avoid cross- cap on tightly.
it is not necessary to contamination between
transfer the filter to the samples, clean forcep tips
digestion vial. between samples by
wiping with a clean towel
or rinsing with deionized
water.
5. Insert the vials in the 6. Insert the vials in a 7. Cool the vials to room 8. Remove the vials from
DRB200 Reactor at cooling rack for two temperature in a cool the water and wipe with a
150 °C. Close the minutes. water bath or with running clean, dry paper towel.
protective cover. Digest for If the solution develops a tap water for several
one hour. colorless upper layer and minutes.
To oxidize resistant a purple lower layer, invert
organics, samples can be the vial several times to
digested for up to four mix and proceed to the
hours. Digest the blank for next step.
the same time period as
the samples.

Page 889
Sample preparation and measurement (continued)
Stored Programs
432 COD Mn III

Zero
Start
9. Select the test. 10. Insert the blank into 11. ZERO the instrument. 12. Make sure the filter
Insert an adapter or a light the 16-mm cell holder. The display will show: disc is not suspended in
shield if required (see the middle of the vial; it
0 mg/L COD Mn can interfere with the
Instrument-specific
information). instrument reading.
The disc must be more
than 20 mm (0.8") or less
than 10 mm (0.4"), from
the bottom of the vial.
Move it by gently swirling
or by lightly tapping the
vial on the table top.
Read
13. Wipe the prepared 14. READ the results in

sample and insert it into mg/L COD Mn.
the .
Interferences
Inorganic materials may also be oxidized by trivalent manganese and constitute a positive
interference when present in significant amounts. Chloride is the most common interference and is
removed by sample pretreatment with the Chloride Removal Cartridge. If chloride is known to be
absent or present in insignificant levels, the pretreatment can be omitted. A simple way to
determine if chloride will affect test results is to run routine samples with and without the chloride
removal, then compare results. Other inorganic interferences (i.e., nitrite, ferrous iron, sulfide) are
not usually present in significant amounts. If necessary, these interferences can be corrected after
determining their concentrations with separate methods and adjusting the final COD test results
accordingly.
Ammonia nitrogen is known to interfere in the presence of chloride; it does not interfere if chloride
is absent.

Page 890

• Use plastic bottles only if they are known to be free of organic contamination.
• Test biologically active samples as soon as possible.
• Samples treated with concentrated sulfuric acid to a pH of less than 2 (about 2 mL per liter)
and refrigerated at 4 °C may be stored up to 28 days.
Accuracy check

• COD standard solution, 800 mg/L
3. Use 0.60 mL of the 800-mg/L solution in place of the sample.
Method performance

432 DR 5000 600 mg/L COD 576–624 mg/L COD 8 mg/L COD
Estimated detection limit (EDL)

The EDL for program 432 is 4 mg/L COD. The EDL is the calculated lowest average concentration
in a deionized water matrix that is different from zero with a 99% level of confidence.
Summary of method
Chemical Oxygen Demand (COD) is defined as “... a measure of the oxygen equivalent of the
organic matter content of a sample that is susceptible to oxidation by a strong chemical oxidant”
(APHA Standard Methods, 19th ed., 1995). Trivalent manganese is a strong, non-carcinogenic
chemical oxidant that changes quantitatively from purple to colorless when it reacts with organic
matter. It typically oxidizes about 80% of the organic compounds. Studies have shown that the
reactions are highly reproducible and test results correlate closely to Biochemical Oxygen
Demand (BOD) values and hexavalent chromium COD tests. None of the oxygen demand tests
provide 100% oxidation of all organic compounds.
A calibration is provided which is based on the oxidation of Potassium Acid Phthalate (KHP). A
different response may be seen in analyzing various wastewaters. The KHP calibration is
adequate for most applications. The highest degree of accuracy is obtained when test results are
correlated to a standard reference method such as BOD or one of the chromium COD methods.
Special waste streams or classes will require a separate calibration to obtain a direct
mg/L COD reading or to generate a correction factor for the precalibrated KHP response. The

Page 891
sample digestion time can be extended up to four hours for samples that are difficult to oxidize.
Required reagents
Manganese III COD Reagent Vials, 20–1000 mg/L COD 1 25/pkg 2623425
Chloride Removal Cartridge (CRC) 1 25/pkg 2661825
Sulfuric Acid, concentrated, ACS 1 mL 2.5 L 97909
Required apparatus
Blender, 120 VAC 1 each 2616100
Cap, with inert Teflon liner, for mixing bottle varies 12/pkg 2401812
Forceps, extra fine point 1 each 2669600
Pipet Tips for TenSette Pipet 19700-10 2 50/pkg 2199796
Vacuum Pretreatment Device (VPD) 1 each 4900000
Vacuum Pump, 1.2 CFM 115V 1 each 2824800
Vial, glass, for sample plus acid 2 each 2427700
COD Standard Solution, 800-mg/L COD 200 mL 2672629
Oxygen Demand Standard for BOD, COD, TOC, 10-mL ampules 16/pkg 2833510
Potassium Acid Phthalate, ACS 500 g 31534
Wastewater Standard, Influent Inorganics, for NH3–N, NO3–N, PO4, COD, SO4, TOC 500 mL 2833149

Dispenser, automatic, 1.0–5.0 mL each 2563137
Titrator Strips, Quantab®, for low range chloride 40 tests 2744940
Finger cots 2/pkg 1464702
Flask, volumetric class A, 1 L each 1457453

Page 892

Weighing Paper, 76x76 mm 500/pkg 1473800
Oven, Laboratory 120 VAC/60Hz each 1428900
COD Standard Solution, 300-mg/L 200 mL 1218629
Standard Methods book, most recent edition each 2270800
Pipet Tips for TenSette Pipet 19700-10 250/pkg 2199725

Page 893
Oxygen Demand, Chemical without chloride removal, 10067

(Manganese III Reactor Digestion Method)
Manganese III Reactor Digestion Method
Method 10067
(without chloride removal)1
30 to 1000 mg/L COD Mn
1 U.S. Patent 5,556,787
Test preparation


DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
If the sample contains chloride, use the chloride removal method. To determine if the sample contains chloride, use
Quantab® Titrator Strips for low range chloride.
If the sample COD value is not between 30 and 1000 mg/L, dilute the sample with deionized water to obtain this range.
Multiply the final result by the dilution factor.
Homogenize the sample for even distribution of solids and better accuracy and reliability.
Stability of the reagent blank allows for reuse. Verify the reagent blank quality by measuring the absorbance of the blank vs.
a clean COD vial filled with deionized water. The absorbance range should be about 1.4–1.5.
If the sample boils during the digestion, the vial is not properly sealed. Test results will be invalid.
Use finger cots to handle hot sample cells.
Spilled reagent will affect test accuracy and is hazardous. Do not run tests with spilled vials.
See the DRB200 User Manual for selecting pre-programmed temperature applications.

Page 895
Blender 1
DRB200 Reactor 1
Light Shield 1
Manganese III COD Reagent Vials, 20–1000 mg/L COD 1
Pipet, TenSette® (0.1 to 1.0 mL) 1
Pipet tips for TenSette Pipet 2
Test Tube Rack 1
Sample preparation and measurement
Stored Programs
432 COD Mn III
Start
15. Select the test. 16. Turn on the DRB200 17. Homogenize 100 mL Pipet 0.5 mL of
Insert an adapter or a light Reactor and heat to of sample for 30 seconds homogenized sample into
shield if required (see 150 °C or use the COD in a blender. one Mn III COD vial (the
Instrument-specific program. If suspended solids are prepared sample) and
information). present, continue to mix 0.5 mL of deionized water
the sample while pipetting. into another Mn III COD
vial (the blank).

Page 896
Sample preparation and measurement (continued)
18. Cap and invert several 19. Insert the vials in the 20. Remove the vials and 21. Cool the vials to room
times to mix. DRB200 Reactor at place them in a cooling temperature in a cool
150 °C. Close the rack for two minutes. water bath or with running
protective cover. Digest for If a vial develops a tap water. This takes
one hour. colorless upper layer and several minutes.
Digest more resistant a purple lower layer, invert
organics and the blank for the vial several times to
up to four hours. mix and proceed.
Zero
22. Invert the vials several 23. Wipe the blank and 24. ZERO the instrument. 25. Wipe the sample and
times to mix. insert it into the 16-mm The display will show: insert it into the 16-mm
round cell holder. round cell holder.
0 mg/L COD Mn
COD Mn.
Interferences
Inorganic materials may also be oxidized by trivalent manganese and constitute a positive
interference when present in significant amounts. Chloride is the most common interference. If
chloride is known to be present in significant levels, the chloride needs to be removed with the
vacuum pretreatment device. A simple way to determine if chloride will affect test results is to run
routine samples with and without the chloride removal, then compare results. Other inorganic
interferences (i.e., nitrite, ferrous iron, sulfide) are not usually present in significant amounts. If
necessary, these interferences can be corrected after determining their concentrations with
separate methods and adjusting the final COD test results accordingly.
Ammonia nitrogen is known to interfere in the presence of chloride; it does not interfere if chloride
is absent.

Page 897

• Samples treated with concentrated sulfuric acid to a pH of less than 2 (about 2 mL per liter)
and refrigerated at 4 °C may be stored up to 28 days.
Accuracy check

• COD Standard Solution, 800 mg/L
6. Use 0.50 mL of the 800-mg/L solution in place of the sample.
Method performance
432 600 mg/L COD 576–624 mg/L COD 8 mg/L COD

Page 898
Estimated detection limit (EDL)

The EDL for program 432 is 4mg/L COD. The EDL is the calculated lowest average concentration
in a deionized water matrix that is different from zero with a 99% level of confidence.
Summary of method
Chemical Oxygen Demand (COD) is defined as “... a measure of the oxygen equivalent of the
organic matter content of a sample that is susceptible to oxidation by a strong chemical oxidant”
(APHA Standard Methods, 19th ed., 1995). Trivalent manganese is a strong, non-carcinogenic
chemical oxidant that changes quantitatively from purple to colorless when it reacts with organic
matter. It typically oxidizes about 80% of the organic compounds. Studies have shown that the
reactions are highly reproducible and test results correlate closely to Biochemical Oxygen
Demand (BOD) values and hexavalent chromium COD tests. None of the oxygen demand tests
provide 100% oxidation of all organic compounds.
A calibration is provided which is based on the oxidation of Potassium Acid Phthalate (KHP). A
different response may be seen in analyzing various wastewaters. The KHP calibration is
adequate for most applications. The highest degree of accuracy is obtained when test results are
correlated to a standard reference method such as BOD or one of the chromium COD methods.
Special waste streams or classes will require a separate calibration to obtain a direct
mg/L COD reading or to generate a correction factor for the precalibrated KHP response. The
sample digestion time can be extended up to four hours for samples that are difficult to oxidize.
Required reagents
Manganese III COD Reagent Vials, 20–1000 mg/L COD 1 25/pkg 2623425
Required apparatus
Blender, 2-speed, 120 VAC 1 each 2616100

Page 899

COD Standard Solution, 800-mg/L COD 200 mL 2672629
Oxygen Demand Standard for BOD, COD, TOC, 10-mL ampules 16/pkg 2833510
Wastewater Standard, Influent Inorganics, for NH3–N, NO3–N, PO4, COD, SO4, TOC 500 mL 2833149
Goggles, Safety vented each 2550700
Gloves, Chemical Resistant, size 9-9½1 1 pair 2410104

Titrator Strips, Quantab®, for low range chloride 40 tests 2744940

Flask, volumetric class A, 1L each 1457453
Standard Methods book, most recent edition each 2270800

Oxygen, COD, MR, 8000
USEPA1 Reactor Digestion Method2 Method 8000

0.7 to 40.03
mg/L COD (ULR); 3 to 150 mg/L COD (LR);
20 to 1500 mg/L COD (HR); 200 to 15,000 mg/L COD (HR Plus)
Scope and Application: For water, wastewater; digestion is required
1 Ranges 3 to 150 mg/L COD and 20 to 1500 mg/L COD are USEPA approved for wastewater analyses (Standard Method 5220 D),
Federal Register, April 21, 1980, 45(78), 26811-26812.
2 Jirka, A.M.; Carter, M.J., Analytical Chemistry, 1975, 47(8), 1397
3 The ULR range is not available on the DR 2700 or the DR/2400.
Test preparation

information required to perform this test

DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800 LZV646
Some of the chemicals and apparatus used in this procedure may be hazardous to the health and safety of the user if
inappropriately handled or accidentally misused. Please read all warnings and associated MSDS sheets.
Run one blank with each set of samples. Run all tests (the samples and the blank) with the same lot of vials. The lot number
appears on the container label. See Blanks for colorimetric determination.
Spilled reagent will affect test accuracy and is hazardous to skin and other materials. Be prepared to wash spills with
running water.
Wear appropriate eye protection and clothing for adequate user protection. If contact occurs, flush the affected area with
running water. Review and follow reagent MSDS safety instructions carefully.
Store unused (light sensitive) vials in a closed box.
If high chloride samples are being tested, refer to the Alternate reagents section.

Page 901
Beaker, 250-mL 1
Blender 1
COD Digestion Reagent vials varies
DRB200 Reactor 1
Magnetic stirrer and stir bar 1
Opaque shipping container for storage of unused, light-sensitive reagent vials varies
Pipet, TenSette®, 0.1 to 1.0 mL, with tips (for 200–15,000 mg/L range) 1
Pipet, volumetric, 2.00 mL 2
Pipet Filler, safety bulb 1
Test Tube Rack 2
Reactor digestion procedure
1. Homogenize 100 mL 2. For the 200– 3. Turn on the DRB200 4. Remove the caps from
of sample for 30 seconds 15,000 mg/L range or to Reactor. Preheat to two COD Digestion
in a blender. For samples improve accuracy and 150 °C. Reagent Vials. (Be sure to
containing large amounts reproducibility of the other See the DRB200 User use vials for the
of solids, increase the ranges, pour the Manual for selecting pre- appropriate range.)
homogenization time. homogenized sample into programmed temperature
If the sample does not a 250-mL beaker and applications.
contain suspended solids, gently stir with a magnetic
omit steps 1 and 2. stir plate.

Page 902
Reactor digestion procedure (continued)
5. Prepared Sample: 6. Blank Preparation: 7. Cap the vials tightly. 8. Hold the vials by the
Hold one vial at a Hold a second vial at a Rinse them with water and cap over a sink. Invert
45-degree angle. Use a 45-degree angle. Use a wipe with a clean paper gently several times to
clean volumetric pipet to clean volumetric pipet to towel. mix. The sample vials
add 2.00 mL of sample to add 2.00 mL of deionized become very hot during
the vial. water to the vial. mixing.
For the 200–15,000 mg/L For the 200–15,000 mg/L Insert the vials in the
vials: Use a TenSette® vials: Use a TenSette preheated DRB200
Pipet to add 0.20 mL of Pipet to add 0.20 mL of Reactor. Close the
sample to the vial. deionized water to the vial. protective lid.
9. Heat the vials for two 10. Turn the reactor off. 11. Invert each vial 12. Place the vials into a
hours. Before removing the vials, several times while still rack and cool to room
wait about 20 minutes for warm. temperature.
the vials to cool to 120 °C Proceed to Colorimetric
or less. determination.

Page 903
Colorimetric determination
Stored Programs
431 COD ULR

430 COD LR Zero
435 COD HR
Start
1. Select the test. 2. Clean the outside of 3. Insert the blank into 4. ZERO the instrument.
Insert an adapter or light the vials with a damp towel the 16-mm cell holder. The display will show:
shield if required (see followed by a dry one.
0 mg/L COD
Instrument-specific or
information). 0.0 mg/L COD
for orientation.
Read
5. Insert the sample vial 6. READ the results in 7. If using High Range
into the 16-mm . mg/L COD. Plus COD Digestion
Reagent Vials, multiply the
result by 10.
For most accurate results
with samples near 1500 or
15,000 mg/L COD, repeat
the analysis with a diluted
sample.
Blanks for colorimetric determination

The blank may be used repeatedly for measurements using the same lot of vials. Store the blank
in the dark.
1. Monitor decomposition by measuring the absorbance at the appropriate wavelength. Refer to

the Range-specific test wavelengths table.
2. Zero the instrument in the absorbance mode. Use a vial containing 5 mL of deionized water
and measure the absorbance of the blank. Record the value.
3. Prepare a new blank when the absorbance has changed by about 0.01 absorbance units.

Page 904
Interferences
Chloride is the primary interference when determining COD concentration. Each COD vial
contains mercuric sulfate that will eliminate chloride interference up to the level specified in
Column 1 of the Interfering substances table. Dilute samples with higher chloride concentrations.
Dilute the sample enough to reduce the chloride concentration to the level given in Column 2.
Note: For best results, use the low range and ultra-low range test for samples with high chloride
concentrations (approaching maximum concentration) and low COD concentrations.
If sample dilution will cause the COD concentration to be too low for accurate determination, add
0.50 g of mercuric sulfate (HgSO4) to each COD vial before the sample is added.
The additional mercuric sulfate will raise the maximum chloride concentration allowable to the
level given in Column 3.

Column 2 Column 3
Column 1
(Suggested chloride (Maximum chloride
Vial range (Maximum chloride
concentration for concentration with
concentration )
diluted samples) mercuric sulfate)
Ultra Low Range 1 2000 1000 N/A

(0.7–40.0 mg/L)
Low Range 2000 1000 8000
(3–150 mg/L)
High Range 2000 1000 4000
(20–1500 mg/L)
High Range Plus 20,000 10,000 40,000
(200–15,000 mg/L)
1 Ultra Low Range is not available on the DR 2700.

• Collect samples in glass bottles.
• Samples treated with sulfuric acid* to a pH of less than 2 (about 2 mL per liter) and refrigerated
at 4 °C can be stored up to 28 days.

Page 905
Accuracy check

• 1000 mg/L COD Standard Solution
OR
• Potassium acid phthalate (KHP), dried overnight at 120 °C

• Deionized water, organic free
• Class A volumetric flasks
• Class A volumetric pipet
0.7 to 40.0 mg/L range
1. Prepare a 30-mg/L COD standard solution as follows:
e. Pipet 3.00 mL of the 1000 mg/L standard into a 100-mL volumetric flask.
f. Dilute to volume with deionized water and mix well.
2. Use 2 mL of the 30 mg/L COD solution in place of the sample. Follow the Colorimetric
determination test procedure. The result should be 30 mg/L. Refer to the Standard adjust
instructions in this procedure to adjust the curve with the reading obtained from the standard.
3 to 150 mg/L range
1. Prepare a 100-mg/L COD standard solution as follows:
a. Pipet 10 mL of the 1000 mg/L standard into a 100-mL volumetric flask.

b. Dilute to volume with deionized water and mix well.
2. Use 2 mL of the 100-mg/L solution in place of the sample. Follow the Colorimetric
determination test procedure. The result should be 100 mg/L. Refer to the Standard adjust
instructions in this procedure to adjust the curve with the reading obtained from the standard
20 to 1500 mg/L range
Use 2 mL of 300 mg/L, 800 or 1000 mg/L COD standards for accuracy check.

Page 906
200 to 15,000 mg/L range

1. Prepare a 10,000-mg/L COD standard solution as follows:
a. Dissolve 8.500 g of dried KHP in 1000-mL of organic-free deionized water.
2. Use 0.2 mL of the 10,000 mg/L COD solution in place of the sample. Follow the Colorimetric
determination test procedure. The result should be 10,000 mg/L (after multiplying by 10).
Refer to the Standard adjust instructions in this procedure to adjust the curve with the reading
obtained from the standard.
Standard adjust
Alternate reagents
Mercury-free COD2 Reagents can provide a mercury-free testing option for non-reporting
purposes. For process control applications, COD2 Reagents will eliminate mercury waste and
save on disposal costs. These reagents are fully compatible with test procedures and calibration
curves programmed into the spectrophotometer. Determine chloride and ammonia for
accurate results.
Important Note: COD2 reagents are not approved for USEPA reporting purposes. Because
COD2 reagents do not contain mercury as a masking agent, they exhibit a positive
interference from chloride. Request a copy of the COD2 Reagent Vial Information Brochure,
Lit. No. 1356, for more information about specific applications.

Page 907
Method performance
430 77–83 mg/L COD 3 mg/L COD
(Range, 80 mg/L COD (Low Range)
3–150 mg/L)
431 28.8–31.2 mg/L COD 0.5 mg/L COD
30 mg/L COD (Ultra Low
(Range,
Range)
0.5–40.0 mg/L)
435 785–815 mg/L COD 23 mg/L COD
(Range, 800 mg/L COD (High Range)
20–1500 mg/L)
435 7850–8150 mg/L COD 230 mg/L COD
8000 mg/L COD (High Range
(Range,
Plus)
200–15,000 mg/L)
Summary of method
The results in mg/L COD are defined as the milligrams of O2 consumed per liter of sample under
the conditions of this procedure. The sample is heated for two hours with sulfuric acid and a strong
oxidizing agent, potassium dichromate. Oxidizable organic compounds react, reducing the
dichromate ion (Cr2O72–) to green chromic ion (Cr3+).
When the 0.7–40.0 or the 3–150 mg/L colorimetric method is used, the amount of Cr6+ remaining
is determined. When the 20–1500 mg/L or 200–15,000 mg/L colorimetric method is used, the
amount of Cr3+ produced is determined. The COD reagent also contains silver and mercury ions.
Silver is a catalyst, and mercury is used to complex chloride interferences.
Test results are measured at the wavelengths specified in the Range-specific test
wavelengths table.
Table 277 Range-specific test wavelengths

Range in mg/L COD Wavelength
0.7 to 40.0 mg/L1 350 nm
3 to 150 mg/L 420 nm
20 to 1500 620 nm
2000 to 15,000 mg/L 620 nm
1 Not available on the DR 2700

Page 908
Required reagents
Select the appropriate COD Digestion Reagent Vial:
Ultra Low Range, 0.7 to 40 mg/L COD 1–2 vials 25/pkg 2415825
Low Range, 3 to 150 mg/L COD 1–2 vials 25/pkg 2125825
High Range, 20 to 1500 mg/L COD 1–2 vials 25/pkg 2125925
High Range Plus, 200 to 15,000 mg/L COD 1–2 vials 25/pkg 2415925
Alternate reagents1
Select the appropriate COD Digestion Reagent Vial:
COD2, Low Range, 0 to 150 mg/L COD 1–2 vials 25/pkg 2565025
COD2, High Range, 0 to 1500 mg/L COD 1–2 vials 25/pkg 2565125
COD2, High Range, 0 to 1500 mg/L COD 1–2 vials 150/pkg 2565115
COD2, High Range Plus, 0 to 15,000 mg/L COD 1–2 vials 25/pkg 2834325
COD Digestion Reagent Vials, 3 to 150 mg/L COD 1–2 vials 150/pkg 2125815
COD Digestion Reagent Vials, 200 to 1500 mg/L COD 1–2 vials 150/pkg 2125915
COD Digestion Reagent Vials, ULR 0.7-40.0 mg/L 1-2 vials 150/pkg 2415815
COD Digestion Reagent Vials, HR plus,200-25,000 mg/L 1-2 vials 150/pkg 2415915
1 These reagents are not approved for USEPA reporting purposes. Request a copy of the COD2 Reagent Vial Information Brochure,
Lit. No. 1356, for more information about specific applications.
Required apparatus
OR
OR
Pipet, Volumetric, Class A, 2.00 mL 1 each 1451536

Page 909

COD Standard Solution, 300-mg/L 500mL 1218649
Oxygen Demand Standard (BOD, COD, TOC), 10-mL ampules 16/pkg 2833510
Stir Bar, 28.6 x 7.9 mm each 2095352
Stirrer, Electromagnetic, 120 VAC, with electrode stand each 4530001
Stirrer, Electromagnetic, 230 VAC, with electrode stand each 4530002
Test Tube Rack, 16 mm each 1864100
Wipes, Disposable 70/pkg 2096900

Flask, volumetric, 1000 mL Class A each 1457453
Flask, volumetric, 100-mL Class A each 1457442
Mercuric Sulfate 28 g 191520
Pipet, volumetric, 3-mL, Class A each 1451503
Pipet, volumetric, 10-mL, Class A each 1451538
Sulfuric Acid, conc 500 mL 97949
Wastewater Influent Standard for mixed parameters NH3–N, NO3–N, PO4, COD, SO4, 500mL 2833149
TOC
Wastewater Effluent Standard, for mixed parameters NH3–N, NO3–N, PO4, COD, SO4, 500mL 2833249
TOC
Gloves, chemical resistant 9-9 ½”1 1 pair 2410104
Safety goggles, vented each 2550700
EZ CODTM Recycling Service with 5-gal2 bucket–mail back3 each 2895405
EZ CODTM Recycling Service with 5-gal bucket–pick up3 each 2895405P
2 20 and 55 gal are available.
3 ez COD available to U.S. customers only

Page 910

Page 911
Oxygen, Dissolved, HR, 8166
Oxygen, Dissolved DOC316.53.01096
HRDO Method Method 8166

HR (0.3 to 15.0 mg/L O2) AccuVac® Ampuls
Test preparation


AccuVac Ampuls
Instrument
Sample cell Adapter
DR 6000 2427606 —
DR 5000 2427606 —
DR 3900 2427606 LZV846 (A)
DR 3800, DR 2800, DR 2700 2122800 LZV584 (C)
Analyze samples on-site. Do not store for later analysis
High Range Dissolved Oxygen AccuVac® Ampuls with reusable Ampul caps 1
Polypropylene Beaker, 50-mL 1
Oxygen, Dissolved
Page 913
Oxygen, Dissolved
HRDO Method
Stored Programs
445 Oxygen, Dis HR AV
Start
1. Select the test. 2. Blank Preparation: 3. Fill a blue Ampul cap 4. Prepared Sample: Fill
Insert an adapter if Fill a sample cell with 10 with sample. a High Range Dissolved
required (see Instrument- mL of sample. Oxygen AccuVac Ampul
specific information). with sample. Keep the tip
fills completely.
5. Hold the Ampul with 6. Shake the Ampul for 7. Start the instrument 8. When the timer
the tip pointing down and 30 seconds. timer. expires, shake the Ampul
immediately insert the A small amount of A two-minute reaction for 30 seconds.
Ampul into the Ampul cap. undissolved reagent will period will begin. This Allow any bubbles to
The cap prevents not affect results. enables the oxygen that dissipate before
contamination from was degassed during proceeding.
atmospheric oxygen. aspiration to redissolve
and react.
Zero Read
9. Insert the blank in the 10. ZERO the instrument. 11. Insert the prepared 12. READ the results in
cell holder. The display will show: sample into the cell holder. mg/L O2.
0.0 mg/L O2
Oxygen, Dissolved
Page 914
Oxygen, Dissolved
Interferences

Cr3+ Greater than 10 mg/L
Cu2+ Greater than 10 mg/L
Magnesium is commonly present in seawater and causes a negative interference. If the
sample contains more than 50% seawater, the oxygen concentration obtained by this
Mg2+
method will be 25% less than the true oxygen concentration. If the sample contains less than
50% seawater, the interference will be less than 5%.
Ni2+ Greater than 10 mg/L
NO2- Greater than 10 mg/L

The main consideration in sampling with the High Range Dissolved Oxygen Ampul is to prevent
the sample from becoming contaminated with atmospheric oxygen between breaking open the
Ampul and reading the absorbance. This is accomplished by capping the Ampul with an Ampul
cap. If the Ampul is securely capped, the Ampul should be safe from contamination for several
hours. The absorbance will decrease by approximately 3% during the first hour and will not change
significantly afterwards.
Sampling and sample handling are important considerations in obtaining meaningful results. The
dissolved oxygen content of the water being tested may change with depth, turbulence,
temperature, sludge deposits, light, microbial action, mixing, travel time and other factors. A single
dissolved oxygen test rarely reflects the accurate overall condition of a body of water. Several
samples taken at different times, locations and depths are recommended for most reliable results.
Samples must be tested immediately upon collection, although only a small error results if the
absorbance reading is taken several hours later.
Accuracy check
The results of this procedure may be compared with the results of a titrimetric procedure or by
using a dissolved oxygen meter.*
Method performance
95% Confidence Limits of Sensitivity—ΔConcentration

Program Standard
445 6.7 mg/L O2 6.2–7.3 mg/L O2 0.09 mg/L O2
Summary of method
The High Range Dissolved Oxygen AccuVac Ampul contains reagent vacuum-sealed in a 14-mL
Ampul. When the AccuVac Ampul is opened in a sample containing dissolved oxygen, it forms a
yellow color which turns purple. The purple color development is proportional to the concentration
of dissolved oxygen. Test results are measured at 535 nm.
* See Optional reagents, apparatus and meters.
Oxygen, Dissolved
Page 915
Oxygen, Dissolved
Required reagents
High Range Dissolved Oxygen AccuVac® Ampuls 1 25/pkg 2515025
Required apparatus
Polypropylene Beaker, 50-mL 1 each 108041
Optional reagents, apparatus and meters

AccuVac® snapper each 2405200

AccuVac sampler each 2405100
AccuVac stoppers 6/pkg 173106
HQ30d Meter with Standard LDO Dissolved Oxygen Probe1 (1 meter cable) each HQ30d53301000
HQ40d Meter with Standard LDO Probe1 (1 meter cable) each HQ40d53301000
1 Additional probes and cable lengths are available.

Oxygen, Dissolved, LR, 8316
Indigo Carmine Method Method 8316

LR (6 to 800 µg/L O2) AccuVac® Ampuls
Scope and Application: For boiler feedwater
Test preparation


AccuVac Ampuls
Instrument
Sample cell Adapter
DR 6000 2427606 —
DR 5000 2427606 —
DR 3900 2427606 LZV846 (A)
DR 3800, DR 2800, DR 2700 2122800 LZV584 (C)
Low Range Dissolved Oxygen AccuVac® Ampuls 1
Oxygen, Dissolved
Page 917
Oxygen, Dissolved
Indigo Carmine method for AccuVac® Ampuls
Stored Programs
446 Oxygen, Dis LR AV

Zero
Start
1. Select the test. 2. Blank Preparation: 3. Insert the blank into 4. ZERO the instrument.
Insert an adapter if Fill a sample cell with the cell holder. The display will show:
required (see Instrument- 10 mL of sample.
0 µg/L O2
Read
5. Fill a Low Range 6. Immediately insert the 7. READ the results in Use the initial reading. The
Dissolved Oxygen Ampul Ampul into the cell holder. µg/L O2. reading is stable for 30
with sample. Keep the tip seconds. After 30 seconds
immersed while the Ampul the Ampul solution will
fills completely. absorb oxygen from
the air.
Interferences
Excess amounts of thioglycolate, ascorbate, ascorbate + sulfite, ascorbate + cupric sulfate, nitrite,
sulfite, thiosulfate and hydroquinone will not reduce the oxidized form of the indicator and do not
cause significant interference.

Hydrazine 100,000 fold excess will begin to reduce the oxidized form of the indicator solution.
Sodium hydrosulfite Reduces the oxidized form of the indicator solution and will cause a significant interference.
Oxygen, Dissolved
Page 918
Oxygen, Dissolved

The main consideration in this procedure is to prevent contaminating the sample with
atmospheric oxygen.
• For best results, sample from a stream of water that is hard plumbed to the sample source.
• Use a funnel to maintain a continual flow of sample and yet collect enough sample to immerse
the Ampul.
• Do not introduce air in place of the sample.
• Rubber tubing, if used, will introduce unacceptable amounts of oxygen into the sample unless
the length of tubing is minimized and the flow rate is maximized.
• Flush the sampling system with sample for at least 5 minutes.
Accuracy check
The reagent blank for this test can be checked by following these steps:
1. Fill a 50-mL beaker with sample and add one sodium hydrosulfite powder pillow.
2. Immerse the tip of a Low Range Dissolved Oxygen AccuVac Ampul in the sample into the tip.
Aspirate the sample into the Ampul.
3. Determine the dissolved oxygen concentration according to the preceding procedure. The
result should be 0 ± 6 µg/L.
Method performance
Sensitivity
Sensitivity—ΔConcentration
Program
per 0.010 ΔAbs
446 6 µg/L O2
Summary of method
The Low Range Dissolved Oxygen AccuVac Ampul contains reagent vacuum-sealed in an Ampul.
When the AccuVac Ampul is broken open in a sample containing dissolved oxygen, the yellow
solution will turn blue. The blue color development is proportional to the concentration of dissolved
oxygen. Test results are measured at 610 nm.
Oxygen, Dissolved
Page 919
Oxygen, Dissolved
Required reagents
Low Range Dissolved Oxygen AccuVac® Ampuls 1 25/pkg 2501025
Required apparatus
Hydrosulfite reagent powder pillows 100/pkg 2118869


Oxygen, Dissolved, UHR, 8333
Ultra High Range Method Method 8333

UHR (1.0 to 40.0 mg/L O2) AccuVac® Ampuls
Scope and Application: For aquaculture
Test preparation


AccuVac Ampuls
Instrument
Sample cell Adapter
DR 6000 2427606 —
DR 5000 2427606 —
DR 3900 2427606 LZV846 (A)
DR 3800, DR 2700, DR 2800 2122800 LZV584 (C)
High Range Dissolved Oxygen AccuVac®Ampuls, with reusable Ampul caps 1
Oxygen, Dissolved
Page 921
Oxygen, Dissolved
UHR Method for AccuVac® Ampuls
Stored Programs
448 Oxygen, Dis UHR
Start
1. Select the test. 2. Blank Preparation: 3. Fill a blue Ampul cap 4. Prepared Sample: Fill
Insert an adapter if Fill a sample cell with 10 with sample. a High Range Dissolved
required (see Instrument- mL of sample. Oxygen AccuVac Ampul
specific information). with sample. Keep the tip
fills completely.
5. Hold the Ampul with 6. Shake the Ampul for 7. Start the instrument 8. When the timer
the tip pointing down and 30 seconds. timer. expires, shake the Ampul
immediately insert the A small amount of A two-minute reaction for 30 seconds.
Ampul into the Ampul cap. undissolved reagent will period will begin. This Allow any bubbles to
The cap prevents not affect results. enables the oxygen that dissipate before
contamination from was degassed during proceeding.
atmospheric oxygen. aspiration to redissolve
and react.
Zero Read
9. Insert the blank in the 10. ZERO the instrument. 11. Insert the prepared 12. READ the results in
cell holder. The display will show: sample into the cell holder. mg/L O2.
0.0 mg/L O2
Oxygen, Dissolved
Page 922
Oxygen, Dissolved
Interferences

Cr3+ Greater than 10 mg/L
Cu2+ Greater than 10 mg/L
Magnesium is commonly present in seawater and causes a negative interference. If the
sample contains more than 50% seawater, the oxygen concentration obtained by this
Mg2+
method will be 25% less than the true oxygen concentration. If the sample contains less than
50% seawater, the interference will be less than 5%.
Ni2+ Greater than 10 mg/L
NO2- Greater than 10 mg/L

The main consideration in sampling with the High Range Dissolved Oxygen Ampul is to prevent
the sample from becoming contaminated with atmospheric oxygen between breaking open the
Ampul and reading the absorbance. This is accomplished by capping the Ampul with an Ampul
cap. If the Ampul is securely capped, the Ampul should be safe from contamination for several
hours. The absorbance will decrease by approximately 3% during the first hour and will not change
significantly afterwards.
dissolved oxygen content of the water being tested may change with depth, turbulence,
temperature, sludge deposits, light, microbial action, mixing, travel time and other factors. A single
dissolved oxygen test rarely reflects the accurate overall condition of a body of water. Several
samples taken at different times, locations and depths are recommended for most reliable results.
Samples must be tested immediately upon collection, although only a small error results if the
absorbance reading is taken several hours later.
Accuracy check
The results of this procedure may be compared with the results of a titrimetric procedure (request
Lit. Code 8042) or by using a dissolved oxygen meter.*
Method performance
448 26.4 mg/L O2 23.6–29.2 mg/L O2 0.34 mg/L O2
0.45 mg/L O2
0.68 mg/L O2
* See Optional reagents, apparatus and meters.
Oxygen, Dissolved
Page 923
Oxygen, Dissolved
Summary of method
The High Range Dissolved Oxygen AccuVac Ampul contains reagent vacuum-sealed in an Ampul.
When the AccuVac Ampul is opened in a sample containing dissolved oxygen, it forms a yellow
color which turns purple. The purple color development is proportional to the concentration of
dissolved oxygen. Test results are measured at 680 nm.
Required reagents
High Range Dissolved Oxygen AccuVac® Ampuls with 2 reusable 1 25/pkg 2515025
Ampul caps



HQ30d Meter with Standard LDO Dissolved Oxygen Probe (1 meter cable)1 each HQ30d53301000
HQ40d Meter with Standard LDO Probe (1 meter cable)1 each HQ40d53301000
1 Additional probes and cable lengths are available.

Oxygen, Dissolved, DT, 8215 and 8332
Azide Modification of Winkler Method Method 8215 and 8332

1 to greater than 10 mg/L Digital Titrator
Test preparation
Use the TitraStir apparatus for best results.
Dissolved Oxygen Reagent Set 1
Sodium Thiosulfate Titration Cartridge, 2.00 N varies
Bottle, with stopper, BOD, 300-mL 1
Clippers, for opening pillows 1
Digital Titrator 1
Dissolved Oxygen 1 Reagent Powder Pillows 1
Sodium Thiosulfate Titration Cartridge, 0.2000 N 1
Bottle, with stopper, BOD, 60-mL 1
Polypropylene Beaker, 50-mL, Low Form, with pour spout 1
Oxygen, Dissolved
Page 925
Oxygen, Dissolved
Method 8215, 300-mL BOD bottle
1. Collect a water sample 2. Add the contents of 3. Immediately and 4. Again, invert the bottle
in a clean 300-mL BOD one Manganous Sulfate without trapping air in the several times and wait
bottle. Allow the sample to Powder Pillow and one bottle, insert the stopper. until the floc settles and
overflow the bottle for 2–3 Alkaline Iodide-Azide Invert the bottle several the top half of the solution
minutes to make sure that Reagent Powder Pillow. times to mix. is clear again.
a representative sample is A flocculent precipitate will Wait until the floc settles
available. form. It will be orange- the second time to make
brown if oxygen is present sure the reaction of the
or white if oxygen is sample and reagents is
absent. The floc settles complete.
slowly in salt water. The
settling normally takes
about five minutes. When
the floc settles, proceed to
step 4.
5. Remove the stopper 6. Select a sample 7. Insert a clean delivery 8. Turn the delivery knob
and add the contents of volume and Sodium tube into the titration to eject a few drops of
one Sulfamic Acid Powder Thiosulfate Titration cartridge. titrant. Reset the counter
Pillow. Replace the Cartridge from the Volume Attach the cartridge to the to zero and wipe the tip.
stopper without trapping multipliers table that titrator body.
air in the bottle. Invert the corresponds to the
prepared sample several expected dissolved
times to mix. oxygen (DO)
The floc will dissolve and concentration.
leave a yellow color if
oxygen is present.
Oxygen, Dissolved
Page 926
Oxygen, Dissolved
Method 8215, 300-mL BOD bottle (continued)
9. Use a graduated 10. Place the delivery tube 11. Add two 1-mL 12. Continue the titration
cylinder to measure the tip into the solution and droppers of Starch to a colorless end point.
sample volume from the swirl the flask while Indicator Solution and Record the number of
Volume multipliers table. titrating with sodium swirl to mix. digits required.
Transfer the sample into a thiosulfate to a pale yellow A dark blue color will
250-mL Erlenmeyer flask. color. develop.
13. Calculate:
Digits Required x
Digit Multiplier =
mg/L Dissolved Oxygen
Table 284 Volume multipliers

Range (mg/L DO) Volume (mL) Titration Cartridge (N Na2S2O3) Digit Multiplier
100–400 150 0.200 0.01

200–800 75 0.200 0.02
600–2400 25 2.000 0.10
Oxygen, Dissolved
Page 927
Oxygen, Dissolved
Method 8332, 60-mL BOD bottle
1. Collect a water sample 2. Add the contents of 3. Immediately and 4. Again invert the bottle
in a clean 60-mL BOD one Dissolved Oxygen 1 without trapping air in the several times and wait
bottle. Allow the sample to Powder Pillow and bottle, insert the stopper. until the floc settles and
overflow the bottle for 2–3 one Dissolved Oxygen 2 Invert the bottle several the top half of the solution
minutes to make sure that Powder Pillow. times to mix. is clear again.
a representative sample is A flocculent precipitate will Wait until the floc settles
obtained. form. It will be orange- the second time to make
brown if oxygen is present sure the reaction of the
or white if oxygen is sample and reagents is
absent. The floc settles complete.
slowly in salt water. The
settling normally takes
about five minutes. When
the floc settles, proceed to
step 4.
5. Remove the stopper 6. Accurately measure 7. Insert a clean straight- 8. Turn the delivery knob
and add the contents of 20 mL of the prepared stem delivery tube to a to eject a few drops of
one Dissolved Oxygen 3 sample and transfer it to a 0.200 N Sodium titrant. Reset the counter
Powder Pillow. Replace 50-mL Erlenmeyer flask. Thiosulfate Titration to zero and wipe the tip.
the stopper without Cartridge. Attach the
trapping air in the bottle cartridge onto the titrator
and invert several times to body.
mix.
The floc will dissolve and
oxygen is present.
Oxygen, Dissolved
Page 928
Oxygen, Dissolved
Method 8332, 60-mL BOD bottle (continued)
9. Titrate the prepared 10. Add two 1-mL 11. Continue the titration 12. Calculate:
solution with 0.2000 N droppers of Starch to a colorless end point. Digits Required x 0.1 =
Sodium Thiosulfate until Indicator Solution and Record the number of mg/L Dissolved Oxygen
the sample changes to a swirl to mix. digits required.
pale yellow color. Record A dark blue color will
the number of digits. develop.
Interferences
Nitrite interference is eliminated by the azide in the reagents. Other reducing or oxidizing
substances may interfere. If these are present, use an alternate method, such as the High Range
Dissolved Oxygen Method (colorimetric, Method 8166) or a dissolved oxygen electrode (4500
OG).

Sampling and sample handling are important in obtaining meaningful results. The dissolved
oxygen content of the sample changes with depth, turbulence, temperature, sludge deposits, light,
microbial action, mixing, travel time and other factors. A single dissolved oxygen test rarely reflects
the over-all condition of a body of water. Several samples taken at different times, locations and
depths are recommended for most reliable results.
• Collect samples in clean BOD Bottles.
• If storage is necessary, run steps 1 through 4 of the procedure and store in the dark at
10–20 °C.
• Seal the bottle with water by pouring a small volume of water into the flared lip area of a
stopper bottle.
• Use a BOD Bottle Cap over the flared lip.
• Samples preserved like this can be held 4–8 hours. Begin with step 5 when analyzing.
Accuracy check
Check the strength of the Sodium Thiosulfate Solution by using an Iodate-Iodide Standard
Solution, 10 mg/L as DO.
1. For the 300-mL procedure, begin at step 5, adding the Sulfamic Acid Powder Pillow to a
200-mL volume of Iodate-Iodide Standard Solution.
2. Use a 100-mL sample volume with the 0.200 N Sodium Thiosulfate Titration Cartridge. This
titration should take 500 digits. If more than 525 digits are required to reach the end point,
replace the Sodium Thiosulfate Cartridge.
Oxygen, Dissolved
Page 929
Oxygen, Dissolved
3. Use a 200-mL sample volume with the 2.00 N Sodium Thiosulfate Titration Cartridge. This
For the 60-mg/L procedure:
1. Begin the analysis at step 5, adding the Dissolved Oxygen 3 Powder Pillow to a 60 mL volume
of Iodate-Iodide Standard Solution.
2. Use a 20 mL sample volume with the 2.00 N Sodium Thiosulfate Titration Cartridge. This
Summary of method
Samples are treated with manganous sulfate and alkaline iodide-azide reagent to form an orange-
brown precipitate. Upon acidification of the sample, this floc reacts with iodide to produce free
iodine as triiodide in proportion to the oxygen concentration. The iodine is titrated with sodium
thiosulfate to a colorless end point.

Required reagents for 300-mL BOD bottle
Dissolved Oxygen Reagent Set (about 50 tests) 2272200
Includes:
(2) Alkaline Iodide-Azide Powder Pillows 50/pkg 107266
(2) Manganous Sulfate Powder Pillows 50/pkg 107166
(1) Sodium Thiosulfate Titration Cartridge, 0.2000 N each 2267501
(1) Starch Indicator Solution 100 mL MDB1 34932
(2) Sulfamic Acid Powder Pillows 50/pkg 2076266
Sodium Thiosulfate Titration Cartridge. 2.00 N each 1440101
1 MDB is Marked Dropper Bottle
Required apparatus for 300-mL BOD bottle

Bottle, with stopper, BOD, 300-mL each 62100
Clippers, for opening pillows each 96800
Delivery tubes w/ 180° hook each 1720500
Oxygen, Dissolved
Page 930
Oxygen, Dissolved
Required reagents for 60-mL BOD bottle

Dissolved Oxygen 1 Reagent Powder Pillows 100/pkg 98199
Sodium Thiosulfate Titration Cartridge, 0.2000 N each 2267501
Required apparatus for 60-mL BOD bottle

Bottle, with stopper, BOD, 60-mL each 190902
Clippers, for opening pillows each 96800
Polypropylene Beaker, 50-mL, Low Form, with pour spout each 108041
Required standards
Iodate-Iodide Standard Solution, 10-mg/L as DO 500 mL 40149
Thermometer -10 –225 °C 405 mm each 2635700
Cap, BOD Bottle Snap-over 6/pkg 241906
BOD Bottle, Serialized (#1-24) 24/pkg 2898700
Oxygen, Dissolved
Page 931
Oxygen, Dissolved, BT, 8229
USEPA1 Azide Modification of Winkler Method2 Method 8229

1 to more than 10 mg/L DO Buret Titration
1 USEPA approved.
2 Adapted from Standard Methods for the Examination of Water and Wastewater, (Standard Method 4500 O C)
Test preparation
Dissolved oxygen can be lost from the sample during sample collection. Review the precautions in Sample collection,
preservation and storage before the test is started.
Standard APHA solutions for dissolved oxygen can be used in place of the powder pillow reagents by substituting 1 mL of
Manganous Sulfate Solution, 1 mL of Alkaline Iodide-Azide Reagent and 1 mL of Sulfuric Acid (concentrated) in place of the
powder pillows. These solutions must be dispensed below the surface of the liquid.
BOD bottle, 300-mL 1
Alkaline Iodide-Azide Reagent Powder Pillow 1 pillow
Manganous Sulfate Powder Pillow 1 pillow
Sulfamic Acid Powder Pillow 1 pillow
Sodium Thiosulfate Standard Solution (titrant), 0.025 N 1 bottle
Oxygen, Dissolved
Page 933
Oxygen, Dissolved
Buret titration
1. Collect a water sample 2. Add the contents of 3. Immediately insert the 4. Again invert the bottle
in a clean, 300-mL, glass one Manganous Sulfate stopper so that no air is several times and wait
stoppered BOD bottle. Powder Pillow and one trapped in the bottle. Invert until the floc has settled
Overflow the bottle for two Alkaline Iodide-Azide several times to mix. and the top half of the
or three minutes to remove Reagent Powder Pillow. A flocculent precipitate will solution is clear again.
any trapped air bubbles to form. It will be Waiting until floc has
make sure that a orange-brown if oxygen is settled twice makes sure
representative sample is present or white if oxygen that the reaction is
available. is absent. complete. Results will not
The floc will settle very be affected if the floc does
slowly in salt water. Wait not completely settle.
five more minutes before
proceeding to step 4.
5. Fill a 25-mL buret to 6. Remove the stopper 7. Pour the prepared 8. Pour the contents of
the zero mark with 0.025 N and add the contents of sample into a 250-mL the graduated cylinder into
Sodium Thiosulfate one Sulfamic Acid Powder graduated cylinder to the a 250-mL Erlenmeyer
Solution. Pillow. Replace the 200-mL mark. flask.
stopper without trapping
air in the bottle and invert
several times to mix. This
is the prepared sample.
The floc will dissolve and
oxygen is present.
Oxygen, Dissolved
Page 934
Oxygen, Dissolved
9. Titrate the sample 10. Add two 1-mL 11. Continue the titration
while gently swirling the droppers of Starch until the solution changes
flask until it turns a very Indicator Solution. Swirl to from dark blue to
pale yellow color. mix. colorless.
The solution will turn dark The amount of titrant used
blue. to reach the end point is
equal to the concentration
of dissolved oxygen in the
sample.
mL titrant used = mg/L DO
Interferences
Nitrite interference is eliminated by the azide in the reagents. Other reducing or oxidizing
substances may interfere. If these are present, use an alternate method, such as the High Range
Dissolved Oxygen Method, (Hach method 8166 - colorimetric) or a dissolved oxygen electrode
(Standard Method 4500 O G).
Pretreatment procedure for activated sludge samples
A sample pretreatment is necessary for activated sludge samples.
1. Add 10 mL of Copper Sulfate-Sulfamic Acid Inhibitor Solution to a clean 1000-mL graduated
cylinder.
2. Fill the cylinder with the sample using a tube that empties near the bottom of the cylinder and
allow the sample to overflow by about 200 mL.
3. Swirl the cylinder to mix the contents. Allow the suspended solids to settle.
4. Siphon the relatively clear top layer into a BOD bottle through a siphon tube extended to the
bottom of the bottle. Withdraw the siphon tube while the water is flowing. Make sure that no air
bubbles are trapped in the bottle. Continue with step 2–step 11 of the test procedure.

dissolved oxygen content of the water being tested varies with depth, turbulence, temperature,
sludge deposits, light, microbial action, mixing, travel time and other factors. A single dissolved
oxygen test rarely reflects the overall condition of a body of water. Several samples taken at
different times, locations and depths are recommended for most reliable results.
Collect samples in a clean BOD bottle as described in step 1. If storage is necessary, complete
steps 1–4 of the test procedure and store in the dark at the temperature of the water source or
water seal at 10–20 °C. (Sealing with water is done by pouring a small amount of water into the
Oxygen, Dissolved
Page 935
Oxygen, Dissolved
flared lip area of a stoppered bottle. Snap a BOD bottle cap over the flared lip). Samples preserved
in this manner can be held four to eight hours. Start the test at step 6.
Accuracy check
The standard solution method can be used to confirm analytical technique and reagent
performance.
Complete the following test to make sure the concentration of the titrant is accurate.
• Iodate-Iodide Standard Solution, 0.00125 N (equivalent to 10 mg/L as O2)
1. Add 200.0 mL of Iodate-Iodide Standard Solution, 0.00125 N, to an Erlenmeyer flask.
2. Add one Sulfamic Acid Powder Pillow and swirl to mix.
3. Follow steps 9–11 of the test procedure to titrate the standard to the end point. The titration
should use 10.0 mL of the titrant solution. If more than 10.5 mL is used, discard the titrant and
replace it with a fresh supply.
Summary of method
The Azide Modification of the Winkler Method is the standard test for dissolved oxygen. In the
analysis, manganous ion reacts with the dissolved oxygen present in the alkaline solution to form a
manganese (IV) oxide hydroxide flocculent. Azide is then added to suppress interference from any
nitrite, which would react with the iodide. The solution is then acidified and the manganese (IV) floc
is reduced by iodide to produce free iodine as I3– in proportion to the oxygen concentration. The
liberated iodine is then titrated to the starch-iodide end point.
Oxygen, Dissolved
Page 936
Oxygen, Dissolved
Required reagents
Alkaline Iodide-Azide Reagent Powder Pillows 1 pillow 50/pkg 107266
Manganous Sulfate Powder Pillows 1 pillow 50/pkg 107166
Sodium Thiosulfate Standard Solution (titrant), 0.025 N varies 1L 2409353
Sulfamic Acid Powder Pillows 1 pillow 100/pkg 107399
Required apparatus
Bottle, glass-stoppered, BOD, 300-mL 1 each 62100
Iodate-Iodide Standard Solution, 0.00125 N 500 mL 40149

APHA reagents:
Alkaline Iodide-Azide Reagent Solution 500 mL 27749
Manganous Sulfate Solution 500 mL 27549
Graduated cylinder, 1000 mL each 50853
BOD bottle caps 6/pkg 241906
BOD bottles, serialized 24/pkg 2898700
Copper Sulfate-Sulfamic acid inhibitor 100 mL 35732
Copper Sulfate-Sulfamic acid inhibitor 500 mL 35749
Oxygen, Dissolved
Page 937
Oxygen Scavengers, 8140
Oxygen Scavengers DOC316.53.01105
Iron Reduction Method for Oxygen Scavengers Method 8140

5 to 600 µg/L carbohydrazide;
3 to 450 µg/L DEHA;
9 to 1000 µg/L hydroquinone; Powder Pillows
13 to 1500 µg/L iso-ascorbic acid [ISA];
15 to 1000 µg/L methylethyl ketoxime [MEKO]
Scope and Application: For testing residual corrosion inhibitors (oxygen scavengers)
in boiler feed water or condensate
Test preparation


The sample temperature should be 25 ± 3 °C (77 ± 5 °F).
Soak glassware with 1:1 hydrochloric acid solution. Rinse several times with deionized water. These two steps will remove
iron deposits that can cause slightly high results.
To determine ferrous iron concentration, repeat the procedure, but do not add DEHA Reagent 2. Correct for the ferrous iron
concentration: OPTIONS>MORE>REAGENT BLANK>ON. The reading attributed to the ferrous iron concentration will appear.
Oxygen Scavengers
Page 939
Oxygen Scavengers
Bottle, glass mixing, with 25-mL mark 2
DEHA Reagent 1 Powder Pillow 2
DEHA Reagent 2 Solution 1 mL
Dropper, 0.5 and 1.0 mL marks 1
Hydrochloric Acid, 1:1, 6.0 N varies
Iron reduction method for Oxygen Scavengers
180 O Scav-Carbohy
181 O Scav-DEHA
182 O Scav-Hydro
183 O Scav-ISA
184 O Scav-MEKO
1. Select the test. 2. Prepared Sample: Fill 3. Blank Preparation: 4. Add the contents of
Insert an adapter if a mixing bottle with 25 mL Fill a second mixing bottle one DEHA Reagent 1
required (see Instrument- of sample. with 25 mL of deionized Powder Pillow to each
specific information). When determining oxygen water. mixing bottle. Swirl to mix.
scavengers that react
quickly with oxygen at
room temperature, cap the
bottle.
Oxygen Scavengers
Page 940
Oxygen Scavengers
Iron reduction method for Oxygen Scavengers (continued)
5. Add 0.5 mL of DEHA 6. Start the instrument 7. When the timer 8. Immediately after
Reagent 2 Solution to timer. expires, transfer the blank transferring to the 10-mL
each bottle. Mix. Place A ten-minute reaction and prepared samples into cell, wipe the blank and
both sample cells in the period (or a two-minute the 10-mL sample cells. insert it into the cell holder.
dark. reaction period for Close the cover.
A purple color will develop hydroquinone) will begin.
if an oxygen scavenger is Keep the sample cells in
present. the dark during the
reaction period.
Zero Read
9. ZERO the instrument. 10. Immediately wipe the 11. READ the results
For greater accuracy, read prepared sample and in µg/L.
the result immediately insert it into the cell holder.
after the timer expires.
Interferences
Substances which reduce ferric iron will interfere. Substances which complex iron strongly may
also interfere.

Borate (as Na2B4O7) Greater than 500 mg/L
Cobalt Greater than 0.025 mg/L
Copper Greater than 8.0 mg/L
All levels.
Ferrous Iron
Note: Determine and subtract (see Before starting the test:)
Hardness (as CaCO3) Greater than 1000 mg/L
Oxygen Scavengers
Page 941
Oxygen Scavengers

Light Light may interfere. Keep sample cells in the dark during color development.
Lignosulfonates Greater than 0.05 mg/L
Manganese Greater than 0.8 mg/L
Molybdenum Greater than 80 mg/L
Nickel Greater than 0.8 mg/L
Phosphate Greater than 10 mg/L
Phosphonates Greater than 10 mg/L
Sulfate Greater than 1000 mg/L
Temperature Sample temperatures below 22 °C or above 28 °C (72 °F or 82 °F) may affect test accuracy.

• Collect samples in clean, dry, plastic or glass containers.
• Avoid excessive agitation or exposure to sunlight when sampling.
• Rinse the container several times with the sample prior to collection.
• Allow the container to overflow and cap the container so that there is no headspace above the
sample.
• Rinse the sample cell several times with the reacted sample, then carefully fill to the 10-mL
mark. Perform the analysis immediately.
Method performance
Program Standard 95% Confidence Limits of ΔConcentration change
Distribution per 0.010 ΔAbs change
180 299 µg/L 295–303 µg/L 4 µg/L
181 226 µg/L 223–229 µg/L 3 µg/L
182 600 µg/L 591–609 µg/L 8 µg/L
183 886 µg/L 873–899 µg/L 12 µg/L
184 976 µg/L 962–990 µg/L 14 µg/L
Summary of method
Diethylhydroxylamine (DEHA) or other oxygen scavengers present in the sample react with ferric
iron in DEHA Reagent 2 Solution to produce ferrous ion in an amount equivalent to the DEHA
concentration. This solution then reacts with DEHA 1 Reagent, which forms a purple color with
ferrous iron proportional to the concentration of oxygen scavenger. Test results are measured at
562 nm. This method reacts with all oxygen scavengers and does not differentiate samples
containing more than one type of oxygen scavenger.
Oxygen Scavengers
Page 942
Oxygen Scavengers
Required reagents
Oxygen Scavenger Reagent Set, includes: — — 2446600
(2) DEHA Reagent 1 Powder Pillows 2 100/pkg 2167969
(1) DEHA Reagent 2 Solution 1 mL 100 mL 2168042
Hydrochloric Acid, 1:1, 6.0 N varies 500 mL 88449
Required apparatus
Bottle, glass mixing, with 25-mL mark 2 each 1704200
Dropper, 0.5 and 1.0-mL marks 1 20/pkg 2124720
Optional apparatus
Thermometer, Non-Mercury, –10 to 225 °C each 2635700
Oxygen Scavengers
Page 943
Ozone, 8311
Ozone DOC316.53.01106
Indigo Method Method 8311

LR (0.01 to 0.25 mg/L O3),
MR (0.01 to 0.75 mg/L O3), AccuVac® Ampul
HR (0.01 to 1.50 mg/L O3)
Test preparation

Instrument Adapter
DR 6000 —
DR 5000 —
DR 3900 LZV846 (A)
DR 3800, DR 2800, DR 2700 LZV584 (C)
Analyze sample immediately. Do not preserve for later analysis.
Use tap water or deionized water for the blank (ozone-free water)
The sequence of measuring the blank and the sample is reversed in this procedure.
Select Ozone AccuVac® Ampuls based on range:
0–0.25 mg/L 2
0–0.75 mg/L 2
0–1.50 mg/L 2
Beaker, Polypropylene, 50-mL, low form with pour spout 1
Water, ozone-free varies
Ozone
Page 945
Ozone
Method Name for powder pillows
Stored Programs
454 Ozone LR AV
455 Ozone MR AV
456 Ozone HR AV
Start
1. Select the test. 2. Blank Preparation: 3. Prepared Sample: 4. Fill one Indigo Ozone
Insert an adapter if Collect at least 40 mL of Gently collect at least Reagent AccuVac Ampul
required (see Instrument- ozone-free water in a 40 mL of sample in with the sample and
specific information). 50-mL beaker. another 50-mL beaker. another with the blank.
Keep the tip immersed
while the Ampul fills.
Zero
5. Quickly invert both 6. Wipe the Ampuls with 7. ZERO the instrument. 8. Insert the blank into
Ampuls several times to a cloth to remove The display will show: the cell holder.
mix. fingerprints or other Press READ. Results are in
marks. 0.00 mg/L O3
Some of the blue color will mg/L O3.
be bleached if ozone is Insert the sample into the
present. cell holder.

The most important consideration when collecting a sample is to prevent the escape of ozone from
the sample. The sample should be collected gently and analyzed immediately. Warming the
sample or disturbing the sample by stirring or shaking, will result in ozone loss. After collecting the
sample, do not transfer it from one container to another unless absolutely necessary.
Stability of Indigo reagent

Because indigo is light-sensitive, the AccuVac Ampuls should be kept in the dark at all times. The
indigo solution, however, decomposes slowly under room light after filling with sample. The blank
Ampul can be used for multiple measurements during the same day.
Ozone
Page 946
Ozone
Method performance
Program Standard 95% Confidence Limits of ΔConcentration change
Distribution per 0.010 ΔAbs change
454 0.15 mg/L 0.14–0.16 mg/L O3 0.01 mg/L O3
455 0.45 mg/L 0.43–0.47 mg/L O3 0.01 mg/L O3
456 1.00 mg/L 0.97–1.03 mg/L O3 0.01 mg/L O3
Summary of method
The reagent formulation adjusts the sample pH to 2.5 after the Ampule has filled. The indigo
reagent reacts immediately and quantitatively with ozone. The blue color of indigo is bleached in
proportion to the amount of ozone present in the sample. Other reagents in the formulation prevent
chlorine interference. No transfer of sample is needed in the procedure, therefore ozone loss due
to sampling is eliminated. Test results are measured at 600 nm.
Required reagents
Select one or more Ozone AccuVac® Ampules based on range:

0–0.25 mg/L 2 25/pkg 2516025
0–0.75 mg/L 2 25/pkg 2517025
0–1.5 mg/L 2 25/pkg 2518025
Ozone
Page 947
Ozone
Required apparatus
Polypropylene Beaker, 50-mL, Low Form each 108041

Snapper, AccuVac® each 2405200

Water, demineralized 4L 27256
SpecCheck Gel Secondary Standard Kit, Ozone, 0 – 0.75 mg/L set each 2708000
Test tube stopper 6/pkg 173106

PCB in Soil, 10050
Polychlorinated Biphenyls (PCB)

in Soil DOC356.53.01107
Immunoassay Method1 Method 10050

Scope and Application: For soil
Test preparation


Instrument Adapter
DR 6000 —
DR 5000 A23618
DR 3900 LZV846 (A)
DR 3800, DR 2800, DR 2700 LZV583
This method analyzes for PCB that has been extracted from soil samples. Sample extracts, calibrators and reagents are
added to cuvettes coated with PCB-specific antibodies. The color that develops is then measured and compared with the
color measurements of the calibrators. The test requires about 20 minutes for complete analysis. As many as 10 cuvettes
can be run simultaneously
Read the entire procedure before starting. Identify and make ready all the necessary reagents, cuvettes and other
rack and mixing as described in Use the 1-cm MicroCuvette rack. Cuvettes can be mixed individually, but test results may not
be as consistent.
There are two protocols in this procedure, one for levels of 1 ppm and 5 ppm and another for 10 ppm and 50 ppm. Each uses
a different quantity of calibrator and sample extract See PCB protocols for more information.
Store the reagents at 4 °C when they are not in use. Allow the reagents to reach room temperature before using them in an
analysis. Actual testing may be done at temperatures ranging from 1– 38 °C.
The Soil Extractant contains methyl alcohol which is poisonous and flammable. Before using this and other reagents, read
the Material Safety Data Sheet (MSDS) for proper use of protective equipment and other safety information.
Polychlorinated Biphenyls (PCB) in Soil

Page 949
PCB Reagent Set 1
Caps, flip spout 1
Wipes, disposable 1
Pipet, TenSette®, 0.1–1.0 mL and pipet tips 1
Soil Extraction Kit and Soil Scoop 1
Analytical Balance 1
Cylinder, graduated 1
Scoop, 5 g 1
Soil extraction procedure
1. Weigh out 5 g of soil in 2. Carefully pour the soil 3. Use the 5-gram scoop 4. Use the graduated
the plastic weighing boat. into an extraction vial. to add one scoop of cylinder to transfer 10 mL
sodium sulfate to the of Soil Extractant into the
extraction vial. extraction vial.

Page 950
5. Cap the extraction vial 6. Allow to settle for at 7. Using the disposable 8. Insert the filtration
tightly and shake least one minute. Carefully bulb pipet, withdraw 1.0– plunger into the filtration
vigorously for one minute. open the extraction vial. 1.5 mL from the liquid barrel. Press firmly on the
layer at the top of the plunger until the sample
extraction vial. extract is forced upward
Transfer it into the filtration into the center of the
barrel (the bottom part of plunger.
the filtering assembly into Use the resultant filtrate
which the plunger inserts). for the immunoassay in
Do not use more than Immunoassay for soil
1.5 mL. The pipet is extracts.
marked in 0.25-mL It may be necessary to
increments. place the filtration
assembly on a table and
press down on the
plunger.
Immunoassay for soil extracts
Single Wavelength
4 5 0
OK
SINGLE WAVELENGTH Cuvette for each calibrator the rack snugly. diluent solution into each
and each sample to be cuvette. The same pipet
Press OPTIONS and the λ tested. tip can be used repeatedly
button. for this step.
Enter 450 nm and press Have the necessary
OK. apparatus at hand for
Insert an adapter if the next four steps; they
required (Instrument- must be done without
specific information). delay.
for orientation.

Page 951
Immunoassay for soil extracts (continued)
5. Use a Wiretrol® pipet 6. Immediately pipet 7. Set the instrument 8. After 5 minutes mix
to transfer the appropriate 0.5 mL of PCB Enzyme timer for 10:00 minutes. the contents of the rack for
volume of calibrator or Conjugate into each Start the reaction period. 30 seconds (Use the 1-cm
sample extract into each calibrator and sample A 10-minute reaction time MicroCuvette rack.)
cuvette (see the PCB cuvette. The same pipette will begin. Immediately
protocols table). tip can be used to add the begin mixing the cuvettes
Use a separate capillary enzyme conjugate to each for 30 seconds. See Use
tube for each solution. cuvette. the 1-cm MicroCuvette
rack.

The sample PCBs and Make sure that most of the
calibrator PCBs remain water is drained from the
attached to the cuvette cuvettes by turning the
walls. cuvettes upside down and
paper towel.

Page 952
Immunoassay for soil extracts (continued)

Color Development
11. With the cuvettes still 12. Set the instrument 13. After 2.5 minutes, mix 14. At the end of the
held snugly in the rack, timer for 5:00 minutes. the contents of the rack a 5-minute reaction period,
pipet 0.5 mL of Color Start the reaction period. second time for a period of pipette 0.5 mL of Stop
Developing Solution into Mix, using the instructions 30 seconds using the Solution into each cuvette
each cuvette. in Use the 1-cm same technique as in the same order as the
Use a new pipette tip for MicroCuvette rack. step 12. Color Developing Solution
each cuvette. Solutions will turn blue in was added in step 11.
some or all of the cuvettes. Slide the rack for 20
seconds (see Use the 1-
cm MicroCuvette rack.)
the Stop Solution.
Use the same pipette tip
Measuring the Color
Zero
water. Wipe the outside of cell holder see Table 0.000 Abs holder.
all the cuvettes with a Instrument- Record the results (ABS)
tissue to remove water, specific information for cell for each calibrator
smudges and fingerprints. orientation. and sample.
Orient the arrow in the See Interpreting and
same direction for all reporting results for help
cuvettes. with interpretation of
results.

Page 953
PCB protocols
There are two protocols in this procedure, one for levels of 1 ppm and 5 ppm and another for 10
ppm and 50 ppm. Each uses a different volume of calibrator and sample extract. Refer to the PCB
protocols table for range and volume information.
Table 289 PCB protocols
Range (as Arochlor 1248) Volume of calibrator and sample extract used
1 ppm and 5 ppm 50 µL
10 ppm and 50 ppm 10 µL
To test across ranges, such as 1 and 50 ppm, test the lower concentration first. If the result is
positive then test at the higher level. If the result of the test at the lower concentration is negative,
the higher range test will be negative also and need not be performed.
The same filtered extract can be used for both protocols if it is tightly capped between assays. The
maximum time between assays cannot exceed one-half hour.
Using the Wiretrol®* pipet

The Wiretrol Pipet can accurately measure small quantities of liquids. It consists of two parts: a
Teflon®-tipped plunger and a calibrated capillary tube. The plunger can be reused; the capillary
tubes must be discarded after one use.
1. Wet the orange 2. Push the tip to the 3. Submerge the 4. To discharge the pipet,
Teflon® tip of the Wiretrol other end of the capillary capillary tube below the place the tip of the
plunger in the sample and tube until it barely extends surface of the liquid to be capillary tube below the
carefully insert it into the beyond the end of the pipetted. Slowly and surface of the solution
end of the capillary tube capillary tube. smoothly draw the Wiretrol and push the Wiretrol
with the colored band. plunger up until the bottom plunger down in one
of the plunger tips reaches smooth motion. Change
the appropriate volume capillary tubes for each
line. calibrator and sample.
Touch the end of the tube
to the side of the vessel to
release remaining drops
on the capillary tube tip.
* Wiretrol is a registered trademark of Drummond Scientific.

Page 954
Use the 1-cm MicroCuvette rack

The MicroCuvette rack (refer to The 1-cm MicroCuvette rack figure) has been designed
specifically to aid in achieving precise and accurate results when using the immunoassay
technique to analyze several samples at the same time.
Figure 1 The 1-cm MicroCuvette rack
Loading the rack—The cuvette rack is designed so that it may be inverted with the cuvettes in

Page 955

There is an inverse relationship between the concentration of PCB and the reading. In other
words, the higher the reading, the lower the concentration of PCB.
Table 290 Relative PCB Concentration
If the sample reading is... the sample PCB Concentration is...
Example
Readings:
1 ppb PCB Calibrator: 0.775 Abs
5 ppb PCB Calibrator: 0.430 Abs
Interpretation for a soil sample
concentration of PCB is greater than both 1 ppm and 5 ppm as Aroclor 1248.
Sample #2—Sample reading is between the readings for the 1 ppm and 5 ppm PCB calibrators.
Therefore the sample concentration of PCB is between 1 ppm and 5 ppm as Aroclor 1248.
sample concentration of PCB is less than both 5 ppm and 1 ppm as Aroclor 1248.

• Wear protective gloves and eyewear.
• When storing reagent sets for extended periods of time, keep them out of direct sunlight. Store
• Keep the foil pouch containing the Antibody Cuvettes sealed when not in use.
• If Stop Solution comes in contact with eyes, wash thoroughly for 15 minutes with cold water

Page 956
Sensitivity
The PCB immunoassay cannot differentiate between the various Arochlors, but it detects their
presence in differing degrees.
Table 291 Various PCBs in soil
Concentration (ppm) to give a positive result at
Compound
1 ppm 5 ppm 10 ppm 50 ppm
1248 1 5 10 50
1016 2 9 20 67
1242 1.2 6 14 50
1254 1.4 4.6 11 28
1260 1.1 4.9 11 38
Table 292 Compounds not detectable at 1000 ppm

Biphenyl 2,4,6-trichlorophenyl 1,3-dichlorobenzene
2,4-dichlorophenyl pentachlorophenol 1,4-dichlorobenzene
2,4,5-trichlorphenyl 1,2-dichlorobenzene 1,2,4-trichlorobenzene

• Analyze the samples as soon as possible after collection.
• If the samples must be stored, collect them in glass or Teflon® containers that have been
washed with soap and water and rinsed with methanol. The container should be capped with a
Teflon-lined cap.
• If a Teflon cap is not available, aluminum foil rinsed in methanol may be used as a substitute
cap liner.
Summary of method
and soil. Antibodies specific for PCB are attached to the walls of plastic cuvettes. They selectively
bind and remove PCB from complex sample matrices. A prepared sample and a reagent
containing enzyme-conjugate molecules (analyte molecules attached to molecules of an enzyme)
are added to the Antibody Cuvettes. During incubation, enzyme-conjugate molecules and PCB
compete for binding sites on the antibodies. Samples with higher levels of analyte will have more
antibody sites occupied by PCB and fewer antibody sites occupied by the enzyme-conjugate
molecules.
color. Therefore, there is an inverse relationship between color intensity and the amount of PCB in
the sample. The resulting color is then compared with a calibrator to determine whether the PCB
concentration in the sample is greater or less than the threshold levels. The PCB concentration is
inversely proportional to the color development: the lighter the color, the higher the PCB
The method reacts with all PCBs and cannot differentiate samples containing more than one type
of PCB.

Page 957

Required reagents
PCB Reagent Set1 20 cuvettes 2773500

Deionized Water 500 mL 27249
Required apparatus
Wipes, disposable 280/box 2097000
Balance, analytical, 80 g capacity, 100–240 VAC each 2936701
Graduated cylinder, 10-mL each 108138
Soil Scoop, 5-g, 4.25-cc each 2657205
Soil Extraction Refill Kit, includes: each 2775200
Dropper, LDPE, 0.5 and 1.0-mL 20/pkg 2124720
Filter and Barrel Assembly 20/pkg 2567620
Sodium Sulfate, anhydrous 250 g 709929
Soil Extractant Solution 200 mL 2567729
Soil Sample Container 20/pkg 2592920
Weighing Boat, 8.9-cm square 20/pkg 2179020
Spatula, disposable 2/pkg 2569320

Gloves, Disposable, Nitrile, Medium1 100/pkg 2550502

1 Other sizes are available.

Phenols, 8047
Phenols DOC316.53.01108
USEPA1 4-Aminoantipyrine Method2 Method 8047

0.002 to 0.200 mg/L
Scope and Application: For Water and Wastewater
1 USEPA accepted (distillation required); procedure is equivalent to USEPA method 420.1 for wastewater
Test preparation


Analyze samples within four hours to avoid oxidation.
Spilled reagent affects test results and is hazardous to skin and other materials.
Use chloroform only with proper ventilation.
Phenol 2 Reagent Powder Pillows contain potassium ferricyanide. Both chloroform (D022) and cyanide (D001) solutions are
regulated as hazardous waste by the Federal RCRA. Do not pour these materials down the drain. Chloroform solutions
and the cotton plug used in the delivery tube of the separatory funnel should be collected for disposal as a reactive waste. Be
sure that cyanide solutions are stored in a caustic solution with a pH >11 to prevent release of hydrogen cyanide gas. Refer
to a current MSDS for safe handling and disposal information.
Phenols
Page 959
Phenols
Chloroform, ACS 60 mL
Clippers 1
Cotton Balls 1
Funnel, separatory, 500-mL 2
Hardness 1 Buffer Solution, pH 10.1 10 mL
Phenol 2 Reagent Powder Pillows 2
Phenol Reagent Powder Pillows 2
Pipet, volumetric, Class A, 5.00-mL 1
Ring, support, 4-inch 2
Support for Ring Stand, 5 x 8 inch base 1
4-Aminoantipyrine Method
Stored Programs
470 Phenol
Start
1. Select the test. 2. Measure 300 mL of 3. Blank Preparation: 4. Measure 300 mL of

Insert an adapter if deionized water in a Pour the measured sample in a 500-mL
required (see Instrument- 500-mL graduated deionized water into a graduated cylinder.
specific information). cylinder. 500-mL separatory funnel.
Phenols
Page 960
Phenols
4-Aminoantipyrine Method (continued)
5. Prepared Sample: 6. Add 5 mL of Hardness 7. Add the contents of 8. Add the contents of
Pour the measured Buffer to each separatory one Phenol Reagent one Phenol 2 Reagent
sample into another funnel. Stopper and shake Powder Pillow to each Powder Pillow to each
500-mL separatory funnel. to mix. separatory funnel. Stopper separatory funnel. Stopper
and shake to dissolve. and shake to dissolve.
9. Add 30 mL of 10. Invert each funnel and 11. Remove the stoppers. 12. Insert a large,
chloroform to each temporarily vent. Shake Allow both funnels to stand pea-sized cotton plug into
separatory funnel. Stopper each funnel briefly and until the chloroform settles the delivery tube of each
each funnel. vent. Then vigorously to the bottom of the funnel. funnel.
shake each funnel for a The chloroform layer will Filtering the chloroform
total of 30 seconds be yellow to amber if layer through the cotton
(venting if necessary). phenol is present. removes suspended water
or particles. The volume of
Note: Make sure that steps chloroform extract will be
12 through 16 are performed about 25 mL.
quickly because chloroform
will evaporate, causing high
readings.
Phenols
Page 961
Phenols
4-Aminoantipyrine Method (continued)
Zero
13. Drain the chloroform 14. Wipe the blank and 15. ZERO the instrument. 16. Wipe the prepared
layers into separate insert it into the cell holder The display will show: sample and insert it into
sample cells (one for the the cell holder
blank and one for each 0.000 mg/L Phenol
sample). Phenol.
Stopper the cells.
The water phase contains
chloroform, which is
hazardous. Dispose
properly.
Interferences

pH The sample pH must be between 3 and 11.5 for best results.
Oxidizing or reducing agents May interfere. Distill samples (see procedure below).
1. Distillation or the following pretreatment is necessary:
2. Fill a clean 500-mL graduated cylinder with 350 mL of sample. Pour the sample into a
Sulfides or suspended matter clean 500-mL Erlenmeyer flask.
4. Filter 300 mL of the sample through a folded filter paper1. Use this solution in step 4.
1 See Distillation reagents and apparatus.

Most reliable results are obtained when samples are analyzed within four hours after collection.
Use the following storage instructions only if prompt analysis is not possible:
1. Collect 500 mL of sample in clean glass containers and add the contents of two Copper
Sulfate Powder Pillows.
2. Adjust the pH to 4 or less with 10% Phosphoric Acid Solution. Store at 4 °C (39 °F) or lower
and analyze within 24 hours.
Phenols
Page 962
Phenols
Accuracy check
Note: For greater accuracy, analyze standard solutions when new lots of reagent are first used.

• Phenol, ACS
• Deionized water
• 1000-mL Class A volumetric flasks
• 10 mL Class A volumetric pipet and safety bulb
1. Prepare a 1000-mg/L phenol stock solution as follows:
a. Weigh 1.000 g of Phenol ACS.

b. Transfer the phenol to a 1000-mL volumetric flask.
c. Dilute to the mark with freshly boiled and cooled deionized water and mix to dissolve.
2. Prepare a 10-mg/L working phenol solution as follows:
a. Pipet 10.0 mL of the 1000-mg/L stock solution to a 1000-mL volumetric flask.

b. Dilute to the mark with deionized water.
3. Prepare a 0.200-mg/L standard solution as follows:
a. Pipet 10.0 mL of the 10-mg/L working solution to a 500-mL volumetric flask.

b. Dilute to the mark with deionized water.
4. Use this solution in place of the sample. Follow the 4-Aminoantipyrine Method test procedure.
to Standard Adjust in the software: OPTIONS> MORE>STANDARD ADJUST.
Distillation
Sample distillation as described in the following steps will eliminate interferences. The sample pH
must be between 3 and 11.5 for the best results. See the Interfering substances table for
pretreatment guidelines.
1. Set up the Distillation Apparatus* by assembling the general purpose apparatus as shown in
the Distillation Apparatus Manual. Use the 500-mL Erlenmeyer flask to collect the distillate. It
may be necessary to use a laboratory jack to elevate the flask.
2. Place a stirring bar into the flask.
3. Measure 300 mL of water sample in a clean 500-mL graduated cylinder. Pour it into the
distillation flask.
* See Distillation reagents and apparatus.
Phenols
Page 963
Phenols
4. For proof of accuracy, use a 0.200-mg/L phenol standard (see Accuracy check) in addition to
the sample.
5. Using a serological pipet, add 1 mL of Methyl Orange Indicator to the distillation flask.
6. Turn on the stirrer power switch. Set the stir control to 5.
7. Add 10% Phosphoric Acid Solution drop-wise until the indicator changes from yellow
to orange.
8. Add the contents of one Copper Sulfate Powder Pillow and allow to dissolve (omit this step if
copper sulfate was used to preserve the sample). Cap the distillation flask.
9. Turn the water on and adjust it so a constant flow is maintained through the condenser. Set the
heat control to 10.
10. Collect 275 mL of distillate in the Erlenmeyer flask, then turn the heat off.
11. Fill a 25-mL graduated cylinder to the 25-mL mark with deionized water. Add the water to the
distillation flask.
12. Turn the still back on. Heat until another 25-mL of distillate is collected.
13. Using a clean graduated cylinder, re-measure the distillate to make sure that 300 mL has been
collected. The distillate is ready for analysis.
Method performance
470 0.100 mg/L phenol 0.093–0.107 mg/L phenol 0.002 mg/L phenol
Phenols
Page 964
Phenols
Summary of method
The 4-aminoantipyrine method measures all ortho- and meta-substituted phenols. These phenols
react with 4-aminoantipyrine in the presence of potassium ferricyanide to form a colored antipyrine
dye. The dye is then extracted from the aqueous phase with chloroform and the color is measured
at 460 nm. The sensitivity of the method varies with the type of phenolic compound. Because
water samples may contain various types of phenolic compounds, the test results are expressed
as the equivalent concentration of phenol.
Required reagents
Phenols Reagent Set (100 Tests), includes: — — 2243900
(2) Chloroform, ACS 60 mL 4L 1445817
(3) Hardness 1 Buffer Solution, pH 10.1 10 mL 500 mL 42449
(2) Phenol 2 Reagent Powder Pillows 2 100/pkg 183699
(2) Phenol Reagent Powder Pillows 2 100/pkg 87299
Required apparatus
Cotton Balls 1 100/pkg 257201
Pipet Bulb, safety 1 each 1465100
Ring, support, 4-inch 2 each 58001
Support for Ring Stand, 5 x 8 inch base 2 each 56300

Balance, analytical, 80 g capacity, 100–240 VAC each 2936701
Copper Sulfate Powder Pillows 50/pkg 1481866
Distillation Heater and Support Apparatus, 115 VAC each 2274400
Distillation Heater and Support Apparatus, 230 VAC each 2274402
Distillation Apparatus Set, general purpose each 2265300
Filter Paper, 12.5 cm 100/pkg 189457
Funnel, 65 mm poly each 108367
Methyl Orange Indicator Solution, 0.5-g/L 100 mL MDB 14832
Phenol, ACS 113 g 75814
Phenols
Page 965
Phenols
Distillation reagents and apparatus (continued)

Phosphoric Acid Solution, 10% 100 mL MDB 1476932
Gloves, Chemical Resistant, 9–9½ inch1 1 pair 2410104

Phosphonates, 8007
Phosphonates DOC316.53.01109
Persulfate UV Oxidation Method1 Method 8007

Multiple Ranges from 0.02 to 125.0 mg/L Powder Pillows
Scope and Application: For boiler and cooling water, wastewater and seawater
1 Adapted from Blystone, P., Larson, P., A Rapid Method for Analysis of Phosphate Compounds, International Water Conference, Pittsburgh,
PA. (Oct 26-28, 1981)
Test preparation


Clean glassware with 1:1 Hydrochloric Acid Solution, followed by a distilled water rinse. Do not clean glassware with
commercial detergent.
Wear UV safety goggles while the UV lamp is on.
Do not handle the UV lamp surface. Fingerprints will etch the glass. Wipe the lamp with a soft, clean tissue between samples
The digestion in step 7 is normally completed in less than 10 minutes. However, high-organic loaded samples or a weak
lamp can cause incomplete phosphate conversion. To check conversion efficiency, perform a longer digestion and make
sure the readings do not increase.
Bottle, square, with 25-mL mark 1
Cylinder, mixing, graduated, 50-mL 1
Goggles, UV safety 1
PhosVer® 3 Phosphate Reagent Powder Pillows 2
Potassium Persulfate Powder Pillow for Phosphonate 1
Phosphonates
Page 967
Phosphonates
Safety bulb 1
UV Lamp with Power Supply 1
Persulfate UV Oxidation method for powder pillows
Stored Programs
501 Phosphonates
Start
1. Select the test. 2. Choose the 3. Blank Preparation: 4. Digested Sample:

Insert an adapter if appropriate sample size Fill a sample cell to the Fill a sample mixing bottle
required (see Instrument- from the Expected ranges 10-mL mark with diluted to the 25-mL mark with
specific information). with multipliers table. Pipet sample from step 2. diluted sample from
the chosen volume into a step 2.
50-mL graduated cylinder.
If necessary, dilute the
sample to 50-mL with
deionized water and mix
well.
5. Add the contents of 6. Insert the ultraviolet 7. Turn on the UV lamp. 8. When the timer
one Potassium Persulfate (UV) lamp into the sample Start the instrument timer. expires, turn off the UV
for Phosphonate Powder bottle. lamp and remove it from
Pillow to the bottle A ten-minute reaction the sample.
containing 25 mL of WARNING period will begin.
sample. Wear UV safety goggles Phosphonates are
Swirl to dissolve the while the lamp is on. converted to
powder. orthophosphate in this
step.
Phosphonates
Page 968
Phosphonates
Persulfate UV Oxidation method for powder pillows
9. Prepared Sample: 10. Add the contents of 11. Start the instrument 12. When the timer
Fill a second sample cell one PhosVer 3 Phosphate timer. expires, insert the blank
to the 10-mL mark with the Reagent Powder Pillow to A two-minute reaction into the cell holder.
digested sample. the blank and prepared period will begin. Complete steps 13–16
sample. Immediately swirl within three minutes after
vigorously 20–30 seconds If the sample is colder than
15 °C, allow four minutes the timer expires.
to mix. Some powder may
not dissolve. for color development.
A blue color will develop if

phosphate is present. Both
sample and blank cells
may develop color. The
increase in sample color is
proportional to
the phosphonate
concentration.
Zero Read Read
13. ZERO the instrument. 14. Wipe the prepared 15. READ the results in 16. Multiply the value in
The display will show: sample and insert it into mg/L PO43–. step 15 by the appropriate
0.00 mg/L PO43– the cell holder. multiplier in the Expected
ranges with multipliers
table to obtain the actual
phosphonate
concentration.
Table 296 Expected ranges with multipliers

Expected range
Sample volume (mL) Multiplier
(mg/L phosphonate)
0 – 2.5 50 0.1
0–5 25 0.2
Phosphonates
Page 969
Phosphonates
Table 296 Expected ranges with multipliers

Expected range
Sample volume (mL) Multiplier
(mg/L phosphonate)
0 – 12.5 10 0.5
0 – 25 5 1.0
0 – 125 1 5.0
To express results in terms of active phosphonate, multiply the final value in step 16 by the
appropriate conversion factor list in the Conversion factors by phosphonate type table.
Table 297 Conversion factors by phosphonate type

Phosphonate type Conversion factor
PBTC 2.84
NTP 1.050
HEDPA 1.085
EDTMPA 1.148
HMDTMPA 1.295
DETPMPA 1.207
HPA 1.49
active phosphonate (mg/L) = phosphonate concentration from step 16 x conversion factor
Phosphonates
Page 970
Phosphonates
Interferences
Interference levels decrease as the sample size increases. For example, copper does not interfere
at or below 100 mg/L for a 5.00 mL sample. If the sample volume is increased to 10 mL, copper
will begin to interfere above 50 mg/L.

Interfering substance Interference level (using 5 mL of sample)
Aluminum 100 mg/L
Arsenate Interferes at all levels
Benzotriazole 10 mg/L
Bicarbonate 1000 mg/L
Bromide 100 mg/L
Calcium 5000 mg/L
CDTA 100 mg/L
Chloride 5000 mg/L
Chromate 100 mg/L
Copper 100 mg/L
Cyanide 100 mg/L (Increase the UV digestion to 30 minutes.)
Diethanoldithiocarbamate 50 mg/L
EDTA 100 mg/L
Iron 200 mg/L
Nitrate 200 mg/L
NTA 250 mg/L
Orthophosphate 15 mg/L
Phosphites and
organophosphorus Reacts quantitatively. Meta- and polyphosphates do not interfere.
compounds
Silica 500 mg/L
Silicate 100 mg/L
Sulfate 2000 mg/L
Sulfide Interferes at all levels
Sulfite 100 mg/L
Thiourea 10 mg/L
extreme sample pH

• Collect samples in acid-cleaned (1:1 HCl) plastic or glass bottles that have been rinsed with
distilled water. Do not use a commercial detergent.
• If prompt analysis is impossible, preserve the sample by adjusting to pH 2 or less with Sulfuric
Acid (about 2 mL per liter).
• Store at 4 °C (39 °F). Preserved samples may be stored up to 24 hours.
Phosphonates
Page 971
Phosphonates
Accuracy check
Ideally, prepare a solution containing the exact phosphonate product to be tested. This will check
the UV conversion of phosphonate to orthophosphate. Alternatively, a phosphate standard can be
used to check the accuracy of the colorimetric part of the method.

• Phosphate Standard Solution, 1 mg/L
• Deionized water
Use 10 mL of a Phosphate Standard Solution, 1 mg/L solution in place of the sample

beginning at step 9 of the Persulfate UV Oxidation method for powder pillows test procedure.
Use deionized water for the blank. A multiplier value from the Expected ranges with multipliers
table is not needed. The expected result is 10.0 mg/L phosphate, due to a factor of 10
in the calibration.
Method performance
Precision—95% Confidence Limits of

Distribution
501 DR 5000 2.00 mg/L PO43– 1.97–2.03 mg/L PO43–
Sensitivity
The sensitivity depends on the sample volume. Sensitivity is expressed as PO43– in this table. Use
the Conversion factors by phosphonate type table to express as a specific phosphonate.
Range (mg/L) Volume (mL) Change in concentration per 0.010 Abs
0–2.5 50 0.02 mg/L PO43–

0–5 25 0.04 mg/L PO43–
0–12.5 10 0.10 mg/L PO43–
0–25 5 0.20 mg/L PO43–
0–125 1 1.00 mg/L PO43–
Summary of method
This method is directly applicable to boiler and cooling tower samples. The procedure is based on
a UV-catalyzed oxidation of phosphonate to orthophosphate. The orthophosphate reacts with the
molybdate in the PhosVer 3 reagent to form a mixed phosphate/molybdate complex. This complex
is reduced by the ascorbic acid in the PhosVer 3, yielding a blue color that is proportional to the
phosphonate present in the original sample. The orthophosphate present in the original sample is
subtracted out by preparing the blank and using it to set zero concentration. Test results are
measured at 880 nm.
Phosphonates
Page 972
Phosphonates
Required reagents
Phosphonate Reagent Set for 10-mL sample (100 tests), includes: — — 2429700
PhosVer® 3 Phosphate Reagent Powder Pillows, 10-mL 2 100/pkg 2106069
Potassium Persulfate Powder Pillow for Phosphonate 1 100/pkg 2084769
Required apparatus
Bottle, square, with 25-mL mark 1 each 1704200
Polypropylene Beaker, 50-mL, low form 1 each 108041
Cylinder, mixing, graduated, 50-mL 1 each 189641
Goggles, UV safety 1 each 2113400
Pipet, serological, graduated, 10-mL 1 each 53238
Safety Bulb 1 each 1465100
UV Lamp with Power Supply, 115 VAC 1 each 2082800
OR
UV Lamp with Power Supply, 230 VAC 1 each 2082802
Phosphate Standard Solution, 1-mg/L 500 mL 256949

Hydrochloric Acid Solution, 1:1 500 mL 88449
Sulfuric Acid, ACS Grade 500 mL 97949
Thermometer, –10 to 225 °C, 405 mm each 2635700
pH paper, 0–14 pH 100/pkg 2601300
Phosver 3 Phosphate Reagent Powder Pillows, 10 mL 1000/pkg 2106028
UV Lamp, shortwave, pencil type each 2671000
Power Supply, 115V/60Hz each 2670700
Power Supply, 220V/50Hz each 2670702
Phosphonates
Page 973
Phosphonates
Optional standards
Phosphate Standard Solution, 3 mg/L 946 mL 2059716
Phosphate Standard Solution, 50 mg/L, 10 mL ampules 16/pkg 17110
Phosphate Standard Solution, 500 mg/L, 10 mL ampules 10/pkg 1424210
Phosphate Standard Solution 100 mL 1424232

Phosphorus, Acid Hydrolyzable Digestion, 8180
Phosphorus, Acid Hydrolyzable

Digestion DOC316.53.01110
USEPA1 Acid Digestion Method2 Method 8180

Scope and Application: For water, wastewater, and seawater
1 USEPA Accepted for wastewater analyses
2 Adapted from Standard Methods for the Examination of Water and Wastewater 4500-P B & E
Test preparation
Rinse all glassware with 1:1 hydrochloric acid. Rinse again with deionized water.
The results of the reactive phosphorus test after the digestion will include the orthophosphate and the acid-hydrolyzable
(condensed) phosphate. The condensed phosphate concentration is determined by subtracting the result of an
orthophosphate test from this result. Make sure that both results are in the same units, either mg/L PO43– or mg/L P before
subtracting. The result from this Acid Hydrolyzable test is subtracted from the result of a total phosphorus test to determine
organic phosphorus.
Sodium Hydroxide Solution, 5.0 N 2 mL
Sulfuric Acid Solution, 5.25 N 2 mL
Hot Plate 1
Phosphorus, Acid Hydrolyzable Digestion

Page 975
Acid digestion
1. Use a graduated 2. Use a 1-mL calibrated 3. Place the flask on a 4. Cool the sample to
cylinder to measure 25 mL dropper to add 2.0 mL of hot plate. Boil gently for 30 room temperature.
of sample. Pour the 5.25 N Sulfuric Acid minutes. Do not boil dry.
sample into a 125-mL Solution to the flask. Concentrate the sample to
Erlenmeyer flask. less than 20 mL for best
recovery. After
concentration, maintain
the volume near 20 mL by
adding small amounts of
deionized water. Do not
exceed 20 mL.
480 P React. Mo
482 P React. Mo. AV
485 P React. Amino
490 P React. PV
492 P React. PV AV
535 P React. PV TNT
540 P React. HT TNT
5. Use a 1-mL calibrated 6. Pour the sample into a 7. Proceed with a

dropper to add 2.0 mL of 25-mL graduated cylinder. reactive phosphorus test
5.0 N Sodium Hydroxide Adjust the volume to 25 of the expected acid
Solution to the flask. Swirl mL with deionized water hydrolyzable phosphorus
to mix. rinsings from the flask. concentration range.
Extend the color
development time to
10 minutes for the
PhosVer 3 method.

Page 976
Interferences

Alkaline or highly buffered
It may be necessary to add additional acid in step 2 to drop the pH of the solution below 1.
samples
Use 50 mL of sample and double the reagent quantities. Use a portion of the digested
Turbidity sample to zero the instrument in the reactive phosphorus procedure. This compensates for
any color or turbidity destroyed by this procedure.

• Analyze the samples immediately for the most reliable results.
• If prompt analysis is not possible, samples may be preserved up to 28 days. Filter immediately
and store at 4 °C (39 °F).
Summary of method
Phosphates present in condensed inorganic forms (meta-, pyro- or other polyphosphates) must be
converted to reactive orthophosphate before analysis. Pretreatment of the sample with acid and
heat hydrolyzes the condensed inorganic forms to orthophosphate.
This procedure must be followed by one of the reactive phosphorus (orthophosphate) analysis
methods for determining the phosphorus content of the sample. If the ascorbic acid (PhosVer 3)
method is used to measure the reactive phosphorus, this method is USEPA accepted for NPDES
reporting.

The following reagents and apparatus are required in addition to those required for the active
phosphorus test.
Required reagents
Sodium Hydroxide Solution, 5.0 N 2 mL 100 mL MDB 245032
Sulfuric Acid Solution, 5.25 N 2 mL 100 mL MDB 244932
Required apparatus
Hot Plate, 7” x 7” Digital, 120 VAC 1 each 2881500
Hot Plate Stirrer, 7” x 7” Digital, 220–240 VAC 1 each 2881602

Page 977
Required apparatus (field applications)

Heatab Cookit, with 1 box Heatabs each 220600
Heatab Replacements 21/pkg 220700

Sodium, Hydroxide, 5.0 N 1000 mL 245053
Filter Paper, folded 12.5 cm 100/pkg 69257
Filter Funnel, Analytical, 65 mL each 108367

Phosphorus, Acid Hydrolyzable, 8180
Phosphorus, Acid Hydrolyzable DOC316.53.01111
PhosVer™ 3 with Acid Hydrolysis Method Method 8180

0.06 to 3.50 mg/L PO43– Test ‘N Tube™ Vials
Test preparation


DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
adjust.
Clean glassware with 1:1 Hydrochloric Acid Standard Solution. Rinse with deionized water. Do not use detergents that
contain phosphate to clean glassware.
Final samples will contain molybdenum. In addition, final samples will have a pH less than 2 and are considered corrosive
(D002) by the Federal RCRA. Refer to the current MSDS for safe handling and disposal instructions.
Total and Acid Hydrolyzable Phosphorus Reagent Set 1
DRB200 Reactor 1
Funnel, micro 1
Pipet, TenSette®, 1 to 10 mL, plus tips
Test Tube Rack 1

Page 979
Acid Hydrolysis, TNT method
Stored Programs
536 P Total/AH PV TNT
Start
1. Turn on the DRB200 2. Select the test. 3. Use a TenSette Pipet 4. Insert the vial into the
Reactor. Preheat to Insert an adapter or light to add 5 mL of sample to a preheated DRB200
150 °C. shield if required (see Total and Acid reactor. Close the
See the DRB200 User Instrument-specific Hydrolyzable Test Vial. protective cover.
Manual for selecting pre- information). Cap and mix.
programmed temperature
applications.
5. Start the instrument 6. After the timer expires, 7. Using a TenSette 8. Clean the outside of
timer. carefully remove the vial Pipet, add 2 mL of 1.00 N the vial with a towel to
A 30-minute heating from the reactor. Insert it in sodium hydroxide to the remove fingerprints or
period will begin. a test tube rack and cool to vial. Cap tightly and shake other marks.
room temperature. to mix.
Zero
9. Insert the sample vial 10. ZERO the instrument. 11. Using a funnel, add 12. Immediately cap
into the 16-mm round cell The display will show: the contents of one tightly and shake to mix for
holder. PhosVer 3 Powder Pillow 10–15 seconds. The
0.00 mg/L PO43– to the vial. powder will not completely
dissolve.

Page 980
Acid Hydrolysis, TNT method (continued)
Read
13. Start the instrument 14. Clean the outside of 15. Wipe the prepared 16. READ the results in
timer. the vial with a towel to sample and insert it into mg/L PO43–.
A two-minute reaction remove fingerprints or the 16-mm round cell
period will begin. other marks. holder.
Read results within two to

eight minutes after adding
the PhosVer 3 reagent.
Interferences

Arsenate All levels
Silicate Greater than 10 mg/L
Greater than 9 mg/L. Remove sulfide interference as follows:
1. Measure 25 mL of sample into a 50-mL beaker.
2. Swirling constantly, add Bromine Water drop-wise until a permanent
Sulfide
yellow color appears.
3. Swirling constantly, add Phenol Solution drop-wise just until the yellow color disappears.
Proceed with step 1.
Large amounts may cause inconsistent results in the test because the acid present in the
Turbidity powder pillows may dissolve some of the suspended particles and because of variable
desorption of orthophosphate from the particles.
extreme sample pH

Page 981

Collect samples in plastic or glass bottles that have been acid washed with 1:1 Hydrochloric Acid
Solution and rinsed with deionized water. Do not use commercial detergents containing phosphate
for cleaning glassware used in this test.
Analyze samples immediately for best results. If prompt analysis is not possible, preserve samples
by filtering immediately and storing the sample at 4 °C (39 °F) for up to 48 hours.
Accuracy check
• Phosphate 2-mL Ampule Standard, 50-mg/L as PO43–
• Ampule breaker

ADDITIONS.
6. Follow the Acid Hydrolysis, TNT method test procedure for each of the spiked samples,


• Phosphate Standard Solution. 3.0-mg/L
1. Use the Phosphate Standard Solution. 3.0-mg/L solution in place of the sample. Follow the
Acid Hydrolysis, TNT method test procedure.
to Standard Adjust in the softwarE: OPTIONS>MORE>STANDARD ADJUST. .

Page 982
Method performance
Precision—95% Confidence Sensitivity—DConcentration

Limits of Distribution per 0.010 DAbs
536 DR 5000 3.00 mg/L PO43– 2.93–3.07 mg/L PO43– 0.06 mg/L PO43–
Summary of method
Phosphates present in condensed inorganic forms (meta-, pyro-, or other polyphosphates)
must be converted to reactive orthophosphate before analysis. Pretreating the sample with
acid and heat hydrolyzes the condensed inorganic forms to orthophosphate.
Orthophosphate reacts with molybdate in an acid medium to produce a mixed phosphate/

molybdate complex. Ascorbic acid then reduces the complex, giving an intense molybdenum
blue color. Test results are measured at 880 nm.

Page 983
Required reagents
Total and Acid Hydrolyzable Phosphorus Reagent Set, includes: 50 tests 2742745
PhosVer 3 Phosphate Reagent Powder Pillows 1 pillow 50/pkg 2106046
Potassium Persulfate Powder Pillows 1 pillow 50/pkg 2084766
Sodium Hydroxide, 1.54 N varies 100 mL 2743042
Sodium Hydroxide Standard Solution, 1.00 N 2 mL 100 mL 104542
Total and Acid Hydrolyzable Test Vials1 1 vial 50/pkg —
Water, deionized varies 100 mL 27242
Required apparatus
Pipet, TenSette®, 1 to 10 mL 1 each 1970010
Drinking Water Standard, Mixed Parameter, Inorganic for F–, NO3, PO4, SO4 500 mL 2833049
Phosphate Standard Solution, 10-mL Voluette® Ampule, 50-mg/L as PO4 3– 16/pkg 17110
Phosphate Standard Solution, 1-mg/L as PO43– 500 mL 256949
Phosphate Standard Solution, 3 mg/L as PO4 3– 946 mL 2059716

Wastewater Standard, Effluent Inorganics, for NH3–N, NO3–N, PO4, COD, SO4, TOC 500 mL 2833249

Page 984

Hydrochloric Acid Solution, 6.0 N, 1:1 500 mL 88449
Thermometer, Non-Mercury, –10 to 225° C each 2635700
Pipet Tips for TenSette Pipet 19700–10 50/pkg 2199796
Beaker, 50 mL, Glass each 50041H
Optional standards
Phosphate, Standard Solution, 3 mg/L 946 mL 2059716
Phosphate, Standard Solution, 50 mg/L, 10 mL ampules 16/pkg 17110
Phosphate, Standard Solution, 500 mg/L, 10 mL ampules 10/pkg 1424210
Phosphate, Standard Solution 100 mL 1424232

Page 985
Phosphorus, Reactive, RSAV, 8114
(Orthophosphate) DOC316.53.01115
Molybdovanadate Method1 Method 8114

0.3 to 45.0 mg/L PO43– Reagent Solution or AccuVac® Ampuls
Test preparation


Instrument
For best results, sample temperature should be 20–25 °C (68–77 °F).
After adding reagent, a yellow color will form if phosphate is present. The blank will be slightly yellow because of the reagent.
Liquid Reagent Test:
Molybdovanadate Reagent 1.0 mL
AccuVac Test:
Molybdovanadate Reagent AccuVac® Ampuls 2
Beaker, 50-mL 2
Phosphorus, Reactive (Orthophosphate)

Page 987
Molybdovanadate reagent solution
Stored Programs
480 P React. Mo.
Start
1. Select the test. 2. Blank Preparation: 3. Prepared Sample: Fill 4. Add 0.5 mL of
Insert an adapter if Fill a sample cell with a second sample cell with Molybdovanadate
required (see Instrument- 10 mL of deionized water. 10 mL of sample. Reagent to each sample
specific information). cell. Swirl to mix.
Zero
5. Start the instrument 6. When the timer 7. ZERO the instrument. 8. Wipe the prepared
timer. expires, wipe the blank The display will show: sample and insert it into
A 7-minute reaction period and insert it into the cell the cell holder.
holder. 0.0 mg/L PO43–
will begin. READ the results in mg/L
If the sample PO43–.
concentration is greater
than 30 mg/L PO43–, read
at exactly seven minutes
or make a 1:1 dilution of
the sample and repeat the
test

Page 988
Molybdovanadate for AccuVac® Ampuls
Stored Programs
482 P React. Mo. AV
Start
1. Select the test. 2. Prepared Sample: 3. Blank Preparation: 4. Start the instrument
Insert an adapter if Collect 40 mL of sample in Collect 40 mL of deionized timer.
required (see Instrument- one 50-mL beaker. Fill a water in another 50-mL A 7-minute reaction period
specific information). Molybdovanadate beaker. will begin.
Reagent AccuVac Ampul Fill another Ampul with
Refer to the user manual with sample. If the sample
for orientation. deionized water. Keep the concentration is greater
tips immersed while the than 30 mg/L PO43–, read
Ampuls fill completely. at exactly seven minutes
or make a 1:1 dilution of
the sample and repeat the
test.
Zero Read
5. When the timer 6. ZERO the instrument. 7. Wipe the prepared 8. READ the results in
expires, wipe the blank The display will show: sample and insert it into mg/L PO43–.
and insert it into the cell the cell holder.
Interferences
The Interfering substances table shows interference levels and types of interference. The
Noninterfering substances at low concentrations (less than 1000 mg/L) table shows substances
that do not interfere in concentrations less than 1000 mg/L.
Arsenate Only interferes if sample is heated.
Iron, ferrous Blue color caused by ferrous iron does not interfere if concentration is less than 100 mg/L.
Molybdate Causes negative interference above 1000 mg/L.
Silica Only interferes if sample is heated.

Page 989

Causes a negative interference.
1. Measure 50 mL of sample into an Erlenmeyer flask.
2. Add Bromine Water1 drop-wise with constant swirling until a permanent yellow color
Sulfide
develops.
3. Add Phenol Solution1 drop-wise until the yellow color just disappears. Proceed with
step 3 (step 2 if using AccuVac procedure).
pH, extreme or
May exceed buffering capacity of reagents. May require pretreatment. pH should be about 7.
highly buffered samples
Fluoride, thorium, bismuth,
Cause negative interference.
thiosulfate or thiocyanate
Table 304 Noninterfering substances at low concentrations (less than 1000 mg/L)
Pyrophosphate Tetraborate Benzoate
Citrate Lactate Formate
Oxalate Tartrate Salicylate
Al3+ Fe3+ Mg2+
Ca2+ Ba2+ Sr2+
Li+ Na+ K+
NH4+ Cd2+ Mn2+
NO3– NO2– SO42–
SO32– Pb2+ Hg+
Hg2+ Sn2+ Cu2+
Ni2+ Ag+ U4+
Zr4+ AsO3– Br–
CO3 2– ClO4 – CN–
IO3– SiO44– Selenate

• Collect samples in clean plastic or glass bottles that have been cleaned with 1:1 Hydrochloric
Acid Solution* and rinsed with deionized water.
• Do not use a commercial detergent. The phosphate content will contaminate the sample.
• Analyze samples as soon as possible for best results.
• If samples cannot be analyzed promptly, store the sample for up to 48 hours at 4 °C (39 °F) or
below.
• Warm stored samples to room temperature before analyzing.

Page 990
Accuracy check
• Phosphate 2-mL Ampule Standard, 500-mg/L PO43–
• Ampule breaker

ADDITIONS.
6. Pour 10 mL of the spiked samples into three 10 mL sample cells or for AccuVacs, pour 10 mL
of the spiked samples into a beaker.
7. Follow the Molybdovanadate for AccuVac® Ampuls test procedure for each of the spiked
instrument.

• Phosphate standard solution, 10-mg/L
1. Use the Phosphate standard solution, 10-mg/L, in place of the sample. Follow the
Molybdovanadate for AccuVac® Ampuls test procedure.
Method performance
480 30.0 mg/L PO43– 29.6–30.4 mg/L PO43– 0.3 mg/L PO43–

Page 991
Summary of method
In the molybdovanadate method, orthophosphate reacts with molybdate in an acid medium to
produce a mixed phosphate/molybdate complex. In the presence of vanadium, yellow
molybdovanadophosphoric acid is formed. The intensity of the yellow color is proportional to the
phosphate concentration. Test results are measured at 430 nm.
Required reagents
Molybdovanadate Reagent 1.0 mL 100 mL MDB 2076032
OR
Molybdovanadate Reagent AccuVac® Ampuls 2 25/pkg 2525025
Required apparatus (reagent liquid)

Required apparatus
Sample cell, 10 mL, round, 25 x 54 mm 1 each 2122800
Sample cell, 10 mL, round, 25 x 60 mm 1 6/pkg 2427606

Phosphate Standard Solution, 10-mL PourRite® Ampule, 500 mg/L as PO4 3– 16/pkg 1424210
Wastewater Influent Standard, for mixed parameters NH3–N, NO3–N, PO4, COD, SO4,
500 mL 2833149
TOC

Page 992

Hydrochloric Acid, 6.0 N 1:1 500 mL 88449
AccuVac ampule drainer each 4103600
Optional standards
Phosphate, Standard Solution, 50 mg/L, 10 mL Voluette Ampules 16/pkg 17110

Page 993

Phosphorus, Reactive, Amino Acid, 8178
Amino Acid Method1 Method 8178

0.23 to 30.00 mg/L PO43–
Test preparation


adjust
The contents of one Amino Acid Reagent Powder Pillow may be substituted for 1 mL of amino acid reagent solution in
step 4.
Amino Acid Reagent 1 mL
Cylinder, 25-mL, graduated, mixing 1
Molybdate Reagent 1 mL

Page 995
Amino Acid Method
Stored Programs
485 P React. Amino
Start
1. Select the test. 2. Fill a 25-mL mixing 3. Add 1 mL of 4. Prepared Sample: Add
Insert an adapter if cylinder with 25 mL of Molybdate Reagent using 1 mL of Amino Acid
required (see Instrument- sample. a 1-mL calibrated dropper. Reagent Solution. Stopper
specific information). and invert several times to
mix.
A blue color will form if
phosphate is present.
Zero
5. Start the instrument 6. Blank Preparation: 7. When the timer 8. ZERO the instrument.
timer. Fill a sample cell with expires, wipe the blank The display will show:
A 10-minute reaction untreated sample. and insert it into the cell
period will begin. Continue
with step 6 while the timer
is running.
Read
9. Fill a second cell with 10. Wipe the prepared 11. READ the results in
prepared sample. sample and insert it into mg/L PO43–.
the cell holder.

Page 996
Interferences

Calcium Greater than 10,000 mg/L as CaCO3
Chloride Greater than 150,000 mg/L Cl–
Add 1 mL of 10 N Sulfuric Acid Standard Solution1 to another 25-mL sample. Use this
Colored samples instead of untreated sample as the blank to zero the instrument. Use a pipet and pipet filler to
measure the sulfuric acid standard.
May cause low results. To eliminate this interference, dilute the sample until two successive
High salt levels (Na+)
dilutions yield about the same result.
Magnesium Greater than 40,000 mg/L as CaCO3
Bleaches the blue color. Remove nitrite interference by adding 0.05 g of sulfamic acid to the
Nitrite (NO2–)
sample. Swirl to mix. Continue with step 4.
As the concentration of phosphate increases, the color changes from blue to green, then
Phosphates, high levels
to yellow and finally to brown. The brown color may suggest a concentration as high as
(PO43–)
100,000 mg/L PO43–. If a color other than blue is formed, dilute the sample and retest.
Sulfide interferes. For samples with sulfide concentration less than 5 mg/L sulfide
interference may be removed by oxidation with Bromine Water as follows:
1. Measure 50 mL of sample into an Erlenmeyer flask.
Sulfide (S2–) 2. Add Bromine Water1 drop-wise with constant swirling until permanent
yellow color develops.
3. Add Phenol Solution1 drop-wise until the yellow color just disappears. Use this solution
in steps 2 and 6.
Temperature For best results, sample temperature should be 21 ±3 °C (70 ±5 °F).
May give inconsistent results for two reasons. Some suspended particles may dissolve
because of the acid used in the test. Also, desorption of orthophosphate from particles may
Turbidity occur. For highly turbid samples, add 1 mL of 10 N Sulfuric Acid Standard Solution1 to
another 25-mL sample. Use this instead of untreated sample as the blank to zero the
instrument. Use a pipet and pipet filler to measure the sulfuric acid standard.
extreme sample pH

Acid Solution and rinsed with deionized water.
• Do not use a commercial phosphate-based detergent for cleaning glassware. The phosphate
content will contaminate the sample.
• Analyze samples immediately for best results.
• If prompt analysis is not possible, preserve samples by filtering immediately and store at 4 °C
(39 °F) for up to 48 hours.
• The sample should have a neutral pH (6–8) and be at room temperature before analysis.

Page 997
Accuracy check
• Ampule breaker

ADDITIONS.
6. Follow the Amino Acid Method test procedure for each of the spiked samples, starting with the


• 10-mg/L Phosphate Standard Solution
1. Use the 10-mg/L Phosphate Standard Solution in place of the sample. Follow the Amino Acid
Method test procedure.
Method performance

485 DR 5000 10.00 mg/L PO43– 9.86–10.14 mg/L PO43– 0.20 mg/LPO43–
Summary of method
In a highly acidic solution, ammonium molybdate reacts with orthophosphate to form
molybdophosphoric acid. This complex is then reduced by the amino acid reagent to yield an
intensely colored molybdenum blue compound. Test results are measured at 530 nm.

Page 998
Required reagents
High Range Reactive Phosphorus Reagent Set, includes: — 100 tests 2244100
Amino Acid Reagent 1 mL 100 mL MDB 193432
Molybdate Reagent 1 mL 100 mL MDB 223632
Required apparatus
Cylinder, 25-mL, graduated, mixing 1 each 189640
Phosphate Standard Solution, 2-mL PourRite® Ampule, 500-mg/L PO43– 16/pkg 1424220
Wastewater Effluent Standard, for mixed parameters NH3–N, NO3–N, PO4, COD, SO4, 500 mL 2833249
TOC
Wastewater Influent Standard for mixed parameters NH3–N, NO3–N, PO4, COD, SO4, 500 mL 2833149
TOC
PourRite Ampule breaker, 2 mL each 2484600

Page 999

Amino Acid Reagent Powder Pillow 100/pkg 80499
Hydrochloric Acid Solution, 1:1, 6.0 N 500 mL 88449
Phenol Solution, 30g/L 29 mL 211220
Sulfamic Acid 454 g 234401
Sulfuric Acid Standard Solution, 10 N 1000 mL 93153
Thermometer, Non-Mercury, -10 to 225°C each 2635700
Optional Standards

Phosphorus, Reactive HR, 8114
Phosphorus, Reactive DOC316.53.01114
Molybdovanadate Rapid Liquid Method1 Method 8114

HR (0.3 to 45.0 mg/L PO4 3–) Pour-Thru Cell
Scope and Application: For treated and natural waters
Test preparation

Instrument Pour-Thru Kit Pour-Thru Cell Orientation Adapter
DR 5000 LZV479 — —

DR 3900
compartment
5940400 1-inch (round) path aligned with arrow on LZV585 (B)

DR 3800, DR 2800, DR 2700
the adapter
See the User Manual for Pour-Thru Module installation instructions.
Clean the Pour-Thru cell and all labware as specified in Analysis labware treatment.
Make sure the PourThru cell is completely seated in the sample cell compartment.
(D002) by the Federal RCRA. Refer to the current MSDS for safe handling and disposal information.
Molybdovanadate Reagent 2 mL
Dispenser, fixed volume, 1.0-mL, w/bottle 1
Flask, Erlenmeyer, 125-mL, PMP w/cap 2
Instrument-specific information 1
Page 1001
Molybdovanadate Rapid Liquid method
Stored Programs
489 P React. Mo. HR RL
Start
1. Select the test. 2. I 3. Rinse a clean plastic 4. Blank Preparation:

Insert an adapter if 125-mL Erlenmeyer flask Measure 25 mL of
required (see Instrument- and a 25-mL graduated deionized water in the
specific information). cylinder with deionized graduated cylinder. Pour
water. the water into the flask.
5. Rinse another clean 6. Prepared Sample: 7. Add 1.0 mL of 8. Start the instrument
plastic 125-mL Erlenmeyer Measure 25 mL of sample Molybdovanadate timer.
flask with deionized water. in the graduated cylinder. Reagent to each flask A 7-minute reaction period
Pour the water into the using a Repipet Jr. will begin.
flask. Dispenser. Swirl to mix.
If the sample
A yellow color will develop concentration is greater
in the sample if phosphate than 30 mg/L PO43–, read
is present. A small amount at exactly seven minutes
of yellow may be present or make a 1:1 dilution of
in the blank due to the the sample and begin the
reagent. test again.
Page 1002
Molybdovanadate Rapid Liquid method (continued)
Zero Read
9. When the timer 10. ZERO the instrument. 11. Pour the prepared 12. READ the results in
expires, pour the blank The display will show: sample from the flask into mg/L PO43–.
from the flask into the the Pour-Thru Cell. Flush the Pour-Thru Cell
Pour-Thru Cell. 0.0 mg/L PO43–
with 50 mL of deionized
water.
Interferences
See the Interfering substances table for a list of substances, interference levels and type of
interference. See the Noninterfering substances at low concentrations (less than 1000 mg/L) table
for a list of substances that do not interfere in concentrations less than 1000 mg/L.
Arsenate Negative interference. Positive interference if sample is heated.
Bismuth Negative interference.
Fluoride Negative interference.
Blue color is caused by ferrous iron but this does not affect results if the ferrous iron
Iron, Ferrous
concentration is less than 100 mg/L.
Molybdate Negative interference.
Silica Positive interference if sample is heated.
Negative interference. Sulfide interference may be removed by oxidation with Bromine Water
as follows:
1. Measure 25 mL of sample into a flask.
Sulfide 2. Add Bromine Water1 drop-wise with constant swirling until permanent yellow color
develops.
3. Add Phenol Solution1 drop-wise until the yellow color just disappears. Proceed with
step 7.
Thiocyanate Negative interference.
Thiosulfate Negative interference.
Thorium Negative interference.
extreme sample pH
Page 1003
Al3+ Selenate Mg2+
Ca2+ Ba2+ Sr2+
Li+ Na+ K+
NH4+ Cd2+ Mn2+
NO3 – NO2 – SO42–
SO32– Pb2+ Hg+

Hg2+ Sn2+ Cu2+
Ni2+ Ag+ U
Zr4+ AsO3– Br–
CO32– ClO4– CN–
IO3– Fe3+ SiO44–

• Do not use detergents that contain phosphate for cleaning labware.
• If prompt analysis is not possible, preserve samples by filtering immediately and storing the
sample at 4 °C (39 °F) or below for up to 48 hours.
Analysis labware treatment

1. Clean containers by normal means (do not use detergents containing phosphorus), then rinse
2. Soak for several minutes in a 1:25 dilution of Molybdovanadate Reagent in deionized water.
3. Rinse well with deionized water. Dedicate these containers for HR PO43– analysis.
4. Fill the Pour-Thru Cell with this same mixture of Molybdovanadate reagent and deionized
water and let stand for several minutes.
5. Rinse the Pour-Thru Cell with 50 mL of deionized water.
Page 1004
How to clean the Pour-Thru Cell

The Pour-Thru Cell may accumulate a buildup of colored products, especially if the reacted
solutions are allowed to stand in the cell for long periods after measurement. To clean the Pour-
Thru Cell:
1. Remove the color by rinsing with a 1:5 dilution of ammonium hydroxide*
2. Follow with several deionized water rinses.
3. Invert a beaker over the glass funnel of the Pour-Thru Cell when not in use.
Accuracy check
• Phosphate Voluette® Ampule Standard Solution, 500-mg/L as PO43–
• Ampule breaker

ADDITIONS.
standard to three 25-mL mixing cylinders and fill to 25 mL with the fresh sample. Mix
thoroughly.
6. Follow the Molybdovanadate Rapid Liquid method test procedure for each of the spiked
instrument.
* Optional reagents and apparatus.
Page 1005


• 10.0-mg/L Phosphate Standard
1. Use the 10.0-mg/L Phosphate Standard solution in place of the sample. Follow the
Molybdovanadate Rapid Liquid method test procedure.
Method performance
Summary of method
In the molybdovanadate method, orthophosphate reacts with molybdate in an acid medium to
produce a phosphomolybdate complex. In the presence of vanadium, yellow
vanadomolybdophosphoric acid is formed. The intensity of the yellow color is proportional to the
phosphate concentration. Test results are measured at 430 nm.
Page 1006
Required reagents
Molybdovanadate Reagent 2 mL 500 mL 2076049
Required apparatus
Dispenser, adjustable 1 each 2563137
Flask, Erlenmeyer, 125-mL, PMP w/cap 2 each 2089843
Phosphate Standard Solution, 10-mg/L as PO4 3– 946 mL 1420416

Phosphate Standard Solution, Voluette® ampule, 10-mL, 500-mg/L as PO43– 16/pkg 1424210
Wastewater Influent Standard for NH3–N, NO3–N, PO4, COD, SO4, TOC 500 mL 2833149

Ammonium Hydroxide, ACS 500 mL 10649
Page 1007
Optional standards

Phosphorus, Reactive, RL, LR, 10055
Phosphorus, Reactive DOC316.53.01117
Ascorbic Acid Rapid Liquid Method1 Method 10055

LR (19 to 3000 µg/L PO43–) Pour-Thru Cell
Scope and Application: For treated and natural waters
Test preparation

Instrument Pour-Thru Kit Pour-Thru Cell Orientation Adapter
DR 5000 LZV479 — —

DR 3900
compartment
5940400 1-inch (round) path aligned with arrow on the LZV585 (B)
DR 3800, DR 2800, DR 2700
adapter
See the user manual for Pour-Thru Module installation instructions.
Clean the Pour-Thru cell and all labware as specified in Analysis labware treatment.
See Reagent preparation for preparing the Ascorbic Acid reagent.
Reaction time depends on sample temperature. For most accurate results, samples should be at room temperature
(about 20 °C).
Obtain a reagent blank for each lot of reagent when the normal sample phosphate concentration is less than 750 µg/L.
Follow the procedure using deionized water in place of the sample. Subtract the reagent blank value from the final results.
Page 1009
Ascorbic Acid Reagent Dilution Solution 1 mL
Ascorbic Acid Reagent Powder varies
Cylinder, graduated, 25 mL, poly 2
Dispenser, bottle top 2
Flask, Erlenmeyer, 125-mL, PMP w/cap 2
Molybdate Reagent Solution 2 mL
Pour-Thru Cell Assembly 1
Ascorbic acid method, rapid liquid
Stored Programs
488 P React. LR RL
Start
1. Select the test. 2. Rinse two clean 3. Rinse a clean 25-mL 4. Fill the rinsed cylinder
Insert an adapter if Erlenmeyer flasks three plastic graduated cylinder to the 25-mL mark with
required (see Instrument- times with the sample. three times with the sample.
specific information). sample.
5. Pour the contents of 6. Measure a second 7. Add 1.0 mL of 8. Prepared Sample:

the 25-mL cylinder into 25-mL portion of sample Molybdate reagent to each Add 1.0 mL of prepared
one of the flasks. into the graduated cylinder flask using a bottle-top Ascorbic Acid reagent to
and pour the contents into dispenser. Swirl to mix. one of the flasks with a
the second flask. bottle-top dispenser. Swirl
to mix. The remaining flask
will be the blank.
Page 1010
Ascorbic acid method, rapid liquid (continued)
Zero
9. Start the instrument 10. When the timer 11. After the flow stops, 12. Pour the prepared
timer. expires, pour the contents ZERO the instrument. sample into the Pour-Thru
of the flask that contains The display will show: Cell.
A five-minute reaction
the blank into the
period will begin. 0 µg/L PO43–
Pour-Thru Cell.
Read
13. READ the results in 14. Flush the Pour-Thru

µg/L PO43–. Cell with at least 50 mL of
deionized water
Interferences

Aluminum 200 mg/L
Arsenate Interferes
Chromium 100 mg/L
Copper 10 mg/L
Hydrogen sulfide Interferes
Iron 100 mg/L
Nickel 300 mg/L
Silica 50 mg/L
Silicate 10 mg/L
Samples with large amounts of turbidity may give inconsistent results because the acid
Turbidity present in the reagents may dissolve some of the suspended particles and variable
desorption of orthophosphate from the particles may occur.
Zinc 80 mg/L
Page 1011

extreme sample pH
Analysis labware treatment

All labware used in this test must be thoroughly cleaned to remove all traces of phosphate.
1. Clean containers with a non-phosphate detergent followed by a rinse with deionized water.
2. Fill and soak for 10 minutes with a 1:25 dilution of Molybdate Reagent in deionized water.
3. Rinse well with deionized water. Keep containers tightly closed when not in use.
4. Treat the Pour-Thru Cell with this same mixture of molybdate and water followed by thorough
rinsing with deionized water.
Dedicate these containers for low-level phosphate analysis. If these containers are rinsed and
capped after use, only occasional pre-treatment is necessary.
How to clean the Pour-Thru cell

solutions are allowed to stand in the cell for long periods after measurement. To remove color:
1. Rinse the Pour-Thru Cell with a 1:5 dilution of Ammonium Hydroxide.
2. Follow the Ammonium Hydroxide rinse with deionized water rinses.
3. Invert a beaker over the glass funnel of the cell when the Pour-Thru Cell is not in use.
Reagent preparation
The Ascorbic Acid reagent must be prepared before use.
1. Using a powder funnel, add the contents of one 48 g bottle of Ascorbic Acid Reagent Powder*
to one 450 mL bottle of Ascorbic Acid Reagent Dilution Solution.
2. Invert several times and swirl until the powder is completely dissolved.
3. Attach dispensers to the top of this bottle and the Molybdate Reagent bottle. This solution may
develop a yellow color with time but will still give accurate results for up to one month after
mixing if stored at 20–25 °C.
4. Record the date of preparation on the bottle and discard any remaining solution after one
month.
Do not add fresh reagent to previously mixed reagent. Use of this reagent after one month may
result in high reagent blanks and low values at high concentrations.
Page 1012

• Do not use detergents that contain phosphate for cleaning labware.
• If prompt analysis is not possible, preserve samples by filtering immediately and storing at 4
°C (39 °F) for up to 48 hours.
Accuracy check
• Phosphate Standard Solution Ampule, 50-mg/L (50,000 µg/L) as PO43– (for concentrations
greater than 1000 µg/L)
• Phosphate Standard Solution Ampule, 15-mg/L (15,000 µg/L) as PO43– (for concentrations
less than 1000 µg/L)
• Ampule breaker
• Flask, Erlenmeyer, 125-mL, PMP w/cap

ADDITIONS.
4. Open the standard solution ampule needed for the expected concentration.
5. Measure three 25 mL portions of fresh samples into three flasks.
standard to the three 25-mL portions of fresh sample.
7. Follow the Ascorbic acid method, rapid liquid test procedure for each of the spiked samples,
Page 1013


• 1000-µg/L (1.000-mg/L) phosphate standard solution
1. Use the 1000-µg/L (1.000-mg/L) phosphate standard solution in place of the sample. Follow
the Ascorbic acid method, rapid liquid test procedure.
Method performance

488 DR 5000 1000 µg/L PO43– 970–1030 µg/L PO43– 21 µg/L PO43–
Summary of method
Orthophosphate reacts with molybdate in an acid medium to produce a phosphomolybdate
complex. Ascorbic acid then reduces the complex, giving an intense molybdenum blue color.
Reactive phosphorus includes existing orthophosphate in the sample plus a small fraction of
condensed phosphate that may be hydrolyzed to orthophosphate during the test. Test results are
measured at 880 nm.
Page 1014
Required reagents
Rapid Liquid Low Range Phosphorus Reagent Set, includes: 2678600
Ascorbic Acid Reagent Dilution Solution 1 mL 450 mL 2599949
Ascorbic Acid Reagent Powder varies 48 g 2651255
Molybdate Reagent Solution 2 mL 500 mL 2599849
Required apparatus
Adjustable Digital Dispenser 2 each 2563137
Flask, Erlenmeyer, 125-mL, PMP w/cap 2 each 2089843
Powder funnel, 65 mm top ID 1 each 2264467
Drinking Water Standard, Mixed Inorganics for NO3, PO4, SO4 500 mL 2833049
Phosphate Standard Solution, 1.00-mg/L as PO43– 500 mL 256949
Phosphate Standard Solution, Voluette® ampule, 10-mL, 50-mg/L PO43– 16/pkg 17110
Phosphate Standard Solution, 15-mg/L PO43– 100 mL 1424342

Wastewater Effluent Standard for NH3–N, NO3–N, PO4, COD, SO4, TOC 500 mL 2833249

Ammonium Hydroxide, ACS 500 mL 10649
Hydrochloric Acid Solution,6.0 N, 1:1 500 mL 88449
Filter Paper, fold 12.5 cm 100/pkg 69257
Page 1015
Phosphorus, Reactive, AVPP, 8048
USEPA1 PhosVer 3 (Ascorbic Acid) Method2 Method 8048

0.02 to 2.50 mg/L PO43– Powder Pillows or AccuVac® Ampuls
1 USEPA Accepted for reporting for wastewater analyses. Procedure is equivalent to USEPA and Standard Method 4500-P-E for wastewater.
Test preparation


Instrument
adjust.
Powder Pillow Test:
PhosVer® 3 Phosphate Reagent powder pillow 1
AccuVac Test:
PhosVer® 3 Phosphate Reagent AccuVac® Ampul 1
Beaker, 50-mL 1
Sample Cell, 10-mL round 1

Page 1017
Stopper for 18-mm Tube (supplied with PhosVer AccuVacs) 1
PhosVer 3 (Ascorbic Acid) method for powder pillows
Stored Programs
490 P React. PV
Start
Insert an adapter if 10-mL of sample. Add the contents of one timer.
required (see Instrument- PhosVer 3 phosphate A two-minute reaction
specific information). Powder Pillow to the cell. period will begin. If the
Immediately stopper and sample was digested
shake vigorously for 30 using the Acid Persulfate
seconds. digestion, a ten-minute
reaction period is required.
Zero
5. Blank Preparation: 6. When the timer 7. ZERO the instrument. 8. Wipe the prepared
Fill a second sample cell expires, wipe the blank The display will show: sample and insert it into
with 10 mL of sample. and insert it into the the cell holder.
cell holder. 0.00 mg/L PO43–
PO43–.

Page 1018
PhosVer 3 (Ascorbic Acid) method for AccuVac® Ampuls
Stored Programs
492 P React. PV AV
Start
1. Select the test. 2. Blank Preparation: 3. Prepared Sample: 4. Secure an Ampul cap
Insert an adapter if Fill a sample cell with Fill a PhosVer 3 over the tip of the Ampul.
required (see Instrument- 10-mL of sample. Phosphate AccuVac Shake the Ampul for
specific information). Ampul with sample. Keep approximately
the tip immersed while the 30 seconds.
Refer to the user manual Ampul fills completely.
for orientation. Accuracy is unaffected by
undissolved powder.
5. Start the instrument 6. When the timer 7. Wipe the prepared

timer. expires, wipe the blank sample and insert it into
A two-minute reaction and insert it into the cell the cell holder.
period will begin. If the holder. READ the results in
sample was digested ZERO the instrument. mg/L PO43–.
using the Acid Persulfate The display will show:
digestion, a ten-minute
reaction period is required. 0.00 mg/L PO43–

Page 1019
Interferences

Arsenate Interferes at any level.
Hydrogen Sulfide Interferes at any level
pH, excess buffering
reagents and require sample pretreatment. pH 2–10 is recommended.
May cause inconsistent results because the acid in the powder pillow may dissolve some of
the suspended particles and because of variable desorption of orthophosphate from the
Turbidity or color particles. For highly turbid or colored samples, add the contents of one Phosphate
Pretreatment1 Powder Pillow to 25 mL of sample. Mix well. Use this solution to zero the
instrument.

• Collect sample in plastic or glass bottles that have been cleaned with 1:1 Hydrochloric Acid
Solution* and rinsed with deionized water.
• Do not use commercial detergents containing phosphate for cleaning glassware used in
phosphate analysis.
• For best results, analyze samples immediately.
• If prompt analysis is not possible, preserve samples by filtering immediately and storing at 4
°C (39 °F) for up to 48 hours.
• Return the sample to room temperature before analysis.
Accuracy check
• Ampule breaker
2. Select OPTIONS>MORE>STANDARD ADDITIONS from the instrument menu.

Page 1020
5. Prepare a 0.1-mL sample spike by adding 0.1 mL of standard to the unspiked sample. Press
the timer icon. After the timer expires, read the result.
6. Prepare a 0.2-mL sample spike by adding 0.1 mL of standard to the 0.1-mL sample spike.
Press the timer icon. After the timer expires, read the result.
7. Prepare a 0.3-mL sample spike by adding 0.1 mL of standard to the 0.2-mL sample spike.
Press the timer icon. After the timer expires, read the result. Each addition should reflect

1. Fill three mixing cylinders each with 50-mL of sample and spike with 0.2 mL, 0.4 mL and
0.6 mL of standard.
3. Analyze each standard addition sample as described in the PhosVer 3 (Ascorbic Acid) method
for AccuVac® Ampuls.
recovery.


• Phosphate standard solution, 50 mg/L
• Deionized water
• 4 mL Class A volumetric pipet and pipet bulb
1. Prepare a 2.00 mg/L phosphate standard solution as follows:
a. Pipet 4.00 mL of Phosphate Standard, 50-mg/L, into a 100-mL volumetric flask.

b. Dilute to volume with demineralized water. Mix well. Prepare this solution daily.
Note: Alternately, use one of the mixed parameter standards listed in Recommended standards. These
contain 2.0 mg/L phosphate.
2. Use this solution in place of the sample. Follow the PhosVer 3 (Ascorbic Acid) method for

Page 1021
Method performance

Program Standard
Summary of method
molybdate complex. Ascorbic acid then reduces the complex, giving an intense molybdenum blue
color. Test results are measured at 880 nm

Page 1022
Required reagents
PhosVer® 3 Phosphate Reagent Powder Pillows, 10-mL 1 100/pkg 2106069

OR
PhosVer® 3 Phosphate Reagent AccuVac® Ampuls 1 25/pkg 2508025

Required apparatus
Phosphate Standard Solution, 10-mL Voluette® Ampul, 50-mg/L as PO4 16/pkg 17110
Phosphate Standard Solution, 50-mg/L as PO4 500 mL 17149
Phosphate Standard Solution, 1-mg/L as PO4 500 mL 256949
Standard, Drinking Water, Mixed Parameter, Inorganic: F, NO3, PO4, SO4 500 mL 2833049
Wastewater Effluent Standard, for mixed parameters: NH3–N, NO3–N, PO4, COD, SO4, 500 mL 2833249
TOC

Hydrochloric Acid Solution, 6.0N, 1:1 500 mL 88449
Mixing Cylinder, 50 mL each 189641
Phosphate Treatment Powder Pillow 100/pkg 1450199
Pipet, TenSette, Pipet, 1.0 - 10.0 mL each 1970010

Page 1023


AccuVac snapper each 2405200
AccuVac ampule blanks 25/pkg 2677925
AccuVac ampule drainer each 4103600
Optional standards
Phosphate; Standard Solution, 100 mg/L 100 mL 1436832
Phosphate; Standard Solution, 500 mg/L, 10 mL Voluette Ampules 16/pkg 1424210
Phosphate; Standard Solution, 500 mg/L 100 mL 1424232

Phosphorus, Reactive Orthophosphate, TNT, 8048
USEPA1 PhosVer® 3 Method Method 8048

(0.06 to 5.00 mg/L PO43–
Test ‘N Tube™ Vials
or 0.02 to 1.60 mg/L P)
Scope and Application: For water, wastewater and seawater;
1 USEPA accepted for reporting wastewater analysis. Procedure is equivalent to USEPA and Standard Method 4500-P E for wastewater.
Test preparation


DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
DR 3900, DR 3800, DR 2800 and DR 2700 only: Install the light shield in Cell Compartment #2 before performing this test.
blank adjust.
PhosVer® 3 Reagent Powder Pillow 1
Reactive Phosphorus Test ‘N Tube Vial 1
Light Shield 1
Micro funnel 1
Pipet, TenSette®, 1–10 mL 1
Pipet tIps for TenSette Pipet 1
Test Tube Rack varies

Page 1025
PhosVer 3 Method, TNT
Stored Programs
535 P React. PV TNT
Start
1. Select the test. 2. Use a TenSette® Pipet 3. Wipe the outside of 4. Insert the vial into the
Insert an adapter if to add 5.0 mL of sample to the vial with a damp towel, 16-mm round cell holder.
required (see Instrument- a Reactive Phosphorus followed by a dry one, to
specific information). Test ‘N Tube Dilution Vial. remove fingerprints or
Cap and mix. other marks.
Zero
5. ZERO the instrument. 6. Using a funnel, add 7. Immediately cap the 8. Start the instrument
The display will show: the contents of one vial tightly and shake for at timer.
0.00 mg/L PO43– PhosVer 3 Phosphate least 20 seconds. The A two-minute reaction
Powder Pillow to the vial. powder will not dissolve period will begin. Read
completely. samples between two and
eight minutes after adding
the PhosVer 3 reagent.
Read
9. Wipe the outside of 10. When the timer READ the

the vial with a damp towel, expires, insert the vial into
followed by a dry one, to the 16 mm round cell.
remove fingerprints or
other marks.

Page 1026
Interferences

Arsenate All levels
Greater than 6 mg/L. Remove sulfide interference as follows:
2. Swirling constantly, add Bromine Water drop-wise until a permanent yellow color
Sulfide
appears.
3. Swirling constantly, add Phenol Solution drop-wise just until the yellow color disappears.
Proceed with step 1 of the PhosVer 3 Method, TNT test.
Large amounts may cause inconsistent results in the test because the acid present in the
Turbidity powder pillows may dissolve some of the suspended particles and because of variable
desorption of orthophosphate from the particles.
extreme sample pH

• Collect samples in plastic or glass bottles that have been acid cleaned with 1:1 Hydrochloric
• Do not use commercial detergents containing phosphate for cleaning glassware used in this
test.
• Analyze samples immediately after collection for best results.
• If prompt analysis is impossible, preserve samples up to 48 hours by filtering immediately and
storing at 4 °C.
Accuracy check
• Ampule breaker
2. Select standard additions from the instrument menu: Options>More>Standard Additions.

Page 1027
6. Follow the PhosVer 3 Method, TNT test procedure for each of the spiked samples, starting


• 3.0-mg/L phosphate standard solution
1. Use the 3.0-mg/L phosphate standard solution in place of the sample. Follow the PhosVer 3
Method, TNT test procedure.
to Standard Adjust in the software: Options>More>Standard Adjust.
Method performance

535 DR 5000 3.00 mg/L PO43– 2.94–3.06 mg/L PO43– 0.06 mg/L PO43–
Summary of method
color. Test results are measured at 880 nm.
Required reagents
Reactive Phosphorus Test ’N Tube™ Reagent Set (50 tests), includes: — — 2742545
PhosVer® 3 Phosphate Reagent Powder Pillows 1 50/pkg 2106046
Reactive Phosphorus Test ‘N Tube Dilution Vials 1 50/pkg NA1
1 Not available separately

Page 1028
Required apparatus

Page 1029
Phosphate Standard Solution, PouRite® Ampule, 50-mg/L as PO43–, 2-mL 20/pkg 17120H
Phosphate Standard Solution, 3 mg/L as PO43– 946 mL 2059716
Wastewater Effluent Standard, for mixed parameters: NH3–N, NO3–N, PO4, COD, SO4, 500 mL 2833249
TOC
PouRite Ampule breaker, 2 mL each 2484600

Bromine Water 30 g/L 29 mL 221120
Phenol Solution 30 g/L 29 mL 211220
Optional standards

Phosphorus, Reactive Orthophosphate, HR, TNT, 8114
Molybdovanadate Method1 Method 8114

HR (1.0 to 100.0 mg/L PO4 3–) Test ‘N Tube™ Vials
Test preparation

DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
DR 3900, DR 3800, DR 2800 and DR 2700 only: Install the light shield in Cell Compartment #2 before performing this test.
Reagent blanks for each lot of reagents may be used more than once. At room temperature, the reagent blank is stable for
up to three weeks.
The seven-minute reaction time in step 5 is for samples at 23 °C. For samples at 13 °C, wait 15 minutes. For samples at
33 °C, wait two minutes.
(D002) by the Federal RCRA. Refer to the current MSDS for safe handling and disposal information.
High Range Reactive Phosphorus Test ’N Tube Vials 1
Light Shield 1
Pipet, TenSette®, 1 to 10 mL 1
Pipet Tips, for TenSette Pipet 1
Test Tube Rack 1

Page 1031
Molybdovanadate method, TNT
Stored Programs
540 P React. HR TNT
Start
1. Select the test. 2. Blank Preparation: 3. Prepared Sample: 4. Start the instrument
Insert an adapter if Use a TenSette® Pipet to Use a TenSette Pipet to timer.
required (see Instrument- add 5.0 mL of deionized add 5.0 mL of sample to a A seven-minute reaction
specific information). water to a Reactive High Reactive High Range period will begin.
Range Phosphorus Test ‘N Phosphorus Test ‘N Tube
Tube Vial. Vial. Read the sample within
two minutes after the timer
Cap and invert to mix. Cap and invert to mix. expires.
Zero Read
5. Wipe the reagent 6. ZERO the instrument. 7. Wipe the prepared 8. READ the results in
blank and insert it into the The display will show: sample vial and insert it mg/L PO43–.
16-mm round cell holder. into the 16-mm round cell
0.0 mg/L PO43– holder.

Page 1032
Interferences
The Interfering substances table lists interference types and levels. The Noninterfering substances
at low concentrations (less than 1000 mg/L) table shows substances that do not interfere in
concentrations less than 1000-mg/L.
Arsenate Only interferes if the sample is heated.1
Blue color caused by ferrous iron does not interfere if iron concentration is less than
Iron, ferrous
100 mg/L.
Silica Only interferes if the sample is heated.1
Causes a negative interference. Remove interference as follows:
2. Add Bromine Water2 drop-wise with constant swirling until a permanent yellow
Sulfide
color develops.
3. Add Phenol Solution2 drop-wise until the yellow color just disappears.
Proceed with step 3.
Extreme pH or highly buffered May exceed buffering capacity of the reagents. Samples may require pretreatment. Sample
samples pH should be about 7.
Cause a negative interference.
Temperature, Cold (less than
Causes a negative interference.
20 °C)
Temperature, Hot (greater
Causes a positive interference.
than 25 °C)
1 Gentle warming of the sample to room temperature will not cause this substance to interfere.
Al3+ Selenate Mg2+
Ca2+ Ba2+ Sr2+
Li+ Na+ K+
NH4+ Cd2+ Mn2+
NO3– NO2– SO42–
SO32– Pb2+ Hg+

Hg2+ Sn2+ Cu2+
Ni2+ Ag+ U
Zr4+ AsO3– Br–
CO32– ClO4– CN–
IO3 – Fe3+ SiO44–

Page 1033

• Collect samples in plastic or glass bottles that have been cleaned with 1:1 Hydrochloric Acid
Solution* and rinsed with deionized water.
• Do not use commercial detergents containing phosphate for cleaning glassware used in
this test.
• For best results, analyze the samples immediately after collection.
• If prompt analysis is impossible, preserve the samples for up to 48 hours by filtering
immediately and storing at 4 °C.
• Bring the stored sample to room temperature before analysis.
Accuracy check
• 10-mL Voluette® Ampule of Phosphate Standard Solution, 500-mg/L PO43–
• 1:1 Hydrochloric acid solution
• Deionized water
• Ampule breaker
1. Clean the glassware with 1:1 hydrochloric acid solution. Rinse again with deionized water. Do
not use detergents containing phosphate to clean the glassware.
3. Select standard additions from the instrument menu:OPTIONS>MORE>STANDARD

ADDITIONS.
7. Follow the Molybdovanadate method, TNT test procedure using 5 mL of each of the spiked
instrument.


Page 1034
• 50-mg/L PO43– standard

1. Use the 50-mg/L PO43– standard solution in place of the sample. Follow the Molybdovanadate
method, TNT test procedure.
Method performance
Summary of method
molybdate complex. In the presence of vanadium, yellow molybdovanadophosphoric acid forms.
The intensity of the yellow color is proportional to the phosphate concentration. Test results are
measured at 420 nm.

Page 1035
Required reagents
High Range Reactive Phosphorus Test ’N Tube™ Reagent Set,
— 50 vials 2767345
includes:
(1) Reactive High Range Phosphorus Test ’N Tube Vials1 1 50/pkg —
(2) Water, deionized 5 mL 100 mL 27242
1 Not available separately.
Required apparatus

Phosphate Standard Solution, 50-mg/L, as PO4 3– 500 mL 17149

Phosphate Standard Solution, Voluette® ampule, 500-mg/L as PO43–, 10-mL 16/pkg 1424210
Wastewater Influent Standard for NH3–N, NO3–N, PO4, COD, SO4, TOC 500 mL 2833149

Ammonium Hydroxide 500 mL 10649
Hydrochloric Acid Solution 6.0 N, 1:1 500 mL 88449
Dropper, LDPE, 0.5–1.0 mL 20/pkg 2124720
Beaker, 50-mL each 50041H -

Page 1036
Optional standards
Phosphate, Standard Solution, 500 mg/L; 10 mL Voluette Ampules 16/pkg 1424210

Page 1037
Phosphorus, Total, 8190
Phosphorus, Total DOC316.53.01121
USEPA1
PhosVer® 3 with Acid Persulfate Digestion Method
Method 8190
0.06 to 3.50 mg/L PO43– or
Test ‘N Tube™ Vials
0.02 to 1.10 mg/L P
1 USEPA Accepted for reporting wastewater analyses (Standard Methods 4500 P-E).
Test preparation


DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
adjust.
The test range for total phosphate is limited to 0.06 to 3.5 mg/L PO43–. Values greater than 3.5 mg/L may be used to
estimate dilution ratios, but should NOT be used for reporting purposes. If the value is greater than 3.5 mg/L, dilute the
sample and repeat the digestion and the colorimetric test.
(D002) by the Federal RCRA. Refer to the current MSDS for safe handling and disposal instructions.
Phosphorus, Total
Page 1039
Phosphorus, Total
Total Phosphorus Test ‘N Tube™ Reagent Set 1
DRB200 Reactor 1
Funnel, micro 1
Pipet, TenSette®, 1 to 10 mL, plus tips 1
Test Tube Rack 1
PhosVer 3, acid persulfate digestion
Stored Programs
536 P Total/AH PV TNT
Start
1. Turn on the DRB200 2. Select the test. 3. Use a TenSette® Pipet 4. Use a funnel to add
Reactor. Preheat to Insert an adapter if to add 5.0 mL of sample to the contents of one
150 °C. required (see Instrument- a Total Phosphorus Test Potassium Persulfate
specific information). Vial. Powder Pillow for
Phosphonate to the vial.
5. Cap tightly and shake 6. Insert the vial into the 7. Set the instrument 8. When the timer
to dissolve. DRB200. Close the timer to 30 minutes and expires, carefully remove
protective cover. start. the hot vial from the
A 30-minute heating reactor. Insert it in a test
period will begin. tube rack and cool to room
temperature.
Phosphorus, Total
Page 1040
Phosphorus, Total
PhosVer 3, acid persulfate digestion (continued)
Zero
9. Use a TenSette Pipet 10. Wipe the outside of 11. Insert the vial into the 12. ZERO the instrument.
to add 2 mL of 1.54 N the vial with a damp cloth 16 mm cell holder. The display will show:
Sodium Hydroxide followed by a dry one.
Standard Solution to the 0.00 mg/L PO43–
vial. Cap and mix.
13. Use a funnel to add 14. Immediately cap 15. Start the instrument 16. After the timer expires,
the contents of one tightly and shake to mix for timer. wipe the outside of the vial
PhosVer 3 Powder Pillow 20–30 seconds. A two-minute reaction with a wet towel, then a
to the vial. The powder will not period will begin. dry one. Insert the
dissolve completely. prepared sample vial into
Read the sample within the 16 mm cell.
2–8 minutes after the timer
expires. READ the results in mg/L
PO43–.
Interferences

Arsenate Interferes at any level
pH, excess buffering
Phosphorus, Total
Page 1041
Phosphorus, Total

Sulfide Greater than 90 mg/L
May cause inconsistent results because the acid in the powder pillow may dissolve some of
Turbidity or color the suspended particles and because of variable desorption of orthophosphate from
the particles.

• Collect samples in plastic or glass bottles that have been acid washed with 1:1 Hydrochloric
• Do not use commercial detergents containing phosphate for cleaning glassware used in this
test.
• If prompt analysis is not possible, samples may be preserved up to 28 days by adjusting the
pH to 2 or less with concentrated Sulfuric Acid* (about 2 mL per liter) and storing at 4 °C.
• Warm stored samples to room temperature and neutralize with 5.0 N Sodium Hydroxide*
before analysis.
Accuracy check
• Ampule breaker
• Mixing cylinders, (3)
1. Clean glassware with 1:1 Hydrochloric Acid Standard Solution. Rinse again with deionized
water. Do not use phosphate detergents to clean glassware.

ADDITIONS.
7. Use a 5-mL aliquot of the spiked sample in place of the sample. Follow the PhosVer 3, acid
persulfate digestion test procedure for each of the spiked samples, starting with the 0.1 mL
sample spike. Measure each of the spiked samples in the instrument.
Phosphorus, Total
Page 1042
Phosphorus, Total


• Phosphate standard solution, 3.0-mg/L
1. Use the 3.0 mg/L phosphate standard solution in place of the sample. Follow the PhosVer 3,
acid persulfate digestion test procedure.
Method performance
Summary of method
Phosphates present in organic and condensed inorganic forms (meta-, pyro- or other
polyphosphates) must be converted to reactive orthophosphate before analysis. Pretreatment of
the sample with acid and heat provides the conditions for hydrolysis of the condensed inorganic
forms. Organic phosphates are converted to orthophosphates by heating with acid and persulfate.

'
Required reagents
Total Phosphorus Test ’N Tube™ Reagent Set, 50 tests, includes: — — 2742645
PhosVer® 3 Phosphate Reagent Powder Pillows 1 50/pkg 2106046
Potassium Persulfate Powder Pillows 1 50/pkg 2084766
Sodium Hydroxide Solution, 1.54 N 2 mL 100 mL 2743042
Total and Acid Hydrolyzable Test Vials1 1 50/pkg —
1 Not sold separately
Required apparatus
Phosphorus, Total
Page 1043
Phosphorus, Total
Required apparatus
Light shield, DR 3900 1 each LZV849
Light shield, DR 3800, DR 2800, DR 2700 1 each LZV646
Pipet, TenSette®, 1.0 to 10 mL 1 each 1970010
Drinking Water Standard, Mixed Parameter, Inorganic for F-, NO3, PO4, SO4 500 mL 2833049
Phosphate Standard Solution, 10-mL Voluette® Ampule, 50-mg/L as PO43– 16/pkg 17110
Phosphate Standard Solution, 3 mg/L as PO43– 946 mL 2059716

Wastewater Standard, Effluent Inorganics, for NH3–N, NO3–N, PO4, COD, SO4, TOC 500 mL 2833249

Pipet, TenSette® Pipet, 0.1–1.0 mL each 1970001
Optional standards
Phosphorus, Total
Page 1044
Phosphorus, Total
Optional standards
Phosphorus, Total
Page 1045
Phosphorus, Total Digestion, 8190
Phosphorus, Total, Digestion DOC316.53.01112
USEPA1 Acid Persulfate Digestion Method2 Method 8190

1 USEPA Accepted for wastewater analyses when used with the ascorbic acid (PhosVer 3) method.
2 Adapted from Standard Methods for the Examination of Water and Wastewater 4500-P B & E
Test preparation
Rinse all glassware with 1:1 hydrochloric acid. Rinse again with deionized water.
Potassium Persulfate Powder Pillows 1
Sodium Hydroxide Solution, 5.0 N 2 mL
Sulfuric Acid Solution, 5.25 N 2 mL
Hot Plate 1
Phosphorus, Total, Digestion

Page 1047
Acid Persulfate Digestion
17. Use a graduated 18. Add the contents of 19. Use a 1-mL calibrated 20. Place the flask on a
cylinder to measure 25 mL one Potassium Persulfate dropper to add 2.0 mL of hot plate. Boil gently for 30
of sample. Pour the Powder Pillow. 5.25 N Sulfuric Acid minutes. Do not boil dry.
sample into a 125-mL Swirl to mix. Solution to the flask. Concentrate the sample to
Erlenmeyer flask. less than 20 mL for best
recovery. After
concentration, maintain
the volume near 20 mL by
adding small amounts of
deionized water. Do not
exceed 20 mL.
480 P React. Mo
482 P React. Mo. AV
485 P React. Amino
490 P React. PV
492 P React. PV AV
535 P React. PV TNT
540 P React. HT TNT
21. Cool the sample to 22. Use a 1-mL calibrated 23. Pour the sample into a 24. Proceed with a
room temperature. dropper to add 2.0 mL of 25-mL graduated cylinder. reactive phosphorus test
5.0 N Sodium Hydroxide Adjust the volume to 25 of the expected total
Solution to the flask. Swirl mL with deionized water phosphorus concentration
to mix. rinsings from the flask. range.
Extend the color
development time to
10 minutes for the
PhosVer 3 (ascorbic acid)
method.

Page 1048
Interferences

Alkaline or highly buffered
It may be necessary to add additional acid in step 19 to drop the pH of the solution below 1.
samples
Use 50 mL of sample and double the reagent quantities. Use a portion of the reacted sample
Turbidity to zero the instrument in the reactive phosphorus procedure. This compensates for any color
or turbidity destroyed by this procedure.

• If prompt analysis is not possible, samples may be preserved up to 28 days.
• To preserve samples. adjust the pH to 2 or less with Concentrated Sulfuric Acid* (about 2 mL
per liter). Store at 4 °C.
• Warm the sample to room temperature and neutralize with 5.0 N Sodium Hydroxide before
analysis.
Summary of method
forms. Organic phosphates are converted to orthophosphate by heating with acid and persulfate.
Organically bound phosphates are thus determined indirectly by subtracting the result of an acid
hydrolyzable phosphorus test from the total phosphorus result.
This procedure must be followed by one of the reactive phosphorus (orthophosphate) analysis
methods for determining the phosphorus content of the sample. If the ascorbic acid (PhosVer 3)
method is used to measure the reactive phosphorus, this method is USEPA accepted for NPDES
reporting.
The following reagents and apparatus are required in addition to those required for the active
phosphorus test.
* See Optional reagents.

Page 1049

Required reagents
Potassium Persulfate Powder Pillows 1 100/pkg 245199
Sodium Hydroxide Solution, 5.0 N 2 mL 100 mL MDB 245032
Sulfuric Acid Solution, 5.25 N 2 mL 100 mL MDB 244932
Required apparatus
Hot Plate, 7” x 7” digital, 115 VAC 1 each 2881500
Hot Plate, Stirrer, 7” x 7” digital, 230 VAC 1 each 2881602
Optional reagents
Sodium, Hydroxide, 5.0 N 1000 mL 245053
Thermometer, Non-Mercury, –10 to 225°C each 2635700
Sampling bottle with cap, low density polyethylene, 250 mL 12/pkg 2087076

Phosphorus, Total, TNT, 10127
Phosphorus, Total DOC316.53.01123
Molybdovanadate Method with Acid

Method 10127
Persulfate Digestion1
HR (1.0 to 100.0 mg/L PO43–) Test ‘N Tube™ Vials
1 Adapted from Standard Methods for the Examination of Water and Wastewater., (4500 B-C)
Test preparation


DR 6000 —
DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
Reagent blanks can be used more than once, but should not be used more than one day.
The final samples will contain molybdenum. In addition, the final samples will have a pH less than 2 and are considered
corrosive (D002) by the Federal RCRA. Refer to the current MSDS for safe handling and disposal instructions.
Total High Range Phosphorus Test ’N Tube™ Reagent Set 1
DRB200 Reactor, 15 x 16 mm 1
Pipet, TenSette®, 1 to 10 mL, plus tips 1
Test Tube Rack 1
Funnel, Micro 1
Phosphorus, Total
Page 1051
Phosphorus, Total
Molybdovanadate method, acid persulfate digestion
Stored Programs
542 P Total HR TNT
Start
1. Turn on the DRB200 2. Select the test. 3. Blank Preparation: 4. Prepared Sample:
Reactor. Heat to 150 °C. Insert an adapter if Use a TenSette® Pipet to Use a TenSette Pipet to
required (see Instrument- add 5.0 mL of deionized add 5.0 mL of sample to a
specific information). water to a Total Total Phosphorus Test ‘N
Phosphorus Test ‘N Tube Tube Vial.
Vial.
5. Use a funnel to add 6. Insert the vials in the 7. Set the instrument 8. After the timer expires,
the contents of one DRB 200 Reactor. timer to 30 minutes and carefully remove the hot
Potassium Persulfate Close the protective cover. start the timer. vials from the reactor.
Powder Pillow to each vial. A 30-minute heating Insert them in a test tube
Cap tightly and shake to period will begin. rack and allow to cool to
dissolve. room temperature
(18–25 °C).
Phosphorus, Total
Page 1052
Phosphorus, Total
Molybdovanadate method, acid persulfate digestion (continued)
9. Use a TenSette Pipet 10. Use a polyethylene 11. Start the instrument 12. Wipe the vials with a
to add 2.0 mL of 1.54 N dropper to add 0.5 mL of timer. damp towel, followed by a
sodium hydroxide to each Molybdovanadate A 7-minute reaction period dry one, to remove
vial. Reagent to each vial. will begin. Read the fingerprints or other
Cap and invert to mix. Cap and invert to mix. sample between seven marks.
and nine minutes after
adding the
Molybdovanadate
Reagent.
Zero Read
13. When the timer 14. ZERO the instrument. 15. Insert the prepared 16. READ the results in
expires, insert the blank The display will show: sample into the 16-mm cell mg/L PO43–.
into the 16-mm cell holder. holder.
0.0 mg/L PO43–
Interferences
Sample turbidity may cause inconsistent results in the test because the acid present in the
reagents may dissolve some of the suspended particles and because of variable desorption of
orthophosphate from the particles.
The Interfering substances table shows interference levels and types. The Noninterfering
substances, (at concentrations less than 1000 mg/L) table shows substances that do not interfere
in concentrations less than 1000 mg/L.

Causes positive interference if the sample is warm when the molybdovanadate reagent is
Arsenate
added (after the digestion).1 Cool the sample after digestion before adding reagent.
Blue color caused by ferrous iron does not interfere if iron concentration is less than
Iron, ferrous
100 mg/L.
Phosphorus, Total
Page 1053
Phosphorus, Total

Causes positive interference if the sample is warm when the molybdovanadate reagent is
Silica
added (after the digestion). Cool the sample after digestion before adding reagent.
Extreme pH or highly buffered May exceed buffering capacity of the reagents. Samples may require pretreatment. Sample
samples pH should be about 7.
Temperature, Cold (less than
18 °C)
Causes a positive interference.
Temperature, Hot (greater
Post-digestion samples should be brought to room temperature (18–25 °C) before the
than 25 °C)
addition of the Molybdovanadate Reagent or sodium hydroxide.
1 Gentle warming of the sample to reach room temperature will not cause this substance to interfere.
Table 324 Noninterfering substances, (at concentrations less than 1000 mg/L)
Pyrophosphate Tetraborate Selenate Benzoate
Citrate Oxalate Lactate Tartrate
Formate Salicylate Al3+ Fe3+
Mg2+ Ca2+ Ba2+ Sr2+
Li+ Na+ K+ NH4+
Cd2+ Mn2+ NO3– NO2–
SO42– SO32– Pb2+ Hg+
Hg2+ Sn2+ Cu2+ Ni2+
Ag+ U4+ Zr4+ AsO3–
Br – CO3 2– ClO4– CN–
IO3– SiO44– —

• Collect samples in plastic or glass bottles that have been acid washed with 1:1 Hydrochloric
• Do not use commercial detergents containing phosphate for cleaning the glassware used in
this test.
• Analyze samples immediately after collection for best results.
• If prompt analysis is impossible, preserve samples up to 28 days by adjusting the pH to 2 or
less with concentrated Sulfuric Acid* (about 2 mL per liter) and storing at 4 °C.
• Warm stored samples to room temperature and neutralize with 5.0 N Sodium Hydroxide*
before analysis.
Phosphorus, Total
Page 1054
Phosphorus, Total
Accuracy check
• 10-mL Voluette® Ampule of Phosphate Standard Solution, 500 mg/L as PO43–
• Ampule breaker
1. Clean glassware with 1:1 Hydrochloric Acid Standard Solution. Rinse again with deionized
water. Do not use detergents containing phosphate to clean glassware.

ADDITIONS
standard to three 10-mL portions of fresh sample in 25 mL mixing cylinders.
7. Follow the Molybdovanadate method, acid persulfate digestion test procedure for each of the
spiked samples, starting with the 0.1 mL sample spike. Measure each of the spiked samples in
the instrument.
Phosphorus, Total
Page 1055
Phosphorus, Total


• Phosphate standard solution, 50-mg/L
1. Use the 50-mg/L phosphate standard solution in place of the sample. Follow the
Molybdovanadate method, acid persulfate digestion test procedure.
Method performance
542 50 mg/L PO43– 49.4–50.6 mg/L PO43– 0.7 mg/L PO43–
Summary of method
forms. Organic phosphates are converted to orthophosphates by heating with acid and persulfate.
molybdate complex. In the presence of vanadium, yellow molybdovanadophosphoric acid forms.
The intensity of the yellow color is proportional to the phosphate concentration. Test results are
measured at 420 nm.
Phosphorus, Total
Page 1056
Phosphorus, Total
Required reagents
Total High Range Phosphorus Test ’N Tube™ Reagent Set, includes: — 50 vials 2767245
(1) Molybdovanadate Reagent1 0.5 mL 25 mL —
(1) Potassium Persulfate powder Pillows 1 50/pkg 2084766
(1) Sodium Hydroxide Solution, 1.54 N 2 mL 100 mL 2743042
(1) Total Phosphorus Test Vials1 1 50/pkg —
(2) Water, deionized 5 mL 100 mL 27242
1 Not available separately.
Required apparatus
Dropper, 0.5 and 1.0 mL marks, plastic 1 20/pkg 2124720
Light shield, DR 3900 1 each LZV849
Light shield, DR 3800, DR 2800, DR 2700 1 each LZV646
Phosphate Standard Solution, Voluette® ampule, 500-mg/L as PO43–, 10-mL 16/pkg 1424210

Wastewater Standard, Influent Inorganics for NH3–N, NO3–N, PO4, COD, SO4, TOC 500 mL 2833149

Molybdovanadate Reagent 100 mL 2076032
Phosphorus, Total
Page 1057
Phosphorus, Total


Funnel, micro each 2584335
Optional standards

Potassium, 8049
Potassium DOC316.53.01127
Tetraphenylborate Method Method 8049

Test preparation


Program # 905 has a calibration curve for potassium; however, due to potential variation between lots of Potassium 3
Reagent, it is recommended that a new calibration for each lot of reagent is performed to obtain best accuracy. Prepare and
store the calibration as directed under Calibration.
Filter highly colored or turbid samples before analysis.
Refer to a current MSDS for pollution prevention and waste management information.
After the test, clean the cells with soap and a brush.
Potassium Reagent 1 Powder Pillow 1
Clippers 1
Cylinder, mixing, 25-mL 1
Flask, volumetric, 100-mL Class A 8
Pipet, TenSette®, 1–10 mL, plus tips varies
Potassium
Page 1059
Potassium
Tetraphenylborate method for powder pillows
Stored Programs
905 Potassium
Start
1. Select the test. 2. Fill a graduated mixing 3. Add the contents of 4. Add the contents of
Insert an adapter if cylinder with 25 mL of one Potassium 1 Reagent one Potassium 3 Reagent
required (see Instrument- sample. Pillow. Add the contents of Pillow after the solution
specific information). one Potassium 2 Reagent clears. Stopper and shake
Pillow. Stopper and invert the solution for 30
several times to mix. seconds.
A white turbidity will form if
potassium is present.
5. Start the instrument 6. Prepared Sample: 7. Blank Preparation: 8. Wipe the blank and
timer. Pour at least 10-mL of the When the timer expires, fill insert it into the cell holder.
A three-minute reaction solution from the cylinder the second sample cell
period will begin. into a sample cell. with 10 mL of unreacted
sample.
Zero Read
9. ZERO the instrument. 10. Within seven minutes 11. READ the results in
The display will show: after the timer expires, mg/L K.
wipe the prepared sample
0.0 mg/L K and insert it into the cell
holder.
Potassium
Page 1060
Potassium
Interferences
The substances in the Interfering substances table have been tested and will not interfere at or
below the levels stated. If these substances are present at higher levels, conduct interference
studies at the higher levels to determine if the substance interferes.

Ammonium Nitrogen 15 mg/L as N
Chloride 15,000 mg/L

• Adjust the pH to 2 or less with Nitric Acid (about 2 mL per liter)*.
• Preserved samples may be stored at least six months at room temperature.
• Before analysis, adjust the pH to 4–5 with 5.0 N Sodium Hydroxide*.
• Do not measure pH in the sample container with a pH electrode, as this will introduce
potassium from the filling solution. Use pH Paper* or pour off sample and test pH in a separate
beaker.
Accuracy check
Important Note: This procedure is applicable only to Stored Program 905 and not to User
Programs.

• Potassium Voluette® Ampule Standard, 250-mg/L K
• Ampule breaker

ADDITIONS.
Potassium
Page 1061
Potassium
6. Follow the Tetraphenylborate method for powder pillows test procedure for each of the spiked
instrument.
Calibration
An approximate calibration curve is preprogrammed within Program 905. For improved accuracy,
a new calibration should be performed with each new lot of reagents. Prepare calibration
standards containing 1, 2, 3, 4, 5, 6, 7 and 8 mg/L potassium as follows:
1. Into eight different 100-mL Class A volumetric flasks, pipet 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and
8.0 mL of the 100-mg/L Potassium Standard Solution using class A glassware or TenSette
Pipet.
2. Dilute to the mark with deionized water. Mix thoroughly.
3. Use deionized water for the 0-mg/L potassium standard.
User-entered Program
Refer to the user manual for instrument-specific information on user-entered programs.
1. Access the User Program feature.
2. For the initial potassium calibration, assign a new program number.
3. Enter a name for the potassium test.
4. Set up the following parameters
• Program Type: Single wavelength • Concentration Resolution: 0.1

• Units: mg/L • Chemical Form: K
• Wavelength l (nm): 650 • Calibration: Read Standards
5. Set up the following parameters:
• Upper Limit: On, 8.0 • Timer 1: Timer 3:00

• Lower Limit: On, -0.2 • Press Calibration: C=a + bA >Edit>OK
6. Enter the concentrations for the calibration, starting with 0.0, in the left column.
7. When all standard concentrations have been entered, navigate to the 0.0 line.
8. Insert the cell containing the blank (deionized water) and zero the instrument.
9. Perform the potassium test on each standard and insert the first prepared standard into the
cell holder. Navigate to the line corresponding to this standard concentration. Read the result.
Repeat for each standard concentration.
10. If the graph result is acceptable, exit the program. It may be possible to obtain a better fit to the
data by reading another curve. The curve which results in the highest r2 value is generally the
best fit. After selection of the best curve, exit the program.
11. Save the calibration.
Potassium
Page 1062
Potassium
Method performance

Program Standard
905 5.0 mg/L K 4.7–5.3 mg/L K 0.1 mg/L K
Summary of method
Potassium in the sample reacts with sodium tetraphenylborate to form potassium
tetraphenylborate, an insoluble white solid. The amount of turbidity produced is proportional to the
potassium concentration. Test results are measured at 650.

Required reagents
Potassium Reagent Set: — — 2459100
Potassium Reagent 1 Powder Pillow 1 25/pkg 1432198
Required apparatus
Cylinder, mixing, 25-mL 1 each 189640
Flask, volumetric, 100-mL Class A 8 each 1457442
Pipet, TenSette®, 1–10 mL 1 each 1970010
Potassium Standard Solution, 10-mL Voluette® Ampule, 250 mg/L 16/pkg 1479010
Potassium Standard Solution, 100-mg/L 500 mL 2351749

Nitric Acid, 1:1 500 mL 254049
Potassium
Page 1063
Potassium

Pipet Tips, for TenSette® Pipet 19700101 250/pkg 2199725

Pipet, TenSette, Pipet, 0.1–1.0 mL each 1970001
Brush, test tube each 69000
Liqui-Nox detergent 946 mL 2088153

Quaternary Ammonium Compounds, 8337
Quaternary Ammonium
Compounds DOC316.53.01128
Direct Binary Complex Method Method 8337

0.2 to 5.0 mg/L as CTAB Powder Pillows
Scope and Application: For cooling tower water and pool/spa water
Test preparation


QAC Reagent 1 Powder Pillows 2 pillows
QAC Reagent 2 Powder Pillows 2 pillows
Bottle, square, with 25 mL mark 2
Clippers, for opening powder pillows 1
Sample Cells, 1-inch, 10 mL 2
Quaternary Ammonium Compounds

Page 1065
Direct Binary Complex Method
Stored Programs
401 QAC
Start
1. Select the test. 2. Blank Preparation: 3. Prepared Sample: 4. Add the contents of
Insert an adapter if Fill one 25-mL mixing Fill another mixing bottle one QAC Reagent 1
required (see Instrument- bottle with 25 mL of with 25 mL of sample. Powder Pillow to each
specific information). deionized water. bottle.
5. Swirl the bottles to 6. Add the contents of 7. Swirl the bottles to 8. Start the instrument
dissolve the reagent. one QAC Reagent 2 dissolve the reagent. Do timer.
Do not shake! Shaking Powder Pillow to each not shake. A two-minute reaction
creates air bubbles that bottle. A purple color will form if period will begin.
interfere with test results. a quaternary ammonium
compound is present
Zero
9. Pour at least 10 mL of 10. When the timer 11. ZERO the instrument. 12. Insert the prepared
the solutions from the expires, insert the blank The display will show: sample into the cell holder.
bottles into the sample into the cell holder. READ the results in mg/L
cells. 0.0 mg/L CTAB
CTAB (cetyl-
trimethylammonium
bromide).

Page 1066
Interferences
Interference studies were conducted by preparing a CTAB standard solution of approximately
3 mg/L as well as a solution of the potential interference. The constituent was said to interfere
when the resulting concentration changed by 10%. The Interfering substances table shows
interfering substances and levels. The Noninterfering substances table shows substance that do
not interfere up to the tested concentrations.
After several samples have been analyzed, the sample cells may exhibit a build-up of a pink or
purple color. A rinse with 1.0 N Sodium Hydroxide Solution followed by an Alconox™ detergent
wash and deionized water rinse will eliminate the build-up when it occurs.

Calcium (as CaCO3) Positive interference above 1350 mg/L
Chlorine, HOCl and OCl– Positive interference above 7 mg/L
Cyanuric acid Negative interference above 70 mg/L
Igepal™ nonionic surfactant Positive interference above 3 mg/L
Iodine, I3– Positive interference above 3 mg/L
Iron, Fe3+ Positive interference above 80 mg/L
Liquimine™ 14–P,
Positive interference above 1825 mg/L
filming amine
Magnesium, Mg2+ Positive interference above 1350 mg/L
Niaproof™ anionic surfactant Negative interference above 11 mg/L
Polyacrylic acid Negative interference above 16 mg/L
Sodium lauryl sulfate Negative interference above 8 mg/L
Sodium polyphosphate Positive interference above 1325 mg/L
Tribenzylamine Positive interference above 7 mg/L
Triton X-100™ nonionic
Positive interference above 4 mg/L
surfactant
Urea Positive interference above 8 mg/L
May exceed the buffering capacity of the reagents and require sample pretreatment. Adjust
the sample pH between 3 and 5 by using a pH meter or pH paper and adding dropwise an
appropriate amount of acid or base such as 1.0 N Sulfuric Acid Standard Solution or 1.0 N
extreme sample pH
Sodium Hydroxide Standard Solution. If significant volumes of acid or base are used, a
volume correction should be made.
Table 329 Noninterfering substances

Non-interfering Substance Highest Concentration Tested (mg/L)
Silica, SiO2 400
Potassium alum, AlKS2O8 500
Sodium thiosulfate, Na2S2O3 30

Page 1067

• Collect samples in glass bottles that have been rinsed several times with sample before final
sample filling.
• Do not use plastic containers; plastic adsorbs QACs.
• Acidify the sample to a pH of less than 2.
• Store at 4 ± 2 °C.
Accuracy check
• QAC Standard Solution, 100-mg/L CTAB
• Mixing bottles (3)

ADDITIONS.
6. Follow the Direct Binary Complex Method test procedure for each of the spiked samples,


• QAC Standard, 100-mg/L as CTAB
• Deionized water
• Class A 5 mL volumetric pipet and pipet bulb
1. Prepare a 5.0 mg/L CTAB standard solution as follows:
a. Pipet 5.0 mL of QAC Standard, 100-mg/L as CTAB, into a 100-mL volumetric flask.
2. Use this solution in place of the sample. Follow the Direct Binary Complex Method test
procedure.
to Standard Adjust in the software: OPTIONS>MORE>STANDARD ADJUST

Page 1068
Method performance

401 DR 5000 3.0 mg/L CTAB 2.7–3.3 mg/L CTAB 0.04 mg/L CTAB
Summary of method
The test method makes use of a colorimetric chemistry in which a quaternary ammonium
compound reacts with an indicator to produce a color change from pale pink to vivid purple. The
test is conducted in a stabilized, acid-buffered solution containing a masking agent to eliminate
potential interferences. This test is applicable to the monitoring of QACs in swimming pools and
cooling towers. Test results are measured at 575 nm.

Page 1069
Required reagents
Quaternary Ammonium Compounds Reagent Set (100 tests), includes: — — 2459200
(4) QAC Reagent 1 Powder Pillows 2 pillows 50/pkg 2401066
(8) QAC Reagent 2 Powder Pillows 2 pillows 25/pkg 2401268

Bottle, square, with 25 mL mark 2 each 1704200
QAC Standard Solution, 100-mg/L as CTAB 100 mL 2415342

Alconox™ detergent 1.8 kg 2088000
Sodium Hydroxide Solution, 1.0 N 1000 mL 104553

Salinity, DT, 10073
Salinity DOC316.53.001180
Mercuric Nitrate Method Method 10073

0–100 ppt1 Salinity Digital Titrator
Scope and Application: For seawater and brackish water
1 parts per thousand (ppt)
Test preparation
ppt salinity x 569 = mg/L chloride (Cl–).
ppt salinity x 940 = mg/L sodium chloride (NaCl)
Use the TitraStir apparatus for best results
Both the sample and the blank will contain mercury (D009) at a concentration regulated as a hazardous waste by the Federal
RCRA. Do not pour these solutions down the drain. Refer to the MSDS for more information on proper disposal of these
materials.
Diphenylcarbazone Reagent Powder Pillows 1
Deionized Water varies
Digital Titrator 1
Mercuric Nitrate Titration Cartridge, 2.570 N varies
Syringe, 3-cc, Luer lock tip 1
Vial with 2-, 5-, 10-, 15-, 20- and 25-mL marks 1
Salinity
Page 1071
Salinity
Iodate-Iodide Method
1. Insert a clean delivery 2. Turn the delivery knob 3. Use the 3-mL syringe 4. Fill the vial to the
tube into the Mercuric to eject a few drops of to collect a 2.0-mL water 10-mL mark with
Nitrate Titration Cartridge. titrant. Reset the counter sample. Add to the vial, deionized water.
Attach the cartridge to the to zero and wipe the tip. provided.
Note: 2 mL = 2 cc
titrator body.
5. Add the contents of 6. Titrate the sample with 7. Calculate sample

one Diphenylcarbazone mercuric nitrate until the salinity in parts per
Reagent Powder Pillow to color changes from yellow thousand:
the vial and mix. to light pink. Digits Required x 0.1 =
ppt Salinity
A small amount of Record the number of
undissolved reagent will digits.
not affect results.
Summary of method
The mercuric nitrate method of chloride analysis has become popular due to the sharp yellow to
pinkish-purple end point of diphenylcarbazone. A single, stable powder has been developed,
combining the color indicator with an appropriate buffer to establish the correct pH.
Salinity
Page 1072
Salinity

Required reagents
Diphenylcarbazone Reagent Powder Pillows 100/pkg 96799
Mercuric Nitrate Titration Cartridge, 2.570 N each 2393701
Water, Deionized 4L 27256
Chloride Standard, 12,500 mg/L as Cl-, (22 ppt Salinity) 10 mL AMP 16/pkg 1425010
Sodium Chloride standard, 10,246 mg/L as NaCl, 100 mL AMP 10.9 ppt salinity 2307442
Required apparatus
Delivery tubes w/ 180 ° hook each 1720500
Digital Titrator2 each 1690001
Syringe, 3-cc, Luer lock tip each 4321300
Vial with 2-, 5-, 10-, 15-, 20- and 25-mL marks each 219300
Salinity
Page 1073
Selenium, 8194
Selenium DOC316.53.01129
Diaminobenzidine Method1 Method 8194

0.01 to 1.00 mg/L
Test preparation


Distillation is required for determining total selenium. See Distillation at the end of the procedure. Use the distillate as the
sample in step 2.
Acetone1 is a suitable solvent for removing toluene from glassware after results are measured.
Toluene (F005) solutions are regulated as hazardous waste by the Federal RCRA. Do not pour these materials down the
drain. Water saturated with toluene, toluene solutions and the cotton plug used in the delivery tube of the separatory funnel
should be collected for disposal with laboratory solvent wastes. Refer to the current MSDS for safe disposal and handling
information.
If there are visible water bubbles on the bottom of the cell, decant the top portion into a clean, dry cell prior to reading the
sample.
Selenium
Page 1075
Selenium
Buffer Solution, sulfate type, pH 2.0 10 mL
Cotton Ball 1
Cylinder, Graduated: 50- and 100-mL 1 of each
Diaminobenzidine, tetrahydrochloride 0.1 g
Distillation reagents and apparatus —
Dropper, 0.5 and 1.0 mL marks, one glass and one plastic 1 of each
Funnel, separatory, 250 mL 2
Hot Plate, 7-inch 1
Pipet, volumetric, 5-mL, plus safety bulb filler 1
Ring support (3-inch) and stand 2
Spoons, measuring, 0.2 and 0.05 g 1 of each
TitraVer® Hardness Reagent 0.4 g
Toluene, ACS 60 mL
Water, Deionized 100 mL
Diaminobenzidine method
Stored Programs
640 Selenium
Start
1. Select the test. 2. Measure 100 mL of 3. Add a 0.2-g spoonful 4. Add a 0.05-g spoonful
Insert an adapter if deionized water into a of TitraVer® Hardness of diaminobenzidene
required (see Instrument- 500-mL Erlenmeyer flask. Reagent to each flask. tetrahydrochloride to each
specific information). Label the flask “blank”. Swirl to mix. flask. Swirl to mix.
Measure 100 mL of
sample into a 500-mL
Erlenmeyer flask. Label
the flask “sample”.
Selenium
Page 1076
Selenium
Diaminobenzidine method (continued)
5. If you have not 6. Heat each flask on a 7. Start the instrument 8. When the timer
distilled the sample, add hot plate. Bring the timer. expires, remove both
5.0 mL of Buffer Solution, contents to a gentle boil. A five-minute reaction flasks. Cool to room
sulfate type, pH 2.0 to period will begin. Continue temperature using a water
each flask. Swirl to mix. to boil the contents gently bath.
If the sample has been during this time period. Do not boil more than one
distilled, adjust the pH of A yellow color will develop minute after the timer
the distillate to pH 2.7 ± if selenium is present. expires.
0.2 using 5 N Sodium
Hydroxide Standard
Solution. Adjust the blank
to pH 2.7 ± 0.2 using 5.25
N Sulfuric Acid Standard
Solution.
9. Transfer the contents 10. Add 2.0 mL of 12 N 11. Add 30-mL of toluene 12. Start the instrument
of each flask to separate Potassium Hydroxide to each funnel. Stopper. timer.
250-mL separatory Standard Solution to each Swirl and invert each A 30-second reaction
funnels. Label the funnels funnel using a calibrated funnel, then open the period will begin. During
“blank” and “sample”. 1.0-mL plastic dropper. stopcock to vent the this time, vigorously shake
Place each flask in a Stopper. Shake each funnel. Close the the funnel that contains
support ring on a stand. funnel to mix. stopcock. Repeat twice the blank.
with each funnel.
Use toluene only with
adequate ventilation.
Selenium
Page 1077
Selenium
Diaminobenzidine method (continued)
13. Start the instrument 14. Start the instrument 15. When the timer 16. Insert a cotton plug
timer. timer. expires, remove the into the delivery tube of
A 30-second reaction A four-minute reaction stopper and drain the each separatory funnel.
period will begin. During period will begin. lower water layer from Slowly drain the toluene
this time, vigorously shake each funnel and discard. into respective sample
the funnel that contains Complete steps 16–19 cells labeled “blank” and
the sample. within five minutes after “sample”. Stopper the
the timer expires. The sample cells.
developed color is stable, Filtering the toluene
but should be measured through dry, absorbent
as soon as possible. cotton will remove water or
suspended particles.
Zero Read
insert it into the cell holder. The display will show: sample and insert it into mg/L Se.
the cell holder.
0.00 mg/L Se
Interferences
There are no positive inorganic interferences with this method. Any other interferences can be
removed by distilling the sample.

Ferric iron Up to 2.5 mg/L. Distill sample to eliminate interference.
Manganese Will not interfere.
Strong oxidizing agents (i.e.,
Can react with the indicator to give low results. Distill sample to eliminate interference.
iodine, bromine or chlorine)
Selenium
Page 1078
Selenium

• Collect samples in clean glass or plastic containers.
• Adjust the pH to 2 or less with Nitric Acid* (about 1.5 mL per liter).
• Preserved samples can be stored for up to six months at room temperature.
Distillation
CAUTION
Always perform this procedure under a fume hood.
This distillation involves the use of a strong acid and oxidizer at high temperatures. To avoid
personal injury, observe all laboratory safety precautions when operating the distilling apparatus.
2. Add 1 mL of Methyl Orange Indicator Solution. Stir with a glass rod.
3. Use a dropper to add 0.1 N Hydrochloric Acid Standard Solution dropwise until the solution
becomes pink. Then add an additional 2 mL.
4. Use a pipet to add 5.0 mL Calcium Chloride Solution. Mix well.
5. Use a dropper to add 1-g/L Potassium Permanganate Standard Solution drop-wise until the
solution is purple.
6. Place the beaker on a hot plate. Evaporate the solution to approximately 250 mL. Periodically
add 1-g/L Potassium Permanganate Solution to keep the solution purple.
7. Any precipitate formed at this step is manganese dioxide and may be ignored.
8. Cool the solution. While cooling, set up the distillation apparatus for the general purpose
distillation as shown in the distillation manual.
9. Pour the treated sample solution into the distillation flask. Add a stirring bar to the flask.
10. Pipet 5.0 mL of 0.1 N Sodium Hydroxide Standard Solution into the flask. Turn the stirrer
power switch to ON. Set the stir control to 5.
11. Turn on the water and adjust so a constant flow is maintained through the condenser. Set the
heat control to 10.
12. When only a few milliliters are left in the distillation flask, turn the power switch off. The
distillate in the Erlenmeyer flask may be discarded.
CAUTION
Perform step 13 under a fume hood.
13. When the flask has cooled, add 50 mL of 19.2 N Sulfuric Acid Standard Solution to the flask.
Add 10 grams of Potassium Bromide† to the flask.
14. Fill a 250-mL beaker to the 75-mL mark with deionized water. Place it under the drip tube.
Elevate the beaker with a laboratory jack so the tube extends below the level of the water.
† Purchase Potassium Bromide from a local chemical supplier.
Selenium
Page 1079
Selenium
15. Add 1.0 mL of 30% hydrogen peroxide solution to the flask. Turn the stir control to 5 and the
heat control to 10. Cap the distillation flask.
16. Heat the distillation flask until the yellow color is gone from the complete distillation apparatus,
including the J-tube and condenser. Remove the beaker from under the drip tube.
17. Turn off the heater switch. When the J-tube and condenser have cooled, rinse them with
deionized water. Add the washings to the 250-mL beaker. Total volume in the beaker should
be approximately 100 mL.
18. Add the Phenol Solution drop-wise to the distilled sample to discharge the bromine color (a
white precipitate of tribromophenol will form).
19. Allow the precipitate to settle. Using a dropper, collect about 5 mL of the clear, colorless
distillate and transfer to a test tube.
20. Test the solution for completeness of precipitation by adding 2 drops of Phenol Solution. If the
solution becomes cloudy or white precipitate forms, residual bromine is still present (proceed
to next step). If no cloudiness occurs, the sample is ready for analysis.
21. Transfer the 5-mL aliquot back to the beaker and continue to add Phenol Solution until no
turbidity is formed in subsequent 5-mL aliquots.
22. Transfer the entire sample into a 500-mL volumetric flask. Rinse the beaker with deionized
water and add to the flask.
23. Dilute to volume with deionized water, stopper and mix well. The distillate is now ready
for analysis.
Accuracy check
• Selenium Standard Solution, 1000 mg/L
• 10 mL volumetric pipet and pipet bulb
• Deionized water

ADDITIONS.
4. Prepare a 100-mg/L selenium standard solution:
a. Pipet 10 mL of 1000-mg/L selenium standard solution into a 100-mL volumetric flask.

b. Dilute to volume with demineralized water.
prepared standard to three 100-mL portions of fresh sample.
Selenium
Page 1080
Selenium
6. Follow the Diaminobenzidine method test procedure for each of the spiked samples, starting


• 1000 mg/L Selenium Standard Solution
• Deionized water
• 500-mL Erlenmeyer flask
• Volumetric pipet
• TenSette Pipet
1. Prepare a 0.5 mg/L Selenium standard solution:
a. Prepare a 100-mg/L selenium standard solution: Pipet 10 mL of 1000-mg/L selenium

standard solution into a 100-mL volumetric flask. Dilute to volume with demineralized
water.
b. Pipet 1.00 mL of the 100-mg/L solution into a 200-mL volumetric flask. Dilute to volume
c. Transfer 100 mL of the standard into a 500-mL Erlenmeyer flask
2. Use this solution in place of the sample. Follow the Diaminobenzidine method test procedure.
Method performance
640 0.50 mg/L Se 0.47–0.53 mg/L Se 0.01 mg/L Se
Summary of method
An EDTA masking agent is added to the sample to remove interferences such as iron prior to the
test. The addition of a sulfate buffer adjusts the sample to the optimum pH of 1 to 2. Under these
conditions, diaminobenzidine reacts with all selenium present as selenite (Se4+) to give a yellow-
colored piazselenol complex which is extracted and the color intensity measured colorimetrically.
Selenium present as Se2+ and Se6+ is not detected unless the sample is distilled. Test results are
measured at 420 nm.
Selenium
Page 1081
Selenium
Required reagents
Selenium Reagent Set (100 tests1), includes: 2244200

(1) Buffer Solution, sulfate type, pH 2.0 10 mL 500 mL 45249
(1) Diaminobenzidine, tetrahydrochloride 0.1 g 5g 706222
(2) Potassium Hydroxide Standard Solution, 12 N 4 mL 100 mL 23032
(1) TitraVer® Hardness Reagent, ACS 0.4 g 100 g 20426
(1) Toluene, ACS 60 mL 4L 1447017
1 This test requires a reagent blank. The number of tests shown refers to any combination of samples and blanks.
Required apparatus
Dropper, 0.5 & 1.0 mL marks, glass 1 5/pkg 1419705
Dropper, 0.5 & 1.0 mL marks, plastic 1 20/pkg 2124720
Hot Plate, 7x7-inch, 120 VAC 1 each 2881500
Hot plate stirrer 7x7-inch, 220-240 VAC 1 each 2881602
Pipet, volumetric, 5-mL 1 each 1451537
Pipet filler, safety bulb 1 each 1465100
Ring, support, (3-inch) 83-mm 2 each 58000
Sample Cells, 1-inch square, 25 mL with stopper, matched pair 2 2/pkg 2612602
Support, ring stand, (5 x 8 inch) 127 x 203 mm 2 each 56300

Calcium Chloride Solution 1000 mL 42853
Hydrochloric Acid Standard Solution, 0.1 N 1000 mL 1481253
Methyl Orange Indicator Solution, (0.50-g/L) 500 mL 14849
Potassium Permanganate Solution, 1-g/L 100 mL 1416442
Potassium Bromide, ACS grade1 — —
Selenium
Page 1082
Selenium

Distillation Apparatus Set, general purpose each 2265300
Distillation Apparatus Heater, 115 VAC each 2274400
Distillation Apparatus Heater, 230 VAC each 2274402
Beaker, 1000 mL each 50053
Glass stirring rod 3/pkg 177001
Stir bar, magnetic each 2095351
Test tubes, 24 mL, 16 mm diameter 10/pkg 56524
1 Purchase Potassium Bromide from a local chemical supplier
Selenium Standard Solution, 1000-mg/L 100 mL 2240742

Selenium
Page 1083
Selenium

Silica, HR, 8185
Silica DOC316.53.01133
Silicomolybdate Method Method 8185

HR (1 to 100 mg/L) Powder Pillows
Scope and Application: For water and seawater
Test preparation


Sample temperature should be 15–25 °C (59–77 °F)
This method is very sensitive to small differences in the measurement wavelength. For best results, use the Standard Adjust
feature (as described in Accuracy check, Standard solution method) to optimize results on each instrument.
High Range Silica Reagent Set 1
Silica
Page 1085
Silica
Silicomolybdate method for powder pillows
Stored Programs
656 Silica HR
Start
1. Select the test. 2. Fill a sample cell with 3. Prepared Sample: 4. Add the contents of
Insert an adapter if 10-mL of sample. Add the contents of one one Acid Reagent Powder
required (see Instrument- Molybdate Reagent Pillow for High Range
specific information). Powder Pillow for High Silica. Swirl to mix.
Range Silica to the sample A yellow color will develop
cell. Swirl until completely if silica or phosphorus is
dissolved. present.
5. Start the instrument 6. When the timer 7. Start the instrument 8. Blank Preparation:
timer. expires, add the contents timer. Fill a second sample cell
A ten-minute reaction of one Citric Acid Powder A two-minute reaction with 10 mL of the original
period will begin. Pillow to the sample cell. period will begin. sample.
Swirl to mix.
Perform steps 3–11 within
Any yellow color due to three minutes after the
phosphorus is removed in timer expires.
this step.
Zero Read
insert the blank into the The display will show: sample and insert the mg/L SiO2.
cell holder. prepared sample into the
0 mg/L SiO2 cell holder.
Silica
Page 1086
Silica
Interferences
Occasionally a sample contains silica which reacts very slowly with molybdate. The nature of
these “molybdate-unreactive” forms is not known. A pretreatment with Sodium Bicarbonate*, then
Sulfuric Acid* will make these forms reactive to molybdate. The pretreatment is given in Standard
Methods for the Examination of Water and Wastewater under Silica-Digestion with Sodium
Bicarbonate. A longer reaction time with the sample and the molybdate and acid reagents (before
adding citric acid) may help instead of the bicarbonate treatment.

Color Eliminated by zeroing the instrument with the original sample.
Iron High levels of Fe2+ and Fe3+ interfere.
Does not interfere below 50 mg/L PO43–. At 60 mg/L PO43–, a negative 2% interference
Phosphate
occurs. At 75 mg/L PO43–, the interference is negative 11%.
Sulfides (S2–) All levels interfere.
Turbidity Eliminated by zeroing the instrument with the original sample.

• Collect samples in clean plastic bottles.
• If prompt analysis is not possible, store samples at 4 °C (39 °F) for up to 28 days.
Accuracy check
• Silica Standard Solution, 1000 mg/L
chemical form.

ADDITIONS.
6. Follow the Silicomolybdate method for powder pillows test procedure for each of the spiked
instrument.
Silica
Page 1087
Silica


• Silica Standard Solution, 50-mg/L
1. Use the Silica Standard Solution, 50-mg/L in place of the sample. Use deionized water as the
blank. Follow the Silicomolybdate method for powder pillows test procedure.
Method performance

Program Standard
656 50 mg/L SiO2 48–52 mg/L SiO2 1.0 mg/L SiO2
Summary of method
Silica and phosphate in the sample react with molybdate ion under acidic conditions to form yellow
silicomolybdic acid complexes and phosphomolybdic acid complexes. Addition of citric acid
destroys the phosphate complexes. Silica is then determined by measuring the remaining yellow
Required reagents
High Range Silica Reagent Set for 10-mL samples (100 tests), includes: 2429600
Acid Reagent Powder Pillows for High Range Silica 1 100/pkg 2107469
Citric Acid Powder Pillows 1 100/pkg 2106269
Molybdate Reagent Powder Pillows for High Range Silica 1 100/pkg 2107369
Silica Standard Solution, 50-mg/L 200 mL 111729
Silica Standard Solution, 1000-mg/L 500 mL 19449
Silica
Page 1088
Silica

Sodium Bicarbonate 454 grams 77601
Sulfuric Acid 1.00 N 100 mL 127032
Silica
Page 1089
Silica, LR, 8186
Heteropoly Blue Method1 Method 8186

LR (0.010 to 1.600 mg/L as SiO2) Powder Pillows
Scope and Application: For boiler and ultrapure water
Test preparation

Powder pillows
Instrument
Sample cell Cell orientation
The four-minute reaction time in step 4 is for samples at 20 °C; for samples at 10 °C, wait eight minutes; for samples at
The one-minute reaction time in step 6 is for samples at 20 °C; for samples at 10 °C, wait two minutes; for samples at 30 °C,
wait 30 seconds.
If testing for very low levels of silica, use Method 8282.
For best results, use matched cells.
Amino Acid F Reagent Powder Pillows (for 10-mL sample) 1 pillow
Citric Acid Powder Pillows 2 pillows
Molybdate 3 Reagent Solution 1 mL
Sample Cells see Instrument-specific information 2
Silica
Page 1091
Silica
Heteropoly Blue
Stored Programs
651 Silica LR
Start
1. Select the test. 2. Insert an adapter if 3. Add 14 drops of 4. Start the timer. A four-
required (Instrument- Molybdate 3 Reagent to minute reaction period will
specific information). each sample cell. Swirl to begin.
Fill two sample cells mix.
(Instrument-
specific information) with
10 mL of sample.
5. When the timer 6. Start the timer. A one- 7. Prepared Sample: 8. Start the timer.
expires, add the contents minute reaction period When the timer expires, A two-minute reaction
of one Citric Acid Reagent will begin. add the contents of one period will begin.
Powder Pillow to each The destruction of Amino Acid F Reagent
sample cell. Swirl to mix. Powder Pillow to one of A blue color will develop if
possible phosphate silica is present.
interference occurs during the sample cells. Swirl to
this period. mix.
Blank Preparation: The
sample without the Amino
Acid F Reagent is the
blank.
Silica
Page 1092
Silica
Heteropoly Blue (continued)
Zero Read
9. Wipe the blank and 10. Wipe the prepared 11. READ the results in
insert it into the cell holder. sample and insert it into mg/L (SiO2)
ZERO the instrument. the cell holder with the fill
line facing the user.
The display will show
0.000 mg/L SiO2.
Interferences
Color Eliminated by zeroing the instrument with the original sample.
Iron Large amounts interfere.
Does not interfere at levels less than 50 mg/L PO4. At 60 mg/L PO4, an
Phosphate
interference of –2% occurs. At 75 mg/L PO4 the interference is –11%.
Occasionally a sample contains silica which reacts very slowly with
molybdate. The nature of these “molybdate-unreactive” forms is not known. A
pretreatment with Sodium Bicarbonate, then Sulfuric Acid will make these
forms reactive to molybdate. The pretreatment is given in Standard Methods
Slow reacting forms of silica
for the Examination of Water and Wastewater under Silica-Digestion with
Sodium Bicarbonate. A longer reaction time with the sample and the
molybdate and acid reagents (before adding citric acid) may help instead of
the bicarbonate pretreatment.
Sulfides Interfere at all levels.
Turbidity Eliminated by zeroing the instrument with the original sample.

Collect samples in clean plastic bottles. Analyze samples as soon as possible after collection. If
prompt analysis is not possible, store samples for up to 7 days by cooling to 4 °C (39 °F) or below.
Warm samples to room temperature before analysis.
Silica
Page 1093
Silica
Accuracy check
• Silica standard solution, 25 mg/L
ADDITIONS.
default values for standard concentration, sample volume, and spike volumes. After the values
5. Follow the test procedure for each of the spiked samples starting with the 0.1 mL sample

1. Use a 1.00-mg/L SiO2 Standard Solution in place of the sample. Perform the silica procedure.
Solution, navigate to Standard Adjust in the software OPTIONS>MORE>STANDARD
ADJUST.
Method performance
651 1.000 mg/L SiO2 0.990–1.010 mg/L SiO2 0.012 mg/L SiO2
Summary of method
Silica and phosphate in the sample react with molybdate ion under acidic conditions to form yellow
silicomolybdic acid complexes and phosphomolybdic acid complexes. Addition of citric acid
destroys the phosphate complexes. An Amino Acid is then added to reduce the yellow
silicomolybdic acid to an intense blue color, which is proportional to the silica concentration. Test
Silica
Page 1094
Silica

Required reagents
Description Quantity/Test Unit Catalog Number
Low Range Silica Reagent Set (100 tests), includes: — — 2459300
Amino Acid F Reagent Powder Pillows (for 10-mL sample) 1 100/pkg 2254069
Citric Acid Powder Pillows 2 100/pkg 2106269
Molybdate 3 Reagent Solution 1 mL 50 mL 199526
Recommended Standard
Silica Standard Solution, 1-mg/L SiO2 500 mL 110649
Silica Standard Solution, 25-mg/L as SiO2 236 mL 2122531
Optional Reagents and Apparatus

Sodium Bicarbonate 454 g 77601
Silica
Page 1095
Silica, Rapid Liquid, 8282
Heteropoly Blue Rapid Liquid Method1 Method 8282

ULR(3 to 1000 µg/L as SiO2) Pour-Thru Cell
Scope and Application: For testing trace levels of soluble silica in pure and ultrapure water
Test preparation


Instrument Pour-Thru Cell Pour-Thru Cell Orientation Adapter
DR 5000 LZV479 — —

DR 3900
compartment
5940400 1-inch (round) path aligned with arrow on the LZV585 (B)
DR 3800, DR 2800, DR 2700
adapter
Clean the Pour-Thru cell and all labware as specified in Labware
See Reagent preparation for instructions on preparing the Amino Acid F Reagent.
The one-minute reaction time in step 12 is for samples at 20 °C; for samples at 10 °C, wait two minutes; for samples at
30 °C, wait 30 seconds.
Silica
Page 1097
Silica
Amino Acid F Reagent Powder varies
Amino Acid Reagent Dilution Solvent varies
Citric Acid F Reagent 1 mL
Molybdate 3 Reagent 1 mL
Dispenser, fixed volume, 1.0-mL, w/bottle 3
Flask, Erlenmeyer, 250-mL, PMP, w/cap 2
Pour-Thru Cell Module (see Instrument-specific information) 1
Heteropoly Blue Rapid Liquid method
Stored Programs
645 Silica ULR Reagent Blank

ON
Start
1. Select the test. 2. Activate the reagent 3. Use the numeric 4. Fill two clean 250-mL
Insert an adapter if blank option to account for keypad on the instrument Erlenmeyer flasks to
required (see Instrument- the Molybdate 3 reagent to manually adjust the overflowing with sample.
specific information). blank. The value is printed reagent blank value.
on the bottle label.
Repeat
Steps 5—7
5. Fill a clean 50-mL 6. Fill the rinsed cylinder 7. Pour the contents of 8. Repeat steps 5
plastic graduated cylinder to the 50-mL mark with the 50-mL cylinder back through 7 for the second
with sample from one of sample from the same into the original flask. flask containing sample.
the flasks; then discard the flask. Discard any
contents of the cylinder. remaining sample in the
Repeat three times. flask.
Silica
Page 1098
Silica
Heteropoly Blue Rapid Liquid method (continued)
9. Add 1.0 mL of 10. Start the instrument 11. When the timer 12. Start the instrument
Molybdate 3 Reagent to timer. expires, add 1.0 mL of timer.
each flask. Swirl to mix. A four-minute reaction Citric Acid F Reagent to A one-minute reaction
period will begin. each flask. Swirl to mix. period will begin. The
destruction of possible
phosphate interference
occurs during this period.
Zero
13. When the timer 14. After the flow stops, 15. Add 1.0 mL of Amino 16. Wait at least 15
expires, pour the contents ZERO the instrument. Acid F Reagent to the seconds, then pour the
of one flask into the The display will show: remaining flask. Swirl to contents of the second
Pour-Thru Cell. mix. flask into the Pour-Thru
0 µg/L SiO2 Cell.
A faint blue color will
develop if silica is present.
Read

mg/L SiO2. Cell with at least 50 mL of
deionized water
Silica
Page 1099
Silica
Interferences

Color Eliminated by zeroing the instrument with the blank (follow procedure).
Iron Interferes at high levels.
pH (extreme) Adjust pH to less than 7.
Phosphate (PO43–) Interferes at levels greater than 50 mg/L PO43–.
Turbidity Eliminated by zeroing the instrument with the blank (follow procedure).

• Use only plastic containers with tight-fitting closures. Do not use glass containers; they will
contaminate the sample with silica.
• Soak sampling containers with a solution made of one part Molybdate 3 Reagent to 50 parts of
high quality deionized water of low silica concentration. Fill the containers completely and let
them stand for several hours. Rinse thoroughly with low-level silica water, drain and close.
Repeat this cleaning periodically.
• Allow the sample stream to flow for 1–2 minutes before collection. Do not adjust the flow
during the sampling period as this may introduce particulates.
• Rinse the container well with sample before collecting the portion for analysis.
• Analyze samples as soon as possible.
Reagent preparation
1. Dissolve the contents of one bottle of Amino Acid F Reagent Powder in one bottle of Amino
Acid Reagent Dilution Solvent.
2. Install a bottle-top dispenser on this bottle, as well as on the Molybdate 3 Reagent and Citric
Acid Reagent bottles.
3. Prepare smaller volumes of Amino Acid F Reagent by dissolving Amino Acid F Reagent
Powder in Amino Acid F Reagent Solvent at a ratio of 11 grams per 100 mL of reagent solvent.
This prepared solution has limited stability; test routinely with the 1-mg/L (1000 µg/L) Silica
Standard Solution to confirm performance.
Reduced sensitivity at high concentrations (1000 µg/L) indicates reagent instability. If the
concentration is less than 950 µg/L, use fresh Amino Acid F Reagent Solution.
Labware
All containers used in this test must be cleansed thoroughly to remove any traces of silica. Use
plastic containers for all analysis and storage because glass can contaminate the sample with
silica. Small bottles or flasks with screw-type closures work well.
1. Clean containers by normal means (do not use phosphate detergents), then rinse with high
quality deionized water of low-level silica concentration.
2. Soak for 10 minutes with a 1:50 dilution of Molybdate 3 Reagent in low-level silica water.
3. Rinse repeatedly with either low-level silica water or the sample before use. Keep containers
tightly closed when not in use.
Silica
Page 1100
Silica
4. Fill the Pour-Thru Cell with this same mixture of Molybdate 3 and water and let stand for
several minutes before use.
5. Rinse with low-level silica water.

solutions are allowed to stand in the cell for long periods after measurement.
1. Remove the color by rinsing with a 1:5 dilution of ammonium hydroxide and deionized water.
2. Rinse several times with deionized water.
3. Cover the Pour-Thru Cell when it is not in use.
Accuracy check
• 1-mg/L (1000-µg/L) Silica standard
• TenSette Pipet and Pipet Tips.
• 250-mL plastic Erlenmeyer flasks (3)
chemical form.

ADDITIONS.
4. Prepare three samples. Fill three plastic Erlenmeyer flasks with 50 mL of prepared sample.
1-mg/L standard to each flask and mix thoroughly.
6. Follow the Heteropoly Blue Rapid Liquid method test procedure for each of the spiked
instrument.
Silica
Page 1101
Silica


• 500-µg/L SiO2 Standard Solution
1. Use a 500-µg/L SiO2 Standard Solution in place of the sample. Follow the Heteropoly Blue
Rapid Liquid method test procedure.
to Standard Adjust in the software:OPTIONS>MORE>STANDARD ADJUST.
Method performance

Program Standard
645 500 µg/L silica 496–504 µg/L silica 13 µg/L silica
Summary of method
A number of modifications are necessary to adapt the Low Range Silica method for analyzing
trace levels in the Ultra Low Range method. It is absolutely necessary to use the one-inch Pour-
Thru Cell and liquid reagents. The Pour-Thru Cell increases the reproducibility of the optics and
reduces the instability of the readings that result from moveable sample cells. Liquid reagents
produce more reproducible readings and lower blank values by eliminating slight turbidity that may
remain when using powdered reagents. Use of liquid reagents in continuous monitors for silica
provides a means of confirming the analyzer performance.
Silica and phosphate in the sample react with molybdate ions under acidic conditions to form
yellow silicomolybdic acid complexes and phosphomolybdic acid complexes. Addition of citric acid
destroys the phosphate complexes. Amino Acid F Reagent is then added to reduce the yellow
Silica
Page 1102
Silica
Required reagents
Rapid Liquid ULR Silica Reagent Set, includes: 2678500
Amino Acid F Reagent Powder varies 55 g 2651155
Amino Acid Reagent Dilution Solvent varies 475 mL 2353011
Citric Acid F Reagent 1 mL 500 mL 2254249
Molybdate 3 Reagent 1 mL 500 mL 199549
Required apparatus
Dispenser, fixed volume, 1.0-mL, w/bottle 3 each 2111302
Flask, Erlenmeyer, 250-mL, PMP, w/cap 2 each 2089846
Funnel, powder 1 each 2264467
Silica Standard Solution, 500-µg/L SiO2 3.78 L 2100817

Ammonium Hydroxide, 58% 500 mL 10649
Molybdate 3 Reagent 2.9 L 199503
Molybdate 3 Reagent 100 mL 199532
Molybdate 3 Reagent 1L 199553
Pipet tips for TenSette Pipet 1970001 50/pkg 2185696
Silica
Page 1103
Silica, 8282
Heteropoly Blue Method1 Method 8282

ULR(3 to 1000 µg/L as SiO2) Pour-Thru Cell
Scope and Application: For testing trace levels of soluble silica in pure and ultrapure water
Test preparation


Instrument Pour-Thru Kit Cell Orientation Adapter
DR 5000 LZV479 — —

DR 3900
compartment
5940400 1-inch (round) path aligned with arrow on LZV585 (B)

DR 3800, DR 2800, DR 2700
the adapter

Clean the Pour-Thru cell and all labware as specified in Labware.
See Reagent preparation for instructions on preparing the Amino Acid F Reagent.
The one-minute reaction time in step 12 is for samples at 20 °C; for samples at 10 °C, wait two minutes; for samples at
30 °C, wait 30 seconds.
Amino Acid F Reagent Solution 1 mL
Citric Acid F Reagent 1 mL
Molybdate 3 Reagent 1 mL
Flask, Erlenmeyer, 250-mL, PMP, with cap 2
Pipet, TenSette®, 0.1 to 1.0 mL with tips 1
Silica
Page 1105
Silica
Heteropoly Blue method
Stored Programs
645 Silica ULR Reagent Blank

ON
Start
1. Select the test. 2. Activate the reagent 3. Use the numeric 4. Fill two clean 250-mL
Insert an adapter if blank option to account for keypad on the instrument Erlenmeyer flasks to
required (see Instrument- the Molybdate 3 reagent to manually adjust the overflowing with sample.
specific information). blank. The value is printed reagent blank value.
on the reagent blank.
Repeat
Steps 5—7
5. Fill a clean 50-mL 6. Fill the rinsed cylinder 7. Pour the contents of 8. Repeat steps 5
plastic graduated cylinder to the 50-mL mark with the 50-mL cylinder back through 7 for the second
with sample from one of sample from the same into the original flask. flask containing sample.
the flasks; then discard the flask. Discard any
contents of the cylinder. remaining sample in the
Repeat three times. flask.
Silica
Page 1106
Silica
Heteropoly Blue method (continued)
9. Use a TenSette® Pipet 10. Start the instrument 11. When the timer 12. Start the instrument
to add 1.0 mL of timer. expires, add 1.0 mL of timer.
Molybdate 3 Reagent to A four-minute reaction Citric Acid F Reagent to A one-minute reaction
each flask. Swirl to mix. period will begin. each flask. Swirl to mix. period will begin. The
destruction of possible
phosphate interference
occurs during this period.
Zero
13. When the timer 14. After the flow stops, 15. Add 1.0 mL of Amino 16. Wait at least 15
expires, pour the contents ZERO the instrument. Acid F Reagent to the seconds, then pour the
of one flask into the The display will show: remaining flask. Swirl to contents of the second
Pour-Thru Cell. mix. flask into the Pour-Thru
0 µg/L SiO2 Cell.
A faint blue color will
develop if silica is present.
Read

mg/L SiO2. Cell with at least 50 mL of
deionized water
Silica
Page 1107
Silica
Interferences

Color Eliminated by zeroing the instrument with the blank (follow procedure).
Iron Interferes at high levels.
pH (extreme) Adjust pH to less than 7.
Phosphate (PO43–) Interferes at levels greater than 50 mg/L PO43–.
Turbidity Eliminated by zeroing the instrument with the blank (follow procedure).
Silica
Page 1108
Silica

• Use only plastic containers with tight-fitting closures. Do not use glass containers; they will
contaminate the sample with silica.
• Soak sampling containers with a solution made of one part Molybdate 3 Reagent to 50 parts of
high quality deionized water of low silica concentration. Fill the containers completely and let
stand for several hours. Rinse thoroughly with low-level silica water, drain and close. Repeat
this cleaning periodically.
• Allow the sample stream to flow for 1–2 minutes before collection. Do not adjust the flow
during the sampling period as this may introduce particulates.
• Rinse the container well with sample before collecting the portion for analysis.
• Analyze samples as soon as possible.
Reagent preparation
Amino Acid F Reagent Solution is available in either 100-mL bottles or a package of 20 unit-dose
ampules. The bottled reagent is stable for up to one year if the bottle is kept closed when not in
use. The ampuled reagent is sealed under argon and is more stable with a shelf life greater than 1
year. Reduced sensitivity at high concentrations (1000 µg/L) indicates reagent instability. Check
the bottled reagent on a routine basis by performing an analysis on a 1-mg/L (1000 µg/L) Silica
Standard Solution. If the concentration is less than 950 µg/L, use a fresh bottle of Amino Acid F
Reagent Solution.
Prepare larger or smaller volumes of Amino Acid F Reagent by dissolving Amino Acid F Reagent
Powder in Amino Acid F Reagent Solvent at a ratio of 11 grams per 100 mL of reagent solvent.
These reagents are available as the Amino Acid F Reagent Package. This prepared solution has
limited stability; test routinely with the 1-mg/L Silica Standard Solution.
Labware
All containers used in this test must be cleansed thoroughly to remove any traces of silica. Use
plastic containers for all analysis and storage because glass can contaminate the sample with
silica. Small bottles or flasks with screw-type closures work well.
1. Clean containers by normal means (do not use phosphate detergents), then rinse with high
quality deionized water of low-level silica concentration.
2. Soak for 10 minutes with a 1:50 dilution of Molybdate 3 Reagent in low-level silica water.
3. Rinse repeatedly with either low-level silica water or the sample before use. Keep containers
tightly closed when not in use.
4. Fill the Pour-Thru Cell with this same mixture of Molybdate 3 and water and let stand for
several minutes before use.
5. Rinse with low-level silica water.

solutions are allowed to stand in the cell for long periods after measurement.
1. Remove the color by rinsing with a 1:5 dilution of ammonium hydroxide and deionized water.
2. Rinse several times with deionized water.
3. Cover the Pour-Thru Cell when it is not in use.
Silica
Page 1109
Silica
Accuracy check
• 1-mg/L (1000-µg/L) Silica standard
• TenSette Pipet and Pipet tips.
• 250-mL plastic Erlenmeyer flasks (3)
chemical form.

ADDITION.
4. Prepare three samples. Fill three plastic Erlenmeyer flasks with 50 mL of prepared sample.
1-mg/L standard to each flask and mix thoroughly.
6. Follow the Heteropoly Blue method test procedure for each of the spiked samples, starting


• 500-µg/L SiO2 Standard Solution
1. Use a 500-µg/L SiO2 Standard Solution in place of the sample. Follow the Heteropoly Blue
method test procedure.
Method performance

645 DR 5000 500 µg/L silica 496–504 µg/L silica 13 µg/L silica
Summary of method
A number of modifications are necessary to adapt the Low Range Silica method for analyzing
trace levels in the Ultra Low Range method. It is absolutely necessary to use the one-inch Pour-
Thru Cell and liquid reagents. The Pour-Thru Cell increases the reproducibility of the optics and
reduces the instability of the readings that result from moveable sample cells. Liquid reagents
produce more reproducible readings and lower blank values by eliminating slight turbidity that may
Silica
Page 1110
Silica
remain when using powdered reagents. Use of liquid reagents in continuous monitors for silica
provides a means of confirming the analyzer performance.
Silica and phosphate in the sample react with molybdate ions under acidic conditions to form
yellow silicomolybdic acid complexes and phosphomolybdic acid complexes. Addition of citric acid
destroys the phosphate complexes. Amino Acid F Reagent is then added to reduce the yellow
Required reagents
ULR Silica Reagent Set (using Amino Acid F solution, 100 tests) — — 2553500
Includes: (2) 199532, (2) 2254232, (1) 2386442
ULR Silica Reagent Set (using Amino Acid F ampules, 40 tests) — — 2581400
Includes: (1) 199532, (1) 2254232, (2) 2386420
Amino Acid F Reagent Solution 1.0 mL 100 mL 2386442
OR
Amino Acid F Reagent Solution, 1.2-mL Ampules 1 20/pkg 2386420
Citric Acid Reagent Solution 2 mL 500 mL 2254249
Molybdate 3 Reagent Solution 2.0 mL 500 mL 199549
Required apparatus
Flask, Erlenmeyer, 250-mL, PMP, w/cap 2 each 2089846
Silica Standard Solution, 500-µg/L SiO2 3.78 L 2100817
Silica
Page 1111
Molybdate 3 Reagent 100 mL 199532
Molybdate 3 Reagent 1L 199553
PourRite® ampule breaker each 2484600
Amino Acid F reagent package each 2254117

Silver, 8120
Silver DOC316.53.01134
Colorimetric Method Method 8120

Test preparation


Instrumetn Sample cell Cell orientation
DR 3800, DR 2800 DR 2700 2495402 Fill line faces user
Digestion is required for samples with interferences. See Digestion.
For best results, measure a reagent blank value for each new lot of reagent (follow the procedure using deionized water in
place of the sample). Subtract the reagent blank value from the final results or enter the value as a reagent blank adjust for
automatic subtraction.
The graduated cylinder must be completely dry before beginning the test. If the Silver 1 Powder becomes moist, it
will not dissolve completely, which will inhibit color development.
The sample pH for this test must be between 9 and 10. Do not use a pH meter to adjust the sample pH as it will contaminate
the sample. See Digestion for the procedure to adjust pH.
The Pour-Thru Cell cannot be used with this procedure.
Silver 1 Reagent Powder Pillow 1
Silver 2 Reagent Powder Pillow 1
Sodium Thiosulfate Powder Pillow 1
Clippers 1
Silver
Page 1113
Silver
Colorimetric Method
Stored Programs
660 Silver
Start
1. Select the test. 2. Add the contents of 3. Add the contents of 4. Use a 50-mL
Insert an adapter if one Silver 1 Powder Pillow one Silver 2 Reagent graduated cylinder to add
required (Instrument- to a dry 50-mL graduated Solution Pillow to the 50 mL of sample to the
specific information). mixing cylinder. cylinder. Swirl to 50-mL graduated mixing
If the Silver 1 Powder completely wet the cylinder. Stopper and
becomes wet at this powder. invert repeatedly for one
point, the powder will If clumps of dry powder minute.
not dissolve completely, are present when the Be sure to first adjust the
which will inhibit color sample is poured in, the pH of samples if they were
development. powder will not dissolve preserved (see Sample
completely, which will collection, preservation
inhibit color development. and storage).
5. Prepared Sample: Fill 6. Blank Preparation: 7. Add the contents of 8. Start the instrument
a sample cell to at least Discard all but 25 mL of one Sodium Thiosulfate timer.
the 10 mL mark with the the sample from the Reagent Powder Pillow to A two-minute reaction time
mixture. mixing cylinder. the sample in the mixing will start.
cylinder. Stopper and
invert to mix.
Be sure to prepare a blank
for each sample.
Silver
Page 1114
Silver
Colorimetric Method (continued)
Zero Read
9. Fill a second sample 10. When the timer 11. ZERO the instrument. 12. Insert the prepared
cell to at least the 10 mL expires, insert the blank in The display will show: sample in the cell holder.
mark with the mixture. the cell holder. READ the results in
0.00 mg/L Ag
mg/L Ag.
Immediately rinse the
cells.
Interferences
Standard solutions of approximately 0.4 mg/L Ag with different concentrations of a potential
interfering ion were prepared. The concentration of silver was measured. Interfering substances
shows the ions that caused a change in the silver concentration of more than ten percent (±10%).

Aluminum Negative interference above 30 mg/L
Ammonia Negative interference above 750 mg/L
Cadmium Negative interference above 15 mg/L
Calcium Positive interference above 600 mg/L
Chloride Negative interference above 19 mg/L
Chromium6+ Negative interference above 90 mg/L
Copper Negative interference above 7 mg/L
Iron Negative interference above 30 mg/L
Lead Negative interference above 13 mg/L
Manganese Negative interference above 19 mg/L
Magnesium Positive interference above 2000 mg/L
Mercury Positive interference above 2 mg/L
Nickel Negative interference above 19 mg/L
Zinc Negative interference above 70 mg/L
Silver
Page 1115
Silver

• Collect samples in acid-cleaned glass or plastic bottles.
• Use pH paper to adjust the pH to 2 or less with concentrated Nitric Acid (about 2 mL/liter).
• Store preserved samples at room temperature for up to 6 months.
• If the sample contains particulates or for a dissolved metal analysis, filter through a 0.45 µm
filter at collection. After filtration, adjust the pH to 2 or less for storage.
• Before analysis, adjust the pH to 9–10 with 5.0 N sodium hydroxide. (See steps 11–12 of
Digestion.)
• Do not use a pH meter because the pH electrode will contaminate the sample.
Accuracy check
• Silver Standard Solution, 1000 mg/L Ag
• 5.0 mL Class A volumetric pipet and pipet bulb
• TenSette Pipet, 0.1–1.0 mL and tips
5. Select OPTIONS>MORE>STANDARD ADDITIONS from the instrument menu. Accept the

are accepted, the unspiked sample reading will appear in the top row. See the user manual for
more information.
6. Prepare a 50.0 mg/L silver standard solution as follows. Add 5.00 mL of 1000 mg/L Silver
Standard Solution to a 100-mL volumetric Class A flask. Dilute to volume with deionized water.
This is a 50.0 mg/L standard solution.
7. Use the TenSette Pipet to prepare spiked samples: add 0.1 mL, 0.2 mL and 0.3 mL of the 50.0
mg/L standard to three 50-mL portions of fresh sample. Mix thoroughly.
8. Follow the Colorimetric Method test procedure for each of the spiked samples, starting with
the 0.1 mL spiked sample. Measure each of the spiked samples in the instrument.
Silver
Page 1116
Silver

• Silver Standard Solution, 1000 mg/L Ag
1. Prepare a 0.5 mg/L silver standard solution as follows: Pipet 0.5 mL of silver standard,
1000 mg/L as Ag, into a 1000-mL (1 liter) volumetric flask. Dilute to the mark with deionized
water. Mix well. Prepare this solution daily.
2. Use the 0.5-mg/L silver standard solution in place of the sample. Follow the Colorimetric
Method test procedure.
navigate to Standard Adjust in the software: OPTIONS>MORE>STANDARD ADJUST.
4. Turn on the Standard Adjust option and accept the displayed concentration. If an alternate
Digestion
If the sample contains organic matter, thiosulfate or cyanide, digest the sample before analysis.
Possible sources for these compounds are wastewater, silver electroplating baths and silver strike
solutions. Use the Digesdahl Digestion Apparatus.
DANGER
Chemical hazard. Poisonous hydrogen cyanide gas may be generated during the digestion.
Operate the Digesdahl in a closed fume hood.
CAUTION
Chemical hazard. Always wear safety glasses and use a safety shield or operate the
Digesdahl in a closed fume hood. Follow the safety precautions in the Digesdahl Digestion
Apparatus Manual.
1. Add an appropriate size sample to the 100-mL digestion flask for use with the Digesdahl. Add
several boiling chips to prevent bumping.
Note: Appropriate sample size is determined experimentally. The final sample concentration (after dilution to
100 mL) should be 0–0.6 mg/L. Larger dilutions may be necessary for electroplating baths and silver
strike solutions. Do not exceed the maximum sample volume of 25 mL. Several 25-mL aliquots may be
digested in succession to concentrate a very dilute sample.
2. Turn on the water aspirator and make sure there is suction in the fractionating head.
3. Carefully add 3 mL of concentrated sulfuric acid to the sample in the volumetric flask.
Immediately place the head on the digestion flask. Never use less than 3 mL of acid.
4. Place the digestion flask on the heater. Turn the temperature dial to 440 °C (825 °F).
5. When the sulfuric acid reflux line becomes visible, wait 3–5 minutes.
6. Make sure there is acid in the flask before adding hydrogen peroxide!
7. Place the capillary funnel on the fractionating head. Fill the funnel to the 10-mL line with
50% hydrogen peroxide.
Note: If the sample completely evaporates, turn the Digesdahl off and cool completely. Carefully add water to
the flask before handling. Start the digestion over with a fresh sample.
Silver
Page 1117
Silver
8. Use the capillary funnel to add 5 mL of hydrogen peroxide. Check the solution in the flask for
digestion completion. If digestion is not complete, continue adding hydrogen peroxide in 5 mL
to 10 mL portions. Several portions may be necessary.
Note: The digestion is complete when the digestate is colorless or the color of the digestate does not change
after hydrogen peroxide is added. A completely digested sample will not cause foam to form.
9. After digestion is complete and all the hydrogen peroxide has boiled away, reduce the volume
of the digestate to near dryness. Do not allow the sample to become completely dry! Remove
the flask from the heater. Cool to room temperature.
10. Slowly add approximately 25 mL of deionized water to the cooled flask. Swirl to mix.
11. Add 2 drops of 1 g/L Phenolphthalein Indicator Solution. Add 2 drops of 1 g/L Thymolphthalein
Indicator Solution.
12. Use sodium hydroxide to adjust the pH of the solution to 9–10. The solution will be pink in this
pH range.
Note: A purple color indicates a pH greater than 10. If this occurs, add a drop of sulfuric acid and 2 drops of
each indicator and repeat the pH adjustment. Initially, use 50% sodium hydroxide, then 1 N sodium
hydroxide as the end point is approached.
13. Filter turbid digestates. Quantitatively transfer the filtrate (or unfiltered sample) to a clean
100-mL volumetric flask. Dilute to the mark with deionized water and mix. Follow the
Colorimetric Method for silver.
Method performance
Standard 95% Confidence Limits of Concentration change
0.50 mg/L Ag 0.49–0.51 mg/L Ag 0.005 mg/L Ag
Summary of method
Silver ions in basic solution react with cadion 2B to form a green to brown to red-purple complex.
The sodium thiosulfate acts as a decolorizing agent for the blank. The Silver 1 and Silver 2
reagents contain the buffer, indicator and masking agents. Organic extractions are not necessary
and this method does not have as many interferences as the traditional dithizone method. Test
Required reagents
Silver Reagent Set (50 tests), includes: 2296600
Silver 1 Reagent Powder Pillow 1 50/pkg 2293566
Silver 2 Reagent Solution Pillow 1 50/pkg 2293666
Sodium Thiosulfate Powder Pillow 1 50/pkg 2293766
Silver
Page 1118
Silver
Required apparatus
Silver Standard Solution, 1000 mg/L Ag 100 mL 1461342
Digestion reagents and apparatus

Hydrogen Peroxide, 50% 490 mL 2119649
Phenolphthalein Indicator Solution, 1 g/L 15 mL SCDB 189736
Sodium Hydroxide Solution, 50% 500 mL 218049
Sodium Hydroxide Solution, 1.00 N 100 mL MDB 104532
Sulfuric Acid, ACS, concentrated 2.5 L 97909
Thymolphthalein Indicator Solution, 1 g/L 15 mL SCDB 2185336
Boiling Chips, silicon carbide 500 g 2055734
Digesdahl Digestion Apparatus, 115 VAC, 50/60 Hz each 2313020
Digesdahl Digestion Apparatus, 230 VAC, 50/60 Hz each 2313021
Safety Shield, for Digesdahl each 5003000
Silver
Page 1119
Silver

Nitric Acid, concentrated ACS 500 mL 15249
pH paper, 0–14 100/pkg 2601300
Membrane filter paper, 0.45 μm 100/pkg 2618800
Volumetric pipet, Class A, 0.5 mL each 1451534

Solids, Settleable Matter, 8165
Solids, Settleable Matter DOC316.53.001202
Direct Measurement1 Method 8165

Scope and Application: For sewage and wastewater.
1 Adapted from Standard Methods for the Examination of Water and Wastewater, Section 2540E.
Test preparation
Imhoff Cone 2
Imhoff Cone Brush 1
Imhoff Cone Support 1
Direct Measurement
1. Fill an Imhoff cone to 2. Wait 45 minutes for 3. Spin the cone forward 4. Wait 15 minutes for
the 1-liter mark with a the undisturbed sample to and backward several the sample to settle.
thoroughly mixed sample. settle. times to dislodge materials
on the inclined side of the
cone.
5. Read the graduated

scale on the Imhoff cone
at the top of the solids
layer as mL/L settleable
matter.
Solids, Settleable Matter

Page 1121
Solids, Settleable Matter

• Refrigerate at 4 °C up to the time of analysis to minimize microbiological decomposition of
solids.
• Analyze within 24 hours. Bring to room temperature before analysis.
Summary of method
The amount of settleable matter in sewage treatment plant influent and effluent gives an empirical
estimate of the type and extent of treatment required and the general quality of the water being
discharged.
Required apparatus
Imhoff Cone 2 each 206700
Imhoff Cone Brush 1 each 68800
Imhoff Cone Support 1 each 57200
Optional apparatus
Beaker, Glass, low form 2000 mL each 50054
Gloves, Chemical resistant, size 9–9 1/2 pair each 2410104
Pitcher, Graduated 2000 mL each 2612854
Sampler, Dipper 3–9 ft handle each 2929501

Solids, Nonfilterable; Total and Volatile, 8158 and 8164
Solids, Nonfilterable Suspended

Solids; Total and Volatile DOC316.53.001204
USEPA1 Gravimetric Method2 Methods 8158 and 8164

1 USEPA accepted.
2 Adapted from Standard Methods for the Examination of Water and Wastewater Section 2450
Test preparation
The Total Nonfilterable Solids are the same as the Total Suspended Solids (TSS)
Filter flask 1
Filter holder 1
Filter, 47-mm 1
Tongs 1
Tweezers 1
Watch glass 1
Desiccator with desiccant 1
Muffle Furnace 1
Drying Oven 1
Solids, Nonfilterable Suspended Solids; Total and Volatile

Page 1123
Gravimetric Method—Total Nonfilterable Residue, Method 8158
1. Use tweezers to place 2. Place the filter holder 3. Slowly release the 4. Place the disc in a
a 47-mm glass fibre filter assembly in the filtering vacuum from the filtering preheated drying oven at
disc in the filter holder. flask and add 100 mL of system and remove the 103 °C for one hour.
Always use tweezers to deionized water. Apply disc from the filter holder
handle filter discs. vacuum to the flask until and transfer to a watch
Moisture from fingers can all the water is drawn glass.
add moisture to the disc through the filter.
and cause a weighing
error.
5. If volatile nonfilterable 6. Use metal tongs to 7. Remove the watch 8. Again, place the disc
solids are also being remove the disc and watch glass and disc from the in the filter holder/flask
measured, use tongs to glass from the oven or desiccator as a unit and assembly. Wet the disc
place the watch glass with furnace and place in a place beside the analytical with deionized water to
the disc into a muffle desiccator. Cover balance. ensure adhesion to the
furnace and ignite at immediately. Allow the Use plastic tweezers to holder.
550 °C for 15 minutes. If watch glass to cool slightly remove the disc from the
not, omit this step. before sealing the watch glass and weigh to
Partially preheat the muffle desiccator as pressure the nearest 0.1 mg
furnace before inserting from the heated air inside (0.0001 g). Record this
the watch glass. Placing the desiccator can force value as B.
the watch glass in a the cover off.
550 °C furnace could Allow the filter and glass to
cause it to shatter. Bring cool to room temperature.
the temperature up to
550 °C 15 minutes after
placing the filter and watch
glass in the furnace.

Page 1124
Gravimetric Method—Total Nonfilterable Residue, Method 8158 (continued)
9. Filter 100 mL (or 10. Remove any residue 11. Place the watch glass 12. Use metal tongs to
more, if solids content is that remains on the sides and filter in a drying oven remove the disc and watch
low) of well-mixed, or bottom lip of the filter at 103 °C for one hour. glass from the oven or
representative water holder. A rubber furnace and place in a
sample by applying policeman on the end of a desiccator. Cover
vacuum to the flask. stirring rod is very helpful immediately. Allow the
Follow with three separate to scrape the residue watch glass to cool slightly
10-mL washings of loose. Small amounts of before sealing the
deionized water. deionized water will help desiccator as pressure
For greatest accuracy, wash the residue down from the heated air inside
filter as much sample as onto the filter disc. the desiccator can force
possible. However, using a Slowly release the vacuum the cover off.
sample that contains more from the filtering system Allow the filter and glass to
than 15 mg of solids will and gently remove the cool to room temperature.
clog the filter prematurely. filter disc from the holder.
Adjust the exact volume of Place the disc on a watch
the water sample to glass. Inspect the filtrate
achieve the optimum (filtered water in flask) to
condition. Several make sure that the solids
completed tests will show are properly trapped on
whether any adjustment is the disc.
necessary.

Page 1125
Gravimetric Method—Total Nonfilterable Residue, Method 8158 (continued)
13. Remove the watch 14. Return the disc to the 15. Calculate Total Non-filterable Residue (TNR):
glass and disc from the watch glass if the mg/L A–B
----------------------------------------------------------------- = mg/L TNR
desiccator as a unit and Volatile Nonfilterable Sample Volume in Liters
place beside the analytical Residue is to be
balance. determined. If not, discard Where:
Use plastic tweezers to the disc. A = Weight (mg) of disc with residue
remove the disc from the If Volatile Nonfilterable B = Weight (mg) of disc
watch glass and weigh to Residue is to be
the nearest 0.1 mg determined, do not lose Example:
(0.0001 g). Record this mg any of the suspended A = 95.5 mg
value as A. matter on the disc. B = 81.5 mg
Volume of sample = 0.1 L
95.5 mg – 81.5 mg
------------------------------------------------- = 140 mg/L TNR
0.1 L

Page 1126
Gravimetric Method—Volatile Nonfilterable Solids, Method 8164
1. Place the watch glass 2. Use metal tongs to 3. Remove the watch 4. Calculate Volatile
and filter disc from the remove the disc and watch glass and disc from the Non-filterable Residue
Total Nonfilterable glass from the oven or desiccator as a unit and (VNR):
Residue procedure furnace and place in a place beside the analytical A–C
----------------------------------------------------------------- =
(step 14) in the muffle desiccator. Cover balance. Sample Volume in Liters
furnace and ignite at immediately. Allow the Use plastic tweezers to mg/L VNR
550 °C for 15 minutes. watch glass to cool slightly remove the disc from the where:
Partially preheat the muffle before sealing the watch glass and weigh to
desiccator as pressure A = Weight (mg) of disc
furnace before inserting the nearest 0.1 mg with residue
the watch glass. However, from the heated air inside (0.0001 g). Record this mg
placing the watch glass in the desiccator can force value as C. C = Weight (mg) of disc
a 550 °C furnace could the cover off. and residue after ignition
cause it to shatter. Bring Allow the filter and glass to Example:
the temperature up to cool to room temperature. A = 95.5 mg
550 °C 15 minutes after
placing the filter and watch C = 91.2 mg
glass in the furnace. Volume of sample = 0.1 L
95.5 mg – 91.2 mg
-------------------------------------------------- =
0.1 L
43 mg/L VNR

• Samples should be analyzed as soon as possible after collection but can be stored up to
seven days by cooling to 4 °C (39 °F).

Page 1127
Required apparatus
Aspirator, vacuum each 213100
Balance, Analytical, 115 VAC, 60 Hz each 2936701
Bottle, wash, 500-mL each 62011
Desiccant, indicating Drierite each 2088701
Desiccator Plate, ceramic each 1428400
Filter disc, glass fiber, 47-mm 100/pkg 253000
Filter Holder, magnetic each 1352900
Furnace, muffle 240 VAC, 50/60 Hz each 1429624
Furnace, muffle, 120 VAC, 50/60 Hz each 1429600
Oven, laboratory, 240 VAC, 50 Hz each 1428902
Stopper, rubber, one-hole, No. 8 6/pkg 211908
Tongs each 56900
Tubing, rubber, 7.9 x 2.4 mm 3.6 m 56019
Tweezers, plastic each 1428200
Watch Glass, 100-mm each 57870

Ammonium Hydroxide, approx.. 58% ACS 500 mL 10649
Bottle, w/cap, wide mouth 500 mL poly 12/pkg 2087079
Brush each 68700
Pump, vacuum, hand-operated each 1428300
Pump, vacuum, 1.2 CFM, 220 VAC, w/Europeon plug each —
Pump, vacuum, 1.2 CFM, 115 VAC, 60 Hz each 2824800
Rubber policeman for 3/16" rod each 1430900
Stirring rod, glass 3/pkg 177001

Solids, Total Filterable, 8163
Solids, Total Filterable

(Total Dissolved Solids) DOC316.53.001246
USEPA1 Gravimetric Method2 Method 8163

1 USEPA accepted.
2 Adapted from Standard Methods for the Examination of Water and Wastewater, Part 2540C
Test preparation
Total Filterable Solids = Total Dissolved Solids (TDS)
Samples with high bicarbonate concentrations may require increased drying at 180 °C to make sure that the conversion of
bicarbonate to carbonate is complete.
Limit sample size to no more than 200 mg residue for best results.
When measuring volatile dissolved solids, heat the evaporating dish to 550 °C for 1 hour before beginning the test.
Sample residue from this procedure can be used directly in Method 8277, Solids, Total Volatile and Fixed.
Evaporating dish 1
Filter flask 1
Filter holder 1
Filter, 47-mm 1
Hot Plate 1
Steam Bath, 8" diameter 1
Analytical Balance 1
Desiccator 1
Tongs 1
Tweezers 1
Drying Oven 1
Solids, Total Filterable (Total Dissolved Solids)

Page 1129
USEPA Gravimetric Method—Total Filterable Solids, Method 8163
5. Heat a clean 6. Assemble the filter 7. Place a 47-mm filter 8. Reconnect vacuum to
evaporating dish in a holder/flask assembly disc in the filter holder. the filter holder/flask
drying oven at 180 °C for using a clean filter flask. With vacuum applied to assembly. Use a clean
one hour. Before using the flask, the flask, wash the filter 100-mL graduated cylinder
Using metal tongs, remove remove all residue from with three separate 20-mL to filter 100 mL (or more if
the evaporating dish from the flask by thoroughly volumes of deionized solids content is low) of a
the furnace and place in a cleaning with a dilute water. Remove all traces well-mixed representative
desiccator. Cover solution of ammonium of water by continuing water sample.
immediately. Allow the hydroxide and rinsing with vacuum for two to three For greatest accuracy,
dish to cool slightly before deionized water. minutes after the water filter as much sample as
sealing the desiccator as has passed through the possible. However, using a
pressure from the heated filter. Disconnect the sample that contains more
air inside the desiccator vacuum and discard these than 15 mg of solids will
can force the cover off. washings from the flask. clog the filter prematurely.
Adjust the exact volume of
the water sample to
achieve the optimum
condition. Several
completed tests will show
whether any adjustment is
necessary.
Apply vacuum
9. Apply vacuum for two 10. Use tongs to transfer 11. Place the steam bath 12. Pour the 100-mL
to three minutes after the the evaporating dish from on the hot plate, add water filtrate sample from the
sample has passed the desiccator to the and transfer the filter flask into the
through the filter. balance. Weigh to the evaporating dish from the evaporating dish.
Disconnect the vacuum. nearest 0.1 mg (0.0001 g) balance to the steam bath. Evaporate to dryness (this
and record this weight may take up to four hours).
as B. Periodically check the
reservoir of the water bath.
Add more water if needed.

Page 1130
USEPA Gravimetric Method—Total Filterable Solids, Method 8163 (continued)
Repeat steps
9 and 10
13. Use tongs to transfer 14. Weigh the evaporating 15. Repeat steps 13 and 16. Calculate the Total
the evaporating dish dish to the nearest 0.1 mg 14 until a constant weight Filterable Residue (TFR).
residue to a drying oven (0.0001 g) on an analytical is obtained or until the Refer to Total filterable
and dry at 180 °C for one balance and record this weight change is less than residue calculation.
hour. Transfer to a weight as A. 4% of the previous weight
desiccator and cool. or 0.5 mg, whichever is
Allow the dish to cool less.
slightly before sealing the The solid residue can be
desiccator; pressure from used directly in method
the heated air inside the 8277 to determine Volatile
desiccator can force the Dissolved Solids.
cover off.
Total filterable residue calculation

To calculate TFR:
A–B
-------------------------------------------------------------- = mg/L TFR
Sample volume in liters
Where:
A = Weight (mg) of residue and dish after drying
B = Weight (mg) of dish Sample Volume = 0.1 L
Example:
A = 20187.3 mg
B = 20140.1 mg
20187.3 – 20140.1
------------------------------------------------- = 472 mg/L TFR
0.1

• Samples should be analyzed as soon as possible after collection but can be stored up to

Page 1131
Summary of method
A well-mixed sample is filtered through a standard glass fiber filter. The filtrate is evaporated to
dryness in a weighed dish and dried to a constant weight at 180 °C. The increase in the weight of
the dish after drying represents the total filterable solids (total dissolved solids).
Required apparatus
Evaporating dish, porcelain, w/lip, 120-mL, 90-mm
Filter disc, glass fiber, 47-mm 100/pkg 253000
Filter Holder, magnetic each 1352900
Hot plate/stirrer, 7 x 7 inch, 115 VAC each 2881600
Hot plate/stirrer, 7 x 7 inch, 220–240 VAC each 2881602
Steam bath, 8-inch diameter 2347900
Tongs each 56900
Tubing, rubber, 7.9 x 2.4 mm 3.6 m 56019
Tweezers, plastic each 1428200

Ammonium Hydroxide, 58% ACS 500 mL 10649
Bottle, w/cap, wide mouth 500 mL poly 12/pkg 2087079
Brush each 68700
Pump, vacuum, hand-operated each 1428300
Pump, vacuum, 1.2 CFM, 220 VAC, w/Europeon plug each —
Pump, vacuum, 1.2 CFM, 115 VAC, 60 Hz each 2824800
Rubber policeman for 3/16" rod each 1430900
Stirring rod, glass 3/pkg 177001

Solids, Volatile Dissolved and Fixed Dissolved, 8277
Solids, Volatile Dissolved and

Fixed Dissolved DOC316.53.001206
Gravimetric Method1 Method 8277

Scope and Application: For wastewater.
Test preparation
The residue from Method 8163—Solids, Total Filterable can be used in this method. Start at Step 12..
Filter flask 1
Filter holder assembly with stopper 1
Filter, 47-mm, glass fiber 1
Tongs 1
Evaporating dish 1
Analytical balance 1
Muffle furnace 1
Vacuum sourceand tubing 1
Deionized water 1
Steam bath 1
Hot plate 1
See Sample collection, preservation and storage for reorder information.
Solids, Volatile Dissolved and Fixed Dissolved

Page 1133
Gravimetric Method
1. Heat an evaporating 2. Assemble the filter 3. Place a 47-mm filter 4. Reconnect vacuum to
dish at 550 °C for one holder/flask assembly disc in the filter holder. the filter holder/flask
hour. Cool and store the using a clean filter flask. With vacuum applied to assembly. Using a clean
dish in a desiccator until All residue should be the flask, wash the filter 100-mL graduated
needed. Allow the dish to removed from the flask by with three separate 20-mL cylinder, filter 100 mL (or
cool slightly before sealing cleansing thoroughly with volumes of deionized more if solids content is
the desiccator, as a dilute solution of water. Remove all traces low) of a well-mixed
pressure from the heated ammonium hydroxide and of water by continuing representative water
air can force the cover off. rinsing with deionized vacuum for two to three sample.
water. minutes after the water For greatest accuracy,
has passed through the filter as much sample as
filter. Disconnect the possible. However, using a
vacuum and discard these sample that contains more
washings from the flask. than 15 mg of solids will
clog the filter prematurely.
Adjust the exact volume of
the water sample to
achieve the optimum
condition. Several
completed tests will show
whether any adjustment is
necessary.
Apply Vacuum
5. Apply vacuum for two 6. Use tongs to transfer 7. Place the steam bath 8. Pour the 100-mL
to three minutes after the the evaporating dish from on the hot plate, add water filtrate sample from the
sample has passed the desiccator to the and transfer the filter flask into the
through the filter. balance. Weigh to the evaporating dish from the evaporating dish.
Disconnect the vacuum. nearest 0.1 mg (0.0001 g) balance to the steam bath. Evaporate to dryness (this
and record this weight as may take up to four hours).
A. Periodically check the
reservoir of the water bath.
Add more water if needed.

Page 1134
Gravimetric Method (continued)
9. Place the dish in a 10. Take the dish out of 11. Use tongs to transfer 12. Transfer the dish into
pre-heated oven at 103- the oven and allow it to the evaporating dish to the a preheated muffle
105 °C and dry to a cool to room temperature balance. Weigh to the furnace at 550 °C for
constant weight. This will in a desiccator. nearest 0.1 mg (0.0001 g). 30 minutes.
take 30–60 minutes. Allow the dish to cool Record this as B.
slightly in the desiccator
before sealing the
desiccator, as pressure
from the heated air can
force the cover off.
Repeat
Steps 9–10
13. Take the dish out of 14. Repeat the ignition 15. The loss of weight is Calculate:
the furnace with tongs and (steps 12. and 13.) until total volatile solids.
mg/L Volatile Diss. Solids =
cool to room temperature two successive weighings Weighed residue is total
in a desiccator. Weigh the do not differ by more than fixed solids. ( B – D ) × 1000
---------------------------------------
dish to the nearest 0.1 mg 4% or 0.5 mg, whichever C
mg/L Fixed Diss. Solids =
(0.0001 g) using an is greater.
analytical balance. Record ( A – D ) × 1000
---------------------------------------
this mg value as D. C
Where:
B = Weight (mg) of solids
+ dish before ignition
D = Weight (mg) of solids
+ dish after ignition
C = Volume in mL of
filtrate transferred to the
aluminum dish
A = Weight (mg) of dish

Page 1135

• Samples should be analyzed as soon as possible after collection, but can be stored up to
Summary of method
A well-mixed sample is filtered through a glass fiber filter. An aliquot of the filtrate is evaporated in
a weighed dish and dried to constant weight in a 103–105 °C oven. The dish and sample residue
are ignited at 550 °C for 30 minutes. The loss of sample mass upon ignition represents the volatile
dissolved solids. The remaining residue after ignition represents the fixed dissolved solids.
Required apparatus
Balance, Analytical, SA80, 115 VAC, 60 Hz each 2936701
Evaporating dish, porcelain, w/lip, 120-mL, 90-mm each 52561
Furnace, muffle 120 VAC 50/60 Hz each 1429600
Furnace, muffle 240 VAC 50/60 Hz each 1429624
Steam bath, 8” diameter each 2347900
Tongs each 56900
Filter disc, 47 mm, glass fiber 100/pkg 253000
Filter Holder, 47 mm magnetic each 1352900
Flask, filtering 1000 mL each 54653
Hot plate/stirrer, 7 x 7 inch, 115 VAC each 2881600
Hot plate/stirrer, 7 x 7 inch, 220-240 VAC each 2881602
Stopper, rubber, one-hole No. 8 6/pkg 211908

Page 1136

Dish, aluminum (63 x 17.5 mm) 100/pkg 2164000
Blender,115 VAC, 1.2 L each 2616100
Blender, 240 VAC, 1.2 L each 2616102
Bottles, w/cap, wide mouth, 500 mL poly 12/pkg 2087079
Beaker, glass, 250 mL each 50046H
Pump, Vacuum, 1.2 cfm, 115 VAC each 2824800
Pump, Vacuum, 1.2 cfm, 220 VAC, European plug each 2824802
Pump, Vacuum, Hand-operated each 1428300
Stir Bar, 22 x 8 mm each 2095350
Stirrer, magnetic, 4 x 4, 115 VAC each 2881200

Page 1137
Solids, Total Volatile and Fixed, 8276
Solids, Total Volatile and Fixed DOC316.53.001205
Gravimetric Method1 Method 8276

Scope and Application: For potable, surface and saline water and domestic and industrial wastewater.
Test preparation
When measuring volatile solids, ignite the aluminum dishes for one hour at 550 °C prior to use.
Larger sample sizes may be used. For larger samples, use an evaporating dish and a steam bath to evaporate the liquid.
Samples from Method 8271 may be used directly in this procedure. Use the resulting sample and start this method at step 7.
Weighing dish, aluminum 1
Drying oven 1
Graduated cylinder, 50 mL 1
Desiccator and desiccant 1
Muffle furnace 1
Solids, Total Volatile and Fixed

Page 1139
Gravimetric Method
1. Heat an aluminum 1. Weigh an aluminum 2. Mix the sample and 3. Place the sample in a
dish to 550 °C for one dish to the nearest 0.1 mg add 50 mL to the preheated oven and
hour. Store and cool the (0.0001 g). Record this mg aluminum dish. Use a evaporate at 103–105 °C
dish in a desiccator until value as C. blender or a beaker with for approximately six
needed. stir bar and stir plate to mix hours.
If a sample from Method the samples. Highly mineralized water
8271 is used, omit steps may prolong the drying
2–6. Proceed to step 7. time.
Note: A steam bath and
evaporating dish may be
used in place of the drying
oven for larger samples.
After evaporation on the
steam bath, dry the dish to
constant weight in a
103–105 °C drying oven.
4. Take the dish out of 5. Weigh the dish to the 6. Transfer the aluminum 7. Weigh the dish to the
the oven and allow it to nearest 0.1 mg (0.0001 g) dish into a pre-heated nearest 0.1 mg using an
cool to room temperature using an analytical muffle furnace at 550 °C analytical balance.
in a desiccator. balance. Record this mg for 30 minutes. Repeat ignition until two
value as A. After 30 minutes, take the successive sample
dish out of the furnace with weighings (A – B) do not
tongs and cool to room differ by more than 4% or
temperature in a 0.5 mg, whichever is less.
desiccator. Record this mg value as B.

Page 1140
8. The loss of weight is

total volatile solids.
Weighed residue is total
fixed solids.
Calculate:
mg/L Volatile Solids
( A – B ) × 1000
= ---------------------------------------------------------------
sample in volume in mL
mg/L Fixed Solids

( B – C ) × 1000
= --------------------------------------------------------
sample volume in mL
where:
A = Weight (mg) of solids
+ dish before ignition at
550 °C
B = Weight (mg) of solids
+ dish after ignition at
550 °C
C = Weight (mg) of empty
dish

Summary of method
A well-mixed sample is evaporated in a weighed dish and dried to a constant weight in a
103–105 °C oven. The dish and sample are ignited at 550 °C for 30 minutes. The loss of sample
mass upon ignition represents the volatile solids. The remaining sample after ignition represents
the fixed solids.

Page 1141
Required apparatus
Furnace, muffle, 120 Vac, 50/60 Hz each 1429600
Furnace, muffle, 240 Vac, 50/60 Hz each 1429624
Oven, laboratory, 240 Vac, 50 Hz each 1428902
Tongs each 56900

Blender, 1.2 liter, 120 VAC each 2616100
Stirrer, Magnetic, 4 x 4, 115 VAC each 2881200
Beaker, Glass, 250 mL each 50046H
Steam Bath, 8 inch diameter each 2347900
Evaporating Dish, porcelain, 120 mL each 52561

Solids, Total, 8271
Solids, Total DOC316.53.001203
USEPA1 Gravimetric Method2 Method 8271

Scope and Application: For potable, surface and saline water and for domestic and industrial wastewater.
1 USEPA Accepted.
2 Adapted from Standard Methods for the Examination of Water and Wastewater, Section 2540B
Test preparation
Test results may be used directly in continuation with Method 8276—Solids, Total Volatile and Fixed
Dry the aluminum dishes at 103-105C for one hour prior to use. Store dried dishes in a desiccator.
Larger samples can be used. For larger samples, use a steam bath and evaporating dishes in place of the aluminum dishes.
Weighing dish, aluminum 1
Drying oven 1
Graduated cylinder, 50 mL 1
Tongs 1
See Sample collection and storage for reorder information.
Solids, Total
Page 1143
Solids, Total
Gravimetric Method
1. Heat an aluminum 2. Weigh the aluminum 3. Mix the sample and 4. Place the sample in a
dish at 103–105 °C for one dish to the nearest 0.1 mg add 50 mL to the preheated oven and
hour. Store and cool the (0.0001 g). Record the mg aluminum dish. Use a evaporate at 103–105 °C
dish in a dessicator. value as B. blender or a beaker with for approximately six
stirbar and stir plate to mix hours.
samples. Highly mineralized water
may prolong the drying
time.
Note: A steam bath and
evaporating dish may be
used in place of the drying
oven for larger samples.
After evaporating on the
steam bath, dry the dish to
constant weight in a 103-
105C drying oven.
Repeat
Steps 4–6
5. Take the dish out of 6. Weigh the dish to the 7. Repeat step 3–step 5
the oven and allow it to nearest 0.1 mg (0.0001 g) until results do not differ by
cool to room temperature using an analytical more than 0.4 mg.
in a desiccator. balance. Record this mg Successive weighings that
value as A. are identical for some
wastewater samples are
unlikely due to slow
organic volatilization.
Solids, Total
Page 1144
Solids, Total
8. Calculate:
( A – B ) × 1000
mg/L Total Solids = ----------------------------------------------------------
Sample Volume in mL
Where:
A = Weight (mg)1 of Note: Continue with Method
sample + dish 8276 if Volatile and Fixed
Solids results are required.
B = Weight (mg) of dish
1 Weight in mg = grams x 1000

Summary of method
A well-mixed sample is evaporated in a weighed dish and dried to constant weight in an oven at
102–105 °C. The increase in weight over that of the empty dish represents the total solids.
Required apparatus
Cylinder, 50-mL each 50841
Tongs each 56900
Solids, Total
Page 1145
Solids, Total

Stirrer, Magnetic, 4 x 4, 115 VAC each 2881200
Beaker, Glass, 250 mL each 50046H
Steam Bath, 8 inch diameter each 2347900
Evaporating Dish, porcelain, 120 mL each 52561

Suspended Solids, 8006
Suspended Solids DOC316.53.01139
Photometric Method1 Method 8006

(5 to 750 mg/L)
1 Adapted from Sewage and Industrial Wastes, 31, 1159 (1959).
Test preparation

TheInstrument-specific information table displays information that may vary from instrument to
instrument. Select your spectrophotometer from the instrument column on the left. Read across to
find the corresponding sample cells and adapters required to perform this test on
your spectrophotometer.
Beaker, 600-mL, polypropylene 1
Blender 1
Cylinder, 500-mL polypropylene, graduated 1
Sample Cells (see the Instrument-specific information table) 2
Suspended Solids
Page 1147
Suspended Solids
Photometric Method
Stored Programs
630 Suspended Solids
Start
1. Select the test. 2. Blend 500 mL of 3. Pour the blended 4. Prepared Sample:
Insert an adapter if sample in a blender at sample into a 600-mL Stir the sample and
required (see the high speed for exactly two beaker. immediately pour 10 mL of
Instrument-specific minutes. the blended sample into a
information table). sample cell.
for orientation.
Zero
5. Blank Preparation: 6. Wipe and insert the 7. ZERO the instrument. 8. Swirl the prepared
Fill a second sample cell blank into the cell holder. The display will show: sample to remove any gas
with 10 mL of tap water or bubbles and uniformly
deionized water. 0 mg/L TSS suspend any residue.
Read
9. Wipe and insert the 10. READ the results in

prepared sample into the mg/L TSS.
cell holder.
Interferences
Samples that absorb strongly at 810 nm, such as blue dyes, may give false, high-bias readings. A
user-entered calibration is advised for these samples.
Suspended Solids
Page 1148
Suspended Solids

Collect samples in clean plastic or glass bottles. Analyze samples as soon as possible after
collection. The sample may be stored for seven days by cooling to 4 °C (39 °F).
Accuracy check
Calibration for this test is based on parallel samples using the gravimetric technique on sewage
samples from a municipal sewage plant. For most samples, this calibration will provide satisfactory
results. When higher accuracy is required, run parallel spectrophotometric and gravimetric
determinations with portions of the same sample. Make the new calibration on the particular
sample using a gravimetric technique as a basis.
Summary of method
This method of determining suspended solids is a simple, direct measurement which does not
require the filtration or ignition/weighing steps that gravimetric procedures do. The USEPA
specifies the gravimetric method for solids determinations, while this method is often used for
checking in-plant processes. Test results as mg/L total suspended solids (TSS) are measured at
810 nm.

Required apparatus
Beaker, 600-mL, polypropylene 1 each 108052
Blender, 1.2-L, 120 VAC 1 each 2616100
Blender, 1.2 L, 240 VAC 1 each 2616102
Cylinder, 500-mL graduated, polypropylene 1 each 108149
Suspended Solids
Page 1149
Sulfate, 8051
Sulfate DOC316.53.01135
USEPA1 SulfaVer 4 Method2 Method 8051

(2 to 70 mg/L) Powder Pillows or AccuVac® Ampuls
1 USEPA accepted for reporting wastewater analyses. Procedure is equivalent to USEPA method 375.4 for wastewater.
Test preparation


Instrument
Adjust the standard curve for each new lot of reagent (Standard solution method).
For best results, calibrate the instrument with each new lot of reagent (see Calibration).
Filter highly colored or turbid samples using filter paper and a funnel. Use this sample in step 2 and 5.
SulfaVer® 4 contains barium chloride. The final solution will contain barium chloride (D005) at a concentration regulated as a
hazardous waste by the Federal RCRA. Refer to a current MSDS for safe handling and disposal instructions.
Use a blank AccuVac® Ampule in place of the sample cell in Step 5 .if necessary.
Sulfate
Page 1151
Sulfate
Powder Pillow Test:
SulfaVer® 4 Reagent Powder Pillows 1
AccuVac Test:
SulfaVer® 4 Reagent AccuVac® Ampuls 1
Beaker, 50-mL 1
Stopper 1
SulfaVer 4 powder pillow procedure
Stored Programs
680 Sulfate
Start
1. Select the test. 2. Prepared sample: Fill 3. Add the contents of 4. Start the instrument
Insert an adapter if a sample cell with 10 mL one SulfaVer 4 Reagent timer.
required (see Instrument- of sample. Powder Pillow to the A five-minute reaction time
specific information). sample cell. Swirl will begin. Do not disturb
Refer to the user manual vigorously to dissolve the the cell during this time.
for orientation. powder.
Note: Accuracy is not
White turbidity will form if affected by undissolved
sulfate is present. powder.
Sulfate
Page 1152
Sulfate
SulfaVer 4 powder pillow procedure (continued)
Zero Read
5. Blank preparation: 6. When the timer 7. ZERO the instrument. 8. Within five minutes
Fill a second sample cell expires, wipe the blank The display will show: after the timer expires,
with 10 mL of sample. and insert it in the cell wipe the cell and insert the
holder (fill lines face right.) 0 mg/L SO42– prepared sample in the
cell holder.
READ the results in
mg/L SO42–.
Clean sample cells with
soap and a brush.
SulfaVer 4 AccuVac® Ampuls procedure
Stored Programs
685 Sulfate AV
Start
1. Select the test. 2. Prepared sample: 3. Cap or stopper the 4. Start the instrument
Insert an adapter if Collect at least 40 mL of Ampul and quickly invert timer.
required (see Instrument- sample in a 50-mL beaker. several times to mix. A five-minute reaction time
specific information). Fill a SulfaVer 4 Reagent White turbidity will form if will begin. Do not disturb
Refer to the user manual AccuVac® Ampul with sulfate is present. the cell during this time.
for orientation. sample from the beaker.
Note: Accuracy is not
Keep the tip immersed affected by undissolved
while the Ampul fills powder.
completely.
Sulfate
Page 1153
Sulfate
SulfaVer 4 AccuVac® Ampuls procedure (continued)
5. Blank Preparation: 6. When the timer 7. Within five minutes

Fill a clean sample cell expires, wipe the blank after the timer expires,
with 10 mL of sample. and insert it in the cell wipe the Ampul and insert
holder. it in the cell holder.
ZERO the instrument. READ the results in
The display will show: mg/L SO42–.
0 mg/L SO42–
Interferences

Calcium Greater than 20,000 mg/L as CaCO3
Chloride Greater than 40,000 mg/L as Cl–
Magnesium Greater than 10,000 mg/L as CaCO3
Silica Greater than 500 mg/L as SiO2

Collect samples in clean plastic or glass bottles. Samples may be stored up to 7 days by cooling to
4 °C (39 °F) or lower. Warm to room temperature before analysis.
Accuracy check
• Sulfate Ampule Standard Solution, 2500 mg/L sulfate
• Ampule breaker
• TenSette Pipet and pipet tips
• Mixing cylinder, 25 mL or 50 mL
• Beaker, 50 mL
2. Select standard additions from the instrument menu:

OPTIONS>MORE>STANDARD ADDITIONS.
Sulfate
Page 1154
Sulfate
5. Fill three mixing cylinders with 25 mL of sample. Use the TenSette Pipet to add 0.1 mL, 0.2 mL
and 0.3 mL of standard, respectively, to each mixing cylinder and mix thoroughly. Transfer 10
mL of each sample spike to a clean sample cell.
Note: For AccuVac® Ampuls, fill three mixing cylinders with 50 mL of sample and spike with 0.2 mL,
0.4 mL and 0.6 mL of standard. Transfer 40 mL from each of the three mixing cylinders to three
50-mL beakers.
6. Follow the SulfaVer 4 powder pillow procedure for each of the spiked samples, starting with
Note: For AccuVac Ampuls, follow the SulfaVer 4 AccuVac® Ampuls procedure for each of the spiked
samples, starting with the 0.2 mL sample spike. Measure each of the spiked samples in
the instrument.

• Sulfate standard solution, 1000 mg/L
• Tensette pipet, 1–10 mL and pipet tips
1. Prepare a 70 mg/L sulfate standard solution as follows: use a pipet to add 7.0 mL of sulfate
standard solution, 1000 mg/L as SO42–, to a 100-mL volumetric flask. Dilute to the mark with
deionized water. Mix well. Prepare this solution daily.
2. Follow the SulfaVer 4 powder pillow procedure or SulfaVer 4 AccuVac® Ampuls procedure
and use the 70-mg/L SO42– standard solution in place of the sample.
3. To adjust the calibration curve using the reading obtained with the standard solution, set
standard adjust to on (OPTIONS>(MORE)>STANDARD ADJUST) and accept the concentration.
Calibration
A calibration is recommended for the SulfaVer 4 method for the best accuracy. Complete the
following steps to enter a new calibration curve in the instrument. Perform this procedure for each
new lot of reagent.
Required items:
• Sulfate standard solution, 1000 mg/L
• Seven 100 mL Class A volumetric flasks
• 1–10 mL TenSette pipet and tips
1. Prepare seven calibration standards (10, 20, 30, 40, 50, 60 and 70 mg/L SO42–) as follows.
Use the Tensette pipet to add 1, 2, 3, 4, 5, 6 and 7 mL of the 1000-mg/L sulfate standard
solution to seven different 100-mL Class A volumetric flasks.
2. Dilute each flask to the mark with deionized water. Mix thoroughly.
3. Use each standard solution in place of the sample and follow the SulfaVer 4 powder pillow
procedure or SulfaVer 4 AccuVac® Ampuls procedure.
Sulfate
Page 1155
Sulfate
4. Refer to the user manual (user programs section) to enter the calibration in the instrument as
a user program.
Method performance
680 40 mg/L SO42– 30–50 mg/L SO42– 0.4 mg/L SO42–
685 40 mg/L SO4 2– 32–48 mg/L SO4 2– 0.7 mg/L SO42–
Summary of method
Sulfate ions in the sample react with barium in the SulfaVer 4 and form a precipitate of barium
sulfate. The amount of turbidity formed is proportional to the sulfate concentration. Test results are
measured at 450 nm.
Sulfate
Page 1156
Sulfate
Required reagents
SulfaVer® 4 Reagent Powder Pillows 1 100/pkg 2106769

OR
SulfaVer® 4 Sulfate Reagent AccuVac® Ampuls 1 25/pkg 2509025
Required apparatus
Sample cell, 10 mL, round, 25 x 54 mm 1 each 2122800
Sample cell, 10 mL, square, matched pair 2 2/pkg 24954025
Sulfate Standard Solution, 1000-mg/L 500 mL 2175749
Sulfate Standard Solution, 2500-mg/L, 10-mL Ampules 16/pkg 1425210
Mixed Parameter Standard for sulfate, fluoride, nitrate and phosphate 500 mL 2833049

Blank AccuVac Ampules 25/pkg 2677825
Ampule Breaker, for 10 mL Ampules each 2196800
Tensette Pipet 0.1–1.0 mL each 1970001
Tips for Tensette Pipet 1–10 50/pkg 2185696
Tensette Pipet 1–10 each 1970010
Tips for tensette Pipet 1–10 50/pkg 2199796
Sulfate
Page 1157
Sulfide, 8131
Sulfide DOC316.53.01136
USEPA1 Methylene Blue Method2 Method 8131

(5 to 800 µg/L)
Scope and Application: For testing total sulfides, H2S, HS–, and certain metal sulfides in groundwater,
wastewater, brines and seawater.
1 USEPA approved for reporting wastewater analysis. Procedure is equivalent to Standard Method 4500-S2– D.
Test preparation

Instrument Sample volume Sample cell Cell orientation
DR 6000 10 mL 2495402 Fill line faces right
DR 5000 10 mL 2495402 Fill line faces user
DR 3900 10 mL 2495402 Fill line faces user
DR 3800, DR 2800, DR 2700 10 mL 2495402 Fill line faces right
Avoid excessive agitation of samples to minimize sulfide loss.
Some sulfide loss may occur if dilution is necessary.
Sulfide 2 reagent contains potassium dichromate. The final solution will contain hexavalent chromium (D007) at a
concentration that is regulated as a hazardous waste by Federal RCRA. Refer to the current MSDS for safe handling and
disposal instructions.
Sulfide 1 Reagent 1–2 mL
Sulfide 2 Reagent 1–2 mL
Water, deionized 10–25 mL
Stoppers 2
Sulfide
Page 1159
Sulfide
Methylene Blue Method
Stored Programs
690 Sulfide
Start
1. Select the test. 2. Blank Preparation: 3. Prepared Sample: 4. Use the dropper to
Insert an adapter if Measure 10 mL of Use a pipet to add 10 mL add 0.5 mL Sulfide 1
required (see Instrument- deionized water in a of sample to a second Reagent to each cell.
specific information). Refer sample cell. sample cell. Do not mix Swirl to mix.
to the user manual for the sample more than
orientation. necessary to prevent
sulfide loss.
5. Use the dropper to 6. Cap or stopper the cell 7. Start the instrument 8. When the timer
add 0.5 mL Sulfide 2 and immediately invert to timer. expires, wipe the blank
Reagent to each cell. mix. A five-minute reaction time and insert it in the cell
The solution will turn pink will begin. holder.
initially and then turn blue
if sulfide is present.
Zero Read
9. ZERO the instrument. 10. Wipe the prepared 11. READ the results in
The display will show: sample and insert it in the µg/L S2–.
cell holder.
0.00 µg/L S2–
Sulfide
Page 1160
Sulfide
Soluble sulfides
Complete the following steps to measure soluble sulfides.
1. Centrifuge a sample in completely filled, capped tubes.
2. Use the supernatant in place of the sample and follow the Methylene Blue Method procedure.
To estimate insoluble sulfides, subtract the soluble sulfide concentration from the total sulfide
concentration.
Interferences
Strong reducing substances such as
Interfere by reducing the blue color or preventing its development.
sulfite, thiosulfate and hydrosulfite.
High concentrations of sulfide may inhibit full color development and require sample
Sulfide, high levels
dilution. Some sulfide loss may occur when the sample is diluted.
For turbid samples, prepare a sulfide-free blank as follows. Use this blank in place of
the deionized water blank in the Methylene Blue Method test procedure.
1. Measure 25 mL of sample into a 50-mL Erlenmeyer flask.
2. Add bromine water by drops with constant swirling until a permanent yellow color
just appears.
Turbidity
3. Add phenol solution by drops until the yellow color just disappears. Use this
solution to replace the deionized water in step 2 of the procedure.
This pretreatment procedure removes sulfide from the sample, but the turbidity and
any color will remain. The interference from turbidity or color will be corrected when
the instrument is set to zero with this solution (step 9).

Collect samples in clean plastic or glass bottles. Fill completely and cap tightly. Prevent excessive
shaking or prolonged exposure to air. Analyze samples immediately.
Method performance
690 DR 5000 520 µg/L S2– 504–536 µg/L S2– 5µg/L S2–
Summary of method
Hydrogen sulfide and acid-soluble metal sulfides react with N,N-dimethyl-p-phenylenediamine
sulfate to form methylene blue. The intensity of the blue color is proportional to the sulfide
concentration. High sulfide levels in oil field waters may be determined after proper dilution. Test
Sulfide
Page 1161
Sulfide
Required reagents
Sulfide Reagent Set, includes: — — 2244500
Sulfide 1 Reagent 1 mL 100 mL MDB 181632
Sulfide 2 Reagent 1 mL 100 mL MDB 181732
Water, deionized 10 mL 4 liters 27256
Required apparatus
Stopper, for 18-mm Tube 2 6/pkg 173106

Stopper, for 18-mm Tube 25/pkg 173125

Sulfite, BT, 8071
Sulfite DOC316.53.01162
Iodate-Iodide Buret Titration Method1 Method 8071

0 to 500 mg/L as SO3 2– (or 0 to > 500 mg/L) Buret Titration
Scope and Application: For boiler water.
Test preparation
Sulfite is easily destroyed by atmospheric oxygen. Violent shaking or swirling will cause low results.
Analyze samples immediately. Cool hot samples to 50 °C (122 °F) or lower before analysis.
Dissolved Oxygen Reagent Powder Pillow 1 pillow
Potassium Iodide-Iodate Standard Solution, 0.0125 N 1 bottle
Graduated cylinder (see Range-specific information) 1
Buret titration
See
Table 1
1. Select a sample 2. Fill the buret to the 3. Use a graduated 4. Add the contents of
volume from the Range- zero mark with 0.0125 N cylinder or pipet to one Dissolved Oxygen 3
specific information. Potassium Iodide-Iodate measure the sample Powder Pillow and mix
Standard Solution. volume. Add the sample to gently.
the Erlenmeyer flask.
If the sample volume is
less than 50 mL, gently
dilute to approximately
50 mL with deionized
water. Do not agitate.
Sulfite
Page 1163
Sulfite
5. Add one full dropper of 6. Titrate the sample 7. Use the multiplier in
Starch Indicator Solution. while gently swirling the the Range-specific
Swirl gently to mix. flask until the color information to calculate
changes to a permanent the concentration:
blue color (end point). mL titrant used x multiplier
Write down the volume of = mg/L sulfite (SO32–)
titrant that was used. Example: 50 mL of sample
was titrated and 5 mL of
titrant was used to reach
the end point. The
concentration is 5 x 10 =
50 mg/L as SO32–

Range (mg/L as SO32–) Sample volume (mL) Multiplier
0–100 50 10
40–200 25 20
100–500 10 50
Over 500 5 100
Interferences
Nitrite Nitrite will react with sulfite to cause a negative error in the titration.
Some metals, especially copper, catalyze the oxidation of sulfite to sulfate. Immediate fixing
Metals of sample with the contents of one Sulfamic Acid Powder Pillow per liter of sample will help
minimize the interference.
Organic compounds Oxidizable organic compounds could cause high results.
Oxidizable compounds Will cause high results.
Sulfide Will cause high results.
Sulfite
Page 1164
Sulfite

• Avoid excessive agitation or prolonged exposure to air. Complete the test procedure as soon
as possible after collection for best accuracy. Samples cannot be preserved or stored.
• Cool hot samples to 50 °C (122 °F) or lower.
Accuracy check
The standard additions method can be used to find if the sample has an interference. The
standard solution method can be used to confirm analytical technique and reagent performance.
• Sulfite Voluette Ampule Standard, 5,000-mg/L SO32–
• Ampule breaker
• Pipet filler
8. Each 0.1 mL of standard that was added will use 1.0 mL of titrant to reach the endpoint. If

Complete the following test to make sure the concentration is accurate.
• 0.025 N Sodium Thiosulfate Standard Solution
• 10-mL volumetric pipet, Class A
1. Use a pipet to add 10.0-mL of 0.025 N Sodium Thiosulfate Standard Solution to a 250-mL
volumetric flask. Dilute to the mark with deionized water. The diluted standard is equivalent to
40 mg/L sulfite.
2. Use a graduated cylinder to measure 50 mL of the diluted standard. Add the standard to the
Erlenmeyer flask.
3. Titrate the standard to the end point with the titrant solution. The result should be close to
40 mg/L sulfite.
Sulfite
Page 1165
Sulfite
Summary of method
The water sample is acidified, treated with a starch indicator and titrated with a potassium iodide-
iodate standard solution. The acid releases free iodine, which is reduced to colorless iodide by the
sulfite in the sample. When all of the sulfite is gone, excess iodine will react with the starch to form
a blue color.

Required reagents
Dissolved Oxygen 3 Reagent Powder Pillows 1 pillow 100/pkg 98799
Potassium Iodide-Iodate Standard Solution, 0.0125 N varies 1L 1400153
Reagent set, Sulfite Buret Titration — 100 tests 2459800
Required apparatus
Buret, Class A, Teflon Stopcock plug, Certified 10-mL 1 each 2636538
Clippers, for opening pillows 1 each 96800
Pipet, volumetric, Class A, 5 mL 1 each 1451537
Pipet, volumetric, Class A, 10 mL 1 each 1451538
Pipet filler, Safety Bulb 1 each 1465100
Sulfite Standard Solution, Voluette® Ampule, 5,000-mg/L SO3 10-mL 16/pkg 1426710

Ampule breaker each 2196800

Sulfite, DT, 8216
Iodate-Iodide Method Method 8216

4 to greater than 400 mg/L as SO3 2– Digital Titrator
Scope and Application: For boiler water
Test preparation
Analyze samples immediately. Cool hot samples to 50 °C (122 °F) or lower before analysis.
Sulfite is easily destroyed by atmospheric oxygen. Violent shaking or swirling will cause low results. Avoid unnecessary
agitation throughout the procedure.
The Dissolved Oxygen 3 Reagent Powder Pillow can be replaced with 0.5 mL of 19.2 N Sulfuric Acid Standard Solution.
mg/L Sulfite (SO32–) x 1.01 = Bisulfite, Hydrogen Sulfite (HSO3–)
mg/L Sulfite (SO32–) x 1.30 = Sodium Bisulfite, Sodium Hydrogen Sulfite (NaHSO3)
mg/L Sulfite (SO32–) x 2.37 = Sodium Metabisulfite, Sodium Pyrosulfite (Na2S2O5)
mg/L Sulfite (SO32–) x 1.58 = Sodium Sulfite (Na2SO3)
Sulfite Reagent Set 1
Clippers 1
Digital Titrator 1
Graduated Cylinder, 10-, 25- or 50-mL, based on sample concentration varies
Sulfite
Page 1167
Sulfite
Iodate-Iodide Method
8. Select a sample 9. Insert a clean delivery 10. Turn the delivery knob 11. Transfer the sample
volume from the Volume tube into the Iodate-Iodide to eject a few drops of volume into a clean,
multipliers table that Titration Cartridge titrant. Reset the counter 125-mL Erlenmeyer flask.
corresponds to the (KIO3–KI). to zero and wipe the tip. Dilute to the 50-mL mark
expected sulfite (SO32–) Attach the cartridge to the with deionized water.
concentration. titrator body.
Use a graduated cylinder
to measure the sample
volume from the Volume
multipliers table.
12. Add the contents of 13. Add one dropperful of 14. Place the delivery tube 15. Calculate:
one Dissolved Oxygen 3 Starch Indicator Solution tip into the solution and Digits Required x
Reagent Powder Pillow and swirl to mix. swirl the flask while Digit Multiplier =
and swirl gently to mix. titrating with the mg/L Sulfite (SO32–)
iodate-iodide to a
permanent blue end point.
Record the number of
digits required.

Range (mg/L as SO32–) Volume (mL) Cartridge Strength Digit Multiplier
Up to 160 50 0.3998 N 0.4

100–400 20 0.3998 N 1.0
Over 400 5 0.3998 N 4.0
200–800 10 0.3998 N 2.0
Sulfite
Page 1168
Sulfite
Accuracy check
This accuracy check should be performed when interferences are suspected or to verify analytical
technique.
1. Snap the top off a Sulfite Voluette Ampule Standard, 5,000-mg/L SO32–.
2. Use a TenSette Pipet to add 0.1 mL of standard to the sample titrated. Resume titration back
to the same end point. Record the number of digits required.
3. Repeat, using additions of 0.2 and 0.3 mL, titrating to the end point after each.
4. Each 0.1-mL addition of standard should require 25 additional digits of titrant. If these uniform
increases do not occur, determine the cause.
A standard solution equivalent to 40-mg/L sulfite can be prepared by diluting 10.0 mL of 0.025 N
Sodium Thiosulfate Titrant to 250 mL in a volumetric flask. Titrate a 50-mL sample, using the
above procedure.
Interferences
• Sulfide, organic matter and other oxidizable substances will cause positive error in the titration.
• Nitrite will react with sulfite to cause low results.
• Some metals, especially copper, catalyze the oxidation of sulfite to sulfate.
• Addition of one Dissolved Oxygen 3 Powder Pillow per liter of sample immediately upon
sampling will help eliminate the effects of nitrite and copper.
Summary of method
Sulfite ion is titrated with potassium iodate-iodide standard solution under acidic conditions to a
blue starch end point. The volume of titrant used is proportional to the sulfite concentration.

Required reagents
Sulfite Reagent Set (about 100 tests), includes: 2272300
Iodate-Iodide Titration Cartridge, 0.3998 N each 1496101
Starch indicator Solution 100 mL MDB 34932
Sulfite
Page 1169
Sulfite
Required apparatus
Clippers for opening pillows each 96800
Thermometer -10–225 °C, 405 mm each 2635700
Volumetric Pipet, 10 mL each 1451538
Volumetric flask, 250 mL each 1457446
Tensette Pipet each 1970001
Required standards
Sodium Thiosulfate Standard Solution, 0.025 N 1000 mL 2409353
Sulfite Standard Solution, Voluette® Ampule, 5,000-mg/L SO3 10-mL 16/pkg 2267410
Sulfite Standard Solution, 15 mg/L 500 mL 2408449

Sulfite, Europe only
Colorimetric Method1
(0.10 to 5.00 mg/L)
Scope and Application: For boiler water and foodstuffs.
1 Reagent sets for this method are only available in Europe.
Test preparation

information required to perform this test

Samples must be analyzed immediately.
The temperature of samples and reagents must be between 15–25 °C (59–77 °F).
Adjust the pH of the sample to a value between 3–10.
Sulfite Reagent A 5 drops
Sulfite Reagent B 2 drops
Pipet, 10-mL serological 1
Sulfite
Page 1171
Sulfite
Colorimetric Method
Stored Programs
692 Sulfite HPT 430
Start
1. Select the test. 2. Blank Preparation: 3. Prepared Sample: 4. Add 5 drops of Sulfite
Insert an adapter if Fill a clean sample cell Pipet 10 mL of sample into Reagent A (HPT 430 A) to
required (see the with 10 mL of sample. a second clean sample the prepared sample.
Instrument-specific cell.
information table). Refer to
the user manual for
orientation.
5. Swirl to mix. 6. Add 2 drops of Sulfite 7. Start the instrument 8. Wipe the blank and
Reagent B (HPT 430 B) to timer. insert it in the cell holder.
the prepared sample. Swirl A 3-minute reaction time
to mix. will begin. Do not disturb
the cell during this time.
Zero Read
9. ZERO the instrument. 10. When the timer 11. READ the results in
The display will show: expires, wipe the prepared mg/L SO32–.
sample and insert it in the
0.00 mg/L SO32– cell holder.
Sulfite
Page 1172
Sulfite
Interferences

Sulfide Greater than 5 mg/L

Collect samples in clean plastic or glass bottles. Analyze immediately.
Method performance
692 DR 5000 3.00 mg/L SO32 2.51–3.49 mg/L SO32– 0.04 mg/L SO32–
Summary of method
The reagents react with sulfite to form a yellow complex. The color is measured at 435 nm.
Required reagents
Sulfite Colorimetric Reagent Set, includes: — 100/pkg HPT430
Sulfite Reagent A1 5 drops 28 mL —
Sulfite Reagent B1 2 drops 8.7 mL —
1 Not available separately. Reagent sets for this method are only available in Europe.
Required apparatus
Pipet, 10-mL serological 1 each 53238
Sulfite
Page 1173
Surfactants, Anionic (Detergents), 8028
Surfactants, Anionic (Detergents) DOC316.53.01138
Crystal Violet Method1 Method 8028

(0.002 to 0.275 mg/L as LAS)
1 Adapted from Analytical Chemistry, 38, 791 (1966).
Test preparation

The Instrument-specific information table displays information that may vary from instrument to
instrument. Select the spectrophotometer from the instrument column on the left. Read across to
the spectrophotometer.
Use benzene only in a well-ventilated area.
Benzene (D018) solutions are regulated as hazardous waste by the Federal RCRA. Do not pour these materials down the
drain. Collect water that is saturated with benzene and benzene solutions for disposal with laboratory solvent wastes. Refer
To prevent water droplets from forming in the sample cells, use only dry sample cells and discard the first few mL of benzene.
Additionally, it can help to transfer the liquid from the funnel to a sample cell, let it sit for a few seconds and decant to a
second cell for reading.
Excessive shaking may cause an emulsion to form, which makes the phases separate more slowly. If this occurs, remove
most of the water layer, then gently mix the contents of the funnel with a clean Teflon®-coated rod or other inert tool.
Spilled reagent will affect test accuracy and is hazardous to the skin and other materials.
Acetone may be used to clean benzene from glassware.
In bright light conditions (e.g. direct sunlight) be sure to cover the cell compartment during measurements.
Surfactants, Anionic (Detergents)

Page 1175
Benzene, ACS 55 mL
Buffer Solution, sulfate-type 10 mL
Detergent Reagent Powder Pillows 1 pillow
Clippers, for opening powder pillows 1
Funnel, separatory, 500-mL 1
Support Ring, 4-inch 1
Support, Ring Stand, 5 x 8 inch base 1
Crystal Violet Method
Stored Programs
710 Surfactants
Start
1. Select the test. 2. Fill a clean 500-mL 3. Pour the sample into a 4. Add 10 mL of Sulfate
Insert an adapter if graduated cylinder to the clean 500-mL separatory Buffer Solution. Stopper
required ( Instrument- 300 mL mark with sample. funnel. the funnel. Shake the
specific information table). funnel for five seconds.

for orientation.

Page 1176
Crystal Violet Method (continued)
5. Add the contents of 6. Stopper the funnel 7. Add 30 mL of benzene 8. Place the separatory
one Detergents Reagent and shake until the powder to the funnel. Stopper the funnel in a support stand.
Powder Pillow to the dissolves completely. The funnel and shake gently
funnel. powder will dissolve for one minute.
slowly.
9. Start the instrument 10. After the timer expires, 11. Prepared Sample: 12. Blank Preparation:
timer. remove the stopper and Drain the top benzene Fill another sample cell to
A 30-minute reaction drain the bottom water layer into a clean 25-mL the 10-mL mark with pure
period will begin. layer. Discard this layer. sample cell. Stopper the benzene. Stopper the cell.
cell.
Do not filter the benzene
layer before color
measurement. Filtration
removes the blue color.
Zero Read
13. Wipe and insert the 14. ZERO the instrument. 15. Wipe and insert the 16. READ the results in
blank cell into the cell The display will show: sample cell into the cell mg/L LAS.
holder. holder.
0.000 mg/L LAS

Page 1177
Interferences
High amounts of chloride, such as those levels found in
Chloride
brines and seawater, will cause low results.
Perchlorate ions Interferes at all levels.
Periodate ions Interferes at all levels.

Collect samples in clean plastic or glass bottles. Analyze samples as soon as possible. Samples
may be stored at least 24 hours by cooling to 4 °C (39 °F). Warm to room temperature
before testing.
Accuracy check
• Detergent Voluette® Ampule Standard, 60-mg/L LAS
• Ampule breaker
• TenSette Pipet, 0.1–1.0 mL and Tips

ADDITIONS.

5. Prepare three sample spikes as follows. Measure 300 mL of sample into each of the three
beakers. Use the TenSette Pipet to add 0.1 mL, 0.2 mL and 0.3 mL of standard, respectively,
to each sample and mix thoroughly.
6. Follow the test procedure for each of the spiked samples, starting with the 0.1 mL sample

Page 1178

• Detergent Voluette® Ampule Standard, 60-mg/L LAS
1. Prepare a 0.180 mg/L LAS standard solution as follows: Pipet 3.0 mL of Detergent Standard,
60-mg/L as LAS, into a 1000-mL (1 liter) volumetric flask. Dilute to the mark with deionized
water. Mix well. Prepare this solution daily.
2. Follow the Crystal Violet Method test procedure.
3. To adjust the calibration curve with the reading obtained with the standard solution, navigate to
Standard Adjust in the software: OPTIONS>MORE>STANDARD ADJUST.
4. Turn on the Standard Adjust option and accept the displayed concentration. If an alternate
Method performance
710 0.180 mg/L LAS 0.172–0.188 mg/L LAS 0.002 mg/L LAS
Summary of method
Detergents, ABS (alkyl benzene sulfonate) or LAS (linear alkylate sulfonate) are determined by
association with crystal violet dye and extraction of the ion-pair complex into benzene. Test results
Required reagents
Detergents Reagent Set, includes: — — 2446800
Benzene, ACS 40 mL 4 liters 1444017
Buffer Solution, sulfate-type 10 mL 500 mL 45249
Detergent Reagent Powder Pillows 1 pillow 25/pkg 100868
Required apparatus
Support Ring, 4-inch 1 each 58001
Support, Ring Stand, 5 x 8 inch base 1 each 56300

Page 1179
Detergent Standard Solution, 10-mL Voluette® Ampule, 60-mg/L LAS 16/pkg 1427110

Beaker, 600 mL each 50052
Pipet filler bulb each 1465100
Pipet tips for TenSette Pipet 19700-01 50/pkg 2185696

Tannin and Lignin, 8193
Tannin and Lignin DOC316.53.01140
Tyrosine Method1 Method 8193

(0.1 to 9.0 mg/L)
Scope and Application: For water, wastewater and boiler water.
1 Adapted from Kloster, M.B., Journal American Water Works Association, Vol. 66, No. 1, p. 44 (1974).
Test preparation


Filter turbid samples and report results as mg/L soluble tannic acid.
For best accuracy, use a pipet to add the TanniVer® 3 solution.
The Pour-Thru Cell can be used as an alternative to the sample cells, when using a full 25 mL sample..
Results will be given in mg/L tannins (as tannic acid).
Tannin and Lignin Reagent Set:
Sodium Carbonate Solution 10 mL
TanniVer® 3 Tannin-Lignin Reagent 1 mL
Pipet Filler 1
Pipet, volumetric Class A, 5.0-mL 1
Pipet, volumetric Class A, 0.5-mL 1
Tannin and Lignin

Page 1181
Tannin and Lignin
Tyrosine Method
Stored Programs
720 Tannin & Lignin
Start
1. Select the test. 2. Blank Preparation: 3. Prepared Sample: 4. Pipet 0.5 mL of

Insert an adapter if Fill a 25-mL graduated Fill a second 25-mL TanniVer® 3 Tannin-Lignin
required (Instrument- mixing cylinder to the graduated mixing cylinder Reagent into each
specific information). 25-mL mark with to the 25-mL mark with cylinder.
deionized water. sample.
5. Insert the stopper and 6. Pipet 5.0 mL of 7. Pour 10 mL of each 8. Start the instrument
invert to mix. Sodium Carbonate solution into two sample timer.
Solution into each cylinder. cells. A 25-minute reaction
Insert the stopper and period will begin.
invert to mix.
A blue color will develop if
tannins and/or lignins are
present.
Zero Read
9. When the timer 10. ZERO the instrument. 11. Insert the sample into 12. READ the results in
expires, insert the blank The display will show: the cell holder. mg/L Tannins
into the cell holder. (as Tannic Acid).
0.0 mg/L Tannins
(as Tannic Acid).
Tannin and Lignin

Page 1182
Tannin and Lignin
Interferences
Causes a positive interference. (2 mg/L of ferrous iron produces a color equivalent to
Ferrous iron about 1 mg/L of tannic acid.) To eliminate interference of ferrous iron up to 20 mg/L,
add one 0.2 g scoop of Sodium Pyrophosphate1 to the sample before testing.
To eliminate sulfite interference, add 1 mL of formaldehyde1 to the sample before
Sulfite
testing the sample.

Accuracy check

• Tannic acid, analytical grade
• 15-mL Class A volumetric pipet and pipet bulb
• Deionized water
1. Prepare a tannic acid stock solution as follows: dissolve 0.200 grams of tannic acid in
deionized water and dilute to 1000 mL. Prepare this solution monthly.
2. Prepare a 6.0-mg/L tannic acid standard solution by diluting 15.00 mL of the stock solution to
500 mL with deionized water. Prepare this standard daily.
3. Follow the tannin and lignin procedure as described above.
4. To adjust the calibration curve using the reading obtained with the 6.0-mg/L Standard Solution,
navigate to Standard Adjust in the software:OPTIONS>MORE>STANDARD ADJUST.
Method performance
720 6.0 mg/L tannic acid 5.8–6.2 mg/L tannin 0.1 mg/L tannin
Summary of method
This test measures all hydroxylated aromatic compounds, including tannin, lignin, phenol and
cresol. This method produces a blue color proportional to the amount of these compounds present
Tannin and Lignin

Page 1183
Tannin and Lignin
in the sample. The results are reported as total tannin and lignin and expressed as mg/L tannic
acid. Test results are measured at 700 nm.

Required reagents
Tannin and Lignin Reagent Set (up to 100 tests), includes: 2244600
(2) Sodium Carbonate Solution 10 mL 500 mL 67549
(1) TanniVer® 3 Tannin-Lignin Reagent 1 mL 100 mL 256032
Required apparatus
Cylinder, mixing, with stopper, 25-mL 2 each 2088640
Pipet filler, safety bulb 1 each 1465100
Tannic Acid, Analytical Grade 113 g 79114

Formaldehyde 100 mL 205932
Pipet, volumetric, 15.0 mL each 1451539
Sodium Pyrophosphate 50 g 1429525
Spatula, micro each 1225600

Toxicity, 10017
Toxicity DOC316.53.01141
ToxTrak™ Method 1, 2 Method 10017

(0 to 100% Inhibition)
Scope and Application: For drinking water, wastewater and natural waters.
1 Liu, D., Bull. Environ. Contm. Toxicol. 26, 145-149 (1981)
2 Environmental Technology Verification ETV Program evaluated, November, 2003
Test preparation

DR 6000, DR 5000 —
DR 3900 LZV849
DR 3800, DR 2800, DR 2700 LZV646
Do not leave the cells in the instrument during incubation. All samples and control cells should be allowed to react under
similar conditions of temperature and light.
If testing chlorinated samples, add two drops of sodium thiosulfate to each blank and sample to remove the chlorine.
If testing drinking water, take the control sample from a reservoir of tap water known to be free of toxins, if possible.
Bacterial Count Broth Tube 1 tube

Pipet, transfer, sterile 2
Reaction tubes, with cap 1
Sodium Thiosulfate varies
ToxTrak™ Reagent Powder Pillows 2
ToxTrak™ Accelerator Solution 4 drops
Clippers 1
Incubator 1
Pipet, volumetric, Class A, 5.00 mL and pipet filler 1
Toxicity
Page 1185
Toxicity
Inoculum development using indigenous biomass
1. Using one of the 2. Incubate the tube

dropper pipets provided, contents at 35 °C (95 °F)
add 1.0 mL of source until the broth is visibly
culture (indigenous turbid (approximately 12
biomass) to a Total hours).
Bacteria Count Broth
Note: The culture can be kept
Tube. for several days in the
Note: Commercial sources of incubator or at room
freeze-dried bacteria may temperature. Use before
also be used. 72 hours for best results.
Reaction tube colorimetric test
Single Wavelength
λ
Zero
6 0 3
OK
1. Press 2. Blank Preparation: 3. Wipe the blank and 4. ZERO the instrument.
Single Wavelength. Fill an empty reaction tube insert it into the cell holder. The display will show:
Press OPTIONS and the λ 0.000 Abs
button.
Enter 603 nm and press
OK.
required ( Instrument-
Toxicity
Page 1186
Toxicity
Reaction tube colorimetric test (continued)
5. Label a reaction tube 6. For each sample or 7. Add 5.0 mL of 8. Add 5.0 mL of sample
“control”. Open one dilution, label a reaction deionized water to the (or dilution) to each
ToxTrak Reagent Powder tube with the sample control tube. sample tube.
Pillow and add the number. Use deionized water that To find the approximate
contents to the empty Open one ToxTrak is free of toxicity or threshold level of toxicity
tube. Reagent Powder Pillow another water source that for a sample, see No
and add the contents to represents baseline observed effect
each empty sample tube. toxicity. concentration (NOEC).
9. Add two drops of 10. Add 0.5 mL of 11. Insert the control in 12. Repeat step 11 for all
Accelerator Solution to inoculum (previously the cell holder. Record the samples and dilutions. Be
each vial. Cap and shake prepared) to each tube. absorbance. sure to record each
to mix. Cap and invert to mix. absorbance.
Shaking fully oxygenates
the samples and assures
that the oxygen
concentration is not a
factor in determining the
respiration rate.
Toxicity
Page 1187
Toxicity
Reaction tube colorimetric test (continued)
Zero Read
13. Allow the solutions in 14. After the absorbance 15. ZERO the instrument. 16. Insert the control into
the tubes to react until the of the control has The display will show: the cell holder.
absorbance of the control decreased by 0.60 (± 0.10) READ the results. The
has decreased by Abs. Remove the control 0.000 Abs
display will give an
0.60 (± 0.10) Abs. This and insert the blank into absorbance reading.
takes 45–75 minutes. the cell holder. Record this value.
Invert occasionally.
The reaction time varies
according to temperature,
age of the culture, bacteria
concentrations, etc.
17. Insert each sample or 18. Calculate the % Inhibition:

dilution into the cell holder ΔA sample
% I = 1 – ⎛⎝ ----------------------⎞⎠ × 100
and READ the results. ΔA control
Record each absorbance where:
value.
ΔA = Initial absorbance value – Final absorbance value
Example:
Absorbance of control: initial = 1.500 abs, final = 0.900 abs; ΔAcontrol = 0.600
Absorbance of sample: initial = 1.700 abs, final = 1.300 abs; ΔAsample = 0.400
% I = 1 – ⎛⎝ ---------------⎞⎠ × 100 = 33% I

0.400
0.600
Interpreting results
The results as percent inhibition (% I) are a relative measurement. They do not represent a true
quantitative measurement of toxic concentration. The percent inhibition does not necessarily
increase in direct proportion to the concentration of toxins.
Results below 10% are not reliable, but can be used to make an estimate of toxicity when the
results are consistent. If a sample shows less than 10% inhibition, repeat the test several times.
Toxicity
Page 1188
Toxicity
Look at the series of data points to find the likelihood of toxicity

(refer to the Interpreting results that are less than 10% inhibition table).
Some toxins will increase respiration and give a negative percent inhibition on this and all other
respiration-based toxicity tests. After repeated testing, samples that always give a percent
inhibition that is more negative than –10% should be considered toxic.
Table 357 Interpreting results that are less than 10% inhibition
Data points: percent inhibition Conclusion
7%, 9%, 5%, 8%, 5% May be slightly toxic
7%, –4%, –5%, 5%, 1% Most likely not toxic
–7%, –9%, –5%, –8%, –5% May be slightly toxic
No observed effect concentration (NOEC)

To determine the minimum inhibition concentration of a toxin:
1. Dilute 1 mL of sample to 10 mL with deionized water.
2. Run the test and find the percent inhibition for the dilution.
3. Dilute 1 mL of the sample dilution from step 1 to 10 mL with deionized water.
4. Run the test and find the percent inhibition for the dilution.
5. Continue to make serial 1:10 dilutions of the sample (1:10, 1:100, 1:1000, etc.) until a level is
reached that gives 0% inhibition in the final calculation.
When 0% inhibition is found, the dilution represents the approximate threshold level of toxicity
for a sample. This is the No Observed Effect Concentration (NOEC).
Lowest observable effect concentration (LOEC)

Due to the many variables involved in the test, the limit of detection is approximately
10% inhibition. This correlates to the Lowest Observable Effect Concentration (LOEC).
Disposal of test cultures

Use one of the following methods to dispose of active bacterial cultures:
• Autoclave used test containers at 121 °C (250 °F) for 15 minutes at 15 pounds of pressure.
Once the containers are sterile, pour the contents down the drain with running water. The
reaction tubes may be washed and re-used.
• Sterilize test containers by using a 1:10 dilution of commercial laundry bleach. Pour the test
container contents and test containers into the bleach solution. Allow 10–15 minutes of contact
time with the bleach solution. Then pour the liquid down the drain and wash the reaction tubes
for reuse.
Summary of method
This method is based on the reduction of resazurin, a redox-active dye, by bacterial respiration.
When it is reduced, resazurin changes color from blue to pink. Toxic substances can inhibit the
rate of resazurin reduction. A chemical accelerant has been added to shorten the reaction time.
The absorbance of the color change is measured at 603 nm.
Toxicity
Page 1189
Toxicity

Required reagents
ToxTrak™ Reagent Set, includes: 1 25/set 2597200
Media Set, Total Bacteria Count Tubes 1 15/pkg 2277700
Pipet, transfer, sterile 1 15/pkg 2232512
Sodium Thiosulfate Standard Solution, 0.0246 N varies 100 mL 2409232
ToxTrak Reagent Powder Pillows 2 50/pkg 2560766
ToxTrak Accelerator Solution 4 drops 15 mL SCDB 2560836
Tubes, 16 x 100 mm (sold as 6/pkg) 1 30 tubes 2275806
Tube caps for 22758-00 (sold as 6/pkg) 1 30 caps 2241106
Required apparatus
Clippers each 93600
Dropper, 0.5 and 1.0 mL marks 20/pkg 2124720
Forceps, flat square tip each 1453700
Incubator, Dri-Bath, 12 well, 120 VAC each 2281400


Rack, test tube each 1864100
BOD seed (polyseed) 50/pkg 2918700
Laboratory pen, permanent marker each 2092000

TPH, 10050
TPH (Total Petroleum Hydrocarbons)

DOC316.53.01142
Immunoassay1 Method 10050
Scope and Application: For soil and water
Test preparation

Instrument Adapter
DR 6000 —
DR 5000 A23618
DR 3900 LZV846 (A)
DR 3800, DR 2800, DR 2700 LZV583
The TPH test can be used for both soil and water testing. When testing soil, start with the Soil Extraction Procedure. When
testing water samples only, start with the Immunoassay Procedure for Soil Extracts and Water Samples. The test requires
about 20 to 30 minutes for complete analysis. As many as 10 cuvettes can be run simultaneously.
Read the entire procedure before starting. Identify and make ready all the necessary reagents, cuvettes and other
Store the reagents at 4 °C when they are not in use. Allow the reagents to reach room temperature before using them in an
analysis. Actual testing may be done at temperatures ranging from 1– 38 °C.
The Soil Extractant contains methyl alcohol which is poisonous and flammable. Before using this and other reagents, read
the Material Safety Data Sheet (MSDS) for proper use of protective equipment and other safety information.

Page 1191
TPH Reagent Set 1
Caps, flip spout 1
Wipes, disposable 1
Pipet Tips, for TenSette Pipet 19700-01 1
1. Weigh out 10 g of soil 2. Carefully pour the soil 3. Use the 5-gram scoop 4. Use the graduated
in the plastic weighing into an extraction vial. to add one scoop of cylinder to transfer 10 mL
boat. sodium sulfate to the of Soil Extractant into the
extraction vial. extraction vial.

Page 1192
Soil extraction procedure (continued)
5. Cap the extraction vial 6. Allow to settle for at 7. Using the disposable 8. Insert the filtration
tightly and shake least one minute. Carefully bulb pipet, withdraw 1.0– plunger into the filtration
vigorously for one minute. open the extraction vial. 1.5 mL from the liquid barrel. Place the filtration
layer at the top of the assembly on a table and
extraction vial. press firmly on the plunger
Transfer it into the filtration until the sample extract is
barrel (the bottom part of forced upward into the
the filtering assembly). center of the plunger.
Do not use more than Use the resultant filtrate as

1.5 mL. The bulb is the sample in the
marked in 0.25-mL Immunoassay procedure
increments. for soil extracts and water
samples.
Immunoassay procedure for soil extracts and water samples
Single Wavelength
4 5 0
OK
1. Press 2. Label an Antibody 3. Insert the cuvettes into 4. Pipet 0.5 mL of Diluent
Single Wavelength. Cuvette for each calibrator the rack snugly. Solution into each
and each sample to be Calibrator cuvette.
Press OPTIONS and the λ tested.
button. The same pipette tip can
be used repeatedly for this
Enter 450 nm and press step.
OK.
required (Instrument-

Page 1193
Immunoassay procedure for soil extracts and water samples (continued)
5. If testing soil: Pipet 6. Have the necessary 7. If testing soil: Use a 8. Immediately pipet
0.5 mL of Diluent Solution apparatus at hand for the Wiretrol pipet to transfer 0.5 mL of TPH Enzyme
into each sample cuvette. next four steps as they 50 µL of the filtered extract Conjugate into each
If testing water: Pipet must be done without from step 8 of the Soil calibrator and sample
0.5 mL of each water delay. extraction procedure into cuvette.
sample into each sample Use a Wiretrol® pipet to the appropriately labeled The same pipette tip can
cuvette. Use a new pipette transfer 50 µL of each cuvette. Use a separate be used repeatedly for this
tip for each sample. calibrator to be used into capillary tube for each step.
the calibrator cuvettes. solution.
Mix the contents of the Mix the contents of the

cuvettes after each cuvettes after the addition
addition. Use a separate of each sample.
capillary tube for each If testing water: Use a
solution. Wiretrol pipet to transfer
50 µL of methanol into
each sample cuvette.
9. Start the instrument 10. After 5 minutes, mix 11. At the end of the 12. Wash each cuvette
timer for 10 minutes. the contents of the rack a 10-minute period, discard forcefully and thoroughly
A 10-minute reaction time second time for a period of the contents of all the four times with deionized
will begin. Immediately mix 30 seconds using the cuvettes into an water. Empty the rinse
the contents of the same technique. appropriate waste water into the waste
cuvettes for 30 seconds container. container.
using the technique The simple TPH and Make sure that most of the
described in Using the 1- calibrator TPH will remain water is drained from the
cm MicroCuvette Rack. attached to the cuvette cuvettes. Turn the
walls. cuvettes upside down and
tap them lightly on a paper
towel.

Page 1194
Immunoassay procedure for soil extracts and water samples (continued)

Color Development
13. With the cuvettes still 14. Start the instrument 15. After 5 minutes, mix 16. At the end of the
held snugly in the rack, timer for 10 minutes. the contents of the rack a 10-minute reaction period,
pipet 0.5 mL of Color A 10-minute reaction time second time for a period of pipette 0.5 mL of Stop
Developing Solution into will begin. Immediately mix 30 seconds using the Solution into each cuvette
each Cuvette. the contents of the same mixing technique in the same order that was
Use a new pipette tip for cuvettes for 30 seconds that was used in step 14. used in step 13. The same
each cuvette. using the technique Solutions will turn blue in pipette tip can be used
described in Using the 1- some or all of the cuvettes. repeatedly for this step.
cm MicroCuvette Rack. Mix the contents of the
cuvettes for 20 seconds
using the same mixing
technique as in step 14.
yellow.
Zero
water. Wipe the outside of cell holder. holder.
all the cuvettes with a 0.000 Abs
Refer to Instrument- READ the results. The
tissue to remove water, specific information for cell display will give an
smudges and fingerprints. orientation. absorbance reading.
Record the results for
each calibrator and
sample.
reporting results to find the
relative concentration.

Page 1195
Using the Wiretrol®* Pipet

The Wiretrol Pipet can accurately measure small quantities of liquids. It consists of two parts: a
Teflon®-tipped plunger and a calibrated capillary tube. The plunger can be reused. The capillary
tubes must be discarded after one use.
1. Wet the orange 2. Push the tip to the 3. Submerge the 4. To discharge the pipet,
Teflon® tip of the Wiretrol other end of the capillary capillary tube below the place the tip of the
plunger in the sample and tube until it barely extends surface of the liquid to be capillary tube below the
carefully insert it into the beyond the end of the pipetted. Slowly and surface of the solution
end of the capillary tube capillary tube. smoothly draw the Wiretrol and push the Wiretrol
with the colored band. plunger up until the bottom plunger down in one
of the plunger tips reaches smooth motion. Change
the appropriate volume capillary tubes for each
line. calibrator and sample.
Touch the end of the tube
to the side of the vessel to
release remaining drops
on the capillary tube tip.
* Wiretrol is a registered trademark of Drummond Scientific.

Page 1196

The MicroCuvette rack (Figure 1) has been designed to aid in achieving precise and accurate
results when using the immunoassay technique to analyze several samples at the same time.
Loading the Rack—The cuvette rack is designed so that it may be inverted with the cuvettes in

Page 1197

There is an inverse relationship between the concentration of TPH and the absorbance reading. In
other words, the higher the absorbance reading, the lower the concentration of TPH:
• If sample reading < calibrator reading, TPH concentration in sample > calibrator reading
• If sample reading > calibrator reading, TPH concentration in sample < calibrator reading
Example:
Readings
TPH Calibrator #1: 0.480 Abs
TPH Calibrator #2: 0.360 Abs
Interpretation for a Soil Sample
Sample #1—The sample reading (0.200 Abs) is less than the readings for both calibrators.
The concentration of TPH in the sample is greater than 50 ppm diesel fuel.
Sample #2—The sample reading (0.400 Abs) is between the readings for the TPH calibrators.
The concentration of TPH in the sample is between 20 ppm and 50 ppm diesel fuel.
Sample #3—The sample reading (0.550 Abs) is greater than the readings for both calibrators.
The concentration of TPH in the sample is less than 20 ppm diesel fuel.
Interpretation for a Water Sample
Sample #1—The sample reading (0.200 Abs) is less than the readings for both calibrators.
The concentration of TPH in the sample is greater than 5 ppm diesel fuel.
Sample #2—The sample reading (0.400 Abs) is between the readings for the TPH calibrators.
The concentration of TPH in the sample is between 2 ppm and 5 ppm diesel fuel.
Sample #3—The sample reading (0.550 Abs) is greater than the readings for both calibrators.
The concentration of TPH in the sample is less than 2 ppm diesel fuel.

1. Wear protective gloves and eyewear.
2. When storing reagent sets for extended periods of time, keep them out of direct sunlight. Store
3. Keep the foil pouch containing the Antibody Cuvettes sealed when not in use.
4. If Stop Solution comes in contact with eyes, wash thoroughly for 15 minutes with cold water

Page 1198
Sensitivity
The antibodies used in the TPH Test Kit react with a variety of compounds found in petroleum
fuels; however, each TPH calibrator has been formulated to represent a specific concentration of
diesel fuel. To use the calibrators for other TPH compounds, see TPH compounds in soil for soil or
TPH compounds in water for water to select the proper TPH calibrator for the compound, sample
and range you want to test.
Example:
To use the TPH calibrators for gasoline, find “Gasoline” in the first column of theTPH compounds
in soil table or theTPH compounds in water table. Read across the column to find the ppm
represented by each calibrator. For gasoline, calibrator #1 = 15 ppm, calibrator #2 = 35 ppm, etc.
Table 359 TPH compounds in soil

TPH calibrator #1 TPH calibrator #2 TPH calibrator #3 TPH calibrator #4
Compound
(ppm) (ppm) (ppm) (ppm)
Diesel fuel 20 50 100 200
Gasoline 15 35 70 140
Kerosene 35 75 140 250
Benzene 20 45 85 160
Toluene 15 30 50 90
Ethylbenzene 5 15 35 75
m-Xylene 9 20 35 70
o-Xylene 10 20 40 80
p-Xylene 3 5 9 16
BTEX 5 15 25 45
Table 360 TPH compounds in water

TPH calibrator #1 TPH calibrator #2 TPH calibrator #3 TPH calibrator #4
Compound
(ppm) (ppm) (ppm) (ppm)
Diesel fuel 2 5 10 20
Gasoline 1.5 3.5 7 14
Kerosene 3.5 7.5 14 25
Benzene 2 4.5 8.5 16
Toluene 1.5 3 5 9
Ethylbenzene 0.5 1.5 3.5 7.5
m-Xylene 0.9 2 3.5 7
o-Xylene 1 2 4 8
p-Xylene 0.3 0.5 0.9 16
BTEX 0.5 1.5 2.5 4.5

Page 1199
Diluting water samples

To test for TPH in water at concentrations that are higher than those shown in theTPH compounds
in water table, dilute the sample with deionized water. Add a volume of sample from the Dilution
multipliers table to a graduated cylinder and dilute to 50 mL with deionized water. Run the test.
Multiply the calibrator levels shown in theTPH compounds in water table by the dilution multiplier in
theDilution multipliers table.
Example:
If a 0.5 mL water sample is diluted to 50 mL and tested, the calibrator levels shown in theTPH
compounds in water table for diesel fuel would represent 200, 500, 1000 and 2000 ppm
respectively.
Table 361 Dilution multipliers

mL Sample Dilution multiplier
0.5 100
1.0 50
2.0 25
5.0 10
10.0 5
25.0 2
Interferences
Chlorine in water samples Interferes above 2 ppm. Remove with 1 drop per 100 mL sodium thiosulfate (0.1 N).

• Analyze the samples as soon as possible after collection.
• To store the samples, collect them in glass or Teflon® containers that have been washed with
soap and water and rinsed with methanol. The container should be capped with a Teflon-lined
cap. If a Teflon cap is not available, aluminum foil rinsed in methanol may be used as a
substitute cap liner.
• When collecting water samples, fill the container completely (no head space) and cover the
container with a tightly-sealed lid immediately after collection.
• Soil—Store the samples at 4 °C (40 °F) for no longer than 14 days.
• Water—Chill the sample in an ice bath or refrigerator to limit the loss of volatile compounds.
Store samples no longer than 24 hours.

Page 1200
Summary of method
This method provides semi-quantitative screening for TPH based on thresholds as diesel fuel in
the following concentrations:
• Soil—20, 50, 100, 200 ppm as diesel fuel
• Water—2, 5, 10, 20 ppm as diesel fuel
and soil. Antibodies specific for TPH are attached to the walls of plastic cuvettes. They selectively
bind and remove TPH from complex sample matrices. A prepared sample and a reagent
containing enzyme-conjugate molecules (analyte molecules attached to molecules of an enzyme)
are added to the Antibody Cuvettes. During incubation, enzyme-conjugate molecules and TPH
compete for binding sites on the antibodies. Samples with higher levels of analyte will have more
antibody sites occupied by TPH and fewer antibody sites occupied by the enzyme-conjugate
molecules.
color. Therefore, there is an inverse relationship between color intensity and the amount of TPH in
the sample. The resulting color is then compared with a calibrator to determine whether the TPH
concentration in the sample is greater or less than the threshold levels. The TPH concentration is
inversely proportional to the color development: the lighter the color, the higher the TPH
Required reagents
TPH Reagent Set1 20 cuvettes 2774300

Deionized water 500 mL 27248
Required apparatus

Page 1201
Soil extraction reagents and apparatus

Balance, AccuLab Pocket Pro 250 B each 2796900
Gloves, disposable, Nitrile, medium1 each 2550502
Pipet tips for 1970001 Ten Sette Pipet 50/pkg 2185696
Soil Scoop, 5-g, 4.25-cc 20/pkg 2657205
Soil Extraction Refill Kit, includes: each 2775200
Dropper, LDPE, 0.5 and 1.0-mL 20/pkg 2124720
Filter and Barrel Assembly 20/pkg 2567620
Soil Extractant Solution 200 mL 2567729
Soil Sample Container 20/pkg 2592920
Weighing Boat, 8.9-cm square 20/pkg 2179020
Spatula, disposable 2/pkg 2569320
Sodium Sulfate, anhydrous 250 g 709929

Safety goggles, vented each 2550700
Graduated cylinder, 10 mL each 108138
Pipet, Wiretrol®, 10–50 μL 20/pkg 2852200
Pipet, Wiretrol, 50–1000 μL 250/pkg 2568905
Sodium Thiosulfate standard solution, 0.1 N 100 mL MDB 32332

Trihalomethanes, 10132
Trihalomethanes DOC316.53.01143
THM Plus™ Method Method 10132

(10 to 600 ppb as Chloroform) Water Bath Method
Scope and Application: For screening THMs in drinking water samples and Formation Potential tests.
Test preparation


DR 6000 2427606 and 2495402 Sample cell faces right
DR 5000 2427606 and 2495402 Sample cell faces user
DR 3900 2427606 and 2495402 Sample cell faces user
DR 3800, DR 2800, DR 2700 2427606 and 2495402 Sample cell faces right
Analyze the samples immediately after collection or refrigerate the samples until the analysis is complete.
If the samples were refrigerated after collection, do not warm the samples to room temperature prior to analyzing. This will
minimize volatilization of the disinfection by-products (DBPs). If refrigerated samples are analyzed, heat the samples for an
additional two minutes (total of seven minutes) in step 12 of the procedure.
If analyzing more than four samples, use 450 mL of water in the water bath.
THM Plus Reagent 2 must be at room temperature before use.
A bottle-top dispenser may be used in place of the TenSette® Pipet.
Trihalomethane compounds are extremely volatile. Immediately cap sample cells after filling with sample.
Reagent blank is stable for 1–2 hours and need not be prepared for each test.
Do not mark below the 10 mL fill line.
Trihalomethanes
Page 1203
Trihalomethanes
THM Plus Reagent Set varies
Beaker, 600-mL 1
Cell Holder Assembly, TTHM 1
Evaporating Dish, 125 mm x 65 mm 2
Hot Plate, 7 x 7 inch 1
Pipet, TenSette®, 0.1–1.0 mL and tips 1
Pipet, TenSette®, 1–10 mL and tips 1
Sample Cells, 10-mL (see the Instrument-specific information table) 2
Wipers, disposable varies
Ice1 varies

1 Depending on the temperature of the tap water, ice may be needed for the cooling baths used in steps 14 and 17.
THM Plus Method

Important Note: Perform steps 4–9 rapidly to avoid loss of THMs from the sample. When testing more than one
sample, complete steps 4–9 for one sample before going on to another. If dispensing sample with a pipette, the
pipette must dispense quickly without causing aeration or back pressure.
Stored Programs
725 THM Plus
Start
1. Select the test. 2. Prepare a hot water 3. Prepare a cooling bath 4. Prepared Sample: Fill
Insert an adapter if bath by adding 500 mL of by adding 500 mL of cold one round sample cell to
required (see water to an evaporating (18–25 °C) tap water to a the 10-mL mark with
the Instrument-specific dish. Put the dish on a hot second evaporating dish. sample. Cap and label as
information table). plate and turn the heater Maintain the temperature “sample”.
on high. in this range.
for orientation.
Trihalomethanes
Page 1204
Trihalomethanes
THM Plus Method (continued)
5. Blank Preparation: 6. Add three drops of 7. Cap tightly and mix 8. Use a TenSette® Pipet
Fill the second sample cell THM Plus Reagent 1 to gently by swirling each cell to add 3 mL of THM Plus
with deionized water. Cap each cell. three times. Reagent 2 to each cell.
and label as “blank”. Vigorous shaking can Avoid excess agitation of
cause loss of THMs into the sample when
the sample cell dispensing the reagent.
headspace. The reagent is viscous and
a small amount may
remain on the tip after
dispensing. This will not
affect the results.
9. Cap tightly and mix by 10. Place the sample cells 11. Place the assembly in 12. Start the instrument
shaking. in the cell holder the hot water bath when timer.
Thorough mixing makes assembly. the water is boiling rapidly. A five-minute reaction
sure that all of the THM Do not allow the water to period will begin.
goes into the liquid and rise above the white Heat for 7 minutes if
does not accumulate in the “diamond” near the top of refrigerated samples are
air above the sample. the sample cells. being analyzed.
Trihalomethanes
Page 1205
Trihalomethanes
13. When the timer 14. Start the instrument 15. Use a TenSette Pipet 16. Replace the cooling
expires, remove the timer. to add 1 mL of THM Plus water with fresh, cold tap
assembly and sample A three-minute cooling Reagent 3 to each cell. water. Place the assembly
cells from the hot water period will begin. The sample and blank will that contains the sample
bath. Place in the cooling become warm. and blank cells into the
bath. When the timer expires, cooling bath.
remove the cells from the
Use ice to cool the tap cooling bath. Use ice to cool the tap
water if necessary. water if necessary.
Invert each cell a few
times to make sure that a
uniform temperature of the
sample is maintained.
17. Start the instrument 18. Add the contents of 19. Cap each cell tightly 20. Start the instrument
timer. one THM Plus Reagent 4 and mix by shaking until all timer.
A three-minute cooling Powder Pillow to the the powder dissolves. A 15-minute development
period will begin. sample cell and one to the The powder dissolves time will begin.
blank. slowly. Intermittent
When the timer expires, The color is stable for at
remove the cells from the shaking during the first five least 30 minutes after the
cooling bath. minutes of the color 15-minute development
development period will time.
The temperature of the help dissolve the reagent
sample should be powder.
15–25 °C.
Trihalomethanes
Page 1206
Trihalomethanes
Zero Read
21. After the timer expires, 22. When the timer 23. ZERO the instrument. 24. Wipe the prepared
pour the prepared sample expires, wipe the blank The display will show: sample and insert it into
and prepared blank into and insert it into the cell the cell holder.
two square sample cells. holder. 0 ppb CHCl3
READ the results in ppb
Allow the solution to settle chloroform (CHCl3).
in the square cells for 30
seconds to enable any
turbidity that may be
present to settle.
Interferences
Table 364 Interfering substances1
Chlorine 10 mg/L
Copper 1000 mg/L
1000 mg/L as CaCO3
Hardness, Ca
May have some turbidity until Reagent 3 is added
4000 mg/L as CaCO3
Hardness, Mg
May have some turbidity until Reagent 3 is added
Iron 10 mg/L
Lead 2 mg/L
Mercury 10 mg/L
Monochloramine 20 mg/L
Nickel 10 mg/L
Sodium Bisulfite 100 mg/L
EDTA Interferes negatively at all levels
1 The substances in the Interfering substances table have been tested and found to cause no interference up to the indicated levels.
Table 365 Additional disinfection by-products (DBPs) that are included in results
Compound Effect
1,1,1-trichloro-2-propanone Interferes positively
1,1,1-tricholoacetonitrile Interferes positively
Chloral hydrate Interferes positively
Dibromochloroacetic acid Interferes positively
Trihalomethanes
Page 1207
Trihalomethanes
Table 365 Additional disinfection by-products (DBPs) that are included in results
Compound Effect
Dichlorobromoacetic acid Interferes positively
Tribromoacetic acid Interferes positively
Trichloroacetic acid Interferes positively

• Collect samples in 40-mL glass bottles sealed with Teflon®-lined septa caps.
• Fill the bottles slowly to overflowing so that no air is included with the sample.
• Seal the bottles tightly and invert to check that no air has been trapped.
• Because trihalomethane compounds (THMs) are extremely volatile, immediate analysis yields
the greatest accuracy. If the samples cannot be analyzed immediately, cool samples to 4 °C.
This will slow the formation of any additional THM compounds in chlorinated samples.
• Store the samples at 4 °C in an atmosphere free of organic vapors. Samples should not be
held more than 14 days. 0.1 N Sodium Thiosulfate can be used to dechlorinate samples for
longer storage.
• Add 1 drop of 0.1 N Sodium Thiosulfate to dechlorinate a finished or distribution system
sample collected in a 125 mL bottle.
Trihalomethanes
Page 1208
Trihalomethanes
Accuracy check
• THM Standard Ampule, 10 mg/L as chloroform
• Ampule breaker
• Wiretrol™ Pipet
Note: Make sure that the chloroform is not lost to volatilization when attempting to add the chloroform to
the solution. Make sure that the chloroform ampule is kept cold (can use a small ice-bath).
1. Open a THM Standard Ampule, 10 ppm as chloroform.
2. Use a Wiretrol Pipet to transfer 0.100 mL (100 µL) of the chloroform standard into a fresh
10 mL portion of sample.
3. Immerse the end of the pipet tip under the water and slowly dispense the chloroform.
4. Cap the sample cell immediately and swirl three times to mix.
Note: The accuracy check methods require careful attention to technique, for it is very easy to lose the
chloroform to volatilization when attempting to add it to the solution. Make sure the chloroform ampule is
kept cold (may wish to use a small ice-bath)
5. Immediately start steps 6–24 of the procedure to analyze the spiked sample.
6. The value of the spiked sample should increase 100 +/- 20 ppb over the value obtained on the
original unspiked sample.
7. Calculate the % Recovery:
ppb THMs Spiked Sample – ppb THMs Unspiked Sample

% Recovery = -------------------------------------------------------------------------------------------------------------------------------------------------------- × 100
100 ppb THM Added
1. Prepare a 99 ppb chloroform standard by pipetting 10.0 mL of organic-free water into a sample
cell. Open a THM Standard Ampule, 10 ppm as chloroform. Use a Wiretrol Pipet to transfer
0.100 mL (100 µL) of the chloroform standard into the organic-free water. When adding the
standard into the sample, discharge the pipet slowly at or near the bottom of the sample cell
with a slight swirling motion.
Note: If the aliquot of the standard is discharged too quickly, the solution will form a single bubble which
will rise to the top of the solution and volatilize, without being absorbed in the solution.
2. Cap the sample cell immediately and swirl three times to mix.
3. Immediately start steps 6–24 of the procedure. Do not make up the standard in advance. Use
the standard immediately upon preparation.
4. To adjust the calibration curve using the reading obtained with the 99 ppb Standard Solution,
navigate to Standard Adjust in the software: OPTIONS>MORE>STANDARD ADJUST
Trihalomethanes
Page 1209
Trihalomethanes
Method performance
725 66 ppb CHCl3 53–79 ppb CHCl3 19 ppb CHCl3
Summary of method
The THM Plus method reacts with the trihalogenated disinfection by-products formed as the result
of the disinfection of drinking water with chlorine in the presence of naturally occurring organic
materials. These disinfection by-products (DBPs) may be produced in the treatment plant or the
distribution system as long as the water is in contact with free chlorine residual. The formation of
the DBPs is influenced by chlorine contact time, chlorine dose and residual, temperature, pH,
precursor concentration, and bromide concentration.
The predominant DBPs formed by the chlorination of drinking water are the trihalomethanes or
THMs. The four trihalogenated compounds that form are chloroform, bromoform,
dichlorobromomethane, and dibromochloromethane. These four compounds comprise the Total
Trihalomethanes (TTHMs) group which is regulated under the Safe Drinking Water Act. The
combined concentration of the TTHMs, is regulated in drinking water samples. Other DBPs that
may be present and react under the conditions of the THM Plus method are listed in Interferences.
In the THM Plus method, THM compounds present in the sample react with
N, N,-diethylnicotinamide under heated alkaline conditions to form a dialdehyde intermediate. The
sample is then cooled and acidified to pH 2.5. The dialdehyde intermediate formed is then reacted
with 7-amino-1,3 napthalene disulfonic acid to form a colored Schiff base. The color formed is
directly proportional to the total amount of THM compounds present in the sample. Test results are
measured at 515 nm and reported as ppb chloroform.

Required reagents
Reagent Set (50 tests1), includes: 2790800

THM Plus™ Reagent 1 6 drops 15 mL 2753929
THM Plus™ Reagent 2 6 mL 330 mL 2754048
THM Plus™ Reagent 3 2 mL 110 mL 2754142
THM Plus™ Reagent 4 2 pillows 100 pillows 2756699
1 Fifty tests equals 25 samples and 25 individual blanks. Additional tests can be obtained when multiple samples are run using a single blank.
Required apparatus
Beaker, 600-mL 1 each 50052
Cell Holder Assembly, TTHM 1 each 4788000
Evaporating Dish, 125 mm x 65 mm 2 each 2764700
Hot Plate, 7 x 7 in., 115 VAC, digital 1 each 2881600
Hot Plate, 7 x 7 in., 230 VAC, digital 1 each 2881602
Trihalomethanes
Page 1210
Trihalomethanes
Required apparatus
Pipet Tips for TenSette Pipet 19700-01 varies 50/pkg 2185696
Pipet, TenSette®, 1–10 mL 1 each 1970010
Wipers, disposable varies 280/pkg 2097000
Chloroform, 10-ppm ampule 7/pkg 2756707
Water, Reagent, Organic-free 500 mL 2641549

Pipet, filler, safety bulb each 1465100
Pipet, volumetric, class A, 10 mL each 1451538
Pipettes, Wiretrol™, 50–100 µL 250/pkg 2568905
Vials, glass, 40-mL, with Septa cap 5/pkg 2794005
Sodium thiosulfate standard solution, 0.1 N 100 mL 323-32
Trihalomethanes
Page 1211
Trihalomethane Formation Potential
Trihalomethane Formation
Potential (THMFP) DOC316.53.01147
Method 102241
THM Plus
Scope and Application: For determining the potential of potable source waters to form trihalomethanes and other
disinfection by-products when under the influence of direct chlorination. For evaluating water treatment processes,
water sources or for predicting THM concentrations in a distribution system. For running Simulated Distribution
System Trihalomethanes (SDS-THM) studies.
1 Adapted from Standard Methods for the Examination of Water and Wastewater, Section 5710
Test preparation
Develop a formation potential test plan. See Trihalomethane formation potential test plan on page 1216.
Precondition sample containers, test bottles and glassware to be chlorine demand free. See Treating analysis labware on
page 1217.
Bring sample to the temperature prescribed in the formation potential plan before preparing the samples.
DPD Free Chlorine Reagent Varies
Chlorine Dosing Solution Ampules Varies
THM Plus Reagent Varies
Sample Bottles/Caps 6
Bottle Labels 6
pH Meter 1
Thermometer 1
Pipet, TenSette®, 0.1 to 1.0 mL and tips 1
Sample Cells, 10 mL, with caps 2
Stirrer/Hotplate 1
Stir Bar Magnet 1
Cell Holder Assembly and Evaporating Dish (water bath) 1
Spectrophotometer 1
Trihalomethane Formation Potential (THMFP)

Page 1213
THM Plus
1. Complete a 2. Prepare six chlorine 3. Do not handle the 4. Open a Chlorine

Trihalomethane formation demand-free bottles. stir bar with fingers. Dosing Solution Ampule.
potential test plan Rinse each bottle with Use tweezers or tongs to Using a TenSette Pipet,
(page 1216). sample and fill each insert a stir bar magnet add 0.2 mL of the chlorine
Measure and record the 118-mL bottle (bottles into each bottle. Set Bottle solution to Bottle #1 while
temperature and pH of the contain 125 mL when filled #1 on a stir plate and stir stirring. Immerse the end
sample water to be tested. to overflowing) with gently. A small vortex of the pipet tip under the
approximately 100 mL of should be visible on the water to dispense the
If the test plan temperature the sample to be tested. chlorine.
is different from the surface of the liquid.
Label the bottles 1 Mixing while adding the
collected sample, through 6.
condition the sample to the chlorine is imperative to
test plan temperature If the study is run with avoid highly localized
before continuing the test. fixed pH, add the pH buffer areas of chlorine
to the sample before filling concentration.
the bottles.
Repeat steps 4–5
5. Turn off the stirrer and 6. Calculate the actual 7. Repeat Steps 4–5 for 8. Incubate or refrigerate
fill the bottle until it is amount of chlorine added bottles 2 through 6. the bottles at the
overflowing with sample. (Equation 1 on page Increase the amount of temperature and contact
Cap in a manner to avoid 1215). chlorine added in time specified in the
trapping any air bubbles increments of 0.2 mL test plan.
and invert to mix. Put the (Table 366).
sample bottle in the dark The amounts added may
or wrap with foil. be increased or decreased
Each 0.2 mL of Dosing based on the expected
Solution added will add chlorine demand of the
approximately 2.0 mg/L sample water and chlorine
Cl2 to the sample. contact time.

Page 1214
THM Plus
Method 8021
or Method 10132
Method 10069
9. After the prescribed 10. Select the sample

chlorine contact time is bottle or bottles having the
completed, analyze the prescribed chlorine
samples for residual Free residual from the test plan
Chlorine using DPD Free and analyze for THMs
Chlorine Reagent (Method using the
8021 or Method 10069). THM Plus method
Follow the chlorine (Method 10132)1, 2.
procedure supplied with Follow the procedure
the spectrophotometer supplied with the
being used. spectrophotometer being
used. Results are µg/L
(ppb) chloroform.
1 Recheck the pH of the sample remaining in the analyzed bottle to check for pH shifts that may occur in low alkalinity waters.
2 The selected bottle or bottles to be analyzed for THMs should be analyzed as soon as possible, especially bottles that have had sample
removed when testing for chlorine residual. If this is not possible, add 1 drop of 0.1 N sodium thiosulfate and store the samples at 4 °C for up to
14 days.
Chlorine addition calculation

Use Equation 1 to calculate the concentration of the chlorine added in step 6.
Equation 1
0.2 mL 〈 volume of standard added〉 × ampule certificate value 〈 mg/L Cl 2〉
mg/L Cl 2 = -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
-
125 mL
Example:
0.2 mL × certificate value of 1250 mg/L Cl
mg/L Cl 2 = ----------------------------------------------------------------------------------------------------------------2-
125 mL
mg/L Chlorine = 2.0

Page 1215
Incremental reagent addition

Table 366 defines the incremental addition of chlorine dosing solution.
Table 366 Incremental addition of a 1250 mg/L Cl2 dosing solution

Bottle # Cl2 dosing solution added (mL) Increases sample concentration (in mg/L Cl2) by
1 0.2 2.0
2 0.4 4.0
3 0.6 6.0
4 0.8 8.0
5 1.0 10.0
6 1.2 12.0
Trihalomethane formation potential test plan

Formation potential tests are run to determine the potential of a water to form THMs or other
disinfection by-products under a standard or a user-specified set of reaction conditions. The goals
of the test plan should be well defined and the reaction conditions clearly documented to support
the goals of the plan.
Formation potential plans are often structured to have a fixed sample pH, sample temperature,
chlorine dose, chlorine contact time and a desired chlorine residual after the contact time.
Formation plans with fixed pH, temperature, chlorine concentration and chlorine contact times are
designed to compare the variability of various waters while holding the variables responsible for
THM formation constant. This allows the evaluation of different source waters and allows the
results to be duplicated at different utilities or by different analysts under similar reaction
conditions. Some standard or fixed reaction condition plans available in the published literature or
in Standard Methods are listed here as examples.
Reaction conditions such as the standard 7-day reaction (incubation) period at pH 7.0 ± 0.2 with
phosphate buffer at 25 ± 2 °C with a remaining chlorine residual between 3.0 and 5.0 mg/L are not
intended to simulate water treatment processes. These conditions are most useful for estimating
the concentration of THM precursors in the raw water or for measuring the effectiveness of a
treatment change under fixed standard conditions. A second standard method using a 3-day
reaction time has also been used. Additionally, a Uniform Formation Conditions (UFC) test is
available that uses a contact time of 24 ± 1 hour at 20.0 ± 1.0 °C, a borate buffer at pH 8.0 ± 0.2
with a target free chlorine residual of 1.0 ± 0.4 mg/L after 24 hours as the sample bottle to be
measured for THMs. The Formation Potential Plan can also be constructed to develop a
Simulated Distribution System Trihalomethanes (SDS-THM) study. The goal of this plan is to use
the existing treated water pH with variable chlorine doses and contact times that simulate the
chlorine levels and water age found in a distribution system. This will estimate the THM formation
occurring in the distribution system.
Another important use of Formation Potential Test plans is to use a single water source and vary
temperature, pH or chlorine contact times to meet specific treatment optimization goals. It is again
important to document all reaction conditions used in the test plan.
Examples of additional formation potential studies are:
• Formation potential studies on settled waters from jar tests to evaluate the effectiveness of
coagulant dosage on removing organics responsible for THM formation.
• Formation potential studies based on chlorine dosage rates and location of
chlorine application.
• Formation potential studies on the rate of THM formation at variable locations within the
treatment stream.

Page 1216
• Formation potential studies to correlate THM levels to UV-254, TOC or SUVA values.
• Formation potential studies to study the effects of using alternative oxidants.
Formation potential procedure modification

Use the following guidelines whenever modifications are made:
• Make smaller chlorine concentration additions by using a larger sample size or smaller
chlorine additions. A 237-mL bottle (contains 250 mL when filled to overflowing) is available for
low chlorine demand applications. Each 0.1 mL of chlorine standard added will add
approximately 0.5 mg/L of chlorine. Substitute 250 mL for 125 mL in Equation 1. A lower
concentration Chlorine Standard Solution, 50–75 mg/L as Cl2 is available for testing low
organic waters.
• High organic waters require larger additions of chlorine. Use 0.5 mL, 1.0 mL, 1.5 mL, etc., to
spike the bottles in steps 4 and 7 in the test procedure.
• Wrap sample bottles made of clear colorless glass in foil to protect the sample from light, or be
kept in the dark during the contact time.
• Sample pH can be modified or standardized by adding a fixed amount of a pH buffer solution
to each bottle. Prepare a reagent blank bottle using organic free water. Add the same amount
of buffer to this blank and carry the blank through the procedure. This will check the formation
potential (if any) that was added by the buffer. Subtract the formation potential of the blank
from the sample THM values.

Collect samples in glass bottles sealed with TFE-lined screw caps. Use only freshly collected
samples and process immediately. If this is not possible, store samples a 4 °C and analyze as
soon as possible. Significant sample degradation can occur in unpreserved samples within
24 hours. Add one drop of thiosulfate solution per 125 mL sample bottle if the samples are
chlorinated and cannot be analyzed immediately.

Glassware used in this test must be chlorine demand-free. Treat all glassware with a dilute
solution of chlorine bleach prepared by adding 0.5 mL of commercial bleach to 1 liter of water.
Alternatively, the sample bottles may be treated by adding 2.0 mL of the Chlorine Dosing Solution
to each 125-mL bottle and filling to overflowing with deionized water. Soak glassware in this
solution for at least one hour. After soaking, rinse the glassware with copious amounts of chlorine
demand-free water before filling with sample.
Summary of method
Organic matter present in drinking water source waters reacts with chlorine to form chlorinated
organic species, some of which may be trihalomethanes or other regulated disinfection
by-products. The potential of various source or treated waters to form disinfection by-products can
be determined by adding chlorine and controlling dosage rate, pH, temperature and contact time.
The THMs formed under these user-defined conditions are determined using the
THM Plus method.

Page 1217
Required reagents
Chlorine Dosing Solution Ampules, 1190–1310 mg/L as Cl2, 10-mL ampules, 16/pkg 2504810
Chloroform, 10 ppm ampule, 7/pkg 2756707
THM Plus Reagent Set 2790800
Water, Organic Free, 500 mL 2641549
Required apparatus
Caps, Black, PP Teflon liner, 12/pkg 2401812
Cell Holder assembly, TTHM 4788000
Evaporating Dish, 125 mm x 65 mm 2764700
Hot Plate/Stirrer, 7 x 7 inch, 110 V 2881600
Sample cells, 10 mL w/caps 2427606
Stir Bar, Teflon-coated, 2.22 x 0.48 cm 4531500
Ampule Breaker, for Voluette® Ampules 2196800
Buffer Solution, pH 7.0, Demand Free, 500 mL 2155353
Chlorine Standard Solution Ampule, 50–75 mg/L as Cl2, 10 mL, 16/pkg 1426810
DPD Free Chlorine AccuVacs®, 25/pkg 2502025
DPD Free Chlorine Swiftest Dispenser w/reagents 2802300
Free Chlorine, Swiftest Dispenser Reagent (refill) 2105560

Page 1218
Incubator, Model 205, 110 V, 0 to 40 °C 2616200
Labels, PolyPaper, 1.5 x 3 " 120/pkg 2091502
Pipettes, Wiretrol, 50–100 µL 2568905
Sension 2 Portable pH/ISE Meter w/electrode 5172510
Sodium Hydroxide Standard Solution, 0.100N, 500 mL 19153
Sodium Thiosulfate Solution, 0.1 N, 100 mL 32332
Standard Methods Handbook 2270800
Sulfuric Acid Standard Solution, 0.100 N, 500 mL 20253
Thermometer, Double Scale, –20 to 110 °C (0 to 230 °F) 2095911
TenSette Pipet, 0.1 to 1.0 mL 1970001
Tips for TenSette Pipet, 0.1 to 1.0 mL, 50/pkg 2185696
Tweezers 1428200

Page 1219
Volatile acids, 8196
Volatile Acids DOC316.53.01144
Esterification Method1 Method 8196

(27 to 2800 mg/L) Reagent solution
Scope and Application: For digestor sludges.
1 Adapted from The Analyst, 87, 949 (1962).
Test preparation

DR 6000 2401906 and 2495402 Fill line faces right

DR 5000 2401906 and 2495402 Fill line faces user
DR 3900 2401906 and 2495402 Fill line faces user
DR 3800, DR 2800, DR 2700 2401906 and 2495402 Fill line faces right

Centrifuge 1
Centrifuge Tubes and Caps 2
Ethylene Glycol 3 mL
Ferric Chloride-Sulfuric Acid Solution 20 mL
Funnel and Filter Paper —
Hot Plate 1
Hydroxylamine Hydrochloride Solution, 100-g/L 1 mL
Pipet Filler 1
Pipet, 2 mL1 1
Pipet, volumetric, Class A, 0.50-mL1 1
Pipet, volumetric, Class A,10-mL1 1

Sample Cells, 10-20-25 mL 2
Sodium Hydroxide Standard Solution, 4.5 N 4 mL
Sulfuric Acid Standard Solution, 19.2 N 0.4 mL
Water Bath and Rack 1
Water, deionized 20.5 mL
1 The TenSette Pipet can be used in place of individual pipets in this procedure.
Volatile Acids
Page 1221
Volatile Acids
Esterification Method
Stored Programs
770 Volatile Acids
Start
1. Select the test. 2. Blank Preparation: 3. Filter or centrifuge 4. Prepared Sample:

Insert an adapter if Pipet 0.5 mL of deionized 10 mL of sample. Pipet 0.5 mL of the filtrate
required (Instrument- water into a dry 25-mL Centrifuging is faster than or supernatant into a
specific information). sample cell. filtration. second dry 25-mL sample
cell.
for orientation.
5. Pipet 1.5 mL of 6. Pipet 0.2 mL of 19.2 N 7. Insert both cells into a 8. Start the instrument
ethylene glycol into each Sulfuric Acid Standard boiling water bath. timer.
sample cell. Swirl to mix. Solution into each cell. Alternatively, the cells may A three-minute reaction
Swirl to mix. be boiled in a 500-mL period will begin.
beaker.
9. When the timer 10. Using a pipet filler, 11. Using a pipet filler, 12. Add 10 mL of Ferric
expires, cool the solutions pipet 0.5 mL of pipet 2.0 mL of 4.5 N Chloride Sulfuric Acid
to 25 °C (until the cell feels Hydroxylamine Sodium Hydroxide Solution to each cell. Swirl
cold) with cool water bath. Hydrochloride Solution Standard Solution into to mix.
into each cell. Swirl to mix. each cell. Swirl to mix.
Volatile Acids
Page 1222
Volatile Acids
Esterification Method (continued)
13. Add 10 mL of 14. Transfer 10 mL of the 15. Transfer 10-mL of the 16. Immediately start the
deionized water to each blank solution from the sample solution from the instrument timer.
cell. Cap and invert to mix. round 25-mL cell to a round 25-mL cell to a Another three-minute
clean dry square sample clean dry square sample reaction period will begin.
cell. cell. During this time, complete
steps 17 and 18.
Zero Read
17. Blot each sample cell 18. ZERO the instrument. 19. Wipe the prepared 20. READ the results in
dry. Immediately insert the The display will show: sample and insert it into mg/L HOAC.
blank into the cell holder. the cell holder.
0 mg/L HOAC

• Analyze as soon as possible after collection.
• Samples can be stored for up to 24 hours by cooling to 4 °C (40 °F) or below.
Accuracy check
• Volatile Acid Voluette® Ampule Standard, 62,500-mg/L as acetic acid
• Ampule breaker
• TenSette Pipets and tips
• Three 25-mL mixing cylinders
Volatile Acids
Page 1223
Volatile Acids

ADDITIONS. Default values for standard concentration, sample volume, and spike volumes
can be accepted or edited. After values are accepted, the unspiked sample reading will
appear in the top row. See the user manual for more information.


1. Prepare a 500-mg/L volatile acid standard solution as follows. Pipet 4.00 mL of Volatile Acid
Standard Solution, 62,500-mg/L, into a 500-mL volumetric flask. Dilute to the mark with
deionized water. Prepare this solution daily.
2. Use the 500-mg/L Volatile Acid Standard Solution in place of the sample. Follow the volatile
acid test procedure.
3. To adjust the calibration curve using the reading obtained with the 500-mg/L standard solution,
navigate to Standard Adjust in the software OPTIONS>MORE>STANDARD ADJUST.
Method performance
770 1350 mg/L as acetic acid 1218–1482 mg/L HOAC 27 mg/L as HOAC
(HOAC)
Summary of method
The volatile acids test is designed specifically for determining volatile acids in digestor sludges.
The method is based on esterification of the carboxylic acids present in the sample and
subsequent determination of the esters by the ferric hydroxamate reaction. All volatile acids
present are reported as their equivalent mg/L as acetic acid. Test results are measured at 495 nm.
Volatile Acids
Page 1224
Volatile Acids

Required reagents
Volatile Acid Reagent Set (90 tests), includes: 2244700
(1) Ethylene Glycol 3 mL 1000 mL 203953
(2) Ferric Chloride-Sulfuric Acid Solution 20 mL 1000 mL 204253
(1) Hydroxylamine Hydrochloride Solution, 100-g/L 1 mL 100 mL 81842
(1) Sodium Hydroxide Standard Solution, 4.5 N 4 mL 1000 mL 204053
(1) Sulfuric Acid Standard Solution, 19.2 N 0.4 mL 1000 mL 203832
Water, deionized 20.5 mL 4L 27256
Required apparatus
Centrifuge, 115 VAC, 60 Hz. 1 each 2676500
Centrifuge Tubes, 15-mL 2 10/pkg 2278739
Centrifuge Tube Caps 2 20/pkg 2585220
Filter Paper, folded, 12.5-cm 1 100/pkg 189457
Funnel, poly, 65-mm 1 each 108367
Hot Plate, 7-inch digital, 120 VAC 1 each 2881500
Hot Plate, 7-inch digital, 240 VAC 1 each 2881502
Cell holder assembly 1 each 4788000
Evaporating dish, 125 mm x 65 mm 1 each 2764700
Volatile Acids Standard Solution, 10-mL Voluette® Ampule, 62,500-mg/L as HOAC 16/pkg 14270-10

Water Bath and Rack each 195555
Volatile Acids
Page 1225
Volatile Acids


Volatile Acids, DT, 8218
Sodium Hydroxide Method Method 8218

100 to 2400 mg/L CH3COOH Digital Titrator
Scope and Application: For wastewater
Test preparation
Distill the sample according to the Volatile Acids Procedure, Sample Distillation instructions in the General Purpose
Distillation Apparatus Set or use the distillation procedure described in Standard Methods for the Examination of Water and
Wastewater.
The final result has been adjusted to give the correct answer based on a 70% correction factor. For higher recoveries, use
the esterification method.
Volatile Acid Reagent Set 1
Digital Titrator 1
Graduated Cylinder 1
Sodium Hydroxide Method
See the
Volume multipliers
table
1. Collect 150 mL of 2. Insert a clean delivery 3. Turn the delivery knob 4. Select the distillate
distillate. tube into a 0.9274 N to eject air and a few drops volume that corresponds
Sodium Hydroxide titration of titrant. Reset the to the expected volatile
cartridge. counter to zero and wipe acids concentration in
Attach the cartridge to the the tip. acetic acid from the
titrator body. Volume multipliers table.
Volatile Acids
Page 1227
Volatile Acids
Sodium Hydroxide Method (continued)
5. Using a graduated 6. Add the contents of 7. Place the delivery tube 8. Calculate:
cylinder, transfer the one Phenolphthalein tip into the solution and Digits Required x
distillate volume into a Indicator Powder Pillow swirl while titrating with Digits Multiplier =
clean, 250-mL Erlenmeyer and swirl to mix. sodium hydroxide until a mg/L Volatile Acids
flask and dilute to light pink color appears. (as acetic acid, CH3COOH)
approximately the 150-mL Record the number of
mark with deionized water. digits required.

Range (mg/L as CH3COOH) Volume (mL) Digit Multiplier
100–400 150 1
200–800 75 2
600–2400 25 6
Summary of method
A sample acidified with sulfuric acid is distilled and the distillate is then titrated to the
phenolphthalein end point with sodium hydroxide standard.
Volatile Acids
Page 1228
Volatile Acids

Required reagents
Volatile Acid Reagent Set, includes: 2460200
Phenolphthalein Indicator Powder Pillows 100/pkg 94299
Sodium Hydroxide Titration Cartridge, 0.9274 N each 1484201
Required apparatus
Cylinder, Graduated 100 mL, TD White each 50842
Cylinder Graduated, 25 mL TD White each 50840
Delivery Tube, 180° hook 5/pkg 1720500
Volatile Acids
Page 1229
Volatile Acids, BT, 8291

100 to 2400 mg/L as CH3COOH Buret Titration
Test preparation
The sample must be distilled before this test can be started. Follow the distillation procedure that is given in the distillation
manual or the distillation procedure that is given in Standard Methods for the Examination of Water and Wastewater.
This method recovers approximately 65 to 95% of the volatile acids in the sample, but 70% is the accepted correction factor.
The final result has been adjusted to give the correct answer based on a 70% correction factor. For higher recoveries, use
the esterification method.
Phenolphthalein Indicator Powder Pillow 1
Sodium Hydroxide Standard Solution, 0.100 N 1 bottle
Buret titration
See
Table 1
1. Distill the sample and 2. Select a sample 3. Fill a 50-mL buret to 4. Use a graduated
collect 150 mL of distillate. volume from the Range- the zero mark with 0.100 N cylinder to measure the
Fully mix the distillate. specific information. Sodium Hydroxide selected volume of
Standard Solution. distillate. Add the sample
to a 250-mL Erlenmeyer
flask.
Volatile Acids
Page 1231
Volatile Acids
5. If the sample volume 6. Add the contents of 7. Titrate the sample 8. Use the multiplier in
is less than 150 mL, dilute one Phenolphthalein while swirling the flask the Range-specific
to approximately 150 mL Indicator Powder Pillow. until a light pink color information to calculate
with deionized water. Swirl to mix. forms and persists for 30 the concentration:
seconds. mL titrant x 86 x multiplier
= mg/L as CH3COOH
was titrated and 3 mL of
titrant was used to reach
the endpoint. The
concentration is 3 x 86 x 6
= 1550 mg/L CH3COOH

Range (mg/L as CH3COOH) Sample volume (mL) Multiplier
100–400 150 1
200–800 75 2
600–2400 25 6

Collect samples in clean plastic or glass bottles. Analyze the sample as soon as possible after
collection. Samples can be stored for up to 24 hours by cooling to 4 °C (39 °F) or below. Warm to
room temperature before starting the test.
Summary of method
A sample acidified with sulfuric acid is distilled and the distillate titrated to the phenolphthalein end
point with sodium hydroxide. The volume of titrant that is necessary to reach the end point is
proportional to the volatile acids concentration. The results are in mg/L as acetic acid (CH3COOH).
Volatile Acids
Page 1232
Volatile Acids
Required reagents
Required apparatus
Bottles, sampling, poly, 250-mL 1 each 2087076
Volatile Acids
Page 1233
Zinc, 8009
Zinc DOC316.53.01145
USEPA1 Zincon Method2 Method 8009

Scope and Application: For water and wastewater. Digestion is required for a total zinc analysis (see Digestion).
1 USEPA approved for wastewater analyses 3500 Zn B: Federal Register, 45(105) 36166 (May 29, 1980).
Test preparation

The Instrument-specific information table displays information that may vary from instrument to
instrument. Select the spectrophotometer from the instrument column on the left. Read across to
the spectrophotometer.
Powder pillows
Instrument
Sample cell Cell orientation
Use only glass-stoppered mixing cylinders in this procedure.
Wash glassware with 1:1 HCl1 and rinse with deionized water before use.
Use a plastic dropper in step 6 of this procedure. Droppers with rubber bulbs may contaminate the reagent.
ZincoVer® 5 reagent contains potassium cyanide. Cyanide solutions are regulated as hazardous waste by the Federal
RCRA. Cyanide should be collected for disposal as a reactive (D003) waste. Be sure that cyanide solutions are stored in a
caustic solution with pH >11 to prevent release of hydrogen cyanide gas. Refer to the current MSDS for handling and
disposal information.
The Pour-Thru Cell cannot be used with this test.

Cyclohexanone 0.5 mL
ZincoVer 5 Reagent Powder Pillow 1
Zinc
Page 1235
Zinc
Zincon method
Stored Programs
780 Zinc
Start
1. Select the test. 2. Fill a 25-mL graduated 3. Add the contents of 4. Invert several times to
Insert an adapter if mixing cylinder with 20 mL one ZincoVer 5 Reagent dissolve the powder
required (the Instrument- of sample. Powder Pillow to the completely. Inconsistent
specific information table). mixing cylinder. Stopper. readings may result if all
the particles are not
Refer to the user manual dissolved.
for orientation.
The sample should be
orange. If the sample is
brown or blue, the zinc
concentration is too high
or an interfering metal is
present. Dilute the sample
and repeat the test.
5. Blank preparation: 6. Prepared sample: 7. Start the instrument 8. Start the instrument
Pour 10 mL of the solution Use a plastic dropper to timer. A 30-second timer.
into a sample cell. add 0.5 mL of reaction period will begin. A three-minute reaction
cyclohexanone to the During the reaction period, period will begin. During
remaining solution in the stopper the mixing cylinder this reaction period,
mixing cylinder. and vigorously shake the complete step 9.
prepared sample.
The sample will be
reddish-orange, brown, or
blue, depending on the
zinc concentration.
Zinc
Page 1236
Zinc
Zincon method
Zero Read
9. Pour the prepared 10. When the timer 11. ZERO the instrument. 12. Wipe the prepared
sample solution from the expires, wipe the blank The display will show: sample and insert it into
mixing cylinder into a and insert it into the cell the cell holder.
second sample cell. holder. 0.00 mg/L Zn
READ the results in
mg/L Zn.
Interferences
Cadmium Greater than 0.5 mg/L
Iron (ferric) Greater than 7 mg/L
Manganese Greater than 5 mg/L
Organic Material Large amounts may interfere. Pretreat the sample with a mild
digestion.
Highly buffered or extreme sample pH May exceed the buffering capacity of the reagents and
require sample pretreatment. Adjust pH to 4–5.
Samples containing AMP cause a negative interference.
Digest the sample to eliminate this interference (follow the
total phosphorus hot plate digestion, Method 8190).
Amino-tri(methylene phosphonic acid) (AMP) Important Note: Be sure to adjust the pH of the
sample after the digestion to pH 4–5 with sodium
hydroxide before the zinc analysis. Correct the pH level
for volume changes.

Collect samples in acid-cleaned plastic or glass bottles. If prompt analysis is impossible, preserve
the sample by adjusting to pH 2 or less with nitric acid (about 2 mL per liter). Preserved samples
may be stored up to six months at room temperature.
Before analysis, adjust the pH to 4–5 with 5.0 N Sodium Hydroxide. Do not exceed pH 5 as zinc
may precipitate. Correct the test result for volume additions.
Accuracy check
Zinc
Page 1237
Zinc
• Zinc Voluette® Ampule Standard, 25 mg/L Zn

• Ampule breaker
• TenSette Pipet 0.1 - 1.0 mL and tips
• 25-mL mixing cylinders
2. Select OPTIONS>MORE>STANDARD ADDITIONS from the instrument menu.
3. Press OK to accept the default values for standard concentration, sample volume, and spike
volumes. Press EDIT to change these values. After values are accepted, the unspiked sample
reading will appear in the top row.

• 100 mg/L zinc standard solution
• 10.00 mL Class A pipet
1. Prepare a 1.00-mg/L zinc standard solution as follows. Pipet 10.00 mL of Zinc Standard
Solution, 100-mg/L, into a 1000-mL volumetric flask. Dilute to the mark with deionized water.
2. Follow the zinc procedure.
3. To adjust the calibration curve using the reading obtained with the 1.00-mg/L standard
solution, navigate to Standard Adjust in the software (OPTIONS>(MORE)>STANDARD ADJUST).
Digestion
A sample digestion is required before an analysis for total zinc can be started. A digestion will
make sure that all zinc compounds in the sample are in a chemical form that will be measured.
Complete the following steps to digest the sample.
Note: The following procedure is the USEPA mild digestion. See the Water Analysis Guide for more
digestion procedures.
1. If nitric acid has not been added to the sample previously, add 5 mL of concentrated nitric acid
to one liter of sample (use a glass serological pipet and pipet filler). If the sample was acidified
at collection, add 3 mL of nitric acid to one liter of sample.
2. Transfer 100 mL of acidified sample to a 250-mL Erlenmeyer flask.
Zinc
Page 1238
Zinc
3. Add 5 mL of 1:1 hydrochloric acid*.

4. Heat the sample on a hot plate* at 95 °C (203 °F) until 15-20 mL remain. Make sure the
sample does not boil.
5. Filter the cooled sample with 0.45 µm filter to remove any insoluble material.
6. Adjust the pH of the digested sample to pH 4–5 with 5.0 N sodium hydroxide. See Sample
collection, preservation and storage for instructions.
7. Quantitatively transfer the sample to a 100-mL volumetric flask and dilute to the mark with
deionized water.
Method performance
780 1.00 mg/L Zn 0.97–1.03 mg/L Zn 0.013 mg/L Zn
Summary of method
Zinc and other metals in the sample are complexed with cyanide. Adding cyclohexanone causes a
selective release of zinc. The zinc reacts with 2-carboxy-2'-hydroxy-5'-sulfoformazyl benzene
(zincon) indicator to form a blue-colored species. The blue color is masked by the brown color
from the excess indicator. The intensity of the blue color is proportional to the amount of zinc
present. Test results are measured at 620 nm.
Zinc
Page 1239
Zinc
Required reagents
Zinc Reagent Set, 20-mL sample size, includes: — — 2429300
Cyclohexanone 0.5 mL 100 mL MDB 1403332
ZincoVer® 5 Reagent Powder Pillows 1 100/pkg 2106669
Required apparatus
Sample cell, 10 mL, square, matched pair 2 2/pkg 2495402
Zinc Standard Solution, 100-mg/L 100 mL 237842
Zinc Standard Solution, 10-mg/L Voluette® Ampule, 25-mL as Zn 16/pkg 1424610
Zinc Standard Solution, 1000-mg/L 100 mL 1417742

Hot Plate, 120 V each 1206701
Hydrochloric Acid 6.0 N, 1:1 500 mL 88449
Nitric Acid, concentrated, ACS 500 mL 15249
Sodium Hydroxide 5.0 N 50 mL SCDB 245026
Tensette Pipet, 0.1–1.0 each 1970001
Tips for Tensette Pipet 1970001 50/pkg 2185696
Pipet, Filter, Safety bulb each 1465100
Filter paper 0.45 µm 100/pkg 1353000
pH paper test strips, 3.0–5.5 pH range 15’ roll 37333
Filtration apparatus, glass each 234000

Microbiology
Page 1241
Page 1242
Bacteria Test Guidelines
Bacteria Test Guidelines DOC316.53.01188
All tests for bacteria use a nutritional broth or agar and incubation at a specific temperature to grow
the target organism. Sterile equipment and careful handling techniques are necessary to prevent
contamination of the sample.
Methods for bacteria testing

The following descriptions give a general overview of the different methods for bacteria tests.
• Presence/absence (P/A)—the sample is added to a bottle containing broth and incubated. A
color change indicates the presence of the target bacteria.
• Most Probable Number (MPN)—the sample is diluted and added to a series of tubes
containing broth. The tubes are incubated and then examined for the presence of gas.
• Membrane Filtration (MF)—the sample is filtered and the filter is placed on a pad containing
growth media. After incubation, the filter is examined for the growth of the target organism.
• Plate count agar—the sample is mixed with an agar in a large petri dish and incubated. After
incubation, the agar is examined for bacteria colonies. This test is usually used for total or
heterotrophic bacteria.
Presumptive and confirmation tests

Two tests are necessary for most methods, a presumptive test and a confirmation test.
• Presumptive test—uses growth media that facilitates the growth of the target organism. A
positive result is an indication of the target organism but can include a false positive result.
The P/A, MPN and MF methods are presumptive tests.
• Confirmation test—uses media that is more selective for the target organism and sometimes
uses a higher incubation temperature. Some media, such as the m-ColiBlue24® broth used
with the MF method, is selective enough for the target organism (E. coli) that no confirmation
test is required.
Techniques for microbiological tests

Good laboratory technique is essential for microbiological tests. To make sure the results are
reliable, collect and preserve samples carefully. Use high-quality laboratory equipment and ready-
to-use media to save time and minimize errors.
Prepare the work area

• Start the incubator while preparing other materials. Set the incubator to the temperature
required by the specific procedure (usually 35 ± 0.5 °C for total coliforms and enterococci and
44.5 ± 0.2 °C for fecal coliforms).
• Disinfect the work bench with a germicidal cloth, dilute bleach solution, bactericidal spray or
dilute iodine solution. Wash hands thoroughly with soap and water.
• Mark each sample container with the sample number, dilution, date and other necessary
information. Avoid contaminating the inside of the sample container in any way.
• Use presterilized Whirl-Pak® bags or bottles for sample collection. If the sample has been
disinfected, use bags or bottles that contain a dechlorinating agent.

Page 1243
Prepare sample containers

To collect samples, use any of the following: sterilized plastic bags, sterilized disposable bottles,
autoclavable glass or plastic bottles.
Sterilized plastic bags or disposable bottles
Presterilized plastic bags and bottles are available with or without dechlorinating agent. The
bottles are available with a 100-mL fill-to line.
Dechlorinating reagent should be used with potable or chlorinated water samples. It is not
necessary for unchlorinated or non-potable water samples. However, dechlorinating reagent will
not interfere with unchlorinated samples so, for simplicity, plastic bags containing dechlorinating
reagent may be used for all samples.
Autoclavable glass or plastic bottles:
Glass or plastic bottles (125-mL size) may be used instead of sterilized plastic bags or disposable
bottles. These containers should be prepared as follows:
1. Wash in hot water and detergent.
2. Thoroughly rinse with hot tap water, followed by a distilled water rinse to make sure that all
detergent is removed.
3. If dechlorinating agent is needed (for chlorinated, potable water), add the contents of one
Dechlorinating Reagent Powder Pillow for each 125-mL of container volume. (A 250-mL
sample container will require two powder pillows.)
4. Steam sterilize glass and autoclavable plastic containers at 121 °C (250 °F) for 15 minutes.
Glass sample containers may be sterilized by hot air at 170 °C (338° F) for one hour.
5. Store sterile containers, tightly capped, in a clean environment until needed.
Autoclave option for sterilization

Use presterilized laboratory equipment and media to save time and minimize errors. When
numerous samples must be run on a routine basis, sterilization of non-disposable materials with
an autoclave may be preferable.
Procedure
1. Wash sample bottles, pipets, petri dishes, filter holder with stopper and graduated cylinder (if
needed) with hot water and detergent.
2. Rinse several times with tap water and then with demineralized water. Dry thoroughly.
3. Prepare all equipment for the autoclave as follows:
• Loosely thread caps on sample bottles and cover caps and bottle necks with foil or paper.
• Cover the openings of graduated cylinders with foil or paper.
• Insert the filter funnel base into an autoclavable rubber stopper that will fit the filter flask.
• Wrap the two parts of the filter funnel assembly separately in heavy wrapping paper and
seal with masking tape.
• Wrap petri dishes (borosilicate glass) in paper or place in aluminum or stainless cans.
4. Sterilize equipment in an autoclave at 121 °C (250 °F) for 15 minutes. Borosilicate glass items
may be sterilized with dry heat at 170 °C (338 °F) for a minimum of 1 hour.

Page 1244
Collect and preserve samples

Collect a sufficient volume of sample for analysis (usually a minimum of 100 mL of sample). World
Health Organization guidelines prescribe 200 mL per sample while Standard Methods for the
Examination of Water and Wastewater prescribes 100 mL per sample. Avoid sample
contamination.
No dechlorination is necessary if the sample is added directly to the growth medium on site.
Otherwise, treat the samples to destroy chlorine residual. Sodium thiosulfate that has been
sterilized within the collection vessel is used to remove chlorine residual. Transport for analysis
immediately after collection.
Analyze as soon as possible after collection. Allow no more than 6 hours to elapse between
collection and examination for non-potable water samples and 30 hours for potable water
samples. For best results, maintain the sample at or below 10 °C, but do not freeze. Failure to
properly collect and transport samples will cause inaccurate results.
Collect at least 100 mL of sample in a presterilized plastic bag or bottle or in a sterile glass or
plastic bottle. Do not fill sample containers completely. Maintain at least 2.5 cm (approximately one
inch) of air space to allow adequate space for mixing the sample prior to analysis.
Faucets, spigots, hydrants or pumps
Collect representative samples by allowing the water to run from a faucet, hydrant or pump at a
moderate rate for 2 to 3 minutes before sampling. Adjust the flow rate before sample collection to
avoid splashing during collection. Do not adjust the rate of flow while collecting the sample. Avoid
valves, spigots and faucets that swivel or leak or those with attachments such as aerators and
screens or remove the attachments prior to sample collection.
Handle the sample containers carefully. Open the sample containers just prior to collection and
close immediately following collection. Do not lay the lid or cap down. Do not touch the lip or inside
surface of the container. Do not rinse the containers before use. Label the sample containers
immediately and analyze promptly.
Rivers, lakes and reservoirs
When sampling a river, lake or reservoir, do not sample near the edge or bank. Remove the cap,
grasp the sample container near the bottom and plunge the container, mouth down, into the water
in order to exclude any surface scum. Fill the container entirely under water by positioning the
mouth into the current or, in non-flowing water, by tilting the container slightly and allowing it to fill
slowly. Do not rinse the container before use. Label the sample containers immediately and
analyze promptly.
Dilute non-potable samples

Non-potable water samples must be diluted to a level at which the bacteria can be measured.
Procedure
1. Wash hands.
2. Open a bottle of sterile Buffered Dilution Water.
3. Invert the sample container in a Belt to Ear motion, approximately 25 times for 30 seconds.
4. Use a sterile pipet to add 11 mL of sample into the dilution water bottle.
5. Put the cap on the dilution water bottle and invert the sample container in a Belt to Ear motion
25 times for 30 seconds. This is a 10-fold or 10x dilution (sample is diluted by a factor of 10).
6. Add 11 mL of the 10x dilution to another dilution bottle and mix well (100x dilution).
7. Add 11 mL of the 100x dilution to a third bottle and mix well (1000x dilution).
8. Continue to make dilutions until the necessary dilution level has been reached.

Page 1245
Dispose of bacteria cultures

To safely dispose of bacterial cultures left in the broth tubes, use one of the following methods:
Bleach
Sterilize used test containers with household bleach. Add 1–2 mL of the bleach to each test tube.
Allow 10 to 15 minutes contact time with the bleach. Pour the liquid down a drain.
Autoclave
Place used test tubes in a contaminated-items bag or a biohazard bag to prevent leakage into the
autoclave. Autoclave the used test tubes in the unsealed bag at 121 °C for 30 minutes at 15
pounds pressure. When cool, seal the bag, place it in another garbage bag and tie tightly.
The use of indicator organisms in water tests

Many serious diseases, such as typhoid fever and dysentery, can be traced directly to pathogenic
microorganisms in polluted water. These disease-producing organisms are discharged in fecal
wastes and are difficult to detect in water supplies. People may come in contact with these
pathogens in drinking water or in recreational waters such as swimming pools, rivers, streams and
bathing beaches.
Testing directly for bacterial pathogens is impractical for many reasons, not the least of which is
the need for lengthy and involved test procedures. It has become customary to use indicator
organisms such as coliform bacteria instead. Indicator organisms are usually not pathogenic and
are present when pathogens are present and absent when pathogens are absent. Indicator
organisms are usually of fecal origin as well.
No one organism or group of organisms satisfies all of the criteria for an indicator. For example, in
temperate climates total coliform bacteria are commonly used as indicator organisms in potable
water supplies. In many tropical climates, however, indigenous Escherichia coli (E. coli) bacteria
are present in pristine water sources where no fecal contamination exists, yet they will produce
positive results in total coliform tests.
In such cases, other bacteria, known to be associated with fecal contamination, can be used as
indicator organisms in place of the coliforms. Hydrogen sulfide-producing bacteria have been
shown to be associated with the presence of fecal contamination and total coliform bacteria and
they may be used as an indicator organism in place of coliforms.
Total coliform tests are used for potable water supplies. Fecal coliform tests are used on untreated
(non-potable) water, wastewater, bathing water and swimming water.

Membrane Filtration Guidelines
Membrane Filtration Guidelines DOC316.53.01190
The Membrane Filtration (MF) method is used to estimate bacterial populations in water that is low
in turbidity. This method is especially useful for large sample volumes or for many daily tests.
Overview
The following basic steps are necessary for a membrane filtration test.
1. Non-potable water samples are diluted.
2. The sample or dilution is filtered through a membrane filter that retains the bacteria.
3. The filter is put in a petri dish on an absorbent pad that contains a nutritional broth or agar that
is selective for the growth of a specific organism.
4. The petri dish containing the filter and pad is incubated for 24 hours at a specific temperature.
5. After incubation, the colonies that have grown are identified and counted.
Sample collection and sterilization

Refer to Bacteria Test Guidelines for instructions on sample collection and equipment sterilization.

Samples that contain a high level of bacteria must be diluted so that the bacteria that grows on the
filter is at a density that can be measured. Table 372 and Table 373 list recommended sample
volumes for various types of samples.
Select a sample size to give 20 to 200 colony-forming units (CFU) per filter. The ideal sample
volume for non-potable water or wastewater yields 20–80 coliform colonies per filter. For finished,
potable water, the volume to be filtered will be 100 mL.
When the sample volume is less than 20 mL (diluted or undiluted), add 10 mL of sterile dilution
water to the filter funnel before vacuum is applied. The dilution water will help to distribute the
bacteria uniformly across the membrane filter.
Table 372 Sample volume by sample type—total coliform test 1

Sample type 100 mL 50 mL 10 mL 1 mL 0.1 mL 0.01 mL 0.001 mL 0.0001 mL
Drinking water X
Swimming pools X
Wells, springs X X X
Lakes, reservoirs X X X
Water supply intake X X X
Bathing beaches X X X
River water X X X X
Chlorinated sewage X X X
Raw sewage X X X X
1 Standard Methods for the Examination of Water and Wastewater, 19th ed., Table 9222:I, page 9–56.

Page 1247
Table 373 Sample volume by sample type—fecal coliform test1

100 mL 50 mL 10 mL 1 mL 0.1 mL 0.01 mL 0.001 mL
Lakes, reservoirs X X
Wells, springs X X
Water supply intake X X X
Natural bathing waters X X X
Sewage treatment plant, X X X
secondary effluent
Farm ponds, rivers X X X
Storm water run-off X X X
Raw municipal sewage X X X
Feedlot run-off X X X
1 Standard Methods for the Examination of Water and Wastewater, 19th ed., Table 9222:III, pages 9–61.
Sample dilution
Non-potable water samples must be diluted to a level at which the bacteria can be measured. The
ideal sample volume for total coliform testing yields approximately 20 to 80 coliform colonies and
not more than 200 colonies of all types per filter. Ideal sample volumes for fecal coliform testing
yield approximately 20 to 60 coliform colonies per filter. Analyze three different sample volumes
when the coliform number is uncertain.
Procedure
1. Wash hands.
3. Invert the sample container in a waist-to-ear motion, approximately 25 times (for 30 seconds).
5. Put the cap on the dilution water bottle. Invert the bottle in a waist-to-ear motion,
approximately 25 times (for 30 seconds). This is a 10-fold or 10x dilution (sample is diluted by
a factor of 10).
8. Continue to make dilutions until the necessary dilution level has been reached.
Dilution series
d. 10-mL sample: Transfer 11 mL of sample into 99 mL of sterile Buffered Dilution Water.
e. 1-mL sample: Transfer 11 mL of the 10-mL dilution from step d into 99 mL of sterile
Buffered Dilution Water.
f. 0.1-mL sample: Transfer 11 mL of the 1-mL dilution from step e into 99 mL of sterile
g. 0.01-mL sample: Transfer 11 mL of the 0.1-mL dilution from step f into 99 mL of sterile
h. 0.001-mL sample: Transfer 11 mL of the 0.01-mL dilution from step g into 99 mL of sterile

Page 1248
i. 0.0001-mL sample: Transfer 11 mL of the 0.001-mL dilution from step h into 99 mL of

sterile Buffered Dilution Water.
Field filtration apparatus

The field filtration apparatus consists of disposable funnels, a portable funnel base (vacuum
support) and hand pump for convenient filtration in the field. A portable incubator can be used for
incubation or for transport to the laboratory.
1. Flame sterilize the top surface of the stainless steel field vacuum support.
2. Attach the syringe tip to the vacuum support tubing.
3. Using sterile forceps, place a membrane filter, grid side up, on the center of the vacuum
support.
Note: To sterilize forceps, dip forceps in alcohol and flame in an alcohol burner. Cool before use.
4. Remove a funnel (base first) from the package.
5. Place the funnel onto the vacuum support. Do not touch the inside of the funnel. Push evenly
on the funnel’s upper rim to snap it on the vacuum support.
6. Pour the sample into the funnel.
7. Use the hand pump to draw the sample through the filter apparatus.
Note: See specific procedures for the sample volume required.
8. Remove the funnel.
9. Press the lever on the vacuum support stem to lift the membrane filter from the vacuum
support surface.
10. Use sterile forceps to remove the membrane filter.
11. Place the membrane filter into a prepared petri dish and incubate at the specified temperature.
12. Disconnect the syringe tip from the vacuum support tubing. Dispose of the liquid in the
syringe.
13. Follow step 2 through step 12 to filter remaining samples.
Accuracy check
Positive and negative controls are important. Pseudomonas aeruginosa is recommended as a
negative control, and a Escherichia coli is recommended as a positive control for total and fecal
coliforms. Escherichia coli is recommended as a negative control and Enterococcus faecalis is
recommended as a positive control for the enterococci. Escherichia coli is recommended as a
negative control and Pseudomonas aeruginosa is recommended as a positive control for
pseudomonas.
Note: Potable water samples from municipal treatment facilities should be negative for total coliforms and
fecal coliforms.

Page 1249

Report test results as the number of colonies per 100 mL of sample.
Single filter test
Use the following equation to calculate the result from a single membrane filter. Note that
“mL sample” in the equation refers to the actual sample volume and not the diluted volume.
Bacterial colonies counted

Bacterial colonies per 100 mL = ------------------------------------------------------------------------- × 100
mL of sample
• Indistinct colonies—If growth covers the entire filtration area of the membrane or a portion of
it, and colonies are not discrete, report the test results as “Confluent growth with or without
coliforms.”
• High colony density—If the total number of colonies exceeds 200 per membrane or the
colonies are too indistinct for accurate counting, report the results as “Too numerous to count”
(TNTC).
In either case, run a new sample using a dilution that will give about 50 to 200 colonies of all types.
When testing non-potable water, if no filter meets the desired minimum colony count, use the
equation under Multiple filter test to calculate the test result.
Multiple filter test
Use the following equation to calculate the result from multiple membrane filters such as duplicate
samples or multiple dilutions of the same sample.
Sum of colonies in all samples

Bacterial colonies per 100 mL = ----------------------------------------------------------------------------------------------------- × 100
Sum of volumes (in mL) of all samples
Prepared broth and agar

Prepared broth or agar is ready to use and is available in broth ampules or agar plates. The
ampules or agar plates contain enough medium for one test. Prepared media is shipped with a
Certificate of Analysis and has an expiration date printed on the label.
The ampules are available in glass or plastic. Open the ampule and pour the broth on the
absorbent pad in a petri dish. Open the plastic ampules by unscrewing the top of the ampule.
Open the glass ampules with an ampule breaker.
Refer to Prepared media for membrane filtration for a list of prepared media that is available for
microbiological tests.

Page 1250
Table 374 Prepared media for membrane filtration
Media Description Cat. No. Selectivity Incubation Shelf life Approval Citations Sample
2812750 Enterococci 48 hours at 1 year at — Drinking water
35 ± 0.5 °C 2–8 °C Ground water
KF-Streptococcus Broth in plastic ampules
Surface water
Recreational water
Prepared agar plate 2805215 Total Coliform 24 hours at 1 year at 40CFR 141.21 (f) (3) Drinking water
Broth in glass ampules 2608420 and Escherichia 35 ± 0.5 °C 2–8 °C footnote (11) and Beverages
coli Bacteria 40CFR 136.3 Ground water
m-ColiBlue24® Broth in plastic ampules 2608450 Surface water
Broth in 100-mL glass 2608442 Does not apply to Agar Recreational water
bottle plate Wastewater
2811715 Enterococci 24 hours at 3 months at — Drinking water
41 ± 0.5 °C 2–8 °C Ground water
m-EI Prepared agar plate
Surface water
Recreational water
Prepared agar plate 2811615 Total Coliform 24 hours at 1 year at Standard Method 18th
Drinking water
Broth in glass ampules 2373520 Bacteria 35 ± 0.5 °C 2–8 °C 9222 A, B and Federal
Beverages
Register V 68; #139 (7/
m-Endo Broth in plastic ampules 2373550 Ground water
21/2003)
Surface water
Broth in 100 mL glass 2373542
Recreational water
bottle
Prepared agar plate 2811515 Fecal Coliform 24 hours at 1 year at 40CFR 141.21 (f) (5),
Ground water
Broth in glass ampules 2373220 Bacteria 44.5 ± 0.2 °C 2–8 °C Standard Method 18th
Surface water
m-FC 9222 D and Federal
2373250 Recreational water
Broth in plastic ampules Register V 68; #139 (7/
Wastewater
21/2003)
Broth in glass ampules 2428520 Fecal Coliform 24 hours at 1 year at 40CFR 141.21 (f) (6) Ground water
m-FC with 2428550 Bacteria 44.5 ± 0.2 ° 2–8 °C (i) and Standard Surface water
Rosolic Acid Broth in plastic ampules Method 18th 9221 D Recreational water
Wastewater
Broth in glass ampules 2428320 Yeast and Mold 48 hours at 1 year at —
m-Green YM 35 ± 0.5 °C 2–8 °C Beverages
Broth in plastic ampules 2428350
Prepared agar plate 2811415 Heterotrophic 48 hours at 1 year at Standard Method 18th Drinking water
2812450 Bacteria 35 ± 0.5 °C 2–8 °C 9215 A, D Beverages
m-HPC Ground water
Broth in plastic ampules Surface water
Recreational water
Page 1251 of 1254

Table 374 Prepared media for membrane filtration
Media Description Cat. No. Selectivity Incubation Shelf life Approval Citations Sample
Agar Tubes/2 tests per 2561106 Escherichia coli 2 hours at 1 year at Federal Register V 68;
m-TEC
tube 35 ± 0.5 °C 2–8 °C #139 (7/21/2003)
2811815 then Recreational water
m-TEC, modified Prepared agar plate 22 hours at (applies to m-TEC and
Page 1252 of 1254

44.5 ± 0.2 °C Modified m-TEC)
Broth in glass ampules 2373820 Heterotrophic 24 hours at 1 year at — Drinking water
2373850 Bacteria 35 ± 0.5 °C 2–8 °C Beverages
m-TGE Ground water

Recreational water
Broth in glass ampules 2428420 Heterotrophic 24 hours at 1 year at — Drinking water
Bacteria 35 ± 0.5 °C 2–8 °C Beverages
2428450
m-TGE with TTC Ground water
Recreational water
2812650 Heterotrophic 24 hours at 1 year at — Drinking water
Bacteria 35 ± 0.5 °C 2–8 °C Beverages
m-TSB/USP Broth in plastic ampules Ground water
Surface water
Recreational water
2437306 Escherichia coli 4 hours at 1 year at — Drinking water
Agar Tubes/2 tests per
(confirmation) 35 ± 0.5 °C 2–8 °C Beverages
tube
Nutrient Agar/MUG 2812115 Ground water
Surface water
Prepared agar plate
Recreational water
2812250 Pseudomonas 24 hours at 1 year at — Drinking water
35 ± 0.5 °C 2–8 °C Beverages
Pseudomonas Broth in plastic ampules Ground water
Surface water
Recreational water
2724106 Stressed At least 1 year at Standard Method 18th
Heterotrophic 72 hours at 2–8 °C 9215 A, D
Agar Tubes/2 tests per
Bacteria 35 ± 0.5 °C
tube
(7 days Drinking water
R2A maximum) Beverages
Prepared agar plate 2814215 At least 1 year at —
2812350 72 hours at 2–8 °C
Broth in plastic ampules 35 ± 0.5 °C
Dehydrated media
Refer to the Dehydrated media and reagents table for a list of dehydrated media that can be
prepared. The media must be measured, mixed with water and sterilized before use.
Dehydrated media and reagents

Brain Heart Infusion Agar 500 g 2405634
Brain Heart Infusion Broth 500 g 2815534
Magenta GIcA (5-bromo-6-chloro-3indoyl-beta-D-glucuronide) 2g 2815035
M-E Agar 100 g 2281226
M-E Agar 500 g 2281234
m-TEC Agar, dehydrated 100 g 2281126
Nalidixic Acid 25 g 2407124
Oxidase Reagent 0.5 mL 2622500
Tryptic Soy Agar 100 g 2565926
Tryptic Soy Broth, ampules 50/pkg 2812650
Enrichment technique for total coliforms

Stressed coliforms require an enrichment technique, such as the one using Lauryl Tryptose (LT)
Broth Ampules described here, to get complete recovery with the MF Method. Consult your local
or state authorities about approved test methods for your application.
Procedure
1. Place a sterile absorbent pad in the lid of a sterile petri dish.

2. Saturate the pad with one LT Broth Ampule. Pour off any excess liquid.
3. Filter the sample through a membrane filter.
4. Place the membrane filter onto the saturated pad.

5. Incubate the filter without inverting the petri dish for 1.5 to 2 hours at
35 ± 0.5 °C in a relative humidity of at least 90 percent.
6. Remove the petri dish from the incubator and open it.
7. Place a sterile absorbent pad in the bottom half of the petri dish.
8. Pour the contents of one m-Endo Broth Ampule into the bottom half of the petri dish.
9. Carefully remove the filter from the lid and roll it onto the new pad to avoid trapping air
between the filter and the nutrient pad.
10. Discard the old pad (the enrichment pad saturated with LT Broth). Replace the culture dish lid.
11. Invert the culture dish and incubate at 35 ±0.5 °C for 20 to 22 hours.
12. After incubation, use an illuminated magnifier or a 10 to 15X microscope to count the colonies
with a greenish-gold metallic sheen.

Page 1253
MF-Bacteria, Pre-poured agar plate methods
Bacteria DOC316.53.01189
Membrane Filtration Method Pre-poured Agar Plate

Scope and Application: water and wastewater
Test preparation
Prepared Agar Plates contain prepared media. The plates are ready to use without the need for further preparation and
contain enough medium for one test.
The shelf life of the prepared agar plates varies from 3 months to 1 year (see the Table of Specifications). Plates are shipped
with a Certificate of Analysis and have an expiration date printed on the label.
To sterilize forceps, dip them in alcohol and flame in an alcohol or Bunsen burner. Let the forceps cool before use.
Disinfect the work bench with a germicidal cloth, dilute bleach solution, bactericidal spray or dilute iodine solution. Wash
hands thoroughly with soap and water.
Agar plate membrane filtration
1. Set up the membrane 2. Invert the sample for 3. Turn off the vacuum 4. With a slight rolling
filter apparatus. 30 seconds to mix. Pour and lift off the funnel top. motion, place the filter, grid
With sterile forceps, place 100 mL of sample into the Using sterile forceps, side up, on the agar.
a membrane filter, grid funnel. Apply vacuum and transfer the filter to the Check for trapped air
side up, into the assembly. filter the sample1. Rinse prepared agar. under the filter and make
the funnel walls with 20 to sure the filter touches the
30 mL of sterile Buffered entire surface of the agar.
Dilution Water. Apply Replace the lid on the
vacuum. Repeatedly rinse petri dish.
the funnel walls two more
times.
Bacteria
Page 1255
Bacteria
Agar plate membrane filtration (continued)
5. Invert the petri dish 6. Remove the petri dish

and incubate. Refer to the from the incubator and
Table of Specifications for count the colonies using a
temperature and time. 10 to 15X stereoscopic
microscope.
1 Release vacuum once dry so that the filter does not dry out and tear.

Report the density as number of colonies per 100 mL of sample. Use samples that produce about
50 and not more than 200 colonies per membrane to compute density.
Use Equation A to calculate density. Note that “mL sample” refers to actual sample volume and not
the volume of the dilution.
Equation A — Density on a single membrane filter.
Colonies counted
Colonies per 100 mL = ------------------------------------------------- × 100
mL sample filtered
• If growth covers the entire filtration area of the membrane or a portion of it, ad colonies are not
discrete, report the results as “Confluent Growth.”
• If the total number of colonies exceeds 200 per membrane or the colonies are too indistinct for
accurate counting, report the results as “Too Numerous To Count” (TNTC).
In either case, a new sample must be run using a dilution that will give about 50 and not more than
200 colonies.
When testing nonpotable water, if no filter meets the desired minimum colony count, the average
density can be calculated with Equation B.
Equation B — Average density for duplicates, multiple dilutions or more than one filter/
sample.
Colonies per 100 mL = ----------------------------------------------------------------------------------------------------- × 100
Bacteria
Page 1256
Bacteria
Specifications
Table 375 Table of Specifications

Prepared
Incubation Sensitivity Selectivity Shelf Life
Agar Plate
m-HPC 24 hours at 35 ± 0.5 °C 1 CFU/ 100 mL Heterotrophic Bacteria 1 year, 2–8 °C
m-Endo 24 hours at 35 ± 0.5 °C 1 CFU/ 100 mL Total Coliform Bacteria 1 year, 2–8 °C
m-FC 24 hours at 44.5 ± 0.2 °C 1 CFU/ 100 mL Fecal Coliform Bacteria 1 year, 2–8 °C
m-ColiBlue24® 24 hours at 35 ± 0.5 °C 1 CFU/ 100 mL Total Coliforms and E. coli 1 year, 2–8 °C
m-EI 24 hours at 44.5 ± 0.5 °C 1 CFU/ 100 mL Enterococci 3 months, 2–8 °C
Modified m-TEC 2 hours at 35 ± 0.5 °C 1 CFU/ 100 mL E. coli 1 year, 2–8 °C
then 22 hours at 44.5 °C
Nutrient Agar/ 24 hours at 35 ± 0.5 °C 1 CFU/ 100 mL E. coli (confirmation) 1 year, 2–8 °C
MUG
Required media and reagents

Agar plates, pre-poured:
m-HPC 15/pkg 2811415
m-Endo 15/pkg 2811615
m-FC 15/pkg 2811515
m-ColiBlue24® 15/pkg 2805215
m-EI 15/pkg 2811715
m-TEC modified 15/pkg 2811815
Nutrient Agar/MUG 15/pkg 2182115
Bacteria
Page 1257
Bacteria
Required apparatus
Aspirator, water each 213102
Bags, Whirl-Pak1 with dechlorinating agent, 180-mL 100/pkg 2075333
Counter, hand tally each 1469600
Filter Holder, magnetic coupling each 1352900
Filter Funnel Manifold, aluminum, 3-place each 2486100
Filtering Flask, 1000-mL each 54653
Filters, Membrane, 47-mm, 0.45-µm, gridded, sterile, Pall Gelman 200/pkg 1353001
Forceps, stainless steel each 2141100
Incubator, Culture, 120 VAC, 50/60 Hz each 2619200
Incubator, Water Bath, 120 VAC, 50/60 Hz each 2616300
Microscope, compound, 10X (15X available) each 2942500
Pipet, serological, 10–11 mL, sterile, disposable 25/pkg 209798
Pump, Vacuum, hand-operated 1 1428300
Tubing, rubber, 0.8 cm (5/16 in.) ID 3.7 m (12 ft.) 55919
1 Whirl-Pak is a registered trademark of Nasco, Inc.
Optional apparatus
Autoclave, Automatic, 120 VAC, 50/60 Hz each 2898600
Bottles, sample, glass, with cap, 118-mL 3/pkg 2163103
Bottles, sample, sterilized, 100-mL fill-to line, disposable 12/pkg 2495012
Bottles, sample, sterilized, 100-mL fill-to line, disposable with dechlorinating agent 12/pkg 2599112
Filter Unit, sterile, disposable with gridded membrane (use with 2656700) 12/pkg 2656600
Filtration Support (for field use), stainless steel each 2586200
Funnels, Push-Fit and membrane filters (use with 2586200) 72/pkg 2586300
Germicidal Cloths 50/pkg 2463200
Incubator, portable, 12 VDC each 2569900
Incubator, Water Bath, with gable cover, 110 VAC, 50/60 Hz each 2616300
Incubator, Water Bath, with gable cover, 220 VAC, 50/60 Hz each 2616302
Magnifier, illuminated, 2.5X and 5X, each 2585400
Magnifier, illuminated, 10X, portable each 2585300
Microscope, Compound, 10X (15X available) each 2942500
Pad, absorbent, with dispenser 1000/pkg 1491800
Pen, colony counter (Felt-tip pen attached to a counter, that marks, beeps and registers each 2613200
accumulative count on an LCD display)
Bacteria
Page 1258
Bacteria
Optional apparatus (continued)

Pump, Vacuum/Pressure, 115 VAC, 60 Hz each 2824800
Pump, Vacuum/Pressure, 230 VAC, 50 Hz each 2824802
Syringe, 140-mL, polypropylene (use with 2586200) each 2586100
Wicks, replacement (use with 2087760) 10/pkg 2097810
Isopropyl alcohol 500 mL 1445949
Alcohol burner each 2087742
Battery eliminator each 2580400
Bags, Whirl-Pak®, without dechlor 207 mL 100/pkg 2233199
Bags, Whirl-Pak, without dechlor 720 mL 10/pkg 1437297
Rechargeable battery pack, for portable incubator 12V DC / 115 V AC adapter each 2580300
230V AC Rechargeable batter pack adapter (for 2580300) each 2595902
Sterilization Indicator, Sterikon® 15/pkg 2811115
Optional media and reagents

Dechlorinating Reagent Powder Pillows 100/pkg 1436369
Dilution Water, buffered, sterile, 99-mL bottles 25/pkg 1430598

Peptone Powder Pillows, 1-g 30/pkg 2142964
Potassium Dihydrogen Phosphate and Magnesium Chloride Powder 50/pkg 2143166
Pillows for buffered dilution water (25 of each)
Tryptic Soy Agar 100 g 2565926
Bacteria
Page 1259
Coliforms—Total, Fecal and E. coli, MF, m-endo, 8074
Coliforms—Total, Fecal and E. coli

DOC316.53.001224
USEPA Membrane Filtration Method1 Method 8074
m-Endo
Scope and Application: For potable water, nonpotable water, recreation water and wastewater
1 Adapted from Standard Methods for the Examination of Water and Wastewater, 9222 B and 9221 B.
Introduction
The Membrane Filtration (MF) method is a fast way to estimate bacterial populations in water. The
MF method is especially useful when evaluating large sample volumes or performing many
coliform tests daily.
Method
In the initial step, an appropriate sample volume passes through a membrane filter with a pore size
small enough (0.45 micron) to retain the bacteria present. The filter is placed on an absorbent pad
(in a petri dish) saturated with a culture medium that is selective for coliform growth. The petri dish
containing the filter and pad is incubated, upside down, for 24 hours at the appropriate
temperature. After incubation, the colonies that have grown are identified and counted using a low-
power microscope.
PourRite™ Ampules contain prepared selective media. This eliminates the measuring, mixing, and
autoclaving needed when preparing dehydrated media. The ampules are designed with a large,
unrestrictive opening that allows media to pour out easily. Each ampule contains enough medium
for one test.
Test preparation
When the sample is less than 20 mL (diluted or undiluted), add 10 mL of sterile dilution water to the filter funnel before
applying the vacuum. This aids in distributing the bacteria evenly across the entire filter surface.
The volume of sample to be filtered will vary with the sample type. Select a maximum sample size to give 20 to 200
colony-forming units (CFU) per filter. The ideal sample volume of nonpotable water or wastewater for coliform testing yields
20–80 coliform colonies per filter. Generally, for finished, potable water, the volume to be filtered will be 100 mL.
If using PourRite® ampules, allow the media to warm to room temperature before opening.
Potable water must not contain any coliform bacteria. Do not dilute potable water samples.
Prepared m-Endo agar plates may be used instead of m-Endo broth.
Potable water procedures

To test potable water with the MF Method, examine a 100-mL sample for total coliforms by
incubating a filter at 35 ± 0.5 °C for 22–24 hours on m-Endo Broth. Coliforms ferment lactose in the
medium and produce an acid-aldehyde complex. This complex combines with Schiff’s Reagent
(also in the medium) to form an iridescent green coating over the colonies. When magnified 10 to
15 times, coliforms appear as dark red colonies with a greenish-gold sheen.
For replacement items, see m-Endo Broth for presumptive total coliforms.

Page 1261
Presumptive test for total coliforms (m-Endo), method 8074
1. Place a sterile 2. Invert an m-Endo 3. Set up the Membrane 4. Invert the sample for
absorbent pad in a sterile Broth PourRite Ampule 2 Filter Assembly. Use 30 seconds to mix. Pour
petri dish using sterilized to 3 times to mix the broth. sterilized forceps to place 100 mL of sample into the
forceps. Replace the lid. Use the ampule breaker a membrane filter, grid funnel. Apply vacuum and
Do not touch the pad or to break open the ampule. side up, into the assembly. filter the sample. Release
the inside of the petri dish. Carefully pour the Alternatively, a sterile the vacuum. Rinse the
contents evenly over the disposable filter unit may funnel walls with 20 to
To sterilize forceps, dip absorbent pad. Replace 30 mL of sterile buffered
forceps in alcohol and be used.
the petri dish lid. Repeat dilution water. Apply
flame in an alcohol or steps 1 and 2 for each vacuum. Repeat rinsing
Bunsen burner. Let petri dish being prepared. step 2 more times.
forceps cool before use.
Release the vacuum when
For ease of use, petri the filter is dry to prevent
dishes with pads are damage to the filter.
available.
5. Turn off the vacuum 6. With a slight rolling 7. Invert the petri dish 8. After incubating, use a
and lift off the funnel top. motion, center the filter, and incubate at 10 to 15X microscope to
Using sterilized forceps, grid side up, on the 35 ± 0.5 °C for 22–24 count the red colonies that
transfer the filter absorbent pad. Check for hours. have a greenish-gold
immediately to the air trapped under the filter metallic sheen.
previously prepared and make sure the filter The sheen may extend
petri dish. touches the entire pad. over the entire colony, or it
Replace the petri dish lid. may be localized to the
edge or to the center.

Page 1262
Presumptive test for total coliforms (m-Endo), method 8074 (continued)
9. Record the results of Depending on the test protocol, confirm positive results.
the test. See Interpreting To confirm total coliforms, follow Confirmation of total coliforms (LT and BGB),
and Reporting Results method 8074.
To confirm fecal coliforms, follow Confirmation of fecal coliforms (EC medium),
method 8074.
To confirm E. coli, follow Confirmation of E. coli (EC or EC/MUG), method 8074.
Confirmation of total coliforms (Lauryl Tryptose and Brilliant Green Bile)

For potable water samples, confirm typical colonies to ensure they are coliforms. (Confirm sheen
colonies, up to a maximum of five.) Inoculate parallel tubes of Lauryl Tryptose (LT) single-strength
(SS) Broth and Brilliant Green Bile (BGB) Broth by transferring growth from each colony. Growth
and gas production in both tubes verifies that the suspect organisms are coliforms. Most Probable
Number (MPN) coliform tubes are ideal for this purpose.
Use the swabbing technique for fecal coliforms or E. coli:
• When determining only the presence or absence of total coliforms
• When inoculating EC or EC/MUG media
Inoculate in this order:
1. EC or EC/MUG
2. LT SS Broth
3. BGB
For replacement items, see Confirmation of total coliforms (brilliant green bile broth and lauryl
tryptose broth).

Page 1263
Confirmation of total coliforms (LT and BGB), method 8074
1. Sterilize an inoculating 2. Touch the needle to 3. Again touch the same 4. Invert both tubes to
needle, or use a sterile, the coliform (sheen) coliform colony with the eliminate any air bubbles
disposable inoculating colony grown on m-Endo needle. Transfer to a trapped in the inner vials.
needle. plate. Transfer to a single- Brilliant Green Bile (BGB) Incubate the tubes at
To sterilize an inoculating strength Lauryl Tryptose Broth tube. 35 ± 0.5 °C. After one
needle, heat to red hot in (LT) Broth tube. hour, invert the tubes to
an alcohol or Bunsen remove trapped air in the
burner. Let the needle cool inner vial, then continue
before use. incubation.
5. After 24 ± 2 hours, 6. If no gas is present in Confirm positive results. If

check the inner vials for the LT Broth tube after growth and gas are
growth and gas bubbles. 48 hours, the colony is not produced in the LT Broth
Growth (turbidity) and gas a coliform and additional tube but not in the BGB
bubbles in both the LT and testing is unnecessary. Broth tube, inoculate
BGB Broth tubes verify Record the results of the another BGB tube from the
that the colonies are test. See Interpreting and gas-positive LT Broth tube.
coliforms. If one or both Reporting Results Incubate this BGB Broth
tubes do not show gas, tube and check for growth
continue incubating both and gas after 24 hours
tubes for an additional and/or after 48 hours. If
24 hours growth and gas are
produced within
48 ± 3 hours, the colony is
confirmed as coliform.

Page 1264
Confirmation of fecal coliforms (EC medium)

Analyze total-coliform-positive potable water samples for the presence of fecal coliform or E. coli.
Confirm fecal coliforms from a membrane filter positive for total coliforms by swabbing the
membrane with a sterile cotton swab and inoculating a tube of EC Medium Broth. Growth and gas
production in the EC Medium confirms the presence of fecal coliforms.
For replacement items, see Confirmation of fecal coliforms (EC medium broth).
Confirmation of fecal coliforms (EC medium), method 8074
1. Use a sterile cotton 2. Swirl the cotton swab 3. Invert the tubes to 4. After 24 ± 2 hours,
swab or inoculating loop to in an EC Medium Broth eliminate any air bubbles check the inner vial for gas
swab the entire surface of tube to transfer the trapped in the inner vial. bubbles. Growth
the total coliform-positive colonies collected from the Incubate the tube at and gas bubbles in the EC
membrane filter (colonies filter. Remove the cotton 44.5 ± 0.2 °C. After one Medium Broth tube
grown on m-Endo Broth). swab from the medium. hour, Invert the tubes to confirm the presence of
Use the same cotton swab remove trapped air in the fecal coliforms.
to transfer colonies from inner vial and continue
the same petri dish to incubation.
other broth media if
desired.
5. Record the results of

the test. See Interpreting
and Reporting Results.

Page 1265
Confirmation of E. coli (EC or EC/MUG)

Potable water samples that test positive for total coliforms may be analyzed for the presence of
E. coli in lieu of fecal coliforms. Use either EC medium or EC with MUG broth to confirm the
presence of E. coli on a membrane filter positive for total coliforms.
For replacement items, see E. coli confirmation with EC/MUG and E. coli confirmation with nutrient
agar.
Confirmation of E. coli (EC or EC/MUG), method 8074
1. Use a sterile cotton 2. Swirl the cotton swab 3. Invert the tubes to 4. After 24 ± 2 hours,
swab or inoculating loop to in an EC/MUG Broth tube eliminate any air bubbles use a long-wave UV lamp
swab the entire surface of to transfer the colonies trapped in the inner vial. to check the tube for
the membrane filter that is collected from the filter. Incubate the tube at fluorescence. Growth and
positive for total coliforms Remove the cotton swab 44.5 ± 0.2 °C. After one fluorescence indicate the
(colonies grown on m- from the medium. hour, invert the tubes to presence of E. coli.
Endo Broth). Use the same cotton swab remove trapped air in the Some glass will auto-
to transfer colonies from inner vial and continue fluoresce. Use Hach brand
the same petri dish to incubation. MPN tubes for best
other broth media if results.
desired. Do not look directly
through the MPN tube into
the UV lamp. View the
tube at 90° from the lamp.
Examine the tubes in a
darkened area.
Have a fluorescent-
positive and a fluorescent-
negative tube, both with
turbidity, to compare with
the sample tube.
Alternatively use an E. coli
presence standard.

Page 1266
Confirmation of E. coli (EC or EC/MUG), method 8074 (continued)
5. Record the results of

the test. See Interpreting
and Reporting Results.
Confirmation of E. coli (Nutrient Agar/MUG), method 8074
1. Heat a beaker of 2. Loosen the cap on 3. Using sterile 4. Use sterilized forceps
water, or a water bath, but one or more NA/MUG technique, pour half of the to lift the membrane filter
do not allow it to boil. nutrient agar tubes. Place contents of the tube into a with total coliform colonies
the tubes into hot water. sterile 47-mm petri dish. off the m-Endo absorbent
When the agar melts, Immediately replace petri pad.
carefully remove the tubes dish lid and allow agar to To sterilize forceps, dip
from the water with tongs. solidify undisturbed. forceps in alcohol and
Pre-poured agar plates flame in an alcohol or
can also be used. Bunsen burner. Let

Page 1267
Confirmation of E. coli (Nutrient Agar/MUG), method 8074 (continued)
5. Immediately transfer 6. Invert the petri dish. 7. Using a long-wave UV 8. Record the results of
the membrane filter to the Incubate for 4 hours at lamp, examine the the test. See Interpreting
petri dish containing NA/ 35 ± 0.5 °C. colonies for fluorescence. and Reporting Results.
MUG. With a slight rolling Fluorescence indicates the
motion, center the filter, presence of E. coli.
grid side up, on the agar. Make sure to examine the
Check for air trapped petri dish in a darkened
under the filter and make area.
sure the entire filter
touches the agar. Replace Some UV lamps do not
the petri dish lid. use the correct wattage
and can give false results.
Be sure to use the UV
lamps and replacement
bulbs that are specified in
Consumables and
replacement items.
Interpreting and Reporting Results

Report coliform density as the number of colonies per 100 mL of sample. For total coliforms, use
samples that produce 20 to 80 coliform colonies, and not more than 200 colonies of all types, per
membrane to compute coliform density. For fecal coliform testing, samples should produce 20 to
60 fecal coliform colonies.
Use Equation A to calculate coliform density. Note that “mL sample” refers to actual sample
volume, and not volume of the dilution.
Equation A—Coliform density on a single membrane filter
Coliform colonies counted
Coliform colonies per 100 mL = --------------------------------------------------------------------- × 100
mL of sample filtered
• If growth covers the entire filtration area of the membrane, or a portion of it, and colonies are
not discrete, report results as “Confluent Growth With or Without Coliforms.”
• If the total number of colonies (coliforms plus non-coliforms) exceeds 200 per membrane or
the colonies are too indistinct for accurate counting, report the results as “Too Numerous To
Count” (TNTC).
In either case, run a new sample using a dilution that will give about 50 coliform colonies and not
more than 200 colonies of all types.
When testing nonpotable water, if no filter meets the desired minimum colony count, calculate the
average coliform density with Equation B.

Page 1268
Equation B—Average coliform density for 1) duplicates, 2) multiple dilutions, or 3) more

than one filter/sample
Coliform colonies per 100 mL = ----------------------------------------------------------------------------------------------------- × 100
Controls:
negative control and Escherichia coli as a positive control. Use the AQUA QC-STIK™ Device for
quality control procedures. Instructions for use come with each AQUA QC-STIK Device.
Potable water samples from municipal treatment facilities should be negative for total coliforms
and fecal coliforms.

m-Endo Broth for presumptive total coliforms

m-Endo prepared agar plates 15/pkg 2811615
m-Endo Broth Ampules, plastic 50/pkg 2373550
m-Endo Broth, 100 mL glass bottle 1 each 2373542
m-Endo Broth PourRite™ Ampules, glass (for total coliform presumptive) 20/pkg 2373520
Dilution Water, buffered, sterile, 99 mL 25/pkg 1430598
Required apparatus
Alcohol Burner 1 2087742
Ampule Breaker, PourRite™ each 2484600
Counter, hand tally 1 1469600
Dish, Petri, with pad, 47-mm, sterile, disposable, Gelman 100/pkg 1471799
Dish, Petri, with pad, 47-mm, sterile, disposable, Millipore 150/pkg 2936300
Filter Holder, magnetic coupling (use with 24861-00) 1 1352900
Filters, Membrane, 47-mm, 0.45-µm, gridded, sterile, Gelman 200/pkg 1353001
Filters, Membrane, 47-mm, 0.45-µm, gridded, sterile, Millipore 150/pkg 2936100
Filtering Flask, 1000-mL 1 54653
Forceps, stainless steel 1 2141100
Loop, inoculating, disposable 25/pkg 2749125
Microscope, compound 1 2942500
Pump, vacuum/pressure, portable, 115 VAC, 60 Hz each 2824800
Tubing, rubber, 0.8 cm (5/16 in.) ID 3.7 m (12 ft) 56019

Page 1269
Optional media, reagents and apparatus

Adapter for rechargeable battery pack, 230 VAC (for 2580300) each 2595902
Autoclave, 120 VAC, 50/60 Hz each 2898600
Bag, for contaminated items 200/pkg 2463300
Bags, Whirl-Pak®, without dechlorinating agent, 207 mL 100/pkg 2233199
Bags, Whirl-Pak®, with dechlorinating agent, 180 mL 100/pkg 2075333
Battery pack, rechargeable, for portable incubator 12 VDC each 2580300
Bottle, sample, sterilized, 100-mL, disposable with dechlorinating agent 12/pkg 2599112
Bottle, sample, sterilized, 100-mL, disposable 12/pkg 2495012
Dish, Petri, 47-mm, sterile, disposable 100/pkg 1485299
Incubator, water bath, 120 VAC, 50/60 Hz each 2616300
Powder Pillows for buffered dilution water (25 of each)1 50/pkg 2143166
Pump, hand vacuum each 1428300
1 Add the contents of one potassium dihydrogen phosphate and one magnesium chloride powder pillow to one liter of distilled water and
autoclave (sterilize) to prepare American Public Health Association buffered dilution water.
Confirmation of total coliforms (brilliant green bile broth and lauryl tryptose broth)
Note: Many of the confirmation products are also listed under the m-Endo presumptive products.

Brilliant Green Bile Broth Tubes (for total coliform confirmation) 15/pkg 32215
Lauryl Tryptose Broth Tubes, single-strength (for total coliform confirmation) 15/pkg 2162315
Required apparatus

Page 1270


Burner, Bunsen each 2162700
Inoculating Needle, disposable 25/pkg 2748925
Lauryl Tryptose Broth Ampules, sterile (for enrichment technique) 20/pkg 1472520
Rack, coliform tube each 221500
Wicks, replacement (used with Alcohol Burner 20877-42) 10/pkg 2097810
Confirmation of fecal coliforms (EC medium broth)


EC Medium Broth Tubes (for fecal coliform confirmation) 15/pkg 1410415
Required apparatus
Swabs, cotton, sterile (for confirmation) 100/pkg 2554300

Incubator, portable, 12 VDC 1 2569900
Incubator, 12-Well Dri-Bath, 115/230 VAC, 50/60 Hz with North American style plug each 2281400
Incubator, 12-Well Dri-Bath, 115/230 VAC, 50/60 Hz with European style plug each 2281402
Inoculating Loop, nichrome wire, with handle 1 2112100

Page 1271
E. coli confirmation with EC/MUG


EC Medium with MUG Broth Tubes (for E. coli confirmation) 15/pkg 2471515
Required apparatus
Lamp, long-wave, ultraviolet, 115 VAC, 60 Hz 1 2184300
Lamp, long-wave, ultraviolet, 230 VAC, 50/60 Hz 1 2184302
Swabs, cotton, sterile (for confirmation) 100/pkg 2554300

Ecoli Fluorescence standard each 2361100
Incubator, portable, 12 VDC 1 2569900
Incubator, 12-Well Dri-Bath, 115/230 VAC, 50/60 Hz with European style plug each 2281402
Inoculating Loop, nichrome wire, with handle 1 2112100
Lamp, long-wave, ultraviolet, portable, 4 watt 1 2415200

Page 1272
E. coli confirmation with nutrient agar


Nutrient Agar with MUG prepared plates 15/pkg 2182115
Nutrient Agar with MUG Tubes, 2 tests/tube (for E. coli confirmation) 6/pkg 2437306
Required apparatus
Lamp, long-wave, ultraviolet, 115 VAC, 60 Hz 1 2184300
Lamp, long-wave, ultraviolet, 230 VAC, 50/60 Hz 1 2184302

Beaker, 600 mL 1 50052
Lamp, long-wave, ultraviolet, portable, 4 watt 1 2415200

Page 1273
Coliforms—Fecal, MF, m-FC and m-FC/RA 8074
Coliforms—Fecal DOC316.53.001209
USEPA Membrane Filtration Method Method 80741

m-FC and m-FC/RA
Scope and Application: For potable water, nonpotable water, recreation water and wastewater.
1 USEPA approved 9222 D.
Introduction
The Membrane Filtration (MF) method is a fast way to estimate bacterial populations in water. The
MF method is especially useful when evaluating large sample volumes or performing many
coliform tests daily.
Method
In the initial step, an appropriate sample volume passes through a membrane filter with a pore size
small enough (0.45 micron) to retain the bacteria present. The filter is placed on an agar plate
prepared with a culture medium that is selective for coliform growth. The petri dish is incubated,
upside down, for 24 hours at the appropriate temperature. After incubation, the colonies that have
grown are identified and counted using a low-power microscope.
PourRite™ Ampules contain prepared selective media. This eliminates the measuring, mixing, and
autoclaving needed when preparing dehydrated media. The ampules are designed with a large,
unrestrictive opening that allows media to pour out easily. Each ampule contains enough medium
for one test.
Test preparation
If using PourRite™ ampules, allow the media to warm to room temperature before opening.
Nonpotable waters procedures

Wastewater, river, bathing, and other nonpotable waters usually are tested for fecal coliforms. In
testing for fecal coliforms, a special medium and an elevated incubation temperature inhibit growth
of nonfecal coliforms. Fecal coliforms growing on the membrane form an acid that reacts with an
aniline dye in the medium, producing a blue color.
Use m-FC Broth with Rosolic Acid to increase specificity when high levels of non-coliform bacteria
may be present, unless all the organisms in the sample are stressed or injured.
Coliforms—Fecal
Page 1275
Coliforms—Fecal
Confirmation of fecal coliforms (m-FC or m-FC/RA), method 8074
1. Place a sterile 2. Invert an m-FC Broth 3. Set up the Membrane 4. Prepare the necessary
absorbent pad in a sterile PourRite Ampule 2 to 3 Filter Assembly. Use dilutions to obtain the
petri dish using sterilized times to mix the broth. Use sterilized forceps to place proper sample size. Invert
forceps. Replace the petri the ampule breaker to a membrane filter, grid the sample for 30 seconds
dish lid. open an ampule. Carefully side up, into the assembly. to mix. Pour sample into
Do not touch the pad or pour the contents evenly the funnel. Apply vacuum
the inside of the petri dish. onto the absorbent pad. and filter the sample.
Replace the petri dish lid. Rinse the funnel walls with
To sterilize forceps, dip 20 to 30 mL of sterile
forceps in alcohol and Use m-FC Broth with
Rosolic Acid to increase buffered dilution water.
flame in an alcohol or Apply vacuum. Repeat
Bunsen burner. Let specificity when high
levels of non-coliform rinsing step, two more
forceps cool before use. times.
bacteria may be present,
Petri dishes with pads are unless the organisms are Release the vacuum when
available. stressed or injured. the filter is dry to prevent
damage to the filter.
5. Turn off the vacuum 6. With a slight rolling 7. Invert the petri dish 8. After incubating, count
and lift off the funnel top. motion, center the filter, and incubate at the blue colonies using a
Use sterile forceps to grid side up, on the 44.5 ± 0.2 °C for 10 to 15X microscope.
transfer the membrane absorbent pad. Check for 24 ± 2 hours.
filter to the previously air trapped under the filter To eliminate environmental
prepared petri dish. and make sure the entire Klebsiella from the fecal
filter touches the pad. coliform population elevate
Replace the petri dish lid. the temperature to
45.0 ± 0.2 °C.
Alternatively, a water bath
with rack may be used for
incubation by placing the
petri dishes into a sealed
bag.
Coliforms—Fecal
Page 1276
Coliforms—Fecal
Confirmation of fecal coliforms (m-FC or m-FC/RA), method 8074 (continued)
9. Record the results of the test. See Interpreting and

reporting results.
To verify results, follow Verifying fecal coliforms, method
8074
Confirmation of total coliforms (Lauryl Tryptose and Brilliant Green Bile)

For potable water samples, confirm typical colonies to ensure they are coliforms. (Confirm sheen
colonies, up to a maximum of five.) Inoculate parallel tubes of Lauryl Tryptose (LT) Single Strength
(SS) Broth and Brilliant Green Bile (BGB) Broth by transferring growth from each colony. Growth
and gas production in both tubes verifies that the suspect organisms are coliforms. Most Probable
Number (MPN) coliform tubes are ideal for this purpose.
Use the swabbing technique for fecal coliforms or E. coli:
• When determining only the presence or absence of total coliforms
• When inoculating EC or EC/MUG media
Inoculate in this order:
1. EC or EC/MUG
2. LT SS Broth
3. BGB
Coliforms—Fecal
Page 1277
Coliforms—Fecal
Confirmation of total coliforms (LT and BGB), method 8074
1. Sterilize an inoculating 2. Touch the needle to 3. Again touch the same 4. Invert both tubes to
needle, or use a sterile, the coliform (sheen) coliform colony with the eliminate any air bubbles
disposable inoculating colony grown on m-Endo needle. Transfer to a trapped in the inner vials.
needle. Broth. Transfer to a single- Brilliant Green Bile (BGB) Incubate the tubes at
To sterilize an inoculating strength Lauryl Tryptose Broth tube. 35 ± 0.5 °C. After one
needle, heat to red hot in (LT) Broth tube. hour, invert the tubes to
an alcohol or Bunsen remove trapped air in the
burner. Let the needle cool inner vial, then continue
before use. incubation.
5. After 24 ± 2 hours, 6. If no gas is present in Confirm positive results. If

check the inner vials for the LT Broth tube after growth and gas are
growth and gas bubbles. 48 hours, the colony is not produced in the LT Broth
Growth (turbidity) and gas a coliform and additional tube but not in the BGB
bubbles in both the LT and testing is unnecessary. Broth tube, inoculate
BGB Broth tubes verify Record the results of the another BGB tube from the
that the colonies are test. See Interpreting and gas-positive LT Broth tube.
coliforms. If one or both reporting results Incubate this BGB Broth
tubes do not show gas, tube and check for growth
continue incubating both and gas after 24 hours
tubes for an additional and/or after 48 hours. If
24 hours growth and gas are
produced within
48 ± 3 hours, the colony is
confirmed as coliform.
Coliforms—Fecal
Page 1278
Coliforms—Fecal
Verifying fecal coliforms, method 8074
1. Sterilize an inoculating 2. Touch the needle to a 3. Invert the tubes to 4. Invert the tubes to
needle, or use a sterile, typical blue colony and eliminate air trapped eliminate air trapped
disposable inoculating transfer to a Lauryl inside the inner vials. inside the inner vials.
needle. Tryptose (LT) Broth tube. Incubate the tubes at Incubate the EC Medium
To sterilize an inoculating Repeat steps 1 and 3 for 35 ± 0.5 °C. After one tubes at 44.5 ± 0.2 °C for
needle, heat to red hot in each test being verified. hour, invert the tubes to 24 ±2 hours. After one
an alcohol or Bunsen Steps 3 and 4 can be remove trapped air in the hour, invert the tubes to
burner flame. Let the performed simultaneously inner vial and continue remove trapped air in the
needle cool before use. if multiple incubators are incubation. Check tubes inner vial.
available. for growth and gas
production at 24 hours.
If no change has occurred,
continue incubation for
another 24 hours.
If growth and gas are not
produced in 48 ± 3 hours,
the colony was not
coliform. If growth and gas
are produced in
48 ± 3 hours, use a sterile
loop to inoculate one
EC Medium Broth tube
from each gas-positive LT
Broth tube.
5. Growth and gas production at 44.5 °C within

24 ± 2 hours confirms the presence of fecal coliforms.
Record the results of the test. See Interpreting and
reporting results.
Coliforms—Fecal
Page 1279
Coliforms—Fecal

Count” (TNTC).
Controls:

Confirmation of fecal coliforms (m-FC or m-FC/RA)

m-FC prepared agar plates 15/pkg 2811515
m-FC Broth Ampules, plastic 50/pkg 2373250
m-FC w/Rosolic Acid Broth Ampules, plastic 50/pkg 2428550
m-FC Broth PourRite™ Ampules (for fecal coliform presumptive) 20/pkg 2373220
m-FC with Rosolic Acid Broth PourRite™ Ampules (fecal coliform presumptive) 20/pkg 2428520
Coliforms—Fecal
Page 1280
Coliforms—Fecal
Required apparatus
Filter Funnel Manifold, aluminum, 3-place (use with 13529-00) 1 2486100
Incubator, Culture, low profile, 110 VAC, 50/60 Hz each 2619200
Microscope, compound each 2942500


Confirmation of total coliforms (brilliant green bile broth and lauryl tryptose broth)

Brilliant Green Bile Broth Tubes (for total coliform confirmation) 15/pkg 32215
Lauryl Tryptose Broth Ampules, sterile (for enrichment technique) 20/pkg 1472520
Lauryl Tryptose Broth Tubes, single-strength (for total coliform confirmation) 15/pkg 2162315
Required apparatus
Burner, Bunsen each 2162700
Coliforms—Fecal
Page 1281
Coliforms—Fecal

Filter Funnel Manifold, aluminum, 3-place (use with 13529-00) each 2486100
Tubing, rubber, 0.8 cm ID 3.7 m (12 ft) 56019
Wicks, replacement, for alcohol burner 2087742 10/pkg 2097810

Coliforms—E. coli, MF, m-TEC, 8367
Coliforms—E. coli DOC316.53.001210

m-TEC
1 USEPA accepted.
Test preparation
m-TEC Agar confirmation for presumptive E. coli test

The m-TEC method detects E. coli in recreational fresh water samples with a two step process.
First, membrane filters are incubated on m-TEC Agar for 2 hours at 35 °C to resuscitate injured
organisms. The thermotolerant organisms are then selected by fermentation of lactose at an
elevated temperature of 44.5 °C. The second step uses a substrate medium containing urea to
distinguish urease-negative E. coli from other thermotolerant coliforms that hydrolyze urea. Yellow
or yellow-brown urease-negative colonies are positive for E. coli.
Presumptive E. coli test (m-TEC Agar), method 8367
1. Heat a beaker of water 2. Place m-TEC Agar 3. Using sterile 4. Set up the Membrane
or water bath but do not tubes into hot water. When technique, pour half of the Filter Assembly. Using
allow it to boil. agar melts, carefully contents of the tube into a sterilized forceps, place a
remove tubes from hot sterile, 47-mm petri dish. membrane filter, grid side
water with tongs. Immediately replace the up, into the assembly.
petri dish lid and allow To sterilize forceps, dip
agar to solidify forceps into alcohol and
undisturbed. flame in an alcohol or
Bunsen burner. Let
Coliforms—E. coli
Page 1283
Coliforms—E. coli
5. Prepare the necessary 6. Turn off the vacuum 7. With a slight rolling 8. Invert the petri dish.
dilutions to obtain the and lift off the funnel top. motion, place the filter, grid Incubate at 35 ± 0.5 °C for
proper sample size. Invert Using sterilized forceps, side up, on the agar. 2 hours and then at
the sample for 30 seconds transfer the membrane Check for air trapped 44.5 ± 0.2 °C for 22 hours.
to mix. Pour sample into filter to the previously under the filter and make
the funnel. Apply vacuum prepared petri dish. sure the entire filter
and filter the sample. touches the agar. Replace
Rinse the funnel walls with the petri dish lid.
20 to 30 mL of sterile
buffered dilution water.
Apply vacuum. Repeat the
rinsing step, two more
times.
the filter is dry to prevent
Coliforms—E. coli
Page 1284
Coliforms—E. coli
9. To confirm, use 10. After 15 minutes, 11. Record the results of

sterilized forceps to count yellow or yellow- the test. See Interpreting
transfer the filter to a pad brown colonies by using a and reporting results.
saturated with at least 10 to 15X microscope. The
2 mL of urea substrate. presence of yellow or
Preparing the urea yellow-brown colonies
substrate: confirms E. coli.
a. Dissolve 2.0 g of urea in

100 mL of deionized water.
b. Add 10 mg of phenol
red sodium indicator to the
urea solution.
c. Use 0.02 N sulfuric acid
to adjust the pH to
between 3 and 4. The
solution will turn yellow.
d. Store solution at 2 to
8 °C. Use within one
week.

Count” (TNTC).
Coliforms—E. coli
Page 1285
Coliforms—E. coli
Controls:
negative control and Escherichia coli as a positive control.

m-TEC Agar Tubes, 2 tests/tube (for E. coli determination) 6/pkg 2561106
Phenol Red Sodium Salt 5g 2563922
Urea Reagent, ACS 100 g 1123726
Required apparatus
Filter Holder, magnetic coupling (use with 24861-00) each 1352900
Microscope, Compound each 2942500
Coliforms—E. coli
Page 1286
Coliforms—E. coli

Bunsen burner with tubing each 2162700
Filter Funnel Manifold, aluminum, 3-place (use with 1352900) each 2486100
m-ColiBlue24® Broth, 100 mL glass bottle 1 each 2608442
Wicks, replacement, for alcohol burner 2087742 — 2097810
Coliforms—E. coli
Page 1287
Coliforms—E. coli, MF, mod m-TEC, 8367
Coliforms—E. coli DOC316.53.001211

modified m-TEC
1 USEPA accepted
Test preparation
Modified m-TEC prepared Agar Plates for E. coli in recreational waters

A revised single-step method uses modified m-TEC Agar to detect E. coli in recreational fresh
water samples. The modified m-TEC Agar contains an enzymatic substrate which is cleaved to
produce colonies. Red or magenta colored colonies specifically represent the presence of E. coli.
Confirmation steps are not required with this method.
Coliforms—E. coli
Page 1289
Coliforms—E. coli
Modified m-TEC for E. coli in recreational waters, method 8367
1. Set up the Membrane 2. Prepare the necessary 3. Turn off the vacuum 4. With a slight rolling
Filter Assembly. Refer to dilutions to obtain 20–80 and lift off the funnel top. motion, place the filter, grid
Introduction to Coliforms. E. coli colonies on the Using sterilized forceps, side up, on the agar plate.
Using sterilized forceps, membranes. transfer the membrane Check for air trapped
place a membrane filter, Invert the sample for 30 filter to the modified m- under the filter and make
grid side up, into the seconds to mix. TEC prepared agar plate. sure the entire filter
assembly. touches the agar. Replace
Pour the sample into the the petri dish lid.
To sterilize forceps, dip funnel. Apply vacuum and
forceps into alcohol and filter the sample. Rinse the
flame in an alcohol or funnel walls with 20 to
Bunsen burner. Let 30 mL of sterile buffered
forceps cool before use. dilution water. Apply
vacuum. Repeat rinsing
step, two more times.
5. Invert the petri dish. 6. Count the number of 7. The presence of these
Incubate at 35 ±0.5 °C red or magenta colonies colonies confirms E. coli.
for 2 hours, then by using a 10 to 15X Record the results of the
at 44.5 ±0.2 °C for microscope. test. See Interpreting and
22 hours. reporting results.
Coliforms—E. coli
Page 1290
Coliforms—E. coli

Count” (TNTC).
Controls:
Coliforms—E. coli
Page 1291
Coliforms—E. coli

m-TEC, modified, prepared agar plates, 47-mm 15/pkg 2811815
Required apparatus

Coliforms—E. coli
Page 1292
Coliforms—E. coli
Optional media, reagents and apparatus (continued)

Coliforms—E. coli
Page 1293
Coliforms—Total and E. coli, MF, m-ColiBlue, 10029
Coliforms—Total and E. coli DOC316.53.001213

m-ColiBlue24®
1 USEPA approved.
Test preparation
If using PourRite™ ampules, allow the media to warm to room temperature before opening.
m-ColiBlue24 Broth PourRite Ampules

The m-ColiBlue24 Broth can be used to analyze drinking water, bottled water, beverages; surface,
well, and groundwater, waste water, recreational waters and process water for ultrapure, chemical
processing and pharmaceutical applications.
Coliforms—Total and E. coli

Page 1295
Simultaneous total coliform and E. coli screening, method 10029
1. Use sterilized forceps 2. Invert ampules two or 3. Set up the Membrane 4. Invert the sample for
to place a sterile, three times to mix broth. Filter Apparatus. With 30 seconds to mix. Pour
absorbent pad in a sterile Break open an ampule of sterile forceps, place a 100 mL of sample or
petri dish. Replace the lid m-ColiBlue24 Broth using membrane filter, grid side diluted sample into the
on the dish. an ampule breaker. Pour up, into the assembly. funnel. Apply vacuum and
Do not touch the pad or the contents evenly over Alternatively, a sterile filter the sample. Rinse the
the inside of the petri dish. the absorbent pad. disposable filter unit may funnel walls with 20 to
Replace the petri dish lid. be used. 30 mL of sterile buffered
To sterilize the forceps, dip dilution water. Apply
them in alcohol and flame vacuum. Rinse again two
in an alcohol or Bunsen more times.
burner. Let the forceps
cool before use. Release the vacuum when

Page 1296
Simultaneous total coliform and E. coli screening, method 10029 (continued)
5. Turn off the vacuum 6. With a slight rolling 7. Invert the petri dish 8. Remove the petri dish
and lift off the funnel top. motion, place the filter, grid and incubate at from the incubator and
Using sterile forceps, side up, on the absorbent 35 ± 0.5 °C for 24 hours. examine the filters for
transfer the filter to the pad. Check for trapped air colony growth. Colonies
previously prepared petri under the filter and make are typically readily visible;
dish. sure the filter touches the however, a microscope or
entire pad. Replace the other 10–15X magnifier
petri dish lid. may be useful. Red and
blue colonies indicate total
coliforms and blue
colonies specifically
indicate E. coli.
Sometimes only the center
of a colony will show color.
Therefore, a colony with
any amount of red color
should be counted as red
and a colony with any
amount of blue should be
counted as a blue colony.
Red colonies may vary in
color intensity. Blue
colonies may appear blue
to purple. Count all the red
and blue colonies as total
coliforms. Count all the
blue to purple colonies as
E. coli.
Optional testing of red colonies

The m-ColiBlue24 Broth is formulated so that coliforms other than E. coli grow as red colonies.
The percentage of red colonies that are false positives (non-coliforms) is comparable to the
percentage of sheen colonies grown on m-Endo Broth that are false positives (non-coliforms);
therefore, confirmation is not required.
A few varieties of the non-coliform bacteria Pseudomonas, Vibrio, and Aeromonas spp. may grow
on m-ColiBlue24 Broth and form red colonies. Such bacteria can be readily distinguished from
total coliforms by the oxidase test. Pseudomonas, Vibrio, and Aeromonas spp. are oxidase-
positive. Total coliforms and Escherichia coli are oxidase-negative. If your sample contains high
levels of interfering bacteria, you can perform an oxidase test to confirm which red colonies are
total coliforms.

Page 1297
Two oxidase procedures are provided. Count the red and blue colonies on the m-ColiBlue24 Broth
membrane filter before starting the oxidase test.
Oxidase method 1
This method enables you to conveniently and rapidly evaluate membrane filters that have
numerous colonies. Use this method after 24 hours of incubation on m-ColiBlue24 Broth.
Research* shows that the oxidase test cannot be performed on media that undergoes acidification
during bacterial growth. The m-ColiBlue24 Broth is formulated so that the medium does not
undergo such acidification. Consequently, many colonies can be simultaneously tested for their
oxidase reaction using the following procedure.
1. Remove the lid from the petri dish containing the m-ColiBlue24 Broth membrane filter, invert
the lid, and place it on the bench top.
Controls: Positive and negative controls are important. Pseudomonas aeruginosa is

recommended for positive controls and Escherichia coli for negative controls. Use Aqua
QC-Stiks™ for quality control procedures.
2. Drop approximately 0.5 mL of Difco SpotTest™ Oxidase Reagent into the center of the
inverted lid.
3. Using sterile forceps, transfer the membrane filter from the pad and place the filter upright in
the inverted lid.
4. Within 10 to 15 seconds, the oxidase reagent will soak into the filter and cause the oxidase-
positive colonies to turn purple. This purple color may be visible in the colony itself or adjacent
to the colony. Oxidase-negative colonies will retain the red color they developed when
incubated on m-ColiBlue24 Broth.
5. After the initial 10 to 15 second reaction time, start counting the red colonies that turn purple.
Count individual colonies by using a microscope with
10–15X magnification
Note: To simplify colony counting place a spare lid on the lid containing the oxidase reagent and
membrane filter. Use a felt-tip pen to mark the lid as you identify the purple colonies. After 30
seconds, you can count marks that indicate purple (oxidase-positive) colonies.
6. Stop counting 30 seconds after initial 10 to 15 second reaction time, because

oxidase-negative colonies will start to develop a purple color.
Note: Bacteria are not killed with this procedure, so colonies may be selected for streaking and for
additional testing.
Colonies that are blue after the initial 24-hour incubation on m-ColiBlue24 Broth are almost always
E. coli and do not need confirmation with the oxidase procedure.
Oxidase method 2
This method is the official oxidase test described in Standard Methods for the Examination of
Water and Wastewater, 18th edition, 1992.
1. Select red colonies from an m-ColiBlue24 Broth membrane filter and streak onto Tryptic
Soy Agar.
2. Incubate Tryptic Soy Agar plates at 35 °C (95 °F) for 18–24 hours or until isolated colonies are
obtained.
* A.H. Havelaar et al. 1980. False-negative oxidase reaction as a result of medium acidification. Antonie van Leeuwenhoek.
46, 301-312. L.K. Hunt et al. 1981. Role of pH in oxidase variability of Aeromonas hydrophila. Journal of Clinical Microbiology.
13: 1054-1059.

Page 1298
Controls: Positive and negative controls are important. Pseudomonas aeruginosa is

recommended for positive and Escherichia coli for negative controls. Use Aqua QC-Stiks™*
for quality control procedures.
3. Saturate a piece of filter paper with Difco SpotTest Oxidase Reagent. (This reagent contains a
stabilized solution of N,N,N’,N’-tetramethyl-p-phenylenediamine dihydrochloride.)
Note: Alternatively, oxidase reagent can be dropped directly onto colonies growing on Tryptic Soy Agar.
Oxidase-positive colonies will turn from pink to purple.
4. Using a sterile nichrome inoculating needle, transfer cellular material from an isolated Tryptic
Soy Agar colony to the moist filter paper.
Note: Do not use iron or other reactive needles for inoculation, because they will cause false-positive
results. Wooden applicator sticks work well.
5. Oxidase-negative colonies will not react with the reagent, but oxidase-positive colonies will
cause the reagent to turn dark purple within 10 seconds.
6. Oxidase-negative colonies should be counted as total coliform bacteria.

Count” (TNTC).
Controls:
* Aqua QC-Stiks is a trademark of MicroBiologics.

Page 1299

m-ColiBlue24® Broth Ampules, glass 20/pkg 2608420

m-ColiBlue24® Broth Ampules, plastic 50/pkg 2608450
m-ColiBlue24® prepared agar plates 15/pkg 2805215
Required apparatus
Tubing, rubber, 0.8 cm (5/16 in.) ID 3.7 m (12 ft) 56019


Page 1300


Page 1301
Enterococci, MF, m-EI, 1600
Enterococci DOC316.53.001212
Membrane Filtration Method Proposed Method 1600

M-EI
Test preparation
colony-forming units (CFU) per filter. The ideal sample volume of nonpotable water or wastewater for bacteria testing yields
20–80 colonies per filter. Generally, for finished, potable water, the volume to be filtered will be 100 mL.
m-EI prepared Agar Plates for Enterococci test in recreational waters

Enterococci are used as an indicator organism for recreational water quality evaluation. The m-EI
Agar specifically detects enterococci in 24 hours after enzymatic cleavage of a substrate
(indoxyl-β-D-glucoside) which results in formation of colonies surrounded by a blue halo.
Enterococci
Page 1303
Enterococci
m-EI for Enterococci, proposed method 1600
1. Set up the Membrane 2. Prepare the necessary 3. Turn off the vacuum 4. With a slight rolling
Filter Assembly. Using dilutions to obtain the and lift off the funnel top. motion, place the filter, grid
sterilized forceps, place a proper sample size. Invert Using sterilized forceps, side up, on the prepared
membrane filter, grid side the sample for 30 seconds transfer the membrane m-EI agar plate. Check for
up, into the assembly. to mix. Pour sample into filter to the previously air trapped under the filter
To sterilize forceps, dip the funnel. Apply vacuum prepared agar plate. and make sure the entire
forceps into alcohol and and filter the sample. filter touches the agar.
flame in an alcohol or Rinse the funnel walls with Replace the petri dish lid.
Bunsen burner. Let 20 to 30 mL of sterile
forceps cool before use. buffered dilution water.
Apply vacuum. Rinse
again two more times.
5. Invert the petri dish. 6. Count any colonies 7. Record these colonies
Incubate at 41 ± 0.5 °C for (regardless of color) with a as enterococci. See
24 hours. blue halo. Use a 10 to 15X Interpreting and reporting
microscope. results.
Optional verification of Enterococci

Several media can be used to verify the presence of enterococci grown on m-EI Agar.
Gram-positive cocci, such as enterococci, will grow and hydrolyze esculin on Bile Esculin Agar,
which results in the formation of a black or brown precipitate. Enterococci can also be confirmed
by their ability to grow in Brain Heart Infusion Broth at an elevated incubation temperature of 45 °C
and in Brain Heart Infusion Broth containing 6.5% NaCl.
Enterococci
Page 1304
Enterococci
Brain and heart infusion broth for optional detection
1. Use a sterile 2. Incubate broth tubes 3. After 24 hours, 4. After 48 hours

inoculating needle to for 24 hours and agar transfer a loopful of broth observe all media for
transfer cells from the slants for 48 hours at from the BHIB tube to growth and apply a Gram
centers of at least 10 well- 35 ± 0.5 °C. each of the following stain to the growth from
isolated typical colonies media and incubate for each BHIA slant.
into a Brain Heart Infusion 48 hours:
Broth (BHIB) tube and Bile Esculin Agar (BEA)
onto a Brain Heart Infusion and incubate at
Agar (BHIA) slant. 35 ± 0.5 °C.
BHIB and incubate at
45 ± 0.5 °C.
BHIB with 6.5% NaCl and
incubate at 35 ± 0.5 °C.
5. Gram-positive cocci that grow and hydrolyze

esculin on BEA (i.e. produce a black or brown
precipitate) and grow in BHIB and BHIB with salt are
verified as Enterococci.
Record the results of the test. See Interpreting and
reporting results
Enterococci
Page 1305
Enterococci

Report enterococci density as the number of colonies per 100 mL of sample. Use samples that
produce 20 to 80 enterococci colonies and not more than 200 colonies of all types per membrane.
Use Equation A to calculate enterococci density. Note that “mL sample” refers to actual sample
volume and not volume of the dilution.
Equation A—Enterococci density on a single membrane filter
Colonies counted
Colonies per 100 mL = ------------------------------------------------------- × 100
• If growth covers the entire filtration area of the membrane or a portion of it and colonies are not
discrete, report results as “Confluent Growth With or Without Enterococci.”
In either case, run a new sample using a dilution that will give about 50 enterococci colonies and
not more than 200 colonies of all types.
average enterococci density with Equation B.
Equation B—Average enterococci density for 1) duplicates, 2) multiple dilutions or 3) more
Colonies per 100 mL = ----------------------------------------------------------------------------------------------------- × 100

m-EI prepared agar plates, 47-mm 15/pkg 2811715
Enterococci
Page 1306
Enterococci
Required apparatus

Bile Esculin Agar 500 g 2815634
Brain Heart Infusion Agar 500 g 2405634
Brain Heart Infusion Broth 500 g 2815534
Enterococci
Page 1307
Enterococci

Incubator, 12-well Dri-Bath, 120 VAC 50/60 Hz each 2281400

Heterotrophic Bacteria MF, m-TGE with TTC, 8242
Heterotrophic Bacteria DOC316.53.01195
Membrane Filtration Method Method 8242

m-TGE Broth with TTC Indicator
Test preparation
Introduction
The Membrane Filter (MF) Heterotrophic Plate Count Method is a way to estimate bacterial
populations in water. Since no single medium can satisfy the growth requirements of all bacteria,
several types of media are offered for detecting heterotophic bacteria in water. The m-HPC
medium, available in both the broth and agar formats, is a high-nutrient medium used to
enumerate heterotrophs in treated potable water samples. The m-TGE broth, originally developed
for use with dairy products, is now commonly used to determine bacterial counts in water by
membrane filtration. The m-TGE broth with TTC contains a redox dye, triphenyltetrazolium
chloride, which colors the colonies red, thus enhancing their visibility. The m-TSB/USP broth is a
general purpose medium which was designed to conform with the formula specified in the
USEPA’s Code of Federal Regulations (21 CFR) for sterility testing of pharmaceutical products.
Convenient packaging
Hach’s m-TGE and m-TGE Broth with TTC come prepared and packaged in glass or plastic
ampules. Prepared ampules eliminate measuring, mixing and autoclaving steps necessary for
preparing dehydrated medium. Break off the top of the ampule and pour the medium onto an
absorbent pad in a petri dish.
Each ampule contains enough selective medium for one test. Packaged medium, when stored
under prescribed conditions, has a shelf-life of one year. Ampules are shipped with a Certificate of
Analysis and have an expiration date printed on the label.
If using PourRite® ampules, all the media should be warm to room temperature before opening.
Heterotrophic Bacteria
Page 1309
m-TGE broth with TTC indicator
1. Place a sterile, 2. Open an ampule of 3. Set up the Membrane 4. Mix the water sample
absorbent pad in a sterile m-TGE with TTC Indicator Filter Assembly. Use by inverting for 30
petri dish (use sterile and carefully pour the sterile forceps to place a seconds. Filter the
forceps to do this) and contents evenly over the membrane filter in the appropriate volume
replace the lid or use absorbent pad. assembly, making sure the through the sterile 47-mm,
a sterile petri dish grid side is up. 0.45-µm, gridded
with pad. Alternatively, a sterile, membrane filter, under
Be careful not to touch the disposable filter unit may partial vacuum1. Rinse the
pad or the inside of the be used. funnel, using 20 to 30 mL
petri dish. portions of sterile buffered
dilution water. Apply
For ease of use, vacuum. Rinse the funnel
petri dishes with pads again for two more times.
are available.
5. Turn off the vacuum 6. With a slight rolling 7. Label the petri dish 8. Remove the dish from
and lift off the funnel top. motion, center the filter, with the sample number, the incubator. Count
Remove the membrane grid side up, on the dilution and date. Invert colonies on membrane
filter, using sterile forceps. absorbent pad. Be sure air the petri dish and incubate filters using a 10 to 15X
Still using the forceps, is not trapped under the at 35 ±0.5 °C for 24 hours. microscope.
transfer the filter filter and that the filter Colonies grown with m-
immediately to the touches the pad at all TGE with TTC Indicator
previously prepared points. Replace the petri appear pink to red aid
petri dish. dish lid. visibility.
1 Release the vacuum once dry so that the filter does not dry out or tear.
Page 1310

Optimal colony density per filter is 20 to 200. Report all colonies counted as colony forming-units
(CFU)/mL. Include in the report the method used, the incubation temperature and time and the
medium. For example: 98 CFU/mL, membrane filter method, 35 °C, 24 hours, m-TGE Broth.
Follow these counting and recording guidelines, based on the number of colonies (on the average)
per square:
1 to 2 or fewer colonies per square:
Count all of the colonies on the filter and divide the results by the volume of original sample used.
Example: if there are 122 colonies on the filter and the volume of original sample used was 10 mL,
compute results as follows:
122 colonies
------------------------------------- = 12.2 CFU/mL
10 mL sample
3 to 10 colonies per square—Count all colonies in 10 representative squares and divide by 10 to
obtain an average number of colonies per square. Multiply this number by 100 and divide by the
volume of original sample used. Example: if you calculated an average of 8 colonies per square
and the volume of original sample used was 0.1 mL, compute results as follows:
8 colonies/square x 100
--------------------------------------------------------------- = 8000 CFU/mL
0.1 mL sample
volume of original sample used. Example: if there are an average of 17 colonies per square and
the volume of original sample used was 0.1 mL, compute results
colonies/square × 100-
as follows: 17
----------------------------------------------------------------- = 17,000 CFU/mL
0.1 mL sample
More than 20 colonies per square—If there are more than 20 colonies per square, record the
count as >2000 divided by the volume of original sample used.
Example—If the original sample volume was 0.01 mL, results would be >2000/0.01 or
>200000 CFU/mL.
Report averaged counts as estimated CFU/mL. Make estimated counts only when there are
discrete, separated colonies without spreaders.
Summary of method
In the initial step, an appropriate sample volume is passed through a membrane filter with a
pore size small enough (0.45 microns) to retain the bacteria present. The filter is placed either
on an absorbent pad (in a petri dish) saturated with a culture medium or on an agar medium
that is selective for heterotrophic bacteria growth. The petri dish containing the filter and pad is
incubated, upside down, for 24 to 48 hours, depending on the medium used, at the
appropriate temperature. After incubation, the colonies which have developed are identified
and counted by using a low-power microscope. The MF method is especially useful for testing
drinking water because large volumes of sample can be analyzed in a short time.
Page 1311

m-TGE Broth with TTC PourRite Ampules 20/pkg 2428420
Dilution Water, Buffered, sterile, 99 mL1 25/pkg 1430598
1 Buffered Dilution Water is prepared with magnesium chloride and potassium dihydrogen phosphate
Required apparatus
Alcohol Burner each 2087742
Ampule Breaker, PourRite each 2484600
Aspirator each 213102
Bags, Whirl-Pak with dechlorinating reagent, sterile, 180-mL 100/pkg 2075333
Filter Holder, magnetic coupling (use with 2486100) each 1352900
Membrane Filters, 0.45-µm, gridded, sterile, Gelman 200/pkg 1353001
Microscope Compound each 2942500
Pump, Vacuum, hand-operated each 1428300
Stopper, Rubber, one hole, No. 8 6/pkg 211908
Tubing, Rubber, 0.8 cm (5/16") ID, 3.7 m (12 ft.) each 56019
Petri dish with pad, 47-mm, Millipore 150/pkg 2936300
Millipore membrane Filters, 0.45-m, gridded, sterile 150/pkg 2936100
Pump, Vacuum/Pressure, Portable, 115 V 60 Hz each 2824800
Page 1312

Potassium Dihydrogen Phosphate and Magnesium Chloride Powder Pillows for buffered 50/pkg 2143166
dilution water (25 of each)1
Sodium Thiosulfate, ACS grade 454 g 46001

Replacement wicks for 2087742 each 2097810
Sterilization Indicator, Sterikon 100/pkg 2811199
Petri dish, 47 mm, sterile, disposable 500/pkg 1485200
Rechargeable battery pack, for portable incubator 12 V DC / 115 V AC adapter each 2580300
230 V AC Rechargeable batter pack adapter (for 2580300) each 2595902
Page 1313
Heterotrophic Bacteria MF, m-TGE Broth, 8242

m-TGE Broth
Test preparation
Introduction
The Membrane Filter (MF) Heterotrophic Plate Count Method is a fast, simple way to estimate
bacterial populations in water. Since no single medium can satisfy the growth requirements of all
bacteria, several types of media are offered for detecting heterotophic bacteria in water. The m-
HPC medium, available in both the broth and agar formats, is a high-nutrient medium used to
preparing dehydrated medium. Simply break off the top of the ampule and pour the medium onto
an absorbent pad in a petri dish.
If using PourRite® ampules, all the media should be warm to room temperature before opening.
Page 1315
m-TGE broth
absorbent pad in a sterile m-HPC and carefully pour Filter Assembly. Use by inverting for 30
petri dish (use sterile the contents evenly over sterile forceps to place a seconds. Filter the
forceps to do this) and the absorbent pad. membrane filter in the appropriate volume
replace the lid or use assembly, making sure the through the sterile 47-mm,
are available.
Alternatively, a prepoured
m-HPC agar plate may be
used.
and lift off the funnel top. motion, center the filter, with the sample number, the incubator. Count the
Remove the membrane grid side up, on the dilution and date. Invert clear to cream colored
filter, using sterile forceps. absorbent pad. Be sure air the petri dish and incubate colonies on membrane
Still using the forceps, is not trapped under the at 35 ±0.5 °C for 48 hours. filters using a 10 to 15X
transfer the filter filter and that the filter microscope.
immediately to the touches the pad at all
previously prepared points. Replace the petri
petri dish. dish lid.
1 Release the vacuum once dry so that the filter does not dry out or tear.
Page 1316

per square:
122 colonies
------------------------------------- = 12.2 CFU/mL
10 mL sample
--------------------------------------------------------------- = 8000 CFU/mL
0.1 mL sample
as follows: 17
----------------------------------------------------------------- = 17,000 CFU/mL
0.1 mL sample
>200000 CFU/mL.
Summary of method
Page 1317

m-TGE Broth PourRite Ampules 50/pkg 2373850
Required apparatus
Ampule Breaker, PourRite each 2484600
Gelman Membrane Filters, 0.45-µm, gridded, sterile 200/pkg 1353001
Pipets, serological, 10–11 mL, sterile, disposable 25/pkg 209798
Page 1318

Potassium Dihydrogen Phosphate and Magnesium Chloride Powder Pillows for 50/pkg 2143166
buffered dilution water (25 of each)1
1 Add the contents of one potassium dihydrogen phosphate and one magnesium chloride powder pillow to one liter of distilled water. Autoclave
(sterilize) to prepare the American Public Health Association buffered dilution water.
Page 1319
Heterotrophic Bacteria MF, m-HPC, 8242

m-HPC, m-TSP-USP, m-TGE Broth or
m-HPC
m-TGE Broth with TTC Indicator
Test preparation
Introduction
The Membrane Filter (MF) Heterotrophic Plate Count Method* is a fast, simple way to estimate
enumerate heterotrophs in treated potable water samples. The m-TGE broth originally developed
preparing dehydrated medium. Simply break off the top of the ampule and pour the medium onto
an absorbent pad in a petri dish.
Disinfect the work bench with a germicidal cloth, dilute bleach solution, bactericidal spray or dilute iodine solution.
Wash hands thoroughly with soap and water.
* Method 8242
Page 1321
m-HPC media
absorbent pad in a sterile m-HPC and carefully pour Filter Assembly. Use by inverting for 30
petri dish (use sterile the contents evenly over sterile forceps to place a seconds. Filter the
forceps to do this) and the absorbent pad. membrane filter in the appropriate volume
For ease of use, vacuum. Repeat rinsing
petri dishes with pads two more times.
are available.
Alternatively, a prepoured
m-HPC agar plate may be
used.
filter, using sterile forceps. absorbent pad. Be sure air the petri dish and incubate colonies on the membrane
1 Release vacuum once dry so that the filter does not dry out and tear.
Page 1322

per square:
122 colonies
------------------------------------- = 12.2 CFU/mL
10 mL sample
--------------------------------------------------------------- = 8000 CFU/mL
0.1 mL sample
as follows: 17
----------------------------------------------------------------- = 17,000 CFU/mL
0.1 mL sample
>200000 CFU/mL.
Summary of method
Page 1323


m-HPC Agar Plates 15/pkg 2811415
m-HPC Broth Ampules, plastic, 2-mL 50/pkg 2812450
1 Buffered Dilution Water is prepared with magnesium chloride and potassium dihydrogen phosphate.
Required apparatus
Gelman, Membrane Filters, 0.45-µm, gridded, sterile 200/pkg 1353001
Pump, Vacuum/Pressure, Portable, 115v, 60 Hz each 2824800
Pump, Vacuum/Pressure, Portable, 220v, 50 Hz each 2824802
Page 1324

Potassium Dihydrogen Phosphate and Magnesium Chloride Powder Pillows for buffered 50/pkg 2143166
dilution water (25 of each)1
Sodium Thiosulfate, ACS grade 454 g 46001
Aspirator, water 1445949 - Isopropyl alcohol, 500 mL each 213102
Sterilization Indicator, Sterikon®, 15/pkg 2811115
230V AC Rechargeable batter pack adapter (for 2580300) each 2595902
Page 1325
Heterotrophic Bacteria MF, m-TSB-USP, 8242

m-TSB-USP
Test preparation
Introduction
The Membrane Filter (MF) Heterotrophic Plate Count Method is a fast, simple way to estimate
preparing dehydrated medium. Break off the top of the ampule and pour the medium on an
absorbent pad in a petri dish.
Disinfect the work bench with a germicidal cloth, dilute bleach solution, bactericidal spray or dilute iodine solution. .
Page 1327
m-TSB-USP media
absorbent pad in a sterile m-TSB-USP and carefully Filter Assembly. Use by inverting for 30
petri dish (use sterile pour the contents evenly sterile forceps to place a seconds. Filter the
forceps to do this) and over the absorbent pad. membrane filter in the appropriate volume
are available.
filter, using sterile forceps. absorbent pad. Be sure air the petri dish and incubate colonies on membrane
1 Release vacuum once dry so that the filter does not dry out and tear
Page 1328

per square:
122 colonies
------------------------------------- = 12.2 CFU/mL
10 mL sample
--------------------------------------------------------------- = 8000 CFU/mL
0.1 mL sample
as follows: 17
----------------------------------------------------------------- = 17,000 CFU/mL
0.1 mL sample
>200000 CFU/mL.
Summary of method
Page 1329

m-TSB/USP Broth Ampules, plastic, 2-mL 50/pkg 2812650
Required apparatus
Gelman Membrane Filters, 0.45-µm, gridded, sterile 200/pkg 1353001
Microscope Compound each 2942500
Page 1330

Potassium Dihydrogen Phosphate and Magnesium Chloride Powder Pillows for 50/pkg 2143166
buffered dilution water (25 of each)1
1 Add the contents of one potassium dihydrogen phosphate and one magnesium chloride powder pillow to one liter of distilled water. Autoclave
(sterilize) to prepare the American Public Health Association buffered dilution water.
Page 1331
Pseudomonas, MF, 8026
Pseudomonas DOC316.53.001214
Membrane Filtration Method Methods 8026

Pseudomonas Broth
Test preparation
colony-forming units (CFU) per filter. The ideal sample volume of nonpotable water or wastewater for bacteria testing yields
20–80 colonies per filter. Generally, for finished, potable water, the volume to be filtered will be 100 mL.
Pseudomonas Broth for detection of Pseudomonas species

Some species of pseudomonads are opportunistic pathogens that can inhabit recreational waters
such as swimming pools and hot tubs. Pseudomonas Broth is formulated to isolate species of
pseudomonas. Pseudomonas Broth contains components that promote the growth of the pigment
pyocyanin, which differentiates Pseudomonas aeruginosa from other species by forming a blue or
blue-green color. Other species of pseudomonas grow on this medium without the colony
color formation.
Pseudomonas
Page 1333
Pseudomonas
Pseudomonas broth, method 8026
1. Place a sterile 2. Invert an ampule 3. Set up the Membrane 4. Invert the sample for
absorbent pad in a sterile containing Pseudomonas Filter Assembly. Use 30 seconds to mix. Pour
petri dish using sterilized Broth 2 to 3 times to mix. sterilized forceps to place 100 mL of sample into the
forceps. Replace the petri Twist the cap to break it a membrane filter, grid funnel. Apply vacuum and
dish lid. open. Carefully pour the side up, into the assembly. filter the sample. Rinse the
Do not touch the pad or contents evenly onto the Alternatively, a sterile funnel walls with 20 to
the inside of the petri dish. absorbent pad. Replace disposable filter unit may 30 mL of sterile buffered
the petri dish lid. Repeat be used. dilution water. Apply
To sterilize forceps, dip steps 1 and 2 for each vacuum. Rinse again two
forceps in alcohol and petri dish. more times.
flame in an alcohol or
Bunsen burner. Let Release the vacuum when
forceps cool before use. the filter is dry to prevent
5. Turn off the vacuum 6. With a slight rolling 7. Invert the petri dish 8. After incubating, count
and lift off the funnel top. motion, center the filter, and incubate at the colonies using an
Using sterilized forceps, grid side up, on the 35 ± 0.5 °C for 22–24 illuminated magnifier or a
transfer the filter absorbent pad. Check for hours. 10 to 15X microscope.
immediately to the air trapped under the filter
previously prepared and make sure that the
petri dish. filter touches the entire
pad. Replace the petri dish
lid.
Pseudomonas
Page 1334
Pseudomonas
Pseudomonas broth, method 8026 (continued)
9. Colonies present on
the filter after incubation
are pseudomonas
species. Colonies with a
blue-green, green or
yellow-green color are
Pseudomonas
aeruginosa.
Record the results of the
test. See Interpreting and
reporting results.

Report pseudomonas density as the number of colonies per 100 mL of sample. Use samples that
produce 20 to 80 pseudomonas colonies and not more than 200 colonies of all types, per
membrane to compute pseudomonas density.
Use Equation A to calculate pseudomonas density. Note that “mL sample” refers to actual sample
volume and not volume of the dilution.
Equation A—Pseudomonas density on a single membrane filter
• If growth covers the entire filtration area of the membrane or a portion of it and colonies are not
discrete, report results as “Confluent Growth With or Without Pseudomonas.”
In either case, run a new sample using a dilution that will give about 50 pseudomonas colonies
and not more than 200 colonies of all types.
average pseudomonas density with Equation B.
Equation B—Average pseudomonas density for 1) duplicates, 2) multiple dilutions or 3)
more than one filter/sample
Colonies per 100 mL = ----------------------------------------------------------------------------------------------------- × 100
Pseudomonas
Page 1335
Pseudomonas

Pseudomonas Broth Ampules, plastic 50/pkg 2812250
Required apparatus
Dish, Petri, with pad, 47-mm, sterile, disposable, Gelman® 100/pkg 1471799
Filters, Membrane, 47-mm, 0.45-µm, gridded, sterile, Gelman® 200/pkg 1353001
Microscope, compound each 2942500

Pseudomonas
Page 1336
Pseudomonas

Pseudomonas
Page 1337
MPN Dilution Guidelines
MPN Dilution Guidelines DOC316.53.01231
Non-potable water samples must be diluted so that a MPN test will have three consecutive
dilutions that contain both positive and negative tubes.
If all of the tubes in a MPN test are positive, the sample must be diluted several more times and
the test must be repeated. If all of the tubes in a MPN test are negative, the sample was diluted too
many times. Repeat the test with less serial dilutions.
Sterile buffered dilution water

Use dilution water that is buffered to a neutral pH and sterilized for microbiological testing. Hach
dilution water is recommended for dilution of most non-potable and wastewater samples. This
solution is packaged in 99 mL bottles. Each bottle contains 99 mL of sterile buffered dilution water.
When 11 mL of sample is added to a 99-mL bottle of dilution water, the sample is diluted by a
factor of 10 (10-fold or 10x dilution). Be sure to mix the bottles thoroughly before and after the
sample is added. The dilution factor of an undiluted sample is 1.
Serial sample dilutions

Complete the following procedure to make serial dilutions of the sample. Refer to Serial dilutions
for non-potable water, MPN test for an illustrated dilution procedure.
Procedure
1. Wash hands.

3. Invert the sample container vigorously for 30 seconds, approximately 25 times, using a waist-
to-ear range of motion.
5. Put the cap on the dilution water bottle and invert (for 30 seconds) 25 times. This is a 10-fold
or 10x dilution (sample is diluted by a factor of 10).
8. Continue to make dilutions until there are three bottles that contain the dilutions listed under
Serial dilutions for non-potable water, MPN test.
Note: Shaking the sample too vigorously will injure or stress the organisms.

Page 1339
Serial dilutions for non-potable water, MPN test

Swimming pool water, chlorinated—Lowest dilution factor = 1
A B C
Undiluted Dilution of 10x Dilution of 100x
99 mL 99 mL
Undiluted pipet 11 mL pipet 11 mL
dilution dilution
sample water water
Inoculate 5 tubes Inoculate 5 tubes Inoculate 5 tubes
Bathing beach water; lake water, unpolluted river water—Lowest dilution factor = 10
B C D
Dilution of 10x Dilution of 100x Dilution of 1000x
Prepared 99 mL 99 mL
pipet 11 mL pipet 11 mL
as B dilution dilution
above water water
Final effluent, chlorinated—Lowest dilution factor = 100

C D E
Dilution of 100x Dilution of 1000x Dilution of 10,000x
as C dilution dilution
above water water

Page 1340
Serial dilutions for non-potable water, MPN test (continued)

River water, polluted—Lowest dilution factor = 1,000
D E F
Dilution of 1000x Dilution of 10,000x Dilution of 100,000x
as D dilution dilution
above water water
Storm water, unchlorinated final effluent—Lowest dilution factor = 10,000

E F G
Dilution of 10,000x Dilution of 100,000x Dilution of 1,000,000x
as E dilution dilution
above water water
Raw sewage—Lowest dilution factor = 100,000

F G H
Dilution of 100,000x Dilution of 1,000,000x Dilution of 10,000,000x
as F dilution dilution
above water water

Page 1341
Coliforms-Fecal, MPN, method 8368
Coliforms—Fecal DOC316.53.01215
USEPA1 A-1 Medium Method 8368

Most Probable Number (MPN) Method
Scope and Application: For non-potable water and wastewater.
1 Most Probable Number Method 8368, A-1 Medium, is USEPA-accepted for testing non-potable waters. Method 8368 meets or exceeds the
specification criteria stated in Standard Methods for the Examination of Water and Wastewater, 18th edition, 9221 E. Fecal Coliform
Procedure. USEPA Manual for the Certification of Laboratories Analyzing Drinking Water states “5.5.3. A-1 medium may be used as an
alternative to EC Medium to enumerate fecal coliforms in source water, in accordance with the Surface Water Treatment Rule. A-1 Medium
must not be used for drinking water samples.”
Make sure that all materials used for containing or transferring samples are sterile.
The bottles of dilution water contain 99 mL of sterile buffered dilution water. When 11 mL of sample is added to a 99-mL
bottle of dilution water, the sample is diluted by a factor of 10 (10-fold or 10x dilution). Be sure to mix the bottles thoroughly
before and after the sample is added.
Refer to Sample Dilution in the Introduction to Bacteria section to find the number of dilutions that must be made for the
sample type. For example if Class A sludge, add a 100X sample dilution to five tubes, a 1000X sample dilution to five tubes
and a 10,000X sample dilution to five tubes. If the coliform density is not known, add five different dilutions to 5 sets of MPN
tubes.
If all the tubes are positive, dilute the sample several more times and repeat the test. If all tubes are negative, the sample
was diluted too many times. Repeat the test with less serial dilutions.
If more than three dilutions were made, use three consecutive dilutions that contain both positive and negative tubes.
The dilution factor for an undiluted sample is 1.
No confirmation is needed when using A-1 Medium.
A-1 Medium Broth Tubes 15
Dilution Water, buffered, 99-mL, sterile 3 bottles
Incubator 1
Pipet, serological, 10-11 mL, sterile 3
Pipet filler 1
Coliform tube rack 1
Coliforms—Fecal
Page 1343
Coliforms—Fecal
Fecal Coliforms, A-1 Medium Broth, method 8368
1. Wash thoroughly with 2. Using sterile buffered 3. Remove the caps from 4. Replace the screw cap
soap and water. dilution water, prepare at 15 tubes of A-1 Medium on each tube immediately
Invert the sample for 30 least 3 serial dilutions of Broth. Use a sterile pipet after the sample is added.
seconds, approximately 25 the sample. to transfer 10 mL of the Invert and swirl the tube
times, to make sure it is Refer to Sample dilution first dilution into 5 tubes. several times to
well-mixed. for instructions. Use a sterile pipet to thoroughly mix the sample
transfer 10 mL of the with the nutrient medium.
second dilution into After the last inversion,
5 tubes. Use a sterile pipet make sure that the inner
to transfer 10 mL of the vial is full of liquid with no
third dilution into the air bubbles.
remaining 5 tubes.
Do not touch the open end
of the tubes or the inside
of the caps.
5. Place the tubes in the 6. After 3 hours, invert 7. After 24 ±2 hours, tap 8. Find the MPN index
incubator at a temperature the tubes to remove each tube gently and per 100 mL from the MPN
of 35 ±0.5 °C . trapped air in the inner examine the inner vials for index table.
Any bubbles that form in vials. Loosen the caps gas. If the inner vials Multiply the MPN index by
the inner vials during this slightly before returning contain gas bubbles, the the dilution factor in the
period are not from the tubes to the incubator. test is positive for fecal first set of tubes (lowest
bacteria. Be sure to Increase the temperature coliform bacteria. Count dilution used in the test).
remove the bubbles by to 44.5 ±0.2 °C and the number of tubes that
contain gas. (MPN index x lowest
inverting the tubes. Make incubate for an additional dilution factor = fecal
sure there are no bubbles 21 hours. The tubes must If none of the tubes coliform bacteria per 100
and then carefully return be kept upright for the rest contain gas, the test is mL sample)
the tubes to an upright of the test. negative for fecal coliform
position. Return the tubes bacteria. See the Example
to the incubator. calculation.
Coliforms—Fecal
Page 1344
Coliforms—Fecal
Sample dilution
Complete the following procedure to make serial dilutions of the sample. Refer to the Dilution
guidelines by sample type to find the number of times the sample must be diluted. Use the three
dilutions from the Dilution guidelines by sample type for the test (step 3 of the fecal coliform
procedure).
Procedure
1. Wash hands.
3. Invert the sample container for 30 seconds, approximately 25 times.
5. Put the cap on the dilution water bottle and invert the sample (for 30 seconds) 25 times. This is
a 10-fold or 10x dilution (sample is diluted by a factor of 10).
8. Continue to make dilutions until there are three bottles that contain the dilutions listed in the
Dilution guidelines by sample type.
Table 376 Dilution guidelines by sample type

Sample type Dilution 1 Dilution 2 Dilution 3
Swimming pool water, chlorinated undiluted (1x) 10x 100x
Bathing beach water 10x 100x 1000x
Lake water 10x 100x 1000x
Unpolluted river water 10x 100x 1000x
Final effluent, chlorinated 100x 1000x 10,000x
River water, polluted 1000x 10,000x 100,000x
Storm water 10,000x 100,000x 1,000,000x
Unchlorinated final effluent 10,000x 100,000x 1,000,000x
Raw sewage 100,000x 1,000,000x 10,000,000x
Example calculation
A sample was diluted into 3 different buffered dilution bottles. The dilutions were 10-fold (10x),
100-fold (100x) and 1000-fold (1000x). 5 MPN tubes were filled from each dilution (15 tubes total).
The first set of tubes (10x) had four tubes with gas, the second set (100x) had 2 tubes with gas
and the third set (1000x) had 1 tube with gas.
1. Find the MPN index for the three sets of tubes from the MPN index table.
2. Multiply the MPN index by the lowest dilution factor.
The MPN index from the MPN index table table for 4, 2 and 1 positive tubes is 26. The coliform
result for the sample is 26 x 10 = 260 coliforms per 100 mL of sample.
Coliforms—Fecal
Page 1345
Coliforms—Fecal
Table 377 MPN index table

Number of positive tubes MPN Number of positive tubes MPN
First dilution Second Third index per First dilution Second Third dilution index per
set dilution set dilution set 100 mL set dilution set set 100 mL
0 0 0 <2 4 2 1 26
0 0 1 2 4 3 0 27
0 1 0 2 4 3 1 33
0 2 0 4 4 4 0 34
1 0 0 2 5 0 0 23
1 0 1 4 5 0 1 30
1 1 0 4 5 0 2 40
1 1 1 6 5 1 0 30
1 2 0 6 5 1 1 50
2 0 0 4 5 1 2 60
2 0 1 7 5 2 0 50
2 1 0 7 5 2 1 70
2 1 1 9 5 2 2 90
2 2 0 9 5 3 0 80
2 3 0 12 5 3 1 110
3 0 0 8 5 3 2 140
3 0 1 11 5 3 3 170
3 1 0 11 5 4 0 130
3 1 1 14 5 4 1 170
3 2 0 14 5 4 2 220
3 2 1 17 5 4 3 280
4 0 0 13 5 4 4 350
4 0 1 17 5 5 0 240
4 1 0 17 5 5 1 300
4 1 1 21 5 5 2 500
4 1 1 26 5 5 3 900
4 2 0 22 5 5 4 1600
5 5 5 ≥ 1600

• Collect at least 100 mL of sample in sterilized Whirl-Pak® bags, sterilized disposable bottles or
autoclaved glass or plastic bottles.
• Do not fill sample containers completely. Leave at least 2.5 cm (approximately 1 inch) of air
space to allow adequate space for mixing the sample prior to analysis.
• Make sure that the samples are representative of the sample source. Fill sample containers
from a tank or reservoir entirely under water.
• Start the analysis as soon as possible after collection. Allow no more than 6 hours to elapse
after collection. If the test cannot be started immediately, cool the sample to below 10 °C. Do
not freeze. Failure to properly collect and transport samples will cause inaccurate results.
Coliforms—Fecal
Page 1346
Coliforms—Fecal
Controls
quality control procedures. Instructions for use come with each AQUA QC-STIK Device. Potable
water samples from municipal treatment facilities should be negative for total and fecal coliforms.
Bacteria disposal
Bleach
Sterilize used test containers with household bleach. Add 1–2 mL of the bleach to each test
container. Allow 10 to 15 minutes contact time with the bleach. Pour the liquid down a drain.
Autoclave
Place used test containers in a contaminated-items bag or a biohazard bag to prevent leakage into
the autoclave. Autoclave the used test containers in the unsealed bag at 121 °C for 30 minutes at
15 pounds pressure. When cool, seal the bag, place it in another garbage bag and tie tightly.
Summary of method
The Most Probable Number (MPN) method (also referred to as the Multiple Tube Fermentation
Technique) uses screw-capped tubes containing sterile broth medium. The tubes contain an
inverted inner vial (Durham tube) for gas collection. Sample is diluted, added to the tubes and
incubated. If coliforms are present, gas is produced and is trapped in the inner vial.
The number of tubes that form gas is used to estimate the number of coliform organisms in the
sample. The MPN method allows for the analysis of highly turbid samples by dilution prior to
analysis. No filtering is necessary.

A-1 Medium Broth Tubes 15/pkg 2560915
Dilution Water, buffered, 99-mL, sterile1 25/pkg 1430598
Required apparatus
Bags, Whirl-Pak®, without dechlorinating agent, 207-mL 100/pkg 2233199
Incubator, 12-well Dri-Bath, 120 VAC, 50/60 Hz each 2281400
Pipet, serological, 10-11 mL, sterile, disposable 25/pkg 209798
Pipet safety bulb each 1465100
Coliforms—Fecal
Page 1347
Coliforms—Fecal

Optional apparatus
Bags for contaminated items 200/pkg 2463300
Bags, Whirl-Pak®, with dechlorinating agent, 180-mL 100/pkg 2075333
Bottle, polysulfone, autoclavable (use for buffered dilution water) 12/pkg 2245300
Bottle, sample, sterilized, 100-mL fill-to line, disposable 12/pkg 2495012
Bottle, sample, sterilized, 100-mL fill-to line, disposable with dechlorinating agent 50/pkg 2599150
Incubator, Portable, 12 VDC each 2569900
Marker, Laboratory each 2092000
Pipet, Serological, 1-mL, sterile, disposable, individually wrapped 50/pkg 2092835
Pipet tips, sterile, individually wrapped 200/pkg 2558996
Pipet Aid, 110 VAC Recharger, 4 replacement filters (UL, CSA approved) each 2551701
Rack, Coliform Tube each 221500

Coliforms—Total and E. Coli, MPN, method 8091
Coliforms—Total and E. Coli DOC316.53.01218
Lauryl Tryptose with MUG Broth1 Method 8091

Scope and Application: For potable and non-potable water.
1 Based on publication by Peter C.S. Feng and Paul A. Hartman “Fluorogenic Assays for Immediate Confirmation of Escherichia coli”. Applied
and Environmental Microbiology, Vol. 43, No. 6, pp. 1320-1329, 1982. This method is not USEPA accepted.
Make sure that all materials used for containing or transferring samples are sterile.
Fluorescence without gas production indicates an anaerogenic (non-gas-producing) strain(s) of E. coli.
Disinfect the work bench with a germicidal cloth, dilute bleach solution, bactericidal spray, or dilute iodine solution. Wash
Lauryl Tryptose with MUG Broth Tubes 5–15
Dilution Water, buffered, 99-mL, sterile (for non-potable water samples only) 3 bottles
Incubator 1
Pipet, serological, 10-11 mL, sterile 3
Pipet filler 1
Coliforms—Total and E. Coli

Page 1349
Potable water test for coliforms—total and E. coli, Lauryl Tryptose with MUG Broth
1. Wash thoroughly with 2. Remove the caps from 3. Replace the screw cap 4. Place the tubes in the
soap and water. 5 or 10 tubes of Lauryl on each tube immediately incubator at a temperature
Invert the sample for 30 Tryptose with MUG Broth after the sample is added. of 35 (±0.5) °C .
seconds, approximately 25 one at a time. Use a sterile Invert the tube several
times, to make sure it is pipet to transfer 10 mL of times to thoroughly mix the
well-mixed. sample into each of the sample with the nutrient
tubes. medium. After the last
Do not touch the open end inversion, make sure the
of the tubes or the inside inner vial is full of liquid
of the caps. with no air bubbles.
5. After one hour, invert 6. After 24 (±2) hours, 7. Use a longwave 8. After 48 (±3) hours,
the tubes to remove tap each tube gently and ultraviolet (UV) lamp to use a longwave ultraviolet
trapped air in the inner examine the inner vials for check the tubes for (UV) lamp to check the
vials. gas. If the broth is cloudy fluorescence. Examine the tubes for fluorescence.
Any bubbles that form in and the inner vials contain tubes in a dark area. Examine the tubes in a
the inner vials during the gas bubbles, coliform If the solution shows dark area.
first hour are not from bacteria are likely present. fluorescence, the test is If the solution shows
bacteria. Remove the The presence of gas in positive for E. coli. fluorescence, the test is
bubbles by inverting the any amount is an If the tube does not positive for E. coli.
tubes. Make sure there are indication of coliform fluoresce, return the tubes If there is no fluorescence,
no bubbles and then bacteria. to the incubator and the test is negative for
carefully return the tubes If none of the tubes examine again after a total E. coli.
to an upright position. contain gas, the test is of 48 (±3) hours. Refer to Potable water
Loosen the caps slightly negative for total coliform Compare the fluorescence MPN results to find the
before returning the tubes bacteria. of the sample tubes to a MPN of the sample.
to the incubator. Continue If tubes are cloudy but tube containing a known
incubation. The tubes have no gas bubbles, E. coli culture to make a
must be kept upright for check the tubes for positive confirmation.
the rest of the test. fluorescence (step 7).

Page 1350
Non-potable water test for coliforms—total and E. coli, Lauryl Tryptose with MUG Broth
soap and water. dilution water, prepare at 15 tubes of Lauryl on each tube immediately
Invert the sample for 30 least 3 serial dilutions of Tryptose with MUG Broth. after the sample is added.
seconds, approximately 25 the sample. Use a sterile pipet to Invert the tube several
times, to make sure that it Refer to Sample dilution transfer 10 mL of the first times to thoroughly mix the
is well-mixed. for instructions. dilution into 5 tubes. Use a sample with the nutrient
sterile pipet to transfer medium. After the last
10 mL of the second inversion, make sure the
dilution into 5 tubes. Use a inner vial is full of liquid
sterile pipet to transfer with no air bubbles.
10 mL of the third dilution
into the remaining 5 tubes.
of the caps.

Page 1351
Non-potable water test for coliforms—total and E. coli, Lauryl Tryptose with MUG Broth
(continued)
5. Place the tubes in the 6. After 24 (±2) hours, 7. Use a longwave 8. After 48 (±3) hours,
incubator at a temperature tap each tube gently and ultraviolet (UV) lamp to use a longwave ultraviolet
of 35 ±0.5 °C . examine the inner vials for check the tubes for (UV) lamp to check the
After one hour, invert the gas. If the broth is cloudy fluorescence. Examine the tubes for fluorescence.
tubes to remove trapped and the inner vials contain tubes in a dark area. Examine the tubes in a
air in the inner vials. gas bubbles, coliform If the solution shows dark area.
Loosen the caps slightly bacteria are likely present. fluorescence, the test is If the solution shows
before returning the tubes The presence of gas in positive for E. coli. fluorescence, the test is
to the incubator. any amount is an If the tube does not positive for E. coli.
Continue incubation. The indication of coliform fluoresce, return the tubes If there is no fluorescence,
tubes must be kept upright bacteria. to the incubator and the test is negative for
for the rest of the test. If none of the tubes examine again after a total E. coli.
contain gas, the test is of 48 (±3) hours. Refer to Non-potable
negative for total coliform Compare the fluorescence water MPN results to find
bacteria. of the sample tubes to a the MPN of the sample.
If tubes are cloudy but tube containing a known
have no gas bubbles, E. coli culture to make a
check the tubes for positive confirmation.
fluorescence (step 7).
Sample dilution
dilutions from the Dilution guidelines by sample type for the test.
Procedure
1. Wash hands.

Page 1352

Storm water 10,000x 100,000x 1,000,000x
Raw sewage 100,000x 1,000,000x 10,000,000x
Potable water MPN results

Use the number of positive tubes to find the MPN per 100 mL from the MPN table for 10 tubes.
Example: 6 of the 10 tubes showed a positive response. The MPN per 100 mL is 9.2.
Table 379 MPN table for 10 tubes1

Number of positive tubes MPN per 100 mL
0 < 1.1
1 1.1
2 2.2
3 3.6
4 5.1
5 6.9
6 9.2
7 12.0
8 16.1
9 23.0
10 > 23.0
1 Table is for undiluted samples, 10 mL per tube. Values are 95 percent confidence limits.
5 broth tubes can be used in place of 10 tubes and the MPN table for 5 tubes can be used.

0 < 2.2
1 2.2
2 5.1
3 9.2
4 16.0
5 > 16.0

Page 1353
Non-potable water MPN results

2. Multiply the MPN index by the lowest dilution factor.

First dilution Second Third index per First dilution Second Third dilution index per
set dilution set dilution set 100 mL set dilution set set 100 mL
0 0 0 <2 4 2 1 26
0 0 1 2 4 3 0 27
0 1 0 2 4 3 1 33
0 2 0 4 4 4 0 34
1 0 0 2 5 0 0 23
1 0 1 4 5 0 1 30
1 1 0 4 5 0 2 40
1 1 1 6 5 1 0 30
1 2 0 6 5 1 1 50
2 0 0 4 5 1 2 60
2 0 1 7 5 2 0 50
2 1 0 7 5 2 1 70
2 1 1 9 5 2 2 90
2 2 0 9 5 3 0 80
2 3 0 12 5 3 1 110
3 0 0 8 5 3 2 140
3 0 1 11 5 3 3 170
3 1 0 11 5 4 0 130
3 1 1 14 5 4 1 170
3 2 0 14 5 4 2 220
3 2 1 17 5 4 3 280
4 0 0 13 5 4 4 350
4 0 1 17 5 5 0 240
4 1 0 17 5 5 1 300
4 1 1 21 5 5 2 500
4 1 1 26 5 5 3 900
4 2 0 22 5 5 4 1600
5 5 5 ≥ 1600

Page 1354

Controls
Bacteria disposal
Bleach
Sterilize used test containers with household bleach. Add 1–2 mL of the bleach to each test
container. Allow 10 to 15 minutes contact time with the bleach. Pour the liquid down a drain.
Autoclave
Place used test containers in a contaminated-items bag or a biohazard bag to prevent leakage into
the autoclave. Autoclave the used test containers in the unsealed bag at 121 °C for 30 minutes at
15 pounds pressure. When cool, seal the bag, place it in another garbage bag, and tie tightly.
Summary of method
inverted inner vial (Durham tube) for gas collection. Sample is diluted, added to the tubes and
incubated. If coliforms are present, gas is produced and is trapped in the inner vial.
The number of tubes that form gas is used to estimate the number of coliform organisms in the
sample. The MPN method allows for the analysis of highly turbid samples by dilution prior to
analysis. No filtering is necessary.
The Lauryl Tryptose with MUG Broth will detect total coliforms and E. coli. The results are
comparable to the traditional MPN fecal coliform tests, but obtained in far less time. No transfer
from presumptive to confirmed medium is necessary with the LT/MUG method. The LT/MUG
medium contains lauryl tryptose broth and 4-methylumbelliferyl-ß-D-glucuronide (MUG), a
fluorogenic reagent. Tubes positive for E. coli will fluoresce when the incubated tubes are
examined under long-wave UV light.

Page 1355

Lauryl Tryptose with MUG Broth Tubes 15/pkg 2182115
Dilution Water, buffered, 99-mL, sterile 25/pkg 1430598
Required apparatus

Lamp, long-wave, ultraviolet, 115 VAC, 60 Hz each 2184300
Lamp, long-wave, ultraviolet, 230 VAC, 50/60 Hz each 2184302

Brilliant Green Bile (BGB) Broth Tubes 15/pkg 32215
Bottle, polysulfone, autoclavable (use for buffered dilution water) 12/pkg 2245300
Bottle, sample, sterilized, 100-mL fill-to line, disposable w/dechlorinating agent 50/pkg 2599150
Ecoli Fluorescence standard each 2361100
Incubator, Portable, 12 VDC each 2569900
Marker, Laboratory each 2092000
Pipet Aid, 110 VAC Recharger, 4 replacement filters (UL, CSA approved) each 2551701

Page 1356

Rack, Coliform Tube each 221500

Page 1357
Coliforms—Total, Fecal and E. coli, MPN, method 8001A
Coliforms—Total, Fecal and E. Coli DOC316.53.01217
USEPA1 Lauryl Tryptose Broth presumptive test with BGB, EC

Method 8001A
Medium and EC/MUG confirmation
Scope and Application: For non-potable water and wastewater.
1 Most Probable Number Method 8001A for wastewater is USEPA-accepted. Method 8001A meets or exceeds the specification criteria stated
in Standard Methods for the Examination of Water and Wastewater, 19th edition, 9221 Multiple-Tube Fermentation Technique for Members of
the Coliform Group.
Make sure that all the materials that are used for containing or transferring samples are sterile.
Use the Lauryl Tryptose Broth for the presumptive test. If the presumptive test is positive, use Brilliant Green Bile (BGB)
broth to confirm if the sample has total coliforms, EC Medium for fecal coliforms or EC Medium with MUG for E. coli. The
confirmation test is used to eliminate false-positive results that can occur with the presumptive test.
For USEPA reporting, the confirmation tubes must be inoculated by an inoculation loop. Cap transfer is not allowed.
To sterilize an inoculating needle, heat the needle to red hot in an alcohol or Bunsen burner. Let the needle cool before use.
Lauryl Tryptose Broth Tubes 15
Brilliant Green Bile (BGB) Broth Tubes varies
EC Medium Broth Tubes varies
EC Medium with MUG Broth Tubes varies
Dilution Water, buffered, 99-mL, sterile 3 or more bottles
Incubator 1
Alcohol burner 1
Inoculating loop 1
Pipet, serological, 10–11 mL, sterile 1
Pipet filler 1
Coliforms—Total, Fecal and E. Coli

Page 1359
Presumptive test for coliform bacteria (Lauryl Tryptose Broth)
soap and water. dilution water, prepare at 15 tubes of Lauryl on each tube immediately
Invert the sample for 30 least 3 serial dilutions of Tryptose Broth one at a after the sample is added.
seconds, approximately 25 the sample. time. Use a sterile pipet to Invert the tube several
times, to make sure that it Refer to Sample dilution transfer 10 mL of the first times to thoroughly mix the
is well-mixed. for instructions. dilution into 5 tubes. Use a sample with the nutrient
sterile pipet to transfer medium. After the last
10 mL of the second inversion, make sure the
dilution into 5 tubes. Use a inner vial is full of liquid
sterile pipet to transfer with no air bubbles.
10 mL of the third dilution
into the remaining 5 tubes.
of the caps.
Positive
Confirm
Results
5. Place the tubes in the 6. After one hour, invert 7. After 24 (±2) hours, 8. Count the number of
incubator at a temperature the tubes to remove tap each tube gently and tubes that contain gas in
of 35 (±0.5) °C . trapped air in the inner examine the inner vials for the inner vial.
Any bubbles that form in vials. gas. If the broth is cloudy Complete a confirmation
the inner vials during the Remove the bubbles by and the inner vials contain test for all tubes that
first hour are not from inverting the tubes. Make gas bubbles, coliform contain gas. The
bacteria. sure there are no bubbles bacteria are likely present. confirmation test will
and then carefully return If no gas can be seen, confirm whether total
the tubes to an upright return the tubes to the coliforms, fecal coliforms
position. incubator and examine or E.Coli are present in the
again after a total of sample.
Loosen the caps slightly 48 (±3) hours.
before returning the tubes If none of the tubes
to the incubator. Continue The presence of gas in contain gas, the test is
incubation. The tubes any amount is an negative for coliform
must be kept upright for indication of coliform bacteria.
the rest of the test. bacteria.

Page 1360
Confirmation test for total coliforms (Brilliant Green Bile Broth)
1. From each positive LT 2. Replace the screw 3. Place the BGB tubes 4. After one hour, invert
Broth tube, inoculate a cap on each tube in the incubator at a the tubes to remove
Brilliant Green Bile (BGB) immediately. Invert the temperature of trapped air in the inner
Broth tube. Use a sterile, BGB tubes to remove 35 (±0.5) °C. vials.
disposable loop or a trapped air in the inner Any bubbles that form in
flame-sterilized, nichrome vials. the inner vials during the
wire. first hour are not from
Put the loop into the bacteria. Remove the
positive Lauryl Tryptose bubbles by inverting the
tube and then into a BGB tubes. Make sure the
Broth tube, making sure bubbles are gone and then
not to touch the rim of carefully return the tubes
either tube. to an upright position.
Loosen the caps slightly
before returning the tubes
to the incubator. Continue
incubation. The tubes
must be kept upright for
the rest of the test.
5. After 24 (±2) hours, 6. After 48 (±3) hours, 7. Find the MPN index per 100 mL from the MPN
tap each tube gently and tap each tube gently and index table.
examine the inner vials for examine the inner vials for Multiply the MPN index by the dilution factor in the first
gas. If the inner vial gas. If the inner vials set of tubes (lowest dilution used in the test).
contains gas bubbles, the contain gas bubbles, the
test is positive for total test is positive for total (MPN index x lowest dilution factor = total coliform
coliform bacteria. coliform bacteria. Count bacteria per 100 mL sample)
If no gas can be seen, the number of tubes that See the Example calculation.
return the tubes to the contain gas.
incubator and examine If none of the tubes
again after a total of contain gas, the test is
48 (±3) hours. negative for coliforms.

Page 1361
Confirmation test for fecal coliforms (EC Medium)
1. From each positive LT 2. Replace the screw 3. Place the EC Medium 4. After one hour, invert
Broth tube, inoculate an cap on each tube Broth tubes in the the tubes to remove
EC Medium Broth tube. immediately. Invert the EC incubator at a temperature trapped air in the inner
Use a sterile, disposable Medium Broth tubes to of 44.5 (±0.2) °C. vials.
loop or a flame-sterilized, remove trapped air in the Any bubbles that form in
nichrome wire. inner vials. the inner vials during the
Put the loop into the first hour are not from
positive Lauryl Tryptose bacteria. Remove the
tube and then into a EC bubbles by inverting the
Medium tube, making sure tubes. Make sure the
not to touch the rim of bubbles are gone and then
either tube. carefully return the tubes
to an upright position.
Loosen the caps slightly
before returning the tubes
to the incubator. Continue
incubation. The tubes
must be kept upright for
the rest of the test.
5. After 24 (±2) hours, 6. Find the MPN index per 100 mL from the MPN
tap each tube gently and index table.
examine the inner vials for Multiply the MPN index by the dilution factor in the first
gas. If the inner vial set of tubes (lowest dilution used in the test).
contains gas bubbles, the
test is positive for fecal (MPN index x lowest dilution factor = fecal coliform
coliform bacteria. Count bacteria per 100 mL sample)
the number of tubes that See the Example calculation.
contain gas.
If none of the tubes
contain gas, the test is
negative for fecal coliforms.

Page 1362
Confirmation test for E. coli (EC Medium with MUG)
1. From each positive LT 2. Place the EC Medium 3. After 24 (±2) hours, 4. Count the number of
Broth tube, inoculate an with MUG tubes in the use a longwave ultraviolet tubes that show
EC Medium with MUG incubator at a temperature (UV) lamp to check the fluorescence.
Broth tube. of 44.5 (±0.2) °C for tubes for fluorescence. Find the MPN index per
Use a sterile, disposable 24 (±2) hours. Examine the tubes in a 100 mL from the MPN
loop or a flame-sterilized, dark area. index table.
nichrome wire. Compare the fluorescence Multiply the MPN index by
Put the loop into the of the sample tubes to a the dilution factor in the
positive Lauryl Tryptose tube containing a known first set of tubes (lowest
tube and then into a EC E. coli culture to make a dilution used in the test).
Medium with MUG tube, positive confirmation.
(MPN index x lowest
making sure not to touch If the solution shows dilution factor = E. coli
the rim of either tube. fluorescence, the test is bacteria per 100 mL
Replace the screw cap on positive for E. coli. If there sample)
each tube immediately. is no fluorescence, the test
is negative for E. coli. See the Example
Invert to mix. calculation.

Note: If more than 6 hours will elapse after collection, refer to the preservation and storage requirements
in Standard Methods for the Examination of Water and Wastewater.
Bacteria disposal
Bleach
Sterilize used test tubes with household bleach. Add 1–2 mL of the bleach to each test tube. Allow
10 to 15 minutes contact time with the bleach. Pour the liquid down a drain.

Page 1363
Autoclave
Sample dilution
dilutions from the Dilution guidelines by sample type for the test (step 3 of the Presumptive test for
coliform bacteria (Lauryl Tryptose Broth)).
Procedure
1. Wash hands.

Storm water 10,000x 100,000x 1,000,000x
Raw sewage 100,000x 1,000,000x 10,000,000x
Example calculation
2. Multiply the MPN index by the lowest dilution factor (e.g. 10 for a 10x dilution).

Page 1364

index per index per
Dilution 1 Dilution 2 Dilution 3 100 mL Dilution 1 Dilution 2 Dilution 3 100 mL
0 0 0 <2 4 2 1 26
0 0 1 2 4 3 0 27
0 1 0 2 4 3 1 33
0 2 0 4 4 4 0 34
1 0 0 2 5 0 0 23
1 0 1 4 5 0 1 30
1 1 0 4 5 0 2 40
1 1 1 6 5 1 0 30
1 2 0 6 5 1 1 50
2 0 0 4 5 1 2 60
2 0 1 7 5 2 0 50
2 1 0 7 5 2 1 70
2 1 1 9 5 2 2 90
2 2 0 9 5 3 0 80
2 3 0 12 5 3 1 110
3 0 0 8 5 3 2 140
3 0 1 11 5 3 3 170
3 1 0 11 5 4 0 130
3 1 1 14 5 4 1 170
3 2 0 14 5 4 2 220
3 2 1 17 5 4 3 280
4 0 0 13 5 4 4 350
4 0 1 17 5 5 0 240
4 1 0 17 5 5 1 300
4 1 1 21 5 5 2 500
4 1 1 26 5 5 3 900
4 2 0 22 5 5 4 1600
5 5 5 ≥ 1600
Controls
water samples from municipal treatment facilities should be negative for total and fecal coliforms.
Summary of method
inverted inner vial (Durham tube) for gas collection. Sample is added to the tubes and incubated. If
coliforms are present, gas is produced and is trapped in the inner vial. The number of tubes that
form gas is used to estimate the number of coliform organisms in the sample. When the EC
Medium with MUG broth is used, fluorescence under a longwave UV lamp confirms the presence
of E. coli.

Page 1365

Lauryl Tryptose Broth Tubes 15/pkg 2101415
Brilliant Green Bile (BGB) Broth Tubes 15/pkg 32215
EC Medium Broth Tubes 15/pkg 1410415
EC Medium with MUG Broth Tubes 15/pkg 2471515
Required apparatus
Inoculating Loop, nichrome wire each 2112100

E. coli Fluorescence standard each 2361100
Inoculating Loops, sterile, disposable 25/pkg 2749125
Lamp, long-wave, ultraviolet, portable, 4 watt each 2415200

Page 1366

Pipet, serological, 1-mL, sterile, disposable, individually wrapped 50/pkg 2092835
Pipet Aid, 110 VAC recharger, 4 replacement filters (UL, CSA approved) each 2551701
Wicks, replacement, for alcohol burner 2087742 2097810

Page 1367
Coliforms—Total, Fecal and E. coli, MPN, method 8001
Coliforms—Total, Fecal and E. Coli DOC316.53.01216
USEPA1 Lauryl Tryptose Broth presumptive test with BGB, EC

Method 8001
Medium and EC/MUG confirmation
Scope and Application: For potable water.
1 Most Probable Number Method 8001 for potable water is USEPA-accepted. Method 8001 meets or exceeds the specification criteria stated in
Standard Methods for the Examination of Water and Wastewater, 19th edition, 9221 Multiple-Tube Fermentation Technique for Members of
the Coliform Group. For potable water, confirm fecal coliforms with EC Medium Broth as cited in 40 CFR Part 141.21, Subpart (F)(5); or
confirm E. coli with EC/MUG Medium Broth as cited in 40 CFR Part 141.21, Subpart (F)(6)(i).
Make sure that all materials that are used for containing or transferring samples are sterile.
Use the Lauryl Tryptose Broth for the presumptive test. If the presumptive test is positive, use Brilliant Green Bile (BGB)
broth to confirm if the sample has total coliforms, then EC Medium for fecal coliforms or EC Medium with MUG for E. coli.
Potable water should not contain any coliform bacteria. Samples should not be diluted.
For USEPA reporting, the confirmation tubes must be inoculated by an inoculation loop. Cap transfer is not allowed.
To sterilize an inoculating needle, heat the needle to red hot in an alcohol or Bunsen burner. Let the needle cool before use.
If all 10 tubes (for a 10-tube MPN test) of the confirmed coliform test are negative, the sample is accepted as meeting
bacterial standards. To make sure that sample results are interpreted in accordance with appropriate standards and
regulations, contact the local, county, state or federal regulatory agency.
If the test will not be used for USEPA reporting, 5 broth tubes can be used in place of 10 tubes. Use the MPN table for 5
tubes to find the result of the 5-tube test. The 5-tube test cannot be used for USEPA reporting.
Lauryl Tryptose Broth Tubes 10
Brilliant Green Bile (BGB) Broth Tubes varies
EC Medium Broth Tubes varies
EC Medium with MUG Broth Tubes varies
Incubator 1
Alcohol burner 1
Inoculating loop 1
Pipet, serological, 10–11 mL, sterile 1
Pipet filler 1

Page 1369
Presumptive test for coliform bacteria (Lauryl Tryptose Broth)
1. Wash thoroughly with 2. Remove the caps from 3. Replace and tighten 4. Place the tubes in the
soap and water. 10 tubes of Lauryl the screw cap on each incubator at a temperature
Invert the sample for 30 Tryptose Broth one at a tube immediately after the of 35 (±0.5) °C .
seconds, approximately 25 time. Use a sterile pipet to sample is added. Invert
times, to make sure it is transfer 10 mL of sample and swirl the tube several
well-mixed. into each of the tubes. times to thoroughly mix the
Do not touch the open end sample with the nutrient
of the tubes or the inside medium. After the last
of the caps. inversion, make sure the
inner vial is full of liquid
with no air bubbles.
Positive
Confirm
Results
5. After one hour, invert 6. After 24 (±2) hours, 7. Complete a

the tubes to remove tap each tube gently and confirmation test for all
trapped air in the inner examine the inner vials for tubes that contain gas.
vials. Loosen the caps gas. If the broth is cloudy The confirmation test will
slightly before returning and the inner vials contain confirm whether total
the tubes to the incubator. gas bubbles, coliform coliforms, fecal coliforms
Continue incubation. The bacteria are likely present. or E.Coli are present in the
tubes must be kept upright If no gas can be seen, sample.
for the rest of the test. return the tubes to the The confirmation test is
incubator and examine used to eliminate false-
Any bubbles that form in again after a total of
the inner vials during the positive results that can
48 (±3) hours. occur with the presumptive
first hour are not from
bacteria. Remove the The presence of gas in test.
bubbles by inverting the any amount is an If none of the tubes
tubes. Make sure there are indication of coliform contain gas, the test is
no bubbles and then bacteria. negative for coliform
carefully return the tubes Count the number of tubes bacteria.
to an upright position. that contain gas in the
inner vial.

Page 1370
Confirmation test for total coliforms (Brilliant Green Bile Broth)
1. From each positive LT 2. Replace and tighten 3. Place the BGB tubes 4. After one hour, invert
Broth tube, inoculate a the screw cap on each in the incubator at a the tubes to remove
Brilliant Green Bile (BGB) tube immediately. Invert temperature of trapped air in the inner
Broth tube. Use a sterile, and swirl the BGB tubes to 35 (±0.5) °C. vials. Loosen the caps
disposable loop or a remove trapped air in the slightly before returning
flame-sterilized, nichrome inner vials. the tubes to the incubator.
wire. If Durham tube vials are Continue incubation. The
Put the loop into the used, after the last tubes must be kept upright
positive Lauryl Tryptose inversion, make sure that for the rest of the test.
tube and then into a BGB the inner tube does not Any bubbles that form in
Broth tube, making sure contain any air bubbles. the inner vials during the
not to touch the rim of first hour are not from
either tube. bacteria. Remove the
bubbles by inverting the
tubes. Make sure the
bubbles are gone and then
carefully return the tubes
Positive
Confirm
Results
5. After 24 (±2) hours, 6. After 48 (±3) hours, 7. Count the number of 8. If the test is positive
tap each tube gently and tap each tube gently and tubes that contain gas in for total coliform bacteria,
examine the inner vials for examine the inner vials for the inner vial. complete a confirmation
gas. If the inner vial gas. If the inner vials Find the MPN of the test for fecal coliform or
contains gas bubbles, the contain gas bubbles, the sample (total coliform E.Coli bacteria (USEPA
test is positive for total test is positive for total bacteria per 100 mL requirement).
coliform bacteria. coliform bacteria. sample) from the MPN
If no gas can be seen, If none of the tubes table for 10 tubes.
return the tubes to the contain gas, the test is
incubator and examine negative for coliform
again after a total of bacteria.
48 (±3) hours.

Page 1371
Confirmation test for fecal coliforms (EC Medium)
1. From each positive LT 2. Replace and tighten 3. Place the EC Medium 4. After one hour, invert
Broth tube, inoculate an the screw cap on each Broth tubes in the the tubes to remove
EC Medium Broth tube. tube immediately. Invert incubator at a temperature trapped air in the inner
Use a sterile, disposable and swirl the EC Medium of 44.5 (±0.2) °C. vials. Loosen the caps
loop or a flame-sterilized, Broth tubes to remove slightly before returning
nichrome wire. trapped air in the inner the tubes to the incubator.
vials. Continue incubation. The
Put the loop into the
positive Lauryl Tryptose If Durham tube vials are tubes must be kept upright
tube and then into a EC used, after the last for the rest of the test.
Medium tube, making sure inversion, make sure that Any bubbles that form in
not to touch the rim of the inner tube does not the inner vials during the
either tube. contain any air bubbles. first hour are not from
bacteria. Remove the
bubbles by inverting the
tubes. Make sure the
bubbles are gone and then
carefully return the tubes
5. After 24 (±2) hours, 6. Count the number of

tap each tube gently and tubes that contain gas in
examine the inner vials for the inner vial.
gas. If the inner vial Find the MPN of the
contains gas bubbles, the sample (fecal coliform
test is positive for fecal bacteria per 100 mL
coliform bacteria. sample) from the MPN
If none of the tubes table for 10 tubes.
contain gas, the test is
negative for fecal coliform
bacteria.

Page 1372
Confirmation test for E. coli (EC Medium with MUG)
1. From each positive LT 2. Place the EC Medium 3. After 24 (±2) hours, 4. Count the number of
Broth tube, inoculate an with MUG tubes in the use a longwave ultraviolet tubes that show
EC Medium with MUG incubator at a temperature (UV) lamp to check the fluorescence.
Broth tube. of 44.5 (±0.2) °C for tubes for fluorescence. Find the MPN of the
Use a sterile, disposable 24 (±2) hours. Examine the tubes in a sample (E. coli bacteria
loop or a flame-sterilized, dark area. per 100 mL sample) from
nichrome wire. Compare the fluorescence the MPN table for 10
Put the loop into the of the sample tubes to a tubes.
positive Lauryl Tryptose tube containing a known
tube and then into a EC E. coli culture to make a
Medium with MUG tube, positive confirmation.
making sure not to touch If the solution shows
the rim of either tube. fluorescence, the test is
Replace and tighten the positive for E. coli. If there
screw cap on each tube is no fluorescence, the test
immediately. Invert and is negative for E. coli.
swirl to mix.
If Durham tube vials are
used, after the last
inversion, make sure that
the inner tube does not
contain any air bubbles.


Page 1373
MPN table
Use the number of positive tubes to find the MPN per 100 mL from the MPN table for 10 tubes.
Example: 6 of the 10 tubes showed a positive response. The MPN per 100 mL is 9.2.

0 < 1.1
1 1.1
2 2.2
3 3.6
4 5.1
5 6.9
6 9.2
7 12.0
8 16.1
9 23.0
10 > 23.0
If the test will not be used for USEPA reporting, 5 broth tubes can be used in place of 10 tubes and
the MPN table for 5 tubes can be used. The 5-tube test cannot be used for USEPA reporting.

0 < 2.2
1 2.2
2 5.1
3 9.2
4 16.0
5 > 16.0
1 Table is for undiluted samples, 10 mL per tube. Values are 95 percent confidence limits. The MPN table for 5 tubes
cannot be used for USEPA reporting.
Bacteria disposal
Bleach
Sterilize used test tubes with household bleach. Add 1–2 mL of the bleach to each test tube. Allow
10 to 15 minutes contact time with the bleach. Pour the liquid down a drain.
Autoclave

Page 1374
Summary of method
inverted inner vial (Durham tube) for gas collection. Sample is added to the tubes and incubated. If
coliforms are present, gas is produced and is trapped in the inner vial. The number of tubes that
form gas is used to estimate the number of coliform organisms in the sample. When the EC
Medium with MUG broth is used, fluorescence under a longwave UV lamp confirms the presence
of E. coli.

Lauryl Tryptose Broth tubes 15/pkg 2101415
Brilliant Green Bile (BGB) Broth tubes 15/pkg 32215
EC Medium Broth tubes 15/pkg 1410415
EC Medium with MUG Broth tubes (without Durham tubes) 15/pkg 2471515
EC Medium with MUG Broth tubes (with Durham tubes) 15/pkg 2282415
Required apparatus
Inoculating Loop, nichrome wire each 2112100


Page 1375
Optional apparatus
E. coli Fluorescence standard each 2361100
Inoculating Loops, sterile, disposable 25/pkg 2749125
Lamp, long-wave, ultraviolet, portable, 4 watt each 2415200
Pipet Aid, 110 VAC recharger, 4 replacement filters (UL, CSA approved) each 2551701
Wicks, replacement, for alcohol burner 2087742 2097810

Bacteria, Hydrogen Sulfide Producing, MPN, 10032
Bacteria, Hydrogen Sulfide

Producing DOC316.53.01196
Most Probable Number (MPN) Method Method 10032

Pathoscreen™ Medium
Scope and Application: For the detection of Salmonella, Citrobacter, Proteus, Edwardsiella and Klebsiella (some
spp.) in drinking water, surface water and recreational water
Test preparation
PathoScreen Medium
PathoScreen Medium detects the presence of hydrogen sulfide-producing bacteria including
Salmonella, Citrobacter, Proteus, Edwardsiella and some species of Klebsiella. The sterilized
powder medium is a reliable medium for monitoring drinking water systems in developing tropical
countries, in remote field locations and in disaster or emergency situations.
PathoScreen Medium is dehydrated, sterilized and packaged in powder pillows. Powder pillows
are available for both Presence/Absence (P/A) and Most Probable Number (MPN) testing. Each
powder pillow contains enough medium for one test. The medium is shipped with a Certificate of
Analysis and has an expiration date printed on the label.
For MPN testing, add one MPN Pillow to a 20-mL sample. For MPN testing, inoculate a set of five
tubes.
Conducting MPN tests with PathoScreen Medium

The MPN method can be used for drinking water, as well as marine and fresh recreational waters,
swimming pools, lakes, shellfish-growing waters, heavily polluted waters and wastewater. For
water that is heavily contaminated, use the multiple tube decimal dilution procedure.
Incubate samples 24–48 hours between 25–35 °C, 77–95 °F. (30 °C, 80 °F is considered optimal.)
PathoScreen Medium has a detection sensitivity of 1 CFU/100 mL.
Bacteria, Hydrogen Sulfide Producing

Page 1377
PathoScreen medium MPN pillows, method 10032
1. Wash hands 2. Remove the caps from 3. Swab the end of a 4. Cap and swirl each
thoroughly with soap and five sterile tubes one at a PathoScreen Medium tube and immediately.
water. time and pipet 20 mL of MPN Pillow with alcohol Invert the tubes a few
sample into each of the and aseptically cut it open times to thoroughly mix the
tubes with a sterile pipet. with clippers. Add pillow sample with the medium.
Use aseptic technique to contents to the 20 mL The sample will be yellow
avoid contaminating the sample. in color.
tubes or the caps.
5. Place the tubes in a 7. Note the reaction after 8. Record results. (See Dispose of all completed
location with a constant 24 hours of incubation. the Five-tube MPN values tests appropriately. Refer
temperature of 25–35 °C (See the MPN Results table.) to the MSDS and local
for 24–48 hours. table.) regulations for proper
6. If an incubator is If the temperature varies disposal.
available, incubate the significantly, continue to
sample at 30 ± 0.5 °C for incubate negative tubes
24–48 hours. for an additional day.
Table 386 MPN Results

Hydrogen sulfide producing bacteria
Test Results Positive Negative Follow-up

Color changes from yellow to black X
Black precipitate forms X
X Incubate additional 12–24 hours and re-
No color change evaluate. If there is no color change,
record as negative.

Page 1378
Using statistical methods it is possible to estimate the number of organisms from any combination
of positive and negative test results. The MPN values in the Five-tube MPN values table are based
on 20 mL of undiluted sample in each of five tubes. If the sample is diluted, multiply the result by
the dilution factor.
Example 1:
Five tubes of undiluted sample are inoculated. Positive results are obtained from three of the five
tubes. The result obtained from the Five-tube MPN values table is 4.6.
Example 2:
A river water sample is collected and diluted. A dilution factor of 10,000 is prepared and five tubes
are inoculated. Positive results are obtained from two of the five tubes. The result obtained from
two of the five tubes. The result obtained from the Five-tube MPN values table is 2.6. This result is
multiplied by 10,000 and a result of 26,000 is recorded.
Table 387 Five-tube MPN values

Positive Tubes MPN/100 mL
0 <1.1
1 1.1
2 2.6
3 4.6
4 8.0
5 >8.0
Required reagents
PathoScreen™ Medium, MPN Pillows, 20-mL sample 50/pkg 2610796
Dilution water—media, reagents and apparatus

Buffered Dilution Water, sterile, 99-mL1 25/pkg 1430598

Pipet, sterile, disposable, 11-mL 25/pkg 209798
Pipet, sterile, disposable, individually wrapped, 10-mL 50/pkg 2092628
Pipet, sterile, disposable, 10-mL 50/pkg 2092828
Pipet Filler, portable, with recharger (UL, CSA approved), 110 VAC each 2551701

Page 1379
Required apparatus
Autoclave, Automatic, 120 VAC each 2898600
Clippers, large each 2065800
Contaminated Items Bags 200/pkg 2463300
Germicidal Cloth 50/pkg 2463200
Incubator, Culture, 120 VAC each 2619200
MPN Vials 10/pkg 1497054
Rack for coliform tubes each 2497903
Sampling Containers
Sampling Bags, Whirl-Pak with dechlorinating agent, 180-mL 100/pkg 2075333
Sampling Bottles, autoclavable 6/pkg 2324333
Sampling Bottles, sterilized, 100-mL fill-to line 12/pkg 2495012
Sampling Bottles, sterilized, 100-mL fill-to line, with dechlorinating agent 12/pkg 2599112

Bottle, polysulfone, autoclavable (for preparing buffered dilution water) 12/pkg 2245300
Magnesium Chloride and Potassium Dihydrogen Phosphate Powder Pillows 25 of each 2143166
Incubator, 12-well, Dri Bath each 2281400

Coliforms-Total, Fecal and E. coli, P/A, 8319 and 8364
Coliforms DOC316.53.01191
Presence/Absence Methods 8319 and 8364

P/A Broth (8319)
P/A Broth w/ MUG (8364)
Test preparation
Disinfect the work bench with a germicidal cloth, dilute bleach solution, bactericidal spray, or dilute iodine solution
Introduction
Both P/A Broth and P/A Broth with MUG come packaged in disposable bottles and in glass
ampules. The medium in both bottles and ampules is concentrated and must be diluted with sterile
water 5:1. The medium is sterilized by membrane filtration to prevent degradation.
P/A Broth ampules, method 8319
1. Collect 100 mL of 2. Add sample to the fill- 3. Add the contents of 4. Incubate the sample at
sample in a sterile sample to line of a sample bottle. one P/A Broth ampule to 35 ± 0.5 °C for
container. Use aseptic Sample may be added the 100 mL of sample. 24–48 hours.
technique to avoid from a sterile container, or
contaminating the sample directly from a faucet or
and sample container. spigot.
Coliforms
Page 1381
Coliforms
P/A Broth ampules, method 8319
Confirm
Samples
Confirm Positive
Positive Dispose of all
Samples completed tests
5. Note the reaction after 6. Confirm presumptive 7. Dispose of all

24 hours of incubation. positive samples by completed tests
See Interpret and report inoculating the appropriate appropriately.
results on page 1383. media directly from Dispose of all completed
positive tests
P/A broth cultures. See the
Confirmation Table on
page 1383.
P/A broth with MUG, method 8364
1. Collect 100 mL of 2. Add sample to the fill- 3. Incubate the sample at 4. Note the reaction after
sample in a sterile sample to line of a Broth 35 ±0.5 °C for 24 hours of incubation.
container. Use aseptic Disposable Bottle. 24–48 hours. See Interpret and report
technique to avoid Sample may be added results.
contaminating the sample from a sterile container, or
and sample container. directly from a faucet or
spigot.
Coliforms
Page 1382
Coliforms
P/A broth with MUG, method 8364 (continued)
Confirm Positive Dispose of all

Samples completed tests
5. Confirm presumptive 6. Dispose of all

positive samples by completed tests
inoculating the appropriate appropriately.
media directly from
positive P/A broth cultures.
See the Confirmation
Table.
Interpret and report results

Reactions using P/A Broth
• Color change from reddish purple to yellow or yellow brown—record the test as presumptive
positive for total coliform bacteria.
• No color change—incubate for an additional 24 hours and recheck the sample for
color change.
If after 24 hours of incubation, the color changes from reddish purple to yellow or yellow
brown—record the test as presumptive positive for total coliform bacteria.
If after 48 ±3 hours of incubation, the sample still appears reddish purple—record the test as
negative for total coliform bacteria.
Reactions using P/A Broth with MUG

• Examine the sample under long-wave UV light; if the sample fluoresces—record the test as
positive for E. coli.
Confirm positive samples

Inoculum from incubated presence/absence samples are used to confirm the presence of total
coliforms, fecal coliforms or E. coli in the original water sample. Use a sterile inoculating loop to
transfer sample to an appropriate confirmation medium.
Table 388 Confirmation Table
Bacteria Confirmation Medium Incubation Incubator Positive Result
Total Coliform Brilliant Green Bile Broth 24 to 48 hours. Culture (2619200 or -02) Gas/Turbidity
(322-15) 35 ± 0.5 °C or 12-well Dri-Bath
(2281400)
Fecal Coliform EC Medium Tubes 24 hours 12-well Dri-Bath Gas/Turbidity
(14104-15) 44.5 ± 0.2 °C (2281400)
E. coli EC Medium with MUG Tubes 24 hours 12-well Dri-Bath Fluorescence
(24715-15) 44.5 ± 0.2 °C (2281400)
Coliforms
Page 1383
Coliforms
Controls
water samples from municipal treatment facilities should be negative for total coliforms and fecal
coliforms.
Completed test disposal

Active bacterial cultures grown during incubation must be disposed of safely. Use either Bleach or
an Autoclave to neutralize bacteria.
Bleach
1. Sterilize used test containers with a 10% bleach solution.
2. Add approximately 12 mL of bleach to each test containerand allow to stand for 10 to 15

minutes.
3. Pour the liquid down the drain. Dispose of the test tubes with the normal garbage.
Autoclave
Test containers must be placed in a bag before autoclaving to prevent leakage of solution into the
autoclave.
1. Place used test tubes in a contaminated-items bag or a biohazard bag and seal tightly.
1. Autoclave the used test tubes in the bag at 121 °C for 15 minutes at 15 pounds of pressure.
2. Place the bag of test tubes in a separate garbage bag and tie tightly.
3. Once the test tubes are sterile, dispose of them with the normal garbage.
Summary of method 8319

P/A Broth is ideal for screening drinking water samples for total coliforms. The method is a simple
modification of the multiple-tube method. It uses lactose and lauryl tryptose broths with bromcresol
purple, which detects acidity formed during lactose fermentation by the bacteria.
Simply combine 100 mL of sample and P/A Broth, incubate for 24 hours and check for a color
change. A yellow color indicates the presence of total coliforms.
Summary of method 8364

P/A Broth with MUG allows simultaneous detection of total coliform bacteria and E. coli. In addition
to the lactose and lauryl tryptose broths with bromcresol purple, this medium contains MUG
reagent (4-methylumbelliferyl-b-D-glucuronide). The MUG reagent produces a fluorogenic product
when hydrolyzed by glucuronidase (an enzyme specific to E. coli). MUG detects non gas-
producing (anaerogenic) strains of E. coli and works well when competitive organisms are present.
Combine 100 mL of sample and P/A Broth with MUG, incubate for 24 hours and check for a color
change and fluorescence. A yellow color indicates the presence of total coliforms. To detect E. coli,
examine samples under a long-wave ultraviolet (UV) light. Fluorescence indicates the presence of
E. coli.
Coliforms
Page 1384
Coliforms

P/A Broth Ampules

P/A Broth Ampules 25/pkg 2494925
P/A Broth with MUG Ampules 25/pkg 2495525
P/A Broth in Disposable Bottles
P/A Broth Disposable Bottles 12/pkg 2323212
P/A Broth Disposable Bottles 50/pkg 2323250
P/A Broth with MUG Disposable Bottles 12/pkg 2401612
P/A Broth with MUG Disposable Bottles 50/pkg 2401650
Required apparatus
Bottles, sample, presterilized, 100-mL fill-to line 12/pkg 2495012
Incubator, Culture, low profile, 110/120 VAC, 50/60 Hz each 2619200
Lamp, handheld, long-wave, ultraviolet, 115 VAC, 60 Hz each 2184300
Optional apparatus
Bags, Whirl-Pak®1, with dechlorinating agent 100/pkg 2075333
Bottles, sample, presterilized, with dechlorinating agent, 100-mL fill-to line 12/pkg 2599112
Bottles, sample, presterilized, with dechlorinating agent, 100-mL fill-to line 12/pkg 2599150
Lamp, long-wave, ultraviolet, portable each 2415200
Incubator, Dri-Bath, 12-Well, 120V each 2281400
Bags, Whirl-Pak1, without dechlor 207 mL 100/pkg 2233199
Bags, Whirl-Pak1, without dechlor 720 mL 10/pkg 1437297
Dechlorinating reagent, powder pillows 100/pkg) 1436369
Inoculating loop, nichrome V wire each 2112100
Inoculating loop, disposable 25/pkg 2749125
Alcohol burner each 2087742
Coliforms
Page 1385
Coliforms
Optional apparatus
230V AC Rechargeable batter pack adapter (for 2580300) rach 2595902
Optional media

Bacteria, Hydrogen Sulfide Producing, P/A 8506
Bacteria, Hydrogen Sulfide

Producing DOC316.53.01197
Presence/Absence (P/A) Method Methods 8506

Pathoscreen™ Medium
Scope and Application: For the detection of Salmonella, Citrobacter, Proteus, Edwardsiella and Klebsiella (some
spp.) in drinking water, surface water and recreational water
Test preparation
PathoScreen Medium
PathoScreen Medium detects the presence of hydrogen sulfide-producing bacteria including
Salmonella, Citrobacter, Proteus, Edwardsiella and some species of Klebsiella. The sterilized
powder medium is a reliable medium for monitoring drinking water systems in developing tropical
countries, in remote field locations and in disaster or emergency situations.
PathoScreen Medium is dehydrated, sterilized and packaged in powder pillows. Powder pillows
are available for both Presence/Absence (P/A) and Most Probable Number (MPN) testing. Each
powder pillow contains enough medium for one test. The medium is shipped with a Certificate of
Analysis and has an expiration date printed on the label.
For P/A testing, add one P/A powder pillow to a 100-mL sample.
Incubate samples 24–48 hours between 25–35 °C, 77–95 °F. (30 °C, 80 °F is considered optimal.)
PathoScreen Medium has a detection sensitivity of 1 CFU/100 mL.

Page 1387
PathoScreen medium P/A pillows, method 8506
1. Wash hands 2. Collect 100 mL of 3. Swab the end of the 4. Place the bottle in a
thoroughly with soap and sample in a sterile sample PathoScreen Medium location with a constant
water. container. P/A Pillow with alcohol and temperature between
aseptically cut it open with 25–35 °C for 24–48 hours.
clippers. Add pillow If an incubator is available,
contents to the 100 mL incubate the sample at 30
sample. ±0.5 °C for 24 to 48 hours.
5. Note the reaction after 6. Record the results. Dispose of all completed tests appropriately. Refer to a
24 hours of incubation. (See the P/A results table.) current MSDS and local regulations.
If the temperature varies
significantly, incubation
may be extended an
additional day.
Table 389 P/A results

Hydrogen sulfide producing bacteria
Test Results Positive Negative Follow-up

Color changes from yellow to black X
Black precipitate forms X
X Incubate additional 12–24 hours and re-evaluate. If there is no
No color change
color change, record as negative.

Page 1388
Required reagents
PathoScreen™ Medium, P/A Pillows, 100-mL sample 50/pkg 2610696
Dilution water—media, reagents and apparatus

Buffered Dilution Water, sterile, 99-mL1 25/pkg 1430598
Required apparatus
Autoclave, Automatic, 120 VAC each 2898600
Clippers, large each 2065800
Contaminated Items Bags 200/pkg 2463300
Germicidal Cloth 50/pkg 2463200
Sampling Containers
Sampling Bags, Whirl-Pak with dechlorinating agent, 180-mL 100/pkg 2075333

Magnesium Chloride and Potassium Dihydrogen Phosphate Powder Pillows 25 of each 2143166

Page 1389
Heterotrophic Bacteria, m-HPC, 8242
Pour Plate Method Method 8242

m-HPC
Test preparation
Introduction
The Pour Plate Method, also known as the standard plate count, is simple to perform and is
commonly used to determine heterotrophic bacteria density. This method does, however, have
disadvantages that limit recovery of the maximum number of organisms. Tempered medium at
44–46 °C (111–115 °F) may cause heat shock to stressed bacteria and the nutritionally rich
medium may decrease recovery of starved bacteria.
The standard plate count attempts to provide a standardized means of determining the density of
aerobic and facultatively anaerobic heterotrophic bacteria in water. Bacteria occur singly or in
pairs, chains, clusters or packets, and no single method, growth medium, or set of physical
conditions can satisfy the physiological requirements of all bacteria in a water sample. However,
the heterotrophic plate count is a good measure of water treatment plant efficiency, aftergrowth in
transmission lines, and the general bacterial composition of source water.
See the Introduction to Bacteria for more information about preparing sample containers and collecting and
preserving samples.
To sterilize the forceps, dip them in alcohol and flame in an alcohol or Bunsen burner. Let the forceps cool before use.
Limit the number of samples to be plated at any one time so that no more than 20 minutes (preferably 10 minutes) elapse
between the dilution of the first sample and the pouring of the last plate.
To save time, start the incubator before preparing the other materials. Set the incubator for the temperature required in the
procedure (usually 35 ± 0.5 °C).
Mark each pour plate, membrane filtration petri dish, or other sample container with the sample number, dilution, date, and
any other necessary information. Take care not to contaminate the inside of the sample container in any way.
Page 1391
Pour plate procedure for heterotrophic bacteria m-HPC, method 8242
to place a sterile, three times to mix broth. Filter Assembly. Use 30 seconds, approximately
absorbent pad in a sterile Open an ampule of sterile forceps to place a 25 times, to make sure it is
petri dish. Replace the lid m-HPC Broth, using an membrane filter, grid side well-mixed. Filter the
on the dish. Do not touch ampule breaker if up, in the assembly. appropriate volume
the pad or the inside of necessary. Pour the Alternatively, a sterile, through the sterile 47 mm,
the petri dish. contents evenly over the disposable filter unit may 0.45µm, gridded
To sterilize the forceps, dip absorbent pad. Replace be used. membrane filter. Apply
them in alcohol and flame the petri dish lid. vacuum and filter the
in an alcohol or Bunsen For broth prepared from sample. Rinse the funnel
burner. Let the forceps dehydrated medium, pipet walls three times with
cool before use. approximately 2.0 mL of 20–30 mL of sterile
broth onto the pad using a buffered dilution water.
Alternatively, a prepared
m-HPC agar plate may be sterile pipet. Drain excess
used. medium from the petri dish
and replace the lid.
and lift off the funnel top. motion, place the filter, grid with the sample number, the incubator. Count
Remove the membrane side up, on the absorbent dilution and date. Invert colonies on membrane
filter, using sterile forceps. pad. Check for trapped air the petri dish and incubate filters using a 10–15X
Still using the forceps, under the filter and make at 35 ±0.5 °C for 48 hours. microscope.
transfer the filter sure the filter touches the Bacterial colonies grown
immediately to the entire pad. Replace the on m-HPC medium appear
previously prepared petri dish lid. clear to cream in color.
petri dish.
Page 1392
Diluting the Sample

The pour plate method requires use of 1 mL, 0.1 mL, and 0.01 mL or 0.001 mL of sample. The
difficulty measuring and working with the two smaller volumes, 0.01 and 0.001 mL, require the use
of sample dilutions. These dilutions are prepared by pipetting 1 mL of undiluted sample into 99 mL
of buffered dilution water. Diluting the sample allows 1 mL of diluted sample to be used instead of
0.01 mL of undiluted sample, and 0.1 mL of diluted sample instead of 0.001 mL of undiluted
sample.
Selecting Sample Volumes/Dilutions

Select the sample volumes or dilutions to be used so that the total number of colonies on a plate
will be between 30 and 300. For most potable water samples, plates suitable for counting will be
obtained by plating 1 mL of undiluted sample, 0.1 mL of undiluted sample and 1 mL of diluted
sample (which equals 0.01 mL of undiluted sample). In examining sewage or turbid water, do not
measure a 0.1-mL inoculum of the original undiluted sample, but do prepare an
appropriate dilution.
Counting, Computing and Reporting Results

(CFU)/mL. Include in the report the method used, the incubation temperature and time, and
the medium.
For example: 98 CFU/L, mL, 35 °C, 24 hours, m-HPC broth.
1 to 2, or fewer colonies per square — Count all of the colonies on the filter, and divide the
results by the volume of original sample used.
For example, if there are 122 colonies on the filter, and the volume of original sample used was 10
mL, compute results as follows:
122 colonies
------------------------------------- = 12.2 CFU/mL
10 mL sample
3 to 10 colonies per square — Count all colonies in 10 representative squares and divide by 10
to obtain an average number of colonies per square. Multiply this number by 100 and divide by the
volume of original sample used.
For example, if you calculated an average of 8 colonies per square, and the volume of original
sample used was 0.1 mL, compute results as follows:
--------------------------------------------------------------- = 8000 CFU/mL
0.1 mL sample
10 to 20 colonies per square — Count all colonies in 5 representative squares and divide by 5 to
For example: if there are an average of 17 colonies per square, and the volume of original sample
used was 0.1 mL, compute results as follows:
------------------------------------------------------------------ = 17, 000 CFU/mL
0.1 mL sample
Page 1393
count as > 2000 divided by the volume of original sample used.
For example, if the original volume of sample used were 0.01 mL, results would be > 2000/0.01 or
> 200,000 CFU/mL.
Note: Report averaged counts as estimated CFU/mL. Make estimated counts only when there are discrete,
separated colonies without spreaders.

Dilution Water, Buffered, sterile, 99-mL 25/pkg 1430598
m-HPC Agar Plates 15/pkg 2811415
m-HPC Broth Ampules, plastic, 2-mL 50/pkg 2812450
Required apparatus
Absorbent Pads with dispenser, sterile, Gelman 1000/pkg 1491800
Whirl-Pak Bags with declorinating agent, sterile, 180-mL 100/pkg 2075333
Forceps each 2141100
Incubator, Culture, low profile, 110 VAC each 2619200
Membrane filters, 0.45-µm, gridded, sterile, Gelman 200/pkg 1353001
Membrane filters, 0.45-µm, gridded, sterile, Millipore 150/pkg 2936100
Petri Dish, polystyrene, sterile, disposable, without pad 100/pkg 1485299
Petri Dish, polystyrene, sterile, disposable, w/pad, Gelman 100/pkg 1471799
Petri Dish, polystyrene, sterile, disposable, w/pad, Millipore 150/pkg 2936300
Pump, vacuum, 110/115 VAC each 2824800
Pump, vacuum, 220/230 VAC, Continental European Plug each 2824802
Rubber Stopper, one hole, No. 8 6/pkg 211908
Rubber Tubing, 3.6-m each 56019
Pipets, Serological, 10–11 mL, sterile, disposable 25/pkg 209798

Page 1394

Microscope, Stereo Binocular each 2942600
Page 1395
Heterotrophic Bacteria, m-TGE with TTC, 8242

m-TGE with TTC
Test preparation
Introduction
preserving samples.
Page 1397
Pour plate procedure for heterotrophic bacteria m-TGE with TTC, method 8242
petri dish. Replace the lid m-TGE with TTC or use an membrane filter, grid side well-mixed. Filter the
sterile pipet. Drain excess
medium from the petri dish
Still using the forceps, under the filter and make at 35 ± 0.5 °C for 24 hours. microscope.
immediately to the entire pad. Replace the on m-TGE with TTC
previously prepared petri dish lid. medium appear
petri dish. red to aid visibility.
Page 1398
Diluting the Sample

sample.


the medium.
For example: 98 CFU/L, mL, 35 °C, 24 hours, m-TGE with TTC broth.
1 to 2, or fewer colonies per square—Count all of the colonies on the filter, and divide the results
by the volume of original sample used.
122 colonies
------------------------------------- = 12.2 CFU/mL
10 mL sample
--------------------------------------------------------------- = 8000 CFU/mL
0.1 mL sample
------------------------------------------------------------------ = 17, 000 CFU/mL
0.1 mL sample
Page 1399
> 200,000 CFU/mL.

m-TGE with TTC PourRite™ Ampules, glass, 2-mL 20/pkg 2428420
Required apparatus

Page 1400


Page 1401
Heterotrophic Bacteria, m-TGE, 8242

m-TGE
Test preparation
Introduction
preserving samples.
Page 1403
Pour plate procedure for heterotrophic bacteria m-TGE, method 8242
petri dish. Replace the lid m-TGE or use an ampule membrane filter, grid side well-mixed. Filter the
on the dish. Do not touch breaker if necessary. Pour up, in the assembly. appropriate volume
the pad or the inside of the contents evenly over Alternatively, a sterile, through the sterile 47 mm,
the petri dish. the absorbent pad. disposable filter unit may 0.45µm, gridded
To sterilize the forceps, dip Replace the petri dish lid. be used. membrane filter. Apply
them in alcohol and flame For broth prepared from vacuum and filter the
in an alcohol or Bunsen dehydrated medium, pipet sample. Rinse the funnel
burner. Let the forceps approximately 2.0 mL of walls three times with
cool before use. broth onto the pad using a 20–30 mL of sterile
sterile pipet. Drain excess buffered dilution water.
Still using the forceps, under the filter and make at 35 ±0.5 °C for 24 hours. microscope.
immediately to the entire pad. Replace the on m-TGE medium appear
previously prepared petri dish lid. clear to cream in color.
petri dish.
Page 1404
Diluting the Sample

sample.


the medium.
For example: 98 CFU/L, mL, 35 °C, 24 hours, m-TGE broth.
122 colonies
------------------------------------- = 12.2 CFU/mL
10 mL sample
--------------------------------------------------------------- = 8000 CFU/mL
0.1 mL sample
------------------------------------------------------------------ = 17, 000 CFU/mL
0.1 mL sample
Page 1405
More than 20 colonies per square — If there are more than 20 colonies per square, record the
> 200,000 CFU/mL.

m-TGE PourRite™ Ampules, glass, 2-mL 20/pkg 2373820
Required apparatus

Page 1406


Page 1407
Heterotrophic Bacteria, m-TSB/USP, 8242

m-TSB/USP
Test preparation
Introduction
preserving samples.
Page 1409
Pour plate procedure for heterotrophic bacteria m-TSB/USP, method 8242
petri dish. Replace the lid m-TSB/USP or use an membrane filter, grid side well-mixed. Filter the
sterile pipet. Drain excess
Still using the forceps, under the filter and make at 35 ± 0.5 °C for 24 hours. microscope.
immediately to the entire pad. Replace the on m-TSB/USP medium
previously prepared petri dish lid. appear clear to cream in
petri dish. color.
Page 1410
Diluting the Sample

sample.


the medium.
For example: 98 CFU/L, mL, 35 °C, 24 hours, m-TSB/USP broth.
122 colonies
------------------------------------- = 12.2 CFU/mL
10 mL sample
--------------------------------------------------------------- = 8000 CFU/mL
0.1 mL sample
------------------------------------------------------------------ = 17, 000 CFU/mL
0.1 mL sample
More than 20 colonies per square — If there are more than 20 colonies per square, record the
> 200,000 CFU/mL.
Page 1411

Description óUnit Catalog number
m-TSB/USP Broth Ampules, plastic, 2-mL 20/pkg 2812650
Required apparatus

Page 1412

Page 1413
Heterotrophic Bacteria, Pour Plate, 8241

Plate Count Agar1
1 This method meets or exceeds the specification criteria stated in Standard Methods for the Examination of Water and Wastewater,
19th edition, Method 9215 B. Pour Plate Method.
Test preparation
preserving samples.
Introduction
Page 1415
Pour plate procedure for heterotrophic bacteria, method 8241
1. Melt the sterile solid 2. Keep the melted 3. Pipet the appropriate 4. Pour at least 10 to
agar medium by placing a medium in a water bath, amount of undiluted or 12 mL of liquefied medium
tube of Plate Count Agar between 44–46 °C, until diluted sample (1 mL or (½ of the contents of Plate
in a beaker of boiling used. 0.1 mL—see Diluting the Count Agar tube) into the
water. (Each tube contains Do not depend on the Sample) into the sterile dish by gently lifting the
enough medium for two sense of touch to indicate petri dish. Prepare at least cover just high enough to
plates.) the proper temperature two plates for each pour.
Avoid prolonged exposure when pouring the agar. different volume of Avoid spilling the medium
to unnecessarily high undiluted or diluted on the outside of the
Place a thermometer in sample used.
temperatures during and the water bath to ensure container or on the inside
after melting. correct temperature Thoroughly mix all of the dish lid when
When the medium range—to avoid undiluted and diluted pouring. Replace the lid
is melted in two or more contamination, the samples by inverting about when finished.
batches, use all of each thermometer should be in 25 complete up-and- down
batch in order of melting, a separate container of (or back-and-forth)
provided the contents melted agar or water, not movements. Or, use a
remain fully melted. in the agar tubes to be mechanical shaker to
Discard any melted agar plated. shake samples or dilutions
that contains precipitate. for 15 seconds.
Loosen caps before Lift the lid of the petri dish

heating to make it easier just high enough to insert
to pour just after the media the pipet. Hold the pipet at
has melted. a 45 ° angle, with the tip
just touching the bottom of
the dish. Allow enough
time (at least
2–4 seconds) for the pipet
to drain.
Page 1416
Pour plate procedure for heterotrophic bacteria, method 8241 (continued)
5. Mix the melted 6. Place the plates on a 7. Invert the plates, place 8. Incubate the plates for
medium thoroughly with level surface and let them them in a plastic bag, and 48 ± 3 hours at
the sample in the petri dish solidify. This generally seal the bag. Place the 35 ± 0.5 °C.
by swirling in a figure-eight takes 10 minutes. bag in an incubator that During incubation,
motion with the petri dish has been prewarmed to maintain humidity within
on the bench top. 35 °C. the incubator so that
Do not invert the petri dish plates will not have
to mix. moisture weight loss
greater than 15%. A pan of
water placed at the bottom
of the incubator may be
sufficient. For incubation
in non-humidified
incubators, make certain
that the plastic bags are
tightly sealed.
9. Using a Quebec
Colony Counter, count all
colonies on the plates
promptly after incubation.
Reporting Results.
Page 1417
Diluting the Sample

sample.
Note: Standard microbiological procedures require at least 2 tests per sample.
undiluted pipet 1 mL
sample petri dish petri dish
1 2
Figure 17 Dilution factor 1, 1-mL sample volume
undiluted pipet 0.1mL

1 2
Figure 18 Dilution factor 10, 0.1-mL sample volume
Page 1418
99 mL
undiluted pipet 1 mL sample pipet 1 mL
blank 2
1
99 mL
undiluted pipet 1 mL sample pipet 0.1 mL
blank 2
1

Interpreting and Reporting Results

Count all colonies on selected plates promptly after incubation. If count must be delayed
temporarily, store plates at 5–10 °C for no more than 24 hours, but avoid routine delays.
Quebec Colony Counters feature a built-in grid to simplify counting. The easiest way to count
colonies is to follow a back and forth pattern, moving down the grid. See the Colony counting
technique figure.
Page 1419
Figure 21 Colony counting technique
Report all counts as colony-forming units (CFU)/mL. Include in the report the method used, the
incubation temperature and time, and the medium. For example, 75 CFU/mL, pour plate method,
35 °C/48 hours, plate count agar.
Generally, results are obtained by averaging the number of colonies on all plates from the same
undiluted or diluted sample volume, and multiplying by a dilution (described below). In this case,
results should be rounded to two significant digits to avoid creating false precision. For three-digit
results, raise the middle digit if the last digit is 5 or greater. Retain the middle digit if the last digit is
4 or smaller. The last digit will be zero.
For example, 143 would become 140, 255 would become 260. Two digit numbers require no
rounding.
Be familiar with the following terms before counting and reporting results:
Average number of colonies/plate —The average number of colonies per plate is derived by
dividing the total number of colonies on all plates that were inoculated with the same sample
volume or dilution volume, and dividing that sum by the number of plates used.
For example, if two plates were each inoculated with 1 mL of diluted sample, and there were 89
colonies on one plate and 103 on the other, then the average number of colonies/plate would be:
89 colonies + 103 colonies
---------------------------------------------------------------------- = 96 colonies
2 plates
Colony-forming units (CFU)/mL —This is the unit used for reporting bacterial density. To derive
the number of CFU/mL, multiply the average number of colonies/plate by the dilution factor of
the incubated sample.
Note: In some instances where a large number of colonies are observed, the average number of
colonies/plate is obtained by adding colonies counted only in a specified number of squares on each
plate.
Dilution factor —The dilution factor is the reciprocal of the volume of original, undiluted sample
plated, and is used to standardize the results according to the sample volume.
For example, if 1 mL of original sample was used, the dilution factor is 1.
If 0.1 mL of original sample was used, the dilution factor is 10. The dilution factor for 1 mL of
diluted sample (0.01 mL of original sample) is 100, and the dilution factor for 0.1 mL of diluted
sample (0.001 mL of original sample) is 1000.
Representative colony distribution—When counting colonies in a specified number of squares
(as seen through the colony counter), count those squares that appear to have an average
number of colonies. Avoid counting squares that have many less or many more colonies than most
of the other squares on the plate.
Spreaders—Spreaders are colonies of bacteria which grow in such a way that they appear to be
“spread” across the plate. See the Spreader growth figure.
Page 1420
Figure 22 Spreader growth
It is preferable when counting and recording results, to consider plates having between 30 and 300
colonies. However, this is not always the case, so when counting and recording colonies, choose
the situation that best describes
your results.
If spreaders are encountered on the plates selected, count colonies on representative portions
only when the colonies are well distributed in
spreader-free areas, and the area covered by the spreaders does not exceed
one-half of the plate area.
When spreading colonies must be counted, count each of the following types as one colony:
1. A chain of colonies that appears to be caused by disintegration of a bacterial clump as agar
and sample were mixed.
2. A spreader that develops as a film of growth between the agar and the bottom of the petri dish.
3. A colony that forms in a film of water at the edge or over the agar surface.
Count as individual colonies the similar-appearing colonies growing in close proximity but not
touching, provided that the distance between them is at least equal to the diameter of the smallest
colony.
Also count as individual colonies those colonies which are touching, but are different in
appearance, such as morphology or color.
To obtain results, multiply the average number of colonies/plate by the dilution factor. Report
counts as CFU/mL.
If plates have excessive spreader growth, report as “spreaders” (Spr). When plates are
uncountable because of missed dilution, accidental dropping, or contamination, or the control
plates indicate that the medium or other material or labware was contaminated, report as
“laboratory accident” (LA).
No colonies— If plates from all dilutions of any sample have no colonies, report the count as less
than one (<1) times the dilution factor for the largest volume of original sample used.
For example, if no colonies develop using 0.1 mL of original sample, report the count as less than
10 (<10) estimated CFU/mL.
Less than 30 colonies/plate—Ordinarily, no more than 1.0 mL of sample is plated. Therefore,
when the total number of colonies developing from 1.0 mL is less than 30, record the number of
colonies as CFU/mL.
30 to 300 colonies/plate—Compute bacterial count per mL by multiplying the average number of
colonies/plate by the dilution factor. Report counts as CFU/mL.
Page 1421
For example, 0.1 mL of undiluted sample was used to inoculate two plates. After incubation, the
plates had colony counts of 115 and 145. The CFU/mL value is computed as follows:
115 colonies + 145 colonies

-------------------------------------------------------------------------- × 10 (dilution factor) = 1300 CFU/mL
2 plates
Greater than 300 colonies/plate— If no plate has 30 to 300 colonies, and one or more plates
have more than 300 colonies, use the plates having a count closest to 300 colonies. Compute the
count by multiplying the average number of colonies/plate by the dilution factor, and report as
estimated CFU/mL.
Far more than 300 colonies/plate— If there are greater than 300 colonies/plate,
do not report the result as “too numerous to count” (TNTC). Instead, follow guidelines for reporting
if there are less than 10 colonies/cm2:
Less than 10 colonies/cm2 — If there are fewer than 10 colonies per cm2 (one “square” as seen
through the colony counter), then count colonies in 13 squares having representative colony
distribution.
a. Select seven consecutive squares horizontally across the plate, and six consecutive
squares vertically, being careful not to count a square more than once. See the Colony
counting, > 300 colonies figure.
b. Add the number of colonies in each square.
c. Multiply this sum by 4.38 when the plate area is 57 cm2 (disposable plastic plates).
d. Multiply the sum by 5 when the plate area is 65 cm2 (glass plates). To determine colony-
forming units (CFU)/mL, compute the average number of colonies/plate and multiply the
result by the dilution factor. Report as estimated CFU/mL.
Figure 23 Colony counting, > 300 colonies
Note: One thousand is the dilution factor for 0.1 mL of diluted sample. This is the sample volume that should
be used when bacterial counts are this high.
More than 10 colonies/cm2—When there are more than 10 colonies per cm2 (one “square” as
seen through the colony counter), count colonies in four squares having representative colony
distribution. Add the number of colonies in these four squares, and divide the sum by 4, to get the
average number of
colonies/square. Multiply this number by 57 when the plate area is 57 cm2 (disposable plastic
plates). Multiply this number by 65 when the plate area is 65 cm2 (glass plates). To determine
CFU/mL, compute the average number of colonies/plate and multiply the result by 1000 (see
“Note” below). Report as estimated CFU/mL.
Page 1422
Avoiding Errors
Avoid inaccuracies in counting due to damaged or dirty optics that impair vision, or due to failure to
recognize colonies. Be careful not to contaminate plates due to improper handling. Laboratory
workers who cannot duplicate their own counts on the same plate within 5%, and counts of other
analysts within 10%, should discover the cause and correct such disagreements.

Plate Count Agar Tubes 20/pkg 2406720
Required apparatus
Bags, Whirl-Pak®1 with dechlorinating agent, sterile, 180-mL 100/pkg 2075333

Bags, Whirl-Pak without dechlorinating agent, sterile, 205-mL 100/pkg 2233199
Bags, Whirl-Pak without dechlorinating agent, sterile, 710-mL 10/pkg 1437297
Beaker, 250-mL each 50046
Bottle, sample, glass, with cap, 118-mL 3/pkg 2163103
Clamp, Test Tube each 63400
Colony Counter, Quebec, 110 VAC, 60 Hz each 2252100
Colony Counter, Quebec, 220 VAC, 50 Hz each 2252102
Dish, Petri, 100 x 15 mm, sterile, disposable 20/pkg 2178901
Dish, Petri, 100 x 15 mm, sterile, disposable 500/pkg 2178900
Hot Plate, 120 VAC, 50/60 Hz each 2881500
Hot Plate, 240 VAC, 50/60 Hz each 2881502
Incubator, Culture, Low-Profile, 120 VAC, 50/60 Hz each 2619200
Incubator, Culture, Low-Profile, 220 VAC, 50/60 Hz each 2619202
Pipets, Serological, 1 mL, sterile, disposable 50/pkg 2092835
Thermometer -20 to 110 °C, non-mercury each 2635702
Page 1423
Total Aerobic Bacteria, Yeasts and Molds, Paddle testers
Total Aerobic Bacteria, Yeasts

and Molds DOC316.53.01223
Total Aerobic Bacteria (Amber)/Yeast and Mold (Red)

Total Aerobic Bacteria (Amber)/Total Coliform (Red)
Total Aerobic Bacteria (Amber)/Disinfection Control (Purple)
Paddle Testers
Scope and Application: For detection of contamination of equipment that contacts food, cosmetics or
pharmaceuticals, boiler and cooling tower water, laboratory surfaces requiring aseptic conditions and kitchen
surfaces in restaurants and hotels
Test preparation
To ensure accurate results, read carefully before proceeding.
Paddle testers have a 12-month shelf life from date of manufacture.
Paddle Testers are most suitable for semi-quantitative screening. Monitoring with Paddle Testers over time will provide a
good indication of how the microbial load is changing at a given site.
For greater accuracy when interpreting results, count the colonies with the aid of the molded-in grid.
Paddle testers in liquid
1. Remove the paddle 2. Dip the paddle into a 3. Remove the paddle 4. Incubate the
from the vial. Do not touch liquid sample. Immerse all from the liquid and allow Paddle Tester.
the media. the growth media on the excess liquid to drip off. See the Incubation of
paddle into the liquid. Do not shake. Return the Paddle Testers for
paddle to the vial and incubation times.
screw on the cap.
Total Aerobic Bacteria, Yeasts and Molds

Page 1425
Paddle testers in liquid (continued)
5. Refer to the Bacterial

colony density and Yeast
and mold colony density
tables for results.
Sterilize the paddles
before disposal. (See
Disposing of Paddles
table.)
Paddle testers on solid and flat surfaces
1. Remove the paddle 2. Press one side of the 3. Turn the paddle over 4. Incubate the
from the vial. Do not touch paddle to the surface to be and press the other side to Paddle Tester.
the media. tested. a new area of the test See the Incubation of
surface. Return the paddle Paddle Testers table for
to the vial and screw on incubation times.
the cap.

Page 1426
Paddle testers on solid and flat surfaces (continued)
5. Refer to the Bacterial

colony density and Yeast
and mold colony density
tables for results.
Sterilize the paddles
before disposal. (See
Disposing of Paddles.)
Table 390 Incubation of Paddle Testers

Incubation of Paddle Testers Incubation Temperature Examine at:
Bacteria: 24 to 48 hours
Yeast and Mold 25 to 30 °C
Yeast and Mold: 48 hours and up to 120 hours (5 days)
Total Coliform 35 to 37 °C 24 to 48 hours
Disinfection Control 35 to 37 °C 24 to 48 hours
Disposing of Paddles
To sterilize Paddle Testers before disposal:
• Autoclave for 15 minutes at 121 °C (250 °F) at 103 kPa (15 psi) or
• Pour 10 mL of household bleach (5.25% NaOCl) into the vial and allow it to sit for a minimum
of 30 minutes.
Interpreting the level of Contamination for total bacteria or total coliform bacteria
Identify the slide that most closely matches the sample. Use the density value above the slide.
Table 391 Bacterial colony density

100 (102) 1000 (103) 10,000 (104) 100,000 (105) 1,000,000 (106) 10,000,000 (107)

Page 1427
Table 391 Bacterial colony density
Interpreting the level of contamination for yeast and mold
Table 392 Yeast and mold colony density

10 (101) 100 (102) 1000 (103) 10,000 (104) 100,000 (105)
Required reagents and media

Total Aerobic Bacteria/Yeast and Mold Paddle Testers 10/pkg 2610810
Total Aerobic Bacteria/Total Coliform Paddle Testers 10/pkg 2610910
Total Aerobic Bacteria/Disinfection Control Paddle Testers 10/pkg 2619510
Required apparatus

Page 1428


Page 1429
Electrochemistry
Page 1431
Page 1432
Conductivity, 8160
Conductivity DOC316.53.01199
USEPA1 Direct Measurement Method2 Method 8160

(0.01 µS/cm to 200.00 mS/cm) Conductivity Meter
1 USEPA accepted for reporting for Standard method 2510-B
2 Procedure is equivalent to Standard Method 2510-B for wastewater.
Test preparation

Meter Standard probe Rugged probe1
HQ40d CDC40101 CDC40105

CDC40103 CDC40110
CDC40115
CDC40130
HQ30d CDC40101 CDC40105

CDC40103 CDC40110
CDC40115
CDC40130
HQ14d CDC40101 CDC40105

CDC40103 CDC40110
CDC40115
CDC40130
sension™ 5 5197500 —
5197503
sension™ 7 5197500 —
5197503
1 Designed for field use.
Conductivity
Page 1433
Conductivity
Analyze samples as soon as possible after collection. However, samples may be stored at least 24 hours by cooling to 4 °C
(39 °F) or below (all storage temperatures have changed to 0 to 6 °C as per the EPA MUR, March 2007). When measuring
solutions that are not at the reference temperature, the meter automatically adjusts the conductivity value to the reference
temperature from 20 or 25 °C.
Water samples containing oils, grease, or fats will coat the electrode and affect the accuracy of the readings. If this occurs,
clean the probe with a strong detergent solution, then thoroughly rinse with deionized water.
Mineral build-up on the probe can be removed with a diluted 1:1 Hydrochloric Acid Solution. Refer to the meter user manual.
Calibration instructions are given in the operation section of the meter manual. For most accurate results calibrate before
use, or check the accuracy of the meter with a known conductivity standard.
Measurement errors can occur if the appropriate temperature correction value is not chosen. Refer to the Temperature
correction table for typical correction values.
One of the following meter/sensor combinations: 1
HQd meter and conductivity IntelliCAL™ probe
sension meter and conductivity electrode 1
One of the following Hach standards:
NaCl Standard Solution, 180 ± 10 µS/cm 1
NaCl Standard Solution, 18 ± 10 mS/cm 1
Radiometer Analytical Certified Conductivity Standards:
KCl, 1 Demal, 111.3 mS/cm ± 0.5% at 25 °C 500 mL
KCl, 0.1 Demal, 12.85 mS/cm ± 0.35% at 25 °C 500 mL
KCl, 0.01 Demal, 1408 S/cm ± 0.5% at 25 °C 500 mL
NaCl, 0.05%, 1015 S/cm ± 0.5% at 25 °C 500 mL
KCl Conductivity Standards:
0.1 Molar KCl, 12.88 mS/cm at 25 °C 500 mL
0.01 Molar KCl, 1413 S/cm at 25 °C 500 mL
0.001 Molar KCl, 148 S/cm at 25 °C 500 mL
Beaker, poly, 100-mL 1
Conductivity
Page 1434
Conductivity
Conductivity
1. Refer to the operation 2. Laboratory tests: 3. Turn the meter on. 4. Rinse the probe
section of the meter Immerse the probe in a Make sure that the meter thoroughly with deionized
manual to prepare the beaker containing the is set to measure water after each
conductivity electrode and sample solution. Move the conductivity. measurement.
meter. probe up and down and To display other units such
The meter will select the tap it on the beaker to as TDS, salinity or
range automatically. remove bubbles from the resistivity (HQd only), refer
electrode. to the meter user manual.
For most accurate results,
calibrate the meter before Field tests: Immerse the
use or check the accuracy probe in the sample
of the meter with a known solution. Move the probe
conductivity standard. up and down to remove
Refer to the meter user bubbles from the
manual for calibration and electrode.
measurement options. The vent holes should be
completely submerged.
Conversions
The Unit conversion table provides equations for converting the conductivity readings to other
units of measure.
Table 394 Unit conversion
From To Use this Equation
mS/cm μS/cm mS/cm x 1000
μS/cm mS/cm μS/cm x 0.001
μS/cm μmhos/cm μS/cm x 1
mS/cm mmhos/cm mS/cm x 1
μS/cm mg/L TDS μS/cm x 0.51
g/L TDS mg/L TDS g/L TDS x 1000
mS/cm g/L TDS mS/cm x 0.5
mg/L TDS g/L TDS mg/L TDS x 0.001
mg/L TDS gpg TDS mg/L TDS x 0.05842
g/L TDS gpg TDS g/L TDS x 58.42
μS/cm ohms cm 1,000,000 ÷ μS/cm
mS/cm ohms cm 1,000 ÷ mS/cm
1 TDS is an empirically-derived value from the conductivity measurement. A value of 0.5 is selected here for simplicity and suitability to a wide
variety of waters. The sension 5 uses a more complex algorithm, based on additional factors, such as temperature, to determine TDS.
Conductivity
Page 1435
Conductivity
The Temperature correction table shows typical temperature correction values for selected
solutions using the linear temperature correction option.
Table 395 Temperature correction

Solution Percent per °C
Ultrapure Water 4.55
Salt (NaCl) 2.125
NaOH 1.72
Dilute Ammonia 1.8810
10% HCl 1.325
5% Sulfuric Acid 0.9698
Interferences
When measuring conductivity, the following items should be considered in order to ensure
accurate results:
• If measuring very low levels of conductivity, protect the sample from atmospheric gases
(carbon dioxide, ammonia). These gases dissolve readily in water and may cause a rapid
change in conductivity. To minimize these effects, boil the sample, then place in a covered
container, such as a Low Ionic Strength (LIS) chamber for cooling.
• To remove the conductivity with hydroxide ions, neutralize by adding 4 drops of
Phenolphthalein Indicator Solution to 50 mL of sample, then adding Gallic Acid Solution, drop-
wise, until the pink color completely disappears.
Accuracy check
Pour a Sodium Chloride Standard Solution (with a conductivity value in the same range as the
sample) into a beaker. Perform the conductivity measurements as described above. The
conductivity reading should be the same (within accuracy limits) as listed on the Standard Solution
label if the meter is calibrated correctly. Calibration can be performed using this solution. Refer to
the meter user manual.
Method performance
The accuracy of a conductivity measurement is dependent on many factors associated with the
overall system, including the meter, meter settings, choice of electrode and conductivity standards
being used during calibration. Refer to the appropriate electrode, meter manual and standard
certificate of analysis to help determine system performance.
Summary of method
Electrolytic conductivity is the capacity of ions in a solution to carry electrical current and is the
reciprocal of the solution resistivity. Current is carried by inorganic dissolved solids (e.g., chloride,
nitrate, sulfate, and phosphate anions) and cations (e.g., sodium, calcium, magnesium, iron, and
aluminum). Organic material like oils, phenols, alcohols, and sugars do not carry electrical current
well and thus do not have enough conductivity for a useful estimate of concentration.
Measuring conductivity is done by measuring the resistance occurring in an area of the test
solution defined by the probe’s physical design. Voltage is applied between the two electrodes
immersed in the solution, and the voltage drop caused by the resistance of the solution is used to
calculate conductivity per centimeter. The basic unit of measure for conductivity is the Siemen
(or mho), the reciprocal of the ohm in the resistance measurement. Because ranges normally
found in aqueous solutions are small, milliSiemens/cm (10–3 S or S/cm) and microSiemens/cm
(10–6 S or µS/cm) are most commonly used.
Conductivity
Page 1436
Conductivity
Required apparatus
Select one meter and probe combination:

HQ40d Meter 1 each HQ40d53000000
IntelliCAL Conductivity Probe, standard, with 1m cable 1 each CDC40101
IntelliCAL Conductivity Probe, standard, with 3m cable 1 each CDC40103
IntelliCAL Conductivity Probe, rugged, with 5m cable 1 each CDC40105
IntelliCAL Conductivity Probe, rugged, with 30 m cable 1 each CDC40130
sension meters and probes. Select one meter and probe

combination:
sension 5 1 each 5180000
Conductivity probe, with 1 m cable 1 each 5197500
Conductivity probe, with 3 m cable 1 each 5197503
Hach, NaCl Conductivity Standards:

Sodium Chloride Standard Solution, 180 ±10 mS/cm, 90 ±1 mg/L TDS 100 mL 2307542
Sodium Chloride Standard Solution, 18,000 ±50 mS/cm, 9000 ±25 mg/L TDS 100 mL 2307442
Radiometer Analytical, Certified Conductivity Standards:
KCl, 1 Demal, 111.3 mS/cm ± 0.5% at 25 °C 500 mL S51M001
KCl, 0.1 Demal, 12.85 mS/cm ± 0.35% at 25 °C 500 mL S51M002
KCl, 0.01 Demal, 1408 µS/cm ± 0.5% at 25 °C 500 mL S51M003
NaCl, 0.05%, 1015 µS/cm ± 0.5% at 25 °C 500 mL S51M004
KCl Conductivity Standards:
0.1 Molar KCl, 12.88 mS/cm at 25 °C 500 mL C20C250
0.01 Molar KCl, 1413 µS/cm at 25 °C 500 mL C20C270
0.001 Molar KCl, 148 µS/cm at 25 °C 500 mL C20C280
Conductivity
Page 1437
Conductivity

Beaker, poly, 100-mL each 108042
Gallic Acid Solution 50 mL SCDB 1442326
Hydrochloric Acid Solution, 1:1 500 mL 88449
Low Ionic Strength Chamber (LIS) each 5189900
Phenolphthalein Indicator Solution 15 mL SCDB 16236
Wash Bottle, 125-mL each 62014

Fluoride, acid solutions
Fluoride in Acid Solutions DOC316.53.01236
Direct Measurement ISE Method Method 8323

0.1 to 100.0 mg/L F ISE Electrode
Scope and Application: For industrial waters (solutions below a pH of 5)
Test preparation


Meter Electrode
sension™ 4 meters 5192800
sension™ 2 meters1 5192800
1 The user must construct the calibration curve with the sension 2 meter.
Refer to the meter user manual for meter operation. Refer to electrode manual for electrode maintenance and care.
Prepare the electrode. Refer to Electrode assembly and Condition the electrode in this procedure.
In solutions below a pH of 5, hydrogen ion complexes some of the fluoride ions, forming HF or HF2 –, which the electrode will
not detect. To free the complexed fluoride, adjust the pH of the solution so it is weakly acidic to weakly basic before analysis.
Do not use a strong base (such as sodium hydroxide) for adjustment since the amount of base added will vary from sample
to sample. Using a strong base can also change the ionic strength of the sample and alter measurement accuracy.
Dilution of samples and standards with a large excess of sodium acetate will buffer the pH and help adjust the total ionic
strength of samples and standards to the same level.
Fluoride ISA buffer pillows (TISAB) 1 mL
Sodium acetate, ACS varies
Fluoride standard solutions:
10.00-mg/L F or varies
100.0-mg/L F varies
Potassium Chloride Reference Electrolyte Gel Cartridges 1
Fluoride in Acid Solutions

Page 1439
Tensette pipet, 1.0–10.0 mL or 1
25 mL Class A volumetric pipet 1
Class A 1000 mL volumetric 1
Bottle, wash, 500-mL 1
Platinum Series Fluoride Combination Electrode, BNC 1
sension™2 Portable pH/ISE Meter. 1
OR
sension™4 Laboratory pH/ISE Meter 1
Stir Bar, 7/8 X 3/16 in. (22.2 x 4.8 cm) 4
Stirrer, electromagnetic with stand and stir bar 1
Fluoride in Acid Solutions method
1. Prepare a 15% 2. Using deionized water, 3. For calibration, 4. Dilute each standard
sodium acetate solution by prepare a background prepare fluoride standards 10:1 with sodium acetate
dissolving 150 g of reagent solution containing all the by adding fluoride to the solution (9 parts sodium
grade sodium acetate in sample matrix background solution. acetate solution to 1 part
1000 mL of deionized components except Prepare fresh standards standard) to prepare the
water. Prepare enough fluoride. Use this solution every two weeks if buffered solution. Mix
solution to dilute all the to prepare the standards. standards are less than 10 thoroughly.
samples and standards. If a standard prepared with mg/L F–. For example, measure
background solution gives When testing with a direct 3 mL of standard (with
the same reading (after reading fluoride meter, use background solution) and
sodium acetate dilution) as two standards. When dilute it with 27 mL of
standard prepared with testing with a pH/mV sodium acetate solution.
pure sodium acetate, it is meter, use three
not necessary to prepare a standards.
background solution.

Page 1440
Fluoride in Acid Solutions method (continued)
5. Add the contents of 6. Turn the meter on. Set 7. Place the beaker with 8. Place the electrode
one Fluoride Total Ionic the electrode type to BNC. the 1-mg/L prepared into the standard.
Strength Adjustment Set the units to mg/L. buffered standard on a
Buffer (TISAB) Powder Press CAL. magnetic stirrer. Stir at a
Pillow to 25 mL of the Refer to the meter user moderate rate.
prepared buffered sample. manual for details.
Stir to dissolve.
Stabilizing... Repeat 7—10
9. Edit the display to 10. The display will show 11. Repeat steps 7–10 for 12. Measure the sample
show the concentration of Stabilizing until the the 10-mg/L and 100 mg/L into 50-mL beakers to
the standard. measurement is complete. standards. measure 25 mL of
Press ENTER and accept Remove the electrode After the last standard is prepared buffered sample.
the concentration. from the standard solution measured, store the For example, measure
Rinse with deionized water calibration in the meter. 3 mL of sample and dilute
and blot dry. Refer to the meter user it with 27 mL of sodium
manual for details. acetate solution. Pour
25 mL of this solution into
a 50-mL beaker.
13. Dilute each unknown 14. Place a stir bar in the 15. Rinse the electrode 16. Place the electrode
10:1 with sodium acetate beaker and set the beaker with deionized water and into the sample. When the
solution before on a magnetic stirrer. Stir blot dry. reading is stable, record
measurement. at a moderate speed. the value.

Page 1441
Electrode assembly
1. Remove the cap from the electrolyte cartridge.
2. Visually inspect the Luer tip of the electrolyte cartridge. If air is present, rotate the feed-screw
counter-clockwise until gel expels the air and fills the tip.
3. Fit the cartridge outlet tube firmly onto the inlet tube of the electrode body (Figure 24).
Figure 24 Attach the outlet tube
4. Place the dispenser unit over the electrolyte cartridge. Screw the dispenser unit onto the
electrode body until reaching the stop. Do not over tighten.
5. Dispense the electrolyte gel by pressing the pump button. Repeat this procedure until gel is
visible at the reference outlet (Figure 25).
Figure 25 Dispense the electrolyte gel
6. Rinse the electrode with deionized water. Do not scratch the crystal.
7. To remove an empty cartridge, unscrew the dispenser unit and rotate the cartridge
counterclockwise while gently pulling it out of the electrode.

Page 1442
8. Connect the BNC connector of the electrode to the BNC connector on the meter (Figure 26).
Figure 26 BNC connector
Note: One BNC and one 5-pin connector are on the back of the meter. Choose the BNC for the fluoride
electrode. Disconnect the pH electrode from the 5-pin connector when using the BNC connector.
Condition the electrode

Condition and store the electrode in 1 mg/L Fluoride standard storage with Ionic Strength Adjuster
for 15 to 30 minutes.
For electrode storage procedures, refer to the Fluoride Electrode Instruction Manual.
Clean the Lanthanum Fluoride Crystal

It may be necessary to clean the LaF crystal on the sensing tip of the probe if it becomes covered
with organic film or buildup.
1. Put a small amount of fluoride toothpaste on a soft toothbrush or cloth.
2. Gently rub the LaF crystal with the toothpaste using a circular motion. Rub until the film is
removed.
3. Thoroughly rinse the probe with deionized water and blot dry. Verify the crystal is clean. If not,
repeat cleaning and rinsing until it is clean.
4. If the crystal becomes contaminated by oil, grease, or fingerprints, soak for a few minutes in
isopropyl alcohol then rinse with deionized water.
Interferences

Cations Do not interfere
Cl–, Br–, SO42–, HCO3–,
Do not interfere
PO43–, acetate
Interferes: refer to pH Effects. Some ions, such as CO32– or PO43–, make the sample more
OH– (Hydroxyl ions) basic, which increases OH– interference, but do not directly interfere with the electrode
operation.
CO32– or PO43– Can make the sample more basic and increase OH–

Page 1443
pH Effects
In solutions with a pH below 5, hydrogen ion complexes some of the fluoride ions, forming the
undissociated acid HF and the ion HF2–. Figure 27 shows the proportion of free fluoride ion in acid
solutions.
If the background ionic strength is high and constant in comparison with the ion being measured,
the activity coefficient is constant and activity is directly proportional to ion concentration. Total
ionic strength adjustor is added to standards and samples to make the background ionic strength
high, decomplex fluoride, and adjust the solution pH to 5.0–5.5.
Figure 27 Ratio of free F– in acid solutions

• Collect samples in plastic bottles.
• Samples may be stored up to 28 days.
Accuracy check
To verify measurement accuracy, perform a standard addition spike on the sample. The spike
should roughly double the measured concentration without significantly diluting the sample.
To perform a standard addition sample:
1. Use the Spike volumes table to determine the concentration and volume of standard to spike
the sample. The volume of sample transferred must be accurate.
2. Add the amount and concentration specified in the Spike volumes table to the sample.
3. After adding the standard, proceed with the calculations. Results from 90-110% recovery are
considered acceptable. Calculate percent recovery as follows:
100 ( X s – X u )
% Recovery = ----------------------------------
K
Where:
Xs = measured value for spiked sample in mg/L
Xu = measured value for unspiked sample adjusted for dilution by the spike, in mg/L
K = known value of the spike in the sample in mg/L
Calculations
Xi × Vu
1. X u = -----------------
-
Vu + V

Page 1444
Where:
Xi = measured value of unspiked sample in mg/L
Vu = volume of separate unspiked portion in mL
V = volume of spike in mL
C×V
2. K = -----------------
Vu + V
Where:
C = concentration of standard used in spike in mg/L
Vu = volume of separate portion before spike in mL
s u 100 ( X – X )
3. Final calculation plugging in Xu and K: % Recovery = ----------------------------------
K
Example:
A sample was analyzed and read 5.0 mg/L F–. As directed in the Spike volumes table, a 1.0-mL
spike of 100-mg/L F– standard was added to another 25-mL sample, giving a final standard
addition result of 8.75 mg/L.
Calculate the percent recovery as follows:
5.0 mg/L × 25 mL
1. X u = ---------------------------------------------- = 4.81 mg/L
25 mL + 1 mL
100 mg/L × 1 mL
2. K = --------------------------------------------- = 3.85 mg/L
25 mL + 1 mL
100 × ( X s – X u ) 100 × ( 8.75 – 4.81 )

3. %R = ---------------------------------------- = -------------------------------------------------- = 102.3 % Recovery
K 3.85
Table 398 Spike volumes

Measured Sample Concentration Measured Sample Volume Standard Concentration Standard Volume
(mg/L) (mL) (mg/L) (mL)
0.1–0.6 25 100 0.1
0.6–1.0 25 100 0.2
1.0–1.5 25 100 0.3
1.5–3.0 25 100 0.5
3–6 25 100 1.0
6–10 25 100 2.0
10–15 25 100 3.0
15–25 25 1000 0.5
25–35 25 1000 0.7
35–50 25 1000 1.0
50–100 25 1000 2.0
Summary of method
The fluoride electrode consists of a sensing Lanthanum Fluoride element bonded into an epoxy
body. When the sensing element contacts fluoride ions in a solution, a potential develops across
the sensing element. The potential is proportional to the level of fluoride ions present. The
potential is measured against a constant reference potential with a pH/mV meter or ISE meter.

Page 1445
Required reagents
Fluoride Standard Solutions:
1.00-mg/L F or varies 500 mL 29149
10.00-mg/L F or varies 500 mL 40520
100.0-mg/L F varies 500 mL 35949
Potassium Chloride Reference Electrolyte
Gel Cartridges varies 2/pkg 2546902
Sodium Acetate, ACS varies 454 g 17801H
Required apparatus
Fluoride Combination Electrode, BNC, w/ filling solution 1 each 5192800
sension 2 Portable pH/ISE Meter 1 each 5172500
OR
sension 4 Laboratory pH/ISE Meter 1 each 5177500
Stir Bar, 7/8 x 3/16 in. (22.2 x 4.8 cm) 4 each 4531500
Stirrer, electromagnetic, 115 VAC, with stand and stir bar 1 each 4530001
Tensette pipet 1.0–10.0 mL 1 each 1970010
Class A, 25 mL volumetric pipet 1 each 1451540
Safety bulb pipet filler 1 each 1418900
Class A, 1000 mL volumetric flask 1 each 1457453
Optional apparatus
Electrode Washer each 2704700
Pipet, TenSette, 0.1 to 1. 0 mL each 1970001
Pipet Tips, for 19700-01 TenSette Pipet 50/pkg 2185696

Fluoride, drinking water, 8323
Fluoride in Drinking Water DOC316.53.01237
USEPA1 Direct Measurement ISE Method Method 8323

0.1 to 10.0 mg/L F– Powder Pillow or TISAB Solution
Scope and Application: Drinking water
1 USEPA equivalent method
Test preparation


Meter Electrode
1 The user must construct the calibration curve with the sension 2 meter.
For USEPA reporting, replace the standards in step 1 with a 0.5-mg/L, 1.0-mg/L and 2.0-mg/L fluoride standard solution to
calibrate the electrode.
Prepare the electrode. Refer to Electrode assembly and Condition the electrode in this procedure.
Fluoride ISA buffer pillows (TISAB) 1
OR
Fluoride ISA solution, concentrated (TISAB) 5.0 mL
Sodium acetate, ACS varies
Fluoride standard solutions:
1.00-mg/L F varies
0.5 mg/L F (USEPA) varies
Potassium chloride reference electrolyte gel cartridges varies
Fluoride in Drinking Water

Page 1447
Beaker, 50-mL, polypropylene 3 or 4 (USEPA)
Cylinder, graduated, 25-mL, polypropylene 3
Platinum series fluoride combination electrode, BNC 1
sension™2 Portable pH/ISE Meter. or sension™4 Laboratory pH/ISE Meter 1
Stir Bar, 7/8 X 3/16 in. (22.2 x 4.8 cm) 2 or 3 (USEPA)
Fluoride in drinking water, powder pillows method
1. In 50-mL beakers, 2. Add the contents of 3. Turn the meter on. Set 4. Place the beaker with
prepare two 25-mL one Fluoride Total Ionic the electrode type to BNC. the 1-mg/L standard on a
standard solutions of Strength Adjustment Set the units to mg/L. magnetic stirrer. Stir at a
1-mg/L and 10-mg/L F–. Buffer (TISAB) Powder Press CAL. moderate rate.
Pillow to each standard. Refer to the meter user
Stir to dissolve. manual for details.
Standard Stabilizing... Repeat 4—7
5. Place the electrode 6. Edit the display to 7. The display will show 8. Repeat steps 4–7 for
into the standard. show the concentration of Stabilizing until the the 10-mg/L and 0.5 mg/L
the standard. measurement is complete. (USEPA) standard.
Press ENTER to accept the Remove the electrode After the last standard is
concentration. from the standard solution measured, store the
Rinse with deionized water calibration in the meter.
and blot dry. Refer to the meter user
manual for details.

Page 1448
Fluoride in drinking water, powder pillows method (continued)
9. Transfer 25 mL of the 10. Remove the electrode 11. Add the contents of 12. After the
sample to a 50-mL beaker. from the standard solution. one TISAB Powder Pillow. measurement is stable,
Add a stir bar to the Rinse it with deionized Stir to dissolve. record or store the
beaker. Place the beaker water and blot dry. Place it measurement value.
on a magnetic stirrer and into the sample.
stir at a moderate rate. The sample should be the
same temperature as the
standards, ±1 °C.
Repeat 9—12
13. Repeat steps 9 14. Remove the electrode

through 12 for each after reading the last
sample. sample. Rinse the
electrode. Store in a
fluoride standard of similar
concentration to the
sample that will be
analyzed next.
Refer to the electrode
manual for more
information on electrode
storage.

Page 1449
Fluoride in drinking water, liquid TISAB solution method
1. In 50-mL beakers, 2. Add the contents of 5 3. Turn the meter on. Set 4. Place the beaker with
prepare two 25-mL mL concentrated Fluoride the electrode type to BNC. the 1-mg/L standard on a
standard solutions of Total Ionic Strength Set the units to mg/L. magnetic stirrer. Stir at a
1-mg/L and 10-mg/L F–. Adjustment Buffer (TISAB) Press CAL. moderate rate.
per 25 mL of standard. Stir Refer to the meter user
to mix. manual for details.
Standard Stabilizing... Repeat 4—7
5. Place the electrode 6. Edit the display to 7. The display will show 8. Repeat steps 4–7 for
into the standard. show the concentration of Stabilizing until the the 10-mg/L standard.
the standard. measurement is complete. After the last standard is
Accept the concentration. Remove the electrode measured, store the
from the standard solution calibration in the meter.
Rinse with deionized water Refer to the meter user
and blot dry. manual for details.

Page 1450
Fluoride in drinking water, liquid TISAB solution method (continued)
9. Transfer 25 mL of the 10. Remove the electrode 11. Add 5 mL 12. After the
sample to a 50-mL beaker. from the standard solution. concentrated liquid TISAB measurement is stable,
Add a stir bar to the Rinse it with deionized to the sample. Stir to mix. record or store the
beaker. Place the beaker water and blot dry. Place it measurement value.
on a magnetic stirrer and into the sample.
stir at a moderate rate. The sample should be the
same temperature as the
standards, ±1 °C.
Repeat 9—12
13. Repeat steps 9 14. Remove the electrode

through 12 for each after reading the last
sample. sample. Rinse the
electrode. Store in a
fluoride standard of similar
sample that will be
analyzed next.
manual for more
storage.

Page 1451
Electrode assembly
3. Fit the cartridge outlet tube firmly onto the inlet tube of the electrode body (Figure 28).
visible at the reference outlet (Figure 29).
6. Rinse the electrode with deionized water. Do not scratch the crystal.
7. To remove an empty cartridge, unscrew the dispenser unit and rotate the cartridge
counterclockwise while gently pulling it out of the electrode.

Page 1452
8. Connect the BNC connector of the electrode to the BNC connector on the meter (Figure 30).
Note: One BNC and one 5-pin connector are on the back of the meter. Choose the BNC for the fluoride
electrode. Disconnect the pH electrode from the 5-pin connector when using the BNC connector.
Condition the electrode

Condition and store the electrode in 1 mg/L Fluoride standard storage with Ionic Strength Adjuster
for 15 to 30 minutes.
For electrode storage procedures, refer to the Fluoride Electrode Instruction Manual.
Clean the Lanthanum Fluoride Crystal

It may be necessary to clean the LaF crystal on the sensing tip of the probe if it becomes covered
with organic film or buildup.
1. 1. Put a small amount of fluoride toothpaste on a soft toothbrush or cloth.
2. Gently rub the LaF crystal with the toothpaste using a circular motion. Rub until the film is
removed.
3. Thoroughly rinse the probe with deionized water and blot dry. Verify the crystal is clean. If not,
repeat cleaning and rinsing until it is clean.
4. If the crystal becomes contaminated by oil, grease, or fingerprints, soak for a few minutes in
isopropyl alcohol then rinse with deionized water.
Interferences

Cations Do not interfere
Cl–, Br–, SO42–, HCO3–,
Do not interfere
PO43–, acetate
OH– (Hydroxyl ions) Interferes: refer to

CO32– or PO43– Make the sample more basic and increase OH–

Page 1453
pH Effects
In solutions with a pH below 5, hydrogen ion complexes some of the fluoride ions, forming the
undissociated acid HF and the ion HF2–. Figure 31 shows the proportion of free fluoride ion in acid
solutions.
If the background ionic strength is high and constant in comparison with the ion being measured,
the activity coefficient is constant and activity is directly proportional to ion concentration. Total
ionic strength adjustor is added to standards and samples to make the background ionic strength
high, decomplex fluoride, and adjust the solution pH to 5.0–5.5.
Figure 31 Ratio of free F– in acid solutions

• Collect samples in plastic bottles.
• Samples may be stored up to 28 days.
Accuracy check
Checking electrode response
To verify measurement accuracy, measure the electrode potential of two fluoride standard
solutions that are one decade apart in concentration. For example, use 1-mg/L and 10-mg/L
standards to bracket an expected sample concentration of 3 mg/L. The two standards should have
mV potentials that are 58 ± 3 mV apart at 25 °C. Both solutions must be greater than 0.2 mg/L F–.
Checking calibration accuracy
To verify calibration accuracy, measure the concentration of a known standard (e.g., 2.00 mg/L)
within the calibration range.
Checking the accuracy of the sample reading
To verify sample measurement accuracy, add a spike of standard fluoride solution with a
TenSette® or volumetric pipet. Use the Spike Volumes table and the formulas in.
Table 401 Spike Volumes
Measured sample Volume & Concentration of F– Standard to Add

CxV
Concentration
V C
0.6–1 mg/L 0.3 mL of... ...100-mg/L 30
1–2 mg/L 0.5 mL of... ...100-mg/L 50
3–6 mg/L 1.0 mL of... ...10-mg/L 100

Page 1454
Percent recovery
To calculate the percent recovery (only applicable if sample volume is 25 mL):
M = S × 25 + C × V
M
E = -----------------
25 + V
A
R = ---- × 100%
E
Where:
M = calculated mass of fluoride present after the spike (micrograms)

S = mg/L of F– in sample (before spike)
C = concentration of standard used for spiking (mg/L)
V = spike volume from the Spike Volumes table (mL)
E = expected concentration after spiking (mg/L)
R = percent recovery (should be 95–100%)
A = actual reading on meter after spike (mg/L F–)
Method performance
Precision
Instrument Standard
95% Confidence Limits of Distribution
sension 4 1.6 mg/L 1.595–1.605 mg/L
sension 2
Summary of method
The fluoride electrode consists of a sensing Lanthanum Fluoride element bonded into an epoxy
body. When the sensing element contacts fluoride ions in a solution, a potential develops across
the sensing element. The potential is proportional to the level of fluoride ions present. The
potential is measured against a constant reference potential with a pH/mV meter or ISE meter.
Required reagents
Fluoride ISA buffer pillows 1 100/pkg 258999
OR
Fluoride ISA solution 5 mL 3.78 L 2829017
Fluoride Standard Solutions:
1.00-mg/L varies 500 mL 29149
2.00-mg/L varies 500 mL 40520
10.0-mg/L varies 500 mL 35949
Potassium Chloride Reference Electrolyte Gel Cartridges varies 2/pkg 2546902

Page 1455
Required apparatus
Fluoride Combination Electrode, BNC, w/ filling solution 1 each 5192800
OR
Optional apparatus
Pipet, TenSette, 0.1 to 1. 0 mL each 1970001
Pipet Tips, for 19700-01 TenSette Pipet 50/pkg 2185696

Nitrate, 8358

0.1 to 100.0 mg/L NO3–N Powder Pillow or TISAB Solution
Scope and Application: Water and wastewater
Test preparation


Meter Electrode
Prepare the electrode. Refer to Electrode assembly and Nitrate half-cell preparation in this procedure.
When using the electrode for the first time, condition the reference electrode for eight hours in a 100 mg/L nitrate-nitrogen
standard
Temperature variation causes inaccurate measurements. Calibration and sample measurements should be made at the
same temperature ± 1 °C.
The stable sample reading is the nitrate-nitrogen (NO3––N) concentration expressed as elemental nitrogen (N). The results
can be expressed as mg/L nitrate (NO3–) by multiplying the results by 4.4.
Ammonium sulfate reference electrolyte gel cartridge 1
Nitrate half-cell internal filling solution 0.5 mL
Nitrate ISA powder pillows 1 per sample or standard
OR
Nitrate ISA solution 25 mL per sample or standard
Nitrate-Nitrogen standard solutions:
1-mg/L as NO3––N 25 mL
Nitrate
Page 1457
Nitrate
Combination Nitrate Electrode, BNC 1
pH/ISE meter, sension™2 (portable) or sension™4 (laboratory) 1
Stir Bar, 7/8 X 3/16 in. (22.2 x 4.8 cm) 4
TenSette® pipet, 1.0–10.0 mL 1
OR
Safety bulb pipet filler 1
Membrane Tip, for nitrate electrode 1
Nitrate-Nitrogen, powder pillow ISA method
1. In 100-mL beakers, 2. Use a TenSette® Pipet 3. Turn the meter on. Set 4. Add a stir bar to each
prepare three 50-mL to pipet 25 mL of standard the electrode type to BNC. beaker. Put the beaker
standard solutions of 1, 10 into each beaker. Add the Set the units to mg/L. with the 1-mg/L buffered
and 100 mg/L contents of one Ionic Press CAL. standard on a magnetic
NO3––N. Strength Adjustor (ISA) Refer to the meter user stirrer. Stir at a moderate
powder pillow into each manual for details. rate.
beaker to make the Starting with the lowest
buffered standard. concentration standard
Stir to dissolve. reduces carry-over
contamination and gives
optimal electrode
response.
Nitrate
Page 1458
Nitrate
Nitrate-Nitrogen, powder pillow ISA method (continued)
Repeat 4—7
5. Put the electrode into 6. Edit the display to 7. When the 8. Repeat steps 4–7 for
the 1.0 mg/L buffered show the concentration of measurement stabilizes, the 10-mg/L and 100 mg/L
standard. the standard. remove the electrode from buffered standards.
Press ENTER to accept the the standard solution After the last standard is
concentration. Rinse with deionized water measured, store the
and blot dry. calibration in the meter.
Refer to the meter user
manual for details.
9. Remove the electrode 10. Accurately measure 11. Add the contents of 12. Add a stir bar to the
from the standard solution. 25.0 mL of sample into a one ISA powder pillow into buffered sample. Put the
Rinse it with deionized 100-mL beaker. the beaker with the sample buffered sample on a
water and blot dry. to make the buffered stirrer and stir at a
sample. Stir to dissolve. moderate rate. Put the
A white precipitate will electrode into the buffered
form if chloride or other sample.
ions are present. This will
not harm the electrode or
interfere with the analysis.
Nitrate
Page 1459
Nitrate
Nitrate-Nitrogen, powder pillow ISA method (continued)
13. Press READ. When 14. Remove the electrode

the measurement after reading the sample.
stabilizes, record or store Rinse the electrode. Store
the value. in a nitrate standard
(without ISA) of similar
sample that will be
analyzed next.
manual for more
storage.
Nitrate-Nitrogen, liquid ISA method
1. In 100-mL beakers, 2. Use a TenSette® Pipet 3. Turn the meter on. Set 4. Add a stir bar to each
prepare three 50-mL to pipet 25 mL of standard the electrode type to BNC. beaker. Put the beaker
standard solutions of 1, 10 into each beaker. Pipet Set the units to mg/L. with the 1-mg/L standard
and 100 mg/L 25 mL of liquid Ionic Press CAL. on a magnetic stirrer. Stir
NO3––N. Strength Adjustor (ISA) Refer to the meter user at a moderate rate.
into each beaker to make manual for details. Starting with the lowest
the buffered solution. concentration standard
reduces carry-over
contamination and gives
optimal electrode
response.
Nitrate
Page 1460
Nitrate
Nitrate-Nitrogen, liquid ISA method (continued)
Repeat 4—7
5. Put the electrode into 6. Edit the display to 7. When the 8. Repeat steps 4–7 for
the 1.0 mg/L buffered show the concentration of measurement stabilizes, the 10-mg/L and 100 mg/L
standard. the standard. remove the electrode from buffered standard.
Press ENTER and accept the standard solution After the last standard is
the concentration. Rinse with deionized water measured, store the
and blot dry. calibration in the meter.
Refer to the meter user
manual for details.
9. Remove the electrode 10. Accurately measure 11. Pipet 25.0 mL of liquid 12. Add a stir bar to the
from the buffered 25.0 mL of sample into a ISA into the beaker with buffered sample. Put the
standard. Rinse it with 100-mL beaker. the sample. buffered sample on a
deionized water and blot A white precipitate will stirrer and stir at a
dry. form if chloride or other moderate rate. Put the
ions are present. This will electrode into the buffered
not harm the electrode or sample.
interfere with the analysis.
Nitrate
Page 1461
Nitrate
13. Press READ when the 14. Remove the electrode

measurement stabilizes to after reading the sample.
record or store the value. Rinse the electrode. Store
in a nitrate standard of
similar concentration to
the sample that will be
analyzed next.
manual for more
storage.
Nitrate half-cell preparation

1. Condition the nitrate membrane tip by immersing the membrane tip in beaker with 100 mg/L
NO3–N solution for approximately one hour prior to use. When using the electrode for the first
time, condition the reference electrode for eight hours in a 100 mg/L nitrate–nitrogen standard.
2. Clean all gel from the electrode tip and glass stem. Rinse with Deionized water and dry
completely with a soft paper towel.
3. Use a soft paper towel soaked with isopropyl or rubbing alcohol to wipe the glass stem. Dry
the stem with a dry, soft paper towel. Make sure that alcohol does not get into the glass stem.
4. Use the provided syringe to fill the membrane to with the electrode filling solution. Fill with
solution to the top of the tip. Alternatively, fill the glass stem as shown in the electrode manual.
Do not fill the stem completely or the pressure will cause the membrane tip to fall off.
5. Use a gentle, twisting action to carefully slide a clean, dry nitrate membrane tip over the glass
stem until the end of the stem rests midway through the Nitrate Membrane Tip and some
resistance is met. Leave a 1/8-inch gap between the tip and the electrode body.
6. Shake the electrode as if shaking down mercury in a thermometer to make sure the Nitrate
Electrode Filling Solution contacts the end of the nitrate membrane tip. Verify that air bubbles
are not present in the tip.
Electrode assembly
Nitrate
Page 1462
Nitrate
3. Fit the cartridge outlet tube firmly onto the inlet tube of the electrode body (Attach the outlet
tube).
visible at the reference outlet (Dispense the electrolyte gel). If readings become erratic make
sure that the electrolyte gel is completely purged through the reference line.
6. Rinse the electrode with deionized water. Do not scrub the electrode tip.
7. Connect the BNC connector of the electrode to the BNC connector on the meter (BNC
connector).
Nitrate
Page 1463
Nitrate
Interferences
For the nitrate electrode, the major interferences include perchlorate, iodide, nitrite, bromide and
chloride. The addition of Nitrate ISA will eliminate most of these interferences. The highest level of
chloride the ISA can accommodate is 40 mg/L Cl–. Concentrations greater than 40 mg/L Cl– in the
sample will cause false high nitrate values. For more information on selectivity coefficients without
ISA, refer to the Electrode Characteristics section of the electrode manual.

• Collect samples in clean glass or plastic bottles. Start nitrate analysis promptly after sampling.
• If storage is necessary, store for up to 24 hours at 4 °C or lower (0–6 °C as per USEPA MUR
March 2007).
• For longer storage periods, adjust sample pH to 2 or less with 2 mL sulfuric acid per liter of
sample. Sample refrigeration is still necessary. When sample is preserved with acid, NO3– and
NO2– cannot be determined.
• Before testing a preserved sample, warm to room temperature, then neutralize to
approximately pH 7 with 5.0 N Sodium Hydroxide Standard Solution.
• Do not use mercury compounds as preservatives.
Accuracy check
To verify electrode response, measure the electrode potential (in mV) of two Nitrate–Nitrogen
standard solutions one decade apart in concentration, bracketing the expected sample
concentration. For example, use 10 and 100 mg/L Nitrate–Nitrogen Standard Solutions to bracket
an expected sample concentration of 30 mg/L. The two solutions should have potentials (in mV)
that are 58 ± 3 mV apart at 25 °C. Both solutions must be above 5 mg/L NO3––N.
To verify calibration accuracy, measure the concentration of a known standard within the
calibration range.
To verify sample measurement accuracy, add a spike of standard NO3––N solution with a
TenSette® or volumetric pipet. Use the Spike volumes table and the formulas in Percent recovery.
Table 403 Spike volumes
Measured sample Volume & Concentration of F– Standard to Add

CxV
Concentration
V C
1–2 mg/L 0.5 mL of 100 mg/L 50
3–6 mg/L 1.0 mL of 100 mg/L 100
7–15 mg/L 0.3 mL of 1000 mg/L 300
15–30 mg/L 0.5 mL of 1000 mg/L 500
Nitrate
Page 1464
Nitrate
Percent recovery
To calculate the percent recovery (only applicable if sample volume is 25 mL):
M = S × 25 + C × V
M
E = -----------------
25 + V
A
R = ---- × 100%
E
Where:
M = calculated mass of nitrate as nitrogen present after the spike (micrograms)

S = mg/L of NO3––N in sample (before spike)
V = spike volume from the Spike volumes table (mL)
A = actual reading on meter after spike (mg/L NO3––N)
Method performance
Precision
Instrument Standard
sension 41 5.0 mg/L 4.56–5.44 mg/L
sension 21
1 With a default stabilization criteria of 0.5 mV/min.
Summary of method
Nitrate ions are selectively absorbed by the ISE membrane, establishing a potential (voltage) that
is proportional to the concentration of nitrate in the sample. This potential is compared to the
constant potential of a reference electrode by measuring the potential of known standard. A
calibration curve can be constructed to determine the concentration of nitrate in unknown samples.
The solvent-polymer membrane is a nitrate ion-exchanger in an inert polyvinyl chloride (PVC)
plastic matrix. The nitrate electrode has an internal silver/silver chloride element, which
establishes a fixed potential when in contact with the internal filling solution. The ion selective
membrane undergoes ion exchange with nitrate in the sample, creating a potential across the
membrane which varies with the amount of nitrate ion in the sample. This potential will decrease
by about 58 mV for every tenfold increase in nitrate concentration in the linear operating range
at 25 °C.
Nitrate
Page 1465
Nitrate

Required reagents
Nitrate ISA powder pillows 1 100/pkg 258999
Nitrate half-cell filling solution varies 50 mL 4456369
Nitrate-Nitrogen Standard Solutions:
1-mg/L varies 500 mL 29149
10-mg/L varies 500 mL 35949
100-mg/L varies 500 mL 35949
1000-mg/L varies 500 mL 35949
Ammonium Sulfate Reference Electrolyte Gel Cartridge varies 2/pkg 2597102
Required apparatus
Nitrate Combination Electrode, Platinum series, BNC 1 each 5192000
OR
OR
Class A 25 mL volumetric pipet 1 each 1451540
Membrane Tips, for nitrate electrode (replacement) 1 each 4613300
Nitrate
Page 1466
Nitrate
Optional reagents
Nitrate Ionic Strength Adjustor, powder 454 g 4456301
Optional apparatus
Pipet Tips, for 197001 TenSette Pipet 50/pkg 2185696
Scoop, measuring, 0.5 gram each 90700
Scoop, measuring, 0.2 gram each 63800
Nitrate
Page 1467
Nitrate, drinking water, 8359

0.04 to 4.00 mg/L NO3–N TISAB Solution
Scope and Application: Drinking water
Test preparation


Meter Electrode
1 Only sension 4 meters can be used for this analysis.
When using the electrode for the first time, condition the reference electrode for eight hours in a 100 mg/L nitrate–nitrogen
standard. After initial conditioning (before the first use or after long-term storage), condition the electrode for 60 minutes only,
unless the electrode is stored dry for longer than four weeks. If the electrode is used daily, store the electrode in a nitrate
standard having a concentration similar to the measured samples.
The low range nitrate test requires several conditioning steps and careful attention to temperature. Read through the
procedure steps and the Sample collection, preservation and storage section before proceeding. The 2-hour conditioning
step must be completed before the calibration step.
Sample concentration for this test must be 3.0 mg/L NO3––N or less. Use a test strip to determine nitrate concentration . If
the electrode is conditioned to a low nitrate-nitrogen concentration (as in this procedure), putting it in a concentrated
NO3––N sample (10 mg/L or higher) will swamp the electrode with nitrate ion and the 2-hour conditioning step must
be repeated.
Note A: If the electrode is conditioned properly, the mV potential should not change more than 0.1 mV every five minutes in
step 7. It may be necessary to press the electrolyte dispenser button again to stabilize the potential reading. If the potential
drifts one direction more than 0.2 mV every 5 minutes, leave the electrodes in the 150-mL beaker until the drift has slowed. If
the drift is higher than 0.1 mV per minute, the electrode should be conditioned longer in 100 mL of 0.040 mg/L standard. Do
not put ISA into the standard.
Prepare the electrode. Refer to Electrode assembly and Nitrate half-cell preparation in this procedure.
Use identical amounts of ionic strength adjustor (ISA) in the standard beaker and in sample measurements. Use liquid ISA.
Temperature variation causes inaccurate measurements. Calibration and sample measurements should be made at the
same temperature ± 1 °C.
The stable sample reading is the nitrate-nitrogen (NO3––N) concentration expressed as elemental nitrogen (N). The results
can be expressed as mg/L nitrate (NO3–) by multiplying the results by 4.4.
Nitrate
Page 1469
Nitrate
Ammonium sulfate reference electrolyte gel cartridge 1
Nitrate half-cell internal filling solution 0.5 mL
Nitrate ISA solution 25 mL per sample or standard
Nitrate Electrode Membrane Tip (replacement) 1
Pipet, TenSette, 0.1 to 1.0 mL 1
Pipet, TenSette, 1.0 to 10.0 mL 1
Nitrate-Nitrogen standard solutions:
100-mg/L as NO3 ––N 25 mL
Combination Nitrate Electrode, BNC 1
sension™4 (laboratory) 1
Stir Bar, 7/8 X 3/16 in. (22.2 x 4.8 cm) 1
Thermometer, Digital 1
100 mL plastic volumetric flask 2
Nitrate
Page 1470
Nitrate
Nitrate-Nitrogen, liquid ISA method
1. Condition the 2. Use a TenSette® Pipet 3. Pour the solution from 4. Remove the electrode
electrode in 100 mg/L to pipet 5.0 mL of of nitrate the flask into a 150-mL from the 0.010 mg/L
NO3–N standard for one liquid ISA into a plastic beaker. nitrate–nitrogen standard
hour (refer to Nitrate half- 100-mL volumetric flask. Add a large stir bar (50.8 x and rinse it well with
cell preparation and Bring to the 100-mL mark 7.9 mm) to the beaker, deionized water. Place the
Electrode Assembly in this with deionized water. place the beaker on an electrode in the 150-mL
procedure). Then The deionized water must electromagnetic stirrer and beaker, submerging the tip
condition the electrode in be at room temperature. begin stirring at a below the solution surface.
0.010 moderate rate.
mg/L NO3–N for at least 2
hours.
To make 100 mL of
0.010 mg/L NO3–N, use a
TenSette Pipet to deliver
0.10 mL of 10 mg/L
NO3–N into a 100 mL
volumetric flask and dilute
to the mark. Mix well.
5. Use a TenSette Pipet 6. Turn the meter on. 7. Press ISE MV until the 8. Press ISE MV to toggle
to add 0.4 mL of 10 mg/L Press SETUP, set the mV potential shows on the to concentration units.
nitrate–nitrogen standard electrode type to BNC and display. Refer to Before Press CAL and scroll to
solution to the solution in scroll to Stabilizing. starting the test: Note A. mg/L. Press ENTER and
the beaker (this makes Press ENTER and edit the After the measurement is accept the concentration
100 mL of 0.04 mg/L display to show 0.1 mg/ stable, record the potential units.
nitrate–nitrogen). min. Accept the value and value.
Allow the electrode to EXIT the setup menu. Measure the temperature
condition for 30 minutes in of the standard (°C) with a
this solution before lab grade thermometer.
proceeding. Record the temperature.
Nitrate
Page 1471
Nitrate
Repeat step 10
9. Edit the display to 10. When the 11. Repeat step 10, 12. Remove the electrode
show the concentration of measurement stabilizes, adding the additional from the last standard,
the solution in the 150 mL pipet the corresponding volumes of 10 mg/L and rinse well with deionized
beaker (0.040 mg/L). additional volume from the 100 mg/L NO3––N water, and blot dry.
Refer to the Low level Low level nitrate standard from the Low Save the solution in the
nitrate calibration table. calibration table. Wait the level nitrate calibration 150-mL beaker with the
Press ENTER to accept the amount of time specified table until all seven 3.84 mg/L NO3––N for
concentration. for step 2 in the table to standards have been later calibration checks.
allow the membrane to measured. Store the
respond. calibration and return to
Enter the concentration in measurement mode.
mg/L (0.080 mg/L). Accept
the concentration.
If Error 2 occurs, use
0.08 mg/L as the first
standard for this
calibration. Then try
conditioning in 0.04 mg/L
NO3––N solution for
subsequent attempts.
Nitrate
Page 1472
Nitrate
13. Accurately pipet 5 mL 14. Pour this 100 mL of 15. Put the electrode into 16. Wait 5–10 minutes to
of nitrate liquid ISA into a sample into a 150-mL the sample and press the allow electrode to
Class A 100-mL beaker. Add a magnetic electrolyte dispenser condition to the low level
volumetric flask. Bring to stir bar, place the beaker button once. nitrate in solution.
the mark with the sample on an electromagnetic Read the nitrate
being measured. stirrer. Stir at a concentration from the
The sample must be at the moderate rate. display after it stabilizes.
same temperature as the This is the sample nitrate
standard solution in the concentration expressed
150-mL beaker used to as elemental nitrogen
perform the calibration. (NO3––N ).
A white precipitate will
form if chloride or other Note: Record this value.
ions that react with silver Between uses, the electrode
are present in solution. can be stored in the sample
This will not hurt the (if not an extreme pH). See
electrode or interfere with electrode manual for details.
the analysis.
Calibration
Make sure to measure millivolt potentials of all nitrate standards at the same temperature ± 0.5 °C.
The sample and standards must be measured at the same temperature, ± 1 °C. This procedure
keeps temperature error to a minimum by using a spiked additions method of calibration. Even so,
care should be taken to use the 100-mg/L NO3–N standard at room temperature.
Use a water bath slightly above room temperature (25 °C) to equilibrate the standard temperature
and sample temperature before measuring mV potentials. Use a laboratory-grade thermometer to
monitor the temperature. A one degree centigrade difference may result in as much as a 0.4 mV
inaccuracy. This temperature variation will, in turn, decrease accuracy of concentration
measurements.
Nitrate
Page 1473
Nitrate
Table 405 Low level nitrate calibration table

Volume 10 mg/L NO3––N Volume 100 mg/L NO3––N Concentration
Step
standard added standard added mg/L Time
1 0.4 mL 0.040 30 min.
2 0.4 mL 0.080 10 min.
3 1.2 mL 0.20 5 min.
4 0.2 mL 0.40 5 min.
5 0.4 mL 0.80 5 min.
6 1.2 mL 1.96 5 min.
7 2.0 mL 3.84 5 min.
Nitrate half-cell preparation

1. Condition the nitrate membrane tip by immersing the membrane tip in beaker with 100 mg/L
NO3–N solution for approximately one hour prior to use.When using th electrode for the first
time, condition the reference electrode for eight hours in a 100 mg/L nitrate–nitrogen standard.
2. Clean all gel from the electrode tip and glass stem. Rinse with Deionized water and dry
completely with a soft paper towel.
3. Use a soft paper towel soaked in isopropyl or rubbing alcohol to wipe the glass stem. Dry the
stem with a dry, soft paper towel. Make sure that alcohol does not get in the glass stem.
4. Use the provided syringe to fill the membrane to with the electrode filling solution. Fill with
solution to the top of the tip. Alternatively, fill the glass stem as shown in the electrode manual.
Do not fill the stem completely or the pressure will cause the membrane tip to fall off.
5. Use a gentle, twisting action to carefully slide a clean, dry nitrate membrane tip over the glass
stem until the end of the stem rests midway through the Nitrate Membrane Tip and some
resistance is met. Leave a 1/8-inch gap between the tip and the electrode body.
6. Shake the electrode as if shaking down mercury in a thermometer to make sure the Nitrate
Electrode Filling Solution contacts the end of the nitrate membrane tip. Verify that air bubbles
are not present in the tip.
Electrode assembly
3. Fit the cartridge outlet tube firmly onto the inlet tube of the electrode body.
visible at the reference outlet. If readings become erratic make sure that the electrolyte gel is
completely purged through the reference line.
6. Rinse the electrode with deionized water. Do not scrub the electrode tip.
7. Connect the BNC connector of the electrode to the BNC connector on the meter.
Nitrate
Page 1474
Nitrate
Interferences
For the nitrate electrode, the major interferences include perchlorate, iodide, nitrite, bromide and
chloride. The addition of Nitrate ISA will eliminate most of these interferences. The highest level of
chloride the ISA can accommodate is 40 mg/L Cl–. Concentrations greater than 40 mg/L Cl– in the
sample will cause false high nitrate values. For more information on selectivity coefficients without
ISA, refer to the Electrode Characteristics section of the electrode manual.

• Start nitrate measurements promptly after sampling. If storage is necessary, store for up to 24
hours at 4 °C or lower (as per the USEPA MUR in March 2007, the storage criteria changed
from 4 °C to 0–6 °C).
• Do not adjust sample pH. This method is for low ionic strength use only. Adjusting the pH
with acid will change the ionic strength and make this method unusable.
Accuracy check
To verify electrode response at these low levels of nitrate, the millivolt potential should decrease
upon each addition of 100 mg/L NO3-N. Using the Low Level Nitrate-Nitrogen procedure, at least
a 5.0 mV drop should be observed from step 1 to step 2 (0.040 mg/L to 0.080 mg/L NO3--N). Each
additional spike should decrease the mV reading substantially from the previous change. If this is
not the case, check the purity of the standard. If this is not the problem, use a new membrane.
1. Pipet 1 mL liquid Nitrate ISA into a 100-mL volumetric flask.
2. Fill the 100-mL volumetric flask to the mark with 1.0 mg/L NO3––N. Pour this solution in a
100-mL beaker and add a stir bar.
3. Put the beaker on an electromagnetic stirrer and measure the concentration of the solution
with the calibrated electrode. The measurement should be 1.0 mg/L ± 0.1 mg/L.
Note: The beaker containing the 3.0 mg/L NO3––N standard used in the calibration may also be used as a
check on the calibration. It should read close to 3.0 mg/L NO3––N ± 0.1 mg/L.
Nitrate
Page 1475
Nitrate

To verify sample measurement accuracy, add a spike of standard NO3––N solution with a
TenSette® or volumetric pipet. Use the Spike volumes of 100-mg/L standard table and the
formulas in Percent recovery.
Table 406 Spike volumes of 100-mg/L standard

Measured sample Concentration Volume of 100 ppm Standard to
CxV
NO3––N Add
0.04 to 0.3 0.1 mL 10

0.30 to 0.60 0.3 mL 30
0.6 to 0.9 0.5 mL 50
0.9 to 3.0 1.0 mL 100
Percent recovery
To calculate the percent recovery:
M = S × 100 + ( C × V )
M
E = --------------------
100 + V
A
R = ---- × 100%
E
Where:
M = calculated mass of nitrate as nitrogen present after the spike (micrograms)

S = mg/L of NO3––N in sample (before spike)
V = spike volume from the Spike volumes of 100-mg/L standard table (mL)
A = actual reading on meter after spike (mg/L NO3––N)
Method performance
Precision
Instrument Standard
sension 41 0.1 mg/L 0.095–0.1005 mg/L

1 With a stabilization criteria of 0.1 mV/min
Nitrate
Page 1476
Nitrate
Summary of method
Nitrate ions are selectively absorbed by the ISE membrane, establishing a potential (voltage) that
is proportional to the concentration of nitrate in the sample. This potential is compared to the
constant potential of a reference electrode by measuring the potential of known standard. A
calibration curve can be constructed to determine the concentration of nitrate in unknown samples.
The solvent-polymer membrane is a nitrate ion-exchanger in an inert polyvinyl chloride (PVC)
plastic matrix. The nitrate electrode has an internal silver/silver chloride element, which
establishes a fixed potential when in contact with the internal filling solution. The ion selective
membrane undergoes ion exchange with nitrate in the sample, creating a potential across the
membrane which varies with the amount of nitrate ion in the sample. This potential will decrease
by about 58 mV for every tenfold increase in nitrate concentration in the linear operating range
at 25 °C.
Required reagents
Ammonium Sulfate Reference Electrolyte Gel Cartridge varies 2/pkg 2597102
Nitrate Electrode Membrane Tips varies 16/pkg 4613300
Nitrate-Nitrogen Standard Solutions 10 mg/L as NO3––N varies 500 mL 30749
Nitrate ISA liquid varies 500 mL 2488349

Nitrate half-cell filling solution varies 50 mL 4456369
Nitrate-Nitrogen Standard Solution 100 mg/L NO3 ––N varies 500 mL 194749
Nitrate test strips varies 25/pkg 2745425

Required apparatus
Beaker, 150-mL, polypropylene each 108044
Pipet, TenSette, 0.1–1.0 m each 1970001
Flask, volumetric, poly, 100 mL each 1406042
Thermometer, Digital each 2630600
Flask, volumetric, 100 mL, polypropylene each 1406041
Nitrate Combination Electrode, Platinum series, BNC each 5192000
sension 4 Laboratory pH/ISE Meter each 5177500
Stir Bar, 7/8 x 3/16 in. (22.2 x 4.8 cm) each 4531500
Stirrer, electromagnetic, 115 VAC, with stand and stir bar each 4530001
Nitrate Electrode Membrane Tips (replacements) 6/pkg 4613300
Nitrate
Page 1477
Nitrate
Optional reagents
Nitrate Ionic Strength Adjustor, powder 454 g 4456301
Nitrate Nitrogen Standard Solutions 1 mg/L as NO3––N 500 mL 204649
Optional apparatus
Beaker, 100 mL, polypropylene each 108042
Pipet Tips, for TenSette Pipet 50/pkg 2185696
Water Bath, circulating each 2616300
Pipet, Mohr, 2.00 mL, glass each 2093636

Nitrogen Ammonia ISE, 10001
USEPA1 Direct Measurement ISE Method2 Method 10001

0.1 to 10.0 mg/L NH3-N ISE Electrode
1 USEPA Accepted for reporting wastewater analyses
2 Adapted from Standard Methods for the Examination of Water and Wastewater, 20th Edition, Method 4500NH3E (with distillation). Manual
distillation may not be required if comparability data on representative samples in company files show the distillation is not necessary. Manual
distillation will be required to resolve any controversies.
Test preparation


Meter Electrode
Refer to the meter manual for meter operation. Refer to electrode manual for electrode maintenance and care.
Prepare the electrode. Refer to New electrodes or electrodes stored more than 7 days and Electrodes stored 1 to 7 days for
more information.
After every hour of continuous use, place the electrode in the storage solution for 10 minutes to thoroughly recondition.
Check with a 10 mg/L NH3–N standard for accuracy and calibrate if necessary.
At high pH, ammonia solutions lose ammonia to the atmosphere, lowering the concentration. It is important to take
measurements as soon as possible after the solution is basic. For most wastewater samples, 1 mL of 10 N NaOH (or
equivalent ISA) is sufficient to increase the pH above 11. If in doubt, check the pH with pH paper and add additional NaOH in
0.1 mL increments until the pH exceeds 11.
Nitrogen, Ammonia
Page 1479
Nitrogen, Ammonia
Ammonia electrode filling solution varies
Ammonia electrode storage solution 20 mL
Ammonia nitrogen standard, 100 mg/L NH3–N varies
Ammonia ISA solution 2 mL per 100 mL sample
Ammonia electrode, combination BNC 1
Beaker, 150 mL, polypropylene 4
Wash bottle 1
sension 4 laboratory pH/ISE meter or sension 2 portable pH/SE meter 1
Stir bar, 22.2 x 4.76 mm (7/16 x 3/16 in.) 4
Stirrer, electromagnetic, with stand and stir bar 1
250 mL Class A volumetric flask 3
Nitrogen, Ammonia in wastewater method
1. Rinse the electrode 2. During the 3. Prepare a 1.0-mg/L 4. Prepare a 0.1-mg/L

with deionized water. conditioning period NH3–N standard by NH3–N standard by
Place it in the Ammonia prepare three standards. pipeting 25 mL of the pipeting 25 mL of the
ISE storage solution with Make a 10-mg/L NH3–N 10-mg/L standard into a 1.0-mg/L NH3–N standard
the ammonia membrane standard by pipeting 25 250-mL volumetric flask. into a 250-mL volumetric
sensor module on to mL of 100-mg/L NH3–N Dilute to the mark with flask. Dilute to the mark
condition for at least Standard into a 250-mL ammonia-free with ammonia -free
15 minutes. volumetric flask. Dilute to deionized water, stopper deionized water, stopper
the mark with ammonia - and thoroughly mix. and thoroughly mix.
free deionized water,
stopper and thoroughly
mix.
Nitrogen, Ammonia
Page 1480
Nitrogen, Ammonia
Nitrogen, Ammonia in wastewater method (continued)
5. Connect the Ammonia 6. Turn the meter on. 7. Press CAL. Use the 8. Transfer 100 mL of the
ISE to the BNC connector Press ISE/MV until the ARROW keys to select the 0.1-mg/L NH3–N standard
on the pH/ISE meter. display shows mg/L or desired units. Press to a 150-mL beaker. Add a
Verify that BNC is selected other chosen ENTER and accept stir bar to the beaker. Put
in Setup 1 of the concentration units. the units. the beaker on a magnetic
Setup menu. stirrer and stir at a
moderate rate.
Stabilizing...
9. Remove the electrode 10. Pipet 2.0 mL of 11. Press READ. The 12. The display will show
from the storage solution. Ammonia ISA Solution into display will show the value Stabilizing... until the
Rinse it with deionized the standard. Immediately from the previous reading is stable. The
water and blot dry. Put the proceed to the next step. calibration. Accept the display will show
electrode into the 0.1-mg/L numerical value or use the Standard 2 and _ _ _ _ or
NH3–N standard. number keys to change the value of standard 2
Make sure no air bubbles the value to match the from the previous
are trapped under the tip concentration of the calibration.
of the electrode. standard, then press
ENTER to accept the
change.
Nitrogen, Ammonia
Page 1481
Nitrogen, Ammonia
13. Rinse the electrode 14. After the last standard 15. The display will show 16. Press REVIEW. Use
with deionized water. is measured, press EXIT. Store?. Press ENTER to the Up arrow key to scroll
Place it in the storage store the calibration or to the second slope value.
solution for one minute. EXIT to leave the It should be –57 ± 3 mV/
Repeat steps 8–13 for calibration mode without decade. If the slope is not
substituting the 1.0- and storing the calibration –57 ± 3 mV/decade,
10-mg/L standards. values. recalibrate the electrode. If
the slope is still incorrect
after recalibration, replace
the ammonia membrane
sensor module.
Press EXIT to return to
measurement mode.
17. Remove the electrode 18. Transfer 100 mL of 19. Remove the electrode 20. Pipet 2.0 mL of
from the last standard. sample to a 150-mL from the storage solution. Ammonia ISA solution into
Rinse it with deionized beaker. Add a stir bar to Rinse with deionized water the sample and proceed
water and place it in the the beaker. Put the beaker and blot dry. Put the immediately to the next
storage solution. on a magnetic stirrer and electrode into the sample. step.
stir at a moderate rate.
Nitrogen, Ammonia
Page 1482
Nitrogen, Ammonia
Stabilizing...
21. Press READ. The

display will show
Stabilizing... until the
reading is stable. Record
or store the measurement
value.
Repeat steps 17–21 for
other samples.
Stabilization times will take
longer for lower
concentrations. A slow
downward drift in
concentration indicates
probable loss of ammonia
to the atmosphere. Record
the highest value that is
stable.
Calibration
Prepare ammonia standard working solutions of 10.0, 1.0 and 0.1 mg/L ammonia nitrogen from a
100-mg/L stock solution. Prepare the standards daily before use. Higher or lower concentration
ranges (0.05–1400 mg/L NH3–N) can be obtained by calibrating the meter with different
standard solutions.
Electrode preparation
New electrodes or electrodes stored more than 7 days
Before using a new Ammonia Electrode or an electrode that has been stored dry, remove the
protective cap from the end.
1. Unscrew the top cap. Carefully remove the internal glass electrode from the outer body. A
white membrane is mounted at the tip of the outer body.
2. Fill the outer body with 3.5 mL of Internal Fill Solution.
3. Rinse the internal glass electrode with deionoized water. Blot dry. Return the electrode to the
filled outer body. Make sure that the key pin at the top of the internal glass electrode is seated
in the slot at the top of the outer body.
4. Reinstall the threaded top cap onto the top of the ammonia electrode body. Finger-tighten the
cap until snug. Do not over-tighten.
Nitrogen, Ammonia
Page 1483
Nitrogen, Ammonia
5. Hold the fully assembled electrode securely by one end and shake the electrode with an
abrupt downward motion (like shaking the mercury down in a thermometer) to remove
bubbles.
6. Place the assembled electrode into the Ammonia Electrode Storage Solution or 1000 mg/L
Ammonia Standard for at least 60 minutes.
Electrodes stored 1 to 7 days

• Keep the electrode in 1000 mg/L ammonia standard without Ionic Strength Adjustor (ISA).
• Alternatively, keep the electrode in the Ammonia Electrode Storage Solution.
• Never let the membrane dry out. Cover the storage beaker and electrode body with Parafilm®
to prevent solution evaporation.
Electrodes stored between samples
Place the electrode in Ammonia Electrode Storage Solution for at least one minute to initialize the
electrode for the next measurement.
Interferences
Distillation prior to ammonia analysis removes all inorganic interferences that complex ammonia.

Amines Volatile low molecular weight gives a positive interference
Mercury Complexes with ammonia
Silver Complexes with ammonia

• Collect samples in glass or plastic containers of convenient size. Clean new bottles by
washing with deionized or distilled water. Fill the sample bottle completely and stopper
immediately. Analyze the sample as soon as possible.
• Ammonia may be lost more quickly from samples at temperatures above 50 °C, so it is
important to collect samples at less than 40 °C or use a cooling coil between the bottle and
sampling point if necessary.
• If chlorine is present, treat the sample immediately with sodium thiosulfate. Add one drop of
0.1 N Sodium Thiosulfate Standard Solution for each 0.3 mg of chlorine present in a one
liter sample.
• If prompt analysis is not possible, preserve the sample with 0.8 mL of concentrated sulfuric
acid per liter. Use a sension pH meter to be sure the pH of the preserved sample is between
1.5 and 2. Some wastewater samples may require more sulfuric acid to achieve this pH. Store
the sample at 4 °C. Samples preserved in this manner may be stored up to 28 days.
• Before analysis, neutralize the sample to pH 7 with 5 N sodium hydroxide. Do not let the pH go
above 10. Correct the test results for the volume addition.
• Do not use mercuric chloride as a preservative because ammonia complexes with
mercuric ions.
Nitrogen, Ammonia
Page 1484
Nitrogen, Ammonia
Accuracy check
1. Use the Spike volumes for standard additions table to determine the concentration and volume
of standard to spike the sample. The volume of sample transferred must be accurate.
2. Add the amount and concentration specified in the Spike volumes for standard additions table
to the 100 mL of sample.
3. After adding the standard, proceed with the calculations. Results from 90-110% recovery are
typically considered acceptable. Calculate percent recovery as follows:
100 ( X s – X u )
% Recovery = ----------------------------------
K
Where:
Calculations
Xi × Vu
1. X u = -----------------
-
Vu + V
Where:
C×V
2. K = -----------------
Vu + V
Where:
s100 ( X – X )
u
K
Nitrogen, Ammonia
Page 1485
Nitrogen, Ammonia
Example:
A sample was analyzed and read 5.0 mg/L NH3–N. As directed in the Spike volumes for standard
additions table, a 4.0-mL spike of 100-mg/L NH3–N standard was added to another 100-mL
sample, giving a final standard addition result of 8.75 mg/L.
5.0 mg/L × 100 mL
1. X u = ------------------------------------------------- = 4.81 mg/L
100 mL + 4 mL
100 mg/L × 4 mL
2. K = -------------------------------------------- = 3.85 mg/L
100 mL + 4 mL
100 × ( X s – X u ) 100 × ( 8.75 – 4.81 )

3. %R = ---------------------------------------- = -------------------------------------------------- = 102.3 % Recovery
K 3.85
Table 409 Spike volumes for standard additions

0.1–0.3 100 100 0.2
0.3–0.5 100 100 0.4
0.5–0.7 100 100 0.6
0.7–0.9 100 100 0.8
0.9–1.1 100 100 1.0
1.0–3.0 100 100 2.0
3.0–6.0 100 100 4.0
6.0–10.0 100 100 8.0
Method performance
Precision
Instrument Standard
sension 41 0.80 mg/L 0.78–0.82 mg/L
sension 21
1 With a default stabilization criteria of 0.5 mV/min.
Summary of method
The ammonia electrode measures ammonia gas or ammonium ions in aqueous solutions that
have been converted to gas by the addition of a strong base. The electrode is a complete
electrochemical cell consisting of a glass pH electrode and a reference electrode.
The gas-permeable membrane separates the sample from a thin layer of electrolyte that is
pressed between the pH bulb and the membrane. At high pH, ammonium ion is converted to
ammonia gas.
The gas diffuses through the membrane and causes a pH change in the thin layer of electrolyte.
The potential across the pH glass changes as a result of the pH change and the electrode
measures the change in potential. The measured pH change is proportional to the ammonia
concentration in the solution.
Nitrogen, Ammonia
Page 1486
Nitrogen, Ammonia
Required reagents
Ammonia Electrode Filling Solution varies 60 mL 4447226
Ammonia Electrode Storage Solution 20 mL 500 mL 2541249
Ammonia Nitrogen Standard, 100 mg/L NH3-N 100 mL 500 mL 2406549
2 mL/100 mL 500 mL 2824349
Ammonia ISA Solution
sample
Required apparatus
Ammonia Electrode 1 each 5192700
Beaker, 150 mL, polypropylene 4 each 108044
Bottle, wash, 500 mL 1 each 62011
Flask, volumetric, Class A, 250 mL 3 each 1457446
sension 4 Laboratory pH/ISE Meter or sension 2 pH/ISE (portable) 1 each 5177500
Stir Bar, 22.2 x 4.76 mm 4 each 4531500
TenSette® Pipet, 1.0–10.0 mL 1 each 1970010
Pipet tips for 1970010 TenSette Pipet varies 50/pkg 2199796
Class A 25 mL volumetric pipet 1 each 1451540
Stirrer, electromagnetic 115 V, with stand and stir bar 1 each 4530001
Optional reagents
Ammonia Nitrogen Standard Solution 1000 mg/L NH3-N 1L 2354153
pH Paper, pH 9.0-12.0 5 rolls/pkg 38533
Nitrogen, Ammonia
Page 1487
Nitrogen, Ammonia
Optional apparatus
Air Gap Assembly each 5025300
Ammonia Electrode Membrane Modules 4/pkg 5192711
Cylinder, graduated, glass 100 mL 50842
TenSette® Pipet, 0.1–1.0 mL each 1970001
Pipet tips for 1970001 TenSette Pipet 50/pkg 2185696
sension 2 Portable pH/ISE Meter each 5172500

Nitrogen Ammonia ISE, 10002
USEPA1 Known Addition ISE Method2 Method 10002

≥0.8 mg/L NH3-N ISE Electrode
1 USEPA Accepted for reporting wastewater analyses
2 Adapted from Standard Methods for the Examination of Water and Wastewater, 20th Edition, Method 4500NH3E (with distillation). Manual
distillation is not required if comparability data on representative samples in company files show the distillation is not necessary. Manual
distillation will be required to resolve any controversies.
Test preparation


Meter Electrode
Refer to the meter manual for meter operation. Refer to electrode manual for electrode maintenance and care.
Prepare the electrode:
Refer to the Distillation Apparatus manual to perform sample distillation, if necessary.
Prepare spiking solutions according to the Nitrogen and Ammonia in the Wastewater section.
When there is a linear relationship between concentration and response, the known addition method can be used to
measure occasional samples because calibration is not required. The sample concentration must be known within a factor of
three, in order to get accurate measurements, because the concentration of ammonia in the sample must be approximately
doubled by the standard addition.
Nitrogen, Ammonia
Page 1489
Nitrogen, Ammonia
Ammonia electrode filling solution varies
Ammonia electrode storage solution 20 mL
Ammonia nitrogen standard, 1000 mg/L NH3–N varies
Ammonia ISA solution 2 mL per 100 mL sample
Ammonia electrode, combination BNC 1
Wash bottle 1
sension 4 laboratory pH/ISE meter 1
Stir bar, 22.2 x 4.76 mm (7/16 x 3/16 in.) 1
Select on based on available voltage:
Stirrer, electromagnetic, 115 V, with stand and stir bar 1
Stirrer, electromagnetic, 230 V, with stand and stir bar 1
Nitrogen, Ammonia known addition method
1. Accurately transfer 2. Stir at a constant and 3. Use a TenSette Pipet 4. Remove the electrode
100 mL of sample to a moderate rate on a to add 1.0 mL of 10 N from the storage solution.
150-mL beaker using a magnetic stirrer to improve NaOH solution into the Rinse it with deionized
volumetric pipet or response time and sample. water and blot dry. Put the
graduated cylinder. Add a accuracy. electrode in the sample.
stir bar to the beaker. Make sure that no air
bubbles are trapped under
the tip of the electrode.
Remove bubbles by lightly
tapping the electrode or by
tilting the electrode to 20°.
Nitrogen, Ammonia
Page 1490
Nitrogen, Ammonia
Nitrogen, Ammonia known addition method (continued)
mL, Sample, ? Stabilizing...
5. Turn the meter on. 6. The display will show 7. The meter will prompt 8. The display will show
Press STD ADDN. Use the the slope value for the last for the sample volume Stabilizing... until the
ARROW keys to select the calibration (the default is – (in mL). Enter the sample baseline reading is stable.
required units. Accept the 59.2 mV). Accept the volume and press ENTER The meter will then prompt
units. numerical value or change to accept the volume. for the standard volume.
the slope value.
Standard
9. Enter the volume of 10. Obtain the standard 11. Add the volume of a 12. Enter the
standard to be used (for concentration and volume known standard (listed in concentration of the
example, 1.0 mL). Press from Table 4 on page 48 Table 4) to the beaker and standard used (for
ENTER and accept the after estimating the proceed as quickly as example, 1000 mg/L).
volume. sample concentration. possible through the rest Press ENTER and accept
of the procedure. the concentration.
Nitrogen, Ammonia
Page 1491
Nitrogen, Ammonia
Nitrogen, Ammonia known addition method (continued)
Sample + 20 mg/L
Standard
13. The display will show 14. The meter will

Sample+Standard and calculate and display the
Stabilizing... until the concentration of the
reading is stable. adjusted value for the
original sample in mg/L.
Record or store this value.
The display will show
STANDARD ADDITONS
when data is recalled for
standard additions.
Nitrogen ammonia in wastewater

Known addition is also a convenient check on the results of direct measurement. Because an
accurate measurement requires that the concentration approximately double as a result of the
addition, the approximate sample concentration must be known within a factor of three. Make a
spiking solution before beginning the procedure.
1. Use the Spiking solutions table to determine how to dilute a 1000 mg/L NH3-N stock solution
to use as a spiking solution.
2. Pipet the appropriate amount of 1000-mg/L NH3-N standard into a 100-mL volumetric flask.
3. Dilute to the mark with ammonia-free water.

4. Determine the slope before performing standard additions of the sample.
a. Use the 100 mg/L and 1000-mg/L NH3-N stock solutions to determine the slope.
b. Check the electrode occasionally to determine if it is functioning properly and to determine
its exact slope value. The frequency of this operation depends on the harshness of
the sample.
Table 411 Spiking solutions
Expected Sample Concentration (mg/L) mL of 1000-mg/L NH3-N Standard Concentration
0.8–4.0 2 20 mg/L
2.5–7.5 5 50 mg/L
5–15 10 100 mg/L
12–50 25 250 mg/L
25–75 50 500 mg/L
50–150 100 1000 mg/L
Nitrogen, Ammonia
Page 1492
Nitrogen, Ammonia
Electrode preparation
Before using a new Ammonia Electrode or an electrode that has been stored dry, remove the
protective cap from the end.
1. Unscrew the top cap. Carefully remove the internal glass electrode from the outer body. A
white membrane is mounted at the tip of the outer body.
2. Fill the outer body with 3.5 mL of Internal Fill Solution.
3. Rinse the internal glass electrode with deionoized water. Blot dry. Return the electrode to the
filled outer body. Make sure that the key pin at the top of the internal glass electrode is seated
in the slot at the top of the outer body.
4. Reinstall the threaded top cap onto the top of the ammonia electrode body. Finger-tighten the
cap until snug. Do not over-tighten.
5. Hold the fully assembled electrode securely by one end and shake with an abrupt downward
motion (like shaking the mercury down in a thermometer) to remove bubbles.
6. Place the assembled electrode into the Ammonia Electrode Storage Solution or 1000 mg/L
Ammonia Standard for at least 60 minutes.

• Keep the electrode in 1000 mg/L ammonia standard without Ionic Strength Adjustor (ISA).
• Alternatively, keep the electrode in the Ammonia Electrode Storage Solution.
• Never let the membrane dry out. Cover the storage beaker and electrode body with Parafilm®
to prevent solution evaporation.
Electrodes stored between samples
Place the electrode in Ammonia Electrode Storage Solution for at least one minute. This
reinitializes the electrode for the next measurement.
Interferences
Distillation prior to ammonia analysis removes all inorganic interferences that complex ammonia.

Amines Volatile low molecular weight gives a positive interference
Mercury Complexes with ammonia
Silver Complexes with ammonia

• Collect samples in glass or plastic containers of convenient size. Clean new bottles by
washing with deionized or distilled water. Fill the sample bottle completely and stopper
immediately. Analyze the sample as soon as possible.
• Ammonia may be lost more quickly from samples at temperatures above 50 °C, so it is
important to collect samples at less than 40 °C or use a cooling coil between the bottle and
sampling point if necessary.
• If chlorine is present, treat the sample immediately with sodium thiosulfate. Add one drop of
0.1 N Sodium Thiosulfate Standard Solution for each 0.3 mg of chlorine present in a one
liter sample.
Nitrogen, Ammonia
Page 1493
Nitrogen, Ammonia
• If prompt analysis is not possible, preserve the sample with 0.8 mL of concentrated sulfuric
acid per liter. Use a sension pH meter to be sure the pH of the preserved sample is between
1.5 and 2. Some wastewater samples may require more sulfuric acid to achieve this pH. Store
the sample at 4 °C. Samples preserved in this manner may be stored up to 28 days.
• Before analysis, neutralize the sample to pH 7 with 5 N sodium hydroxide. Do not let the pH go
above 10. Correct the test results for the volume addition.
• Do not use mercuric chloride as a preservative because ammonia complexes with
mercuric ions.
Accuracy check
1. Use the Spike volumes for known additions table to determine the concentration and volume
of standard to spike the sample. The volume of sample transferred must be accurate.
2. Add the amount and concentration specified in the Spike volumes for known additions table to
the sample while performing the standard addition method on the sample. Do not allow the
sample to stand too long before spiking or ammonia will be lost to the atmosphere. T
3. After adding the standard, proceed with the calculations. Results from 90–110% recovery are
typically considered acceptable. Calculate percent recovery as follows:
100 ( X s – X u )
% Recovery = ----------------------------------
K
Where:
Calculations
Xi × Vu
1. X u = -----------------
-
Vu + V
Where:
C×V
2. K = -----------------
Vu + V
Where:
s100 ( X – X )
u
K
Example:
Nitrogen, Ammonia
Page 1494
Nitrogen, Ammonia
A sample was analyzed and read 5.0 mg/L NH3–N. As directed in the Spike volumes for known
additions table, a 4.0-mL spike of 100-mg/L NH3–N standard was added to another 100-mL
sample, giving a final standard addition result of 8.75 mg/L.
5.0 mg/L × 100 mL
1. X u = ------------------------------------------------- = 4.81 mg/L
100 mL + 4 mL
100 mg/L × 4 mL
2. K = -------------------------------------------- = 3.85 mg/L
100 mL + 4 mL
100 × ( X s – X u ) 100 × ( 8.75 – 4.81 )

3. %R = ---------------------------------------- = -------------------------------------------------- = 102.3 % Recovery
K 3.85
Table 413 Spike volumes for known additions

0.8–1.0 100 100 1.0
1–3 100 100 2.0
3–6 100 100 4.0
6–9 100 100 8.0
9–12 100 100 10.0
12–20 100 1000 2.0
20–40 100 1000 4.0
40–60 100 1000 6.0
60–75 100 1000 8.0
Method performance
Precision
Instrument Standard
sension 4 5.00 mg/L 4.81–5.19 mg/L
sension 2 4.81–5.19 mg/L
Summary of method
The ammonia electrode measures ammonia gas or ammonium ions in aqueous solutions that
have been converted to gas by the addition of a strong base. The electrode is a complete
electrochemical cell consisting of a glass pH electrode and a reference electrode.
The gas-permeable membrane separates the sample from a thin layer of electrolyte that is
pressed between the pH bulb and the membrane. At high pH, ammonium ion is converted to
ammonia gas.
The gas diffuses through the membrane and causes a pH change in the thin layer of electrolyte.
The potential across the pH glass changes as a result of the pH change and the electrode
measures the change in potential. The measured pH change is proportional to the ammonia
concentration in the solution.
Nitrogen, Ammonia
Page 1495
Nitrogen, Ammonia
Required reagents
Ammonia Electrode Filling Solution 3 mL 50 mL 4447226
Ammonia Electrode Storage Solution 5 mL 500 mL 2541249
Ammonia Nitrogen Standard, 1000 mg/L NH3-N varies 1L 2354153
Sodium Hydroxide Solution, 10 N 10 mL 500 mL 2545049
Required apparatus
Ammonia Electrode 1 each 5192700
Beaker, 150 mL, polypropylene 1 each 108044
Bottle, wash, 500 mL 1 each 62011
Stir Bar, 22.2 x 4.76 mm 1 each 4531500
TenSette® Pipet, 1.0–10.0 mL 1 each 1970010
Pipet tips for 1970010 TenSette Pipet varies 50/pkg 2199796
Optional reagents
pH Paper, pH 9.0-12.0 5 rolls/pkg 38533
5.0 N NaOH 1000 mL 245053
Optional apparatus
Ammonia Electrode Membrane Modules 4/pkg 5192711
Pipet tips for 1970001 TenSette Pipet 50/pkg 2185696

Sodium ISE, 8359
Sodium DOC316.53.01240

10 to 2000 µg/L Na+ ISE Electrode
Scope and Application: Drinking water and process water application
Test preparation


Meter Electrode
1 Only sension 4 meters can be used for this analysis.
Before the test, prepare a 10 mg/L Na+ standard:

1. Use a Class A pipet to dispense 10 mL of 1000 mg/L Na+ standard into a 1.0 L volumetric flask.
This procedure requires several conditioning steps. Read through all the procedure steps before proceeding.
This test is for low range analysis only. If the electrode is conditioned to a low sodium concentration (as in this procedure),
putting it in a concentrated Na+ (10 mg/L or higher) will swamp the electrode with sodium ion and the overnight conditioning
step must be repeated.
Measure millivolt potentials of all sodium standards at the same temperature ± 0.5 °C. Samples and standards must be
measured at the same temperature, ± 1 °C. Use room temperature 100 mg/L Na+ standard. This procedure keeps
temperature error to a minimum by using a spiked additions method of calibration.
Note A: If the electrode is conditioned properly, the mV potential should not change more than 0.1 mV every minute in step
10. If excessive drift occurs leave the electrode in the 600-mL beaker until the drift slows. If the electrode drifts 10 mV or
more in the positive direction (for example, from –175 to –165 mV), repeat steps 5–10, but do not dispense electrolyte gel
again as directed in step 7.
Potassium chloride electrolyte gel cartridge 1
pH/ISE meter, sension™4 (laboratory) 1
Sodium ISE Electrode, BNC 1
Sodium ISA powder pillows 1
Sodium
Page 1497
Sodium
Sodium standard solutions:
100 mg/L Na+
1000 mg/L Na+
Flask, 100-mL, volumetric 1
Pipet, TenSette™, 0.1 to 1.0 mL, plus tips 1
Stir Bar, 7/8 X 3/16 in. (22.2 x 4.8 cm) 1
Sodium ISE, powder pillow method
1. Install the Potassium 2. Connect the 3. Prime the electrode by 4. Condition the
Chloride Electrolyte Gel combination sodium pushing the dispenser electrode in sodium
Cartridge in the Platinum electrode to the meter. button until gel comes out electrode storage solution
Series Combination Ensure the electrode has of the reference junction. for a minimum of 1 hour
Sodium Electrode. been conditioned for at Rinse excess gel from the before use. Then,
least 8 hours in Sodium tip and the outlet. condition the electrode in
Electrode Storage Solution 0.10 mg/L sodium for at
before its initial use. least 8 hours.
To make 100 mL of 0.10
mg/L Na+ standard, use a
TenSette™ Pipet to put
0.10 mL of 100 mg/L Na+
into a 100-mL volumetric
flask and dilute to the
mark. Mix well.
Sodium
Page 1498
Sodium
Sodium ISE, powder pillow method (continued)
5. Accurately 6. Pour the water in the 7. Remove the electrode 8. Use a TenSette® Pipet
measure 400 mL of cylinder into a 600-mL from the 0.10 mg/L sodium to add 0.4 mL of 10 mg/L
deionized water into a plastic beaker. Add the standard, dispense gel Sodium Standard Solution
plastic 500-mL graduated contents of one Sodium and rinse excess gel away to the solution in the
cylinder. Ionic Strength Adjustor with deionized water. beaker. Refer to the Low
The deionized water must powder pillow to Place the electrode in the level sodium calibration
be at room temperature. the solution. 600-mL beaker, table. (This makes 400 mL
Add a large stir bar (50.8 x submerging the tip below of 0.010 mg/L or 10 µg/L
7.9 mm) to the beaker. the solution surface. sodium.)
Place the beaker on an Allow the electrode
electromagnetic stirrer and to condition for 15 minutes
begin stirring at a in this solution
moderate rate. before proceeding.
9. Turn the meter on. Set 10. Press ISE MV until the 11. Press ISE MV to toggle 12. Edit the display to
the electrode type to BNC. mV potential shows on the to concentration units. show the concentration of
Press SETUP and scroll to display. Refer to Before Press CAL and scroll to the solution in the 600 mL
Stabilizing. starting the test: Note A. µg/L. Press ENTER to beaker (10 µg/L). Refer to
After the measurement is accept the concentration the Low level sodium
Press ENTER and edit the units. calibration table.
display to show 0.1 mg/ stable, record the potential
min. Accept the value and value. Press ENTER to accept the
EXIT the setup menu. Measure the temperature concentration.
of the standard (°C) with a
lab grade thermometer.
Record the temperature.
Sodium
Page 1499
Sodium
Repeat step 13
13. When the 14. Repeat step 13, 15. Remove the electrode 16. Accurately measure
measurement stabilizes, adding the additional from the last standard, 400 mL of sample into a
pipet the corresponding volumes of 10 mg/L and rinse well with deionized 500-mL graduated
additional volume from the 100 mg/L Na+ standard water and blot dry. cylinder.
10-mg/L Na+ standard in from the Low level sodium Save the solution in the The sample must be at the
the Low level sodium calibration table until all 600-mL beaker with the same temperature as the
calibration table. Wait the seven standards have 2.00 mg/L Na+ for later standard solution in the
amount of time specified been measured. Store the calibration checks. 600-mL beaker used to
for step 2 in the table to calibration and return to perform the calibration.
allow the membrane to measurement mode.
respond.
Enter the concentration in
µg/L (20 µg/L). Press
ENTER to accept the
concentration.
Sodium
Page 1500
Sodium
17. Pour this 400 mL of 18. Add the contents of 19. Place the electrode 20. When the
sample into a 600-mL one Sodium Ionic Strength into the sample. measurement stabilizes,
beaker. Add a magnetic Adjustor Powder Pillow. Wait 10 minutes to allow record the sample
stir bar, place the beaker electrode to condition to concentration value.
on an electromagnetic the low level sodium in If the sodium
stirrer. Stir at a moderate solution. concentration is more than
rate. the 10 to 2000 g/L
If the electrode is placed in calibration range, press
a sample of high sodium the ISE mV key. If the mV
concentration, errors may reading is greater than the
result and reconditioning mV at 2000 g/L Na+,
to lower sodium levels will analyze the sample using
be required. the procedure for Potable,
Ground and Irrigation
Water (refer to the Sodium
electrode manual). If the
mV reading is less than
the mV reading of the 10
g/L standard, there is less
than 10 g/L Na+ in the
sample.
Calibration
Use a water bath slightly above room temperature (25 °C) to equilibrate the standard temperature
and sample temperature before measuring mV potentials. Use a laboratory-grade thermometer to
monitor the temperature. A one degree centigrade difference may result in as much as a 0.4 mV
inaccuracy. This temperature variation will, in turn, decrease accuracy of concentration
measurements.
Table 415 Low level sodium calibration

Concentration
Volume 10 mg/L Na+ standard Volume 100 mg/L Na+
Step
added standard added µg/L Time
1 0.4 mL 10 until mV stabilizes
2 0.4 mL 20 15 min.
3 1.2 mL 50 15 min.
Sodium
Page 1501
Sodium
Table 415 Low level sodium calibration

Concentration
Volume 10 mg/L Na+ standard Volume 100 mg/L Na+
Step
added standard added µg/L Time
4 0.2 mL 100 10 min.
5 0.4 mL 200 10 min.
6 1.2 mL 495 10 min.
7 2.0 mL 1000 10 min.
8 4.0 mL 2000 10 min.
Interferences
The Sodium ISA is formulated to remove most interferences. Silver is a major interference.

Analyze immediately after sampling. If immediate analysis is impossible, cool samples to 4 °C and
analyze within six hours
Low ionic strength conditioning

The conditioning steps in the section apply to measurements in samples containing less than
1.0 mg/L Na+.
Initial use
The sensing half-cell is packaged and shipped dry from the factory with a dry cap over the glass
membrane tip.
1. Remove the cap and install the gel cartridge.

2. Click the reference electrolyte dispenser until gel emerges from the tip.
3. Rinse the electrode with a small stream of sample delivered through a disposable plastic
Pasteur pipette or with deionized water from a wash bottle.
4. Place the tip in Sodium Electrode Storage Solution or in 1 M sodium chloride solution and soak
for at least one hour. Condition the electrode in 0.10 mg/L sodium solution for at least 8 hours
before calibrating using the Low Range Sodium Method. Refer to the electrode user manual
for more information.
Sodium
Page 1502
Sodium
Between uses
Between uses, in intervals of up to a few hours, the electrode can be stored in the sample (if not an
extreme pH) or in a neutral low-ionic-strength solution such as tap water. Before measuring a new
sample, refresh the reference electrolyte gel by clicking the dispenser several times. Carefully
rinse the electrode to prevent contaminating the sample.
Accuracy check
Electrode response
To verify electrode response at these low levels of sodium, the millivolt potential should increase
upon each addition of 100 mg/L Na+. Using the Low Level Sodium procedure, at least a 4.0 mV
increase should be observed from step 1 to step 2 (10 µg/L to 20 µg/L Na+). Each additional spike
should increase the mV reading substantially from the previous change. If this is not the case,
check the purity of the standard used. If this is not the problem, the electrode is probably not
conditioned for low sodium levels.
Calibration accuracy
1. Fill the 500-mL graduated cylinder to the 400-mL mark with deionized water.
2. Pour this solution in a 600-mL beaker and add a stir bar.
3. Pipet 0.2 mL of 100 mg/L Sodium Standard into the 600-mL beaker.
4. Add the contents of one Sodium Ionic Strength Adjustor powder pillow and place on an
electromagnetic stirrer.
5. Rinse the calibrated electrode before placing in solution. Measure the concentration of the
solution. The reading should be approximately 50 µg/L.
Note: The beaker with the 2000 µg/L Na+ standard used in the calibration may be used as a check on the
calibration. It should read close to 2000 µg/L.
Accuracy of the sample measurement

To verify sample measurement accuracy, add a spike of standard solution with a TenSette® or
volumetric pipet. Use the Spike volumes of 100-mg/L standard table and the formulas in Percent
recovery.
Table 416 Spike volumes of 100-mg/L standard

Measured sample Concentration Volume of 100 ppm Standard to
CxV
Na+ (µg/L) Add
10 to 49 0.1 mL 10
50 to 99 0.2 mL 20
100 to 299 0.4 mL 40
300 to 599 1.2 mL 120
600 to 2000 2.0 mL 200
Sodium
Page 1503
Sodium
Percent recovery
To calculate the percent recovery:
M = S × 400 + ( C × V )
M
E = --------------------
400 + V
A
R = ---- × 100%
E
Where:
M = calculated mass of sodium present after the spike (micrograms)

S = mg/L of Na+ in sample (before spike)
V = spike volume from the Spike volumes of 100-mg/L standard table (mL)
A = actual reading on meter after spike
Method performance
Precision
Instrument Standard
sension 41 25 µg/L 21.34–28.66 µg/L

1 With a stabilization criteria of 0.1 mV/min
Sodium
Page 1504
Sodium
Required reagents
Potassium Chloride Reference Electrolyte Gel Cartridge varies 3/pkg .2546902
Sodium Ionic Strength Adjustor Powder Pillows varies 100/pkg 4451569
Sodium Standard Solutions 100 mg/L as Na+ varies 1000 mL 2318153
Sodium Standard Solutions 1000 mg/L as Na+ varies 500 mL 1474949
Required apparatus
Cylinder, graduated, 500 mL, polypropylene each 108149
Pipet Tips, for TenSette Pipet 50/pkg 2185696
sension 4 Laboratory pH/ISE Meter each 5177500
Sodium Combination Electrode, Platinum series, BNC each 5192500
Optional reagents
Sodium Ionic Strength Adjustor (ISA), powder 454 g 4451501
Optional apparatus
Water Bath, circulating each 2616300
Sodium
Page 1505
Oxygen, dissolved, 8157
Direct Measurement Method Method 8157

0 to 20 mg/L or 0 to 200% saturation Clark-type Amperometric Sensor
Scope and Application: For water, wastewater and process water applications
Test preparation


Meter Electrode
sension™ 5 meters 5197000, 5197003 or 5197015
sension™ 8 meters 5197000, 5197003 or 5197015
Assemble the probe. Refer to the Probe assembly section in this procedure.
Refer to the sension meter manual for instructions on polarizing and zeroing the meter.
Dissolved oxygen probes are continuously polarized when they are connected to the meter. A steady reading will not be
seen for 30 to 50 minutes when the probe electrolyte is new or when the probe has been unplugged for more than one hour.
Interrupted connections of less than one hour will require 5 to 25 minutes before a stable reading is observed.
While not in use, the probe should be stored with the Calibration and Storage Chamber attached to the end of the probe. The
sponge inside the Chamber should be kept moist.
Keep the DO probe at a uniform temperature. When holding the probe, do not touch the metallic button on the side of the
probe. The button is a temperature sensor. An inaccurate calibration will result if the temperature of the thermistor is different
from the probe membrane.
The displayed % saturation is based on a meter calculation for the equilibrium dissolved oxygen concentration. The
calculation uses the sample temperature, salinity, barometric pressure, altitude and measured concentration in mg/L values.
Changing the entries in setups 4, 5 or 6 will change the displayed mg/L or % saturation. Refer to the sension meter manual
for more information.
Dissolved oxygen probe 1
sension 6 or 8 meter 1
Filling solution, dissolved oxygen 2 mL
Calibration and storage chamber 1
Oxygen, Dissolved
Page 1507
Oxygen, Dissolved
Membrane for dissolved oxygen probe 1
Beaker, polypropylene, 100-, 250-, 400- or 600-mL 1
Dissolved oxygen, amperometric sensor method
1. At least one hour 2. Zero the Dissolved 3. Secure the probe 4. Prepare the calibration
before measurement, Oxygen meter before cable to the calibration and and storage chamber by
polarize the probe by calibration when storage chamber by holding it under water and
connecting it to the meter. measuring dissolved wrapping cable through squeezing it a couple of
oxygen levels less than the bottom of the chamber times to pull water into the
1 mg/L or 10% saturation. lid before filling with water. lower chamber through the
Refer to Zeroing the inlet. Alternately, open the
probe. bottom of the chamber and
insert a damp sponge.
New sponges will be
compressed. Add water to
expand them. Avoid
completely filling the lower
chamber with water.
Oxygen, Dissolved
Page 1508
Oxygen, Dissolved
Dissolved oxygen, amperometric sensor method (continued)
5. Insert the DO probe 6. Allow at least ten 7. Press CAL. Edit the 8. The display will show
into the calibration and minutes for the barometric pressure. the current value for
storage chamber. The atmosphere in the Press ENTER and enter sample salinity (‰).
probe tip must not be chamber to reach a steady the current altitude and Set the salinity to zero
flooded with water or be state. press ENTER. (0‰).
holding a drop of water on To speed up probe Refer to the Adjusting
the membrane. stabilization, squeeze the barometric pressure and
lower chamber a couple of altitude table.
times to force water
saturated air into
the chamber.
9. Press ENTER. The 10. Add the weight 11. If sample salinity has 12. Insert the probe into
display will show 100%. assembly to the probe if been measured using an the sample. The probe
Press READ. When the required (3- or 15-m cable Electrolytic Conductivity must be deep enough to
calibration is complete, the versions only). Meter, enter the value in cover the thermistor
meter will return to parts per thousand. (metallic button) located
measurement mode. on the side of the probe.
Oxygen, Dissolved
Page 1509
Oxygen, Dissolved
Dissolved oxygen, amperometric sensor method (continued)
13. Agitate the probe in 14. Stir the sample 15. When the reading on 16. Change the display
the sample to dislodge air vigorously with the probe the meter stabilizes, from concentration in mg/L
bubbles from the sensing or use a stir stand and stir record or store the value in to % saturation if %
area of the probe tip. bar (for laboratory use). the meter memory. saturation is required.
When measuring deep
bodies of water, create
sufficient flow across the
probe tip by pulling on the
cable to move the probe
up and down (for field
use).
For high Sodium concentration samples
1. Pour the 400 mL 2. Add the contents of 3. Put the electrode into 4. When the
sample into a 600-mL one Sodium Ionic Strength the sample. measurement stabilizes,
beaker. Add a magnetic Adjustor Powder Pillow. Wait 10 minutes to allow record the sample
stir bar and put the beaker electrode to condition to concentration value.
on an electromagnetic the low level sodium in
stirrer. Stir at a moderate solution.
rate.
If the electrode is in a
sample of high sodium
concentration, errors may
result and reconditioning
to lower sodium levels will
be required.
Oxygen, Dissolved
Page 1510
Oxygen, Dissolved
Barometric pressure and altitude adjustment
Table 418 Adjusting barometric pressure and altitude

Enter a new barometric pressure when the barometric pressure or the altitude of the instrument changes using one of the
methods below:
Sea level equivalent True barometric pressure
1. Obtain the sea level equivalent barometric pressure from 1. Obtain the true barometric pressure from a nearby
TV, radio or a local airport. mercury barometer.
2. Enter this value into the meter. Refer to the Changing the 2. Enter this value into the meter. Refer to the Changing the
Barometric Pressure section of the meter manual. Barometric Pressure section of the meter manual.
3. Enter the local altitude. Refer to the Adjusting the Altitude 3. Enter the altitude as 0 feet or meters. Refer to the
section of the meter manual. Adjusting the Altitude section of the meter manual.
Probe assembly
1. Hold the membrane module cap in a vertical position. Fill the module cap about 2/3 of the way
full with Dissolved Oxygen Electrolyte Filling Solution.
2. Hold the DO probe in a vertical position with the tip down. Gently screw the module cap onto
the tip. Electrolyte should leak out of the threads.
Note: If electrolyte does not leak out of the threads, air may remain inside the module cap. Repeat this
procedure using more filling solution.
3. Attach the DO probe cable connector to the input connector at the top of the meter. Refer to
the sension 6 Dissolved Oxygen Meter Instruction Manual for additional information.
Probe polarization
Dissolved oxygen probes are continuously polarized when they are connected to the sension
meter. A steady reading will not be seen for 30 to 50 minutes when the probe electrolyte is new or
when the probe has been unplugged for more than one hour. Interrupted connections of less than
one hour will require 5 to 25 minutes before a stable reading is observed.
While not in use, the probe should be stored with the calibration and storage chamber attached to
the end of the probe. Keep the sponge inside the chamber moist.
Zeroing the probe

Zeroing the sension 6 Dissolved Oxygen meter is necessary only when measuring dissolved
oxygen levels less than 1 mg/L or 10% saturation. A new DO probe can generate a 0.02 to 0.05
mg/L positive error in an oxygen-free (anoxic) solution. If this level of error cannot be tolerated,
zero the meter using the following procedure. Zero the meter also after replacing the sensing
membrane or changing the internal filling solution.
Procedure
1. Measure about 150 mL of sample or deionized water into a 250-mL beaker. Place a magnetic
stir bar in the beaker.
2. Add 0.25 g sodium sulfite or the contents of one Silica 3 Reagent Powder Pillow to the water.
Stir to dissolve the reagent.
3. Catalyze the reduction of dissolved oxygen by adding 0.1 mL of a 1000 mg/L Cobalt Standard
solution to the water.
Oxygen, Dissolved
Page 1511
Oxygen, Dissolved
4. Place the probe in the stirring sample for at least 10 minutes. This solution is effective for 30
minutes or more.
5. Press the CAL key. The CAL icon will appear in the upper left corner of the display.
6. Press the READ ENTER key three times to skip to the display showing 100%.
7. Press the 0 key on the keypad then press READ ENTER.
8. The meter shows Stabilizing... while the readings are taken. When the meter’s zero DO
criteria have been met, it will return to the read mode. The meter will not exit the zeroing
routine until the meter’s zero criteria have been met.
9. When the meter cannot complete the zeroing procedure, it will begin to beep and show the
faulty probe icon. If the meter does not complete the zeroing procedure and exits to the
reading mode, add additional sodium sulfite and cobalt standard solution to the stirring water.
Otherwise, press EXIT to return to one display screen at a time and leave the calibration
routine without completing the zeroing procedure.
Interferences
Oxidizing gases such as chlorine, chlorine dioxide, sulphur trioxide and bromine can react at the
cathode to produce positive interferences. Reducing gases such as hydrogen, hydrogen sulfide,
sulfur dioxide and boranes can react at the anode. After exposure to reducing gases, the user may
need to clean the anode and replace the internal filling solution and membrane cap.
Oxygen, Dissolved
Page 1512
Oxygen, Dissolved

• Analyze samples in-situ, if possible.
• Collect samples in 300 mL glass BOD bottles. Fill the bottles completely.
• Analyze samples immediately. Do not store samples.
Accuracy check
1. Return the electrode to the calibration and storage chamber. The chamber should contain a
wet sponge or a small amount of water.
2. Allow at least 10 minutes for stabilization.
3. Enter the current barometric pressure and altitude into the meter. Refer to the meter manual.
4. The meter should display 100% saturation. If not, recalibrate the meter.
Method performance
Refer to the electrode and meter manual to determine the method performance.
Summary of method
The Dissolved Oxygen Probe is a Clark-type amperometric sensor used to measure dissolved
oxygen in aqueous solutions. It consists of an anode/cathode electrode system and potassium
chloride-based electrolyte, separated from the sample by a replaceable oxygen-permeable
Teflon® membrane. At a constant temperature, the electric current varies linearly with the oxygen
concentration of the solution. A built-in thermistor provides automatic temperature compensation
when using the sension Dissolved Oxygen Meters. The unit % Dissolved Oxygen is dependant
on the temperature and salinity of the sample and the barometric pressure of the environment
where the measurement is taken.

Required reagents
Filling Solution, Dissolved Oxygen 59 mL 2759126
Required apparatus
sension 6 meter, 115 VAC each 5185000
sension 8 meter, 115 VAC each 5455000

Cable, Dissolved Oxygen Probe, 1 meter .each .5197000
Cable, Dissolved Oxygen Probe, 3 meter each 5197003
Cable, Dissolved Oxygen Probe, 15 meter each 5197015
Barometer, Digital .each .2758400
Batteries, AA 4/pkg 1938004
BOD Accessory Kit includes funnel and spacer for Dissolved Oxygen Probe each 5197100
Calibration Storage Chamber, Dissolved Oxygen Probe each 5197400
Dissolved Oxygen Service Kit, includes 2 membranes, fill solution, polishing cloth, each 5196800
2 sponges
Oxygen, Dissolved
Page 1513
Oxygen, Dissolved

Docking Station, external, 115 V, N. American style plug each 5187501
Docking Station, external, 230 V, European style plug each 5187502
Membranes, for Dissolved Oxygen Probe 2/pkg 5197300
Weight Assembly each 5196900

Cobalt Standard Solution, 100 mg/L 100 mL 2150342
Silica 3 Reagent Powder Pillows (contains sodium sulfite) 100/pkg 27169
Sodium Sulfite 454 g 19501

Oxygen Demand, Biochemical, LBOD 10230
Oxygen Demand, Biochemical DOC316.53.01242
Dilution Method1 Method 10230

Scope and Application: For water and wastewater. LBOD Measurement
1 Adapted from Standard Methods for the Examination of Water and Wastewater and from Klein, R.L.; Gibbs, C. Journal of Water Pollution
Control Federation, 1979, 51(9), 2257. USEPA recommended for compliance monitoring. Meets ASTM 888-05 (C)
Test preparation
The BOD test is a 5-day test. Follow all steps carefully to make sure that the test does not have to be repeated. The
IntelliCAL™ LBOD probe measures dissolved oxygen in a 300 mL BOD bottle.
The dilution water for this test must not have an oxygen demand or any toxins. When incubated for 5 days at 20 °C, the
dissolved oxygen concentration in the dilution water must not change by more than 0.2 mg/L.
Carbonaceous BOD (CBOD) can be determined by the addition of nitrification inhibitor. A test for CBOD is recommended for
biologically treated effluents, samples seeded with biologically treated effluents and river water.
BOD bottles, 300-mL, glass, with glass stoppers and plastic caps 6
Dilution water containing nutrient buffer and seed (see Dilution water preparation) varies
HQ40d or HQ30d meter with IntelliCAL LBOD101 probe 1
Nitrification inhibitor (for CBOD only) 1 bottle
Pipet, seriological 1
Incubator 1

Page 1515
Dilution method
select
sample
size
1. Prepare the dilution 2. Select the sample 3. Stir the sample gently 4. Fill an additional BOD
water using a BOD volumes. See Sample size with the pipet. Use the bottle with dilution water
Nutrient Buffer Pillow. See selection. pipet to add the minimum only. This will be the
Dilution water preparation. sample volume to the first dilution water blank.
BOD bottle.
Add the remaining four
sample volumes to four
more BOD bottles.
When analyzing
disinfected samples or
industrial effluents, refer to
Interferences.
5. If the test is for CBOD, 6. Fill each bottle to just 7. Stopper the bottles 8. Probe calibration is
add two portions of below the lip with dilution carefully to prevent air required before initial and
Nitrification Inhibitor water. Allow the dilution bubbles from becoming final BOD readings. Refer
(approximately 0.16 g) to water to flow down the trapped. Press down on to the Calibration section
each bottle. sides of the bottle to the stopper and invert the of this procedure.
The oxidation of nitrogen prevent air bubbles from bottles several times to Be sure to measure the
compounds will be becoming trapped in the mix. DO of the blank.
prevented. Report results bottle.
An initial DO
as CBOD. measurement is not
necessary when the
graphical method (not for
reporting) is used for
calculation.

Page 1516
Dilution method (continued)
Read
9. Rinse the LBOD probe 10. Place the LBOD Probe 11. Engage the stir 12. Press the key under
with deionized water. in the BOD Bottle paddle on the LBOD probe READ. After the
containing the sample. by pushing the button on measurement has
Make sure there are no air the top of the probe. The stabilized, the dissolved
bubbles trapped under the green indicator light on the oxygen value will show on
probe. top of the probe will the display.
illuminate when the stirrer
is running.
Read
DO
13. Record the value. 14. Remove the LBOD 15. After five days, 16. Calculate the BOD
Data is stored Probe from the bottle and measure the remaining value (see BOD
automatically in the Data stopper the bottles dissolved oxygen calculation—standard
Log when Press to Read carefully to prevent air concentration in each methods or BOD
or Interval is selected in bubbles from becoming bottle. calculation—graphical
the Setup Measurement trapped. Add dilution water At least 1.0 mg/L DO method).
Mode. When Continuous to the lip of each BOD should be left in each
is selected, data will only Bottle to make a water BOD bottle.
be stored when the seal.
GREEN/RIGHT key under Repeat Steps 9–14 for
Store is pressed. each BOD bottle.
Calibration
Water-saturated air calibration
1. Fill a BOD bottle ¾ full with water (225 mL). If a 0% calibration point is required, refer to the
Sulfite correction section.
2. Put the BOD stopper in the BOD bottle and shake vigorously for about one minute to saturate
the air with water.

Page 1517
3. Remove the stopper. Put the Intellical LBOD Probe into the BOD bottle for several minutes to
reach equilibrium. Inspect the LBOD probe sensor cap surface to make sure it is dry. If the
sensor cap is wet, carefully dry the cap with a non-abrasive cloth.
4. Make sure the meter is in the measurement screen. Press CALIBRATION.

Note: For the HQ40d meter with two probes connected, make sure the meter is in the single scree
LDO101 mode.
5. Press READ. When the measurement has stabilized, the calibrated measurement will show on
the screen. The standard value will be highlighted.
6. Press DONE to view the calibration summary. The slope value is the comparison between the
latest calibration and the factory calibration expressed as a percentage.
Note: If the calibration slope does not meet the acceptance criteria, the display will show “Slope out of
range”. Let the probe stand in water-saturated air for several minutes. When the probe reaches
equilibrium, press READ.
7. Press STORE to accept the calibration and return to the measurement mode. The calibration
record is stored in the data log.
Note: A successful calibration will show “OK” in the measurement screen.
Sulfite correction
1. Fill a BOD bottle full with deionized water.
2. Add 300 mg of sodium sulfite to the bottle.
3. Add a small crystal of cobalt chloride.
4. Put the stopper in the BOD bottle and invert several times to mix the chemicals.
5. Put the LBOD probe in the bottle and engage the stirrer. This will help speed up the
calibration. When the meter reaches a stable reading, press the calibration button on
the meter.
6. After the 0% saturated message is displayed press STORE. After using sulfite, be sure to clean
the probe thoroughly.
7. To clean the sulfite off of the probe, put the LBOD probe in a BOD bottle full of water, activate
the stirrer and run for 10 minutes to remove sulfite residue.
Dilution water preparation

The dilution water must be prepared very carefully to make sure that no source of oxygen demand
or toxins are added. The water that is used to prepare the dilution water must be of very high
quality. The water must not have any organic compounds or any toxic compounds such as
chlorine, copper and mercury.
Use the following guidelines to make sure the dilution water is of high quality.
Guidelines
• Use distilled water from an alkaline permanganate distillation for the best results.
• Do not use deionized water from ion exchange columns. The resins in the cartridges
(especially new cartridges) will occasionally release organic materials that have an oxygen
demand. In addition, bacteria can grow on the columns and contaminate the dilution water.
• Store the distilled water in clean jugs in an incubator at 20 °C. Shake the jugs to saturate the
water with air or cap the jugs loosely and store for 24 hours or more.

Page 1518
• A small aquarium pump or air compressor can be used to saturate the water with air. Make
sure that the air is filtered and that the filter does not grow bacteria.
• Add the nutrients and seed (if necessary) to the distilled water immediately before the test.
• The dissolved oxygen concentration in the dilution water must not change by more than
0.2 mg/L when incubated for 5 days at 20 °C.
Procedure
1. Prepare and store the distilled water at 20 °C (see Guidelines).
2. Select a BOD nutrient buffer pillow from the BOD nutrient buffer pillows table.
3. Shake the pillow to mix the contents.
4. Add the contents of the pillow to the distilled water. Cap the jug and shake vigorously for one
minute to dissolve the nutrients and to saturate the water with air.
5. If the sample is known to be low in bacteria, for example industrial waste or sewage that has
been disinfected, add 3 mL of bacterial seed to each liter of the dilution water. Use raw
sewage for the bacterial seed. Allow the sewage to stand undisturbed at 20 °C for 24 to
36 hours before use. Pipet from the upper portion of the sewage. Make sure to measure the
BOD of the seed so that it can be subtracted from the BOD of the sample.
Table 419 BOD nutrient buffer pillows
Volume of dilution water to prepare BOD nutrient buffer pillow catalog no.
300 mL (add pillow to each BOD bottle) 1416066
3 liters 1486166
4 liters 2436466
6 liters 1486266
19 liters 1486398
Note: To prepare dilution water by the conventional method, pipet 1 mL of each of the following solutions per
liter of distilled water at 20 °C: Calcium Chloride Solution, Ferric Chloride Solution, Magnesium Sulfate
Solution and Phosphate Buffer Solution. Cap the bottle and shake vigorously for one minute. The
Phosphate Buffer Solution should be refrigerated to decrease the rate of biological growth. Use care with
all solutions to avoid contamination.

Make an estimation of the sample volumes that are necessary for the test. At least 2.0 mg/L of
dissolved oxygen (DO) should be consumed during the test and at least 1.0 mg/L DO should be
left in the BOD bottle.
Samples such as raw sewage will have a high BOD. Small sample volumes must be used
because large samples will consume all of the oxygen. Samples with a low BOD must use larger
sample volumes to make sure that enough oxygen is consumed to give accurate results.
The elevation of the laboratory changes the amount of oxygen that can dissolve in water (refer to
the Oxygen values at various altitudes (20 °C) table). At higher elevations, the amount of oxygen
that can dissolve in water decreases, so less oxygen is available to microorganisms.
Procedure
1. Refer to the Minimum sample volume table to select the minimum sample volume. For
example, if a sewage sample is estimated to contain 300 mg/L BOD, the minimum sample
volume is 2 mL. For sewage effluent with an estimated BOD of 40 mg/L, the minimum sample
volume is 15 mL.

Page 1519
2. Refer to the Maximum sample volume table to select the maximum sample volume. At 1000
feet, with an estimated BOD of 300 mg/L, the largest sample volume is 8 mL. For a BOD of
40 mg/L the maximum volume is 60 mL (also at 1000 feet).
3. Select three other sample volumes between the minimum and maximum volumes so that
there are five sample volumes total.
Table 420 Minimum sample volume

Sample type Estimated BOD (mg/L) Minimum sample volume (mL)
Strong trade waste 600 1
Raw and settled sewage 300 2
200 3
150 4
120 5
100 6
75 8
60 10
Oxidized effluents 50 12
40 15
30 20
20 30
10 60
Polluted river waters 6 100
4 200
2 300
Table 421 Maximum sample volume

BOD at sea level BOD at 1000 ft BOD at 5000 ft Maximum sample volume (mL)
2460 2380 2032 1
1230 1189 1016 2
820 793 677 3
615 595 508 4
492 476 406 5
410 397 339 6
304 294 251 8
246 238 203 10
205 198 169 12
164 158 135 15
123 119 101 20
82 79 68 30
41 40 34 60
25 24 21 100
12 12 10 200
8 8 7 300

Page 1520
Table 422 Oxygen values at various altitudes (20 °C)

Altitude (ft) Oxygen value (mg/L) in water saturated with air
Sea level 9.2
1000 8.9
2000 8.6
3000 8.2
4000 7.9
5000 7.6
6000 7.4
BOD calculation—standard methods

Use the Standard Methods calculation when the results must be reported to a regulatory agency.
When dilution water is not seeded:
D1 – D2
BOD 5, mg/L = -------------------
-
P
When dilution water is seeded:
( D 1 – D 2 ) – ( B 1 – B 2 )f
BOD 5, mg/L = ---------------------------------------------------------
-
P
where:
BOD5 = BOD value from the 5-day test
D1 = DO of diluted sample immediately after preparation, in mg/L
D2 = DO of diluted sample after 5 day incubation at 20 °C, in mg/L
P = Decimal volumetric fraction of sample used
B1 = DO of seed control before incubation, in mg/L
B2 = DO of seed control after incubation, in mg/L
f = ratio of seed in diluted sample to seed in seed control =
(% seed in diluted sample)/(% seed in seed control) OR
If seed material is added directly to sample or to seed control bottles:
f = (volume of seed in diluted sample)/(volume of seed in seed control)
Report results as CBOD5 if nitrification inhibitor was added.
Averaged results are acceptable if more than one sample dilution meets all of the following criteria:
• The remaining DO is at least 1 mg/L
• The final DO value is at least 2 mg/L lower than the initial DO value
• There is no evidence of toxicity at higher sample concentrations
• There are no obvious anomalies

Page 1521
BOD calculation—graphical method

Important Note: The Graphical Method cannot be used when the results must be reported to a
regulatory agency.
1. Plot the mg/L dissolved oxygen (DO) remaining in each diluted sample versus the mL sample
taken. Draw the best straight line through the plotted points. Refer to Dissolved Oxygen per
mL of Sample.
Note: An erroneous point is visually evident at this time and can be disregarded. However, at least three
points should be on the line or very close to it. For unseeded dilution water, the line should cross the
mg/L oxygen remaining scale near or below the oxygen saturation value for the altitude of the laboratory
as discussed in Dilution water preparation.
2. To calculate the BOD, use the following equation which is mathematically equivalent to the
BOD equation in Standard Methods.
mg/L BOD = (A x 300) – B + C
where:
A = the slope
The slope of the line is equal to the mg/L DO consumed per mL of sample taken. Take any
point on the line and subtract the mg/L DO remaining at that point from the mg/L DO where the
line crosses the DO scale (Y intercept, mg/L DO remaining). Divide the difference by the mL of
sample at the point chosen.
300 = the volume of the BOD bottle
B = the Y intercept
This is the DO value where the line crosses the “DO remaining” scale. (This should be very
close to the actual dilution water blank value.)
C = the sample DO
This is the DO of the undiluted sample.
Another way to write this equation is:
mg/L BOD = (Slope x 300) – Y intercept + Sample DO
Note: If the best straight line is obtained by linear regression through use of a calculator, the sign (-) of
the slope must be changed (+) before multiplying by 300.
Example:
The mg/L DO remaining was determined for a series of four dilutions of domestic sewage after five
days of incubation. Results were as follows:
mL of sample taken mg/L DO remaining
2.0 7.50
3.0 6.75
6.0 4.50
9.0 2.25

Page 1522
The DO values were plotted versus the mL of sample taken and a straight line drawn as in
Dissolved Oxygen per mL of Sample. If a set of BOD dilutions is run correctly with a homogeneous
sample, a graph of the mg/L DO remaining versus the sample volume would result in a straight
line. The value where the line intersects the y-axis is equal to the DO content of the dilution water
after incubation, although this is not actually measured. In this case, it was equal to 9.0 mg/L and
the DO of the domestic sewage sample was assumed to be zero. If another type of sample is
used, the DO of an undiluted sample should be measured either by the Winkler titration or
potentiometrically.
The American Public Health Association formula for calculating BOD also can be written as follows
(not approved for reporting purposes):
mg/L DO remaining w/smaller sample volume – mg/L DO remaining w/larger sample volume
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- × 300 – DO D + S = mg/L BOD
mL of larger sample volume – mL of smaller sample volume
Using this information in the example:
mg/L DO remaining with smaller sample volume = 7.50
mg/L DO remaining with larger sample volume = 2.25
mL of larger sample volume = 9.0
mL of smaller sample volume = 2.0
300 = volume (mL) of BOD bottle
DOD = mg/L DO of dilution water = 9.0
S = mg/L DO of sample = assumed in this case to be zero
Therefore:
7.50 – 2.25-
---------------------------- × 300 – 9 + 0 = mg/L BOD = 216 mg/L BOD
9.0 – 2.0
Using the equation below:
(slope x 300) – Y-Intercept + sample DO = mg/L BOD
To determine slope, arbitrarily select point A in Figure 1. At this point the mg/L DO remaining is
equal to 3.0 mg/L. The mL of sample at this point is 8 mL.The difference between the y-
intercept of 9.0 mg/L and 3.0 mg/L equals 6 mg/L; 6 mg/L divided by 8 mL = 0.75 mg/L per mL.
slope = 0.75 mg/L per mL
Y intercept = 9.0 mg/L
sample DO = 0 (Because the sample is domestic sewage, this is assumed to be zero.)
Therefore:
(0.75 x 300) – 9.0 + 0 = mg/L BOD = 216 mg/L BOD

Page 1523
y Intercept
mg/L DO Remaining
mL of Sample
Figure 35 Dissolved Oxygen per mL of Sample
Interferences
Many chlorinated and industrial effluents require special handling to ensure reliable BOD results.
Usually, careful experimentation with the particular sample will indicate what modifications should
be made to the test procedure.
Toxins in the sample will adversely affect any microorganisms present and result in lower BODs.
To eliminate small amounts of residual chlorine, allow the sample to stand for one to two hours
at room temperature. For larger quantities, determine the amount of sodium thiosulfate to add to
the sample as follows:
c. Measure 100 mL of sample into a 250-mL Erlenmeyer flask. Using a 10-mL serological
pipet and a pipet filler, add 10 mL of 0.020 N Sulfuric Acid Standard Solution and 10 mL of
Potassium Iodide Solution, 100-g/L, to the flask.
d. Add three full droppers of Starch Indicator Solution and swirl to mix.
e. Fill a 25-mL buret with 0.025 N Sodium Thiosulfate Standard Solution and titrate the
sample from dark blue to colorless.
f. Calculate the amount of 0.025 N Sodium Thiosulfate Standard Solution to add to the
sample:
mL titrant used x volume of remaining sample
mL 0.025 N sodium thiosulfate required = -------------------------------------------------------------------------------------------------------------------------
100
g. Add the required amount of 0.025 N Sodium Thiosulfate Standard Solution to the sample.
Mix thoroughly. Wait 10 to 20 minutes before running the BOD test.

Page 1524
To eliminate the effect of phenols, heavy metals or cyanide, dilute the sample with high quality
distilled water. Alternately, the seed used in the dilution water may be acclimatized to tolerate such
materials. Acclimatize seed as follows:
a. Fill a one-gallon stainless steel or plastic container with domestic sewage and aerate for
24 hours. Allow the heavier material to settle.
b. After settling for one hour, siphon off three quarts of material and discard.
c. Fill the container with a mixture of 90% sewage and 10% wastes containing the toxic
material.
d. Aerate for 24 hours. Repeat steps b and c with increasing amounts of waste until the
container holds 100% toxic waste material.
Optimum pH for the BOD test is between 6.5 and 7.5. Adjust samples to pH 7.2 with Phosphate
Buffer Solution or 1 N Sulfuric Acid or Sodium Hydroxide Standard Solution if the pH is not in
this range.
Cold samples may be supersaturated with oxygen and will have low BOD results. Fill a one-quart
bottle about halfway with cold sample and shake vigorously for two minutes. Allow sample
temperature to reach 20 °C before testing.
Accuracy check
• BOD Standard Solution, Voluette® Ampule, 300-mg/L, 10-mL (300-mg/L of glucose and
300-mg/L of glutamic acid)
• Seeded dilution water
• 4 BOD bottles
• 1.0–4.0 mL Class A volumetric pipets
• TenSette Pipet
2. Use a pipet to add 1.00, 2.00, 3.00 and 4.00 mL of standard into four BOD bottles.
3. Fill the bottles with seeded dilution water and measure the DO concentration.
4. Incubate the bottles at 20 °C for five days.
5. Measure the DO remaining in each bottle.
6. Calculate the BOD value (refer to BOD calculation—standard methods or BOD calculation—
graphical method).
7. Divide the value by two. The result for comparison with Standard Methods should be
198 (± 30.5) mg/L.
Note: The result must be divided by 2 to correspond with values reported in Standard Methods because
the Standard Methods procedure uses 150 mg/L each of glucose and glutamic acid.

Page 1525
Method performance
The following statements are true for dissolved oxygen when the measurement is below 10 mg/L
DO and the temperature is kept between 10 and 30 °C for a single probe.
Precision
Instrument Standard 95% Confidence Limits of
Distribution
LBOD101 7.94–8.06 mg/L DO 7.97–8.03 mg/L DO
Summary of method
Biochemical Oxygen Demand (BOD) is a measurement of the oxygen requirements of municipal
and industrial wastewaters and sewage. The test results are used to calculate the effect of waste
discharges on the oxygen resources of the receiving waters. The BOD test is of limited value in
measuring the actual oxygen demand because temperature change, biological population, water
movement, sunlight, oxygen concentration and other environmental factors cannot be reproduced
accurately in the laboratory. The BOD test is of greatest value after patterns of oxygen uptake for a
specific effluent and receiving water have been established.
The BOD test is performed by incubating a sealed wastewater sample (or a prepared dilution) for
the standard five-day period and then determining the change in dissolved oxygen content. The
BOD value is then calculated from the results of the dissolved oxygen tests.
Required reagents
BOD Nutrient Buffer Pillows, for 3 liters of dilution water 1 pillow 50/pkg 1486166
Required apparatus
BOD Bottle, glass-stoppered, 300-mL 6 each 62100
BOD Bottle Cap 6 6/pkg 241906
HQ40d meter 1 each HQ40d
OR
HQ30d meter 1 each HQ30d
IntelliCAL LBOD probe 1 each LBOD10101
Pipet, seriological:
Pipet Filler 1 each 1218900

Page 1526
BOD Standard Solution, Voluette® Ampule, 300-mg/L, 10-mL 16/pkg 1486510

BOD Nutrient Buffer Pillows
for 300 mL of dilution water 50/pkg 1486166
Buffer Solution, APHA, for BOD, pH 7.2, phosphate type 1L 43153
Calcium Chloride Solution, APHA, for BOD 1L 42853
Ferric Chloride Solution, APHA, for BOD 1L 42953
Magnesium Sulfate Solution, APHA, for BOD 1L 43053
Dispenser Cap, for Nitrification Inhibitor each 45901
Potassium Iodide Solution, 100-g/L 500 mL 1228949
Sodium Hydroxide, pellets, ACS 500 g 18734
Up-Grade Kit, HQd Accessories, LBOD Probe each LBOD10130
Replacement LBOD Sensor cap, with I-button each 5838000
Replacement Stirrer assembly 5/pkg 5850800
Field Kit, includes: protective glove, 2 standard probe holders and 5 120-mL sample cups each 5825800
Keyboard (HQ40d meter only) each LZV582
Citizen PD-24 USB Handy printer, 115 VAC each 5835800
Color Coded Probe Clips (5 color coded sets), 5 sets 10/pkg 5818400

Page 1527
LDO, 10360

(0.1 to 20.0 mg/L or 1 to 200% saturation) LDO Probe
Scope and Application: For water, wastewater and process water applications
Test preparation


Meter Probe
HQd meters LDO101
Before attaching probes to the HQd meter for the first time, set the meter time and date.
Refer to the probe instructions for probe preparation.
For probes that are continuously immersed in aqueous solutions, condition the sensor cap for 72 hours.
When an IntelliCAL™ probe is connected to a HQ30d or HQ40d meter, the meter automatically recognizes the measurement
parameter and is ready for use.
The IntelliCAL LDO101 probes automatically compensate for barometric pressure, elevation and temperature.
The LDO probe is calibrated at the factory. For more accurate results, manual calibration is recommended. Refer to the
Calibration section of this procedure.
Salinity affects the concentration of dissolved oxygen in the sample. To correct for salinity effects, refer to Modifying LDO
Measurement Options in the meter manual.
HQd meter 1
IntelliCAL LDO101 probe 1
Sensor cap for HQd with I-button 1
Shroud 1
BOD bottle, 300-mL or Erlenmeyer flask, 250-mL 1
Beaker, polypropylene (100-, 250-, 400- or 600-mL) 1
Oxygen, Dissolved
Page 1529
Oxygen, Dissolved
Method Name for powder pillows
1. Prepare the probe. 2. Connect the probe to 3. Calibrate the probe. 4. Laboratory tests:
Refer to the probe the meter. Refer to the Calibration Immerse the probe in the
instructions. section of this procedure. beaker containing the
Go to step 4 for laboratory sample solution. Move the
tests. Go to step 5 for field probe up and down and
tests. tap it on the beaker to
remove bubbles from the
probe.
Read
5. Field tests: Immerse 6. Press READ to store

the probe directly into the data in the data log.
sample. Move the probe
up and down to remove
bubbles from the probe.
Calibration
The LDO probe is calibrated at the factory. For more accurate results, manual calibration
is recommended.
1. Remove the shroud from the probe body.
2. Add a small amount of water (about 1 cm ) to the bottom of narrow-neck bottle, such as a
BOD bottle.
Note: Use a wider neck bottle or flask (for example, a 250-mL Erlenmeyer flask) for the rugged probe.
3. Insert a stopper and shake vigorously for several minutes.
4. Remove the stopper. If the sensor cap surface is wet, carefully dry the cap with a
nonabrasive cloth, then put the probe in the bottle. Allow several minutes for the probe to
reach equilibrium.
Oxygen, Dissolved
Page 1530
Oxygen, Dissolved
5. Make sure the meter is in the measurement screen. Press the CALIBRATION key.
Note: For HQ40d meters with two probes attached, the display must be in the single screen
LDO101 mode.
6. Press READ. When the measurement is stable, the calibrated measurement will show on the
display. The standard value will be highlighted on the display.
7. Press DONE to view the calibration summary. The slope value is the comparison between the
latest calibration and the factory calibration expressed as a percentage.
Note: If the calibration slope does not meet the acceptance criteria, the display will show “Slope out of
range”. Let the probe stand in water-saturated air for several minutes. When the probe reaches
equilibrium, press READ.
8. Press STORE to accept the calibration and return to the measurement mode. The calibration
record is stored in the data log.
Note: A successful calibration will show “OK” in the measurement screen.
Interferences
There are no significant interferences with the LDO technology.
The IntelliCAL LDO101 probes are designed for water and wastewater applications, but can be
used for other applications. Some organic solvents may damage the sensor cap and probe body.

• Analyze samples in-situ, if possible.
• Collect samples in an appropriate container. Fill completely and analyze immediately.
• Do not store samples.
Accuracy check
1. Return the electrode to a water-saturated air environment.
2. Allow at least 10 minutes for stabilization.

3. Read the % saturation on the right side of the measurement mode screen. The meter should
display 100% saturation. If not, allow additional time for the air to reach water saturation or
calibrate the probe.
Method performance
The following statements are true for dissolved oxygen when the temperature is kept between 10
and 30 degrees C.
Precision Accuracy
Method Standard 95% Confidence Limits of Concentration change
10360 8.00 mg/L DO 7.95–8.05 mg/L DO 7.90–8.10 mg/L DO
10360 15.00 mg/L DO 14.90–15.10 mg/L DO 14.80–15.20 mg/L DO
Oxygen, Dissolved
Page 1531
Oxygen, Dissolved
Summary of method
The oxygen sensor is made up of a clear, oxygen impermeable hard substrate. An oxygen
sensitive luminescent dye, along with a scattering agent, is pad-printed on the substrate. A final
overlay of dark pigment is added to prevent stray light from entering the measurement cell. The
luminescent dye emits red light when exposed to blue light. The scattering agent distributes the
emitted light throughout the sensor matrix and contributes to the opacity of the sensor. Pulses from
a red LED serve as an internal reference. The duration of the luminescence is proportional to the
concentration of dissolved oxygen in the sample.

Required apparatus (select one)
HQ40d multi-parameter meter, dual input 1 each HQ40d53000000
HQ30d multi-parameter meter, single input 1 each HQ30d53000000
Required probes (select one)

LDO Probe, standard, with 1 m cable each LDO10101
LDO Probe, standard, with 3 m cable each LDO10103
LDO Probe, rugged, with 5 m cable each LDO10105
Optional apparatus
AC Power Adapter for HQd meters (included w/ HQ40d) each 5826300
BOD bottle, 300 mL each 62100
BOD bottle, 300 mL 6/pkg 62106
Citizen PD-24 USB Handy printer, 115 VAC each 5835800
Color Coded Probe Clips (5 color coded sets) 5 sets 10/pkg 5818400
Depth Markers for Rugged LDO probe only 10/pkg 5828610
Erlenmeyer flask, 250 mL each 2089846
Field Kit (Includes glove kit, 2 probe holders and 5 120 mL sample cups)1 each 5825800
Glove kit only for HQd meters each 5828700
Probe Holder for HQd meter, IntelliCAL Standard probes only each 5829400
Replacement Sensor cap w/ I-button each 5811200
Replacement Shroud kit Rugged LDO probe each 5825900
USB Keyboard for HQd meters (must have 5813400 & 5826300) each LZV582
USB/DC Adapter for HQd meters (must have 5826300, inc w/HQ40d) each 5813400
1 Included with HQ40d

ORP, 10228
Oxidation Reduction Potential

(ORP) DOC316.53.01244

(–2000 mV to 2000 mV) ORP Electrode
Scope and Application: For drinking water, wastewater and process water applications
Test preparation


Meter Platinum-series ORP electrode Gel-filled ORP electrode
sension™ 1 5193700 5193900
sension™ 2 5193700 5193900
sension™ 3 5193700 5193900
sension™ 4 5193700 5193900
sension pH/mV meter 1
Gel-filled or combination ORP electrode 1
Beaker, polypropylene (100-, 250-, 400- or 600-mL) 1
Deionized water
Oxidation Reduction Potential (ORP)

Page 1533
Oxidation reduction potential (ORP)
1. Connect the electrode 2. Turn on the meter. 3. Rinse the electrode in 4. Platinum-series
to the meter. Press PHMV to select mV. deionized water and electrodes only: Press
blot dry. the dispenser button on
top of the electrode until
the electrolyte get is visible
at the reference junction.
5. Put the electrode in 6. When the

the sample. measurement stabilizes,
store or record the mV and
temperature readings.
Standard hydrogen electrode (SHE) calculation

For some applications it is customary to report oxidation reduction (redox) potential measurements
relative to the standard hydrogen electrode (SHE).
1. Select the value that corresponds to the temperature of the solution measured. Refer to the
Reference electrode potentials table.
2. Substitute the electrode potential value (C) in the equation and solve for ESHE:
E SHE = E O + C
Where:
ESHE = oxidation reduction potential of the sample relative to the SHE, following the
international sign convention.
EO = potential developed by the ORP electrode
C = potential developed by the reference electrode relative to the SHE.

Page 1534
The Reference electrode potentials table shows the potentials, C, developed by the reference
electrode relative to the standard hydrogen electrode at various temperatures.
Table 425 Reference electrode potentials

Temperature (°C) Electrode potential in mV (C)
10 221
15 216
20 213
25 208
30 204
35 200
40 196
Interferences
Many factors limit the interpretation of ORP measurements in water. These factors include
irreversible reactions, electrode poisoning, the presence of multiple redox couples, very small
exchange currents and inert redox couples. ORP measurements in the field correlate poorly with
ORP values calculated from the redox couples present. Due to these factors, the interpretation of
ORP measurements will be specific to your particular application.
Sample collection, preservation, storage and cleaning

• Analyze samples immediately after collection.
• For best results, minimize contact with the environment and the time between collection
and measurement.
• If the electrode is not clean, remove inorganic deposits by immersing the electrode tip in room-
temperature 0.1 N HCl for 10 minutes. To remove grease, oil or other organic deposits,
immerse the tip in warm water and detergent and swirl gently. After cleaning, rinse with DI
water. Repeat Procedure A after cleaning.
Accuracy check
Checking the electrode is necessary only when there is evidence of malfunction that cannot be
traced to other causes.
Procedure A
1. Open an ampule of Light's Solution or ORP verification solution*. Pour the contents of the
ampule into a beaker.
2. Immediately place the ORP Electrode tip into the solution.

3. Verify that the potential is 475 ± 10 mV or the specified ORP value.
Note: This potential is the standard reduction potential for Fe2+/3+ with the reference electrode potential
subtracted. The solution is 0.01 M in both Fe2+ and Fe3+.
* See Optional apparatus.

Page 1535
Procedure B
1. Prepare solution A (0.1 M potassium ferrocyanide and 0.05 M potassium ferricyanide) as

follows:
a. Weigh out 4.22 g reagent-grade K4Fe(CN)6•3H2O and 1.65 g reagent-grade K3Fe(CN)6.

Put the solids in a 100-mL volumetric flask.
b. Add about 50 mL deionized water and swirl to dissolve the solids.
c. Dilute to volume with deionized water.
2. Prepare solution B (0.01 M potassium ferrocyanide, 0.05 M potassium ferricyanide and 0.36 M
potassium fluoride) as follows:
a. Weigh out 0.42 g of reagent-grade K4Fe(CN)6? •3H2O, 1.65 g of reagent-grade

K3Fe(CN)6 and 3.39 g of reagent-grade KF•2H2O. Put the solids in a 100-mL volumetric
flask.
b. Add about 50 mL deionized water and swirl to dissolve the solids.
c. Dilute to volume with deionized water.
3. Transfer solution A to a 150-mL beaker. Place the electrode in the solution and wait until the
reading stabilizes. The potential should be about 234 mV.
4. Rinse the electrode and repeat the measurement with solution B. The potential should be
about 66 mV greater in solution B than in solution A.
Summary of method
Redox measurements are made by determining the electron activity of a solution using an inert
indicator electrode and a reference electrode. The potential difference between the indicator
electrode and the reference electrode equals the redox potential of the system. The Gel-filled ORP
and Platinum Series ORP electrodes use a platinum indicator electrode and a silver/silver chloride
reference electrode.
Required apparatus
Select one meter and probe combination: — — —
sension™ 1 Portable pH/mV Meter each 5170010
sension™ 2 Portable pH/ISE Meter each 5172510
sension™ 3 Benchtop pH/mV Meter each 5175010
sension™ 4 Benchtop pH/ISE Meter each 5177510
Gel-filled Combination ORP Electrode, 5-pin 1 5193900
OR
Platinum Series Combination ORP Electrode, 5-pin 1 5193700

Page 1536
Optional reagents
Light's Solution, ampules 20/pkg 26125-20
200 mV ORP Solution 500 mL 25M2A1001-115
600 mV ORP Solution 500 mL 25M2A1002-115
Hydrochloric Acid Standard Solution 0.1 N 1L 1481253
Sodium Hydrochloride Standard Solution, 0.1 N 1L 19153
Optional apparatus
Beaker, 100-mL, polypropylene each .108042
Electrode Holder, with electromagnetic stirrer each 4530001
Electrode Holder each 4530000
Stir Bar, Magnetic, 22.2 x 7.9 mm each 2095350

Page 1537
pH
pH DOC316.53.01245
USEPA Electrode Method Method 8156

pH Meter
Scope and Application: For drinking water1, wastewater2 and process water applications.
1 Based on Standard Method 4500-H+B, ASTM Method D1293-95 and USEPA Method 150.1
2 Based on Standard Method 4500-H+B, ASTM Method D1293-84(90)/(A or B) and USEPA Method 150.1
Test preparation

Meter Standard probe Rugged probe1
HQ40d PHC10101, PHC10103 (gel)

PHC10105, PHC10110, PHC10115, PHC10130
PHC30101, PHC30103 (liquid)

PHC10105, PHC10110, PHC10115, PHC10130

PHC10105, PHC10110, PHC10115, PHC10130
5191000 (platinum)
5193500 (gel)
sension™ 1 —
5194000 (refillable)
5191500 (flat)
5191000 (platinum)
5193500 (gel)
sension™ 3 —
5194000 (refillable)
5191500 (flat)
1 Designed for field use.

For optimal response time, condition the electrode for several minutes in a solution comparable to the sample in terms of pH
and ionic strength.
For rugged IntelliCAL™ electrodes, the shroud may need to be removed before measurement and calibration.
For HQd meters, data is stored automatically when Press to Read or Interval is selected in the Setup Measurement Mode.
When Continuous is selected, data will only be stored when the key under STORE is pressed. For sension meters, the
STORE key must be pressed.
pH meter and probe combination 1
pH buffers (4.0, 7.0, 10.0) 3
Beakers/sample containers 3
pH
Page 1539
pH
Sample pH measurement (calibration required)
1. Refer to the operation 2. Connect the pH 3. Turn the meter on. 4. For setup options such
section of the electrode or electrode to the meter. Make sure that the meter as measurement
meter manual to prepare is set to measure to resolution, temperature
the pH electrode and measure pH. units, calibration buffer set
meter. and other options refer to
appropriate meter or
electrode manual.
5. In three separate 6. Calibrate the pH meter 7. Rinse the electrode in 8. Put the electrode in
beakers or appropriate and electrode as directed deionized water and blot the sample and press
containers, prepare fresh in the instructions in the dry prior to sample READ. For faster
buffers of 4.0, 7.0 and 10.0 meter or probe manual. measurement. Rinse the response, stir at a slow to
pH. Make sure that the electrode between moderate rate.
The sample pH should fall calibration slope is measurements to
within the pH range of the acceptable (typically -58 minimize contamination.
calibration buffers. One, ±3 mV per pH unit at
two or three calibration 25 °C).
buffers may be used to
calibrate.
Other pH calibration
buffers sets may be used.
pH
Page 1540
pH
Sample pH measurement (calibration required) (continued)
9. When the 10. Store the pH electrode

measurement is stable, in pH storage solution
store or record the pH and when not in use. See
temperature values. Sample collection,
For HQd meters, data is preservation, general
stored automatically when storage and cleaning for
Press to Read or Interval more details.
is selected in the Setup
Measurement Mode.
When Continuous is
selected, data will only be
stored when the key under
STORE is pressed. For
sension meters, the
STORE key must be
pressed.
Low Ionic Strength (LIS) or high purity water measurements

Low ionic strength solutions have very low buffering capacity and readily absorb carbon dioxide
from the air. When a sample absorbs carbon dioxide from the atmosphere, carbonic acid forms.
Carbonic acid decreases the sample pH and increases conductivity, causing inaccurate readings.
One solution to this problem is to test the sample in a low volume, airtight sample chamber such
as a Low Ionic Strength (LIS) Chamber. Use refillable or platinum series electrodes for
measurement of pH in LIS or high purity waters.
Initial use
1. Before measuring an LIS sample, soak the electrode in a solution similar to the sample in ionic
strength and pH for 10 to 15 minutes.
2. Rinse the electrode with deionized water from a wash bottle.
3. Blot excess liquid with a soft paper towel.
4. Put the electrode in the sample.
pH
Page 1541
pH
Between uses
Between uses, in intervals of up to a two hours, the electrode can be stored in the sample (if the
sample is not an extreme pH), or in a neutral LIS solution such as tap water. Rinse the electrode
before use to prevent sample contamination.
Important Note: If pH electrodes are stored in LIS samples for a long period of time, the electrode
life may be shortened.
After measuring the LIS samples, put electrode back into the electrode storage solution or
3 M KCl.
Sample collection, preservation, general storage and cleaning

• Analyze samples immediately, preferably in the field.
• Storage of an electrode is based on how long the electrode will be stored, how quickly the
electrode needs to be used and the type of sample being measured. For general storage, use
the Hach storage solution or a 3 M Potasium Chloride (KCl) solution.
• A contaminated glass bulb or fouled electrode may cause slow response times. Do not clean
the bulb too often because the bulb life may shorten.
• To clean an electrode with general contamination, immerse the electrode tip in 0.1 N
hydrochloric acid (HCl). Then, immerse the electrode in 0.1 N sodium hydroxide (NaOH) and
again in 0.1 N hydrochloric acid, each for a 2-minute period. Rinse with deionized water and
soak in deionized water for at least 15 minutes.
• To clean an electrode contaminated with oils and fats, immerse the electrode tip in a detergent
solution. Use a soft brush or ultrasonic bath if necessary. Avoid scratching the glass bulb.
Interferences
• Acid error is negligible.
• Sodium error, usually present in alkaline solutions, is low but increases at pH values higher
than pH 11.
For more detailed information, refer to the meter or electrode manual.
Accuracy check
Check electrode response
An electrode is responding properly if its calibration slope meets the slope specifications of the
electrode (typically -58 ±3 mV at 25 °C).
Check calibration accuracy
Return the electrode to a calibration buffer and measure the pH to test the system. Rinse and
recondition the electrode before measuring subsequent samples.
Method performance
The accuracy of a pH measurement depends on many factors associated with the overall pH
system, including the pH meter, choice of electrode and pH standards or buffers used during pH
calibration. Refer to the appropriate electrode and meter manual to determine method
performance.
pH
Page 1542
pH
Summary of method
pH is a measure of the hydrogen ion activity in a solution and is defined as:
–log10 aH+
Where
aH+ is the activity of the hydrogen ion.
A Combination pH Electrode responds to the hydrogen ion concentration (activity) by developing
an electrical potential at the glass/liquid interface. At a constant temperature, this potential varies
linearly with the pH of the solution being measured.
Water with relatively high conductivity typically has a fairly high buffer capacity. Slight pH changes
due to absorption of carbon dioxide are usually not significant. If the sample conductivity is not
known and high accuracy is desired, follow either the LIS or high purity water methods.

Required apparatus and reagents
HQ meters and probes (select one meter and probe combination)

HQ40d meter 1 each HQ40d53000000
pH Gel Probe, standard, with 1 m cable 1 each PHC10101
pH Gel Probe, standard, with 3 m cable 1 each PHC10103
pH Liquid Probe, standard, with 1 m cable 1 each PHC30101
pH Liquid Probe, standard, with 3 m cable 1 each PHC30103
pH Gel Probe, rugged, with 5 m cable 1 each PHC10105
sension meters and probes (select one meter and probe combination)
Electrolyte cartridge, potassium chloride 1 2/pkg 2546902
Gel Filled pH electrode 1 each 5193500
Refillable pH electrode, platinum series electrode (5191000 1 each 5194000
as #1); flat Platinum series electrode (5195000 as #4)
For LIS and high purity water measurements
Low Ionic Strength (LIS) Chamber 1 each 5189900
pH
Page 1543
pH
Hach Solutions1
pH Color buffer solution kit (NIST), 500 mL, includes: each 2947600
pH 4.01 +/- 0.02 pH buffer (NIST) 500 mL 2283449
pH 7.00 +/- 0.02 pH buffer (NIST) 500 mL 2283549
pH 10.01+/- 0.02 pH buffer (NIST) 500 mL 2283649
Powder pillows1
pH 4.01 +/- 0.02 pH buffer powder pillow (NIST) 50/pkg 2226966
pH 7.00 +/- 0.02 pH buffer powder pillow (NIST) 50/pkg 2227066
pH 10.01+/- 0.02 pH buffer powder pillow (NIST) 50/pkg 2227166
Radiometer Analytical (IUPAC Series certified pH standards):
pH 1.679 ± 0.010 at 25 °C 500 mL S11M001
pH 4.005 ± 0.010 at 25 °C 500 mL S11M002
pH 7.000 ± 0.010 at 25 °C 500 mL S11M004
pH 10.012 ± 0.010 at 25 °C 500 mL S11M007
pH buffer 1.09, technical 500 mL S11M009
Refill Solution and Storage:
pH Filling Solution (for PHC301), 3M KCl, saturated with AgCl 30 mL 2841700
pH Electrode Storage Solution 500 mL 2756549
1 Larger quantities are available

Sample bottle, general purpose with Screw-cap, polypropylene, 500-mL each 2758101
Sample bottle, cleaned and certified, HDPE, suitable for EPA reporting, 500-mL each 2758201
sension 2 meter each 5172511
sension 4 meter each 5177500

Chemical Procedures Explained
Page 1545
Page 1546
Acidity tests explained
Acidity
For water, wastewater and seawater Digital Titration Method
Acidity is the quantitative expression of water’s capacity to neutralize a strong base to a

designated pH and an indicator of how corrosive water is. Acidity can be caused by weak organic
acids, such as acetic and tannic acids, and strong mineral acids including sulfuric and hydrochloric
acids; however, the most common source of acidity in unpolluted water is carbon dioxide in the
form of carbonic acid.
Acidity is classified by the pH value of a titration end point. Acidity caused by mineral acids exhibits
a pH below 4.5. Salts of certain metals, particularly those with trivalent iron and aluminum, may
hydrolyze in water and also contribute to acidity.
Standard Methods for the Examination of Water and Wastewater (Standard Methods)
recommends titration with sodium hydroxide to an end point pH of 3.7 to determine mineral acidity.
Titrate to pH 8.3 to determine total acidity.
Acidity is commonly determined using methyl orange as a color indicator of the pH end point.
Because methyl orange undergoes a color change from red to orange at a pH of 3.7, the results of
the titration are termed Methyl Orange Acidity. Hach procedures for acidity use bromphenol blue
indicator instead of methyl orange because the methyl orange color change is difficult to detect.
The bromphenol blue indicator gives a sharp end point change from yellow to blue-violet.
Total acidity includes acidity caused by mineral acids, weak organic acids, and carbon dioxide (in
the form of carbonic acid). Acidity determined by titrating to a phenolphthalein end point pH of 8.3
corresponds to the neutralization of carbonic acid to bicarbonate. Because carbon dioxide is the
major cause of acidity in natural waters, in most cases the phenolphthalein acidity is equal to the
total acidity. Acidity tests can be performed using a pH meter to detect the end points; however,
methyl orange acidity and phenolphthalein acidity are the terms used to describe the results.
Results of the acidity tests are reported in mg/L CaCO3.
Table 427 Chemical reactions

Methyl orange acidity Phenolphthalein acidity
2NaOH + H 2 SO 4 → Na 2 SO 4 NaOH + H 2 CO 3 → NaHCO 3 + H 2 O
NaOH + HCl → NaCl + H 2 O NaOH + HC2 H 3 O 2 → NaC2 H 3 O 2 + H 2 O
Reactions of indicator Phenolphthein
HO OH
C O
C O
Figure 36 Colorless—pH < 8.3
Acidity
Page 1547
Acidity
HO O
C O
C O¯
Figure 37 Pink—pH > 8.3
Reactions of indicator Bromphenol blue*
Br
Br
HO
OH
Br
Br
C O
O
S
O
Figure 38 Yellow=pH 3
Br
Br
HO O
Br
Br
C
SO3¯
Figure 39 Blue-violet=pH 4.6
* 3’,3”,5’,5”, -Tetrabromophenolsulfonephthalein
Acidity
Page 1548
Alkalinity tests explained
Alkalinity
For water, wastewater and seawater Titration Method
Introduction
Alkalinity is a measure of the capacity of water to neutralize acids. Alkalinity of water is due
primarily to the presence of bicarbonate, carbonate, and hydroxide ions. Salts of weak acids, such
as borates, silicates and phosphates, may also contribute. Salts of certain organic acids may
contribute to alkalinity in polluted or anaerobic water, but their contribution usually is negligible.
Bicarbonate is the major form of alkalinity. Carbonates and hydroxide may be significant when
algal activity is high and in certain industrial water and wastewater, such as boiler water.
Alkalinity is significant in the treatment processes for potable water and wastewater. The alkalinity
acts as a pH buffer in coagulation and lime-soda softening of water. In wastewater treatment,
alkalinity is an important parameter in determining the amenability of wastes to the treatment
process and control of processes such as anaerobic digestion, where bicarbonate alkalinity, total
alkalinity and any fraction contributed by volatile acid salts become considerations.
Alkalinity is expressed as phenolphthalein alkalinity or total alkalinity. Both types can be
determined by titration with a standard sulfuric acid solution to an end point pH, evidenced by the
color change of a standard indicator solution. The pH also can be determined with a pH meter.
Phenolphthalein alkalinity is determined by titration to a pH of 8.3 (the phenolphthalein end point)
and registers the total hydroxide and one half the carbonate present. Total alkalinity is determined
by titration to a pH of 4.9, 4.6, 4.5, or 4.3, depending on the amount of carbon dioxide present. The
total alkalinity includes all carbonate, bicarbonate and hydroxide alkalinity.
The following end points are recommended for determining total alkalinity in water samples of
various compositions and alkalinity concentrations.
Table 428 Determine total alkalinity

Sample Trait End Point
Alkalinity approximately 30 mg/L pH 4.9
Silicates or phosphates known present or suspected pH 4.5
Industrial waste or complex system pH 4.5
Routine or automated analyses pH 4.5
Chemical reactions
Sulfuric acid (hydrochloric acid may be used) reacts with the three forms of alkalinity, converting
them to water or carbonic acid. If hydroxide is present, it reacts to form water:
– 2–
2OH + H 2 SO 4 → 2H 2 O + SO 4
This conversion usually is complete at a pH of about 10. Phenolphthalein alkalinity is determined

by titration to an end point pH of 8.3, which corresponds to the conversion of carbonate to
bicarbonate.
2– – 2–
2CO 3 + H 2 SO 4 → 2HCO 3 + SO 4
If hydroxide is present, titration to pH 8.3 will indicate the alkalinity due to all of the hydroxide plus
one-half of the carbonate. Continued titration to pH 4.5 completes the conversion of carbonate
plus any bicarbonate present to carbonic acid. This value is termed Total Alkalinity.
Alkalinity
Page 1549
Alkalinity
– 2–
2HCO 3 + H 2 SO 4 → 2H 2 CO 3 + SO 4
Methyl red
O
C OH
CH3
N N N
H CH3
Figure 40 Red=pH 4.8
O
C O¯
CH3
N N N
CH3
Figure 41 Yellow=pH 6.0
Bromcresol green
Br Br
OH CH3 O
Br C Br
¯O3S CH3
Figure 42 Blue=pH 5.5
Alkalinity
Page 1550
Alkalinity
Br Br
OH OH
CH3
Br Br
C CH3
S
O
O
Figure 43 Yellow=pH 3.8
Alkalinity
Page 1551
Aluminum tests explained
Aluminum
For water Aluminon Method
Introduction
Aluminum, the earth’s most abundant metal, is present in natural waters through contact with
rocks, soil and clay. Alum coagulation in water clarification systems may also contribute to the
aluminum content of treated water, although only 20–50 µg/L of aluminum remain in the finished
product from a well-controlled operation.
The Aluminon Method is one of the oldest and most thoroughly documented methods available for
determining aluminum in water. The AluVer 3™ Aluminum Reagent used in this method is
packaged in powder pillow form, providing exceptional stability.
Chemical reactions
AluVer is an aluminon reagent in combination with a pH buffer. AluVer 3 reacts with aluminum
present in a sample to form a reddish-colored solution in direct proportion to the aluminum
concentration.
Ascorbic acid is added prior to the addition of AluVer 3 to eliminate interference due to iron. To
establish a reagent blank, the sample is split after the addition of the AluVer 3. Bleaching 3
Reagent is then added to one-half of the split sample to bleach out the color of the aluminum
aluminon complex.
O O
COOH COOH COOH C
HO O HO O
C C
3 +Al3+ Al3+ + 3H+
COOH COOH
OH OH
3
Aurintricarboxylic acid
Figure 44 Chemical reaction
Aluminum
Page 1552
Barium tests explained
Barium
For water, wastewater, oil-field water and
Turbidimetric Method
seawater
Introduction
Although barium is relatively abundant in nature, usually only trace amounts are found in water.
Barium concentrations average about 0.05 mg/L in potable waters, but may range as high as
0.9 mg/L in some natural waters. More than 1 mg/L of barium implies that the water is not suitable
for drinking and is polluted by industrial wastes. Barium and its compounds can be found in
pigments, rat poisons, fireworks, and are used in rubber making, x-ray photography and even as
weighting agents for oil well drilling.
Chemical reactions
Barium is determined by adding sulfate to the water sample to form barium sulfate, which
precipitates. These particles are held in suspension as colloids by the BariVer™ 4 Reagent. The
barium concentration is determined by measuring the resulting turbidity using a spectrophotometer
or colorimeter. The barium concentration is proportional to the increase in turbidity when barium
sulfate precipitates. The Hach procedure uses sodium sulfate, contained in BariVer 4 Reagent
Powder, as the source of sulfate. The BariVer 4 Method is especially useful for brines where
barium and sulfate coexist in solution and precipitation usually cannot be initiated by the simple
addition of more sulfate.
2+ 2–
Ba + SO 4 → BaSO 4
Barium
Page 1553
Boron tests explained
Boron
For water and wastewater Azomethine-H and Carmine Methods
Introduction
Boron normally occurs in natural waters at concentrations less than 1.0 mg/L. Boron in natural
water could be an indicator of sanitary pollution from domestic wastewater, usually in the form of
borates from laundry detergents. In water for human consumption, boron concentrations typically
should be less than 300 µg/L. Large amounts of boron can affect the central nervous system;
when continually ingested over an extended period of time boron can cause a syndrome
called borism.
In the semiconductor industry, boron has been used as an indicator of ion-exchange resin
exhaustion in wafer rinsewater treatment. Boron is routinely monitored in irrigation water since
many varieties of plants are sensitive to excess boron.
Analytical colorimetric methods for boron include the Curcumin Method, the Carmine Method and
the Azomethine-H Method.
Chemical reactions
Azomethine-H method
The Azomethine-H Method involves the coupling of H-acid with an aromatic hydroxyaldehyde,
such as salicylaldehyde, due to the catalytic effect when boron is present. At neutral pH values
and a controlled temperature, the condensation reaction is completed quickly (within 15 minutes).
After product formation, the solution is adjusted to an acidic pH for optimum color measurement at
410 nm (yellow) using a colorimeter or spectrophotometer. The method is sensitive and highly
selective for the determination of dissolved boron in water.
Aromatic Azomethine
H-acid
Hydroxyaldehyde (a Schiff base)
CHO H
HO3S HO3S
OH C
NH2 N
HO
+
B(OH)
OH OH
HO3S HO3S
Figure 45 Chemical reactions for the Azomethine-H method
Boron
Page 1554
Boron
Carmine method
In the presence of concentrated sulfuric acid, boron exists as the cation B3+. The cation
complexes to the carmine indicator causing the solution to change color from red to blue.
The blue-colored complex is read at 605 nm using a spectrophotometer, and the amount of color is
proportional to the dissolved boron concentration.
B2+
CH3 O OH CH3 O O
3+
CO2H B CO2H
H+
HO OH OH OH
CO2H O OH CO2H O OH
Carmine Boron—Carmine Complex

Figure 46 Chemical reactions for the Carmine method
Boron
Page 1555
Benzo- and tolylltriazole tests explained
Benzotriazole and Tolyltriazole

For water Ultraviolet Digestion Method
Introduction
Benzotriazole and tolyltriazole (BZT and TTA) are used extensively as corrosion inhibitors for
copper alloys. In both open and closed recirculating water cooling systems, concentrations of 2 to
10 mg/L provide effective corrosion control after initial passivation
CH3
N
N
N
N
N
N
H
H
Benzotriazole Tolyltriazole
Figure 47 Chemical procedures
The Hach method for BZT and TTA determinations offers substantial improvements over
conventional analytical methods. Time-consuming conventional methods, such as ultraviolet (UV)
spectroscopy, liquid chromatography and potentiometric titration, require expensive equipment
and highly skilled personnel. The Hach method uses simple, inexpensive equipment and can be
performed in less than 10 minutes without loss of accuracy or precision. The analysis range is 0–
15 mg/L at a wavelength of 425 nm.
Chemical reactions
This method of analysis is based on the UV-photolysis of triazole in the presence of a chemical
catalyst to form a dimer or polymer of the triazole. A stoichiometric amount of a soluble yellow-
colored compound is then formed. The calibration follows Beer’s Law throughout the 0–15 mg/L
concentration range, with an estimated detection limit of approximately 0.3 mg/L
N UV
N (Yellow-colored complex)
Chemical
N Catalyst
H
Figure 48 Chemical reaction
Benzotriazole and Tolyltriazole

Page 1556
Carbon dioxide tests explained
Carbon Dioxide
For water and seawater Titration Method
Introduction
Carbon dioxide is present in all surface waters, generally in amounts less than 10 mg/L; however,
higher concentrations are not uncommon in ground waters. Dissolved carbon dioxide has no
harmful physiological effect on humans and is used to recarbonate water during the final stages of
water-softening processes and also to carbonate soft drinks. High concentrations of dissolved
carbon dioxide are corrosive and have been known to kill fish.
The analysis for carbon dioxide is similar to that for acidity. A water sample is titrated to a
phenolphthalein end point with Sodium Hydroxide Standard Solution. Strong mineral acids are
assumed to be absent or to be negligible in effect. Care must be taken during the analysis to
minimize the loss of carbon dioxide from the water sample as a result of aeration during collection
and swirling of the sample.
Chemical reactions
The reaction of sodium hydroxide with carbon dioxide (as carbonic acid) occurs essentially in two
steps; first a reaction from carbonic acid to bicarbonate and then to carbonate.
Because the conversion of carbon dioxide to bicarbonate is complete at pH 8.3, phenolphthalein
can be used as a color indicator for the titration. The sodium hydroxide titrant must be of high
quality and be free from sodium carbonate.
CO 2 + H 2 O → H2 CO 3 (Carbonic acid)
H 2 CO 3 + NaOH → NaHCO 3 + H 2 O
NaHCO 3 + NaOH → Na 2 CO 3 + H 2 O
Carbon Dioxide
Page 1557
Chloramine (Mono) tests explained
Chloramine (Mono)
For water and wastewater Indophenol Method
Introduction
Chloramination disinfection is the practice of forming inorganic chloramines in water to reduce
microbial concentrations to within acceptable limits. The chloramines; monochloramine (NH2Cl),
dichloramine (NHCl2) and nitrogen trichloride (NCl3), form when chlorine and ammonia are
combined in water. Traditionally, treated wastewater, which contains ammonia, is disinfected by
the addition of chlorine. In recent years, many drinking water facilities have converted to
chloramination to disinfect potable water. Roughly 20% of all drinking water facilities in the United
States now use chloramines as the residual disinfectant.
For the chloramination of drinking water, monochloramine is the preferred disinfectant. Formation
of dichloramine and nitrogen trichloride is avoided, since more chlorine is consumed and the
presence of these chloramines can produce odors or off-tastes.
In treated wastewater, any organic nitrogen compounds present will form organic chloramines
during chlorination. Organic chloramines, as a class, are much weaker disinfectants than the
inorganic chloramines. Chlorine overfeeds and ineffective mixing can lead to greater production of
organic chloramines, thereby diminishing the total germicidal activity.
Hach chemists have developed a method for the specific determination of monochloramine in
water. The method is based on the classic indophenol chemistry for determining ammonia. The
chemistry has been improved to increase the specificity of the method for inorganic
monochloramine in the presence of organic chloramines. In addition, the method was modified to
greatly accelerate the color development time and increase the precision of the test. The new test
has been shown to be specific for monochloramine, without interference from organic or inorganic
amines, dichloramines, free chlorine, organic chloramines, nitrites or manganese.
Chemical reactions
Monochloramine reacts specifically with a substituted phenate to form a quinone imine
intermediate. In the presence of a cyanoferrate, the intermediate couples with excess phenate to
form a green-colored indophenol. The amount of indophenol formed is proportional to
concentration of monochloramine in the sample. See the Chemical reactions figure below.
Benzoquinone Monoimine formation
NH2Cl + OH H2N OH HN O
R R R
HN O+ O¯ ¯O N O
R R R R
Indophenol
Formation
Figure 49 Chemical reactions1

1 R= reaction accelerating group
Chloramine (Mono)
Page 1558
Chloramine (Mono) and Free Ammonia explained
Chloramine (Mono);
Indophenol method
For determining free ammonia and monochloramine simultaneously in finished
chloraminated water
Introduction
Chloramination disinfection is the practice of forming inorganic chloramines in water to reduce
microbial concentrations to within acceptable limits. The chloramines—monochloramine (NH2Cl),
dichloramine (NHCl2), and trichloramine (NCl3)—form when chlorine and ammonia are combined
in water. In recent years, many drinking water facilities have converted from free chlorination to
chloramination to disinfect potable water. Chloramines are weaker oxidants than free chlorine and
therefore minimize the formation of harmful disinfection by-products.
A typical chloramination curve is presented in Figure 50. For the chloramination of drinking water,
monochloramine is the preferred disinfectant (Section I of the curve). This is optimized by an
approximate 5:1 ratio (by weight) of chlorine to ammonia. Adding too much chlorine leads to the
decrease of monochloramine and the formation of dichloramine and trichloramine, causing taste
and odor problems (Section II). Adding too little chlorine leaves excess unreacted or "free"
ammonia in the water which acts as a food source and can lead to nitrification and bacterial growth
in the distribution system. At the breakpoint, which is the vertical line between Sections II and III,
no monochloramine remains. Any additional chlorine added will be in the form of free chlorine.
In treated wastewater, any organic nitrogen compounds present will form organic chloramines
during chlorination. Organic chloramines, as a class, are much weaker disinfectants than the
inorganic chloramines. Chlorine overfeeds and ineffective mixing can lead to greater production of
organic chloramines, thus diminishing the total germicidal activity.
Figure 50 Breakpoint curve for chloraminated water

Page 1559
Chemical reactions
Refer to Figure 51 for the indophenol method mechanism.
Added hypochlorite combines with free ammonia to form more monochloramine (1). In the
presence of a cyanoferrate catalyst, monochloramine in the sample reacts with a substituted
phenol to form an intermediate monoimine compound (2). The intermediate couples with excess
substituted phenol to form a green-colored indophenol, which is proportional to the amount of
monochloramine present in the sample (3). Free ammonia is determined by comparing the color
intensities, with and without added hypochlorite.
Figure 51 Chemical reactions for the indophenol method

Page 1560
Chloride tests explained
Chloride
Mercuric Nitrate, Mohr Argentometric and
For water and wastewater
Mercuric Thiocyanate Methods
Introduction
Chlorides are present in all potable water supplies and in sewage, usually as a metallic salt. When
sodium is present in drinking water, chloride concentrations in excess of 250 mg/L give a salty
taste. If the chloride is present as a calcium or magnesium salt, the taste detection level may be as
high as 1000 mg/L chloride.
Chloride is essential in the human diet and passes through the digestive system unchanged,
thereby becoming one of the major components of raw sewage. The wide use of zeolite spheres in
water softeners also contributes a large amount of chloride to sewage and wastewaters.
High chloride concentrations in water are not known to have toxic effects on humans, although
large amounts may act corrosively on metal pipes and be harmful to plant life. The maximum
allowable chloride concentration of 250 mg/L in drinking water has been established for reasons of
taste rather than as a safeguard against physical hazard.
Chemical reactions
Mercuric nitrate method
Mercuric nitrate reacts selectively with all the chloride present in a sample to produce mercuric
chloride and nitrate ions. When all the chloride present in the sample has been complexed, excess
mercuric ions combine with diphenylcarbazone to form a purple-colored complex indicating the
end point. Hach procedures use Diphenylcarbazone Reagent Powder containing the indicator and
a buffer for maximum convenience and reagent stability.
– –
Hg ( NO 3 ) 2 + 2Cl → HgCl 2 + 2NO 3
Silver nitrate method (Mohr Argentometric method)
In the chloride test, using silver nitrate as the titrant and potassium chromate as the indicator,
silver nitrate first reacts selectively with the chloride present in the sample to produce insoluble
white silver chloride. After all the chloride has been precipitated, the silver nitrate then reacts with
the potassium chromate to form an orange-colored silver chromate precipitate, thereby marking
the end point of the titration. Potassium chromate indicator is combined with a buffer in Chloride 2
Indicator Powder.
– –
AgNO 3 + K 2 CrO 4 + Cl → AgCl + NO 3 + K 2 CrO 4
Ag 2 CrO 4
2AgNO 3 + K 2 CrO 4 → + 2KNO 3
(Orange)
Chloride
Page 1561
Chloride
Mercuric Thiocyanate method

Colorimetric determination of chloride by the Mercuric Thiocyanate Method involves reaction of
chloride in the sample with mercuric thiocyanate to produce mercuric chloride and free thiocyanate
ions. In the presence of Fe3+ (ferric ion), the free thiocyanate ion forms highly colored ferric
thiocyanate in proportion to the chloride concentration. Two liquid reagents have been formulated
for this test: Mercuric Thiocyanate Solution and Ferric Ion Solution.
Hg ( SCN ) 2 – –
1. + 2Cl → HgCl 2 + 2SCN
(Mercuric Thiocyanate)
3+ – Fe ( SCN ) 3
2. Fe + 3SCN →
(Red-orange)
Chloride
Page 1562
Chlorine dioxide tests explained
Chlorine Dioxide
For water and wastewater DPD and Chlorophenol Red Methods
Introduction
Chlorine Dioxide is a deep yellow gas that is generated directly for on-site use as a bleaching
agent in industrial processes, such as the manufacture of pulp and paper. It is used increasingly
for special treatment objectives in municipal water treatment because, unlike chlorine, chlorine
dioxide does not form trihalomethanes (THMs) in reaction with certain organic compounds. Two
colorimetric methods for chlorine dioxide at low levels are used in Hach procedures. Hach also
offers a high range method that directly measures the yellow color of the chlorine dioxide gas
dissolved in the sample water.
The DPD method is an extension of the N,N-diethyl-p-phenylenediamine (DPD) method for
determining free and total chlorine. Glycine is used to eliminate chlorine interference.
The Chlorophenol Red (CPR) method reacts specifically with chlorine dioxide.
Chemical reactions
DPD Method
Chlorine dioxide reacts with the DPD (N,N-diethyl-p-phenylenediamine) Indicator Reagent (to the
extent of one-fifth of its total available chlorine content corresponding to the reduction of chlorine
dioxide to chlorite) to form a pink color. The color intensity is proportional to the ClO2 in the
sample. Chlorine interference is eliminated by adding glycine, which converts free chlorine to
chloroaminoascorbic acid, but has no effect on chlorine dioxide at the test pH.
Chlorophenol red method
Chlorophenol Red (CPR) indicator reacts specifically with chlorine dioxide with a distinct color
change; no interference is experienced form other mild oxidants, including hypochlorite, chlorite,
chromate, permanganate, ferric iron, or low levels of chloramines. One mole of CPR reacts with
two moles of chlorine dioxide to form a colorless product with a net decrease in absorbance at
570 nm. The discoloration of CPR is linear to approximately 0.6 mg/L, although concentrations to
approximately 1.0 mg/L are easily determined. The reaction of CPR with ClO2 is reproducible. No
equation for this reaction will be suggested; however, the reaction may result in the formation of an
ion-pair complex.
The reaction of CPR with chlorine dioxide is pH-sensitive. A pH of 7.0 has been suggested for the
spectrophotometric method. Hach researchers found the optimum pH for this reaction is actually
5.2. It was also determined that the sensitivity is improved if the solution is buffered to near pH 10
after the initial reaction. The reagents for this method are contained in three convenient solutions.
Reagent 1 is a buffer which adjusts the sample to the optimum pH, 5.2. Reagent 2 is a special
formulation of CPR which is added after the pH adjustment. Reagent 3 is a pH 10 buffer added
after CPR to increase sensitivity. Blanks for standardizing the spectrophotometer are prepared by
adding dechlorinating agent to a 50-mL sample, thereby destroying up to 35 mg/L of ClO2.
Chlorine Dioxide
Page 1563
Chlorine Dioxide
OH O¯
Cl Cl
O O
Cl Cl
C C
SO3¯ SO3¯
Yellow (acid color) Red (base color)

Figure 52 Chlorophenol Red structures
Chlorine Dioxide
Page 1564
Chlorine, free and total tests explained

For water, wastewater and seawater DPD Method
Introduction
Chlorine is the disinfectant most frequently used for water and wastewater treatment. It was used
first for industrial applications and to control odor in wastewater, in the early 1800s. The
subsequent use of chlorine to disinfect water occurred by the mid-1800s. Industrial uses of
chlorine include applications such as bleaching paper and controlling nuisance organisms in
cooling towers.
Hydrochloric and hypochlorous acids are formed when chlorine is added to water. The disinfectant
and form causing bleaching action, is hypochlorous acid.
Cl 2 + H 2 O → HCl + HOCl (Hypochlorous acid)

Depending upon variables such as pH, temperature and the amount of organic or ammonia
nitrogen, other forms of chlorine in water may include hypochlorite ions (OCl–) and chloramines.
Chlorine existing in water as hypochlorous acid or the hypochlorite ion is termed free available
chlorine. Chloramines, including monochloramine (NH2Cl), dichloramine (NHCl2) and nitrogen
trichloride (NCl3) are referred to as Combined Available Chlorine. Total Chlorine refers to the sum
of free and combined available forms.
Methods for determining free, combined and total chlorine include: amperometric titration,
colorimetric DPD, titrimetric DPD and iodimetric titration. The most widely used method,
colorimetric DPD, is easy to perform, requires little apparatus, is inexpensive and adapts well to
field test situations. DPD (N,N-diethyl-p-phenylenediamine) is oxidized by chlorine, causing a
magenta (red) color. The intensity of color is directly proportional to the chlorine concentration.
DPD reacts in much the same way with other oxidants, including bromine, chlorine dioxide,
hydrogen peroxide, iodine, ozone and permanganate.
Chemical reactions
Free available chlorine
Hypochlorous acid and the hypochlorite ion oxidize DPD causing a magenta color. The reaction is
pH dependent. DPD and appropriate buffer are packaged together in DPD Free Chlorine Reagent
Powder Pillows to handle high levels of hardness without precipitation.
Total chlorine
Potassium iodide is added to the reaction to determine combined available chlorine forms and total
chlorine. Chloramines oxidize the iodide to iodine; then the liberated iodine reacts with DPD to
form the magenta color. DPD Total Chlorine Reagent Powder Pillows from Hach contain DPD,
potassium iodide and a buffer.

Page 1565
+ +
NH3 NH3
+Cl2
I3¯
N+ N+
H 5C 2 C2H5 H 5C 2 C2H5
H
(Colorless) Würster Dye (Red)
Figure 53 DPD chemical reactions

Page 1566
Chromium tests explained
Chromium
For water and wastewater Total and Hexavalent Methods
Introduction
Chromium may be present in water as the hexavalent (chromate) or the trivalent form, although
trivalent chromium rarely occurs in potable water. Hexavalent chromium enters a water supply
through industrial wastes from metal plating baths and from industrial cooling towers where
chromate is used to inhibit metal corrosion. Chromium is an objectionable contaminant in public
drinking water supplies due to its suspected carcinogenic effects. Chromium present in potable
waters above a 3-µg/L level indicates the possible presence of industrial wastes. Concentrations
greater than 50 µg/L are sufficient grounds to reject the water supply.
Chemical reactions
Hexavalent chromium
Hexavalent chromium is determined by the 1,5-Diphenylcarbohydrazide Method using a single dry
powder formulation called ChromaVer 3™ Chromium Reagent. This reagent contains an acidic
buffer combined with 1,5-Diphenylcarbohydrazide which reacts to give a purple color when
hexavalent chromium is present. The method is applicable to fresh water and wastewater
samples. Color development is directly proportional to the amount of hexavalent chromium
present.
N N
H H
R C
N N N O Cr O
2 + Cr6+
N C H C R
H O N N
H H
1,5-diphenylcarbohydrazide
Figure 54 Hexavalent chromium chemical reaction
Total chromium
In the analysis for total chromium, the sample is heated to the boiling point under strong alkaline
conditions in the presence of hypobromite. The trivalent chromium is converted to hexavalent
chromium. The proper chemical conditions for this oxidation are provided by Chromium 1 Reagent
Powder.
After the oxidation is complete, excess hypobromite is destroyed by the addition of Chromium 2
Reagent Powder. Then ChromaVer 3 Chromium Reagent, which contains an acidic buffer
combined with 1,5-Diphenylcarbohydrazide, is added. A purple color develops with an intensity
directly proportional to the total chromium concentration. The trivalent chromium can be
determined by subtracting the hexavalent chromium test results from the results obtained in the
total chromium test.
3+ – – 2– –
2Cr + 3 OBr + 10 OH → 2CrO 4 + 3Br + 5H 2 O
Chromium
Page 1567
Cobalt tests explained
Cobalt
For water 1-(2-Pyridylazo)-2-Naphthol (PAN) Method
Introduction
Cobalt is valuable because of its ability to increase the strength and corrosion resistance of alloys.
It is associated with nickel, silver, lead, copper and iron ores, from which it is most frequently
obtained as a by-product. Cobalt is often found in industrial wastewaters as a corrosion product of
alloys of iron, nickel and cobalt, but it seldom occurs in natural waters.
Toxicity of cobalt to aquatic life varies depending on pH, the species or organism, and synergetic
effects. It is considered to be relatively nontoxic to humans. Methods for detection of low levels of
cobalt historically have been limited to expensive and time-consuming techniques—mainly atomic
absorption. By comparison, cobalt can be determined quantitatively by a simple colorimetric
procedure using a spectrophotometer. Accuracy and precision rivals atomic absorption
measurements. The very sensitive 1-(2-Pyridylazo)-2-Naphthol (PAN) Method is capable of
detecting 0.1 mg/L cobalt. This unique method is relatively free from interferences and provides for
simultaneous determinations of nickel and cobalt on the same sample portion without special
treatments.
Chemical reactions
PAN is suspended in water by use of surfactants to allow it to form complexes with the metals in
the sample. A complexing agent can be used to decompose all PAN chelates except those of
cobalt, nickel and iron. A pH adjustment using the Phthalate-Phosphate Reagent aids in the
masking of iron up to 10 mg/L, and also enhances the rate of development of the colored cobalt
and nickel PAN complexes.
+ Charge for Co
0 Charge for Ni
N N N
2 + Co2+ Co (or Ni) + 2H+

N N N
O
OH
N N N
Figure 55 Formation of Co-PAN or Ni-PAN complex1

1 Cheng, K. L., and Bray, R. H., Journal of Analytical Chemistry, 27, 1955, page 783.
Cobalt
Page 1568
Cobalt
The absorbance of the cobalt PAN complex at 560 nm is the same as at 620 nm; however,
absorbance caused by the nickel PAN complex is zero at 620 nm. This difference in absorbance
wavelengths allows cobalt to be determined without interference from nickel at a wavelength of
620 nm. Therefore, the nickel can be determined on the same sample by measuring the
absorbance at 560 nm and subtracting the absorbance at 620 nm.
Ni
Co
Absorbance
520
530
540
550
560
570
580
590
600
610
620
630
640
650
Nm
Figure 56 Typical absorbance scan for Co and Ni
Cobalt
Page 1569
Copper tests explained
Copper
Bicinchoninate, Porphyrin and
For water, wastewater and seawater
Bathocuproine Methods
Introduction
Although copper comprises only 0.007% of the earth’s crust, it is a very important element. Copper
occurs in both free and combined forms throughout nature in many minerals. Copper may occur in
natural waters, wastewaters, and industrial waste streams as soluble copper salts or as copper
compounds precipitated on suspended solids. Forms of copper in water can be classified as
insoluble, dissolved (free and complexed), and total recoverable. Insoluble copper includes
precipitates such as copper sulfides and hydroxides. All copper in solution is known as dissolved
copper, including Cu1+ (cuprous) and Cu2+ (cupric) ions and copper chelates such as CuEDTA.
Copper concentrations in potable water are usually very low. Copper is not considered a health
hazard to humans although more than 1 mg/L can impart a bitter taste to water and large oral
doses can cause vomiting and may eventually cause liver damage. Copper salts, such as copper
sulfate (CuSO4), may be used to control algae; however, they may also be toxic to fish and wildlife.
Hach’s simplified test procedures for copper use a variety of reagents to satisfy the desired range
of detection and the form of copper to be measured. Hach procedures use primarily the
Bicinchoninate and the Porphyrin Methods.
The Copper reagents and applications table lists proprietary reagents and applications.
Table 429 Copper reagents and applications

Form measured
Reagent Application
Without pretreatment With digestion
CuVer 1™1 Free Total recoverable water, wastewater

CuVer 2™ Total dissolved copper Total recoverable
Free Total recoverable hard water, wastewater,
Free copper reagent
seawater
Free Total recoverable extremely low levels in water,
Porphyrin reagents
wastewater and seawater
1 CuVer is a trademark of Hach Company.
Chemical reactions
Bicinchoninate method
Copper can be determined by the reaction of copper with 2, 2’-biquinoline-4,4’-dicarboxylic acid
(bicinchoninic acid). Bicinchoninate reacts with Cu1+ to produce a purple-colored complex.
Bicinchoninate does not react readily with Cu2+. Determination of Cu2+ begins by reducing it to
Cu1+. The CuVer 1 Reagent combines the bicinchoninate reagent with a buffer and reducing
agent, allowing determination of Cu1+ and Cu2+. Total recoverable copper can be determined with
this method if the sample is first digested to convert all of the copper present (including insoluble
forms and complexed forms) to free copper.
Complexed copper forms such as CuEDTA react directly with CuVer 2™. Digestion is not
necessary, and high levels of hardness do not interfere. The results will be in terms of total
dissolved copper (free and complexed). When using CuVer 1, digestion is necessary and high
levels of hardness interfere.
Copper
Page 1570
Copper
HOOC N HOOC N N COOH
2 + Cu+ Cu
HOOC N HOOC N N COOH
Figure 57 Reaction of Cu1+ and bicinchoninic acid
Use Free Copper Reagent Powder Pillows to determine free copper separately from complexed
copper. The powder pillows contain bicinchoninate, a reducing agent and an inhibitor to eliminate
calcium and magnesium interference. The results will be in terms of free copper. Complexed
copper may then be determined by adding Hydrosulfite Reagent, repeating the analysis and
subtracting the results of the two analyses.
Porphyrin method
The porphyrin method for determining copper is a very sensitive test, capable of detecting free
copper (Cu1+ and Cu2+) and total recoverable copper (with digestion) in the range of 0–150 µg/L.
Because of the sensitivity of the method, it is difficult to obtain water of high enough quality to
establish a blank value. The porphyrin method uses a split sample. One half of the split sample is
treated with a masking agent to complex the free copper forms; then, porphyrin reagent, a buffer,
and a reducing agent is added. This forms a “zero blank” without the need for special copper-free
water. Porphyrin reagent is added to the second half of the split sample, where it reacts with the
free copper.
Interference caused by the reaction of porphyrin with other metals is minimized by using the split
sample because interferences are compensated for in the blank. Porphyrin reacts slowly with
Cu2+, but a special formulation of the porphyrin and addition of a buffer allow the reaction with free
copper to be completed within seconds. A reducing agent is also added in order to destroy
unreacted porphyrin (which would otherwise interfere). An intense absorbance at 425 nm makes
this method very sensitive when using a colorimeter or spectrophotometer. However, strong visual
color development does not occur.
Copper
Page 1571
Copper
CH3
N N
CH3 N Cu N CH3
N N
CH3
Figure 58 Final structure from the porphyrin method
Copper
Page 1572
Cyanide tests explained
Cyanide
For water, wastewater and seawater Pyridine-Pyrazolone Method
Introduction
Cyanide is extremely toxic and occurs primarily in industrial effluents. Metal-cleaning and
electroplating baths, gas scrubbers, gas works, coke ovens and other chemical treatments are the
main sources of the cyanide found in industrial wastes. Natural waters do not contain cyanide; its
presence usually indicates contamination from an industrial source. Proper neutral or alkaline
chlorination of cyanide-containing wastewaters will reduce the level well below toxic limits.
Chemical reactions
The cyanide test involves the following 4 steps:
(1) 2CN¯ + Cl2 2CNCl
Cyanide is reacted with chlorine to produce cyanogen chloride (CNCl); the chlorine is provided by
CyaniVer™ 3 Reagent.
(2) CNCl + N N+ + Cl¯

CN
An intermediate nitrile is then formed by the addition of pyridine; the pyridine is provided by the
addition of CyaniVer 4 Reagent. Excess chlorine is destroyed at this point.
H H
(3) 4H2O + N+ + Cl¯ O C C C C C O + 2NH3 + CO2 + HCl

CN H H H H
The nitrile is hydrolyzed to glutaconaldehyde; the reagent is provided in the CyaniVer 4 Reagent
from the previous step.
H H
(4) O C C C C C O +2 N O
N
H H H H
H3C
O O
H H H H
N N + 2 H2 O
C C C C C
N N
H H
CH3 CH3
Cyanide
Page 1573
Cyanide
Finally, CyaniVer 5 Reagent, containing an excess of pyralozone, is added. The reaction with the
glutaconaldehyde results in a blue color. The intensity of the color is directly proportional to the
amount of cyanide present in the sample.
Cyanide
Page 1574
Fluoride tests explained
Fluoride
SPADNS, SPADNS 2 and Ion-selective
For water and seawater
Electrode Methods
Introduction
Fluoride occurs naturally in some ground waters, and a 1-mg/L level is normally maintained in
public drinking water supplies for the prevention of dental cavities. Excessive amounts of fluoride
cause an objectionable discoloration of tooth enamel called “mottling”. For this reason, a
permissible level in drinking water has been established by the USEPA in accordance with the
Safe Drinking Water Act.
Chemical reactions
SPADNS method
The fluoride analysis involves the reaction of fluoride with a dark red zirconium-dye complex.
Fluoride combines with part of the zirconium to form a colorless zirconium-fluoride complex with
the net effect of bleaching the color. Measurement of the decrease in color intensity provides an
accurate determination of the fluoride concentration. The SPADNS Method is the preferable
colorimetric method due to its rapid reaction with fluoride and the stability of the SPADNS reagent.
HO3S HO3S
OH
OH H+
N Zr + 6F¯ N + ZrF62– + NH2O
O OH OH OH
N N
HO3S SO3H HO3S SO3H
(Red) (Colorless)
Figure 59 Chemical reaction for SPADNS method
SPADNS 2 method
Sodium Arsenite is used in the SPADNS method as a reducing agent to prevent interference from
chlorine and other oxidants that are typically present in drinking water. The SPADNS 2 test
eliminates arsenic from the original SPADNS formulation by using a non-toxic proprietary reducing
agent to achieve identical results and test performance. All other chemistry remains the same as
the SPADNS method.
Method of analysis
Ion-Selective electrode method
The Ion-Selective electrode method requires a Hach sension™ ISE Meter and an electrode
system consisting of a silver/silver chloride reference electrode and a standard fluoride ion-
selective electrode. Fluoride measurement is accomplished when a voltage potential is
established across the lanthanum fluoride crystal on the end of the electrode; this potential is in
direct proportion to the fluoride concentration of the sample. The meter is calibrated with fluoride
standards bracketing the expected range. The concentration may be read directly from the meter.
A total ionic strength adjustment buffer (TISAB) is used to eliminate interferences in the test, to
adjust the pH to an optimum value and to introduce sufficient sodium chloride to mask variations in
Fluoride
Page 1575
Fluoride
ionic strength. TISAB reagent uses sodium 1,2-cyclohexanediaminetetraacetic acid (CDTA) for
chelation of interfering metals, such as Al3+ and Fe3+, as well as other complexing and buffering
agents.
Fluoride
Page 1576
Formaldehyde tests explained
Formaldehyde
For water MBTH Method
Introduction
Formaldehyde is used in the treatment of fabric in textile industries, in metal plating baths, as a
preservative for biological tissues and as a disinfectant in dialysis and reverse osmosis equipment.
The MBTH Method is a sensitive colorimetric test for low range measurement of aldehydes; it is
most sensitive for formaldehyde.
Chemical reactions
MBTH Method
MBTH (3-methyl-2-benzothiazoline hydrazone) is added in excess to a sample containing
formaldehyde, triggering multiple reactions. First, MBTH and formaldehyde react to form an azine
(1). Excess MBTH is oxidized by addition of a developing solution (2). Oxidized MBTH reacts with
the azine to form a species with an intense blue color (3). Intensity of the blue color is proportional
to the original concentration of formaldehyde.
MBTH is contained in MBTH Powder Pillows. Liquid reagent for oxidizing excess MBTH is
contained in the Developing Solution for Low Range Formaldehyde.
CH3 CH3
H
N N
(1) C N NH2 + C O C N N CH2 + H2O

S S
H
CH3
N
(2) C N NH+
S
(Oxidized MBTH)
CH3 CH3
N N +
(3) C N N CH N N C
S S
(Blue-colored complex)
Formaldehyde
Page 1577
Formaldehyde
CH3 CH3
H
N N
(1) C N NH2 + C O C N N CH2 + H2O

S S
H
Figure 60 Chemical reaction for the MBTH method
Formaldehyde
Page 1578
Hardness tests explained
Hardness
EDTA Titration and Calmagite Colorimetric
For water, wastewater and seawater Methods
Introduction
Hardness in water is caused by dissolved minerals, primarily divalent cations, including calcium
(Ca2+), iron (Fe2+), strontium (Sr2+), zinc (Zn2+) and manganese (Mn2+). Calcium and magnesium
ions are usually the only ions present in significant concentrations; therefore, hardness is generally
considered to be a measure of the calcium and magnesium content of water. Considerations
should be given when other cations contributing to hardness are present in significant amounts.
Titration methods
Hardness in water can be determined quickly by titration and the use of color indicators. By proper
choice of pH, total hardness (Ca2+ and Mg2+) or the portion contributed by calcium and
magnesium individually can be measured. The traditional test for hardness involves pH
adjustment to 10.1 with an ammonium buffer, addition of Eriochrome Black T indicator [1-(1-
hydroxy-2-naphthylazo)-6-nitro-2-naphthol-4-sulfonic acid] and then titration with Na2EDTA
(ethylenediaminetetraacetic acid, disodium salt) solution.
HOOCH2C CH2COOH
NCH2CH2N
HOOCH2C CH2COOH
Figure 61 Chemical structure of EDTA-Ethylenediaminetetraacetic acid
Some other indicators are more stable, giving a faster reaction and a more distinct end point than
Eriochrome Black T. One of the best is calmagite, 1-(1-hydroxyl-4-methyl-2-phenylazo)-2-
naphthol-4 sulfonic acid, which is used in Hach total hardness tests.
Colorimetric method
The Colorimetric Method is for low level measurement of hardness. The interference of some
metals with the Titration Methods will be rendered inconsequential after diluting the sample to
bring it into the range of this test. Calmagite indicator and two chelating agents, EGTA and EDTA,
are used in the test.
Chemical reactions
Total hardness
Several solutions including digital titrator cartridges are described in the following section for
titrating prepared water samples containing calmagite indicator. TitraVer™ Hardness Titrant
(0.020 N EDTA) is the most widely used. Other strengths of TitraVer Hardness Titrant are available
for titrating high hardness samples. HexaVer™ Hardness Titrant also is available. HexaVer is
CDTA (cyclohexanediaminetetraacetic acid, disodium salt). It gives slightly sharper end points and
can tolerate higher levels of iron interference than TitraVer.
Hardness
Page 1579
Hardness
CH2COONa
N
CH2COOH
CH2COOH
N
CH2COONa
Figure 62 Chemical structure of CDTA, disodium salt
Calmagite indicator is available in special formulations as ManVer™ and UniVer™. The ManVer
formulations of calmagite have been specially prepared to enhance stability and to be free from
most interferences. Interferences caused by metal ions, such as copper or iron, can be removed or
masked by the use of the magnesium salt of CDTA. It is effective, yet safe to use. Cyanide
compounds also may be used to overcome interferences. Their use is avoided where possible
because of potential environmental and health hazards.
CH3 CH3
O O
N + Mg 2+ N + H+
Mg
N N
OH O
SO3¯ SO3
Calmagite (blue) Calmagite-Mg complex (wine red)
Figure 63 Reaction between magnesium and calmagite indicator
The reaction of calmagite is pH-dependent; it has been determined that a pH of 10.1 is ideal.
Traditionally, ammonia buffers have been used; however, they have a strong odor. Hach methods
use Hardness 1 Buffer (2-amino-2-methyl-1-propanol), which is stable, safe to use and has a less
objectionable odor.
The sequence of analysis in the hardness tests begins with pH adjustment and addition of
inhibitors followed by formation of the Mg2+ and Ca2+ complexes with calmagite. The calcium
forms a weak complex with calmagite at this pH. The solution is titrated with TitraVer (EDTA) or
HexaVer (CDTA). The titrant first complexes any calcium, then magnesium. Color change from
wine red to blue is an indication that all calcium and magnesium have been removed from the
calmagite and complexed with the titrant.
Hardness
Page 1580
Hardness
H2
OOC C C O
C
N
O
C
Mg
C
O
N
C
OOC C C O
H2
Figure 64 Magnesium complexed with TitraVer
Expression of results of the hardness titration is mg/L as CaCO3. The reaction of TitraVer with
Ca2+ and Mg2+ is a 1:1 ratio.
Calcium hardness
The test for calcium hardness is very similar to the total hardness test. Traditionally, either
murexide indicator (ammonium purpurate) or Eriochrome Blue-Black R indicator is followed by
titration with EDTA. CalVer 2 Calcium Indicator has been developed by Hach to replace these
indicators. CalVer 2 (hydroxy naphthol blue) is more sensitive and has a sharper end point color
change.
CalVer 2 Calcium Indicator forms a red-violet complex with calcium and changes to pure blue after
TitraVer removes calcium from the complex. The pH is elevated to at least 13 to precipitate
magnesium. A few drops of Magnesium Standard Solution may be added to the reaction to
sharpen the end point color change. This may seem inconsistent because magnesium is
precipitated by elevating the pH. However, the added magnesium is chelated preferentially by the
dye and the quantity of chelated magnesium is very small; thus any error caused by addition of
magnesium is negligible.
The pH adjustment is accomplished by addition of potassium hydroxide prior to addition of CalVer
2. Potassium cyanide may also be added to complex interfering metals prior to the addition of
CalVer 2.
Calcium hardness and total hardness may be determined sequentially using the same sample.
After the calcium hardness is determined the sample pH can be adjusted downward, using sulfuric
acid. Then Hardness Buffer 1 and ManVer 2 are added and titration with TitraVer is resumed.
Hardness
Page 1581
Hardness
!
DO NOT use this procedure if potassium cyanide has been used in determining
calcium hardness! The addition of sulfuric acid will cause deadly hydrogen cyanide gas
to evolve.
Colorimetric method
Calmagite, contained in Calcium and Magnesium Indicator Solution, is added to a sample and the
pH is elevated to about 12.5 by using a buffer. Adding calmagite prior to pH adjustment prevents
the calcium and magnesium precipitation that ordinarily would occur at this elevated pH. The
sample is then split into three equal portions.
EDTA is added to the first portion to sequester calcium and magnesium, thereby breaking the Ca-
and Mg-calmagite complexes. This solution is used as a zero reference blank to standardize the
spectrophotometer.
EGTA or ethyleneglycol-bis (2-aminoethylether)-N,N,N’,N’-tetraacetic acid, is added to the second
sample portion. EGTA selectively chelates calcium under conditions of the test; only absorbance
due to the Mg-calmagite complex remains to be measured. The result is expressed as mg/L Mg as
CaCO3. After measurement, the spectrophotometer is adjusted to read “zero” on this portion.
Absorbance of the third sample portion (containing no chelant) is measured to determine mg/L Ca
as CaCO3. Adjust the spectrophotometer to a reading of zero after measurement of the second
sample portion to compensate for absorbance due to magnesium in the sample.
¯OOCH2C CH2COOH
N -CH2-CH2-O-CH2-CH2-O-CH2-CH2-N
HOOCH2C CH2COO¯
Figure 65 Chemical structure of EGTA
Hardness
Page 1582
Hydrazine tests explained
Hydrazine
For water and boiler water p-Dimethylaminobenzaldehyde Method
Introduction
Hydrazine is used as an oxygen scavenger for high pressure boilers in power plants and other
industries to reduce corrosion of metal pipes and fittings. The test for hydrazine is a modification of
the p-Dimethylamino-benzaldehyde Method, in which several solutions have been formulated into
a single, stable reagent called HydraVer™ 2 Hydrazine Reagent. The method is both sensitive and
easy to perform. It is used mostly for the determination of small amounts of hydrazine in boiler
feedwater. There are no common interferences.
Chemical reactions
Under acid conditions, hydrazine combines with p-Dimethylaminobenzaldehyde to form a yellow-
colored azine complex. Color development follows Beer’s law and is stable after maximum color is
developed in 10 to 15 minutes.
O
CH3 H H
2 N C H + :N N:
CH3
H H
p-Dimethylaminobenzaldehyde Hydrazine
CH3 CH3
N C N N C N +2H2O
CH3 CH3
H H
Azine complex (yellow in dilute solution)
Figure 66 Chemical reaction for p-Dimethylaminobenzaldehyde method
Hydrazine
Page 1583
Iron tests explained
Iron
1,10-Phenanthroline, FerroZine, TPTZ and
For water and seawater
Titration Methods
Introduction
Natural waters contain variable, but minor, amounts of iron, despite its universal distribution and
abundance. Iron in ground waters is normally present in the ferrous (Fe2+), or soluble state, which
oxidizes easily to ferric (Fe3+) iron on exposure to air. Iron can enter a water system from leaching
of natural deposits, iron-bearing industrial wastes, effluents of pickling operations, or from acidic
mine drainage.
Iron in domestic water supply systems stains laundry and porcelain, causing more of a nuisance
than a potential health hazard. Taste thresholds of iron in water, 0.1 mg/L for Fe2+ and 0.2 mg/L for
Fe3+, result in a bitter or astringent taste. Water used in industrial processes must contain less
than 0.2 mg/L of total iron.
Three methods of colorimetric iron analysis are used in Hach procedures. The 1,10-
Phenanthroline Method is the best-known test for iron. The Fe2+ procedure uses Ferrous Iron
Reagent Powder containing 1,10-Phenanthroline as an indicator. Total iron determination or
analysis uses FerroVer Iron Reagent. FerroVer Iron Reagent contains 1,10-Phenanthroline,
combined with a reducing agent, to convert all but the most resistant forms of iron present in the
sample to Fe2+.
The FerroZine Method for total iron is more than twice as sensitive as the 1,10-Phenanthroline
Method. Researchers at Hach have patented a process to manufacture high purity FerroZine Iron
Reagent, ideal for iron measurement, in economical quantities. FerroZine is highly specific for iron,
forms an intensely-colored stable complex and performs in the pH range of 3–7.5. The FerroZine
Method requires boiling to dissolve rust.
The TPTZ Method for total iron has the advantages of simplicity, sensitivity and freedom from
common interferences. Iron in the sample, including precipitated or suspended iron such as rust, is
converted to Fe2+ by a reducing agent. A highly colored Fe2+-TPTZ complex is formed.
Hach Methods also include a high-range titration procedure utilizing sulfosalicylic acid as the
indicator and EDTA as the titrant.
Chemical reactions
1,10-Phenanthroline method
1,10-Phenanthroline, contained in Ferrous Iron Reagent Powder, reacts with Fe2+ to form a
characteristic orange-colored complex. The intensity of color development is directly proportional
to the amount of Fe2+ in the sample. Total iron also can be determined with FerroVer Iron Reagent.
(When Environmental Protection Agency reporting is necessary, digestion of the sample is
also required)
Iron
Page 1584
Iron
N
N
2+
3 + Fe N Fe N
N N N
N
Figure 67 Chemical reaction for 1,10-Phenanthroline method
FerroZine method
Very low concentrations of iron can be determined using an ultra-sensitive iron indicator, FerroZine
Iron Reagent, 3-(2-pyridyl)-5, 6-bis (4-phensylsulfonic acid)-1, 2, 4-triazine, monosodium salt.
FerroZine Iron Reagent also can be used to analyze samples containing magnetite (black iron
oxide) or ferrites. The test is performed by adding a solution of FerroZine Iron Reagent to the water
sample. The sample is thereby buffered to a pH of 3.5 and a purple-colored complex directly
proportional to the iron concentration is formed. A reducing agent is included to convert any Fe3+
to Fe2+(which forms the colored complex).
Where: SO3H
N N
= SO3¯ Na+
N N N
FerroZine SO3H
N
N
N N Fe N
3 SO3¯ Na++ Fe2+ N
N N N N
Figure 68 Chemical reaction for FerroZine method
Iron
Page 1585
Iron
Titration method
The Titration Method is intended for high iron concentrations, such as oil-field water
determinations. In this method the iron present in the sample is oxidized to Fe3+ by an oxidizing
agent. The Fe3+ is then detected with sulfosalicylic acid, which forms a wine red complex with
Fe3+. The solution is titrated with TitraVer (EDTA) to a colorless to yellow end point. A buffer is
added to stabilize the Fe3+
Sulfosaliclylic acid SO3¯

OH
OH
C SO3¯
O O
CO2 H + Fe 3+
HO C + 6H+
O Fe O
HO3 S O O
C OH
SO3¯
Figure 69 Titration method
Iron
Page 1586
Iron
TPTZ method
TPTZ, 2,4,6-tripyridyl-s-triazine, reacts with Fe2+ to form a deep blue-purple color. Reducing
agents are added to convert iron in the sample to the Fe2+ form. TPTZ, reducing agents and pH
buffers are combined in one simple reagent— TPTZ Iron Reagent Powder Pillows.
TPTZ
N
N N
N N N
+ Fe2+ Fe
N N
2
N N
N N N
N N N
N N
Figure 70 Chemical reaction for TPTZ method
Iron
Page 1587
Langelier and Aggressive indices
Langelier and Aggressive

Indices
Method 8073
Langelier saturation index

The Langelier Saturation Index (LI), a measure of a solution’s ability to dissolve or deposit calcium
carbonate, is often used as an indicator of the corrosivity of water. The index is not related directly
to corrosion, but is related to the deposition of a calcium carbonate film or scale; this covering can
insulate pipes, boilers and other components of a system from contact with water. When no
protective scale is formed, water is considered to be aggressive and corrosion can occur. Highly
corrosive water can cause system failures or result in health problems because of dissolved lead
and other heavy metals. An excess of scale can also damage water systems, necessitating repair
or replacement.
In developing the LI, Langelier derived an equation for the pH at which water is saturated with
calcium carbonate (pHs). This equation is based on the equilibrium expressions for calcium
carbonate solubility and bicarbonate dissociation. To approximate actual conditions more closely,
pHs calculations were modified to include the effects of temperature and ionic strength.
The Langelier Index is defined as the difference between actual pH (measured) and calculated
pHs. The magnitude and sign of the LI value show water’s tendency to form or dissolve scale and
thus to inhibit or encourage corrosion.
Although information obtained from the LI is not quantitative, it can be useful in estimating water
treatment requirements for low pressure boilers, cooling towers and water treatment plants, as
well as serving as a general indicator of the corrosivity of water.
Parameter measurement
The Langelier Saturation Index can be calculated easily by using Hach products to determine the
pH of calcium carbonate saturation (pH) and the actual pH of a solution. Using a simple formula,
the pH is derived from the values for calcium hardness, total alkalinity at pH 4.5, temperature and
total filterable residue (total dissolved solids). All of these procedures are included in this manual.
Temperature:
Temperature can be measured in degrees celsius with a laboratory thermometer
(Catalog. Number. 566-01). If only a Fahrenheit thermometer is available, conversion to degrees
Celsius will be necessary.
[°C = 5/9 x (°F – 32)].
Calculation
After the preceding parameters have been determined, calculate the pH from the following
formula:
pH s = A + B – C – D
Where:
Constant A takes into account the effect of temperature. It is found by selecting the value from the
Water temperature table that corresponds to the measured temperature in degrees Celsius.
Constant B is a correction for the ionic strength of the sample. It is determined using the TDS table
by taking the value that corresponds to the measured total filterable residue or the estimated total
dissolved solids (TDS).
Langelier and Aggressive Indices

Page 1588
Value C is obtained from the Hardness or alkalinity table by reading the value corresponding to the
calcium hardness (in mg/L CaCO3) of the sample.
Value D is obtained from the Hardness or alkalinity table by reading the measured value for total
alkalinity (in mg/L CaCO3) of the sample.
The Langelier Saturation Index is the difference between the actual pH of the solution and pHs
calculated above.
LI = pH – pH
actual s
Interpretation
The LI is a gauge of whether a water will precipitate or dissolve calcium carbonate. If the pHs is
equal to the actual pH, the water is considered “balanced”. This means that calcium carbonate will
not be dissolved or precipitated. If the pHs is less than the actual pH (the LI is a positive number),
the water will tend to deposit calcium carbonate and is scale-forming (nonaggressive). If the pHs is
greater than the actual pH (the LI is a negative number), the water is not saturated and will
dissolve calcium carbonate (aggressive). In summary:
pHS = pHactual, water is balanced
pHS < pHactual, LI = positive number, water is scale forming (nonaggressive)
pHS > pHactual, LI = negative number, water is not scale forming (aggressive)
It is important to remember that the LI value is not a quantitative measure of calcium carbonate
saturation or corrosion.
Because the protective scale formation is dependent on pH, bicarbonate ion, calcium carbonate,
dissolved solids and temperature; each may affect the water’s corrosive tendencies independently.
Soft, low-alkalinity waters with either low or excessively high pH are corrosive, even though this
may not be predicted by the LI. This is because insufficient amounts of calcium carbonate and
alkalinity are available to form a protective scale.
Waters with high pH values and sufficient hardness and alkalinity may also be corrosive, even if
the LI predicts the opposite. This is the result of calcium and magnesium complexes that cannot
actively participate in the scale forming process. Analytical procedures do not distinguish between
these complexes and available calcium and magnesium; therefore, the LI value is not accurate in
such situations.
Corrosive tendencies may also be exhibited by water containing high concentrations of sulfate,
chloride and other ions which interfere with uniform carbonate film formation.
As a result of these and other problems, the LI is useful only for determining the corrosivity of
waters containing more than 40 mg/L of alkalinity, sufficient calcium ion concentration and ranging
between pH 6.5 and 9.5.
Aggressive index
The Aggressive Index (AI), originally developed for monitoring water in asbestos pipe, is
sometimes substituted for the Langelier Index as an indicator of the corrosivity of water. The AI is
derived from the actual pH, calcium hardness and total alkalinity. (Use procedures contained in
this handbook). Where it is applicable, it is simpler and more convenient than the LI. Because the
AI does not include the effects of temperature or dissolved solids, it is less accurate as an
analytical tool than the LI.
Calculation
After obtaining the pH, total alkalinity and calcium hardness, use the following formula to calculate
the AI:
Al = pHactual + C + D

Page 1589
Where:
Value C is obtained from the Hardness or alkalinity table by reading the value corresponding to the
calcium hardness (in mg/L CaCO3) of the sample.
Value D is obtained from the Hardness or alkalinity table by reading the measured value for total
alkalinity (in mg/L CaCO3) of the sample.
Interpretation
As with LI, the AI is not a quantitative measure of corrosion, but is a general indicator of the
tendency for corrosion to occur and as such, should be used with proper reservation. An AI of 12
or above indicates nonaggressive (not corrosive) water. AI values below 10 indicate extremely
aggressive (corrosive) conditions. Values of 10–11.9 suggest that the water is moderately
aggressive. Corrosivity characteristics of water as indicated by the LI and AI are compared in the
Corrosion characteristics table.
Table 430 Water temperature
Water temperature, °C A
0 2.60
4 2.50
8 2.40
12 2.30
16 2.20
20 2.10
25 2.00
30 1.90
40 1.70
50 1.55
60 1.40
70 1.25
80 1.15

Page 1590
Table 431 TDS

TDS, mg/L B
0 9.70
100 9.77
200 9.83
400 9.86
600 9.89
1000 9.90
Table 432 Hardness or alkalinity

Calcium hardness or total alkalinity in mg/L CaCO3 C1 or D2
10 1.00
20 1.30
30 1.48
40 1.60
50 1.70
60 1.78
70 1.84
80 1.90
100 2.00
200 2.30
300 2.48
400 2.60
500 2.70
600 2.78
700 2.84
800 2.90
900 2.95
1000 3.00
1 Factor C is the logarithm (base 10) of the calcium hardness expressed in mg/L
2 Factor D is the logarithm (base 10) of the total alkalinity expressed in mg/L
Table 433 Corrosion characteristics

Corrosive characteristics Langelier index Aggressive index
Highly aggressive < –2.0 < 10.0
Moderately aggressive –2.0 to 0.0 10.00 to 12.0
Nonaggressive >0.0 >12.0

Page 1591
References
1. Langelier, W. F., “The Analytical Control of Anticorrosion Water Treatment” Journal of

American Water Works Association 1936, 28, 1500.
2. Larson, T.E.; Buswell A. M. “Calcium Carbonate Saturation Index and Alkalinity

Interpretations” Journal of American Water Works Association 1942, 34, 1667.
3. Langelier, W. F., “Chemical Equilibria in Water Treatment” Journal of American Water Works
Association 1946, 38, 169.
4. Maguire, J. J.; Polsky, J. W. “Simplified Plant Control Test for Boiler Water Dissolved Solids”
Combustion 1947, May, 35.
5. Betz Handbook of Industrial Water Conditioning 1962, 6th ed., Betz Laboratories: Trevose,
PA.
6. Robinson, R. A.; Stokes, R. H. Electrolyte Solutions 1965, Butterworth & Co. LTD: London.
7. Federal Register 1980, 45 (168), August 27, 1980, p. 57338.

Page 1592
Lead tests explained
Lead
For water and wastewater LeadTrak™ Method
Introduction
Lead is seldom found in ground water in more than trace quantities; it averages around 10 µg/L.
Surface waters contain very low levels of lead because it is precipitated by a variety of substances.
Lead may be found in potable water systems as a result of the corrosion of lead service lines,
lead-based solder joints, or lead-based plumbing fixtures.
Lead and its compounds are poisonous and accumulate in the bone structure when ingested in
amounts exceeding the natural elimination rate of about 300 micrograms per day. Accumulation of
significant amounts of lead in the body may cause severe and permanent brain damage,
convulsions and death. Environmental concern with lead poisoning has resulted in a national
program to reduce the concentration of lead in consumer products.
Chemical reactions
LeadTrak method
The LeadTrak™ Method for determining soluble lead as Pb2+ in potable water first involves the
acidification of the sample to keep all lead ions soluble and to prevent the lead from being lost by
precipitation or absorption on the sample container walls. Complexing and buffering agents are
then added to the sample to modify the lead ions into a form which allows them to be retained by
the cellulose medium in the concentrator column. Other competing ions, such as iron, copper and
zinc, pass through the column and are eliminated.
The lead ions are then eluted from the concentrator column with a nitric acid solution. The nitric
acid eluant is neutralized and reacted with meso-tetra (4-N-methylpyridyl) porphine tetratosylate to
form a faintly colored complex. The absorbance of the complex is measured at 477 nm. EDTA is
then added to the complex. The EDTA complexes with the lead and removes the lead from the
porphine complex. The absorbance of the sample is read again and the lead concentration is
determined by the difference between the two readings.
Lead
Page 1593
Manganese tests explained
Manganese
Periodate Oxidation and PAN Methods
Introduction
Manganese is present in ground waters primarily as the divalent ion (Mn2+), due to the lack of
subsurface oxygen. Surface waters may contain combinations of manganese in various oxidation
states as soluble complexes, or as suspended particles.
The occurrence of manganese in public water supplies presents more of an economic problem
than a potential health hazard. Manganese causes dark stains in laundry and on plumbing fixtures,
tends to deposit in water lines, and imparts an objectionable taste to beverages such as coffee and
tea. Manganese levels in natural waters rarely exceed 1 mg/L, but levels of 0.1 mg/L are sufficient
to cause the taste and staining problems. The recommended allowable manganese level in public
water supplies is 0.05 mg/L.
Two methods for manganese determination are used in test procedures. The Periodate Oxidation
Method gives a simple, rapid test for high levels of manganese. The 1-(2-Pyridylazo)-2-Napthol
(PAN) Method is a sensitive, rapid procedure for low levels of manganese.
Chemical reactions
Periodate oxidation method
Manganese is oxidized to permanganate using periodate in a slightly acidified water sample. No
indicator is necessary. Intensity of the purple color of the permanganate ion is a direct indication of
the amount of manganese present in the sample.
2+ – – – +
3H2 O + 2Mn + 5IO 4 → 2MnO 4 + 5IO 3 + 6H
PAN method
The PAN method employs an Alkaline-Cyanide Reagent. PAN Indicator is added and forms an
orange-red colored complex with the manganese ion.
N O
N
2 + Mn2+ Mn + 2H+
N N N N
O N
OH
N
Figure 71 Chemical reaction for PAN method
Manganese
Page 1594
Mercury cold vapor tests explained
Mercury, Cold Vapor

Introduction
Mercury is sometimes dumped into rivers and lakes by industry. Farmers have also used mercury
compounds to prevent fungi attack on seeds. In nature, bacteria change elemental mercury to
organic mercury compounds that are very toxic to all living organisms. These compounds
bioaccumulate in the food chain and can eventually reach humans. At high enough levels, mercury
causes nerve damage, mutations and death. Because mercury is so toxic, environmental
monitoring is necessary— especially at sources of industrial discharge.
Chemical reactions
The sample is digested to convert all forms of mercury to mercuric ions (Hg2+) and to destroy
interfering substances that may be present. The sample is then treated with a reducing agent to
convert the mercuric ions to elemental mercury (Hg). The elemental mercury is volatilized from the
sample in a semi-closed system by bubbling air through the sample.
The airstream containing the mercury vapor is swept through an absorber column that contains
hypochlorous acid and hypochlorite ions. The vaporized mercury ions react in the column and
form mercuric chloride (HgCl2). The mercuric chloride is then eluted off the column with an acid
solution.
The eluate is treated with HgEx™ Reagent 3 to destroy the excess hypochlorous acid and
hypochlorite, and to make the solution alkaline. A sensitive indicator (HgEx 4) forms a complex
with the mercuric ions, and HgEx 5 maximizes the mercury-indicator reaction, thus increasing the
sensitivity of the test. The spectrophotometer is zeroed on this solution at the absorbance peak
(412 nm) of the unreacted indicator. HgEx 6 is added to break the mercury-indicator bond. The
analyst then measures the absorbance increase of unreacted indicator. This increase is
proportional to the amount of mercury in the sample.
Mercury, Cold Vapor

Page 1596
Molybdenum, molybdate tests explained
Molybdenum, Molybdate
Mercaptoacetic Acid and Ternary Complex
For water
Methods
Introduction
Molybdenum (molybdate) salts are commonly used as corrosion inhibitors in cooling water
systems. There are numerous procedures for determining molybdenum as molybdate (MoO42–) in
water. The Mercaptoacetic Acid Method is one of the most frequently referenced methods for
determining molybdenum. The Hach procedure improves and simplifies this time-proven
procedure.
Chemical reactions
Mercaptoacetic acid
The MolyVer™ (Mercaptoacetic Acid) Method utilizes three reagent powders. First, a MolyVer 1
Reagent Powder Pillow is added. MolyVer 1 contains a buffer to control the pH, in addition to a
chelating agent to mask interferences.
Low test results can be caused from reduction of Mo6+ to Mo5+, because the test is specific for
Mo6+. MolyVer 2 Reagent Powder is added to prevent reduction of the Mo6+ ion. Finally,
mercaptoacetic acid, contained in MolyVer 3 Reagent Powder, is added. The reaction of MolyVer 3
with Mo6+ results in formation of a characteristic yellow color. Development of the yellow color
follows Beer’s Law over the range of the test.
MoO42¯ + 2HSCH2 COOH + 2H+

Molybdate Mercaptoacetic acid
CH2 S Mo S CH2 + 2H2O
HO C O O O C OH
Figure 72 Chemical reaction for the Mercaptoacetic Acid method
Page 1597
Ternary complex method

The ternary complex method is a two-reagent method for molybdenum in the
0–3 mg/L range. First, the molybdenum-containing sample reacts with Molybdenum 1 Reagent,
which contains colorimetric indicator, pH buffer, and reducing agent. The reducing agent
counteracts interference by iron, a common contaminant in boiler and cooling water samples. The
indicator forms a colored, binary complex with molybdenum. Depending on molybdenum levels,
binary-complex color will range from pale yellow to rusty orange.
Second, Molybdenum 2 Reagent will combine with the binary complex to form an intensely
colored, blue, ternary (three-part) complex directly proportional to sample molybdenum
concentration. The eye perceives the color as ranging from yellow to green because the blue color
is superimposed over a yellow background. These colors correspond to 0 mg/L Mo (yellow) up to 3
mg/L Mo (dark green).
Molybdenum 2 Indicator-Molybdate-
Indicator-Molybdate reagent solution Molybdenum 2
MoO4 2¯ + Molybdenum 1 reagent
binary complex reagent Ternary
complex
Sample: Powder Pillow:
Contains · ph Buffer Weak color: Yellow to green
molybdate- · Reducing agent Yellow to orange color
molybdenum · Colorimetric indicator
Figure 73 Chemical reaction pathway for the Ternary Complex method
Page 1598
Nickel tests explained
Nickel
For water Heptoxime and PAN Methods
Introduction
Nickel is seldom found in natural waters, but often present in industrial wastewater as a direct
product of metal plating baths, and as a corrosion product of stainless steel, nickel or cobalt alloys.
Nickel is considered relatively nontoxic to humans. The toxicity of nickel to aquatic life varies
widely and is influenced by species, pH, synergetic effects, and other factors. Nickel salts at
concentrations between 0.5 and 1.0 mg/L have been shown to be toxic to some plant species.
Two methods for determining nickel, the Heptoxime Method and the PAN Method, are used in
Hach procedures. The well-known heptoxime indicator has been formulated into a dry, stable
powder packaged in pillows for the nickel determination. A second powder pillow is used to
overcome interference from other metals present. The heptoxime forms a yellow complex, which is
extracted into chloroform for measurement.
The PAN procedure is a sensitive method for detecting nickel and cobalt in concentrations less
than 1 mg/L. The method is unique because simultaneous determinations of nickel and cobalt
concentrations can be made on the same sample portion without the need for solvent extraction or
sample preconcentration steps.
Chemical reactions
Heptoxime method
Nickel is analyzed quantitatively through its reaction with Heptoxime to form a yellow-colored
complex, which is then extracted into a chloroform layer to concentrate the color and to enable a
more sensitive colorimetric determination. Chelating agents are added to the sample to overcome
the interferences caused by cobalt, copper and iron.
NOH O N N O
2+
2 + Ni H Ni H + 2H+
NOH
O N N O
Figure 74 Chemical reactions for Heptoxime method
PAN method
The PAN method for nickel is discussed in detail in the PAN method for cobalt, because both
cobalt and nickel can be determined on the same sample.
Nickel
Page 1599
Nitrogen, Ammonia tests explained
Nitrogen, Ammonia
For water, wastewater and seawater Nessler Method and Salicylate Method
Introduction
Ammonia is a product of the microbiological decay of animal and plant protein. It can be directly
reused by plants to produce protein. Ammonia and ammonia compounds are applied directly as
fertilizers.
The presence of ammonia nitrogen in surface water usually indicates domestic pollution. Ammonia
in ground water is normal and is due to microbiological processes. Two methods for determining
ammonia, the Nessler method and the Salicylate method, are used in Hach products and
procedures.
Chemical reactions
Nessler method
In the ammonia test, Nessler Reagent (K2HgI4) reacts with the ammonia present in the sample
(under strongly alkaline conditions) to produce a yellow-colored species. The intensity of the color
is in direct proportion to the ammonia concentration.
2K 2 HgI 4 + NH3 + 3KOH → Hg2 OINH 2 + 7KI + 2H 2 O
Salicylate method
The Salicylate method is a variation of the well-known Phenate Method, but it has an advantage of
being free from mercury salts and phenol. This method is most useful for low range ammonia
nitrogen determinations. Although the procedure involves multiple reactions before a final green
color is developed, all reagents are contained in convenient powder pillows (Salicylate Reagent
Powder Pillows and Alkaline Cyanurate Powder Pillows) or a combination of powder pillows and
TNT vials.
Ammonia compounds are initially combined with hypochlorite to form monochloramine (1), which
then reacts with salicylate to form 5-aminosalicylate (2).
(2) NH2Cl + OH H 2N OH + Cl¯
COO¯ COO¯
Oxidation of 5-aminosalicylate is carried out in the presence of a catalyst, nitroprusside or
Fe(CN)5NO2– (also called nitroferricyanide), which results in the formation of indosalicylate, a
blue-colored compound. The blue color is masked by the yellow color (from excess nitroprusside)
causing a green-colored solution. The intensity of the color is directly proportional to the ammonia
concentration in the sample.
O¯ N O
COO¯ COO¯
Figure 75 Chemical structure of Indosalicylate
Nitrogen, Ammonia
Page 1600
Nitrogen, Kjeldahl tests explained
Nitrogen, Kjeldahl
For water and wastewater Peroxide Digestion Method
Introduction
The test for Kjeldahl nitrogen, also referred to as crude protein, is used to determine ammonia and
organic nitrogen present in a sample. Only small fractions of nitrite and nitrate nitrogen are
included in the test. A preliminary digestion is used to oxidize carbon compounds to carbon
dioxide, and to convert organic forms of any nitrogen present (amino acids, proteins, peptides) to
ammonia. The traditional digestion uses sulfuric acid and various combinations of metallic
catalysts and salts. Digestion of at least 2 hours is followed by addition of sodium hydroxide to the
digest and then distillation of the ammonia into a boric acid or buffer solution. Ammonia in the
distillate is measured with back titration or nesslerization. This procedure requires several hours
for reagent preparation, digestion, distillation and final measurement.
The Hach Digesdahl Digestion Apparatus and the Peroxide Digestion Method allow completion of
the Kjeldahl test within 15 minutes or less, depending on the nature of the sample. First, the
sample is charred in concentrated sulfuric acid. Fifty percent hydrogen peroxide is fed into the
reaction mixture, where it oxidizes organic carbonaceous matter and converts organic nitrogen
into ammonium bisulfate. For example, the reaction with glycine, a simple amino acid is:
NH2CH2COOH + 2H2O2 + H2SO4 → NH4HSO4 + CO2 + 2H2O
Glycine
Digestion apparatus
The Digesdahl digestion apparatus includes a fractionating column. Hydrogen peroxide, added
slowly, trickles into the reaction mixture in the flask below. The temperature of the reaction is
maintained near the boiling point of sulfuric acid (300 °C, 572 °F). Vapors from the reaction rise to
the column where SO2 and water vapors are drawn off by an aspirator. Hydrogen peroxide vapors
condense in the column and return to the reaction mixture.
It should be noted that no metal catalysts or salts are used in digestion. The digest is suitable for
nesslerization for final measurement without an intermediate distillation step. The digest is also
suitable for mineral analysis of Ca, Mg, Mn, K, P and Zn. See Nitrogen, Ammonia for more
information about the Nessler method.
Nitrogen, Kjeldahl
Page 1601
Nitrogen, Nitrate tests explained
Nitrogen, Nitrate
For water and wastewater Cadmium Reduction Method
Introduction
Nitrate represents the most completely oxidized state of nitrogen, and is commonly found in water.
Nitrate-forming bacteria convert nitrites into nitrates under aerobic conditions; lightning converts
large amounts of atmospheric nitrogen (N2) directly to nitrates. Many granular commercial
fertilizers contain nitrogen in the form of nitrates.
High levels of nitrate in water may indicate biological wastes in the final stages of stabilization, or
run-off from heavily fertilized fields. Nitrate-rich effluents discharged into receiving waters can
degrade water quality by encouraging excessive growth of algae. Drinking waters containing
excessive amounts of nitrates can cause infant methemoglobinemia (blue babies). For this
reason, a maximum concentration level in drinking water has been established by the USEPA in
accordance with the Safe Drinking Water Act.
Two methods of analysis are used in the high range tests. The NitraVer™5 high range method is a
modification of the Cadmium Reduction Method, using gentisic acid in place of 1-naphthylamine.
All the necessary reagents have been combined into a single stable powder.
The Chromotropic Acid high range nitrate method involves the reaction of nitrate in a strong acid
medium with chromotropic acid. The final reaction mixture is contained in the screw-capped
Test ’N Tube™ vial.
The low range nitrate test is also a modification of the Cadmium Reduction Method, and uses a
very sensitive chromotropic acid indicator. Both methods register nitrate and nitrite nitrogen.
Chemical reactions
High range—NitraVer 5
In the NitraVer 5 high range test, cadmium metal is used to reduce nitrates (NO3–) to nitrites
(NO2–) (reaction 1). Next, the nitrite ions react in an acidic medium with sulfanilic acid to form an
intermediate diazonium salt (reaction 2) which, when coupled with gentisic acid (reaction 3), forms
an amber-colored compound. Color intensity of the compound is directly proportional to the nitrate
concentration of the water sample.
+
(2) NO2¯ + H2N SO3H + 2H+ HO3S N N + 2H2O
Sufanilic Acid Diazonium Salt
Nitrogen, Nitrate
Page 1602
Nitrogen, Nitrate
High range—Chromotropic acid method

In the Chromotropic Acid test, sample is added to a Test ‘N Tube™ vial containing sulfuric acid.
This sample/sulfuric acid mixture is used to zero the spectrophotometer. Chromotropic acid is then
added as NitraVer X Reagent B. Two moles of nitrate react with one mole of chromotropic acid to
form a yellow reaction product, which exhibits maximum absorbance at 410 nm.
OH OH OH OH
2ON NO2
H+
(2) NO3¯ + + 2H+
SO3 SO3
SO3 SO3
Nitrate Chromotropic acid
Yellow color
Low range
In the low range nitrate test, cadmium metal is used to reduce the nitrates to nitrites. The cadmium
is provided in NitraVer 6 Reagent Powder Pillows. Nitrite ions react with sulfanilic acid to produce
an intermediate diazonium salt, as in the high range test. The diazonium salt then forms a red-
orange complex with chromotropic acid. The color intensity is in direct proportion to the nitrate
concentration in the sample (reaction 4). In the low range test the sulfanilic acid and chromotropic
acid are contained in NitriVer 3 Reagent Powder Pillows.
OH OH
+
(3) HO3S N N + COOH HO3S N N COOH + H+
OH OH
Diazonium salt Gentisic acid Amber colored species
OH OH
OH OH
+
(4) HO3S N N + HO3S N N + H+
HO3S SO3H HO3S SO3H
Diazonium Salt Chromotropic Acid Red-Orange color
Nitrogen, Nitrate
Page 1603
Nitrogen, Nitrite tests explained
Nitrogen, Nitrite
For water and wastewater Ferrous Sulfate and Diazotization Methods
Introduction
Nitrite nitrogen occurs as an intermediate stage in the biological decomposition of compounds
containing organic nitrogen. Nitrite-forming bacteria convert ammonia under aerobic conditions to
nitrites. The bacterial reduction of nitrates can also produce nitrites under anaerobic conditions.
Nitrites are often used as corrosion inhibitors in industrial process water and cooling towers; the
food industry uses nitrite compounds as preservatives.
Because nitrites readily oxidize to nitrates, they are not often found in surface waters. The
presence of large quantities of nitrites indicates partially decomposed organic wastes in the water
being tested. Drinking water concentrations seldom exceed 0.1 mg/L of nitrite.
The high range nitrite test is a modification of the classical brown ring test for nitrate using ferrous
sulfate. By controlling the sample pH, the nitrite present is reduced to nitrous oxide, which reacts
with the indicator to form a greenish-brown color. Nitrates are not registered in the test. All
necessary reagents have been combined in a single powder pillow form called NitriVer 2 Nitrite
Reagent Powder. A special agent helps prevent color formation or precipitation of common
interfering ions.
The low range nitrite test uses chromotropic acid and sulfanilic acid as the indicator. The indicator
and a buffer are combined in a single powder NitriVer 3 Nitrite Reagent. The test is sensitive to low
nitrite concentrations.
Chemical reactions
High range, ferrous sulfate method
In an acidic medium ferrous sulfate reduces nitrogen in nitrite (NO2–) to form nitrous oxide (NO).
Ferrous ions combine with the nitrous oxide to form a brown-colored complex ion, the color
intensity of which is in direct proportion to the nitrite present in the water sample. Color
development follows Beer’s Law.
2Fe2+ + 4H+ + 2NO2¯ → 2Fe3+ + 2NO + 2H2O
NO + FeSO4 → FeSO4 · NO
Low range, diazotization method

In the low range nitrite test, nitrite ions react with sulfanilic acid to form an intermediate diazonium
salt. This reacts with chromotropic acid to produce a red-orange complex directly proportional to
the amount of nitrite present. A measurement of the color intensity will provide an accurate
determination of the nitrite concentration in the water sample.
Nitrogen, Nitrite
Page 1604
Nitrogen, Nitrite
+
+
NO2¯ + HO3S NH2 + 2H HO3S N N + 2H2O
Sulfanilic acid
OH OH OH OH
+ N N SO3H + H+
HO3S N N +
HO3S SO3H HO3S SO3H
Chromotropic acid
Figure 76 Chemical reaction for Low Range Diazotization method
Nitrogen, Nitrite
Page 1605
Nitrogen, Total tests explained
Nitrogen, Total
Titanium Chloride and Persulfate Digestion
Methods
Introduction
Total nitrogen methods measure nitrogen loads on influent streams, at intermediate stages of
water treatment for sludge, and on effluent to gauge overall treatment plant efficiency. Assessing
nitrogen levels allows process monitoring, adjustment and nitrogen reduction efficiency throughout
the treatment.
Titanium chloride reduction method (Total Inorganic Nitrogen)
Titanium (III) ions reduce nitrate and nitrite to ammonia in a basic environment. After centrifugation
to remove solids, the ammonia is combined with chlorine to form monochloramine.
Monochloramine reacts with salicylate to form 5-aminosalicylate, a green solution, as in the
salicylate method in Ammonia Nitrogen (see Nitrogen, Ammonia).
Persulfate digestion method (Total Nitrogen)
An alkaline persulfate digestion converts all forms of nitrogen to nitrate. Sodium metabisulfate is
added after the digestion to eliminate interferences from halogen oxides. Under strongly acidic
conditions, nitrate reacts with chromotropic acid to nitrate the biphenyl rings at several locations,
forming several nitrated products (Figure 77). The nitrated products that form are measured
at 410 nm.
HO3S SO3H
OH OH
Figure 77 Chromatropic acid structure, including available reaction sites for nitrate
Nitrogen, Total
Page 1606
Total Organic Carbon tests explained

For water and wastewater Direct Method
Introduction
Total Organic Carbon (TOC) testing is important in drinking water treatment as an indicator of
potential disinfection by-product formation. In wastewater, TOC is valuable as a surrogate for COD
testing and has applications in domestic wastewater pre-treatment standards, effluent discharge
limitations, and industrial process waters.
The colorimetric TOC test measures the total amount of non-volatile organic carbon in a sample.
The method is based on controlled digestion/diffusion in a sealed glass assembly*. Sample carbon
is oxidized to carbon dioxide by persulfate oxidation. The carbon dioxide diffuses into a colored pH
indicator solution where it is converted into carbonic acid. The resulting color change is
proportional to the concentration of carbon present in the sample.
Chemical reactions
Inorganic carbon is removed from the sample by adjusting the sample to pH 2 with a buffer, and
stirring vigorously for 10 minutes:
TOC = Total Carbon – Inorganic Carbon
A suitable volume of treated sample and potassium persulfate is added to a 16-mm screw top
digestion vial containing Acid Digestion Solution Reagent. A 9-mm sealed glass ampule containing
the TOC Indicator Solution is opened and placed inside the digestion vial. The whole assembly is
then sealed with a screw cap and digested at 103–105° C (217–221 °F) for 2 hours.
In the presence of acidic persulfate and with increased pressure and elevated temperature, the
sample’s organic carbon is oxidized to carbon dioxide. For example, in the persulfate digestion of
a sample that contains formate, the chemical reaction is:
S2O82– + HCOO– → HSO4– + SO42– + CO2
The evolved CO2 then diffuses and is trapped in an aqueous solution containing a pH indicator.
The absorbed CO2 forms carbonic acid according to:
CO2 + H2O → 2H+ + CO32–
The pH indicator (prior to CO2 absorption) is in its deprotonated, or basic, form (D–). As the
absorbed CO2 level increases, the hydrogen ion level will also increase, resulting in an increase of
the protonated form of the indicator:
D– (Color A) + H+ → DH (Color B)
The concentration of the carbon in the sample is proportional to the color change, either the
change in Color A (ΔD–), or the change Color B (ΔDH) or the sum (ΔD– + ΔDH).
* U.S. Patent 6,368,870

Page 1607
Oxygen Demand, Mn III tests explained
Oxygen Demand, Chemical, Mn III

Introduction
Chemical oxygen demand (COD) is defined as “a measure of the oxygen equivalent of the organic
matter content of a sample that is susceptible to oxidation by a strong chemical oxidant.*” Trivalent
manganese (Mn III) is a strong, non-carcinogenic chemical oxidant that changes quantitatively
from purple to faint pink when it reacts with organic matter. Manganese III COD results are
measured colorimetrically, and the color intensity is inversely proportional to the amount of COD in
the sample.
The digestion time is 60 minutes, but can be extended when samples are difficult to completely
oxidize. The reagent typically has an oxidation efficiency of about 80% for standards prepared
from potassium acid phthalate and domestic wastewater samples. No oxygen demand test will
oxidize all organic compounds with 100% efficiency. With non-typical samples, standards can be
prepared from other reference materials. Studies have shown that the Mn III COD procedure
correlates very well to biochemical oxygen demand (BOD) and dichromate COD tests.
Many COD reagents contain mercury, chromium and silver. The absence of these materials in the
Mn III COD Reagent significantly minimizes the disposal cost and reduces exposure of the analyst
to hazardous compounds.
Inorganic materials may interfere with the Mn III COD Reagent. Chloride is the most common
interference and is removed by sample pretreatment with the Chloride Removal Cartridge.
Ammonia interferes with the test when present with chloride. The interference is severe at high
ammonia and chloride concentrations.
Chemical reactions
Trivalent manganese oxidizes organic materials in the sample to CO2 and H2O. In the process,
manganese III is reduced to manganese II. The reaction occurring in the Mn III COD Reagent Vial
is represented by the equation below:
2 KC8H5O4 + 30 Mn2(SO4)3 + 24 H2O →16 CO2 + 60 MnSO4 + 28 H2SO4 + 2 KHSO4
Chemical oxygen demand results are usually expressed by the amount of oxygen consumed
during the oxidation of organic matter.
When oxygen is used as the primary oxidant in the oxidation of potassium acid phthalate, the
equation below describes the reaction:
KC8H5O4 + 7.5 O2 → 8 CO2 + 2 H2O + KOH
Seven and one-half molecules of oxygen (O2) consume one molecule of potassium acid phthalate
(KHP). On a weight basis, the theoretical oxygen demand for KHP is 1.175 mg O2 per mg KHP.
The interference from chloride is minimized by sample pre-treatment with the Chloride Removal
Cartridge (CRC). The CRC contains a proprietary reagent to remove chloride from the sample
solution. The flow rate through the CRC has been optimized for chloride removal, while at the
same time minimizing any effect the CRC might have on other sample components.
* APHA Standard Methods for the Examination of Water and Wastewater, 19th ed., 1995

Page 1608
Suspended solids, which may contain oxidizable organic compounds, are filtered out of the
sample with a glass fiber filter located in the upper part of the Chloride Removal Cartridge. After
the sample has been filtered into a Mn III COD Reagent Vial, the glass fiber filter, along with any
suspended solids present, is transferred into the COD Reagent Vial. The glass fiber filter is binder
free and has no oxygen demand.
Comparability of the Mn III COD to other tests
For samples from a specific source, the Mn III COD results can be related empirically to BOD,
dichromate COD, organic carbon, or organic matter. The test is useful for monitoring and control
after correlation has been established. The test can also be used to estimate dilutions for the five
day BOD test. This will ensure reliable BOD data for pre-treatment and compliance monitoring. For
samples with constant chloride concentrations, reliable correlations can be developed without the
chloride removal pre-treatment.
Test oxidation efficiency
Different test methods may oxidize sample components with different efficiencies. The Mn III COD
Reagent will oxidize KHP standards with about 80% efficiency. Many wastewater samples are also
oxidized with 80% efficiency.
For example, an ASTM Wastewater Reference Sample was analyzed for COD using both the
dichromate and Mn III COD. The dichromate COD result was 1018 mg/L and Mn III COD result
was 1008 mg/L. These test results are comparable despite different oxidation efficiencies because
the instrument calibration is based on KHP.
Correlation of test results
To demonstrate how to correlate the results between the two different tests, a glutamic acid
standard was prepared to contain a theoretical chemical oxygen demand of 500 mg/L. The
dichromate COD result was 506 mg/L and the Mn III COD result was 459 mg/L. To correlate the
Mn III COD results with the dichromate COD, a correlation factor is determined.
Cr COD 506 mg/L
------------------------------ = ------------------------- = 1.102 (Correlation Factor)
Mn III COD 459 mg/L
Mn III COD, mg/L × 1.102 = Dichromate COD, mg/L
Calibrations based on reference materials other than KHP

It may be desirable for COD results to closely match the theoretical demand of the sample.
Occasionally, samples contain a major component that is incompletely oxidized. In this situation,
calibration standards can be prepared from known values of that sample component. In the
example above, calibration standards would be prepared from glutamic acid.

Page 1609
COD explained

Reactor Digestion Using Potassium
For wastewater
Dichromate Method
Introduction
The Dichromate Chemical Oxygen Demand (COD) test measures the oxygen equivalent of the
amount of organic matter oxidizable by potassium dichromate in a 50% sulfuric acid solution.
Generally, a silver compound is added as a catalyst to promote the oxidation of certain classes of
organics, and a mercuric compound may be added to reduce interference from the oxidation of
chloride ions by the dichromate. End products are carbon dioxide, water, and various states of the
chromium ion.
After the oxidation step is completed, the amount of dichromate consumed is determined
titrimetrically or colorimetrically. Either the amount of reduced chromium (chromic ion), or the
amount of unreacted dichromate, can be measured. If the latter method is chosen, the analyst
must know the precise amount of dichromate added.
Chemical reactions
In the oxidation of organic materials by dichromate in sulfuric acid, most of the carbon is converted
to carbon dioxide while any hydrogen present in the organic compound is converted to water.
Other elements also may be oxidized.
Chemical oxygen demand results are usually expressed by the amount of oxygen consumed
during the oxidation of organic matter. When oxygen is used as the primary oxidant in the
oxidation of potassium acid pthalate, the equation below describes the reaction.
KC 8 H 5 O 4 + 7.5O 2 → 8CO 2 + 2H 2 O + KOH
Seven and one-half molecules of oxygen (O2) consume one molecule of potassium acid pthalate
(KHP). On a weight basis, the theoretical oxygen demand for KHP is 1.175 mg O2 per mg KHP.
There are two basic methods, titrimetric and colorimetric, for determining the amount of chromium
in a particular valence state. There are several variations of each method.
When titration is used in the measurement process, the amount of Cr6+ left is determined. It is
done in one of two ways; in both cases, the precise initial amount of Cr6+ ion must be known. This
is necessary because one must be able to subtract the final Cr6+ level from the initial level to yield
the amount that was reduced to Cr3+. This difference is used to calculate the COD. The initial
amount is known either through calculation, because primary standard grade potassium
dichromate is readily available, or by testing the bulk solution before running the individual tests.
The final amount of dichromate is most commonly determined by direct titration using ferrous
ammonium sulfate as the titrant and “ferroin” (1,10-phenanthroline ferrous sulfate) as the indicator.
The Fe2+ in the titrant reacts with the chromic ions:
2+ 6+ 3+ 3+
3Fe + Cr → 3Fe + Cr
1,10-phenanthroline forms an intense color with Fe2+ but no color with Fe3+. When reduction of
Cr6+ to Cr3+ is complete, Fe2+ reacts to form the ferroin complex and the solution color changes
sharply from greenish-blue to orange-brown, signaling the end point. The end point also can be
detected potentiometrically.

Page 1610
The colorimetric determination has several advantages over titration. Colorimetric determination is
quicker and easier to run, and does not require additional reagents. In the Reactor Digestion
Method, the digestion vials can be checked during the digestion process while they are hot to
determine when no further oxidation is taking place, resulting in a shorter digestion time. When
using the Reactor Digestion Method the spectrophotometer is set at 420 nm or 365 nm for the low
ranges, and 620 nm for the high ranges. Low range measurement determines the remaining
yellow Cr6+. High range measurement determines the amount of green Cr3+ produced.

Page 1611
Dissolved oxygen tests explained
Oxygen, Dissolved
Azide Modification of Winkler Method and
For water, wastewater, and seawater
Luminescence Measurement (LDO) Method
Introduction
The dissolved oxygen test is one of the most important analyses in determining the quality of
natural waters. The effect of oxidation of wastes on streams, the suitability of water for fish and
other organisms, and the progress of self-purification can all be measured or estimated from the
dissolved oxygen content. In aerobic sewage treatment units, the minimum objectionable odor
potential, maximum treatment efficiency and stabilization of wastewater are dependent on
maintenance of adequate dissolved oxygen. Frequent dissolved oxygen measurement is essential
for adequate process control.
Dissolved oxygen is essential for the survival of aquatic plant and animal life. Generally, 4–5 mg/L
of dissolved oxygen content is a borderline concentration for an extended time period. For
adequate game fish population, the dissolved oxygen content should be in the 8–15 mg/L range.
Dissolved oxygen concentration varies with water depth, sludge deposits, temperature, clarity and
flow rate. Thus a single water sample is rarely representative of the over-all condition of a body of
water.
Chemical reactions
Azide modification of Winkler method
In the analysis, Mn2+ (manganous ion) reacts with the dissolved oxygen present in the alkaline
solution to form a Mn4+ oxide hydroxide floc (1). Azide is added at this time to suppress
interference from any nitrate present (which would react with the iodide). The solution is then
acidified, and the manganese floc is reduced by iodide to produce Mn2+ and free iodine as I3–(I2 +
I – in solution, see equation 2). The iodine gives the clear supernate a brown color. Phenylarsine
oxide (PAO) or thiosulfate is then used to titrate the iodine to a colorless end point (3). (Starch
indicator can be added to enhance the determination of the end point by producing a color change
from dark blue to colorless.) The dissolved oxygen of the sample is then calculated from the
quantity of titrant used.
(2) MnO(OH)2 + 6I¯ + 6H+ Mn2+ + 2I 3̄ + 3H2O

OH
(3) 2H2O + I3¯ As = O 2HI + I¯ + As = O
OH
Oxygen, Dissolved
Page 1612
Oxygen, Dissolved
Luminescence measurement of dissolved oxygen (LDO)

The luminescence-based sensor procedure measures the light emission characteristics from a
luminescence-based reaction at the sensor-water interface. A light emitting diode (LED) provides
incident light required to excite the luminophore substrate. In the presence of dissolved oxygen the
reaction is suppressed. The resulting dynamic lifetime of the excited luminophore is evaluated and
equated to dissolved oxygen concentration.
Figure 78 LDO probe
Dissolved oxygen
The Dissolved oxygen saturation in water (mg/L) table lists the mg/L dissolved oxygen in water at
saturation for various temperatures and atmospheric pressures. The table was formulated in a
laboratory using pure water. The values given are only approximations for estimating the oxygen
content of a particular body of surface water
Table 434 Dissolved oxygen saturation in water (mg/L)

Pressure in millimeters and inches Hg
mm
Temp
775 760 750 725 700 675 650 625
inches
°F °C 30.51 29.92 29.53 28.45 27.56 26.57 25.59 24.61

32.0 0 14.9 14.6 14.4 13.9 13.5 12.9 12.5 12.0
33.8 1 14.5 14.2 14.1 13.6 13.1 12.6 12.2 11.7
35.6 2 14.1 13.8 13.7 13.2 12.9 12.3 11.8 11.4
37.4 3 13.8 13.5 13.3 12.9 12.4 12.0 11.5 11.1
39.2 4 13.4 13.1 13.0 12.5 12.1 11.7 11.2 10.8
41.0 5 13.2 12.8 12.6 12.2 11.8 11.4 10.9 10.5
42.8 6 12.7 12.4 12.3 11.9 11.5 11.1 10.7 10.3
44.6 7 12.4 12.1 12.0 11.6 11.2 10.8 10.4 10.0
46.4 8 12.1 11.8 11.7 11.3 10.9 10.5 10.1 9.8
48.2 9 11.8 11.6 11.5 11.1 10.7 10.3 9.9 9.5
50.0 10 11.6 11.3 11.2 10.8 10.4 10.1 9.7 9.3
51.8 11 11.3 11.0 10.9 10.6 10.2 9.8 9.5 9.1
53.6 12 11.1 10.8 10.7 10.3 10.0 9.6 9.2 8.9
55.4 13 10.8 10.5 10.5 10.1 9.8 9.4 9.1 8.7
57.2 14 10.6 10.3 10.2 9.9 9.5 9.2 8.9 8.5
Oxygen, Dissolved
Page 1613
Oxygen, Dissolved
Table 434 Dissolved oxygen saturation in water (mg/L) (continued)

59.0 15 10.4 10.1 10.0 9.7 9.3 9.0 8.7 8.3
60.8 16 10.1 9.9 9.8 9.5 9.1 8.8 8.5 8.1
62.6 17 9.9 9.7 9.6 9.3 9.0 8.6 8.3 8.0
64.4 18 9.7 9.5 9.4 9.1 8.8 8.4 8.1 7.8
66.2 19 9.5 9.3 9.2 8.9 8.6 8.3 8.0 7.6
68.0 20 9.3 9.1 9.1 8.7 8.4 8.1 7.8 7.5
69.8 21 9.2 8.9 8.9 8.6 8.3 8.0 7.7 7.4
71.6 22 9.0 8.7 8.7 8.4 8.1 7.8 7.5 7.2
73.4 23 8.8 8.6 8.5 8.2 8.0 7.7 7.4 7.1
75.2 24 8.7 8.4 8.4 8.1 7.8 7.5 7.2 7.0
77.0 25 8.5 8.3 8.3 8.0 7.7 7.4 7.1 6.8
78.8 26 8.4 8.1 8.1 7.8 7.6 7.3 7.0 6.7
80.6 27 8.2 8.0 8.0 7.7 7.4 7.1 6.9 6.6
82.4 28 8.1 7.8 7.8 7.6 7.3 7.0 6.7 6.5
84.2 29 7.9 7.7 7.7 7.4 7.2 6.9 6.6 6.4
86.0 30 7.8 7.6 7.6 7.3 7.0 6.8 6.5 6.2
87.8 31 7.7 7.4 7.4 7.2 6.9 6.7 6.4 6.1
89.6 32 7.6 7.3 7.3 7.0 6.8 6.6 6.3 6.0
91.4 33 7.4 7.2 7.2 6.9 6.7 6.4 6.2 5.9
93.2 34 7.3 7.1 7.1 6.8 6.6 6.3 6.1 5.8
95.0 35 7.2 7.0 7.0 6.7 6.5 6.2 6.0 5.7
96.8 36 7.1 6.8 6.9 6.6 6.4 6.1 5.9 5.6
98.6 37 7.0 6.7 6.7 6.5 6.3 6.0 5.8 5.6
100.4 38 6.9 6.6 6.6 6.4 6.2 5.9 5.7 5.5
102.2 39 6.8 6.5 6.5 6.3 6.1 5.8 5.6 5.4
104.0 40 6.7 6.4 6.4 6.2 6.0 5.7 5.5 5.3
105.8 41 6.6 6.3 6.3 6.1 5.9 5.6 5.4 5.2
107.6 42 6.5 6.2 6.2 6.0 5.8 5.6 5.3 5.1
109.4 43 6.4 6.1 6.1 5.9 5.7 5.5 5.2 5.0
111.2 44 6.3 6.0 6.0 5.8 5.6 5.4 5.2 4.9
113.0 45 6.2 5.9 5.9 5.7 5.5 5.3 5.1 4.8
114.8 46 6.1 5.8 5.9 5.6 5.4 5.2 5.4 4.8
116.6 47 6.0 5.7 5.8 5.6 5.3 5.1 4.8 4.7
118.4 48 5.9 5.7 5.7 5.5 5.3 5.0 4.8 4.6
120.2 49 5.8 5.6 5.6 5.4 5.2 5.0 4.7 4.5
122.0 50 5.7 5.5 5.5 5.3 5.1 4.9 4.7 4.4
Oxygen, Dissolved
Page 1614
Oxygen scavenger tests explained
Oxygen Scavengers
For water (DEHA) Iron Reduction Method
Introduction
Diethylhydroxylamin (DEHA) is used as an oxygen scavenger to reduce corrosion in boilers. It is
also used in photographic processes and in the manufacture of certain silicon compounds.
Analytical methods to monitor DEHA concentrations have been limited to complex techniques not
suitable for on-site use. Hach has developed a relatively simple test, available in both laboratory
and field test kit formats.
OH
N
H5C2 C2H5
Figure 79 Chemical structure of N,N-Diethylhydroxylamine (DEHA)
Chemical reactions
DEHA reacts quantitatively with Fe3+ (ferric iron) and reduces it to Fe2+ (ferrous iron). The Fe2+
can then be determined by use of FerroZine™, a sensitive ferrous iron indicator.
A buffer at an optimum pH of 2.9–3.0 enhances color development. Best results are obtained if the
sample temperature is 25 °C (77 °F), and with a reaction time of 10 minutes in the dark. All
chemicals for this procedure are packaged in two reagents. DEHA Reagent 1 Powder Pillows
combine buffer and FerroZine indicator. DEHA Reagent 2 is a liquid formulation containing a
source of Fe3+ for the reaction. An explanation of the reaction between FerroZine and Fe2+ can be
found in the FerroZine Method for Iron explanation.
This method is also applicable to other oxygen scavengers, such as hydroquinone, erythorbic acid
(iso-ascorbic acid) methyl ethyl ketoxime and carbohydrazide, by use of the appropriate
conversion factor.
Oxygen Scavengers
Page 1615
Ozone tests explained
Ozone
For water Indigo Method1
1 Adapted from: Analytical Aspects of Ozone Treatment of Water and Wastewater; Lewis Publishers: Chelsea, Michigan, 1986; pages 153–
156.
Introduction
Ozone (O3), a powerful oxidant, is being increasingly used for water disinfection. It was first used
in the Netherlands in the late 1800’s to disinfect drinking water, and is now used worldwide in
drinking water and wastewater facilities, swimming pools, spas, and in the bottled water and
beverage industries. Ozone quickly provides microbial sterilization and disinfection, organic
compound destruction and conversion of iron or manganese salts to insoluble oxides which can be
precipitated or filtered from the water. The major reaction by-products are oxygen, water and
carbon dioxide. For environmental safety, unreacted or residual ozone should be monitored.
Chemical reactions
As ozone reacts quantitatively with indigo trisulfonate (blue indigo dye), the color of the solution
fades. Color intensity, inversely proportional to the amount of ozone present, is then measured at
600 nm with a photometer (colorimeter or spectrophotometer). The reagent is formulated to
prevent interference from any chlorine residual which may be present.
Traditionally, ozone loss during sampling is a major cause of analytical error. Ozone is liberated
when the sample is transferred from container to container, the loss causing erroneously low
determinations. The evacuated AccuVac™ Ampuls draw the sample directly from the water stream
or source in seconds. Ozone liberated while rushing into the Ampul is trapped there and reacts
immediately with the indigo reagents. The reagent buffers the sample solution to pH 2.5. The
Ampul is then placed directly in a photometer and measurements are taken in the reaction vial,
eliminating cross contamination between samples.
AccuVac Ampuls for analysis cover three ranges: low range (0–0.25 mg/L), medium range (0–0.75
mg/L), and high range (0–1.50 mg/L). The lower ranges are necessary because small amounts of
ozone will bleach the indigo dye only slightly. This slight decrease in color is difficult to detect if the
original blue color is very intense, as it is with the high-range Ampuls.
The low and medium-range tests are designed with less intense color so that a slight bleaching
can be more easily detected, thereby producing results accurate to as little as 0.01 mg/L.
Conceptually the reaction may be described as:
O H SO3K O
KO3S N KO3S
2O3 + 2 O2 + O
4
N N
Ozone Potassium Indigotrisulfonate
O
Potassium Isatin sulfonate
H SO3K SO3K H
Figure 80 Chemical reaction for ozone determination, indigo method
Ozone
Page 1616
pH tests explained
pH
Introduction
pH is a measure of the hydrogen ion activity in a solution and is defined as:
–log10 a H+ where a H+ is the activity of the hydrogen ion. Practically, this means that at pH 0, the
hydrogen ion concentration is 1 x 1014 times greater than at pH 14. This also means the hydroxyl
ion concentration at pH 14 is 1 x 1014 times greater than at pH 0. When the hydrogen and hydroxyl
ions are present in equal numbers (the neutral point), the pH is 7. Values from pH 0 to pH 7 are
termed Acidic and those from pH 7 to pH 14 are termed Basic. It is important to note that a pH
change of one unit (for instance, from pH 6 to pH 7) actually is a factor-of-10 change (decade
difference) in the hydrogen ion concentration.
The pH electrode used in pH measurement consists of a glass sensing half-cell and a reference
half-cell. Together the two half-cells form an electrode system. The sensing half-cell is a thin pH-
sensitive glass membrane separating two solutions. The outer solution is the sample to be tested.
The internal solution is enclosed within the glass membrane and has a known pH. An electrical
potential is developed on the inside surface of the glass membrane with the internal solution and
remains constant. Another electrical potential is developed on the outside surface of the glass
membrane with the sample solution. This potential varies with the pH of the sample solution. The
amount of variation is linear when the temperature is constant. The change in potential per pH unit
is termed the Electrode Slope.
A second half-cell, or reference half-cell, in contact with the sample solution has a constant
potential. Therefore, any changes in the potential of the electrode system at a given temperature
will be due to changes in the pH of the sample solution. Sensing and reference half-cells may be
contained in two separate electrodes, or they may be combined and called a combination
electrode; see the Platinum series combination electrode figure.
Temperature effects on pH measurements depend on the reference electrode used, pH of the
solution within the pH electrode and pH of the test solution. At a certain pH, temperature will have
no effect on the potential of the electrode system. This is known as the isopotential point. Also, at
some pH level, the system will exhibit 0 millivolts. This is known as the zero potential point. Both
the isopotential and zero potential points are features designed into electrodes. Hach electrodes
are designed so the isopotential and zero potential points are at pH 7. This minimizes temperature
effects in the majority of typical samples.
Conventional electrode design

The porous junction of a conventional reference half-cell is made of ceramic or fiber. With time, the
junction will become clogged with silver chloride or contaminants, causing large variation in the
reference potential. In addition, reference solution can be contaminated or diluted by back
diffusion of sample into the junction. Particular contaminants may be introduced into the junction.
Clogged or fouled junctions can cause drift along with inaccurate, noisy, erratic and sluggish pH
measurements. The performance of conventional porous junctions deteriorates as they age
because of clogging; see Figure 81.
pH
Page 1617
pH
New Used
When new, Even after just a few days,
conventional porous conventional reference
reference junction junctions can become
allows electrolyte clogged, causing slow,
solution to flow freely. unstable response and
inaccurate results.
Figure 81 Conventional combination electrode
Platinum series electrode design

Platinum Series Electrodes solve this clogging problem because they use a continually renewed
liquid junction, also known as a free diffusion junction (FDJ); see Figure 82. There is no ceramic or
fiber plug to become clogged and therefore the electrode lasts longer. The free diffusion junction
has been shown to have a faster and more stable response than conventional electrodes
(Figure 81). Figure 84 shows the accuracy and stability of the Platinum Series Electrode.
Figure 85 shows the variation of the Platinum Series Electrode and a conventional electrode. The
Platinum Series Electrode clearly provides repeatable results.
Free flowing reference

junction with reference
element
pH Sensing
half-cell
Figure 82 Platinum series combination electrode
Note: The free-flowing reference junction design prevents clogging. Every measurement is rapid, accurate
and stable, regardless of electrode age.
pH
Page 1618
pH
The Platinum Series Electrode provides stable results in one minute in this deionized water
sample. The conventional ceramic frit junction electrode shows a slow, noisy response.
Platinum series electrode

Actual pH
Conventional ceramic frit electrode
0 10 20 30
Time in minutes
Figure 83 Response times and electrode drift for Platinum Series and conventional electrodes
The initial pH reading obtained with a conventional reference junction for a deionized water
sample was incorrect, and shifted by 0.36 pH units when the sample ionic strength was increased
by adding 50 mg of ultrapure KCl. The same electrode with a Platinum Series reference electrode
showed significantly improved stability and accuracy.
Conventional ceramic frit reference junction
5.0
Platinum series
reference junction
Actual pH
6.0 +KCI
5 10 20 30
Time in minutes
Figure 84 Accuracy of Platinum Series and conventional electrodes
pH
Page 1619
pH
Platinum series pH systems (white) provide repeatable results every time. The conventional
electrode (gray) varies greatly from one measurement to the next.
+0.1
Conventional ceramic
frit electrode
Platinum series electrode

Expected pH
-0.1 Conventional Ceramic Frit

Electrode
Figure 85 Repeatability comparison between Platinum Series and conventional electrodes
pH
Page 1620
pH Indicators explained
pH Indicators
Introduction
Acid-base indicators behave as weak acids or bases in water or solvents. An equilibrium
established between the hydronium ion (H3O)+ and the indicator ion (In– ) can be represented as
follows for an aqueous medium:
H2O + HIn → H3O+ + In– (In as a weak acid)
(acid color) (base color)
In– + H2O → InH+ + OH– (In as a weak base)

(base color) (acid color)
The ratio of HIn to In– or In to InH+ varies as a function of pH. However, the human eye cannot
detect all the color changes as result of the varying concentrations of In in the solution. The color
change between the predominately acid form of the indicator and the predominately base form is
clearly visible, and therefore suitable for visual acid-base titration.
Most of the Hach Acid-Base Indicators can be divided into three classes based on the structure.
The phthalein indicators are sparingly soluble in water but are quite soluble in alcohols. Alcohol is
the preferred solvent for indicator solution preparation. Equilibrium of the phthalein-type indicators
is exemplified by phenolphthalein represented as follows:
HO OH
+ H2O
C O
C O
HO OH
+ H3O+
C OH
O
C
O¯
O¯
O
C
+ H3O+
O
C
O¯
pH Indicators
Page 1621
pH Indicators
Many of the sulfonphthalein indicators exhibit two useful visual transition ranges. These indicators
exhibit good stability toward strong alkali solution. Indicator solutions are usually prepared in 20%
alcohol solution. The equilibria of this type of indicator are exemplified by cresol red, which is
represented as follows:
Acidic transition range
HO OH+
H3C O CH3
SO3¯
+ H2O
(orange)
HO O
H3C C CH3
SO3¯
+ H3O+
(yellow)
Basic transition range
O¯ O
H3C C CH3
SO3¯
+ H3O
pH Indicators
Page 1622
pH Indicators
Most of the azo indicators exhibit a color change from red to yellow. The equilibrium for azo-type
indicators is exemplified by methyl orange, as shown below:
SO3¯ N N N(CH3)2
+
SO3¯ N N N(CH3)2 + H+
H
SO3¯ N N N(CH3)2
+
Indicator choice
The visual transition range is the main factor in selection of pH indicator. Indicator solubility also is
important. When using the indicator for an acid-base titration, ease of identifying the color change
and slope of the titration curve must be considered.
Figure 86 illustrates titration of a strong acid with a strong base. Any of the three indicators would
be used with satisfactory results in the appropriate pH ranges. However, as the slope of the curve
decreases, the selection of an indicator with the proper transition range is more important.
In Figure 87, titration of a weak acid with a strong base; only phenolphthalein would be
satisfactory. Use of methyl red would give an incorrect result because the transition range of the
indicator does not correspond with the actual titration equivalence point.
Phenolphthalein range
Bromthymol Blue range
Methyl Red range
Volume Base (mL)
Figure 86 Titration of a strong acid with a strong base
pH Indicators
Page 1623
pH Indicators
Volume Base (mL)
Figure 87 Titration of weak acid with a strong base
pH Indicators
Page 1624
pH Indicators
The chart below shows the ranges of some pH indicators available from Hach Company.
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Crystal Violet (yellow to blue-violet)
Cresol Red (orange to yellow) (yellow to purple)
(yellow to blue)
Thymol Red (red to yellow)
Erythrosin B (orange to red)
2, 4-Dinitrophenol (colorless to yellow)

Bromphenol Blue (yellow to blue)
Methyl Orange (pink to yellow)
Bromcresol Green (yellow to blue)
Methyl Red (pink to yellow)
Eriochrome* Black T (red to blue)
Bromcresol Purple (yellow to purple)
Alizarin (yellow to red) (red to purple)

Bromthymol Blue (yellow to blue)
Phenol Red (yellow to red)

m-Nitrophenol (colorless to yellow)
o-Cresolphthalein (colorless to red)
The pH ranges shown are approximate.
Specific transition ranges depend on the indicator Phenolphthalein (colorless to pink)
solvent chosen.
Thymolphthalein
(colorless to blue)
Alizarin Yellow GG
* Trademark CIBA GEIGY CORP. (yellow to orange)
pH Indicators
Page 1625
Phenols test explained
Phenols
For water, wastewater and seawater 4-Aminoantipyrine Method
Introduction
Phenols are produced as waste in oil refineries, coke plants, and in some chemical manufacturing
plants. Natural waters normally contain less than 1 µg/L, but concentrations up to 20 µg/L occur in
some areas. Levels of 10 to 100 µg/L phenol are detectable by taste and odor. A 1-µg/L phenol
concentration can impart an objectionable taste to water following slight chlorination.
Chemical reaction
Phenols and all substituted phenols (except those with para substitution), are determined by
buffering the sample to a pH of 10.0 and adding 4-aminoantipyrine to produce a yellow or amber-
colored complex in the presence of ferricyanide ion. The color is intensified through extraction of
the complex into chloroform. Measurement of this color quantitatively determines the phenol
concentration of the sample.
OH
H3 C N O + H3C
N O
N N
H3 C NH2 H3C N O
4-Aminoantipyrine Phenol
Figure 88 Chemical reaction for 4-Aminoantipyrine method
Phenols
Page 1626
Phosphonate tests explained
Phosphonates
For water Ultraviolet Photochemical Oxidation Method
Introduction
Phosphonates are employed as chemical additives to function as threshold antiscalants, corrosion
inhibitors, chelants, sludge conditioners, deflocculants, dispersants and crystal growth modifiers in
various industrial water treatment processes. They are used predominantly as scale and corrosion
preventatives for boiler and cooling tower waters. Phosphonates exist in various formulations as
acids or salts and are marketed in the form of concentrated solutions.
Until recently, analytical methods for phosphonates have been difficult, time consuming and
subject to many interferences. The Ultraviolet (UV) Photochemical Oxidation Method involves a
photochemical oxidation of phosphonate followed by conventional colorimetric determination of
the liberated orthophosphate by the Ascorbic Acid Method. The UV Photochemical Oxidation
Method is rapid, easy to use, relatively free from interferences, and applicable to both field and
laboratory situations.
Chemical reactions
Phosphonic acids are organic compounds of the form R-PO3H2. Structures of two commonly used
treatment chemicals are shown below; the phosphonic acid group is shown in parentheses.
Phosphonates are the corresponding anions formed by ionization of one or more of the acidic
hydrogens.
Aminotri Hydroxyethylene diphosphonic

(methylene phosphonic acid) acid
CH2 (PO3H2 ) (PO3H2)
N CH2 (PO3H2 ) CH3 C OH
CH2 (PO3H2 ) (PO3H2)

Figure 89 Chemical structures of two common phosphonic acids
Decomposition of these compounds by oxidation will liberate the organically bound phosphate as
orthophosphate. The combined action of the UV radiation and oxygen will liberate orthophosphate
rapidly without the necessity of heat or corrosive agents. When the photo-oxidation is carried out in
the absence of acid, no significant degree of depolymerization or hydrolysis of condensed (pyro,
meta or other poly) phosphates occurs, making the method a true test for organic phosphate.
Presence of excess oxygen is ensured by the addition of a small amount of potassium persulfate.
In this oxygen-rich environment UV light will rapidly catalyze the oxidation of the phosphonate C-P
bond.
UV C PO3H2 C + H3PO4
The orthophosphate formed can then be determined colorimetrically using the Ascorbic Acid
method. Reagents for the Ascorbic Acid Method for orthophosphate have been combined into a
single reagent powder, PhosVer™3. Determination of orthophosphate using PhosVer 3 is
described in the Phosphorus methods.
Phosphonates
Page 1627
Phosphorous tests explained
Phosphorous
Amino Acid, Ascorbic Acid and
Molybdovanadate Methods
Introduction
Phosphorus occurs in natural water and wastewaters almost solely as phosphates. Phosphates
may enter water from agricultural run-off and as biological and industrial wastes. They may be
added to water in municipal and industrial water treatment processes to control corrosion. A
certain amount of phosphate is essential for most plants and animals, but too much phosphate in
water can contribute to eutrophication, especially when large amounts of nitrogen are also
present.
Phosphorus can be classified as orthophosphate, condensed phosphate or organically bound
phosphate. Condensed phosphates are formed by dehydrating the orthophosphate radical; they
include metaphosphate, pyrophosphate and polyphosphate. The only form of phosphate
determined directly is orthophosphate; other forms require pretreatment for conversion to
orthophosphate for analysis. When no pretreatment is used, phosphate analyses determine
Reactive Phosphorus. Reactive phosphorus is a measure of orthophosphate, plus a small fraction
of condensed phosphate that may have been hydrolyzed during the test.
Hach offers high and low range tests for reactive phosphorus. High range tests can be completed
with the Amino Acid Method or the Molybdovanadate Method. The Molybdovanadate Method uses
a single reagent and has a faster reaction than the Amino Acid Method. Both methods have a
broad range and are free from most interferences. Low range tests use the Ascorbic Acid Method.
Condensed phosphates plus orthophosphate can be determined by acid hydrolysis using sulfuric
acid, followed by the reactive phosphorus test for the appropriate range. A small amount of
organically bound phosphorus will be included in this measurement. The results of the test are
reported as acid-hydrolyzable phosphorus. Total phosphorus (orthophosphate, condensed and
organically bound) can be determined by acid oxidation with persulfate, followed by the reactive
phosphorus test. Organically bound phosphate can then be determined by subtracting the acid-
hydrolyzable phosphorus.
Chemical reactions
Pretreatment steps
Reactions for pretreatment to determine acid-hydrolyzable and total phosphorus are illustrated
below:
R O P O R' + K2S2O8 + H2SO4
OH
H3PO4 + 2K+ + 3SO42¯ + Various organic fragments
Figure 90 Example of potassium persulfate oxidation of organically bound phosphorus1

1 R and R’ represent various organic groups
Phosphorous
Page 1628
Phosphorous
Amino acid and ascorbic acid methods

Reactive phosphorus is determined in essentially two steps for either the Ascorbic Acid Method
(low range) or the Amino Acid Method (high range). The first step involves reaction of
orthophosphate with molybdate in acid solution, which forms a yellow-colored phosphomolybdate
complex:
12MoO3 + H2PO4– → (H2PMo12O40)–
The phosphomolybdate complex is then reduced by either an amino acid or ascorbic acid, causing
a characteristic molybdenum blue species. Various structures for the molybdenum blue species
have been suggested in the literature. For example, see Killeffer, D. H., Molybdenum Compounds-
Their Chemistry and Technology, Interscience Publishers, 1952.
All reagents for the Ascorbic Acid Method are contained in PhosVer™3 Reagent Powder Pillows.
Reagents for the Amino Acid Method are contained in Amino Acid Reagent Solution and
Molybdate Reagent Solution.
Molybdovanadate method
Reactive phosphorus combines with molybdate in an acid medium to form a phosphomolybdate
complex. Vanadium, contained in Molybdovanadate Reagent, reacts with the complex to form
vanadomolybdophosphoric acid. Intensity of the resulting yellow color is proportional to the
concentration of reactive phosphorus. One possible formula for the complex is suggested below.
The exact structure is not known.
4–
[ PO 4 • VO 3 • 16MoO 3 ]
Phosphorous
Page 1629
Potassium tests explained
Potassium
For water and wastewater Tetraphenylborate Method
Introduction
Potassium, one of the most abundant elements, is found in many minerals. Soils contain
approximately 1 to 4% potassium. Concentrations of potassium in most drinking water is usually
less than 20 mg/L; occasionally brines may contain more than 100 mg/L. The greatest areas of
interest in measurement of potassium levels probably are medicine and agriculture, due to the
importance of potassium as a mineral for plants and animals. Potassium salts, particularly potash,
are common in fertilizers.
The Tetraphenylborate Method for determination of potassium in water is accurate, rapid, and
inexpensive. In the reaction, a precipitate is formed and the resulting increase in turbidity is
measured. All necessary reagents are packaged in three powder pillows to provide reagent
stability, convenience and accuracy.
Chemical reactions
Potassium combines with sodium tetraphenylborate to form potassium tetraphenylborate, a white
precipitate. The precipitate remains in suspension in samples with low concentrations of
potassium, causing an increase in turbidity.
NaB(C6H5)4 + K+ KB(C6H5)4 + Na+

Figure 91 Chemical reaction between potassium and tetraphenylborate
The sodium tetraphenylborate is contained in Potassium 3 Reagent Powder Pillows. Ammonium

salts, magnesium and calcium interfere with the precipitation. Potassium 1 Reagent Powder
Pillows and Potassium 2 Reagent Powder Pillows prevent these interferences.
Potassium
Page 1630
Selenium tests explained
Selenium
For water and wastewater Diaminobenzidine Method
Introduction
Selenium levels in natural waters seldom exceed 0.01 mg/L and concentrations greater than
0.50 mg/L are rare. The appearance of selenium in natural waters may be due to seepage from
soils containing selenium or intrusion of industrial wastes.
Selenium, very toxic to man and animals, exhibits properties similar to those of arsenic. Selenium
is suspected of causing dental caries and of being carcinogenic, but trace amounts also have been
found essential to maintain normal body metabolism. For this reason, a maximum concentration
level in drinking water has been established by the USEPA in accordance with the Safe Drinking
Water Act.
The Diaminobenzidine Extraction Method uses diaminobenzidine indicator, which develops a
yellow color with selenium. This method measures only the tetravalent selenium (Se4+). Selenium
present as Se2+ and Se6+ is not detected unless the sample is first distilled.
Chemical reactions
In the test for selenium, an EDTA masking agent is first added to the sample to remove
interferences such as iron. The addition of formic acid adjusts the sample to an optimum pH range
of 1–2. Under these conditions, diaminobenzidine reacts with all selenium present in the Se4+ form
to give a yellow-colored piazselenol complex. (Selenium existing in the selenate form is not
determined by this method.) The sample is then buffered to a pH of 10–11, where the piazselenol
complex can be extracted into toluene. Measurement of the color intensity directly indicates the
amount of Se4+ in the sample.
H2 N NH2
H2N NH2 + H2SeO3
Diaminobenzidine
H2N
N
Se
H2N N + 3H2O
Selenium
Page 1631
Silica tests explained
Silica
For water and seawater Silicomolybdate/Heteropoly Blue Method
Introduction
Silicon is the second most abundant element in nature. Accordingly, it is not surprising that most
waters contain compounds of silicon, usually as silica (SiO2) or silicates (SiO4– and SiO32–). Silica
concentration in water is commonly less than 30 mg/L, although concentrations greater than
100 mg/L are not unusual; concentrations exceeding 1000 mg/L are possible in brines and
brackish water.
Silica and silicates are added to water for a number of uses, such as water conditioners,
detergents, and corrosion inhibitors. However, silica in water can cause significant problems for
industries, primarily in boiler and turbine applications. High pressures and high temperatures
cause silica deposits on tubes of boilers and heat exchangers. These glassy deposits lower the
efficiency of heat transfer and lead to premature failure. Silica deposits on steam turbine blades
decrease efficiency and necessitate costly downtime for cleaning. Silica levels must be kept below
0.005 mg/L in very high pressure applications.
Measuring silica in water is useful when efficiency of demineralizers is monitored. Testing for silica
(one of the first impurities detected when the exchange capacity of a demineralizer is exhausted)
provides a sensitive check of demineralizer performance.
Analytical procedures for silica include the Silicomolybdate Method for high range measurement
and the Heteropoly Blue Method for low range measurement. The Heteropoly Blue method is an
extension of the Silicomolybdate method to increase sensitivity.
Chemical reactions
High and low ranges
The Silicomolybdate Method involves the reaction of molybdate ion with silica and phosphate
under acid conditions to form a yellow color. Citric acid is added to destroy the phosphomolybdic
acid complex (the yellow color formed due to phosphate), but not the silicomolybdic acid complex.
For large amounts of silica, the remaining yellow color is intense enough to be read directly. For
low concentrations, an amino-naphthol sulfonic acid reducing agent is used to convert the faint
yellow color to a dark heteropoly blue species. The color formed is directly proportional to the
amount of silica present in the original sample; a colorimetric measurement of this intensity
provides an accurate means of determining the silica concentration. Some forms of silica (usually
polymeric) will not react with ammonium molybdate and must be digested with sodium bicarbonate
to be converted to a reactive form.
Silicic acid reacts with water and hydrates as follows:
H2SiO3 + 3H2O → H8SiO6
This hydrated silicic acid reacts with molybdate in the presence of acids to form
silicomolybdic acid.
H8SiO6 + 12(NH4)2MoO4 + 12H2SO4 → H8[Si(Mo2O7)6] + 12(NH4)2SO4 + 12H2O
This silicomolybdic acid is then reduced to a blue color (heteropoly species) by an amino naphthol
sulfonic acid for low concentrations.
Silica
Page 1632
Sulfate tests explained
Sulfate
For water, seawater and oil-field water Turbidimetric Method
Introduction
Sulfate occurs in natural waters in a wide range of concentrations. Mine waters and industrial
effluents frequently contain large amounts of sulfate from pyrite oxidation and the use of sulfuric
acid.
Because of sulfate’s cathartic action, a secondary maximum contaminant level has been
established by the USEPA, in accordance with the Safe Drinking Water Act. The taste threshold of
magnesium sulfate is 400 to 600 mg/L; calcium sulfate is 250 to 800 mg/L. Sulfate may be either
beneficial or detrimental in water used for manufacturing and domestic supply. The presence of
sulfate is advantageous in producing desired flavors in the brewing industry. In domestic water
systems, sulfates do not appear to cause any increased corrosion on brass fittings, but
concentrations above 200 mg/L do increase the amount of lead dissolved from lead pipes.
Sulfate determination is important in oil field applications where two or more waters are mixed.
High concentrations of sulfate, along with barium, calcium, and strontium, can form insoluble
scales.
The procedure for determining sulfate is a modification of the Barium Sulfate Turbidimetric
Method. A single dry powder reagent, SulfaVer™4 Sulfate Reagent, will cause a milky precipitate
to form if sulfate is present. The amount of turbidity formed is proportional to the amount of sulfate
present.
Chemical reactions
Sulfate is determined by its quantitative precipitation with barium chloride. Because the finely
divided barium sulfate turbidity formed is proportional to the amount of sulfate in the sample, a
photometric reading enables the sulfate concentration to be accurately determined.
2+ 2–
Ba + SO 4 → BaSO 4 ¯ ¯
Sulfate
Page 1633
Sulfide tests explained
Sulfide
For water, wastewater and seawater Methylene Blue Method
Introduction
Sulfide is a poisonous by-product of the anaerobic decomposition of organic matter and commonly
is found in sewage and industrial wastewaters. Sulfide can be present as the free sulfide ion (S2¯)
or as dissolved hydrogen sulfide (H2S and HS–). The toxicity of hydrogen sulfide is equivalent to
that of hydrogen cyanide, but its offensive odor is detectable long before toxic levels are reached.
However, at high concentrations hydrogen sulfide quickly deadens the sense of smell; thus toxic
levels may be present but undetected.
Chemical reactions
The sulfide test is based on the ability of hydrogen sulfide and acid-soluble metallic sulfides to
convert N,N-dimethyl-p-phenylenediamine directly to methylene blue in the presence of a mild
oxidizing agent (potassium dichromate). Intensity of the methylene blue color development is
directly proportional to the amount of sulfide present in the original sample. A colorimetric
measurement of this intensity provides an accurate means to determine the sulfide concentration.
All necessary reagents are contained in Sulfide 1 Reagent and Sulfide 2 Reagent.
NH2
4 + 2H2S + Cr2O72¯ + 10H+
(CH3)2N
2 + 2Cr3+ + 2NH4+ + 7H2O

(CH3)2N S N(CH3)2
Figure 92 Chemical reaction for hydrogen sulfide using the Methylene Blue method
Sulfide
Page 1634
Sulfite tests explained
Sulfite
For water, wastewater and seawater Titration Method
Introduction
Sulfite is most commonly found in boilers and boiler feedwater, where it is used to inhibit corrosion
by reducing dissolved oxygen. It may also be found in industrial wastes such as paper mill
effluents. Sulfite normally is not present in natural waters because it readily oxidizes to sulfate.
Chemical reactions
The water sample is acidified by the addition of Dissolved Oxygen 3 Reagent Powder Pillow,
starch indicator is then added and the solution is titrated with Potassium Iodide-Iodate Solution.
The acidified solution releases free iodine, which oxidizes sulfite to form sulfate. The iodine is
reduced to colorless iodide:
KIO3 + 5KI + 6HCI → 6KCI + 3I2 + 3H2O
SO32– + I2 + H2O → SO42– + 2HI

When all sulfite has been converted to sulfate, excess iodine is indicated by a blue color from the
starch-iodine reaction.
Sulfite
Page 1635
Turbidity tests explained
Turbidity
Introduction and definition

Turbidity, a qualitative characteristic which is imparted by solids obstructing the transmittance of
light through a water sample, is an important water quality indicator. Turbidity can be interpreted as
a measure of the relative clarity of water and often indicates the presence of dispersed, suspended
solids; particles not in true solution such as silt, clay, algae and other microorganisms; organic
matter and other minute particles. Turbidity is not a direct measure of suspended particles in water,
but a measure of the scattering effect such particles have on light.
The extent to which suspended solids can be tolerated varies widely, as do the levels at which they
exist. Industrial cooling water, for example, can tolerate relatively high levels of suspended solids
without significant problems. In modern high pressure boilers, however, water must be virtually
free of impurities. Solids in drinking water can support growth of harmful microorganisms and
reduce effectiveness of chlorination, resulting in health hazards. In almost all water supplies, high
levels of suspended matter are unacceptable for aesthetic reasons and can interfere with chemical
and biological tests.
Theory of light scattering

Very simply, the optical property expressed as turbidity is the interaction between light and
suspended particles in water. A directed beam of light remains relatively undisturbed when
transmitted through absolutely pure water, but even the molecules in a pure fluid will scatter light to
a certain degree. Therefore, no solution will have a zero turbidity. In samples containing
suspended solids, the manner in which the sample interferes with light transmittance is related to
the size, shape and composition of the particles in the solution and to the wavelength (color) of the
incident light.
A minute particle interacts with incident light by absorbing the light energy and then, as if a point
light source itself, reradiating the light energy in all directions. This omnidirectional reradiation
constitutes the "scattering" of the incident light. The spatial distribution of scattered light depends
on the ratio of particle size to wavelength of incident light. Particles much smaller than the
wavelength of incident light exhibit a fairly symmetrical scattering distribution with approximately
equal amounts of light scattered both forward and backward (see Figure 93).
As particle sizes increase in relation to wavelength, light scattered from different points of the
sample particle create interference patterns that are additive in the forward direction. This
constructive interference results in forward-scattered light of a higher intensity than light scattered
in other directions (see Figure 94 and Figure 95). In addition, smaller particles scatter shorter
(blue) wavelengths more intensely while having little effect on longer (red) wavelengths.
Conversely, larger particles scatter long wavelengths more readily than they scatter short
wavelengths of light.
Particle shape and refractive index also affect scatter distribution and intensity. Spherical particles
exhibit a larger forward-to-back scatter ratio than coiled or rod-shaped particles. The refractive
index of a particle is a measure of how it redirects light passing through it from another medium
such as the suspending fluid. The particle's refractive index must be different than the refractive
index of the sample fluid in order for scattering to occur. As the difference between the refractive
indices of suspended particle and suspending fluid increases, scattering become more intense.
The color of suspended solids and sample fluid are significant in scattered-light detection. A
colored substance absorbs light energy in certain bands of the visible spectrum, changing the
character of both transmitted light and scattered light and preventing a certain portion of the
scattered light from reaching the detection system.
Light scattering intensifies as particle concentration increases. But as scattered light strikes more
and more particles, multiple scattering occurs and absorption of light increases. When particulate
concentration exceeds a certain point, detectable levels of both scattered and transmitted light
Turbidity
Page 1636
Turbidity
drop rapidly, marking the upper limit of measurable turbidity. Decreasing the path length of light
through the sample reduces the number of particles between the light source and the light detector
and extends the upper limit of turbidity measurement.
Incident beam
Figure 93 Effects of particle size and light wavelength (small particles)1

1 Size: Smaller Than 1/10 the wavelength of light
Description: Scattering symmetric
Incident beam
Figure 94 Effects of particle size and light wavelength (large particles)1

1 Size: Approximately 1/4 the wavelength of light
Description: Scattering concentrated in forward direction
Incident beam
Figure 95 Effects of particle size and light wavelength (larger particles)1

1 Size: Larger Than the wavelength of light
Description: Extreme concentration of scattering in forward direction;
Development of Maxima and Minima of scattering Intensity at wider angles
General instrument description

The instrument optical system typically includes a lamp, lenses and apertures to focus the light, a
90-degree detector to monitor scattered light and optionally, a forward-scatter light detector, a
transmitted-light detector and a back-scatter light detector. The optional detectors may be added to
minimize the impact of color, stray light and lamp and optical variabilities (see Figure 96).
Turbidity
Page 1637
Turbidity
90°
Detector
Transmitted
Light
Detector
Lamp
Lens Sample
Cell
Figure 96 General turbidimeter optical system
Formazin primary standard

The chemically accepted definition of a primary standard is a standard that is prepared by the end
user, on the bench, from traceable raw materials.
Formazin meets that criteria when prepared by accurately weighing and dissolving 5.000 g of
hydrazine sulfate and 50.0 g of hexamethylenetetramine in one liter of distilled water. The solution
develops a white turbidity after standing at 25 °C (77 °F) for 48 hours and can be prepared
repeatably with an accuracy of ±1%. This solution is equal to 4000 NTU. All other turbidity
standards are traced to formazin.
Due to the statistical reproducibility of the nephelometric scatter of white light by the formazin
polymer, instruments with the traditional tungsten filament white light optical designs can be
calibrated with a high degree of accuracy and reproducibility. The randomness of particle shapes
and sizes within formazin standards yield statistically reproducible scatter on all makes and
models of turbidimeters.
Turbidity
Page 1638
Zinc test explained
Zinc
For water and wastewater Zincon Method
Introduction
Zinc concentrations in most water supplies average about 1 mg/L, but may range as high as
50 mg/L in some areas. Although zinc is commonly found in many natural waters, the deterioration
of galvanized iron and leaching of brass can add substantial amounts. Industrial effluents may
contribute large amounts of zinc; high concentrations suggest the presence of lead and cadmium,
both common impurities from the galvanizing process.
Zinc is essential to human metabolism and has been found to be necessary for proper growth.
High concentrations of zinc in water act as stomach irritants but the effects are temporary.
Concentrations above 5 mg/L show no harmful physiological effects but can cause a bitter taste
and/or an opalescence in alkaline drinking water.
A dry powder form of 2-carboxy-2’hydroxy-5’sulfoformazyl benzene indicator, commonly called
zincon, is used in the ZincoVer® Method of determining zinc concentrations. This test has been
approved by the Environmental Protection Agency for National Pollutant Discharge Elimination
System-reporting purposes based on comparability studies if the sample is first digested. Testing
done for non-reporting purposes generally does not require sample digestion.
Chemical reaction
In the analysis of zinc, cyanide is added to a buffered water sample of pH 9 to form a complex with
all heavy metals present in the sample. (1) The addition of cyclohexanone then frees the zinc from
the cyanide complex (2) and enables it to react with the indicator, zincon (3) a blue-colored
complex forms in direct proportion to the amount of zinc in the sample. Measurement of the color
intensity determines the zinc concentration.
(2) Zn(CN)42– + 4 + 4H2 Zn2++ 4 + 4OH¯

N C OH
O
O
C O
O– O - O
¯O
(3) ¯OH + Zn2+ + H
Zn
C + H2 O
¯O3S N N ¯O3S N N
N N N N
C C
Zincon (orange) (Blue)
Zinc
Page 1639
Zinc
Zinc
Page 1640
Technical Support
Page 1641
Contact Hach Company
U.S.A. Customers
By Telephone:
6:30 a.m. to 5:00 p.m. MST
Monday through Friday
(800) 227-HACH (800-227-4224)
By Fax:
(970) 669-2932
By Mail:
Hach Company
P.O. Box 389
Loveland, Colorado 80539-0389 U.S.A.
Visit us on the web:
Order products online at www.hach.com
Information Required
•Hach account number (if available) •Billing address
•Your name and phone number •Shipping address
•Purchase order number •Catalog number
•Brief description or model number •Quantity
International Customers
Hach maintains a worldwide network of dealers and distributors. To locate the representative
nearest you, send an e-mail to: intl@hach.com or contact:
Hach Company World Headquarters; Loveland, Colorado, U.S.A.
Telephone: (970) 669-3050; Fax: (970) 669-2932
Technical and Customer Service (U.S.A. only)

Hach Technical and Customer Service Department personnel are eager to answer questions
about our products and their use. Specialists in analytical methods, they are happy to put their
talents to work for you.
Call 1-800-227-4224 or e-mail techhelp@hach.com
Page 1642

Water Analysis Handbook 7°ed PDF

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Water Analysis Handbook 7°ed PDF

Uploaded by

Copyright:

Available Formats

WATER ANALYSIS

©Hach Company, 2008, 2012.

Applications Guide ........................................................................................................................... 11

Alkalinity, USEPA Buret Titration Method ............................................................................................. 111

Chlorine, Total, USEPA DPD Method ................................................................................................... 363

Iron, TitraVer Titration Method.............................................................................................................. 625

Oxygen, Dissolved, HRDO Method ...................................................................................................... 913

Sulfate, USEPA SulfaVer 4 Method.................................................................................................... 1151

Heterotrophic Bacteria, Pour Plate Method ........................................................................................ 1403

Formaldehyde, For water.................................................................................................................... 1577

Metals/Mining, Mfg & Finishing

Power Plant Utilities

Pulp & Paper Mills

Pools & Spas

Metals/Mining, Mfg & Finishing

Power Plant Utilities

Pulp & Paper Mills

Pools & Spas

Metals/Mining, Mfg & Finishing

Power Plant Utilities

Pulp & Paper Mills

Pools & Spas

1.1 Procedure abbreviations

conc concentrated PCB poly chlorinated biphenyl

DB dropping bottle ppb parts per billion

DBP disinfection by-products ppm parts per million

CFR Code of Federal Regulations RL Rapid Liquid™

EDL Estimated detection limit SCDB self-contained dropping bottle

EPA Environmental Protection Agency THM trihalomethane

F&T free and total TNT Test ‘N Tube™

FM FerroMo® TOC total organic carbon

FV FerroVer® TPH total petroleum hydrocarbons

FZ FerroZine® TPTZ 2,4,6-Tri-(2-Pyridyl)-1,3,5-Triazine

1.2.2 Hardness conversion

Mixing sample in a square sample cell:

Figure 1 Rotate a sample cell

Figure 2 Swirl a cylinder and invert a sample cell

Figure 3 General purpose distillation apparatus

2.5.1 Vacuum filtration

To filter using a vacuum:

Figure 4 Vacuum filtration

2.5.2 Required apparatus for vacuum filtration

2.5.3 Gravity filtration

To filter using gravity:

Figure 5 Gravity filtration

Table 5 Required apparatus for gravity filtration

2.6.2 Reagent blank

2.7 Sample dilution

To dilute the sample:

Table 6 Sample dilution volumes

2.7.1 Sample dilution with interfering substances

Interference Level × Dilution Factor = Interference level in sample

2.8 AccuVac® Ampuls

To use AccuVac Ampuls:

Figure 6 How to use AccuVac Ampuls

2.8.1 Using the AccuVac Snapper

To use the AccuVac Snapper:

How to use the AccuVac snapper

Figure 7 How to use the AccuVac snapper

2.9 PermaChem® pillows

Figure 8 How to open PermaChem pillows

2.10 Sample cells

2.10.1 Orientation of sample cells