CAHCET Water Lab Manual

C ABDUL HAKEEM COLLEGE OF
ENGINEERING AND TECHNOLOGY
Approved by AICTE, New Delhi & Affiliated to Anna University, Chennai
LABORATORY MANUAL
DEPARTMENT OF CIVIL ENGINEERING
CE8512-WATER AND WASTEWATER ANALYSIS

LABORATORY
Faculty in charge HOD

GENERAL INSTRUCTIONS
 Wear the Overcoats and shoes , before entering in to the Laboratory

 Operate the equipment / Instruments only in the presence of Lab
Technician / Lab In charge.
 Have a clear Idea about the experiment, he/she has to do at a
particular session
 Come with the observation of results of the previous experiments
getting signed by the Lab- In charge, well in advance. Those students
who have not got the signature for the particular experiments will not
be allowed to do the next experiment.
 Put his/her signature in the register before getting required
instruments for doing the experiments.
CAHCET/CIVIL ENGG 2 CE8512 - WWW LAB

Name : Reg.No :
INDEX
Signature of
S.NO Date Name of the Experiment Page.No Marks
the staff
1 Sampling and preservation 6
2 Determination of pH 9
3 Determination of Turbidity 11
4 Determination of Total Solids 13
Determination of Total Dissolved

5 Solids 15
Determination of Total Fixed And

6 Volatile Solids 17
7 Determination of Chlorides 20
8 Determination of Hardness 23
9 Determination of Sulphates 27
Determination of Dissolved
10 29
Oxygen

Signature of
S.NO Date Name of the Experiment Page.No Marks
the staff
Determination of Biochemical
11 Oxygen Demand. 33
Determination of Chemical
12 Oxygen Demand 38
Determination of Residual
13 Chlorine 41
Determination of Optimum
14 amount of Coagulant dosage. 43
Of Coagulant of Available
Determination
15 Chlorine 46
16 Determination of Fluoride 49
Determination of Iron
17 52
Determination of Alkalinity
18 42
Determination of Acidity
19 56
Determination of Conductivity
20 59
Determination of Phosphates
21 60
Determination of SVI of
Biological sludge & Microscopic
22 63
examination
Determination of MPN INDEX

23 of water sample 64

Topic Beyond The Syllabus
Determination of Nitrates
1 67
2 Preparation of Nutrient Agar 70
3 Preparation of Nutrient Broth 70

Ex. No: 01 SAMPLING AND PRESERVATION METHODS
Date:
WATER SAMPLING
The significance of a chemical analysis depends to a large extent on the sampling

programme. An ideal sample should be done which is both valid and representative. These
conditions are met by collection of samples through a process of random selection. This
ensures that the composition of the sample is identical to that of the water body from which it
is collected and the sample shares the same physico-chemical characteristics with the
sampled water at the time and site of sampling.
The relevant factors for any sampling programme are (a) frequency of sample collection, (b)
total number of samples, (c) size of each sample, (d) sites of sample collection, (e) method of
sample collection, (f) data to be collected with each sample, and (g) transportation and care
of samples
For analysis of natural and waste water, two principal types of sampling Procedures are
employed:
SPOT OR GRAB SAMPLES:
These samples are discrete portions of water samples taken at a given time. A series of grab
samples, collected from different depths at a given site, reflect variations in constituents over
a period of time. The total number of grab samples should satisfy the requirements of the
sampling programme
COMPOSITE SAMPLES:
These samples are essentially weighted series of grab samples , the volume of each being
proportional to the rate of flow of the water stream at the time and site of sample collection.
Samples may Composed over anytime period, such as 4, 8 or 24 hours, on the purpose of
analysis. Such composite samples are useful for determining the average condition which ,
when correlated with flow, , can be used for computing the material balance of a stream of
water body over a period of time.
It may be stated in general, that it is more meaningful to analyze a large number of separate
samples taken at different times and different locations than to compile and analyse a single
representative sample.
Separate samples must be collected for chemical and biological analysis. , since the sampling
and preservation techniques are quite different. For accurate analysis, it is desirable to allow
a short-time interval between sampling and analysis. As matter of fact temperature, pH and
dissolved gases (D.O.) must be determined in the field and as quickly as possible after
sampling.
Collection of truly representative sample is as important as sample preservation. A

representative single sample is taken from a number of different locations over a long period
of time. In general, it is more significant to analyse a large number of separate samples taken
at different times and different locations than it is to compile and analyse a single
representative sample. It is essential to keep accurate records of the location, time and
conditions of sample collection:
PRESERVATION:
It is essential to protect samples from changes in composition and deterioration with aging
due to various interactions. The optimum sample holding time ranges from zero for
parameters .such as pH, temperature and D.O to one week for metals. The preservation
techniques for various parameters are summarized in the following table. As mentioned
above, these are essential for retarding biological action, hydrolysis of chemical compounds
and complexes and reduction of volatility of constituents. It is desirable for accurate results,
that analysis must be undertaken within 4 hours for some parameters and 24 hours for others,
from the time of collection and it must be concluded within a week.

WATER SAMPLE PRESERVATION:
Parameter Volume Container Preservation

(mL)
PH 100 Polythene
Measure with in 0-4 hours
DO 100 Polythene
COD 500 Polythene Add H2SO4 to pH2 ; refrigerate
Nitrogen
Analyse as soon as possible , add0.8mL conc.
Ammonia
500 Polythene H2SO4 / L Add 40 mg HgCl2 / L and
refrigerate
Nitrate+ Nitrite
Add NaOH to pH 12 and 25mL of 2%
Cyanide 500 Polythene
ascorbic acid and refrigerate
Add 1 mL of 2N Zn(CH3COO) 2 and 2 mL
Sulphide 500 Polythene
of 1M NaOH Stir and refrigerate
Polythene/
Phosphate 500 Add 40 mg HgCl2 / L and refrigerate
Glass bottle
Acidify with H3PO4 to pH 4.0 and add
Polythene/
Phenol 500 1g Cu2SO4 5H2O per L to inhibit
Glass bottle
biodegradation
Tannin & Polythene/
500 Analyse as soon as possible
Lignin Glass bottle
Chromium ,
arsenic , lead , Polythene/
500 Add 5mL conc. HNO3 / L and refrigerate
Xzinc , Glass bottle
Mercury
Sterilize the bottles in auto clave at 121o c at
E.Coli /Tota
15 lb/inch2 pressure for 15 minutes, collect
bacteria/ 100 Glass bottle
the sample in sterilized bottle and refrigerate
actenomycetis
immediately.
Microplankton/
algae
Add 5mL formalin per 100 mL sample
other 500 Glass bottle
and refrigerate immediately.
biological
organisms

Ex. No: 02 DETERMINATION OF pH
Date:
AIM:
To determine pH of the given sample.
PRINCIPLE:
PH is measured by a pH meter using a glass electrode which generates a potential

varying linearly with the pH of the solution in which it is immersed. It is a Nernstain
concentration cell with potential controlled by the activities of H on either side of a very thin
glass membrane. The latter bottom part of a bulb at the end of a glass tube containing a

reference solution of fixed aH

RT a H ( Sample )
E  Cons tan t  In 
nF a H ( S tan dard )
= Constant + 0.058 pH at 20
A calomel or Ag/AgCl/KCI reference electrode is usually located around the glass

electrode stem for sample operation.
APPARATUS:
1. pH meter along with electrodes

2. Buffer Solution
3. Thermometer

PROCEDURE:
1. Calibrate the electrodes with two standard buffer solutions of solutions of pH 4.0 and
9.2 (A buffer solution is a solution offering resistance to change in pH and whose pH
value is known)
2. The sample temperature is determined at the same time and is entered into the meter
to allow for a temperature correction.
3. Rinse the electrodes thoroughly with deionized distilled water and carefully wipe
with a tissue paper.
4. Dip the electrodes into the sample solution, swirl the solution and wait up to one
minute for steady reading. A pH meter reading within  0.1 pH unit will be adequate
for such work.
5. The reading is taken after the indicated value remains constant for about a minute.
OBSERVATIONS:
pH
Description of sample Temperature
pH meter
RESULTS:
The pH of the given sample is ………………………….

Ex.No:03 DETERMINATION OF TURBIDITY
Date:
AIM:
To find the turbidity of the given samples
PRINCIPLE:
When light is passed through a sample having suspended particles, some of the light
is scattered by the particles. The scattering of the light is generally proportional to the
turbidity. The turbidity if the sample is thus measured from the amount of light scattered by
the sample taking a reference with standard turbidity suspension.
APPARATUS:
1. Naphelometric turbidimeter.
2. Sample tubes.
REAGENTS:
1. Dissolve 1.0 gm Hydrazine sulphate and dilute to 100 ml.

2. Dissolve 10 gm Hexamethylene Tetramine and dilute to 100 ml.
3. Mix 5 ml of each of the above solution (1 and 2) in a 100 ml Volumetric flask and
allow to stand for 24 hours at 25  3C and dilute to 1000 ml. This solution has a
turbidity of 40 NTU.
4. This solution can be kept it for about a month.

PROCEDURE:
1. Switch on Nephelometric turbidimeter and wait for few minutes till it warms up.
2. Set the instrument at 100 on the scale with a 40 NTU standard suspension. In this
case, every division on the scale will be equal to 0.4 NTU turbidity.
3. Shake thoroughly the sample, and keep it for sometime to eliminate the air
bubbles.
4. Take sample in Nephelometer sample tube and put the sample in sample chamber
and find out the value on the scale.
5. Dilute the sample with turbidity free water and again read the turbidity.
OBSERVATIONS:
S.NO Sample Details Turbidity
RESULTS:
The Turbidity of the given sample is ………………………………. (NTU)

Ex.No:04 DETERMINATION OF TOTAL SOLIDS
Date:
AIM:
To determine the total Solids of the given sample.
PRINCIPLE:
Total solids are determined as the residue left after evaporation and drying of the
unfiltered sample.
APPARATUS:
1. Evaporating dishes (pyrex, porcelain or platinum)

2. Oven
3. Desicator
4. Water bath
PROCEDURE:
1. A clean porcelain dish is ignited in a furnace and after partial cooling in the air, it
is cooled in a desicator and weighed.
2. A 10 ml of well mixed sample (graduated cylinder is rinsed to ensure transfer of
all suspend matter) is placed in the dish and evaporated at 100C on water bath,
followed by drying in oven at 103C for 1 hour.
3. Dry to a constant weight at 103C, cool in a desicator and weigh.

CALCULATIONS:
(A - B) x 1000
Total solids (mg/I) =
V
A = Final Weight of the dish in mg.

B = Initial weight of the dish in mg.
C = Volume of sample taken in ml.
OBSERVATIONS:
Sample details Volume of Initial weight of Final weight of Total solids
sample (ml) the dish (mg) the dish (mg) (mg/I)
RESULTS:
The Total Solids of the given sample is ----------------------…………mg/L

Ex.No: 05 DETERMINATIONS OF TOTAL DISSOLVED SOLIDS
Date:
AIM:
To find out Total dissolved solids of the given sample.
PRINCIPLE:
Total dissolved solids are determined as the residue left after evaporation and drying
of the filtered sample.
APPARATUS:
1. Evaporating dishes
2. Oven
3. Desiccator
4. Whatman filter paper No. 44
5. Water bath
PROCEDURE:
1. A clean porcelain dish is ignited in a muffle furnace and after partial cooling in
the air, it is cooled in a desiccator and weighed.
2. A 10 ml of filtered sample is placed in the dish and evaporated at 100C on water
bath, followed by drying in oven at 103C, cool in a desiccator and weigh.

CALCULATIONS:
(A - B) x 1000
TDS (mg / L) =
V
A = Final Weight of the dish in mg.

B = Initial weight of the dish in mg.
V = Volume of sample taken in ml.
OBSERVATIONS:
Sample details Volume of Initial weight of Find weight of Total dissolved

sample (ml) the dish (mg) the dish (mg) solids (mg/I)
RESULTS:
The Total dissolved solids of the given sample is …………………… mg/L

Ex.No: 06 DETERMINATION OF TOTAL FIXED AND VOLATILE
Date: SOLIDS
AIM:
To determine the total fixed and volatile solids of the given sample.
PRINCIPLE:
Total volatile solids and fixed solids are determined as residue remaining after
evaporation, drying at 103C and ignition at 600C.
APPARATUS:
1. Evaporating dish.
2. Oven 103C
3. Muffle furnace 600C
4. Desiccator
5. Water bath
PROCEDURE:
1. A clean porcelain dish is lgnited in a muffle furnace and after partial cooling in air
, it is cooled in a desiccator and weigh (W1).
2. A 10 ml of well mixed sample (graduated cylinder in rinsed to ensure transfer of
all suspended matter) is placed in the dish and evaporated at 100C on water bath,
followed by drying in oven at 103C for 1 hour.
3. Dry to a constant weight at 103C, cool In a desiccator and weigh (W2).
4. Ignite the residue on evaporation at 600C in the muffle furnace to constant
weight in 10 to 15 minutes.
5. Allow the dish to cool and moisten the ash with a few drops of distilled water.
6. Dry to a constant weight at 103C, cool in a desiccator and weigh (W3).

CALCULATIONS:
(W2 - W1) x 1000

(a) Total solids (mg /I) =
ml of sample
(W2 - W3) x 1000

b) Total volatile solids (mg/I) =
ml of sample
(c) Total fixed solids = (a) – (b)

OBSERVATIONS:
Type of Sample Volume of Weight of Weight of Residue

solids details sample ml empty dish empty dish (mg/L)
(mg) + Residue W2-W1
W1 (mg)
W2
Total solids
Total volatile
solids
RESULTS:
The Total Volatile solids of the given sample is …………………… mg/L
The Total Fixed solids of the given sample is …………………… …..mg/L

Ex.No: 07 DETERMINATION OF CHLORIDES
Date:
AIM:
To estimate the amount of Chloride present in the given water sample.
PRINCIPLE:
Chloride ion is determined by Mohr’s method, titration with standard silver nitrate
solution in which silver chloride is precipitated at first. The end of titration is indicated by
formation of red silver chromate from excess AgNO3 and potassium chromate used as an
indicator in neutral to slightly alkaline solution.
 
AgNO 3  Cl  AgCl( white)  NO 3
2AgNO 3
 K 2 CrO 4
 Ag 2
CrO 4
(red)  2KNO 3
APPARATUS:
1. Burette
2. Pipettes
3. Conical flask
REAGENTS:
1. Chloride free distilled water

Potassium chromate indicator Dissolve 50 g K2CrO4 in a little distilled water. Add
AgNO3 solution until a definite red precipitate is formed. Let stand 12 h, filter, and dilute to1
L with distilled water.
2. Standard silver nitrates (0.0141 N):
Dissolve 2.395 g AgNO3 in distilled water and dilute to 1000 mL.

3. Standard sodium chloride (0.0141N):
Dissolve 824.0 mg NaCl (dried at 140°C) in distilled water and dilute to 1000
mL; 1.00 mL = 500 µg Cl.
PROCEDURE:
1. Take 25ml of the sample in conical flask.

2. Adjust its pH to be between 7.0 and 8.0 either with sulphuric acid or sodium
hydroxide solution. Otherwise, AgOH is formed at high pH level or CrO
2
4
is
2
converted to Cr 2 O 7
at low pH levels.
3. Add 1 ml of potassium chromate to get light yellow colour.
4. Titrate with standard silver nitrate solution till colour change from yellow to
brick red.
5. Note the volume of silver nitrate added (A).
6. If more quantity of potassium chromate is added, Ag 2
CrO 4
may form too
soon or not soon enough.
7. Instead of sample use Distilled Water (Blank) (B), repeat the step 2 to 6.
CALCULATIONS:
(A - B) x N x 35.36 x 1000
 Chloride in ( mg/l) 
volume of the sample taken
A = mL titration for sample,

B = mL titration for blank, and
N = normality of AgNO3.
OBSERVATIONS:
Sample details Volume of Initial burette Final burette AgNO 3

solution
sample reading (ml) reading (ml) used (ml)
taken(ml)
Water Sample

Blank
RESULTS:
The amount of Chloride present in the given water sample is ………………. mg/L

Ex.No: 08 DETERMINATION OF HARDNESS
Date:
AIM :
To determine the total hardness, calcium and magnesium of the given sample.
PRINCIPLE:
In alkaline condition, EDTA reacts with Ca and Mg to form a soluble chelated

complex. Ca and Mg ions develop wine- red colour with Erio Chromo black T under alkaline
condition. When EDTA is added as a titrant, Ca and Mg divalent ions get complexed
resulting in sharp change from wine – red to blue which indicates end point of the reaction.
  pH 10  0.1
Ca  Mg  EDTA      Ca EDTA  Mg EDTA
2
M  Erio Chromo Black T  ( M Erio Chromo Black T wine - red)
The pH for this titration has to be maintained at 10.0  0.1. At a higher pH i.e. at
++ ++
about 12, Mg ion precipitates and only Ca ion remain in solution. At this pH Murexide
indicator forms a pink colour with Ca++ . When EDTA is added Ca++ gets complexed
resulting in a change from pink to purple which indicates end point of the reaction.
REAGENTS:
(i) Buffer solution:
Dissolve 16.9 g ammonium chloride (NH4Cl) in 143 mL conc ammonium
hydroxide (NH4OH). Add 1.25 g magnesium salt of EDTA (available commercially) and
dilute to 250 mL with distilled water.
(ii) Erio-Chrome Black ‘T’ Indicator:

 Dissolve 0.5g of solid in 100 ml of ethanol or isopropyl alcohol

(iii) Standard EDTA solution 0.01 M
 Dissolve 3.72 gm of disodium salt of EDTA in distilled water and dilute to 1 litre of
solution .
 Store in polythene container
(iv) Murexide indicator
PROCEDURE:
A. TOTAL HARDNESS :
1. Take 100ml well mixed sample in conical flask.

2. Add 1 -2 ml buffer solution followed by 1ml inhibitor
3. Add 2 drops of Erio Chromo Black T and titrate with standard EDTA (0.01M)
till wine – red colour changes to blue.
4. Note down the volume of EDTA required (A).
CALCULATIONS:
A x 1000
Total hardness (mg/L) as CaCO3 =
ml of sample

B. CALCIUM HARDNESS :
1. Take 25 or 30 ml of sample in conical flask.

2. Add 1 ml NaOH to raise pH to 12.0 and a pinch of murexide indicator.
3. Titrate with EDTA till pink colour changes to purple. Note the volume of
EDTA used (A1).
CALCULATIONS:
A1 x 1000
Total hardness (mg/l ) as CaCO3 =
ml of sample
Where, A1 = Volume of EDTA used by the sample.
C.MAGNESIUM HARDNESS:
Magnesium hardness = Total hardness – Calcium hardness

OBSERVATIONS:
Sample Volume of Initial Final burette EDTA Hardness

details sample burette reading (ml) solution (mg/l)
taken (ml) reading(ml) used (ml)
Total
hardness
Calcium
hardness
RESULTS:
The Total hardness present in the given sample is …………….mg/L.

The Calcium hardness present in the given sample is …………….mg/L.
The Magnesium hardness present in the given sample is …………….mg/L.

Ex.No: 09 DETERMINATION OF SULPHATE
Date:
AIM
To find the amount of sulphates in the given sample.
PRINCIPLE
Benzidine hydrochloride reacts with sulphates in HCl solution to form a slightly
soluble compound of benzidine sulphuric acid.
+ C12 H8 (NH2)2 2 HCl C12 H8 (NH2)2 H2SO4
Benzidine Sulphuric acid
+ Ca++ + Mg++ + Cl2
The compound is filtered and washed entirely free of HCl. The amount of H2SO4 in
the compound is determined by titration with standard NaOH (0.05 N)
C12 H8 (NH2)2H2SO4 + 2NaOH Na2 SO4 + C12 H8 (Na2)2 2H2O
APPARATUS
1. Filter paper
2. Beaker
3. Hot pan
4. Burette
5. Pipettes
REAGENTS
1. Hydroxylamine chloride
2. Benzidine hydrochloride
3. NaOH (0.05N)
4. Phenolphthalein indicator
REAGENT PREPARATION
Hydroxylamine HCl solution
Dissolve 10gm of NH2OH. HCl in distilled and dilute to 100 ml of distilled

water.
Sodium hydroxide 0.05N
Dissolve 0.2 gm of NaOH in distilled water and dilute to 100 ml with distilled
water.
PROCEDURE

1. Take 125 ml of sample in a 400 ml beaker.
2. Add 5 ml of hydroxylamine chloride and then add 10 ml benzidine hydrochloride.
3. Stir the mixture vigorously and allow the precipitate to settle.
4. Filter the solution and wash the beaker and the filter paper with cold distilled
water.
5. Pierce the filter paper in the funnel and wash the precipitate formed on the filter
paper to the original beaker with 100 to 150 ml distilled water.
6. Heat the beaker to dissolve the contents for 20 to 30 minutes.
7. Add 2 drops of Phenolpthalein indicator and titrate with 0.05N NaOH until pink
colour is developed.
FORMULA
Concentration of sulphates (mg/l) = X1000
RESULT
The amount of sulphates in the given sample is ------------------------

Ex.No: 10 DETERMINATION OF DISSOLVED OXYGEN
Date: (WINKLER METHOD)
AIM:
To find the quantity of dissolved oxygen present in the given sample.
PRINCIPLE:
Oxygen present in sample oxidizes the divalent manganous to its higher valency
which precipitates as a brown hydrated oxide after addition of NAOH and KI. Upon
acidification, manganese reverts to divalent state and liberates iodine from KI equivalent to
D.O. content in the sample. The liberated iodine is titrated against Na2S2o3 (0.25N), Using
starch as an indicator. If oxygen absent in sample, the MnSO4 react with the alkali to form
precipitate Mn(OH)2 .
APPARATUS:
1. BOD bottles (capacity 300ml)

2. Sampling device for collection of samples
3. Burette
4. Pipettes
REAGENTS:
(i) Sodium thio sulphate solution:

Dissolve 6.2gm of sodium thio sulphate crystal and dilute to one liter. Preserve
the solution by adding 1ml of chloroform and 1 gm of NaoH per liter of
solution.
(ii) Potassium dichromate:
Dissolve 0.3 gm of Potassium dichromate in 500 ml of distilled water.
(iii) Potassium Iodide:
Dissolve 10 gm of potassium Iodide in 100 ml of distilled water.
(iv) Hydrochloric acid:

Dissolve 150 ml of Hcl in one liter of water.
(v) Manganous Sulphate solution:

Dissolve 48 gm of manganous sulphate in 100 ml of distilled water.
(vi) Alkaline potassium iodide:

Dissolve 70 gm of potassium hydroxide or 40 gm of NaOH & 15 gm of
potassium iodide in 100 ml of distilled water.
(vii) Starch Indicator:

Take 0.5 gm of Starch prepare a paste with water & make 100 ml with distilled
water and boil by stirring and finally cool to room temperature.
PROCEDURE:
Estimation for Dissolved oxygen:
1. Take the BOD bottle and collect 300 ml of water sample into it.
2. Add 2 ml of manganous sulphate and 2ml of alkali iodine-azide solution to the BOD
bottle. The tip of the pipette should be below the liquid level, while adding these
reagents.
3. Restopper with care to exclude air bubbles and mix by repeatedly inverting the bottle
2 to 3 times.
4. If no oxygen is present, the manganous ion reacts with hydroxide ion to form white
precipitate of Mn (OH)2. If oxygen is present, some Mn++ is oxidized to M++++ and
precipitates as a brown coloured manganic oxide.
 
Mn  2 ( OH )  Mn ( OH ) 2 ( white )

  1
Mn  2 ( OH )  O 2  MnO 2
( brown )  H 2 O
2
5. After shaking and allowing sufficient time for all oxygen to react, the chemical
precipitates are allowed are allowed to settle leaving clear liquid within the upper
portion.
6. 2 ml of concentrated sulphuric acid is added.
7. The bottle is restoppered and mixed by inverted until the suspension is completely
dissolved and yellow colour is uniform throughout the bottle.
   
MnO 2
 2l  4H  Mn  l2  2 H 2
O
8. A Volume of 203 ml is taken into the conical flask and 2 ml of starch solution is
added
9. Titrated with 0.025 N sodium thiosulphate solutions until yellow colored iodine turns
to a pale straw colour.
10. Continue titration till the disappearance of the blue colour.
CALCULATIONS:
For titration of 200 mL sample, 1 mL 0.025M Na2S2O3 = 1 mg DO/L

OBSERVATIONS:
Sample Temp of Volume of Initial Final ml of D.O. in

details sample C sample burette burette Na2S2O3 mg/l
taken ml reading reading solution
ml ml used
RESULTS:
The amount of Dissolved oxygen present in the given sample is ………… mg/L.

Ex.No:11 DETERMINATION OF BIOCHEMICAL OXYGEN
Date: DEMAND.
AIM:
To determine Biochemical oxygen demand (BOD) exerted by the given waste water
sample.
PRINCIPLE:
The BOD is an empirical biological test. This BOD test may be considered as wet
oxidation procedure in which the living organisms serve as the medium for oxidation of the
organic matter to carbon-dioxide and water.
 a b 3   a 3 
 n    c O 2      nCO    c  H 2 O  cNH
Bacteria
CnH a
ObN c 2 3
 4 2 4   2 2 
On the basis of the above relationship, it is possible to interpret BOD data in terms of
organic matter as well as the amount of oxygen used during its oxidation.
REAGENTS:
a.Phosphate buffer solution:

Dissolve 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4⋅7H2O, and 1.7 g
NH4Cl in about 500 mL distilled water and dilute to 1 L. The pH should be 7.2
without further adjustment. Alternatively, dissolve 42.5 g KH2PO4 or 54.3 g
K2HPO4 in about 700 mL distilled water. Adjust pH to 7.2 with 30% NaOH and
dilute to 1 L.

b. Magnesium sulfate solution:
Dissolve 22.5 g MgSO4⋅7H2O in distilled water and dilute to1 L.
c. Calcium chloride solution:

Dissolve 27.5 g CaCl 2 in distilled water and dilute to 1 L.
d. Ferric chloride solution:

Dissolve 0.25 g FeCl3⋅6H2O in distilled water and dilute to 1 L.
e. Acid and alkali solutions, 1N, for neutralization of caustic or acidic waste samples.
1) Acid—slowly and while stirring, add 28 mL conc sulfuric acid to distilled water.
Dilute to 1L.
2) Alkali—Dissolve 40 g of sodium hydroxide in distilled water. Dilute to 1 L.
f. Sodium sulfite solution:

Dissolve 1.575 g Na2SO3 in 1000 mL distilled water. This solution is not stable;
prepare daily.
h. Glucose-glutamic acid solution:

Dry reagent-grade glucose and reagent-grade glutamic acid at 103°C for 1 h. Add 150
mg glucose and 150 mg glutamic acid to distilled water and dilute to 1 L. Prepare fresh
immediately before use.
i. Ammonium chloride solution:

Dissolve 1.15 g NH4Cl in about 500 mL distilled water, adjust pH to 7.2 with NaOH
solution, and dilute to 1 L. Solution contains 0.3 mg N/mL.
j. Dilution water:
Use demineralized, distilled, tap, or natural water for making sample dilutions.
Aerate the required volume of distilled water in a container by passing compressed air
for 1 to 2 days to obtain the saturation of DO. Try to maintain the temperature at 20oC.
PROCEDURE:
1. Place the desired volume of distilled water in a 5 litre flask. Aeration is done by
bubbling compressed air through water.
2. Add 1 ml of phosphate buffer, 1 ml of magnesium sulphate solution. 1 ml of calcium
chloride solution and 1 ml of ferric chloride solution for every litre of distilled water
(dilution water).
3. In the case of the waste waters which are not expected to have sufficient bacterial
population, add seed to the dilution water. Generally, 2 ml of settled sewage is
sufficient for 1,000 ml of dilution water.
4. Highly acidic or alkaline samples are to be neutralized to a pH of 7.

5. Add 2 or 3 ml of sodium thiosulphate solution to destroy residual chlorine if any.
6. Take the sample as follows
Strong wastes: 0.1, 0.5 or 1%.
Settle domestic sewage: 1, 2.5 or 5%
Treated effluents: 5, 12.5 or 25%
River water 25% to 100%.
7. Dilute the sample with the distilled water and mix the contents well.
8. Take diluted sample with into 2 BOD bottles.
9. Fill another two BOD bottles with diluted (distilled) water alone.
10. Immediately find D.O. of a diluted waste water and diluted water. (distilled water)
11. Incubate the other two BOD bottles at 20C for 5 days. They are to be tightly
stoppered to prevent any air entry into the bottles.
12. Determine D.O. content in the incubated bottles at the end of 5 days (120 hours).

CALCULATIONS:
Let initial D.O. of diluted sample = DO

D.O. at the end of 5 days for the diluted sample = D5
Initial D.O. of dilution water (blank) = CO
D.O at the end of 5 days for the distilled water (blank) = C5

 D.O depletion of dilution water = CO – C5
D.O. depletion of the diluted sample = DO – D5
 D.O. depletion due to microbes = (DO - D5) – (CO - C5)
BOD at 20C of the sample
 (D  D 5 ) x vol .of bottle 

=   (C  C
0
0 5
)
 mlsample 

OBSERVATIONS:
S. No Volume Dilution Initial Initial Final Final 5 days

sample ratio D.O of D.O of D.O. of D.O. of BOD at
(ml) sample blank Sample blank 20C
mg/l mg/l mg/l (mg/l)
mg/l
RESULTS:
The BOD5,27oC of the given sample is ………………… mg/L

Ex.No:12 DETERMINATION OF CHEMICAL OXYGEN
Date: DEMAND
AIM:
To find out chemical oxygen demand (COD) of given waste water sample.
PRINCIPLE:
The organic matter present in sample gets oxidized completely by K2Cr2O7 in the
presence of H2SO4 to produce CO2 and H2O. The excess K2Cr2O7 remaining after the
reaction is titrated with Fe (NH4)2 (SO4)2. The dichromate consumed gives the O2 required to
oxidation of the organic matter.
APPARATUS:
1. Reflux apparatus
2. Hot plate/heating mantle
3. Burette
REGENTS:
1. Standard potassium dichromate solution, 0.04167M:

Dissolve 12.259 g K2Cr2O7, primary standard grade, previously dried at 150°C for 2
h, in distilled water and dilute to 1000 mL. This reagent undergoes a six-electron
reduction reaction; the equivalent concentration is 6 ×0.04167M or 0.2500N.
2. Sulfuric acid reagent:

Add Ag2SO4, reagent or technical grade, crystals or powder, to conc H2SO4 at the rate
of 5.5 g Ag2SO4/kg H2SO4. Let stand 1 to 2 days to dissolve.

3. Ferro in indicator solution:
Dissolve 1.485 g 1,10-phenanthroline monohydrate and 695 mg FeSO4⋅7H2O in
distilled water and dilute to 100 mL. This indicator solution may be purchased
already prepared.
4. Standard ferrous ammonium sulfate (FAS) titrant, approximately 0.25M:

Dissolve 98 g Fe(NH4)2(SO4)2⋅6H2O in distilled water. Add 20 mL conc H2SO4, cool,
and dilute to 1000 L.
5. Mercuric sulfate, HgSO4, crystals or powder
PROCEDURE:
1. Place 0.4 gm of HgSO4 in the reflux flask.

2. Add 20ml of sample (or an aliquot diluted to 20ml)
3. 10ml of more concentrated dichromate solution are placed into flask together with
glass beeds.
4. Add slowly 30ml of H2SO4 containing Ag2SO4 and mix thoroughly.
5. Connect the flask to condenser. Mix the contents thoroughly before heating. Improper
mixing results in bumping and the sample may be blown out.
6. Reflux for a minimum period of 2 hours. Cool and wash down the condenser with
distilled water.
7. Dilute the sample to make up 150ml and cool.
8. Titrate excess K2Cr2O7 with 0.1N Fe (NH4)2 SO4 using ferroin indicator. Sharp colour
change from blue green to wine-red indicates the end point.
9. Reflux the blank in the same manner using distilled water instead of sample.

CALCULATIONS:
A  B  x M x 8 x 1000
 COD =
Quantity of sample ( ml )
Quantity of Fe(NH4)2(SO4) added for blank = A ml

Quantity of Fe(NH4)2(SO4) added for the sample = B ml
Molarity of Fe(NH4)2(SO4) =M
OBSERVATIONS:
S .No Sample Vol. of Initial Final burette Vol. of COD of the

details sample burette reading ml Fe(NH4)2 sample mg/l
taken ml reading ml (SO4)2
RESULTS:
The COD of the given sample is …………………..mg/L

Ex.No:13 DETERMINATION OF RESIDUAL CHLORINE
Date: (STARCH IODIDE METHOD)
AIM:
To determine the amount of residual chlorine present in the given water sample.
APPARATUS REQUIRED:
Burette , pipette, conical flask, std.reagent bottle ,wash bottle and weight box
PRINCIPLE:
Drinking water is chlorinated with free chlorine or with the hypo chlorites
for disinfections. Therefore it contains residual chlorine i.e.excess chlorine not consumed by
the pollutants in water .Starch-oxide method for determination of residual chlorine depends
upon the oxidizing power of free and combined residual chlorine in water to convert iodide
to free iodine . This oxidation is represented as
Cl2 +2I- → I2+2Cl-
I2 + 2Na2S2O3 → Na2S4O6 + 2NaCl
REAGENTS
1. Standard Sodium thio Sulphate Solution 0.025 N:
Dissolve 25 g of sodium thiosulphate in one liter of distilled water
2. Potassium dichromate:
Dissolve 4.9 g of potassium dichromate in 1000 ml of distilled water.
3. Potassium Iodide:
Dissolve 10 g of potassium iodide in 100 ml of distilled water.
4. Hydrchloric acid:
Dissolve 150 ml of conc. Hydrochloric acid in one liter of distilled water.
5. Starch indicator:

Take 0.5 g of starch, prepare a paste with water and make 100 ml with water and
boiled by stirring and finally cooled to room temperature.
PROCEDURE:
Estimation of residual chlorine:
1. Take given sample of water in 200 ml bottle and add 5 ml of Hcl and 5 ml of KI.
2. Add drops of starch shake the bottle and keep it for 5 minutes without disturbance.
3. Take the contents in a conical flask and titrate it against sodium thiosulphate taken in
the burette.
4. End point is the change of color from blue to colorless.
5. Repeat the titration for concordant values.
CALCULATION:
( A  B ) XNX 35 . 45
X 1000  AmountofCh lorine
Quantity of sample ( ml )
A = mL titration for sample,

B = mL titration for blank, and
N = Normality of Na2S2O3.
OBSERVATION:
S .No Sample Vol. of Initial Final burette Vol. of Residual
details sample burette reading ml Na2S2O3. chlorine
taken ml reading ml mg/l
RESULT:

The amount of residual chlorine present in the given sample is ---------------- mg/L

Ex.No:14 DETERMINATION OF OPTIMUM AMOUNT OF
Date: COAGULANT
AIM:
To find the optimum amount of coagulant required to treat the turbid waters.
PRINCIPLE:
Metal salts hydrolyze in presence of the natural alkalinity to form metal hydroxides.
The divalent cations can reduce the zeta – potential, while the metal hydro oxides are good
absorbents and hence remove the suspended particles by enmeshing them.
APPARATUS:
1. Jar test apparatus

2. Beakers
3. Pipettes
4. Colorimeter
5. Turbidity meter
6. pH meter
REAGENTS:
1. Alum solution ( synthetic coagulant)
2. Normal solution (natural coagulant)
PROCEDURE:
1. Measure initial turbidity of the sample.

2. Measure 1 liter quantities of the water to be tested into a series of glass jars.
3. Attach to stirring device.( Jar test apparatus).
4. Add progressive volumes of the chemical solution to each of the jars covering the
range of chemical dosage expected.( 1ml,2ml,3ml,…etc)
5. Mix rapidly each sample for 1 minute.
6. Reduce the speed to about 10 rpm and mix for 15 minutes, (Flocculation).
7. Allow the floes to settle for 15 minutes.
8. Measure turbidity of each settled sample.
9. Plot graph % removal of turbidity Vs. Coagulant dose and select the optimum
dosage.
PRECAUTIONS:
1. Add coagulant doses simultaneously to all glass jars while stirring.

2. It is advisable to siphon out the settled sample from the jars so as not to disturb the
settled floc.

OBSERVATIONS:
Initial Turbidity of the sample --------------NTU
S.No Coagulant Dose Added Final turbidity % Removal
RESULT:
Optimum dose of coagulant =-----------mg/L

Ex.No:15 DETERMINATION OF AVAILABLE CHLORINE
Date:
AIM:
To determine the available chlorine in the given sample of bleaching powder.
PRINCIPLE:
Chlorine is a strong oxidizing agent and liberates iodine from iodide ion.
Cl 2
 2 Kl  l 2  2KCl
Starch give blue colour with iodine
l 2  Starch  Blue colour
The liberated iodine is titrated with standard sodium thiosulphate – a reducing agent.
l 2  2Na 2
S 4O 3
 Na 2
S 4O 6
 2Nal.
The disappearance of blue colour indicates the completion of reaction with free iodine
is converted back to iodide.
APPARATUS:
1. Conical flask
2. Burette
3. Pipette
REAGENTS:
1. Concentrated acetic acid,glacial.

2. Potassium iodide crystals

3. Sodium thiosulphate 0.025N:
Dissolve 25 g of sodium thiosulphate in one liter of distilled water.
4. Starch indicator.
Take 0.5 g of starch, prepare a paste with water and make 100 ml with water
and boiled by stirring and finally cooled to room temperature
PROCEDURE:
1. Take 5gm of fresh bleaching powder. Add small quantity of water to it, and
prepare fine paste. Add some more water, stir and allow settling for a few
minutes. Dilute it with distilled water to make upto 1 litre and stopper the
container.
2. Take 25ml of the bleaching powder solution in a conical flask and add a pinch of
Kl
3. Add 10ml of acetic acid and allow the reaction to complete
4. Titrate the sample with standard sodium thiosulphate solution until the yellow
colour of the liberated iodine is almost faded out.
5. Add 1 ml of starch solution and titrate until the blue colour disappears.
6. Note down the quantity of sodium thiosulphate added(V1).
7. Repeat the same procedure for distilled water.
8. Note down the volume of sodium thiosulphate added (V2).
CALCULATIONS:
( V 1  V 2 ) x N x 35.45 x 1000
Concentration of chlorine =
Volume of bleaching powder solution
V1= Vol of Na3s2o3 (sample)

V2= Vol of Na3s2o3 (Blank)
N = Normality of Na3s2o3

OBSERVATIONS:
Volume of Initial Final Available

Sample
S .No sample burette burette chlorine
details
taken ml reading reading mg/l
RESULTS:
The available chlorine in the given sample is………………….mg/L

Ex.No:16 DETERMINATION OF FLUORIDE
Date:
AIM:
To determine the Fluoride in the given water sample.
PRINCIPLE:
The test is based on the fact that fluoride ion combines with zirconium ion to form a
stable complex ion,ZrF5 and these results in bleaching the reddish colour of Zirconium and
alizarin combination. The decrease in intensity of colour is directly proportional to fluoride
concentration.
EQUIPMENTS:
a. Spectrophotometer, for use at 570 nm, providing a light path of at least 1 cm
REAGENTS:
1. Stock fluoride solution:

Dissolve 221.0 mg anhydrous sodium fluoride, NaF, in distilled water and dilute to
1000 mL; 1.00 mL = 100 µg F–.
2. Standard fluoride solution:

Dilute 100 mL stock fluoride solution to 1000 mL with distilled water; 1.00 mL =
10.0 µg F–.
3. SPADNS solution:
Dissolve 958 mg SPADNS, sodium 2-(parasulfophenylazo)-1,8-dihydroxy-3,6-
naphthalene disulfonate, also called 4,5-dihydroxy-3-(parasulfophenylazo)-2,7-
naphthalenedisulfonic acid trisodium salt, in distilled water and dilute to 500 mL.
This solution is stable for at least 1 year if protected from direct sunlight.

4. Zirconyl-acid reagent:
Dissolve 133 mg zirconyl chloride octahydrate, ZrOCl2⋅8H2O, in about 25 mL
distilled water. Add 350 mL conc HCl and dilute to 500 mL with distilled water.
5. Acid zirconyl-SPADNS reagent:

Mix equal volumes of SPADNS solution and zirconyl-acid reagent. The combined
reagent is stable for at least 2 years.
6. Reference solution:
Add 10 mL SPADNS solution to 100 mL distilled water. Dilute 7 mL conc. HCl to 10
mL and add to the diluted SPADNS solution. The resulting solution, used forsetting
the instrument reference point (zero), is stable for at least 1 year. Alternatively, use a
Prepared standard of 0 mg F–/L as a reference.
7. Sodium arsenite solution:

Dissolve 5.0 g NaAsO2 and dilute to 1 L with distilled water.
PROCEDURE:
a. Preparation of standard curve:
1. Prepare fluoride standards in the range of 0 to 1.40 mg F–/L by diluting appropriate

quantities of standard fluoride solution to 50 mL with distilled water.
2. Pipet 5.00 mL each of SPADNS solution and zirconyl-acid reagent, or 10.00 mL
mixed acid-zirconyl-SPADNS reagent, to each standard and mix well. Avoid
contamination.
3. Set photometer to zero absorbance with the reference solution and obtain absorbance
readings of standards.
4. Plot a curve of the milligrams fluoride-absorbance relationship.
5. Prepare a new standard curve whenever a fresh reagent is made or a different standard
temperature is desired.

b. Sample pretreatment:
If the sample contains residual chlorine, remove it by adding 1 drop (0.05 mL)
NaAsO2 solution/ 0.1 mg residual chlorine and mix. (Sodium arsenite concentrations
of 1300 mg/L produce an error of 0.1 mg/L at 1.0 mg F–/L.)
OBSERVATIONS:
S.NO Concentration Absorbance
RESULTS:
The Concentration of the fluoride in the given sample is …………. (mg/L)

Ex.No:17 DETERMINATION OF IRON
Date:
AIM:
To find the total iron present in the given sample.
APPARATUS:
1. Nesseler’s tubes (100ml)

2. Beaker
3. Pipettes.
REAGENTS:
1. Potassium permanganate, 0.1M: Dissolve 0.316 KMnO4 in reagent water
and dilute to 100 mL.
2. HCL
3. Stock iron solution: Use metal (1) or salt (2) for preparing the stock solution.
1) Use electrolytic iron wire, or ‘‘iron wire for standardizing,’’ to prepare the
solution. If necessary, clean wire with fine sandpaper to remove any oxide
coating and to produce a bright surface. Weigh 200.0 mg wire and place in a
1000-mL volumetric flask. Dissolve in 20 mL 6N sulfuric acid (H2SO4) and
dilute to mark with water; 1.00 mL = 200 µg Fe.
2) If ferrous ammonium sulfate is preferred, slowly add 20 mL conc H2SO4 to
50 mL water and dissolve 1.404 g Fe(NH4)2(SO4)2⋅6H2O. Add 0.1M
potassium permanganate (KMnO4) dropwise until a faint pink color persists.
Dilute to 1000 mL with water and mix; 1.00 mL = 200µg Fe.
4. Standard iron solutions: Prepare daily for use.

1) Pipet 50.00 mL stock solution into a 1000-mL volumetric flask and dilute
to mark with water; 1.00 mL = 10.0 µg Fe.

2) Pipet 5.00 mL stock solution into a 1000-mL volumetric flask and dilute to
mark with water; 1.00 mL = 1.00 µg Fe.
PROCEDURE:
1. Place 100ml of the well shaken sample in a 250ml breaker , and add 5ml HCL.
Reduce the volume of to 250ml beaker, and 40ml by placing on a hot plate cool
and add Potassium permanganate solution drop by drop until a pink colour
persist for atleast 5 minutes. Transfer to a 50ml Nessler tube and make up to the
mark.
2. Pipette 1.0ml, 2.0ml, 3.0ml, 4.0ml ,5.0ml and 6.0ml iron standard solution in to
50ml Nessler tubes , add 1ml dil.HCL and 2 drops of Potassium ermanganate
solution.
3. Mix well and make up to the mark with distilled water.
4. Add to the standards and sample 1ml thiocyanate solution and mix well.
5. Compare the colour of the sample with that of the standards and find out the mg
equivalent of iron present in the sample as follows.
CALCULATION:
Mg/l iron as Fe = Matching std X 0.01 X 1000
Ml of sample taken
RESULT:
The Total iron present in the given sample is ……………………. mg/L

Ex.No:18 DETERMINATION OF ACIDITY
Date:
AIM
To determine Acidity (Base capacity) of the given sample.
PRINCIPLE
The mineral acids present in the sample which are contributing mineral acidity can be
calculated by titrating or neutralizing samples with strong base NaOH to pH 4.3. The CO2
and bicarbonates (carbonic acid) present and contribute CO2 acidity in the sample can be
neutralized completely by continuing the titration to pH 8.2.
APPARATUS
1. Burette
2. Conical flask
3. Pipette
REAGENTS
1. Standard sodium hydroxide (0.02 N)

3. Methyl orange indicator
4. Sodium thiosulphate (0.1 N)
5. Carbon dioxide free distilled water.
REAGENT PREPARATION
Standard sodium hydroxide (0.02N)

0.8 gm of NaOH is dissolved in CO2 free distilled water and diluted to
1000 ml. it is standardized against 0.02N potassium biopthalate,KHC3H4O4.store in air tight,
rubber
stoppered Pyrex/corning glass bottle to protect from atmospheric CO2.
Sodium thiosulphate (0.1N)

Dissolve 24.82 g of A.R. Na2S2O3.5H2O (molecular weight 248.21, eq.wt.
248.21) in 1 liter of boiled out distilled water.
Methyl orange indicator

0.5 gm of methyl orange powder is dissolved in CO2 free distilled water and
diluted to one litre.

Phenolphthalein indicator
5 gm of phenolphthalein powder is dissolved in 500 ml of 95% ethyl-
alcohol, and made up 1 litre with distilled water. Add drop wise 0.02N NaOH till faint
pink colour appears.
PROCEDURE
1. Take 100 ml of the given sample in an Erlenmeyer flask.

2. Add 1 drop of 0.1 N sodium thiosulphate solution to remove the residual chlorine if
present.
3. Add 2 drops of Methyl orange. The sample turns pink.
4. Proceed with titration until the colour changes to yellow.
5. Note down the volume of the NaOH added (V1).
6. Take another conical flask containing 100 ml of water sample; add 2 or 3 drops of
phenolphthalein.
7. Proceed with titration until the sample turns pink.
8. Note down the total volume of NaOH added (V2).
CALCULATIONS
Mineral acidity due to mineral acids (as CaCO3) (mg/l)
=
CO2 acidity due to CO2 (as CaCO2 (mg/l)
OBSERVATIONS AND RESULTS
Methyl orange indicator Phenolphthalein Indicator

Sa Volume Initia Fina NaO Initia Final NaOH
mpl of l l H l burett Used
e buret bure Used buret e
deta Sample te tte te readi (ml)
ils (ml) readi read (ml) readi ng
ng ing ng (ml)
(ml) (ml) (ml)
RESULTS
The acidity of the given sample is ----------------------------

Ex.No:19 DETERMINATION OF ALKALINITY
Date:
AIM
To determine the alkalinity of the given sample.
PRINCIPLE
Alkalinity can be obtained by neutralizing OH--, CO3-- -- and HCO3-- with standard
H2SO4. Titration to pH 8.3 or decolourization of phenolphthalein indicator will show
complete neutralization of OH-- and ½ of CO3-- --, while to complete neutralization of OH--
and ½ of
CO3-- --, while to pH 4.4 sharp change from yellow to pink of methyl orange indicator will
indicate total alkalinity i.e. OH--, CO3-- -- and HCO3—
APPARATUS
1. Burette
2. Conical flask
3. Pipettes
REAGENTS
1. Standard sulphuric acid (0.02 N)

3. Methyl orange
4. Carbon dioxide free distilled water
5. Sodium thiosulphate (0.1N).
REAGENT PREPARATION
Standard sulphuric acid(0.02N)

Prepare 0.1N H2SO4 by diluting 3 ml of conc.H2SO4 to1000 ml. standardize
it
against standard Na2CO3 0.1N.take appropriate volume of H2SO4 (approximately 0.1N) and
dilute with distilled water to make up to 1 litre to obtain standard 0.02N H2SO4
Sodium thiosulphate (0.1N)

Dissolve 24.82 g of A.R. Na2S2O3.5H2O(molecular weight 248.21, eq.wt.
248.21) in 1 liter of boiled out distilled water.
Methyl orange indicator

0.5 gm of methyl orange powder is dissolved in CO2 free distilled water and
diluted to one litre.
Phenolphthalein indicator

5 gm of phenolphthalein powder is dissolved in 500 ml of 95% ethyl-
alcohol, and made up 1 litre with distilled water. Add drop wise 0.02N NaOH till faint
pink colour appears.
PROCEDURE
1. Take 100 ml of the given sample in a conical flask.
2. Add one drop of 0.1N sodium thiosulphate solution to remove the free residual
chlorine if present.
3. Add 2 drops of phenolphthalein indicator. The sample turns pink.
4. Run down 0.02N standard sulphuric acid till the solution turns to colour less.
5. Note down the volume of H2SO4 added (V1).
6. Add 2 drops of methyl orange indicator the sample turns to yellow.
7. Resume titration till the colour of the solution turns to pink.
8. Note down the total volume of H2SO4 added (V2).
CALCULATIONS
1. Phenolphthalein alkalinity (P) (mg/l) as CaCO3
2. Total alkalinity (T) as CaCO3 mg/l
Value of P and Alkalinity due to

T OH -
CO- - HCO3-
P=0 0 0 T
P < 1/2T 0 2P T-2P
P = 1/2T 0 2P 0
P >1/2T 2P-T 2T-2P 0
P=T T 0 0

OBSERVATIONS AND RESULTS
Phenolphthalein Methyl orange
Sa Volume Initia Fina H2SO Initia Final H2SO4
mpl of l l 4 l burett Used
e Sample buret bure Used buret e
deta (ml) te tte te readi (ml)
ils readi read (ml) readi ng
ng ing ng (ml)
(ml) (ml) (ml)
RESULTS
For a given sample,
Hydroxide alkalinity (mg/l ) =
Carbonate alkalinity (mg/l) =
Bicarbonate alkalinity (mg/l) =

Ex.No: 20 DETERMINATION OF CONDUCTIVITY
Date:
AIM
To determine the conductivity of the given sample.
PRINCIPLE
The electrical conductivity is a total parameter for dissolved, dissociated substances. Its
value depends on the concentration and degrees of dissociation of the ions as well as the
temperature and migration velocity of the ions in the electric field.
APPARATUS
1. Conductivity meter with measuring cell
2. Beaker
3. Thermometer
REAGENTS
1. KCl 0.1 N
PROCEDURE
1. Calibrate the cell with standard 0.1 N KCl solution of conductivity 14.12 mmhos at
30 C.
2. Rinse the cell thoroughly with deionized distilled water and carefully wipe with a
tissue paper.
3. Dip the cell into the sample solution, swirl the solution and wait upto 1 minute for a
steady reading.
4. Note down the Instrument reading and also temperature by a thermometer.
OBSEVATIONS
Description Temperature Conductivity Remarks

of sample (mmhos)
RESULT
The conductivity of the given sample is --------------------------

Ex. No: 21 PHOSPHATE DETERMINATION BY ASCORBIC ACID METHOD
Date: (SPECTROPHOTOMETRY)
Aim:
The objective of the experiment is to determine PO4 (phosphate) in water and
wastewater.
Principle:
Phosphate and molybdate ions combine in acidic solution to form 12-
molybdophosphoric acid, which can be measured at 340nm. Alternativelyupon treatment
with a suitable reducing agent such as hydrazine sulphate, thereaction yields a highly
coloured blue product called heteropoly blue. Acidicmolybdate can also be reduced to a blue
substance, but only in less acidicsolutions. Although the blue heterpoly compound has not
been characterizedcompletely, it appears to have a molecular composition similar to that of
theunreduced species, differing only in that some of the covalently boundmolybdenum atoms
are in a +5 rather than +6 oxidation state..
REAGENTS:
Stock Solution:
This solution is only needed to make standard solution.
Dissolve 219.5 mg of anhydrous KH2PO4 and dilute to 1,000 ml. This solution will be 50 ug
P/ ml.
Standard Solution:
Dilute 25 ml of stock solution to 500 ml. This will be 2.5 ug P/ml.
The working standard is 2.5 ug/ml (2500 ppb) of phosphorus in phosphate (PO4-P). Use it to
make a series of standards.

Standard Solutions:
Volume (ul) used of the Standard Conc.

working standard, ( ppb PO4-P )
diluted to 25 ml.
5N Sulufuric Acid:
Dilute 70 ml of conc. sulfuric acid to 500 ml.
Potassium antimonyl tartarate - K(SbO)C4H4O6.1/2 H2O:

The bottle of the solid salt may be labeled as: Potassium antimoy(III) oxitartarate, or
Antimony Potassium Tartarate.
Dissolve 1.3715 g in 400 ml H2O, Dilute to 500 ml. Store in glass bottle.
4% Ammonium Molybdate - (NH4)6 Mo7O24.4H2O:

Dissolve 20 g in 500 ml. Stir for sometime (HARD TO DISSOLVE !!)
Store in glass stoppered bottle.
Ascorbic Acid:
Dissolve 1.76 g in 100 ml H2O.
This solution is stable for ONE WEEK ONLY at 4 degrees C.

Combined Reagent:
For 100 ml of the combined reagent add the following, IN ORDER:
- 50 ml of 5N sulfuric acid.
- 5 ml Potassium antimonyl tartarate. STIRR THOROUGHLY
- 15 ml Ammonium Molybdate. STIRR THOROUGHLY
- 30 ml Ascorbic acid. STIRR THOROUGHLY
- Let the reagent cool down to room temperature.
This combined reagent is stable for 4 HOURS ONLY !!!
PROCEDURE:
- Take 25 ml sample in a 50-ml graduated tube

- Add 4 ml combined reagent.
- Cover the tube with parafilm and shake well.
- Wait for blue color to develop. It needs 10-30 minutes time.
- Measure absorbance on the Spectronic-21 at the wavelength 880 nm.
RESULT:
The amount of Phosphate found in water sample is………………………2.5ug P/ml.

EX.NO: 22 DETERMINATION OF SVI OF BIOLOGICAL SLUDGE AND
MICROSCOPIC EXAMINATION
DATE:
Aim
To determine the sludge volume index in order to find out the physical state of sludge
produced in a biological aeration system.
Apparatus required
Graduated cylinder of 1L, Hot air oven, Weighing balance, Glass beaker.
Theory
Sludge volume index (S.V.I ) is defined as the volume of sludge in ml occupied by 1
gm of solids in the activated mixed liquor after settling for 30 min. In a 1000ml graduated
cylinder.
SVI = Vs (mL/L) X 1000 / MLSS (mg/L)
Vs - Volmue of settled solids in a 1L graduate cylinder after 30 min (mL/L)
MLSS - Mixed Liquor Suspended Solids in mg/L
The amount of recycle flow depends to a great extent on the settling characteristic of
MLSS.
If the SVI is So the recycle ratio required could be about 0.2. The volume of SVI is of
operational importance, since it refers to the treatment system. Any increase in SVI number
increases of MLSS concentration nitrate their settling characteristics of solids.
Procedure
1. Collect 1L of sample of mixed liquor from the aeration tank.
2. Allow the mixed liquor to settle for 30min, and the settled sludge volume is reduced.
3. The above sample of mixed liquor after remaining of settled solids in cylinder is
taken.
4. For further testing of MLSS by available standard method adopted for measuring
suspended solids in sewage.
5. If SVI lies between 50-150 mg/L indicates good settling sludge.
Result
The sludge volume index of given sample is _________________

Ex.No:23 DETERMINATION OF MPN INDEX OF WATER SAMPLE
Date:
Aim:
To analyse water coliforms by MPN.
Principle:
Most Probable Number (MPN) method for coliform bacteria-using the multiple
tube fermentation technique: In this method the MPN of total coliform bacteria, faecal
coliform bacteria, faecal coliform bacteria (or the thermotolerant coliforms) present in the
water sample is determined, along with the presence / absence of Escherichia coli. In the
multiple-tube method, a series of tubes containing a suitable selective broth culture medium
(lactose-containing broth, such as MacConkey broth) is inoculated with test portions of a
water sample. After a specified incubation time at a given temperature, each tube showing
gas formation is regarded as “presumptive positive” since the gas indicates the possible
presence of coliforms. However, gas may also be produced by other organisms, and so a
subsequent confirmatory test is essential. The two tests are known respectively as the
presumptive test and the confirmatory test. For the confirmatory test, a more selective culture
medium (brilliant green bile broth) is inoculated with material taken from the positive tubes.
After an appropriate incubation time, the tubes are examined for gas formation as before. The
most probable number (MPN) of bacteria present can then be estimated from the number of
tubes inoculated and the number of positive tubes obtained in the confirmatory test. Using
specially devised statistical tables. This technique is known as the MPNmethod.
Matrials required:
Durham tubes, Gas burner, Inoculation loop and holder, MacConkey Broth
with neutral red (double strength), MacConkey Broth with neutral red (single strength),
Brilliant Green Blue broth (BGB),Tryptone water/Peptone water (for indole test), Kovac’s
Reagent and Sodium thiosulphate solution.
MEDIA COMPOSITION:
MacConkey broth
Double-strength medium:
i) Dissolve the peptone, sodium chloride and bile salts in the water by heating and
store at 4ºC overnight.
ii) Filter while still cold, add the lactose and dissolve.
iii) Adjust to pH 7.4 ± 0.2 and add the Neutral Red.
Single-strength medium:
• Prepare single-strength medium by dilution of the double-strength medium with an equal
volume of distilled water or make separatory using half the concentration of ingredients.
• Distribute single-strength medium in 5 mL volumes and double-strength medium in 10
mL and 50 mL volumes. Each tube or bottle used should contain an inverted fermentation
(Durham) tube.
• Autoclave at 115ºC for 10 min.
Brilliant Green Lactose Bile Broth:
 Dissolve peptone and lactose in 500 mL distilled water
• Add 20 g dehydrated oxgall dissolved in 200 mL distilled water. The pH of this
solution should be 7.0-7.5.
• Mix and add water to make 975 brilliant green in distilled water
• Add distilled water to make 1 litre
• Dispense into fermentation tubes, making certain that fluid level covers inverted vials.
 Autoclave 15 min at 121ºC. Final pH, 7.2 ± 0.1.
Procedure:
i)With the stopper in position, shake the bottle vigorously to achieve a homogeneous
dispersion of bacteria. (If the bottle is completely full, remove the stopper and discard about
20-30 mL of water; then replace the stopper and shake. This ensures thorough mixing).
ii) Add 50 mL of sample to a tube / flask containing 50 mL of presumptive broth (double

strength). With a sterile 10 mL pipette, inoculate 10 mL of the sample into each the five
tubes containing 5 mL presumptive broth (single strength). It is advisable to shake the tubes
gently to distribute the sample uniformly throughout the medium. Be careful as to not shake
so hard that air is introduced into the inverted tubes.Incubate the tubes at 35º C ± 5ºC for 24
hours.
iii) Incubate the tubes at 35º C ± 5ºC for 24 hours.
iv) At the end of the 24-hour incubation period, examine each tube for the presence of gas. If
present, gas can be seen in the Durham tube. If none is visible, gently shake the tube; if any
effervescence (streams of tine bubbles) is observed, the tubes should be considered
positive.
v) Record the number of positive tubes after 24 hours
vi) Re-incubate negative tubes for a further 24-hour period. At the end of this period, check
the tubes again for gas production as in 5 above. Gas production at the end of either 24 or 48
hours’ incuba-tion is presumed to be due to the presence of coliforms in the sample.
vii) Record the number of positive tubes after 48 hours.
viii) Using a sterile loop, transfer one or two loops-full from each presumptive
positive tube into tow tubes containing respectively confirmatory broth (BGB) and tryptone
water. (Sterilise the inoculation loop before each transfer by flaming and allow cooling). To
confirmthe presence of thermotolerant coliforms, incubate the subculture tubes from each
presumptive positive tube for 24 hours at 44.5 ± 0.5ºC.
[Alternatively, transfer a loopful of a positive MacConkey broth tube into BGB medium and
incubate at 35ºC for 24-48 hours. This will be a better confirmation of Total Coliforms.]
ix) At the end of 24 hours’ incubation, examine each broth tube for growth and the presence
of gas in the Durham tube. Record the results, as done previously.
x) To each tube of tryptone water, add approximately 0.1 mL of Kovacs reagent and mix
gently. The presence of indole is indicated by a red colour in the Kovacs reagent, forming a
film over the aqueous phase of the medium.
xi) Confirmatory tests positive for indole, growth, and gas production show the presence of
E.coli. Growth and gas production in the presence of indole confirms thermo tolerant
coliforms.
Result:
The analyze water coli forms of given sample is _________________

Ex. No: 1 DETERMINATION OF NITRATE
Date:
AIM:
To determine the amount of nitrate in given sample.
PRINCIPLE:
The nitrate ion selective electrode is a combination electrode with a positive and a
negative half cell. The negative half cell is bathed in a fixed level of nitrate (negatively
charged) solution behind a membrane which is selectively sensitive to nitrate ions. The
positive half cell is bathed in ammonium ions from the ammonium sulfate gel solution which
is dispensed by depressing the button on the top of the electrode body.
APPARATUS:
4. Ion selective electrode with meter

5. Buffer Solution
6. Thermometer
PROCEDURE:
6. Calibrate the electrodes with two standard buffer solutions of solutions nitrate
7. The sample temperature is determined at the same time and is entered into the meter
to allow for a temperature correction.
8. Rinse the electrodes thoroughly with deionized distilled water and carefully wipe
with a tissue paper.
9. Dip the electrodes into the sample solution, swirl the solution and wait up to one
minute for steady reading. The reading is taken after the indicated value remains
constant for about a minute.

OBSERVATIONS:
Description of sample Temperature Amount of Nitrate
RESULTS:
The amount of nitrate in given sample is ………………………….

Ex.No: 2 PREPARATION OF MEDIA
Date:
AIM
To prepare nutrient broth and nutrient agar for microbial culture.
INTRODUCTION:
A common liquid medium is used for growing bacteria is nutrient broth medium.
It contains beef extract, peptone and sodium chloride. This medium can be supplemented
with other substances like sugars and organic salts to meet the requirements of any particular
organisms. This media always kept sterile until they are used.
Liquid growth media contains nutrient are usually solidified by the addition of agar. Agar
– agar(often called simply agar) is the complex polysaccharide consist of 3-6 anhydro L-
galactose and D-pyranose free of nitrogen. It produced from various red algae which belongs
to gellatium, gracilaria and others genera. It has no nutritional value.It liquefies on heating at
100°C and hardens into jelly by cooling to 40-45°C.
APPARATUS REQUIRED:
Conical flask, cotton wool, pipettes, measuring cylinder, test tubes,
petriplates and pH meter.
REAGENTS REQUIRED:
Beef extract, peptone, sodium chloride and nutrient agar media.
COMPOSITION OF NUTRIENT BROTH:
Beef extract - 3g
Peptone - 5g
Sodium chloride - 5g
Distilled water - 1L
pH - 7.2 ± 0.2
COMPOSITION OF AGAR MEDIA:
Beef extract - 3g
Peptone - 5g
Sodium chloride - 5g
Agar – 15g
Distilled water - 1L
pH - 7.2 ± 0.2

PROCEDURE:
PREPARATION OF NUTRIENT BROTH:
1. Distilled water is taken in a conical flask and the above ingredients are weighed
and dissolved in the distilled water.
2. The pH of the solution is adjusted to 7.2 ± 0.2.
3. The nutrient broth is distributed in the test tubes and plug tubes with cotton.
4. The medium is sterilized at 121°C for 15minutes in an autoclave.
5. After 15minutes the test tubs are removed and kept it in a sterile condition for
culture of microorganisms.
PREPARATION OF NUTRIENT AGAR MEDIUM:

1. Distilled water is taken in a conical flask and the above ingredients are weighed
and dissolved in the distilled water.
2. The pH of the solution is adjusted to 7.2 ± 0.2.
3. The medium is sterilized at 121°C for 15minutes in an autoclave.
4. The medium is poured into sterilized Petri dishes in laminar air flow chamber.
5. After solidification medium is ready for culturing of microorganisms.
Result:
The Nutrient Broth and Nutrient Agar have been prepared for microbial culture.

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C ABDUL HAKEEM COLLEGE OF

ENGINEERING AND TECHNOLOGY

Approved by AICTE, New Delhi & Affiliated to Anna University, Chennai

DEPARTMENT OF CIVIL ENGINEERING

CE8512-WATER AND WASTEWATER ANALYSIS

Faculty in charge HOD

 Wear the Overcoats and shoes , before entering in to the Laboratory

CAHCET/CIVIL ENGG 2 CE8512 - WWW LAB

1 Sampling and preservation 6

4 Determination of Total Solids 13

Determination of Total Dissolved

Determination of Total Fixed And

CAHCET/CIVIL ENGG 3 CE8512 - WWW LAB

Determination of MPN INDEX

CAHCET/CIVIL ENGG 4 CE8512 - WWW LAB

2 Preparation of Nutrient Agar 70

3 Preparation of Nutrient Broth 70

CAHCET/CIVIL ENGG 5 CE8512 - WWW LAB

The significance of a chemical analysis depends to a large extent on the sampling

SPOT OR GRAB SAMPLES:

Collection of truly representative sample is as important as sample preservation. A

CAHCET/CIVIL ENGG 7 CE8512 - WWW LAB

Parameter Volume Container Preservation

CAHCET/CIVIL ENGG 8 CE8512 - WWW LAB

To determine pH of the given sample.

PH is measured by a pH meter using a glass electrode which generates a potential

= Constant + 0.058 pH at 20

A calomel or Ag/AgCl/KCI reference electrode is usually located around the glass

1. pH meter along with electrodes

CAHCET/CIVIL ENGG 9 CE8512 - WWW LAB

The pH of the given sample is ………………………….

CAHCET/CIVIL ENGG 10 CE8512 - WWW LAB

To find the turbidity of the given samples

1. Dissolve 1.0 gm Hydrazine sulphate and dilute to 100 ml.

CAHCET/CIVIL ENGG 11 CE8512 - WWW LAB

S.NO Sample Details Turbidity

The Turbidity of the given sample is ………………………………. (NTU)

CAHCET/CIVIL ENGG 12 CE8512 - WWW LAB

1. Evaporating dishes (pyrex, porcelain or platinum)

CAHCET/CIVIL ENGG 13 CE8512 - WWW LAB

A = Final Weight of the dish in mg.

The Total Solids of the given sample is ----------------------…………mg/L

CAHCET/CIVIL ENGG 14 CE8512 - WWW LAB

To find out Total dissolved solids of the given sample.

CAHCET/CIVIL ENGG 15 CE8512 - WWW LAB

A = Final Weight of the dish in mg.

Sample details Volume of Initial weight of Find weight of Total dissolved

The Total dissolved solids of the given sample is …………………… mg/L

CAHCET/CIVIL ENGG 16 CE8512 - WWW LAB

CAHCET/CIVIL ENGG 17 CE8512 - WWW LAB

(W2 - W1) x 1000

(W2 - W3) x 1000

(c) Total fixed solids = (a) – (b)

CAHCET/CIVIL ENGG 18 CE8512 - WWW LAB

Type of Sample Volume of Weight of Weight of Residue

The Total Volatile solids of the given sample is …………………… mg/L

The Total Fixed solids of the given sample is …………………… …..mg/L

CAHCET/CIVIL ENGG 19 CE8512 - WWW LAB

To estimate the amount of Chloride present in the given water sample.

1. Chloride free distilled water

CAHCET/CIVIL ENGG 20 CE8512 - WWW LAB

1. Take 25ml of the sample in conical flask.