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Phosphorus Determination Using The Colorimetric Ascorbic Acid Technique
Phosphorus Determination Using The Colorimetric Ascorbic Acid Technique
Introduction
Phosphorus has been identified as a prime nutrient needed for algae growth in inland
environments. In 1992, the EPA reported that accelerated eutrophication was one of the
leading problems facing the Nation's lakes and reservoirs. Eutrophication caused by the
overabundance of nutrients in water can result in a variety of water-quality problems,
including fish kills, noxious tastes and odors, clogged pipelines, and restricted recreation.
In freshwater, phosphorus is often the nutrient responsible for accelerated eutrophication.
Many algae blooms in rivers and lakes are attributed to elevated phosphorus
concentrations resulting from human activities. Phosphorus enters surface waters from
agricultural and urban runoff as well as from industrial and municipal wastewater
treatment plant effluent.
No national criteria have been established for concentrations of phosphorus
compounds in water; however, to control eutrophication, the EPA makes the following
recommendations:
Total phosphates should not exceed 50 g/L (as phosphorus) in a stream at a point
where it enters a lake or reservoir.
Total phosphorus should not exceed 100 g/L in streams that do not discharge
directly into lakes or reservoirs.
Municipal wastewater treatment plants in many areas are required to remove
phosphorous in their treatment process. While the biological treatment process removes
some phosphorus, in most cases precipitation as an insoluble metal phosphate is required
to meet discharge regulations. This precipitation step is normally accomplished with a
metallic salt such as ferric sulfate, ferric chloride or aluminum sulfate. This precipitation
step may be accomplished in the primary or secondary clarifiers.
Another technique that has been used recently for phosphorus removal is contact with
wallastonite mine tailings. The phosphorus removal is presumably by precipitation.
Results obtained to date suggest that a long contact time on the order of 24 hours is
required to obtain reasonable levels of phosphorus removal. This long contact time
requires the construction of large wallastonite beds. The economics could be improved
significantly if the contact time could be reduced. In this experiment we will explore the
effect of contact time using batch tests.
Spectrophotometer Limitations
The diode array spectrophotometer has 316 diodes that cover the wavelength range of
190 nm to 820 nm. Each diode generates a voltage output that is proportional to the
number of incident photons. The voltage is then digitized, but the manufacturer of the
instrument in the Cornell Environmental Laboratory, Hewlett-Packard, doesn't report the
resolution of the analog to digital converter. At very low concentrations the difference
between the intensity of light transmitted through the reference and the intensity of light
transmitted through the sample approaches zero. At some low concentration the
difference in light intensity approaches the resolution of the analog to digital converter.
Another source of instrument error is drift in lamp intensity over time. The lamp intensity
is measured when a reference sample is made. The light intensity recorded by the diodes
will vary proportionally to any lamp intensity drift.
The IDL should decrease as the number of diodes used in the analysis increases (as in
Spectral analysis) for the same reason that replicate analysis of samples decreases the
standard deviation. The "Spectral analysis" feature, which can be used to measure either
single or multiple components, uses as much of the spectrum as the user desires and thus
potentially decreases the IDL. Spectral analysis uses general least squares regression to
add multiples of extinction coefficient arrays for each component to obtain the best curve
fit for the sample. The extinction coefficient arrays are obtained from the slope of the
linear regression line for absorbance as a function of concentration at each wavelength.
Experimental Objectives
1) Measure the concentration of phosphorus in several samples to test the precision of
the ascorbic acid technique.
2) Analyze the data using spectrophotometer software outside the lab.
3) Analyze multiple samples so that confidence intervals can be calculated.
4) Estimate the method detection limit (MDL).
5) Discuss methods to improve the method detection limit.
6) Compare the results obtained using conventional analysis at a single wavelength with
spectral analysis.
Experimental Procedures
Standards Preparation Method
1) Use 100 g P/L stock.
2) Use a digital pipet and prepare 1 mL of each standard.
3) Use E-pure water to dilute the 100 g P/L stock.
Samples Analysis
1) Measure the reference using a reagent blank. (The reagent blank is also the 0 mg/L
standard.)
2) Analyze the reagent blank as a sample and verify that the absorbance deviates less
than 0.004 AU (absorbance units) from zero. If the absorbance deviates more than
0.004 AU reanalyze the reference sample.
3) Analyze 6 standards as standards using the spectrophotometer and save the data
as \\Enviro\enviro\Courses\453\phosphorus\netid_Pstd.
4) Analyze 6 standards as samples using the spectrophotometer and save the data as
\\Enviro\enviro\Courses\453\phosphorus\netid_Pstdsam.
5) Analyze 7 10-g P/L standards as samples and save the data as
\\Enviro\enviro\Courses\453\phosphorus\netid_10Pstdsam.
6) Analyze wallastonite samples as samples using the spectrophotometer and save the
data as \\Enviro\enviro\Courses\453\phosphorus\netid_wall.
7) After you have analyzed all of your samples use the computer at your workstation to
export each of the data files. You will need to use the Spectrophotometer software and
load each of the files in turn and then use the export function. Save the files using the
same naming convention as before, but use “exp” as the last 3 letters in the name.
Prelab Questions
1) You will be creating 1 mL standards by diluting a stock of 100 g P/L. Create a table
showing how you will prepare 1 mL of each of the standards using only pipettes.
2) All of the samples including standards are diluted with a small amount of combined
reagent. How is this dilution accounted for when calculating the concentration of
samples?
Data Analysis
1) Plot the absorbance spectra of the standards.
2) Choose an appropriate wavelength (perhaps an absorbance peak) and use Excel to
create a calibration curve. For the calibration curve, absorbance should be a function
of phosphorus concentration.
3) Use the 10-g/L standards that were analyzed as samples to evaluate the Method
Detection Limit using single wavelength analysis. Use your Spreadsheet to calculate
the concentration of each of the replicates.
4) Create a plot showing phosphorus remaining as a function of time and wallastonite
dose. Report the wallastonite dose in g/L.
Spreadsheet requirements
Your spreadsheet must contain all of the analysis requested above as well as the following
capabilities:
1) A well-marked cell containing the analytical wavelength for single wavelength
analysis. Changes to this cell must be reflected in all calculations and graphs.
2) The graph showing the absorbance spectra of the standards must have a vertical line
indicating the analytical wavelength.
3) All of the graphs must be on the same page as the analytical wavelength control so
the effect of changing the wavelength can be easily observed.
4) A separate sheet where you answer the questions below.
Hints
If you haven't already learned how to use Vlookup() now is the time!
The row() function returns the number of the row. I found it useful for this analysis!
The slope() and intercept() functions eliminate the need to type equations off of graphs!
Questions
1) What is happening in the UV region?
2) Are there any absorbance peaks?
3) Total phosphorus concentration in Cayuga Lake varies between 10 and 50 ppb. Would
the techniques used in this lab be able to measure these phosphorus levels?
4) What did you learn about the effect of time and wallastonite concentration on
phosphorus removal?
References
http://wwwrvares.er.usgs.gov/nawqa/circ-1136/h6.html#PHOS
http://www.epa.gov/glnpo/lmmb/methods/index.html#Volume 3
Standard Methods for the Examination of Water and Wastewater.
Lab Prep Notes
Reagents
A Sulfuric acid solution, 4.9 N: Table . Reagents
Add 136 mL concentrated H2SO4 to
800 mL E-pure water. Cool and
Description Supplier Catalog
dilute to 1 L with E-pure water. number
B Ammonium molybdate solution: concentrated Fisher Scientific
Dissolve 40 g of (NH4 )6 H2SO4
(NH4 )6 Fisher Scientific
Mo7O24•4H2O in 900 mL E-pure
Mo7O24•4H2O
water and dilute to 1 L. Store at 4°C. C6H8O6 Fisher Scientific
C Ascorbic acid: Dissolve 9 g of K(SbO)C4H4O6• Fisher Scientific
ascorbic acid (C6H8O6) in 400 mL E- ½H2O
sodium lauryl
pure water and dilute to 500 mL. sulfate
Store at 4°C. Keep well stoppered. KH2PO4 Fisher Scientific
Prepare fresh monthly or as needed. Table . Equipment list
D Antimony potassium tartrate:
Description Supplier Catalog #
Dissolve 3.0 g of
100-1095 µL Fisher Scientific 13-707-5
K(SbO)C4H4O6•½H2O in 800 mL E- pipette
pure water and dilute to 1 L. Store at 10-109.5 µL pipette Fisher Scientific 13-707-3
4°C. Disposable cuvets Fisher Scientific 14-385-942
Cuvet holder Fisher Scientific 14-385-939
Combined color reagent: Combine
UV-Vis Hewlett-Packard 8452A
the following solutions in order, spectrophotometer Company
mixing (but do not entrain air as
oxygen oxidizes the ascorbic acid) after each addition: (Prepare fresh weekly.
Store at 4°C)
Stock A, (4.9 N H2SO4) 50 mL
Stock B, (Ammonium molybdate solution) 15 mL
Stock C, (Ascorbic acid solution) 30 mL
Stock D, (Antimony-tartrate solution) 5 mL
Water diluent solution: Add 4.0 g sodium lauryl sulfate and 5 g NaCl per L of E-pure
water.
Stock phosphorus standard: Dissolve 0.4394 g of Potassium phosphate monobasic
(KH2PO4) (dried at 105°C for one hour) in 900 mL E-pure water. Add 2 mL of
concentrated H2SO4 and dilute to 1 L. 1.0 mL = 0.100 mg P (100 mg P/L).
Rinse reagent containers with the mixed reagent and then with E-pure water to allow
the reagent to react with all the phosphorus in the containers.