Experiment 9: Environmental II
Phosphate Contamination in Water
A. Purpose
1. To learn about the properties of phosphate salts and their role as contaminants
of natural water.
2. To determine quantitatively (by spectrophotometric means), the phosphate
content in a number of water samples.
B. Theory
You are already familiar with the spectrophotometric determination of the
concentration of a solution in a certain colored substance. This experiment is
designed to use this important quantitative technique for the determination of
phosphate content in water samples. Phosphates, which are essential nutrients for
plant life, are one of several substances that contribute to the growth of algae and
premature aging or eutrophication of lakes. Among the sources of phosphate in
water are household detergents and fertilizers. Although it is possible that other
factors such as nitrate content or dissolved carbon dioxide (CO2) are limiting
factors in the growth of algae, ecologists advise lowering the amounts of
phosphates in surface water. The analytical method to be used has been selected
because of its sensitivity, which makes it possible to detect minute quantities of
phosphate. Under acidic conditions, the orthophosphate ion, PO43, reacts with
ammonium molybdate to form molybdophosphoric acid. A yellow compound
vanadomolybdophosphoric acid, is formed in the presence of vanadium. The color
intensity produced depends on the amount of phosphate present, and the acidity of
the solution. By maintaining the same acidity in all solutions and by adding the
same large excess of vanadate/molybdate reagent to each solution, we can ensure
that the amount of phosphate present controls the intensity of color developed. It
is of course possible to get a rough idea of the relative amount of phosphate in the
solutions by visually comparing their color intensities. By use of the
spectrophotometer, however, we can obtain a more accurate quantitative
measurement.
C. Materials and setup
307 ppm phosphate (100 ppm phosphorous) standard (0.4395 g of KH2PO4 made
up to a total volume of 1.00 L with distilled water); for store preparators:
vanadate/molybdate reagent solution [add 1.25 g of ammonium metavanadate,
NH4VO3, to 300 mL of water with stirring; add 330 mL of 12 M HCl and
continue to stir until all solid has dissolved; cool to room temperature, add 25 g of
ammonium molybdate, (NH4)6Mo7O24.4H2O and stir until dissolved; dilute to 1.0
L]; samples of water (free of suspended materials) from different sources such as
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local lakes or streams, tap water, water from a private well, melted snow, rain
water, etc. Spectronic 20.
All standard solutions and mixtures used for spectrophotometric analysis may be
rinsed down the sink. Unsused portions of ammonium molybdate reagent or
ammonium metavanadate reagent should be poured into the heavy metal waste
container.
D. Procedure (Work in pairs or groups of three)

Preparation of a Set of Standard Phosphate Solutions:
1. Obtain from the stock room a sample of standard phosphate solution
(3.230  103 M KH2PO4), in a clean and dry 50 mL beaker.
2. Pipet a 1.00 mL aliquot of this standard solution and transfer into a clean
and dry 50.0 mL volumetric flask (free of phosphate contaminants).
3. Make up the solution to the mark with distilled water. Stopper the flask
and mix well, then transfer the solution to a clean beaker. Label the
beaker.
4. Repeat steps 2 and 3 using 2.00, 3.00, 4.00 and 5.00 mL aliquots of the
original standard solution. Make sure to rinse the volumetric flask very
thoroughly with distilled, water each time before use.
You have thus prepared five phosphate standards of different (known)
concentrations.
5. To each solution in the beaker, add 10 mL of ammonium
vanadate/molybdate reagent. Swirl each solution well to achieve complete
mixing. You must observe a yellow color whose intensity depends on the
concentration of phosphate in the solution.
6. Measure the absorbance of each of the five solutions (treated with
vanadate/molybdate reagent) at 420 nm in a spectrophotometer as
demonstrated by your instructor.
Note: the blank solution here consists of: 0 mL KH2PO4, 50.0 mL
distilled water and 10.0 mL vanadate/molybdate reagent.
7. Using the graph sheet provided, construct a calibration curve (Absorbance
A versus concentration C). Determine the slope of this curve.

Measurement of Phosphate in an Unknown Water Sample:
8. Prepare for testing two water samples: one is tap water and another is a sea
water sample, preferably green with algae in the summer (because of
excessive phosphate in the water).
9. Measure 50.0 mL aliquots (you can use a graduated cylinder) of the
unknown water samples to be tested into two clean and dry flasks.
10. To each sample, add 10 mL of ammonium vanadate/molybdate reagent.
Swirl each solution well to achieve complete mixing.
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11. Measure the Absorbance of your treated samples in the spectrophotometer
at 420 nm.
12. From the slope of the calibration curve (Part A), determine the
concentration of phosphate in your unknown samples expressed in ppm
and in molar units.
Useful readings: “Phosphate in the Little Miami River”
(http://w3.one.net/~jwclymer/lmr.html); “Phosphate Snap Test Kit”
(http://webpages.charter.net/kwingerden/erhs/aquarium/infophos.htm);
“Water resource characterization DSS – Phosphorous”
(http://h2osparc.wq.ncsu.edu/info/phos.html)
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E. CHEM 203 Lab Report
NAME : _______________________
EXPERIMENT No. : ______________
TITLE : __________________________________
DATE : ________________
Partner : ________________________

Calibration curve
Sample
Volume of phosphate
standard solution (mL)
Concentration
(M)
Concentration
(ppm)
Absorbance
(420 nm)
Blank
1
2
3
4
5
Slope of Calibration Curve: b (M1) = __________________

Analysis of the water samples

Identity of water samples analyzed
1. __________________________________________________________________
2. __________________________________________________________________
3. __________________________________________________________________
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
Concentrations
Water sample
Absorbance
Concentration (M)
Concentration (ppm)
1.
2.
3.
Questions
1. Write a balanced net ionic equation for the reaction used in this analysis.
2. If H+ ion, VO3 ion, and Mo7O246 ion, as well as PO43 ion, are required to
produce the yellow color, why does the analysis measure phosphate ion content of
the water sample?
3. Why is distilled water used for diluting the standard solutions in this experiment?
4. If a given water sample contains 10 parts per million phosphorous, how many
grams of phosphorous would a liter contain?
5. The following terms used in this experiment may be unfamiliar to you. Look up
the definition of any term that you do not recognize: spectrophotometric,
eutrophication, ecologist, sensitivity, diffraction, monochromatic light,
wavelength, nanometer, cuvette.
6. Why are phosphates used in household detergents? What are some of the
substitutes that have been suggested for use instead of phosphates for cleaning
purposes.
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