Experiment 5

Analysis of Phosphate in Water

General Discussion
In the present experiment, we will analyze a series of natural water samples for
their phosphate content. Detergents are among the greatest contributors to phosphate
content in rivers and lakes because phosphate containing compounds are used in
detergent formulation as water softeners (builders). Phosphate is not toxic to animals or
plants. In fact, it is a plant nutrient which stimulates the growth of aquatic Weeds and
algae. This may cause lakes and rivers to become clogged and overrun with plants.
The principle of this method involves the formation of molybdophosphoric acid,
which is reduced to the intensely colored complex, molybdenum blue. This analytical
method is extremely sensitive and is reliable down to concentrations below 0.1 mg
phosphorus per liter.

Apparatus
The Spectronic 20spectrophotometer will be employed in the measurement of
color intensity of the blue solutions. A wavelength of 650 nanometers will be used in
these analyses.

Reagents
a. Ammonium molybdate reagent: (prepared by instructor) The solution is prepared
by dissolving 25g of (NH4)6Mo7O244H2O in 175 mL purified water. Cautiously, 280 mL
of concentrated H2SO4 is added to 400 mL of purified water. The acid solution is cooled,
the molybdate solution is added, and the entire mixture is diluted to 1 liter.
b. Stannous chloride reagent: (prepared by instructor) 2.5 g of SnCl22H2O is dissolved
in 100 mL of glycerine. The mixture is gently heated to hasten dissolution.
c. Stock 20.0 mg/L phosphate solution: (prepared by instructor) To prepare this solution,
0.286 g of KH2PO4 is dissolved in 1.0 liter of water. This is a 200 mg/L stock solution.
Then dilute 100 mL of this solution to 1.0 liter. Then dilute 100 mL of this solution to
1.0 liter. Then dilute 100 mL of this solution to 1.0 liter.

Procedure
Note: Glassware should be washed thoroughly with hot water followed by rinsing with
purified water. Do not use phosphatecontaining detergents to clean equipment for
this experiment.
Prepare the following standard phosphate solutions:
a. 1.0 mg/L standard: Place 2.00 mL of 20.0 mg/L phosphate solution in a 100-mL
graduated cylinder and dilute to 40 mL with purified water. (Save exactly 25 mL in an
erlenmeyer flask for analysis with the spectrophotometer.)
b. 2.0 mg/L standard: Place 4.00 mL of 20.0 mg/L phosphate solution in a 100-mL
graduated cylinder and dilute to 40 mL with purified water. (Save exactly 25 mL in an
erlenmeyer flask for analysis with the spectrophotometer.)
c. 3.0 mg/L standard: Repeat the directions for the 1.0 mg/L standard using 6.00 mL of
20.0 mg/L phosphate. (Save 25 mL)
d. 4.0 mg/L standard: Repeat the directions for the 1.0 mg/L standard using 8.00 mL
of 20.0 mg/L phosphate. (Save 25 mL)
e. 5.0 mg/L standard: Repeat the directions for the 1.0 mg/L standard using 10.00 mL
of 20.0 mg/L phosphate. (Save 25mL)
f. Blank: Set aside 25 mL of purified water which will be treated with the color
developing reagent to serve as a blank.
These five standard solutions and the blank should now be treated according to the
following "color development" procedure. After measuring the absorbance of these
solutions, make a plot of absorbance versus concentration as described by your
instructor.

Color development in sample:

This procedure is used for the five standard solutions and for any river, lake, or
sewage water samples which are to be analyzed for phosphate.
Place in an erlenmeyer flask 25 mL of the water sample to be analyzed. Put 1.00 mL
(using a pipet) of ammonium molybdate solution into the flask and swirl to mix. To the
flask add 2 drops of stannous chloride solution and mix by swirling. If phosphate is
present, a blue color will develop to a maximum in 5 minutes. Note: The time period is
somewhat critical. Measurements should be taken anywhere from 5 to 15 minutes after
addition of stannous chloride.
While you are waiting for the blue color to develop, set the wavelength to 650 nm
on the spectrophotometer. Use the blank solution to set it to read zero absorbance.
Using 650 nanometers wavelength, measure the absorbance (after 5-10 minutes color
development) of the blue sample.*

Waste Disposal
All solutions may be rinsed down the drain with water.
Calculations
From the concentration and the absorbance of the five standards and the blank,
make a plot of absorbance as a function of concentration (a "standard curve" or "Beer's
Law plot"). Use the plot and the absorbance of each river, lake, sewage, or unknown
solution to determine the concentration in milligrams phosphate ion per liter (mg/L
PO43-) in that sample. We sometimes use concentration units of parts per million (ppm)
synonymously with mg/L because 1.0 liter of water at room temperature weighs 1000
grams (to two significant figures). Thus 1.0 mg is one millionth of the weight of one liter.
For the sake of consistency in this manual we use units of mg/L and related units such as
g/mL and g/L.
*Should one of your samples produce a very dark blue color which can not be read
with the spectrophotometer, dilute the original water sample 100 fold. This is
accomplished by placing 1.0 mL of the water sample in a 100-mL graduated cylinder and
then adding enough purified water to bring the volume up to 100 mL. Now 25 mL of this
diluted sample may be analyzed according to directions for color development in sample,
previously given. Remember that the concentration which you ultimately obtain from
this sample will have to be multiplied by 100 because of the 100 fold dilution.
Report the following data:

Sources of Water Samples

1.
2.
3.
4.

Absorbance of Standards:

Blank
1.0 mg/L PO43-
2.0 mg/L PO43-
3.0 mg/L PO43-
4.0 mg/L PO43-
5.0 mg/L PO43-
Note: Prepare a standard curve and turn it in with the report sheet.

Absorbance of Water Samples Dilution Factor (if any)

1.
2.
3.
4.

Concentration of Phosphate in Water Samples

1. 3
2. 4

Discussion

Identify those aspects of the procedure which may have introduced some error
into the final results. In each case indicate whether the error would cause the
measured value to be too high, too low, or either.