In this 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. 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 (eutrophication).

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 ppm (mg) phosphorus per liter.

The Spectronic 20 spectrophotometer will be employed in the measurement of color intensity of the blue solution. A wavelength of 650 nm will be used in these analyses. For additional information on the theory and use of a spectrophotometer follow this link.

Reagents used:
a. Ammonium molybdate reagent: (prepared by the lab assistant)
b. Stannous chloride reagent: (prepared by the lab assistant)
c. Stock 20.0 ppm phosphate solution: (prepared by the lab assistant)

Note: Glassware should be washed thoroughly with hot water followed by a rinsing with distilled water. DO NOT use phosphate-containing detergents to clean equipment for this experiment.

Prepare the following standard phosphate solutions:
a. 1.0 ppm standard: Place 2.00 ml of 20.0 ppm phosphate solution in a 100 ml graduated cylinder and dilute to 40 ml with distilled water.
b. 2.0 ppm standard: Place 4.00 ml of 20.0 ppm phosphate solution in a 100 ml graduated cylinder and dilute to 40 ml with distilled water.
c. 3.0 ppm standard: Place 6.00 ml of 20.0 ppm phosphate solution in a 100 ml graduated cylinder and dilute to 40 ml with distilled water.
d. Blank: Set aside 25 ml of distilled water, which will be treated with the color-developing reagent to serve as a blank.

These three 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 (Y-axis) versus concentration (x-axis).

Color development in sample:
This procedure is used for the three 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 intensity 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 nm wavelength, measure the absorbance (after 5-15 minutes development) of the blue sample*.

If the sample is one of the three standards, the absorbance value will be used to make a plot of absorbance (y-axis) versus the concentration of the standard (x-axis).

If the sample is river, lake, sewage, etc. water, determine the ppm phosphate by comparing the absorbance of the solution to the plot made from the standards.

* Should one of your samples produce a very dark blue color which cannot 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 distilled water to bring the volume up to 100 ml. Now, this diluted sample may be analyzed according to the directions for color development above.

Remember that the concentration that you ultimately obtain from this sample will have to be multiplied by 100 because of the 100 fold dilution.

USING SPECTROPHOTOMETER (with Vernier software):
Start Vernier software
Go to "Collect Absorbance vs. Concentration Data"
Set calibration ranges as appropriate (Abs 0 - 2; Conc. 0 - 3ppm ?)
Follow instructions (Shown on computer screen):
a) Make sure correct wavelength (& filter) are set
b) Remove any cuvette and set %T to 0 using left knob
c) Insert blank and set %T at 100 using right knob
d) Insert first standard and type in concentration (i.e. 1, etc.)
e) Repeat until all standards have been entered
f) Press "ESC"
f) Go to "Interpolate" and insert "Unknown"
g) Record unknown concentration
h) Go to Main Menu & select "File Option"
i) Select "Save data to disk"
j) Select unique file name to save calibration curve

All contents copyrighted (c) 1998
Peter Jeschofnig, Ph.D., Professor of Science, Colorado Mountain College
All Rights reserved

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This page was created by Peter Jeschofnig and was last updated: 1/31/2000