Spring 2008
Determination of Phosphate
Purpose:
To determine the concentration of phosphate (PO4-3) in various water samples.
Materials:
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polyethylene bottles
pipettes (various sizes)
graduated cylinders (various sizes)
volumetric flasks
9 cuvettes
Spectrophotometer
Access to the following reagents:
Reagents:
1. Ammonium molybdate solution
Dissolve 15.0 grams of reagent grade ammonium paramolybdate,
(NH4)6Mo7O24·4H2O, in distilled water and volumetrically dilute to 500 mL.
Store in plastic in the dark.
2. Sulfuric acid solution
While stirring slowly add 140 mL of concentrated sulfuric acid, H2SO4, to 900
mL of distilled water. Use a water bath if necessary. Allow the solution to cool,
store in glass.
3. Ascorbic acid solution
Dissolve 27.0 grams of ascorbic acid in distilled water and volumetrically dilute
to 500 mL. Store in the freezer if held for more than one week.
4. Potassium antimonyl-tartrate solution
Dissolve 0.34 grams of potassium antimonyl-tartrate in distilled water and
volumetrically dilute to 250 mL, warming may be necessary to help dissolution.
Store in glass or plastic, stable for many months.
5. Primary Standard
Dissolve 0.816 grams of anhydrous potassium dihydrogen phosphate, KH2PO4,
in distilled water and volumetrically dilute to 1L. Stable, store in a dark bottle.
Methods:
1. Mixed Reagent solution
a. Mix together in this order: 20 mL of the ammonium molybdate solution, 50
mL of the sulfuric acid dilution, 20 mL of the ascorbic acid solution, and 5
mL of the potassium antimonyl-tartrate solution. LABEL it.
2. Run Standard
a. Using distilled water volumetrically dilute 10 mL of the Primary Standard to 1
L. Pipet 2.5 mL of the Run Standard into three 50 mL graduated cylinders.
Add 47.5 mL of distilled water. LABEL them.
Phosphate Determination
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Spring 2008
3. Reagent Blanks
a. Fill 2 graduated cylinders with 25 mL of DIW. LABEL them.
4. Run Standard and Reagent Blank Analyses
a. Add 5 mL of the Mixed Reagent solution to each Run Standard and Reagent
Blank. Stopper and mix well. Let stand for 5 minutes but not more than 2 to
3 hours.
5. Measure the absorbance at 885 nm using a spectrophotometer that has been blanked
with distilled water. Determine the average absorbance of the 3 Run Standards
(ABSstnd) and the 2 Reagent Blanks (ABSrb) for the next step.
6. F factor or Calibration Factor Calculation: [F1cm should be about 5]
F=
3.00
[(ABSstnd - ABS rb)] x 10
Before the water samples are run, a "F" or Calibration Factor is obtained
by running Standards and Reagent Blanks as a check on the reagents. You
will be graded on the accuracy of your F factor. Record your absorbances
and calculations on your data sheet.
7. Water Sample Analyses
a. Fill 4 graduated cylinders with 50 mL of each water sample. LABEL them.
b. Add 5 mL of the Mixed Reagent to each graduated cylinder. Stopper and mix
well.
c. After 5 minutes but before 2-3 hours measure the absorbance at 885 nm using
a spectrophotometer that has been blanked with distilled water.
a. Do a Turbidity Blank determination for each water sample (read the
Absorbance of untreated water sample at 885 nm). These cuvettes will be
next to the spectrophotometer. Record the values on your data sheet.
8. Calculate the sample's phosphate concentration in microgram atoms of phosphate per
liter of sample:
g-at PO43-/L = Corrected Absorbance x F
Corrected Absorbance = ABSsample - ABSrb - ABSturbidity
Phosphate Determination
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