Principle

Reactive phosphate (soluble and/or total) reacts with molybdic acid, ascorbic acid, and trivalent antimony to form a colored complex. The absorbance of the resulting blue solution is measured at 885 nm against a reagent blank.

Sample collection and handling

Water samples should be collected in acid-washed polyethylene bottles. Samples should be kept cool and dark or frozen until later analysis.

Procedure

Warm the samples to between 15 and 30 oC and measure the absorbance of the sample at 885 nm to obtain a turbidity correction. If absorbance > 0.05, filter the samples.

Add 10 ml of mixed reagent to 100 ml of sample (or 1 ml to 10 ml) and mix at once. After 10 minutes and before 2-3 hr measure absorbance of the colored solution at 885 nm against a reagent blank and distilled water. Absorbance of the reagent blank should not exceed 0.02. Correct the sample's absorbance reading by subtracting both the turbidity and reagent blank's absorbance. Calculate the concentration of reactive phosphate as micromolar phosphate:

F is a calibration factor determined in the Calibration section below.

Illustration of phosphate assay results; blank on the right and blue-colored sample on the left.

Reagents

All reagents should be made using reagent grade chemicals and the cleanest distilled water available. Contamination of glassware and other containers with soap must be avoided!

1. Ammonium molybdate: Dissolve 15 g ammonium paramolybdate, (NH4)6Mo7O24.H2O, in 500 ml distilled water. Store in a plastic bottle.

2. Sulfuric acid solution: Add 140 ml conc. sulfuric acid to 900 ml distilled water, cool, and store in a glass bottle.

3. Ascorbic acid: Dissolve 27 g ascorbic acid in 500 ml distilled water. Decant into vials of sufficient volume for making individual batches of mixed reagent (see below) and keep frozen until used.

4. Potassium antimonyl tartrate: Dissolve 0.34 g potassium antimonyl tartrate (also called antimony potassium tartrate) in 250 ml distilled water. Store in a glass or plastic bottle.

5. MIXED REAGENT: Mix 100 ml ammonium molybdate, 250 ml sulfuric acid solution, 100 ml ascorbic acid, and 50 ml potassium antimonyl tartrate for immediate use. For smaller sample volumes, appropriately smaller volumes of mixed reagent may be prepared. Solution should become yellow after ascorbic acid is added.

Calibration

Prepare a standard phosphate solution by dissolving 0.816 g of anhydrous potassium dihydrogen phosphate, KH2PO4, in 1000 ml distilled water. Dilute 10 ml of this solution to 1000 ml with distilled water. Store both solutions in the dark with 1 ml chloroform added as a preservative.

Prepare four 3 uM standards, each with 5 ml of dilute phosphate solution made up to 100 ml with distilled water and transfer to polyethylene bottles. Make up two blanks with distilled water. Follow the procedure above for reactive phosphate determination. Calculate F from the equation:

Absstd is the average absorbance of the four 3 uM standards and Absbl is the average absorbance of the two blanks. F should be approximately 50.

Determination of other forms of phosphate

Reactive phosphate occurs in both dissolved and absorbed forms, which can be separated analytically by filtration followed by analysis. Polyphosphate may be determined after boiling a water sample. Dissolved organic phosphate analysis requires preliminary digestion of filtered samples with an oxidizer followed by phosphate analysis. Total phosphate may be determined after digestion of a whole sample by an appropriate method.

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