* Approved by Standard Methods Committee, 1997.


4500-P E. Ascorbic Acid Method


1. General Discussion


    a. Principle: Ammonium molybdate and potassium antimonyl tartrate react in acid medium with orthophosphate to form a heteropoly acid - phosphomolybdic acid - that is reduced to intensely colored molybdenum blue by ascorbic acid.

    b. Interference: Arsenates react with the molybdate reagent to produce a blue color similar to that formed with phosphate. Concentrations as low as 0.1 mg As/L interfere with the phosphate determination. Hexavalent chromium and NO2  interfere to give results about 3% low at concentrations of 1 mg/L and 10 to 15% low at 10 mg/L. Sulfide (Na2S) and silicate do not interfere at concentrations of 1.0 and 10 mg/L.

    c. Minimum detectable concentration: Approximately 10 g P/L. P ranges are as follows:


P Range Light Path
mg/L cm

0.30 -2.0 0.5
0.15 -1.30 1.0
0.01 -0.25 5.0



2. Apparatus    


    a. Colorimetric equipment: One of the following is required:

    1) Spectrophotometer, with infrared phototube for use at 880 nm, providing a light path of 2.5 cm or longer.

    2) Filter photometer, equipped with a red color filter and a light path of 0.5 cm or longer.

    b. Acid-washed glassware: See Section 4500-P.C.2b.




  Phosphorus   Standard  


  Concentration, No. of Deviation  


Ascorbic Acid   Dissolved Labora-


Method Orthophosphate tories          
  g/L   Distilled River   Distilled River
      Water Water   Water Water
13th Edition1 228 8 3.87 2.17   4.01 2.08
Current Method2 228 8 3.03 1.75   2.38 1.39


3. Reagents


      a. Sulfuric acid, H2SO4, 5N: Dilute 70 mL conc H2SO4 to 500 mL with distilled water.

    b. Potassium antimonyl tartrate solution: Dissolve 1.3715 g K(SbO)C4H4O61/2H2O in 400 mL distilled water in a 500-mL volumetric flask and dilute to volume. Store in a glass-stoppered bottle.

     c. Ammonium molybdate solution: Dissolve 20 g (NH4)6Mo7O24 4H2O in 500 mL distilled water. Store in a glass-stoppered bottle.

    d. Ascorbic acid, 0.1M: Dissolve 1.76 g ascorbic acid in 100 mL distilled water. The solution is stable for about 1 week at 4oC.

    e. Combined reagent: Mix  the  above  reagents  in  the  following  proportions  for  100  mL of the combined reagent: 50 mL 5N H2SO4, 5 mL potassium antimonyl tartrate solution, 15 mL ammonium molybdate solution, and 30 mL ascorbic acid solution. Mix after addition of each reagent. Let all reagents reach room temperature before they are mixed and mix in the order given. If turbidity forms in the combined reagent, shake and let stand for a few minutes until turbidity disappears before proceeding. The reagent is stable for 4 h.

    f. Stock phosphate solution: See Section 4500-P.C.3e.

   g. Standard phosphate solution: Dilute 50.0 mL stock phosphate solution to 1000 mL with distilled water; 1.00 mL = 2.50 g P.


4. Procedure


    a. Treatment of sample: Pipet 50.0 mL sample into a clean, dry test tube or 125-mL erlenmeyer flask. Add 0.05 mL (1 drop) phenolphthalein indicator. If a red color develops add 5N H2SO4 solution dropwise to just discharge the color. Add 8.0 mL combined reagent and mix thoroughly. After at least 10 min but no more than 30 min, measure absorbance of each sample at 880 nm, using reagent blank as the reference solution.

    b. Correction for turbidity or interfering color: Natural color of water generally does not interfere at the high wavelength used. For highly colored or turbid waters, prepare a blank by adding all reagents except ascorbic acid and potassium antimonyl tartrate to the sample. Subtract blank absorbance from absorbance of each sample.

    c. Preparation of calibration curve: Prepare individual calibration curves from a series of six standards within the phosphate ranges indicated in 1c above. Use a distilled water blank with the combined reagent to make photometric readings for the calibration curve. Plot absorbance vs. phosphate concentration to give a straight line passing through the origin. Test at least one phosphate standard with each set of samples.


5. Calculation


                                                        mg P (in approximately 58 mL

                                                               final volume) X 1000

                                      mg P/L =                                                    

                                                                       mL sample



6. Precision and Bias


    The precision and bias values given in Table 4500-P:I are for a single-solution procedure given in the 13th edition. The present procedure differs in reagent-to-sample ratios, no addition of solvent, and acidity conditions. It is superior in precision and bias to the previous technique in the analysis of both distilled water and river water at the 228-g P/L level (Table 4500-P:II).






7. References

  1. EDWARDS, G.P., A.H. MOLOF & R.W. SCHNEEMAN. 1965. Determination of orthophosphate in fresh and saline waters. J. Amer. Water Works Assoc. 57:917.

  2. MURPHY, J. & J. RILEY. 1962. A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta 27:31.

8. Bibliography


SLETTEN, O. & C.M. BACH. 1961. Modified stannous chloride reagent for orthophosphate determination. J. Amer. Water Works Assoc. 53:1031.


STRICKLAND, J.D.H. & T.R. PARSONS. 1965. A Manual of Sea Water Analysis, 2nd ed. Fisheries Research Board of Canada, Ottawa.



Standard Methods for the Examination of Water and Wastewater. 20th Ed. American Public Health Association, American Water Works Association, Water Environment Federation.