Determination of Phosphorous by Colorimetry


  1. ACID MOLYBDATE STOCK SOLUTION - In a 2 L volumetric flask, dissolve 125 g ammonium molybdate in 400 mL deionized H20 by heating to 60oC until solution is clear. Allow to cool, then dissolve 2.9 g antimony potassium tartrate in the molybdate solution. Place the flask in an ice bath in the sink and very slowly add 1500 mL con. H2S04. Cool in the ice bath and very slowly dilute to 2 L with H20. (It is very important to not let this solution get too hot. It may cause severe burns if mishandled.) Store in brown bottle under refrigeration.
  2. ASCORBIC ACID STOCK SOLUTION - Dissolve 211.2 g ascorbic acid in 1500 mL deionized water and dilute to 2 L. Store in brown bottle under refrigeration.
  3. WORKING SOLUTION (Prepare Fresh Daily) - Add 20 mL acid molybdate stock to about 800 mL water. Mix, then add 10 mL ascorbic acid stock and dilute to 1 L.
  4. STANDARDS - 10, 80 ppm phosphorous. To make 1000 ppm P stock solution, dissolve 4.3937 g of dried KH2P04 in deionized H20 then dilute to 1 L. (10 ppm: 1 mL of 1000 ppm stock diluted to 100 mL dH20. 80 ppm: 8 mL of 1000 ppm stock diluted to 100 mL dH20.)



Procedure for Preparing Samples

  1. Wet or dry ash sample as previously described.
  2. Set diluter to 1:100.
  3. Place hose from diluter into flask containing the P working solution.
  4. Run diluter through several cycles to draw the working solution into diluter.
  5. See diluter manual for instructions on diluting samples.
  6. Use Vortex stirrer to insure proper mixing.
  7. Allow color to develop for 30 minutes.
  8. Set up spectrophotometer as follows.

Set Up of Spectrophometer

  1. Turn power on.
  2. Let instrument run through self test.
  3. Enter wavelength at 660 nm.
  4. Press <Second Function> <Go To >.
  5. Allow for a 30 min. warm up.
  6. Press <T% / A/C> until you get to concentration mode.
  7. Asp Blk by pressing <Sample> (hold under tubing until set up).
  8. Press <Second Function> <100% T / Zero A>
  9. After set up, remove Blk from tubing.
  10. Asp high standard by pressing <Sample> (hold under tubing until set).
  11. Enter value for high standard - Press <%T / A/C> (80 ppm corresponds to 80% in sample).
  12. Let set up then remove tube.
  13. Read cks and samples by pressing <Sample>, record value, remove tube (can read accurately to 140 ppm or 1.40% P in sample).
  14. Clean cell, leaving water in cell.
  15. Disconnect pump.
  16. Turn power off.
  17. Cover instrument.


  1. Murphy, J. and J. P. Riley. 1962. A Modified Single Solution Method for the Determination of Phosphate in Natural Waters. Anal Chim. Acta. 27:31-36.
  2. Nelson, W. L., A. Mehlich and E. Winters. 1953. The Development, Evaluation and Use of Soil Tests for Phosphorus Availability. In W. H. Pierre and A. G. Norman (ed.). Soil and Fertilizer Phosphorus, Vol. IV Agronomy, A Series of Monographs, Academic Press, Inc., NY pp.153-188.
  3. Watanbe, F. S. and S. R. Olsen. 1965. Test of an Ascorbic Acid Method for Determining Phosphorus in Water and NaHC03 Extracts from Soil. Soil Sci. Soc. Amer. Proceedings, 29:677-78.