Measuring total phosphorus of plants and soil.

Total Phosphorus

            Following is a simple, easy and accurate method for total phosphorus in plants and soils.  The basic method is taken form Aspila K.I., H. Agemian and A . S. Y. Chau. 1976. A semi-automated method for the determination of inorganic, organic and total phosphate in sediments. Analyst 101 187-197.  The modification of adding Mg(NO3)2 is take from K.R. Ruttenberg. 1990 Diagenesis and burial of phosphorus in marine sediments: implications for the marine phosphorus budget. Ph.D. dissertation. Yale Univ. New Haven, CT.  See KC Ruttenberg 1992.

            All samples ground at least to 40-60 mesh, either on a Wiley mill or Bob's roller mill.  Weigh about 0.1 gm of sample into a 20 ml glass scint vial.  Then add 0.5 mls of 50% w/v Mg(NO3)2 letting the sample soak up the solution but do not let the sample dry completely before ashing or it will ignite and flare up during ashing. Ashed for 2 hours at 550 C (about 1/2 hour to get to temperature and then 1.5 hours at 550).  The scint vials can be marked with a diamond tipped pen or other high temperature pens. You can use glass petri dishes to stack the vials. After cooling add 10 mls of 1 N HCl and vortex to get all sediment into solution.  Then shake for 16 hours.  If the vials are left over night there is no need to centrifuge or filter the solution.

Dilute subsamples depending on levels expected and measure PO4-P using the method for high concentrations of Harwood, et al. 1969.

For 1000 ml of 1 N HCL

Concentrated Hydrochloric Acid is calculated to be 12.178 N based on a density of 1.2 g/mL, a formula weight of 36.46 g/mol, and a concentration of 37% w/w.

To make a 1 N solution, slowly add 82.117 mL of your stock solution to 250 mL deionized water.

Adjust the final volume of solution to 1000 mL with deionized water.

Harwood, J.E., R.A. van Steenderen and A.L. Kuhn. 1969.  A rapid method for orthophosphate analysis at high concentrations in water.  Water Research 3:417-423.

Ruttenberg, K.C. 1992. Development of a sequential extraction method for different forms of phosphorus in marine sediments. Limnol. Oceanogr. 37(70: 1460-1482.

Phosphorus Assay

A rapid Method for Orthophosphate Analysis at High Concentrations in Water.

Harwood, Steenderen and Kuhn (1969).

A variation of the Murphy and Riley (1962) method in which the calibration graph is linear to 100 um/L.  The range was extended by increasing the strength of the antimony and ascorbic acid.  This method is suitable for both trace and high-level analysis.  This method and Murphy and Riley (1962) method depends on the condensation of orthophosphate and molybdate ions in acid solution to molybdophosphoric acid. On reduction with ascorbic acid a blue complex is formed which is then read with a spectrophotometer at 890 nm.   For a discussion on interfering ions see John (1968).

Reagents:  All reagents are A.R. grade where possible and volumetric flasks are used for all volumes.

  1. 4 N H4SO4:112 ml concentrated sulphuric acid is slowly added to 800ml distilled or DI water.After cooling the solutions diluted to 1 liter with distilled or DI water.
  2. 0.96 % molybate: 4.8 grms ammonium molybate (NH4)6 MO7O24. 4H2O crystals is dissolved in distilled or DI water, with heating.On cooling the solution is diluted to 500ml.Good for approximately 6 weeks.
  3. 10% ascorbic acid: 10 grms asocrbic acid is dissolved in 80 mls distilled or DI water and diluted to 100 mls or 5 grams ascorbic acid is dissolved in 40 mls distilled or DI water and diluted to 50 mls.
  4. K- antimony tartrate:0.667 grams K-antimony tartrate is dissolved in 220 mls of distilled or DI water and then diluted to 250 mls.

Mixed Reagents:

MR1.          Equal volumes of 4N H2SO4 to Ammonium Molybdate.  Use 0.28 mls of this mixed reagent for each ml of sample.

MR2.          Equal volumes of Ascorbic Acid to K-antimony tartrate..  Used 0.11 mls this mixed reagent for each ml of sample.

For Example:   For each 3 mls of sample add 0.84 mls MR1; vortex; wait one minute.

            Then add 0.33 mls MR2; vortex.  Wait at least 30 minutes in cold weather then read at 890 nm in   a 5 cm cell.


Stock  10,000 um/l        1.3609 grams KH2PO4 to 1 liter distilled or DI.

Stock  1,000 um/l          0.1361 grams KH2PO4 to 1 liter distilled or DI.

Standards     in 100ml volumetric flask

(um/l)                            mls of 10,000 um/l                     mls 1,000 um/l

100                               1

80                                 0.8

60                                 0.6

40                                 0.4

20                                 0.2

10                                                                                 1

 5                                                                                 0.5

 3                                                                                 0.3

 1                                                                                 0.1

Harwood, J.E., R.A. van Steenderen and A.L. Kuhn. 1969.  A rapid method for orthophosphate analysis at high concentrations in water.  Water Research 3:417-423.

John, M. K.  1970?? Colorimetric determination of phosphorus in soil and plant materials with ascorbic acid.  Soil Sci.  109:214-220.

Murphy, J., and Riley, J.P. 1962. A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta. 27: 31-36.