Ascorbic Acid Method
Adapted from Kuo S. (1996) Phosphorus. Methods of soil Analysis. Part 3. Chemical Methods-SSSA Book Series no. 5.
Reagents
Solution A
In 500 ml volumetric flask
- 250 ml DI water
- 70 ml Sulfuric acid (18M H2SO4)
- Mix well & then bring to volume with DI water.
Solution B
In 500 ml volumetric flask
- 250 ml DI water
- 20 g Ammonium molybdate [(NH4)6Mo7O24 · 4H2O]
- Mix well & then bring to volume with DI water.
Solution C
In 100 ml volumetric flask
- 50 ml DI water
- 0.2728 g Antimony potassium tartrate [K(SbO) · C4H4O6 · ½ H2O]
- Bring to volume with DI water.
Working Solution - Use within 8 hours
(enough for 50 samples + standards)
In 500 ml volumetric flask
- 250 ml Solution A
- 75 ml Solution B
- 2.64 g Ascorbic acid (1 M C6H8O6)
- 25 ml Solution C Bring to volume with DI water
Standards
P ppm | 20 ppm Stock (ml) | Extract (ml)
0.0 0.0 10.0
0.5 0.25 9.75
1 0.5 9.5
2 1.0 9.0
5 2.5 7.5
10 5.0 5.0
15 7.5 2.5
20 10.0 0.0
In 100 ml volumetric flask
- 50 ml Extraction solution (e.g. 0.5M NaHCO3)
- 2.0 ml Certified standard P solution (1,000 ppm)
- 2 drops 0.25% p-nitrophenol
If acidic, then add 5 M NaOH dropwise until yellow, then add 0.25 M H2SO4; until clear. If alkaline, add acid until clear. Beware, it might strongly effervesce.
Bring to volume to make a 20 ppm working standard. Follow table for standard dilutions.
Suggested Procedure
In 15 or 50 ml centrifuge tube
- Transfer an aliquot of sample solution that contains 2 to 40 µg P.
- Dilute with DI water, if necessary, but record dilution for later calculation.
- It is recommended to use a 6.25:1 ratio between sample/standard and the working solution.
Mix well, incubate for exactly 10 min, dispense 300 µl, 4 times into clear microplate and read at 880 nm. Twenty samples can fit on one microplate. Run standards in duplicate. It is recommended to have sample absorbance between 0.5 and 3.0. If below 0.5, consider using the Spec-20 w/ cuvettes, which has a longer path length. This procedure is sensitive to 0.5 µg P ml1.
Created: 07/02/10