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Adapted from Kuo S. (1996) Phosphorus. Methods of soil Analysis. Part 3. Chemical Methods-SSSA Book Series no. 5.

By Jared L. DeForest

Reagents

Reagent A

In 500 ml volumetric flask

• 400 ml DI water
• 8.8 g ascorbic acid (0.1 M C₆H₈O₆)
• 40.9 g trichloroacetic acid (0.5 M C2HCl₃O₂)
• Bring to volume with DI water.

Reagent B – Make daily (H4)

In 100 ml volumetric flask

• Fill one-third with DI water.
• 1.24 g Ammonium molybdate (0.01 M (NH₄)6Mo₇O₂₄ · 4H₂O)
• Bring to volume with DI water.

Reagent C (H3)

In 1 liter volumetric flask

• Fill one-half with DI water.
• 29.4 g Sodium Citrate (0.01 M Na₃C₆H₅O₇ · 2H₂O)
• 26.0 g Sodium Arsenite (0.1 M NaAsO₂)
• 50 mL 99% Acetic Acid (5% NaAsO₂)
• Bring to volume with DI water.

P ppm P ppm	20 ppm Stock (ml) 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

Standards

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.

Procedure

Add the following in 15 ml centrifuge tube

• 5 ml Reagent A
• 2.5 ml Circumneutral pH (pH 5 – 7.5) aliquot of sample or standard

Add immediately

• 1 ml Reagent B
• 2.5 ml Reagent C

Mix well, incubate for 10 min, centrifuge, dispense 250 µl, 4 times into clear microplate and read at 700 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.

Created: 8/19/09 Revised: 10/10/11