Ascorbic Acid Method

DeForest Lab
Ohio University
Inorganic Phosphorus (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 & 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
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.
P ppm
0.0
0.5
1
2
5
10
15
20
20 ppm
Stock (ml)
0.0
0.25
0.5
1.0
2.5
5.0
7.5
10.0
Extract
(ml)
10.0
9.75
9.5
9.0
7.5
5.0
2.5
0.0
Suggested Procedure
In 15 or 50 ml centrifuge tube
Transfer an aliquot of sample solution that contains 2 to 40 g 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 l, 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 g P ml-1.
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Created: 07/02/10

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