The estimation of phosphorus

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Abstract
When the King-Fiske and Subbarow P method (Biochem. Jour. 26, 292, 1932) was investigated using an absolute photometer, the intensity of the blue color produced does not reach a steady value within a reasonable period; and the intensity at a given time varies markedly with temp. In the procedure now described these disadvantages are eliminated. Orthophosphate P: to an aliquot of test, soln. add 2 c.c. 60% HClO4, 2 c.c. amidol reagent (2 g.2:4-diaminophenol hydrochloride, 40 g. NaHSO2, glass-diSuBed water to 200 c.c.) and 1 c.c. 8.3% NH4 molybdate. Dilute to 25 c.c, measure the color intensity after 5-30 min. and read P conc. from standardisation curve obtained with pure phosphate solns. Total P: digest an aliquot of test soln with 2.2 c.c. 60% HClO4 until colorless, transfer to a volumetric flask, add amidol reagent and molybdate, and proceed as before. To determine orthophosphate P in turbid and/or highly colored solns. proceed as follows: develop the blue color as described above, add 1 c.c. saturated (COOH)2, transfer to separatory funnel, extract the blue color with 2 successive portions (10 c.c. and 5 c.c.) of isobutyl alc., transfer the combined extract to a volumetric flask, dilute to 25 ml. with 95% ethyl alc., measure the color intensity within 24 hrs. and read P conc. from a standardisation (color intensity/mg. P) curve.