The determination of phosphorus and phosphatase with N-phenyl-p-phenylenediamine.

Methods for the determination of phosphorus depend almost entirely upon the reduction of phosphomolybdic acid with the formation of a complex blue pigment which is presumably a mixture of molybdenum oxides. Many variations of methodology revolve about the means by which the colored complex is obtained. The method of Fiske and Subbarow (2), in which aminonaphtholsulfonic acid (ANS) is utilized, has probably received the widest acceptance. It has been our experience that, when submicrogram amounts of phosphorus are involved, this method has shown a disturbing lack of day to day reproducibility. Furthermore, the absorbance of the solutions is such that special glassware and special photometric techniques are required when only small amounts of phosphorus are to be determined. The substitution of stannous chloride for ANS, as suggested by Kuttner and Cohen (3) and by Shinowara et al. (4), gives optical densities which are somewhat greater, but the absorbances are not constant with respect to time. Stannous chloride is sufficiently vigorous to reduce silicomolybdic acid as well as phosphomolybdic acid, unless great care is taken. Recently Beveridge and Johnson (5) proposed a mixed molybdatehydrazine sulfate reagent, but, upon exploration of this method, we were no better satisfied with the results. Like that of Taussky and Shorr (6), the method also requires that the reagent be freshly prepared. The procedure of Berenblum, and Chain (7) solves many of these problems, but demands some inconvenient manipulations for which special extractors are required. The use of Elon, suggested by Gomori (8), is satisfactory, except that almost an hour is required to achieve maximal absorbance of the colored solutions. The present investigation was undertaken in search of a reducing agent possessing the high sensitivity and rapidity of color development characteristic of stannous chloride and the high order of stability characteristic of ANS. Some years ago Feigl (9) pointed out that benzidine will reduce