ADAPTATION OF THE METHOD OF LOWRY AND LOPEZ TO THE ESTIMATION OF INORGANIC AND ORGANIC PHOSPHATE IN PLANT EXTRACTS

Abstract

The method of Lowry and Lopez using ascorbic acid as the reducing agent with the Fiske–Subbarow reagents at pH 4.0 is proposed for the estimation of phosphate in plant extracts. The method has been reinvestigated with respect to its adaptation to the Coleman Universal spectrophotometer, Model 14, to suitability of wave length, pH range, reagent concentration, rate of reaction, color stability, and the effect of silicate. The experiments, in general, confirm those of Lowry and Lopez. A wave length of 650 mμ has been employed. No blue color is formed in the reagent blank between a pH of 3.1 and 4.2; accordingly a pH of 4.0, maintained by a sodium acetate – acetic acid buffer, has been used. A concentration of 0.25% ammonium molybdate and 0.1% ascorbic acid has been shown to be optimum for maximum color development in concentrations of phosphate up to about 25 μgm. P in the final volume. The reduction of phosphomolybdic acid is complete in about five minutes, and the method is characterized by a high degree of color stability. Silicate interferes with the reaction. Where the plant has a liberal silicate economy the cell wall material, with which the silicon is associated, must be removed from the extract. The method has been applied to the estimation of phosphate liberated from organic combination by hydrolysis for specified times in 1 N hydrochloric acid. Total phosphate is estimated by wet ashing with 60% perchloric acid. Recovery tests of known amounts of inorganic phosphate and glucose-1-phosphate added to wheat leaf and seedling extracts give an absolute error within 2%, which is considered permissible. A balance sheet of phosphate fractions in the supernatant, residue, and whole juice of wheat leaves is discussed.