Notes on the Colorimetric Determination of Inorganic Orthophosphate

Abstract

The effect of the amount of the amidol reagent on the rates of hydrolysis of some acid-labile phosphate compounds in the determination of inorganic orthophosphate according to the method of Allen and its modifications by Whelan and Bailey and by Nakamura is reported. It is shown in this paper that the most important factor in these methods is the temperature at which molybdenum blue color is developed. The rate constants of acid hydrolysis of adenosine triphosphate, glucose-1-phosphate, fructose-1,6-diphosphate phosphoryl (enol) pyruvate and inorganic pyrophosphate at various temperatures under the experimental conditions of Nakamura’s method in the presence and absence of molybdate are also presented. In the determination of total phosphorus, there was only a very slight, if any, condensation of orthophosphate to polyphosphates when the sample to be tested was incinerated in perchloric acid or in sulfuric acid. Both colorimetric method and radioactivity measurements were applied for the determination of orthophosphate remained as such in the digest. Also, the loss of phosphoric acid was not observed when care was taken so as not to allow the white vapor of the acid emerge from the digestion flask during incineration.