Inorganic phosphate determination: colorimetric assay based on the formation of a rhodamine B-phosphomolybdate...

A sensitive technique for inorganic phosphate determination was developed. It is based on the formation of an insoluble rhodamine B-phosphomolybdate complex. After it is washed with 1 N HCl the precipitate is dissolved in acetone and rhodamine B is measured spectrophotometrically at 555 nm. In 1 N HCl, the complex is composed of three molecules rhodamine B and one molecule phosphomolybdate. Due to the high molar absorbance of rhodamine B in acetone and to the threefold amplification of dye concentration compared to Pi concentration in the precipitated complex, a molar absorption coefficient of 330,000 +/- 5000 M-1 cm-1 (SD) is obtained. This allows the determination of quantities as low as 1.5 nmol Pi with good precision, while quantities as low as 0.5 nmol Pi are detectable. The effect of anions and buffers was studied. Some possible applications of the method are illustrated, as, e.g., enzyme activity measurement at very low substrate concentration and determination of small quantities of Pi and total phosphate in (biological) samples.