A new method of separating inorganic orthophosphate from phosphoric esters and anhydrides by an immobilized catalyst column.

A column of polyvinylpolypyrrolidone packed in a 1-ml Tuberculin syringe is used as a stationary phase for affinity chromatography of phosphomolybdate. When a mixture of inorganic orthophosphate, phosphoric esters, and phosphoric anhydrides is introduced into such a column in the presence of molybdate (2–3%, pH 3–5), inorganic orthophosphate adsorbs specifically to the column material as phosphomolybdate, while other phosphate compounds, which do not react with molybdate, drain through. Mild centrifugation (8–50g) is used to hasten elution to minimize the hydrolysis of acid-labile phosphates. The method described allows separation of radioactive phosphate compounds from a small amount of solution (0.2–1.0 ml) without either organic solvent extraction or transfer of sample, which may cause error and/or contamination. With 3% molybdate, pH 3.0, 98.5 ± 0.6% of ATP is recovered, while 0.05 ± 0.01% of inorganic orthophosphate is eluted in the effluent. Retained inorganic orthophosphate can be eluted later by 0.5 m ammonium hydroxide with a recovery of 98.2 ± 0.9%. Unlike other methods of separating phosphomolybdate, this one is virtually insensitive to the presence of reducing reagents.