Separation of phosphomolybdate by affinity chromatography

A new method of separating phosphomolybdate from phosphoric esters and anhydrides using a small column of polyvinylpolypyrrolidone is introduced. When a mixture of phosphate compounds and ammonium molybdate (1–3%, pH 3-5) is poured onto such a column, inorganic orthophosphate (Pi)^p1 is selectively adsorbed onto the column material as phosphomolybdate, while other phosphate compounds, which do not react with molybdate, are drained through the column. Using radioisotopes, retention and recovery percentages were measured. At pH 3, 99.99% of Pi^p32 was retained in the column, while 97 ± 1% of ATP^p32 was recovered in the effluent. Retained Pi^p32 was eluted out later with 0.5 M ammonium hydroxide with a recovery percentage of 98 ± 1%. Unlike other methods of separating phosphomolybdate, the separation was little affected by the presence of reducing agents. The use of disposable columns, which can be prepared easily, packed ahead of time, and stored for later use, makes the radioisotopes assay convenient and contamination-free.