Separation of inositol phosphates by high-performance ion-exchange chromatography

Abstract

A method is presented for the liquid chromatographic separation of inositol phosphate (IP) esters on a strong anion-exchange column which eliminates the column hydrolysis that plagued earlier investigations. Acceptable separation conditions that resolve the various IP congeners consisted of a 60-min linear gradient from 0.1 to 0.5 M sodium chloride in 0.5 mM tetrasodium ethylenediaminetetraacetate. The method was applied to an enzymatic hydrolysate of inositol hexaphosphate (phytate), a wheat bran extract and a soil extract. The last sample was pre-treated by alkaline bromination prior to high-performance liquid chromatographic separation. Phosphorus-specific detection was accomplished by discrete fraction collection and manual analysis using persulphate oxidation and the ascorbic acid-molybdate phosphate method.