Simultaneous separation of ribonucleotides, nucleosides and nitrogen bases by ion-pair reversed-phase high-performance liquid chromatography on columns with radial compression

Abstract

A method is described for the simultaneous analysis of twelve major nucleotides, certain biochemically essential nucleosides and nitrogen bases. The separation was achieved using an ion-pair reversed-phase high-performance liquid chromatographic (HPLC) system with a radially compressed NovaPak C18 cartridge and tetrabutylammonium phosphate (TBA) as a mobile phase modifier under acetonitrile gradient conditions. The effects of TBA and acetonitrile on the retention of major nucleotides were studied. The retention of nucleotides increased significantly with an increase in the TBA concentration from 0 to 2 mM and remained constant up to 5 mM TBA. The logarithm of the capacity factor decreased in proportion to the concentration of acetonitrile in the mobile phase. The separation of nitrogen bases, nucleosides and some of their deoxy forms was performed on an isocratic ion-pair reversed-phase HPLC system with pentanesulphonic and heptanesulphonic acid using a mu Bondapak C18 column. The described chromatographic procedures were applied to the separation of various biological extracts.