The Separation of the Major Ribonucleotides, Nucleosides, and Bases in Reverse-Phase Ion-Pair Chromatography

The separation of 12 major nucleotides was achieved on a reversed-phase HPLC system with the radially compressed NovaPak C18 column and tetrabutylammonium phosphate (TBA) ion pairing reagent by acetonitrile gradient elution. The effect of tetrabutylammonium phosphate and acetonitrile on the retention of the major nucleotides in this system was studied. It was found that in k' decreases in a proportion to the concentration of acetonitrile in the mobile phase. The maximum of nucleotide retention was found to be at TBA concentration of about 2 mM, the amount of acetonitrile in the mobile phase being constant. A simultaneous separation of main nucleotides, nucleosides, and bases was achieved under optimum conditions.