Analysis of Ribonucleotides by Reverse-Phase HPLC Using Ion Pairing on Radially Compressed Or Stainless Steel Columns

Abstract

A method is described for the analysis of 16 major nucleotides including NAD, IDP, GDP, cAMP, cGMP and succinyl AMP by reverse phase HPLC. The use of 65mM KH₂PO₄, at pH 3.2, low concentrations of the ion pairing reagent tetrabutylammonium phosphate and acetonitrile allowed the simultaneous separation of these nucleotides by isocratic or gradient elution in 18 and 28 minutes respectively. Stainless steel and radially compressed columns were compared and a similar separation profile was obtained. The latter columns increased retention and improved the efficiency of separation.