High-Performance Liquid Chromatography of Nucleobases, Nucleosides and Nucleotides. I. Mobile Phase Composition for the Separation of Charged Solutes by Reversed-Phase Chromatography

To find optimal conditions for the separation of nucleobases, nucleosides and nucleotides by reversed-phase chromatography, the polarity, pH, ion concentration and polarity of the buffer ions of the mobile phase were varied. A systematic study of the effects of these parameters on retardation led to the formulation of the following simple rules: (1) methanol changes only column capacity ratios, not selectivity; (2) pH influences both column capacity ratios and selectivity; (3) the concentration of buffer ions had hardly any effect on retardation; (4) the hydrophobicity of the buffer ions has strong effects only on column capacity ratios and selectivity of solutes with opposite charge; and (5) a mixture of buffer ions with different hydrophobicities has the chromatographic properties of a buffer ion with intermediate hydrophobicity.