Optimum solvent selectivity and gradient mode for deoxyribonucleosides in reversed-phase high-performance liquid chromatography

Abstract

The separation condition for five deoxyribonucleosides (dCyd, dUrd, dGuo, dThd, and dAdo) was determined by the optimization of mobile phase conditions. In this work, the binary system of water and methanol was applied in RP-HPLC. The elution profiles were calculated using plate theory based on the quadratic equation of retention factor, ln k= A+ BF+ CF 2, and F was the volume percent of methanol. We modified the plate theory to calculate elution profile in both step and linear gradient mode. The optimal mobile phase composition was obtained by comparing the resolutions of the five deoxyribonucleosides and the separation times of last-eluting solute (dAdo) in step gradient elution. Also, in linear gradient elution, resolutions and separation time could be predicted by a slight modification of the relationship used in step gradient mode. The final calculated result for the resolution of five deoxyribonucleosides suggested that the appropriate first mobile phase composition was water/methanol 88/12 vol.%, then after 7 min the second composition of water/methanol, 65/35 vol.% was step-changed. Under experimental conditions, the agreement between the experimental data and the calculated values was relatively good.