Optimal composition of mobile phase for resolution of deoxyribonucleosides by plate theory in RP-HPLC

초록

To determine the separation condition for five deoxyribonucleosides (dCyd, dUrd, dGuo, dThd, dAdo) in RP-HPLC (Reversed-Phase High Performance Liquid Chromatography), the optimization method used in this work was from the relationship of capacity factor and mobile phase composition. In a gradient-elution mode by changing mobile phase compositions, the composition and gradient time were determined in stepwise-gradient mode from the relationship. In this work, the binary systems of mobile phase (water/methanol or water/acetonitrile) were applied. A Waters column (30x0.39cm) packed by micro-Bondapak C18 reversed-phase material of 10um particle size was used, and the flow rate of mobile phase was set at 1.0ml/min. The experimentally measured dead volume of the chromatographic system was 2.7cm3, and the volume of the mixer for the two mobile phases was 6.8cm3. The elution profiles were calculated by the plate theory based on the quadratic equation of capacity factor, lnk' = LF2+MF+N. F denotes the volume fraction of organic modifier in the mobile phase, while L, M, and N the empirical constants. The optimal mobile phase was obtained by comparing the calculated results of the resolutions of the five components and the retention times of last-eluting component (dAdo). The final result showed that the initial mobile phase was pure water, then after 7min. the second composition of water/methanol, 81/19 (v/v, %), was step-changed. The agreement between the experimental data and the calculated values was good.