Optimized Methodolgy for Resolution of Deoxyribonucleosides

초록

To determine the separation condition for five deoxyribonucleosides (dCyd, dUrd, dGuo, dThd, and 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 the stepwise-gradient mode. In this work, the binary systems of mobile phase (water/methanol) were applied. The elution profiles were calculated by the plate theory based on the quadratic equation of capacity factor, lnk'= AF2+BF+C. F denotes the volume fraction of organic modifier in the mobile phase, while A, B, and C 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 7 min. the second composition of water/methanol, 81/19 (vol.%), was step-changed. The agreement between the experimental data and the calculated values was good, so the methodology developed in this work can be used to obtain the optimum separation condition in gradient mode.