Diastereoisomer separation of silybin A and silybin B by semi-preparative liquid chromatography using Box-Behnken design

The effects of the flow rate, the injection volume, and the mobile phase ratio on the retention time and the resolution of separation of silybin were examined using Box-Behnken design on a symmetry prep C18 column. The optimum values of the operating parameters were 8 ml/min for the flow rate, 3 ml for the injection volume, and 40:60 for the mobile phase ratio. Under the optimal conditions, the retention time of silybin B was 59.552 min and the resolution of silybin A and silybin B was 1.0696, where the purities of the obtained silybin A and silybin B were both over 99% by ultra-performance liquid chromatography. The preparative-liquid chromatography simulation was applied with COMSOL. The optimization results of the response surface are consistent with the experiment and the simulation results. The structures of the compounds were identified by ultra-performance liquid chromatography-tandem mass spectrometry and ultra-performance liquid chromatography- time of flight mass spectrometry.