Study On Separation Of Natural Products And Solvent System Selection In High-speed Countercurrent Chromatography

High-speed countercurrent chromatography (HSCCC) is a liquid-liquid chromatography which uses no solid support and has been widely used as an effective means of separation and purification of natural products. Spirodela polyrrhiza (L.) Schleid. and Malus hupehensis are traditional Chinese medicines, whose main active ingredients are flavonoids.In this study, flavonoids were isolated from S. polyrrhiza and Malus hupehensis leaves respectively by HSCCC. Partition coefficients of target components were measured in a series of solvent systems by HPLC to choose the optimal ones. In S. polyrrhiza’s part, hexane-ethyl acetate-methanol-water (1:10:1:10,v/v) was selected and the two un-separated components were successfully separated by recycling HSCCC. In Malus hupehensis leaves’part, hexane-ethyl acetate-methanol-water (1:4:1:4,v/v) was chosen and the un-separated components were successfully isolated by the second HSCCC separation with hexane-ethyl acetate-methanol-0.1mol/L Cu2+(1:4:1:4,v/v). HSCCC conditions: temperature,25℃; revolution speed,900r/min; forward elution mode; flow rate,2.0ml/min; detection,280nm.Nonrandom two-liquid model (NRTL), a thermodynamic model was introduced for fast solvent system selection in HSCCC. The partition behavior of six given solutes in the biphasic solvent system was represented by the NRTL model. The performance of NRTL method was evaluated with the hexane-ethyl acetate-methanol-water solvent family. The calculated partition coefficients of the six model solutes were compared with the experimental data, which shows that the NRTL model can provide fast and reliable predictions of partition coefficient.The results of this study prove that HSCCC is an effective method for isolation of natural products, and the NRTL method is a fast solvent selection which is significant to reduce the workload of the HSCCC experiment.