Studies On Separation And Purification Of High Polar Compounds (Bergenin, Etc) By HSCCC

Active components in natural products can be divided into low polar, polar and medium polar according to the polarity. The active components of high polarity are tend to dissolve in water phase of high speed countercurrent chromatography solvent system, so it is difficult to reach the ideal separationeffect. And this phenomenon has been restricting the separation and purification of active components with high polarity in natural products. In this study, bergenin, paeoniflorin and salvonic acid B were taken as examples to explore high speed countercurrent chromatography solvent system used in the separation and purification of high polar compounds. To research the application of high speed countercurrent chromatography with salt solution solvent system and petroleum ether-ethyl acetate-butanol-water pH-zone refining high speed countercurrent chromatography solvent system in separation and purification of high polar active components in natural products. And this research provids a universal high speed countercurrent chromatography solvent system for the separation and purification of high polar compounds. The main results and conclusions are as fellows:1. The separation was performed with a two-phase system composed of petroleum ether-ethyl acetate-butanol-1%Na2SO4(aq)(1:2:2:3, v/v). The lower-phase was used as the mobile phase with a flow rate of2.0ml/min, the apparatus rotated at850r/min and the effluents were detected at254nm.320.30mg of bergenin was obtained from2.00g of the crude sample in one step. The purity determined by high performance liquid chromatography (HPLC) was96.59%. The structure of the target compound was identified by electrospray ionisation mass spectrometry (ESI-MS),’H-nuclear magnetic resonance (’H-NMR) and13C-nuclear magnetic resonance (13C-NMR).2. The separation was performed with a two-phase system composed of butanol-ethyl acetate-5%Na2SO4(aq)(4:1:5, v/v). The lower-phase was used as the mobile phase with a flow rate of2.0ml/min, the apparatus rotated at800r/min and the effluents were detected at254nm.56.13mg of paeoniflorin was obtained from100.00mg of the crude sample in one step. The purity determined by high performance liquid chromatography (HPLC) was98.67 %. The structure of the target compound was identified by electrospray ionisation mass spectrometry (ESI-MS),1H-nuclear magnetic resonance(1H-NMR) and13C-nuclear magnetic resonance (13C-NMR).3. The separation was performed with a two-phase system composed of petroleum ether-ethyl acetate-butanol-water (2:3:6:9, v/v). The lower-phase was used as the mobile phase with a flow rate of2.0ml/min, the apparatus rotated at850r/min and the effluents were detected at254nm.1419.15mg of salvianolic acid B,171.25mg of rosmarinic acid and52.34mg of methyl lithospermate were obtained from6.00g of the crude sample in one step. The purities determined by high performance liquid chromatography (HPLC)were94.53%,96.36%and96.83%respectively. The structure of the target compound was identified by electrospray ionisation mass spectrometry (ESI-MS),’H-nuclear magnetic resonance (1H-NMR) and13C-nuclear magnetic resonance (13C-NMR).4. The separation was performed with a two-phase system composed of petroleum ether-ethyl acetate-butanol-water (1:4:2:5.5, v/v). The lower-phase was used as the mobile phase with a flow rate of2.0ml/min, the apparatus rotated at850r/min and the effluents were detected at254nm.217.62mg of cichoric acid was obtained from4.00g of the crude sample in one step. The purity determined by high performance liquid chromatography (HPLC) was98.31%. The structure of the target compound was identified by1H-nuclear magnetic resonance(1H-NMR) and13C-nuclear magnetic resonance (13C-NMR).Through the separation and purification of the4high polar compounds, this study provids a method for screening and optimizing the high speed countercurrent chromatography with salt solution solvent system and petroleum ether-ethyl acetate-butanol-water pH-zone refining high speed countercurrent chromatography solvent system, and these solvent system are suitable for preparative separation and purification of high polar compounds. This study provides new ideas and theoretical basis for the separation and purification of high polar compounds.