To Study The Techniques For Purification Of Nature Products By High-speed Counter-current Chromatography

Objective:(1) To develop new methods for single preparing stationary and mobile phases used in high-speed counter-current chromatography (HSCCC). (2) To separate and purify active proteins from natural resources by J-type HSCCC apparatus using reverse micelles solvent systems.Method:(1-1) Gas chromatography (GC)-aid single preparation of solvent system. The content of each solvent in stationary and mobile phases of a solvent system can be determined and calculated by GC analysis. According to the volume ratios of solvents in each phase, the mobile and stationary phases were parepared, respectively. Then, the new developed method was used to separate alkaloids from Coptis chinensis Franch by HSCCC. (1-2) UNIFAC mathematic model for single preparation of solvent system. According to UNIFAC mathematic model, the activity coefficient and molar ratio of solvents in each phase were calculated. Then, the density and molecular mass of each solvent were used to calculate the volume ratios for single preparing the stationary and mobile phases. The examples of separating the active compounds from Dioscorea nipponica Makino and Lophatherum gracile were carried out to demonstrate the pragmatic of the new approach. (2) The reverse micelles solvent system was used to separate proteins by J-type-HSCCC. The stationary phase was reverse micelles phase (0.1 mol/L CTAB/isooctane-hexylalcohol), and the mobile phase was aqueous phase consisted of Na-phosphate buffers and adjusted by HCl and KC1 to afford suitable pH value and ionic strength. Bromelain from pineapple and c-phycocyanin from Spirulina were separated by the method.Result:(1-1) Five alkaloids including palmatine, berberine, epiberber- ine, jatrorrhizine and coptisine were separated from C. chinensis by HSCCC using a solvent system composed of chloroform-methanol-water (2:1:1, v/v/v) single prepared by GC-aid method. The purities and recoveries of all products were over 98.5% and 92%. However,134 mL chloroform,336 mL methanol and 452 mL water were saved in HSCCC procedure with the two phases single prepared. (1-2) Dioscin from D. nipponica Makino and quer-cetin-3-O-glucoside from L. gracile were successfully isolated using the solvent systems composed of n-hexane-ethyl acetate-ethanol-water (2:5:2: 5, v/v/v/v) and ethyl acetate-n-butanol-water (2:1:3, v/v/v), which were single prepared by UNIFAC mathematic model coupled with counter-current chromatography solvent selection software (CCC-SSS). The comparative study between the developed single preparation methods and traditional together preparation method were carried out, and there were no difference. But solvent consume was significant different. Especially, when the solvent system single prepared by UNIFAC mathematic model, much organic solvent were saved. For quercetin-3-O-glucoside purification,356 mL ethyl acetate,182 mL n-butanol and 13 mL water were saved. For dioscin isolation,136 mL n-hexane,315 mL ethyl acetate and 58 mL methanol were saved, but 54 mL water was more used, when the new approach was carried out. (2) Pure bromelain (electrophoresis purity and MS purity,100%; recovery,88.4%) from pineapple and high pure c-phycocyanin (purity, A620/A280=4.25; recovery,85%) from Spirulina were successfully separated by HSCCC using reverse micelles solvent systems.Conclusion:(1) The comparative study of single and together preparation of the two phases of solvent system proved that the single preparation methods are convenient, economic and reliable for natural product separation by HSCCC. And importantly, they can save much solvents, and friendly to our living conditions and natural resource. (2) The HSCCC technique using reverse micelles solvent system can be widely employed in the filed of protein separation and purification.