Separation Of Natural Acidic Compounds By PH-Zone-Refining Countercurrent Chromatography And Its Theory

Countercurrent chromatography is a continuous liquid-liquid distribution technique that does not require a solid carrier and no irreversible adsorption of samples would happen.pH-zone-refining countercurrent chromatography,developed from conventional countercurrent chromatography,is a special preparative method for separation of ionizable constituents,where the amount of injected sample can reach as much as tenfold that of conventional countercurrent chromatography.In this paper,pH-zone-refining countercurrent chromatography was applied to separate acidic components from natural products and its chromatographic retention mechanism was discussed in comparison with pH-peak-focusing mode.The main contents are as follows:（1）The combined use of pH-zone-refining countercurrent chromatography and conventional countercurrent chromatography was successfully employed for the preparative separation and purification of baicalin and wogonoside from the traditional Chinese medicinal herb Scutellaria baicalensis Georgi.A more environmentally friendly biphasic solvent system composed of n-butanol-ethyl acetate-water（2:3:5,v/v）was used for pH-zone-refining countercurrent chromatography,in which trifluoroacetic acid was added to a final concentration of 10mmol/L in the organic phase and ammonia was added to a final concentration of 10mmol/L in the aqueous phase while a two-phase solvent system composed of ethyl acetate-ethanol-3 mmol/L hydrochloric acid（10:1:10,v/v）was used for the conventional countercurrent chromatography.Two elution modes including reverse displacement mode and normal displacement mode were investigated for pH-zone-refining countercurrent chromatography.The first run provided 186.7 mg of baicalin with a purity of 95.3%and 143.4 mg of a mixture of baicalin and wogonoside from 500.0 mg of the crude extracts of Scutellaria baicalensis Georgi by pH-zone-refining countercurrent chromatography with reverse displacement elution mode.The mixture was further separated by means of a second run in conventional countercurrent chromatography,yielding 64.3 mg of baicalin with a purity of 98.2%and 46.1 mg of wogonoside with a purity of 98.9%.（2）On the basis of Pharmacopoeia research,Box-Behnken response surface test was used to investigate the extraction process factors such as extraction time,liquid to solid ratio and the concentration of ethanol.The effects of holding time,holding temperature,settling time and concentration of concentrated extraction water on acid deposition were studied.Finally,the best extraction and purification process of Scutellaria baicalensis was established.（3）pH-zone-refining countercurrent chromatography was investigated in preparative separation of two bioactive components,oleanolic acid and ursolic acid,from three different natural products,Aralia chinensis,apple peels and Eriobotrya japonica Thunb.Oleanolic acid and ursolic acid were structural isomeric pentacyclic triterpene acids,which was widely distributed in many natural products.However,it was difficult to separate these components with high purity by conventional methods.A biphasic solvent system composed of n-hexane-dichloromethane-methanol-water（7:3:2:8,v/v）was selected,in which an optimized concentration of 10 mmol/L trifluoroacetic acid was added in the upper phase as retainer and a 10 mmol/L ammonia（with 25%-28%NH₃）was added in the aqueous phase as eluter.A 38.6 mg of oleanolic acid with 99.01%purity could be separated from 100.0 mg of the crude extract of Aralia chinensis,while 65.6 mg of a mixture of ursolic acid（90.98%）and oleanolic acid（6.51%）and 46.6 mg of a mixture of ursolic acid（74.35%）and oleanolic acid（23.61%）could be separated from 100.0 mg of the crude extract of apple peels and 100.0 mg of the crude extract of Eriobotrya japonica Thunb,respectively,by pH-zone-refining countercurrent chromatography using the above selected biphasic solvent system.Results showed that pH-zone-refining countercurrent chromatography is an efficient method for preparative separation of pentacyclic trterpene acids from natural products.（4）Reverse phase countercurrent chromatographic and reverse phase high performance liquid-liquid chromatographic separation of oleanolic acid and ursolic acid with hydroxypropyl-β-cyclodextrin as additive were investigated,respectively.Results showed that a high peak resolution,R_S=8.143,was achieved for the two structural isomeric compounds by conventional reverse phase high performance liquid chromatography,which was greatly improved compared with the published values（improved about 300%）.Meanwhile,a biphasic solvent system composed of n-hexane-ethyl acetate-0.1 mol/L hydroxypropyl-β-cyclodextrin（9:1:10,v/v）was selected for reverse phase liquid-liquid chromatography,which provided a high peak resolution,R_S=8.500,for semi-preparative apparatus.The formation constants were determined to be 172.521 L/mol for oleanoic acid and 159.908 L/mol for ursolic acid by liquid-liquid chromatography.（5）The problem of peak overlapping and peak sequence occurred in pH-zone-refining countercurrent chromatography were discussed from the aspects of the acidity and hydrophobicity of the target components by measuring p Ka value and oil-water partition coefficient of baicalin and wogonoside.The two components overlapped in pH-zone-refining countercurrent chromatography were separated by pH-peak-focusing countercurrent chromatography,and the separation mechanism of the mode was explained.