An effective high-speed countercurrent chromatography (HSCCC) method was established for further separation and purification of four minor flavonols in addition to five major flavonols which were reported by our previous study from extracts of Flos gossypii. HSCCC was performed with three two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (7.5:15:6:7, v/v), (2.5:15:2:7, v/v), and (0:1:0:1, v/v). The separation was repeated three times, and 3.8mg of 8-methoxyl-kaempferol-7-O-β-D-rhamnoside (HPLC purity 98.27%), 6.7mg of astragalin (HPLC purity 94.18%), 3.3mg of 4'-methoxyl-quercetin-7-O- β-D-glucoside (HPLC purity 94.30%), and 8.2mg of hyperoside (HPLC purity 93.48%) were separated from 150mg of the crude sample. The chemical structures of the flavonols were confirmed by MS, 1H NMR, and 13C NMR. Meanwhile, the results indicated that the target compound with smaller K value (<0.5) can be separated by increasing column length of HSCCC. And, four separation rules of flavonols were established according to the present study and references were summarized, which can be used as a useful guide for separation of flavonols by HSCCC.
Yang Yi,Zhao Yongxin,Gu Dongyu,et al. SEPARATION OF THE MINOR FLAVONOLS FROM FLOS GOSSYPII BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY[J]. JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES,2010,33(16):1502-1515.