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 3.8 mg of 8-methoxyl-kaempferol-7-O-β-D-rhamnoside (HPLC purity 98.27%), 6.7 mg of astragalin (HPLC purity 94.18%), 3.3 mg of 4′-methoxyl-quercetin-7-O-β-D-glucoside (HPLC purity 94.30%), and 8.2 mg of hyperoside (HPLC purity 93.48%) were separated from 150 mg 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.

中文翻译：

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用高速逆流色谱法从棉絮中分离微量黄酮醇

建立了一种有效的高速逆流色谱 (HSCCC) 方法，用于进一步分离和纯化四种次要黄酮醇，以及我们之前研究报告的棉花提取物中的五种主要黄酮醇。HSCCC 使用由正己烷-乙酸乙酯-甲醇-水 (7.5:15:6:7, v/v)、(2.5:15:2:7, v/v)、和（0:1:0:1，v/v）。重复分离3次，8-甲氧基-山奈酚-7-O-β-D-鼠李糖苷3.8mg（HPLC纯度98.27%）、黄芪甲苷6.7mg（HPLC纯度94.18%）、4'-3.3mg甲氧基-槲皮素-7-O-β-D-葡萄糖苷（HPLC 纯度 94.30%）和 8.2 mg 金丝桃苷（HPLC 纯度 93.48%）从 150 mg 粗样品中分离出来。黄酮醇的化学结构经质谱、1H NMR 和 13C NMR 证实。同时，结果表明，通过增加HSCCC的柱长可以分离K值较小(<0.5)的目标化合物。并根据本研究建立了黄酮醇的4个分离规则，并总结了参考文献，可作为HSCCC分离黄酮醇的有益指导。