A practicable strategy for enrichment and separation of four minor flavonoids including two isomers from barley seedlings by macroporous resin column chromatography, medium-pressure LC, and high-speed countercurrent chromatography.

Separation of minor compounds especially with similar polarities and structures from complex samples is a challenging work. In the present study, an efficient method was successfully established by macroporous resin column chromatography, medium-pressure liquid chromatography, and high-speed countercurrent chromatography for separation of four minor flavonoids from barley seedlings. Macroporous resin column chromatography and medium-pressure liquid chromatography were used for enrichment of these four flavonoids. High-pressure liquid chromatography analysis showed the total content of these four flavonoids increased from 2.2% in the crude extract to 95.3% in the medium-pressure liquid chromatography fraction. It was indicated that the combination of macroporous resin column chromatography and medium-pressure liquid chromatography could be a practicable strategy for enrichment of minor compounds from complex sample. Then, high-speed countercurrent chromatography was employed for separation of these four flavonoids using ethyl acetate/n-butanol/water (0.1% glacial acetic acid) (4:1:5, v/v/v) as solvent system. As a result, four flavonoids including two isomers with purities higher than 98% were obtained. Interestingly, two flavonoids existing in one high-pressure liquid chromatography peak were also successfully separated. All these indicated high-speed countercurrent chromatography had great potential for separation of compounds with similar structures and polarities. This study provides a reference for efficient enrichment and separation of minor compounds from complex sample.