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Flavonol glycosides are yellowish compounds which are widespread in the plant kingdom. Apart from the coloring properties, flavonol glycosides are known to possess considerable antioxidative capacities. Investigation into the composition of flavonol glycosides from tea has shown that tea leaves contain a variety of different flavonol-O-glycosides. The aglycons myricetin, quercetin, and kaempferol are glycosylated with one, two, or three sugar moieties. By using HSCCC, the flavonol glycosides were purified in a single run on a preparative scale. Peaks were assigned by means of HPLC-DAD and ESI-MS/MS through comparison with authentic reference compounds. Quercetin-3-O-rutinoside and kaempferol-3-O-rutinoside were successfully purified with a mixture of ethyl acetate/water (1:1, v/v; mobile phase: lower phase). HSCCC was capable of purifying several hundred milligrams of the above-mentioned flavonol glycosides within 2 h.