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After an initial clean-up step on silica gel, a preparative high-speed counter-current chromatography coupled with on-line high-performance liquid chromatography-diode array detection method (HSCCC-HPLC-DAD) was successfully developed for the isolation and determination two polymethoxylated flavones, 3',4',7-trimethoxyquercetin and artemetin, from the aerial part of Taraxacum mongolicum. The HSCCC separation was performed with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (6:5:6:5, v/v/v/v) at a flow rate of 1.5 mL/min and at 800 rpm. The on-line purity monitoring of a representative aliquot from each HSCCC fraction was operated automatically. Using this method, fractions with high purity were collected. The HSCCC purification step was done in 5 h, and afforded 84.2 mg of 3',4',7-trimethoxyquercetin and 52.3 mg of artemetin, with purity over 98% from 200 mg of the enriched extracts of T. mongolicum. The structures were identified by electorspray ionization mass spectrometry and 1H NMR experiments. To our best knowledge, 3',4',7-trimethoxyquercetin was obtained from the plant of genus Taraxacum for the first time by our group. This hyphenated method could be used for the preparation of bioactive compounds with higher purity from natural products.