A comparative study of application high-speed counter-current chromatography (HSCCC), and reversed-phase semi-preparative liquid chromatography (RPLC) for separating chemical compounds from Spiranthes australis (R. Brown) Lindl roots was investigated. Some parameters such as solvent system, flow rate of the mobile phase, separation time, solvent consuming, sample size, yields, recoveries and purities of the products were all investigated. The results indicated that three compounds were separated by HSCCC only in one run and less than 240 min with the purities and recoveries of over 97 and 91%, respectively, which were also isolated by RPLC with the purities of over 98.5% and recoveries of less than 75% using multiple chromatographic steps, and the total separation time and solvent consuming were about 7 and 13 times more than HSCCC. The obtained compounds were elucidated as 5-hydroxyl-7,4'-dimethoxy-flavone (1), 5-hydroxyl-7,3',4'-dimethoxy-flavone (2), and 5-gamma,gamma-dimethylallyi-8-[2-(2,6-dihydroxyphenyl)-3-dimethyl-but-2-enyol]-umbelliferon (3), a novel compound, based on spectroscopic analysis. (C) 2007 Elsevier B.V. All rights reserved.