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Several compounds in the rhizome of S.gracilis displayed thermal instability in the previous isolation experiments.Ascribed to heat-treatment, time-consuming procedure and irreversible adsorption of samples on stationary phase, those compounds were difficult to isolate and purify by the silica gel column chromatography technique.HSCCC combined with HPLC were used to isolation and purification of those thermolabile compounds, two unknown compounds, from the extract of the rhizome of Sphallerocarpus gracilis, were traced to be thermolabile through the analysis of HPLC chromatogram of before and after heat-treated samples.