A method to optimize a high‐speed countercurrent chromatography solvent system from many possible solvent systems was guided by average polarity and K values of crude samples, which was successfully applied in separation of the naphtho‐γ‐pyrones. This method expanded the scope of spectrophotometer application in the solvent systems selection, and showed the merits of simplicity, rapidity, and low reagent consumption. As a consequence of utilizing the method, an efficient separation procedure was established, and two groups of naphtho‐γ‐pyrone isomers (fonsecinone B and asperpyrone F; asperpyrone C, fonsecinone A, and aurasperone A) were obtained from Pleurotus ostreatus by high‐speed countercurrent chromatography with a solvent system composed of petroleum ether/ethyl acetate/methanol/water (5:5:5:5, v/v). The results indicate that high‐speed countercurrent chromatography could be a powerful technology for separation isomers and analogues, and the method guided by average polarity and K values of crude samples is feasible to optimize solvent system.
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