The isolation of minor components from complex natural product matrices presents a significant challenge in the field of purification science due to their low concentrations and the presence of structurally similar compounds. This study introduces an optimized twin-column recycling chromatography method for the efficient and simultaneous purification of these elusive constituents. By introducing water at a small flowing rate between the twin columns

a step solvent gradient is created

by which the leading edge of concentration band would migrate at a slower rate than the trailing edge as it flowing from the upstream to downstream column. Hence

the band broadening is counterbalanced

resulting in an enrichment effect for those minor components in separation process. Herein

two target substances

which showed similar peak position in high performance liquid chromatography (HPLC) and did not exceed 1.8% in crude paclitaxel were selected as target compounds for separation. By using the twin-column recycling chromatography with a step solvent gradient

a successful purification was achieved in getting the two with the purity almost 100%. We suggest this method is suitable for the separation of most components in natural produces

which shows higher precision and recovery rate compared with the common lab-operated separation ways for natural products (thin-layer chromatography and prep-HPLC).