A method for the preparation of pure ziyuglycoside Ⅰ and Ⅱ from the crude extracts of the roots of Sanguisorba officinalis L.has been developed using semi-preparative high performance liquid chromatography coupled with evaporative light scattering detector (SP-HPLC-ELSD).Ziyuglycoside Ⅰ and Ⅱ were extracted from the raw material with 80% ethanol solution,then purified with precipitation method and petroleum ether.Since the semi-preparative HPLC consumes large quantities of solvents and samples,analytical HPLC was first used to investigate the effects of the mobile phase composition,gradient elution conditions and injection volume on the separation efficiency of ziyuglycoside Ⅰ and Ⅱ.Then the flow rate and injection volume were scaled up to semi-preparative HPLC according to the magnification formula with the same gradient elution conditions.For a better separation,the flow rate was reduced from 4.7 to 4 mL/min.The final optimal chromatographic conditions were as follows:mobile phase:a mixture of methanol (A) and 0.1% formic acid in water (B) for gradient elution;flow rate:4 mL/min;and injection volume:0.75 mL;the isolation and preparation of the two major monomers was achieved within 45 min.The two compounds were identified by HPLC-QQQ-MS as ziyuglycoside Ⅰ and Ⅱ,their purity was over 98%,determined by HPLC-ELSD,and their yields were 88.4% and 90.2%,respectively.The preparative method was simple,effective,and economical and could yield to products with high purity,therefore,it is suitable for the preparation of ziyuglycoside Ⅰ and Ⅱ standards on a laboratory scale.For industrial production,further scaling up with the magnification formula to a preparative method could be quickly established using a preparative column with larger inner diameter.