The Construction Of On-line Sephadex LH-20 Liquid Chromatography × Reversed Phase Liquid Chromatography System And The Study On The Chemical Constituents Of Flos Carthami

A novel on-line comprehensive two-dimensional mixed-mode Sephadex LH-20 chromatography x reversed phase liquid chromatography(Sephadex LH-20 x RPLC)was constructed with the development of mixed-mode chromatography technique.Firstly,the mixture of reference compounds was separated on Sephadex LH-20 with methanol or methanol/water as elution solvent.Based on the retention time of these compounds,whose molecular weight and hydrophobicity was different from each other,Sephadex LH-20 was convinced to possess the separation mechanisms of size exclusion,reversed phase partition and adsorption when eluted with methanol/water.Then,the system parameters,including the column length and flow rate of first dimensional Sephadex LH-20,the sample loading,modulation frequency and the interface,were optimized using water extract of Flos Carthami as the sample solvent.At last,the optimized Sephadex LH-20 x RPLC system was used for the analysis of water extract of Flos Carthami,better orthogonality(72.9%)and separation efficiency was achieved when comparing with RPLC x RPLC and HILIC x RPLC.Moreover,the on-line Sephadex LH-20 x RPLC system was applied in the analysis of other complex natural products,such as saffron crocus stigma,ginkgo leaf,cape jasmine and blackberrylily rhizome,all of which showed good separation efficiency.The Sephadex LH-20 x RPLC system was also expanded to the on-line and off-line separation of Flos Carthami.Finally,24 compounds were separated from Flos Carthami,where 11 compounds were obtained from the on-line preparative Sephadex LH-20 x RPLC system,and 22 compounds were separated in the off-line process.In summary,a mixed-mode Sephadex LH-20 x RPLC system was constructed,which provides a novel techique for the analysis of complex water soluble samples.