Separation And Analysis Of Chemical Components Piper Kadsura Based On Supercritical Fluid Chromatography

Supercritical fluid chromatography(SFC)that has the advantages of high efficiency,rapid and green has been paid attention to the area of separation of natural products.Piper kadsura is a kind of rattan medicine rich in lignans and alkaloids.The research of its material base is the foundation for the better clinical application.In this paper,a rapid two-dimensional chromatography method was established based on SFC,separation and purification of lignan analogues were performed using chiral SFC,an overall separation scheme was developed based on the combination of SFC and reversed-phase liquid chromatography(RPLC),and SFC alkyl and phenyl stationary phases were evaluated to explore the separation mechanism of SFC.This work is a basis for expanding the application of SFC,and also provides technical support for solving the separation problem of natural products.First,a 2D RPLC/UHPSFC method was established to rapidly analyze two parts of petroleum ether(PE)and ethyl acetate(EA)of Piper kadsura.21 fractions in PE part and 28 fractions in EA part were separated by RPLC.By the use of optimized SFC,each PE fraction could be separated within 2 min,and each EA fraction could be separated within 3 min with well separation performance,which fully proved the fast and efficient characteristics of SFC.At the same time,UHPSFC also showed high orthogonality with RPLC.204 and 829 peaks were respectively detected in the PE and EA parts within 63 min and 112 min(including the equilibrium time of 1 min in each separation).Through the analysis by the two dimensional system,the complexity of the chemical composition of Piper kadsura was more deeply recognized.Second,chiral SFC was used to separate four diastereomers of lignans.The separation on the CSP-1 chromatographic column was carried out by the use of automatic continuous stacking injection mode.Each cycle was injected four times,and the time period was 32.5 min.After two cycles,710 mg samples could be separated,and the component 1(compounds 1 and 2)and component 2(compounds 3 and 4)were obtained.The CSP-2 column was also performed under the automatically stacking injection mode.Through 14 times' purification,103.1 mg compound 1 and 10 mg compound 2 were obtained from the components 1 within 37 min,while 152.3 mg compound 3 and 178.6 mg compound 4 were obtained from the component 2 within 35 min through 10 times' purification.Chiral SFC is an effective way to separation of structural analogues,and different chiral columns can provide different separation selectivity,which has a good application prospect in the separation of natural products.On the basis of the above work,a four level strategy was established for the systematic separation of Piper kadsura.Based on the advantages of high efficiency,fast,less organic solvent consumption,simple and convenient post-processing of SFC,the first two levels' SFC separations were to realize rapid simplification of samples to solve the problem of insufficient peak capacity of chromatographic separation.The SFC separation was mainly concerned with stability,sample loading,preparative efficiency and collection mode.The third level was to make use of the advantage of orthogonality between SFC and RPLC to purify compounds through prep-RPLC,so the separation selectivity was more concerned.The fourth level using chiral SFC was to purify structural analogues.A demonstrative fraction obtained from the first SFC separation was separated by this strategy,and 28 compounds were prepared,including five new compounds.The prep-SFC successfully improved the efficiency of this multilevel preparation,and this strategy overcame the problem of complex sample separation.Last,the linear solvation energy relationship(LESR)model of five C8 stationary phases and two phenyl stationary phases with different bond density was established.The model parameters quantitatively indicated that the hydrophobic interaction increased with the increase of the bond density,and the variety and proportion of the modifier had an influence on the various interactions.The result of vector angle indicated that the orthogonality of C8 and silica increased with the increase of bonding density.The orthogonality existed among C8 stationary phases with different bonding densities,as well as the phenyl and C8 with higher bonding density.Besides,the higher proportion of modifier was beneficial to improve the separation selectivity.The log k-log k plot of the stationary phases showed that the solute and the stationary phase could produce a variety of interactions which was beneficial to the regulation of separation selectivity.The result of separation of eight amide alkaloids by silica and seven above-mentioned stationary phases showed better retention on silica,while better selectivity on C8 and phenyl.The C8 and phenyl columns could distinguish alkaloids with different saturated carbon chain length,the number of double bonds,and cis-trans isomerization.In this paper,SFC technology was applied to rapid analysis,structural analogues purification and systematic separation of Piper kadsura.And the application of non-polar stationary phase in SFC was also evaluated and expanded.With the systematic work,the paper had a deeper understanding of the separation mechanism and retention behavior of SFC and the chemical composition of Piper kadsura.