Separation Of Active Components From Licorice By Simulated Moving Bed And Two-dimensional Liquid Chromatography

Licorice,an important medicinal plant,has been used in many fields for many years.So far,it has been found that there are many active components in licorice. Most of these active components belong to triterpenoids and flavonoids.The researches on the pharmacological function of the active components show that they possess many important activities,such as anti-HIV,anti-SARS,anti-tumor,anti-oxidative activities,and so on.For a long time,the extraction and separation of active components from licorice are one of important issues in separation science.Because of the complexity of the chemical components of licorice and the lower content of some active components in licorice,the efficient purification of active components from licorice is very difficult using the traditional extraction and adsorption processes.However,simulated moving bed(SMB) chromatography and two-dimensional liquid chromatography(2D LC), which are two new techniques for separation of natural active components,present huge potentials.The continuous preparative separation can be accomplished by SMB, the separation and purification of multi-component can be achieved more effectively by 2D LC.In this work,the separation and purification of active components from licorice by SMB and 2D LC were studied mainly.To gain the sample suitable for feeding to SMB,the pretreatment processes, including extraction and separation by preparative column,were carried out to treat the total flavonoids of licorice and the pretreatment processes were optimized.The optimal conditions for extraction were obtained through orthogonal experiments. Considering the nonlinear chromatographic theory and the experimental analysis,the optimized values of injection concentration and injection volume for preparative chromatography was founded to enrich the content of liquiritin.Under nonlinear conditions,the separation of liquiritin was for the first time studied with the three zones SMB set up in our laboratory.Liquiritin is an important active component in licorice.According to the specific situation of the sample fed into SMB,competitive Langmuir model of two-component approximation was put forwarded to describe adsorption behaviors of liquiritin and its nearest neighbor,the isotherm parameters were determined by inverse method.Based on this,the operational parameters of SMB were determined by Triangle Method and they were listed as follows.The flow rate of zoneⅠwas 2 mL/min,the flow rate of zoneⅡwas 2 mL/min,the flow rate of feed was 2 mL/h,and switching time was 8 min.85%pure of liquiritin product was achieved by three zones SMB.The recovery of liquiritin was 90.4%.The extract from SMB was recrystallized and eventually over 97%pure of liquiritin product was obtained.The separation of liquiritin by 2D LC was firstly explored.In the condition of analytical scale,the purification of liquiritin from extract of licorice by 2D LC was studied under different 2D modes,including SEC×RP,NP×RP and RP×RP.For the 2D modes mentioned above,liquiritin with high purity were gained through the selection of chromatographic conditions and cut time of the fractions.In the condition of semi-preparative scale,the purifications of liquiritin by off-line and on-line NP-RP constructed by myself were performed respectively.The mixed materials of C18 and NH2 stationary phases were used in the second dimension for the on-line 2D separation,to enrich liquiritin in this dimension.In this work,the off-line 2D separation of the mixture of multiple active components was studied systematically.The target active components included glycyrrhizic acid,18β-glycyrrhetinic acid,liquiritin,isoliquiritigenin,and licochalcone B.And the former two of them belong to triterpenoids and the latter three of them to flavonoids.The selected 2D RP-RP system suitable for the separation of the mixture above was as follows.The SB C18 stationary phase was used in the first dimension,CN stationary phase was used in the second dimension,the mobile phase of acitonitrile-water(2:3,v/v) with pH 6 was used in both dimensions.The effects of the influencing factors on 2D separation,including injection concentration, injection volume,temperature and flow rate,were discussed.And the results showed that in the selected range the effects of injection concentration and injection volume on the 2D separation of active components were not remarkable,while the effect of temperature was distinct.The decreasing of flow rate facilitated the gain of active components with high purities.The results of this study show that it is feasible and efficient to separate the active components in licorice by SMB and 2D LC.The obtained results and the used methods could be referred for the separation of other natural active components.