高速逆流色谱原理及应用

张语迟高速逆流色谱原理及应用多级萃取技术离心分配色谱平面图离心分配色谱立体图同步行星式逆流色谱高速逆流色谱运行平面图分离区分离区混合区HSCCC系统连接工作图1、HPLC方法的建立尽可能使各峰得到良好的分离2、溶剂系统的选择根据样品的溶解特性选择溶剂体系测定分配系数，依据分配系数确定溶剂系统K2/K1:0.5-2.53、HSCCC分离条件优化色谱柱转速；固定相的保留；流动相流速；分离温度。4、HPLC测定收集组分的纯度HSCCC方法的建立过程:丁醇-醋酸-水丁醇-甲醇-水(缓冲液)丁醇-乙酸乙酯-水(缓冲液)乙酸乙酯-水(缓冲液)氯仿-甲醇-水(毒)甲基异丁基酮-丙酮-水正己烷-乙酸乙酯-甲醇-水(石油醚-乙酸乙酯-甲醇-水)正己烷-乙腈正己烷-甲醇-水正己烷-乙酸乙酯-甲醇-水(石油醚-乙酸乙酯-甲醇-水)水溶性和强极性中等极性(氯仿)弱极性(正己烷)HSCCC常用溶剂系统（依据组分的极性和溶解性）PreparativeIsolationofSixMajorSaponinsfromPlatycodiRadixbyHigh-speedCounter-currentChromatographyYoungWanHaandYeongShikKimABSTRACT:Introduction–Platycosides,theprimaryconstituentsofPlatycodiRadix,areknowntohavenumerousandvariedbiologicalactivities,exertinganti-inflammation,anti-allergy,anti-tumour,anti-obesityandanti-hyperlipidemiaeffects.However,effectivemethodsforisolatingandpurifyingplatycosidesfromPlatycodiRadixarenotcurrentlyavailable.Objective–TodevelopanefficientmethodforthepreparativeseparationofsixplatycosidesfromPlatycodiRadixbyhighspeedcounter-currentchromatography(HSCCC)coupledwithanevaporativelightscatteringdetection(ELSD)system.Methodology–Preparativeseparationwasperformedbywaterextractionusingreversed-phaseC18columnchromatographyonanHSCCC-ELSDsystem.Atwo-phasesolventsystemcomprisedhexane–n-butanol–water(1:40:20,v/v)and(1:10:5,v/v)wasemployed.Twootherkeyparameters,revolutionspeedoftheseparationcolumnandflow-rateofthemobilephase,werealsoinvestigatedforoptimumHSCCCperformance.EachpeakfractionobtainedfromseparationoftheplatycosideswascollectedaccordingtotheELSDelutionprofileanddeterminedbyHPLC.Results–Usingthedescribedmethod,sixplatycosides,allwithpuritiesofover94%,couldbeisolatedfrom300mgoftheplatycoside-enrichedfraction.Theirstructureswerecharacterizedbyelectrosprayionisationmassspectrometry(ESI-MS),1HNMRand13C-NMR.IntroductionExperimentalApparatus.Reagentsandmaterials.Preparationofcrudesample.MeasurementofpartitioncoefficientsPreparationofthetwo-phasesolventsystemandsamplesolution.HSCCCseparationSelectionofthetwo-phasesolventsystemandHSCCCconditionsResultsandDiscussionHPLCanalysisandidentificationofHSCCCpeakfractionsStructuralidentificationAcknowledgementsReferences高速逆流色谱的延伸应用实例1pH区带CCC分离同分异构体HSCCC条件：溶剂系统:正丁醇-水(1:1)固定相:上相pH=0.5(HCl)流动相:下相pH=10.7(NH3)流速:2.0mlmin-1转速:715rpm检测波长:206nm样品量:230mg粗提物机型:pH-zone-refiningCCC固定相保留：60%1:3-sulfophthalicacid99.0％2:4-sulfophthalicacid99.0％JournalofChromatographyA,966(2002)111–118pH区带逆流色谱分离中药单体中应用实例2CCC分离蛇床子HSCCC条件：溶剂系统:正己烷-乙酸乙酯-乙醇-水(5:5:5:5)流速:0–180min,1mlmin−1180–480min,2mlmin−1转速:1000rpm检测波长:254nm样品量:500mg粗提物机型:CCC-1000相保留:58%A:bergapten96.5%45.8mgB:imperatorin98.2%118.3mg92.1、93.7%JournalofChromatographyA,1061(2004)51–54逆流色谱分离中药应用(V流动相)实例3CCC分离短瓣金莲花黄酮苷HSCCC条件：溶剂系统:乙酸乙酯-正丁醇-水(2:1:3)流速:0–190min,1.5mlmin−1190–480min,2.5mlmin−1转速:800rpm检测波长:254nm样品量:500mg粗提物机型:CCC-1000保留值:49%1:quercetin-3-O-neohesperidoside85.1%9.3mg2:orientin(荭草素)97%95.8mg3:未知化合物97%4:vitexin(牡荆素)97%11.6mgJournalofChromatographyA,1092(2005)216–221逆流色谱分离中药应用(V流动相)1.5ml/min2.5ml/min贯叶连翘中金丝桃苷HSCCC-HPLC-DAD联用分离中药应用三相CCC分离15种化合物逆流色谱分离中药应用(三相溶剂系统)HEX—MeOAc—ACN--Water实例6二维CCC分离冬凌草HSCCC条件：溶剂系统:一维:正己烷-乙酸乙酯-甲醇-水1:5:1:5二维:正己烷-乙酸乙酯-甲醇-水3:5:3:5流速:2mlmin−1转速:800rpm样品量:250mg粗提物机型:2D-CCC保留值:一维:40%二维:63%1:oridonin(甲素)95%2:ponicidin(乙素)95%JournalofChromatographyA,1146(2007)125–130二维逆流色谱分离中药应用实例7分离白花前胡中香豆素HSCCC条件：溶剂系统:石油醚-乙酸乙酯-甲醇-水5:5:6:4流速:2mlmin−1转速:900rpm样品量:250mg粗提物机型:TBE-300保留值:54.3%1:7.0mg99.3%2:7.4mg96.1%3:12.5mg96.5%4:42.1mg99.3%5:9.8mg99.7%6:65.2mg98.1%7:31.7mg,92.8%JournalofChromatographyB,877(2009)2571–2578HSCCC-ESI-MSn分离中药应用扩展和想象湖州:活猪走进去鲜肉运出来全机械化操作《中国猪业网》2010-12-27由于该定点屠宰迁建项目采用自动化同步流水线加工，活猪进去，出来便是新鲜的猪肉，减少了手工的环节和过程，猪肉更卫生，肉品质量也会有大幅提高。Applicationofacceleratedsolventextractioncoupledwithhigh-performancecounter-currentchromatographytoextractionandonlineisolationofchemicalconstituentsfromHypericumperforatumL.YuchiZhang,ChunmingLiu,MinYu,ZhengkunZhang,YanjuanQi,JingWang,GuimeiWu,SainanLi,JingYu,YangHuJournalofChromatographyA,1218(2011)2827–2834HypericumperforatumL.,atypicaltraditionalChinesemedicine,isusedallovertheworldforthetreatmentofdepression.Additionally,ithasbeenreportedthattheextractsofH.perforatummayhaveaninhibitoryeffect,onhumanbladdercancercells,andactasanantioxidantandanticonvulsant.TheextractsofH.perforatumcontainmanyconstituentswithdocumentedbiologicalactivitysuchasquinicacids,phloroglucinolsandabroadrangeofflavonoids.ExposureoftheextractsofH.perforatumtolightmayleadtothedegradationofphloroglucinols,whichareextremelysensitivetooxidationandunstableinsolutiononexposuretoair,thereforephloroglucinolsaredifficulttoseparateandisolatebyconventionalmethod.Inthiscase,amethodcombininganextractionsystemwithanisolationsystemonlinetoavoidtheexposureoftheextractstoairandlightisurgentlyneeded.IntroductionThediagrampresentstwosetups(B)enablinghyphenationofASEandHPCCC电视剧《东北一家人》背景：汽车厂家属区开辟一片荒地，打算承包给个人，由个人决定所要开发的娱乐项目。甲（牛小伟，吕小品扮演）：打算建设过山车。乙（牛继红，金珠扮演）：打算开发滑梯和秋千相关的项目。丙（孙明，巩汉林扮演）：认为甲和乙所开发的项目太过时，小儿科，丙的思路是在过山车上打滑梯，荡秋千。扩展和想象CompletedpreparativeseparationofalkaloidsfromCephaltaxusfortuninebystep-pH-gradienthigh-speedcoun