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-currentChromatography,YoungWanHaandYeongShikKim,ABSTRACT:IntroductionPlatycosides,theprimaryconstituentsofPlatycodiRadix,areknowntohavenumerousandvariedbiologicalactivities,exertinganti-inflammation,anti-allergy,anti-tumour,anti-obesityandanti-hyperlipidemiaeffects.However,effectivemethodsforisolatingandpurifyingplatycosidesfromPlatycodiRadixarenotcurrentlyavailable.ObjectiveTodevelopanefficientmethodforthepreparativeseparationofsixplatycosidesfromPlatycodiRadixbyhighspeedcounter-currentchromatography(HSCCC)coupledwithanevaporativelightscatteringdetection(ELSD)system.MethodologyPreparativeseparationwasperformedbywaterextractionusingreversed-phaseC18columnchromatographyonanHSCCC-ELSDsystem.Atwo-phasesolventsystemcomprisedhexanen-butanolwater(1:40:20,v/v)and(1:10:5,v/v)wasemployed.Twootherkeyparameters,revolutionspeedoftheseparationcolumnandflow-rateofthemobilephase,werealsoinvestigatedforoptimumHSCCCperformance.EachpeakfractionobtainedfromseparationoftheplatycosideswascollectedaccordingtotheELSDelutionprofileanddeterminedbyHPLC.ResultsUsingthedescribedmethod,sixplatycosides,allwithpuritiesofover94%,couldbeisolatedfrom300mgoftheplatycoside-enrichedfraction.Theirstructureswerecharacterizedbyelectrosprayionisationmassspectrometry(ESI-MS),1HNMRand13C-NMR.,Introduction,Experimental,Apparatus.,Reagentsandmaterials.,Preparationofcrudesample.,Measurementofpartitioncoefficients,Preparationofthetwo-phasesolventsystemandsamplesolution.,HSCCCseparation,Selectionofthetwo-phasesolventsystemandHSCCCconditions,ResultsandDiscussion,HPLCanalysisandidentificationofHSCCCpeakfractions,Structuralidentification,Acknowledgements,References,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-sulfophthalicacid99.02:4-sulfophthalicacid99.0,JournalofChromatographyA,966(2002)111118,pH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:0180min,1mlmin1180480min,2mlmin1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)5154,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:0190min,1.5mlmin1190480min,2.5mlmin1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%11.6mg,JournalofChromatographyA,1092(2005)216221,逆流色谱分离中药应用(V流动相),1.5ml/min,2.5ml/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),HEXMeOAcACN-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:2mlmin1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)125130,二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:2mlmin1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)25712578,HSCCC-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,YangHu,JournalofChromatographyA,1218(2011)28272834,HypericumperforatumL.,atypicaltraditionalChinesemedicine,isusedallovertheworldforthetreatmentofdepression.Additionally,ithasbeenreportedthattheextractsofH.perforatummayhaveaninhibitoryeffect,onhumanbladdercancercells,andactasanantioxidantandanticonvulsant.TheextractsofH.perforatumcontainmanyconstituentswithdocumentedbiologicalactivitysuchasquinicacids,phloroglucinolsandabroadrangeofflavonoids.ExposureoftheextractsofH.perforatumtolightmayleadtothedegradationofphloroglucinols,whichareextremelysensitivetooxidationandunstableinsolutiononexposuretoair,thereforephloroglucinolsaredifficulttoseparateandisolatebyconventionalmethod.Inthiscase,amethodcombininganextractionsystemwithanisolationsystemonlinetoavoidtheexposureoftheextractstoairandlightisurgentlyneeded.,Introduction,Thediagrampresentstwosetups(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-speedcounter-currentchromatography,ZhonghuaLiu,QizhenDu,KuiwuWang,LiliXiu,GuangleiSong,JournalofChromatographyA,1216(2009)46634667,Chemicalstructureofcephalotaxine-typealkaloids,TheHSCCCexperimentwasperformedwithaprocedure:ThecolumnwasfirstentirelyfilledwiththeorganicstationaryphasecontainingTFA.Thiswasfollowedbysampleinjection.Thentheaqueousphasecontaining2%NH4OH(eluterbase)waspumpedintotheinletofthecolumnataflow-rateof3.0mL/minintheheadtotailelutionmode,whiletheapparatuswasrotatedat750rpm.AfterpeaksIandIIwereelutedoutofthecolumn,themobilephasewaschangedtotheaqueousphasecontaining0.2%NH4OHuntilpeakIIIwereelutedout.Followingthis,themobilephasewaschangedtotheaqueousphasecontaining0.01