色谱柱的故障诊断与维护2(1)

液相色谱柱色谱柱的故障诊断与维护八方世纪科技有限公司北京总部：010-82656628，82656500，82656399广州办事处：020-87768360，87786522HiThere...AuthoringDivisionNameFileNameSecurityNotice(ifrequired)HLC与GC色谱柱问题用户调查色谱柱问题百分率BackPressure,PluggedFrits24%PoorReproducibility16SampleRecovery14LossofResolution13Instability11Voids8.1Leaks,Fittings4.8pHRange2.0LowPlateCount2.0ColumnOverload2.0Cost1.7Miscellaneous15WewillstartwithsomemoresurveyresultsfromLC/GCtoshowsomeofthemostcommonproblemswithcolumnsandHPLC.Thenumberonproblembyfar,ishighbackpressure.Thiscanbecausedbypluggedfritsaswellascolumncontaminationorsolvent/sampleimmiscibility.Poorreproducibility,lowsamplerecovery,andlossofresolutionarethenextmostcommonproblemsasreportedbychromatographers.InstabilityandleaksareusuallyafunctionoftheHPLChardwareandarenextaswellascolumnvoidswhichcanbecausedbytheequipmentaswell.pHrange,lowplatecountandcolumnoverloadareallcolumnrelatedproblemsduetoadeterioratingphaseorpoorphasestability.Costisalsoreportedasacolumnproblem,andmiscellaneouscomprises15%.介绍提纲I.常见问题，现象及排除 方案 气瓶 现场处置方案 .pdf气瓶 现场处置方案 .doc见习基地管理方案.doc关于群访事件的化解方案建筑工地扬尘治理专项方案下载 -基线漂移/噪音-峰型-压力变化-保留漂移II.日常维护/良好的实践-在线装置-样品制备-操作的限制III.再生/维修已损坏的色谱柱-物理方法-化学方法TheoutlineofthissectionwillbetofirstcoversometroubleshootingoftheHPLCsystemandcolumnproblems.Wewilllookatchromatographicbaselinesymptomsandsolutions.Wewilltalkaboutatroubleshootingguide.WewillthenmoveintoadiscussiononpreventionofHPLCcolumnproblemsusingin-linedevicesandsampleprep.I.常见问题基线噪音可能的原因：流动池脏检测器灯有问题如果是周期性的，则起源于泵的脉冲温度对检测器的影响气泡经过检测器ng,andhighpressureoperationofthecolumn.Degassingsolventspreferablewithaninlinevacuumishighlyrecommended.Also,usingahighpressureflowcellwithpostcellflowrestrictionwillhelptokeepanyresidualgasinsolutionsothereisnooutgassinginthecell.ThiscanbeaccomplishedwithasmallpieceofsmallIDtubing.基线漂移梯度洗脱温度不稳定(示差检测器）流动相中有污染物柱中的流动相没有平衡体系中有污染物流出可能的原因：asswareandsolventsarecleanwhenyoustart.Ifyouaregoingtorecycle,useaverylargevolumeofsolventlike5litersforstandardborecolumnsandbewillingtodealwiththisproblem.Anunequilibratedmobileandstationaryphasesystemcanalsoproducethisdrift,justconsiderthefirstexampleofagradient.Ifyouareoperatinginanisocraticmode,waitlongeruntilyouareequilibrated.Trycleaningthecolumnwithaseriesofsolventsandgradientsbeforeyoustartequilibrating.Thiswillhelpinachievingamorerapidequilibration.Contaminationbleedinthesystemcanalsocausethisdrift,especiallywhenusingagradient.Thecontaminationmayprecipitatefromsolutionatthebeginning,andthenredissolveattheendofthegradient.Startw/clean.鬼峰鬼峰——即使不样也会出现的峰20%-100%MeOH没有进样371517问题1-流动相脏问题2-样品过量(也可能是回收率太差)Howmanypeoplehaveseenthis?Peakswithoutainjection?Wecalltheseghostpeaks,andweusuallyseetheseinablankgradientmethod.Theproblemispronouncedcontaminationinthemobilephaseorthecolumn.Onceuponatimetherewasanenvironmentalfirm,newtoHPLC,thatwasanalyzingforPAH's.Thegradientacetonitrile/watermethodlookedlikethisbaseline,evenwithoutthesamplebeinginjected.Whenaskedaboutthequalityoftheacetonitrileandwater,theyrespondedbyshowingtheirnicebottlesfromareputablesolventvendor.UponfurtherinvestigationandinspectionoftheHPLCsystem,theapplicationchemistforHPsawthattheirsolventreservoirmarked"WATER",wasinfactaJacuzzi.Frothandfoameverywhere!Themoralofthestory,alwayssuspectyourglassware,especiallyifyouareusingadishwasherordishwashingservice.Residualsoapysurfactantshavebeenknowntolingerevenafter2to3rinses,producingprofoundcontaminationandpossibledestroyingorpermanentlymodifyingthecolumnstationaryphase.UseHPLCgrade.Agoodpracticetogetintowithglassware,istoorinsetheglasswear2to3moretimeswithwatertoremoveanysoapfilm,thenrinse2to3timeswithwhateversolventisappropriateforyoursystem.YoumayhavetorinseinbetweenthewaterandsolventwithauniversalsolventsuchasTHF,orIPA.Columncontaminationcanberemediedbycleaningwithaseriesofsolventsandgradients,oranewcolumn.峰型双峰柱塌陷柱中有死体积过滤片部分堵塞只有一个双峰——组分的共洗脱可能原因：样品与溶剂不匹配Therearemanypeakshapeproblemsthatcanoccur,thisoneispeakdoubling.Thisiswhentheoriginalanalysisproducesacertainnumberofpeaksinamixture,thenaftersometime,thesepeaksmultiplyby2,andlookliketheredchromatogram.Thisiscausedbyacolumnvoidformingattheheadofthecolumnorablockedfrit.Thishappensovertimeasthecolumnbeginstofail,butcanhappenmorequicklyiftheflowrateandpressurearealwaysveryhigh.Thiscausesthecolumnbedtosettleandaspacetoform.Contaminationorincompatiblemobilephasescanalsocausethis.Onceuponatimetherewasachromatographernewtotheareaofchiralseparations.Heinjectedmultiplesamplesontoapreparativescale,$25,000,chiralcolumn.Thesesampleshedissolvedinmethylenechloridebecausehecouldnotdissolvetheinthemethanol/hexanemixofthemobilephase.Afterthefirstseparationandpurification,thecolumnperformancebegantofall.Bythefifthsample,therewasnostationaryphaseleft.Thisparticularphasewassolubleinmethylenechloride.Themoralofthestory,checkthevendorspecificationsforthephasetoensurethatthecolumnisdyinganuntimelydeath.Ifthereisonlyonpeakthatisdoubled,theremaybeanewpartiallycoelutingcompoundinthemix.DiodearraydetectionorMSisusefulforthisdetermination.峰型所有的峰都展宽柱效降低——柱死体积大进样量/质量高捻度流动相部分峰展宽前次进样后流出的组分大分子量样品——蛋白或聚合物样品与流动相不匹配Peakbroadeningisanothercommonpeakshapeproblem.Ifallofthepeaksbecomebroadened,thiscouldbeaproblemwithcolumnefficiencyandalossofthisefficiency,possibleduetoacolumnvoid.Largeinjectionvolumeorextracolumnvolumecanleadtodispersionandpeakbroadening.Thethirdpossiblecauseifallpeaksarebroader,ishighviscosityinthemobilephase.Thisconditionofaviscousliquidalsocausesdispersionandbandbroadening.Ifonlyoneorsomepeaksarebroad,itcouldmeanthatthisorthesepeaksarefromapreviousinjectionandareverylateelutingcompoundsfromthepreviousanalysis.Also,highmolecularweightproteinsandpolymerstendtoproducebroadpeaks.Inacomplexmix,somecompoundswithvariousisomericformsthattautomerizeorflipbackandforth,willproducebroadpeaks.Aminesalsomaygivethispeakshapeinsomecases.峰型原因：部分峰拖尾二次保留效应——残留硅醇基的反应小峰紧跟在大峰的后面流出柱头有污染金属所有峰拖尾额外的柱效应柱损坏不适当的样品量/溶剂Tailingpeakshaveasymmetryvaluegreaterthat1.2.Whenonlysomepeakstail,itmaybecausedbyasecondaryretentioneffectsuchasresidualsilanolinteractions.Abaseddeactivated,ahighlycarbonloaded,orapolymericbasematerialcolumnmaysolvethisproblem.Sometimesifonlysomepeakstail,itisanew,smallpeakthatiscoelutingwiththetailofthelargerpeak.Adiodearraydetectorcanhelpdetermineifthecompoundsarethesame.Ifallpeaksaretailing,Itmaybeaproblemwithextracolumneffectsfromtubingorfittings.Check/changetubingandfittings.Itmayalsobeabadcolumnorthewrongphasefortheseparation.Tryadifferentcolumn/phase.Abuildupofcontaminationonthecolumninletcanalsoproducethetailingduetosecondaryinteractionswiththecontaminant.Cleanthecolumnwithaseriesofsolventsandgradients,orchange.Aswesawearlier,heavymetalscancausethisaswell.Tryamorepurephaseoracidwashthecolumn.峰型Theoppositetotailingpeaksarefrontingpeakswithasymmetryvalueoflessthan0.9.Frontingcanhappenwhenacolumnisoverloadedandthecolumncapacityisexceeded.Thinkofawavewashingtoshore.Wavesarefairlygaussianortailslightlywhentheystart.Astheybuildandbecomelarger,theypushthewaterinfrontofthemandbreakinafrontingpattern.Thesamethinghappensonacolumnwithtoomuchcompound.Reducethesampleinjectionsizeordilutethesampleby10or100.Frontingcanalsoindicateasmallpeakelutingtooclosetoalargerpeak.Diodearrayisusefulindeterminingifthisisthecase.峰型负峰正常峰负峰原因 样品的吸收比流动相的吸收值低 当样品组分通过色谱柱时打破了原来的平衡 用示差检测器时是正常的Thelastpeakshapeproblemwewillcovertoday,isnegativepeaks.Thisiswhenthepeakgoesdownbelowthebaselinebackgroundabsorbance.Thismeansthattheadsorbanceofthispeakislessthanthemobilephaseabsorbance.Thisoccurssometimesatthevoidasanequilibriumdisturbancewhenthesamplesolventpassesthroughthecolumn.Bydissolvingthesampleinthemobilephase,thiscanbereduced.NegativepeaksarenormalandmeasuredinRIdetectionbecausetherefractiveindexcanbepositiveornegativewithrespecttothemobilephaseRI.NegativepeakscanbeusefulifthesampledoesnotabsorbintheUVdetector.Anadditivewhichdoeabsorbcanbeputintothemobilephaseandthesamplenegativepeakscanthenbemeasured.ThisiscalledindirectUVdetection.宽峰柱外体积增加*在进样口到柱或柱到检测器之间，使用细的短的连接管*保证所有的管路接头都使用正确*用小体积检测池*减少进样量Extracolumndispersionlookslikethis....Ononesystemwithonecolumn,thepeaksareniceandsharp.Onadifferentsystemorwithadifferentcolumnorchemist,theanalysislooksliketheoneontheright.Tubingisthenumberonecauseofthisbandbroadeningfromdispersion.Makesureyouareusingshort,smallidtubingbetweentheinjectorandcolumnandbetweencolumnanddetector,eliminatingasmuchadditionaldeadvolumeaspossible.Ifusingadifferentsysteminadifferentlab,donotbefooledbythelengthofthetubingalone.ChecktheID!Makesureallconnectionsaremadewithmatchedfittings,aswewillseelater.Thisincludesunions,columnendfittings,andinjector/detectorfittings.Checktovolumeofthedetectorflowcelltomakesureitiscompatiblewiththecolumn.Smalleridcolumnsrequiresmallerflowcellvolumesifyouwouldliketomaintaintheefficiencyoftheseparation.Makesureyourinjectionvolumeshavenotincreasedduetoasoftwareprogrammingerrorinthemethod.Checkthemicrolitersinjected.Alsocheckthesampleconcentrationtomakesureitisnot10or100timesmoreconcentratedthanyouthink,theattenuationwillgivethisoneaway.柱外体积MinimizeLengthandDiameterofTubingDoNotVolumeOverloadInjectionUseAppropriateVolumeFlowCellE.C.V=Vinj.+Vtubing+VcellHPLC使用的接头无死体积接头不适用的接头Previouslywediscussedthemultiplecolumnproblemsassociatedwithextracolumneffectsandmatchedfittings.Thisslideshowstwoexamplesofunionsfortubingconnections.ThezerodeadvolumeunionisagoodmatchforanalyticalHPLC.Youcanseethereisverylittleextravolumeaddedtothesystembythistypeofunion.Onthecontrast,theuniononthebottomisunacceptableforanalyticalworkandwilladdquiteafewmicrolitersofdeadvolumetothesystem.ThismaybeO.K.foranisocraticseparationona4-4.6mmidcolumn,butitisbettertohaveazerodeadvolumeunion,especiallyiftheHPLCsystemwillbeusedforgradientwork.ThebottomunioniscommonlyfoundonPreparativescaleHPLCsystemstoallowforthefasterflowratesandsolventvolumes.HPLC中使用的柱接头0.090in.0.130in.0.090in.0.170in.SwagelokWatersParkerRheodyneValcoUptight0.090in.0.080in.TroubleshootingLCFittings,PartII.J.W.DolanandP.Upchurch.LC/GCMagazine6:788(1988)Tocontinuewiththeconceptofmatchedfittings,thisslideshowsvariouscolumnconnectorsusedinHPLC.Noticethatforeachvendor,thedistancefromtheendofthetubingtowheretheferrulesare"seated"or"swaged"isdifferent?Ifthedistanceistoogreatfortheendfittingofthecolumn,theferrulewillnotsealwiththecolumnandthefittingwillleak.Ifyouthentightendownonthefitting,thetubingwillpuncturethecolumnfrit.Ifthedistanceistooshort,youcansealtheferruleandnothavealeak,buttherewillbealargeextracolumnvolumeforbandbroadeningtooccur.Noticehowtheferrulesareshapeddifferentlyaswell?Somearedesignedtobeusedwithcolumnsandsomearespecifictoinjectorsordetectorsorpumps.DONOTINTERCHANGETHESE!Theymaybecomeapermanentfixture,orimbeddedinyourcolumnorequipment.Thelastpointis,onceyouseattheferruleorswageitintoplace,thatfittingistherightsizeforonlythatcolumntype.Eachcolumnendfittingorholderfromonevendorisnotmachinedthesameasanothervendor.Threadnumberanddepthvary.Acartridgesystemwiththesameendholdersforallcartridgespreventsyoufromneedingtohavemultiplededicatedendfittingsformultiplecolumns.ManyusersarechangingtouniversalendfittingswhichareplasticorPEEK,yetsealathighpressures,areadjustable,andre-usable.Thereislittlechanceofmismatch.柱外体积效应的影响01234567891011柱外体积10µL20µLTime,min.2.1x150mmF=0.2mL/min.(1ul进样)I.不进样时柱压连续升高-泵密封垫-流动相有颗粒-流动相溶解性-流动相的不稳定性(聚合)-形成柱死体积(与使用条件有关)II.进样时压力升高-样品有颗粒-样品在流动相中不溶-样品组分与固定相不兼容压力I.所有峰都向低保留值方向漂移（酸、碱、中性化合物）-键合相流失-流动相不稳定(可能性较小)-溶剂输送系统(流速变化)II.所有峰都向高保留值方向漂移-在水/有机溶剂的混合体系中，有机溶剂含量减少-柱改变(可能性较小)-溶剂输送系统(流速变化)III.离子型化合物的色谱峰的保留值漂移-流动相中挥发性组分损失(离子强度,pH漂移)-柱改变(键合相或污染)保留值漂移I.与流动相有关的问题-使用新鲜的流动相 ?pH ?电导 ?色谱性能测试II.与色谱柱有关的问题-测试新柱-用 标准 excel标准偏差excel标准偏差函数exl标准差函数国标检验抽样标准表免费下载红头文件格式标准下载 的测试混合样测试新柱-“清洗”色谱柱后再测-考虑样品基质与使用条件对柱稳定性的影响保留值漂移良好的柱使用 规范 编程规范下载gsp规范下载钢格栅规范下载警徽规范下载建设厅规范下载 Nowletscoversomegoodcolumnpracticesthatwillhelpyoutoincreaseyourcolumnlifetime.Filtersolventsbeforeyouusethem.ThecolumnistheHPLCsystem'sbestfilter,somakesurethecolumnisn'tperformingthistask.Pretreatsamplesthatcontainstronglyretainedcompoundsofnointerest.Thismeanstoremoveanysamplematrix,orcontaminants,orincompatibleanalytestothemethod/column.Solidphaseextractionisgoodforthiscleanup.Avoidextremecolumntemperatures,greaterthan60degrees,orcheckthevendorlimits.Somecolumnsarenowmadeforhighertemperatures.Flush/cleanthecolumnoftenwithstrongsolvents.Alsousegradientcleaning.KeepthemobilephasepHbetween3and7unlessotherwiseindicatedbythecolumnvendor(polymericphases).Ifitisnecessarytodothis,useaprecolumnandexpectashorterlifetime.Alwaysusefreshbuffersolutionsandaqueousmobilephasesortreatthemwithsodiumazide,thiscanbedangerous.Watersolutionsgrow"bugs"orbacteria,thesewillcontaminateandpermanentlymodifythestationaryphase.Inlinewiththelastpoint,preparecolumnsforstoragebyrinsingallofthebufferoutofthecolumn,thenstoreintheappropriatesolvent,atleast50%organic.Thiswillkeepbacteriafromgrowingonthecolumn.Makesuretorinsethebufferofffirstandwithwater,asmanybuffersaltsprecipitatein100%organic.Thencapthecolumntightlytokeepairout.Avoidphysicallymishandlingthecolumns.Thisincludesbanging,dropping,overtighteningfittings,andhittingyourlabpartner.预柱与保护柱从泵来的流动相预柱进样器保护柱 分析 定性数据统计分析pdf销售业绩分析模板建筑结构震害分析销售进度分析表京东商城竞争战略分析 柱到检测器预柱对流动相而言保护柱对色谱柱而言StrippingorConcentratingColumnHereisaschematicillustratingthedifferencebetweenapre-columnandaguardcolumn.Apre-columnisanin-linefilterdeviceforfilteringsolventspriortotheinjectionvalvewherethesampleisintroduced.Aguardcolumnprotectstheanalyticalcolumnfromsamplecontaminantsthatarestronglyorpermanentlyretainedbythecolumnphase.AuthoringDivisionNameFileNameSecurityNotice(ifrequired)H带可重复使用接头及完整保护柱的卡套柱无保护柱有保护柱AmorerecentcartridgecolumndesignthatweuseatHewlett-Packardisshownhere.Thisdesignhelpstoincorporatein-linesamplefiltrationandcleanupdevices,likefiltersandguardcolumns.Aguardcartridgecanbeeasilyinsertedintothecolumnholdersbyreversingthecolletsatthefrontend.Thisrequiresnoadditionalhardwareandaddsnoadditionaldelayvolumewithrespecttothemoretraditionalapproachoftwocolumnsandtubing.Thisdesignalsoallowsfortrulyresusableendfittings.SincethesamecolumnholderwillfitanysizeHP-cartridgecolumnofanyoftheofferedphases,thereisnoneedtoremaketheendfittingstoaccommodateadifferentcolumn.Theguardscanhelptoautomatesomesampleprep,andareinexpensivecomparedtotraditionalguardcolumns.带Techtron保护柱的Zorbax卡套柱min5101520253035mAU140142.5145147.5150152.5155157.5160min5101520253035mAU140142.5145147.5150152.5155157.5160BDSODSHypersil250-2mm有integratedGuardColumnBDSODSHypersil250-2mm没有GuardColumn2mmID卡套柱有或无保护柱的比较Thisslideshowsthecomparisonofa2mmidHPcartridgecolumnwithandwithouttheintegratedguardcartridge.Noticehowtheruntimesonlyshiftslightly,bylessthan+0.5minuteswiththeguard.Also,resolutionandselectivityarevirtuallythesamebetweenallofthepeaks.Thisdesignprovidesaneasytouse,automatedapproachtoextendinganalyticalcolumnlifetimewithoutlosstoselectivityandresolution.制备样品:流动相过滤样品，以确保其不含有固体微粒用流动相或比流动相极性较弱的溶剂溶解样品进样的样品体积应尽量小样品不溶于流动相真正的环境样品 样品的溶解性 溶剂强度 体积效应Contaminant 微粒 强保留的组分Whenmanuallypreparingthesample,makesuretofilterandremoveparticulates.Useafilterthatiscompatiblewiththesamplesolution.Thereareaqueousandorganicfilters.Dissolvethesampleinthemobilephaseifyoucan.Ifnot,useasolventthatisdifferentbutweakerwithrespecttothemobilephasestrengthinthemodeofHPLCyouareusing.Sometimeswhenthesampleisdissolvedinadifferentsolvent,peakdoublingcanoccur.Keepthesamplevolumeassmallaspossible.Sometimeswhenasampleispartiallysoluble,itisattractivetoaddmoremobilephasetodissolvethesampleandtheinjectmorevolumeforthesameamountofsamplemassoncolumn.Thisonlyworkstoapoint,thenthebandsbecometoobroadandresolutionislost.样品制备方法的进步1.固相萃取（SPE）-硅胶基键合相品种全-可多次使用的聚合物（唯一的选择性）-自动化工艺-玻璃、Teflon基体适合高灵敏度的工作-专业化，滥用药物，儿茶酚胺、脱盐、去金属3.带不同功能团的过滤膜4.SPE薄膜2.屏蔽亲水相/内部多孔载体Somemorerecentimprovementsinsamplepreparecoveredonthisslide.SolidphaseextractionorSPEusesthesamephilosophyasamatchedguardorcleanupcolumnatareducedprice.TheSPEcartridgesordisksaremadeofdifferentphasesdesignedtoextractoutwhatyoudon'twantonyourcolumn.Theycanbemanualorrobotcompatibleforautomatedsamplecleanupinaserialorparallelmode.ThisslideliststhetypesofSPE...readtheslide操作条件--流动相对柱寿命的影响*易水解的硅氧键操作条件--流动相对色谱柱寿命的影响键合相水解速度随温度的升高而加大StableBondCN(diisopropylpropylCN)ConventionalCN(dimethylpropylCN)LowpHeluentatelevatedtemperature(60C)在安装柱前后测试压力差如果柱压力太高，反装色谱柱，并用10-20倍柱体积的流动相冲洗色谱柱，监测压力变化若出现沉淀(如：蛋白凝聚、细胞物、聚合物)设法用相应的可溶性溶剂，如0.1%TFA/80%乙腈,6M盐酸胍,THF,HFIP若仍无效，应考虑更换过滤片色谱柱的修复与再生--物理性阻塞ColumnInletFritCompressionFerruleColumnBodyFemaleEndFittingMaleEndFitting 带手套 填料不能变干 不要接触色谱柱体 加热填料会被挤出 不要拧的过紧色谱柱故障诊断--更换进样口过滤片由于键合相的水解或硅胶的溶解而造成的色谱柱填料的变化是不可逆的由于污染而产生的色谱柱变化可以通过适当的清洗使之修复梯度洗脱的方式(水->强有机溶剂)通常最有效低pH流动相,如0.1%TFAor0.01MH3PO4,也是有效的升高温度（60-80℃）色谱柱修复和再生--化学变化-I6M盐酸胍、异硫氰酸胍、尿素等溶液可用于清除蛋白质的沉积XDB,有机聚合物等填料可使用高pH水溶液/有机溶液，如：0.01MNaOH50%异丙醇/水溶液，硅胶基质应尽量减少接触时间(15-30min).色谱柱的修复和再生--化学变化-IIExample:4.6mmIDx15cmZorbax300SB-C18Flowrate:1mL/min;Temp:~80℃A:0.1%TFAinwaterB:0.1%TFAinacetonitrile100%Afor30min.;0-100%Bover30min.;Hold100%Bfor30min.,Returnto100%Ain5min.Repeat3times. 总结 初级经济法重点总结下载党员个人总结TXt高中句型全总结.doc高中句型全总结.doc理论力学知识点总结pdf 如果使用时加以注意，大部分的液相色谱柱的问题是可以预防的-流动相必须是互溶的，无颗粒-使用保护柱和在线过滤器，并定期更换-使用恰当的样品制备方法-使用恰当的色谱柱清洗程序-不同类型的色谱柱，请选择不同的操作条件 (如：SB:pH1-6,XDB:pH3-11)-保存柱反压及重要的色谱参数的记录(如：R,N,As,k')-色谱柱需保存在有机溶剂中(乙腈)Tosummarizethissectionontroubleshooting,mostHPLCcolumnproblemscanbepreventedprovidedtheproperprecautionsaretaken.Readthelist...andremembertotroubleshootbaselinechanges.Theseareanearlyindicationthattheremaybeacolumnproblem.Thankyouforyourtimetoday!Arethereanyquestions?HiThere...WewillstartwithsomemoresurveyresultsfromLC/GCtoshowsomeofthemostcommonproblemswithcolumnsandHPLC.Thenumberonproblembyfar,ishighbackpressure.Thiscanbecausedbypluggedfritsaswellascolumncontaminationorsolvent/sampleimmiscibility.Poorreproducibility,lowsamplerecovery,andlossofresolutionarethenextmostcommonproblemsasreportedbychromatographers.InstabilityandleaksareusuallyafunctionoftheHPLChardwareandarenextaswellascolumnvoidswhichcanbecausedbytheequipmentaswell.pHrange,lowplatecountandcolumnoverloadareallcolumnrelatedproblemsduetoadeterioratingphaseorpoorphasestability.Costisalsoreportedasacolumnproblem,andmiscellaneouscomprises15%.TheoutlineofthissectionwillbetofirstcoversometroubleshootingoftheHPLCsystemandcolumnproblems.Wewilllookatchromatographicbaselinesymptomsandsolutions.Wewilltalkaboutatroubleshootingguide.WewillthenmoveintoadiscussiononpreventionofHPLCcolumnproblemsusingin-linedevicesandsampleprep.ng,andhighpressureoperationofthecolumn.Degassingsolventspreferablewithaninlinevacuumishighlyrecommended.Also,usingahighpressureflowcellwithpostcellflowrestrictionwillhelptokeepanyresidualgasinsolutionsothereisnooutgassinginthecell.ThiscanbeaccomplishedwithasmallpieceofsmallIDtubing.asswareandsolventsarecleanwhenyoustart.Ifyouaregoingtorecycle,useaverylargevolumeofsolventlike5litersforstandardborecolumnsandbewillingtodealwiththisproblem.Anunequilibratedmobileandstationaryphasesystemcanalsoproducethisdrift,justconsiderthefirstexampleofagradient.Ifyouareoperatinginanisocraticmode,waitlongeruntilyouareequilibrated.Trycleaningthecolumnwithaseriesofsolventsandgradientsbeforeyoustartequilibrating.Thiswillhelpinachievingamorerapidequilibration.Contaminationbleedinthesystemcanalsocausethisdrift,especiallywhenusingagradient.Thecontaminationmayprecipitatefromsolutionatthebeginning,andthenredissolveattheendofthegradient.Startw/clean.Howmanypeoplehaveseenthis?Peakswithoutainjection?Wecalltheseghostpeaks,andweusuallyseetheseinablankgradientmethod.Theproblemispronouncedcontaminationinthemobilephaseorthecolumn.Onceuponatimetherewasanenvironmentalfirm,newtoHPLC,thatwasanalyzingforPAH's.Thegradientacetonitrile/watermethodlookedlikethisbaseline,evenwithoutthesamplebeinginjected.Whenaskedaboutthequalityoftheacetonitrileandwater,theyrespondedbyshowingtheirnicebottlesfromareputablesolventvendor.UponfurtherinvestigationandinspectionoftheHPLCsystem,theapplicationchemistforHPsawthattheirsolventreservoirmarked"WATER",wasinfactaJacuzzi.Frothandfoameverywhere!Themoralofthestory,alwayssuspectyourglassware,especiallyifyouareusingadishwasherordishwashingservice.Residualsoapysurfactantshavebeenknowntolingerevenafter2to3rinses,producingprofoundcontaminationandpossibledestroyingorpermanentlymodifyingthecolumnstationaryphase.UseHPLCgrade.