retentiontimeshiftswithnotes.docx
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retentiontimeshiftswithnotes.docx
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retentiontimeshiftswithnotes
Script
SidebarNotes
Introduction
JohnHenderson:
Hi,I’mJohnHenderson,anapplicationsscientistatAgilentTechnologies,andI’mherewithMaureenJoseph,productmarketingmanagerforAgilentHPLCcolumns.We’regoingtotalkhereaboutchangingretentiontimes,andhowtotroubleshootandsolvethem.
Maureen:
Retentiontimechanges(fromrun-to-run)canbecausedbyanumberofdifferentthings:
includingthecolumnaging,columncontamination,apoorcolumn/mobilephasecombinationforyouranalytes,insufficientcolumnre-equilibration,achangeinflowrateorcolumntemperature,justtostartthelist.Withthismanypotentialcauses,troubleshootingyourissuemayrequireafewtries.
John:
Essentially,thesourceisgoingtocomefromyourcolumn,theinstrument,orthemobilephase.
Maureen:
That’sright.Toshowyouwhatwemean,here’sanexampleweranrecently[insertone_minute_analysis],
wherewesawnotonlychangingretentiontimes,butuglypeakshapes!
Yourfirstresponsewouldlikelybe:
AmIpumpingfromtherightmobilephasebottles?
andafterconfirmingyouare,youmightsay,“thiscolumnmustbebad!
”Well,weconsideredanumberofpossiblecauses,workingfromthemorecommonlistofpossibilities.#1,checkingthatourmethodparametersaresetcorrectlyinChemStation,#2-remakingthemobilephase,#3-changingthecolumn.Ultimately,however,thisproblemwasoneofthelastweconsidered.Itwasaninstrumentissue–[insertALS_Injection_fix.gif]Thecapillariestotheautosamplervalvewereconnectedwrong!
Now,youmaynotchangecapillariesofteninyourlab,butinthisapplicationsdevelopmentlab,nonroutineandoftenverydifferentmethodsarerunfrequentlyonthesameLC.ThemodulardesignandversatilityoftheLCsaregreatforswappingcolumns,andmodules,inthiscasesomeone[pointtosomeoneoffcamera?
]installedthecapillariesincorrectly.
JohnHenderson:
[sarcastically]Thatwasafunonetotroubleshoot.Igotalotofworkdonethatday.
Maureen:
Troubleshootingcanbefrustratingsometimes,especiallyifyougotoofardowntheroadwithanincorrectassumption.Soaswe’vebeenpointingoutinanumberofthesesegments,spendingsometimeevaluatingandnarrowingdownthepotentialcausecansaveyoumoretime.Ofcourse,that’snotalwayseasytodo.Onegoodruletofollowtosaveyourselfsomeworkistostartwiththesimplestthingsfirst.
John:
OK,solet’sgetstarted.We’regoingtoworkthroughthepotentialcausesweseeforretentiontimeshifts,startingwiththemorecommoncausesandtheeasiestfixes,thenworkingthroughtomorecomplexcausesandsolutions.
JohnHendersonisanApplicationsScientistforAgilentChemistriesandSuppliesDivision.
MaureenJosephistheHPLCColumnsProductManager.
Somepossiblecausestoconsiderforretentiontimechangesare:
-Columnaging
-Columncontamination
-Apoorcolumn/mobilephasecombinationforyouranalytes
-Insufficientcolumnre-equilibration
-Changesinflowrateorcolumntemperature
ChangeinColumnDimensionsorFlowRate
Maureen:
Sometimespeopleforgetthatwhenyouchangethecolumndimensions,youneedtoadjustyoursystemandmethodparameterstoachievesimilarresults.Thesearethingslikeflowrateorgradienttimeandinjectionvolume.WerecommendusingtheLCMethodTranslationSoftwaretohelpyouwiththat.Therearedifferentversionsfordifferentinstruments.Findtheoptionsbysearchingfor“LCMethodTranslator”attheAsite.Thissoftwarewillhelpyouquicklycalculateandadjustthesystem/methodparameterstoadapttochangesinyourcolumndimensions;thetranslatorisespeciallyusefulforgradientmethods.
Wetalkaboutgradientanalysesinmoredepthinanothervideosegment,butwewanttomentionithere,too,becauseit’sanissuewhereweseesomeconfusion.
Let’slookatthisexample[showchanging_column_length.gif].
Here,weseeagradientseparationofpesticidesandflameretardantsindrinkingwater.Let’sassumethechromatographerrecognizesthereisalotofextraresolutioninthetopchromatogram,andsubstantialtimesavingscouldberealizedifashortercolumnwasused.Bysimplychangingthecolumntohalfthelength,theanalysistimewouldalsobecutinhalfright?
Welldoingsoproducedthebottomresult--hardlyanytimesavings[pointarrowtotimelineonlowerchromatogram].Whyaren’ttheretentiontimes50%shorterlikethecolumnlength?
Thegradienttimewaskeptthesameat15minutes,andwasnotchangedtoreflectthenewshortercolumnlength.Andasyousee,retentiontimesarenotwhereyouexpectthemtobe.
Becausethecolumnlengthwascutby50%,thegradienttimemustalsobecutinhalftorealizethetimesavingsandkeeptheseparationthesame.
Now,whenwechangeourgradienttime–cuttingitinhalftomatchourcolumnlengthandvolumechange–weseethatouranalysistimeiscutinhalf[showcolumn_length_2.gif]
John:
Hereisanotherexample,whichdemonstratesthesameprinciple,onlywechangedflowratetodecreaseanalysistimeinsteadofcolumnlength.Seethischromatogramofagradientanalysisofalkylphenones[showflow_rate_example.gif].
Whenweincreasedtheflowrate60%tospeedupouranalysis,aswewouldinanisocraticmethod,wefindthatanalysistimedidnotdecrease60%,andretentionfactorshavechanged,infactthey’veincreased!
Hereagain,weneedtoadjustourgradienttomatchourflowratechange.
[Insertgradient_adjusted_flowrate.gif–showarrowto8.2,12.9ineachwhileJohntalks,todemonstratetheyarethesame]Here’sthenewchromatogram,whereyouseeourretentionfactorshaveadjustedcorrectly,andwe’vecutouranalysistime.
Maureen:
Thisexampleisonethatcanbetoughtosortoutandhighlightshowtroubleshootingcanbecomplex.Theflowratedidchangesotheanalysistimeshouldhavechangedbuttheresultswereunexpected.ThisisoneexamplewheremanypeoplemightsuspecttherewasaproblemwiththeLCpumpdeliveringtherightflowrate.Butitwasjustamethodissueandisthefirstthingyoushouldcheck.
John:
Yes.Whenwechangetheflowrateofanisocraticmethod,analysistimechangesproportionallytoflowrate,andretentionfactorsdonotchange.Sowhydoretentionfactorschangefromjustchangingflowrateingradients?
Maureen:
Tounderstandthat,weneedtolookatthegradientequation.[showequation]
Theretentionfactoringradients,k*,dependsonseveralparametersincludingflowrate,gradienttime,andcolumnvoidvolume[pointtopartsofequationastheyarenamed],whichisproportionaltocolumnsize!
Anychangeinflowrate,gradienttimeorvoidvolumewillchangek*orretentionfactor.Vmisproportionaltocolumnlength,soanychangeincolumnlengthwillalsochangek*.
John:
Sotokeepk*formethod1thesamewhenchangingtomethod2,thesefactorsmustbalance[insertequation1.gif]
tG1F1/Vm1=tG2F2/Vm2
Inourfirstexample[insertequation2.gif]
thecolumnlengthwashalvedandthecolumnlengthisdirectlyproportionaltothevoidvolume,sosincethevoidvolumewashalved,thegradientorflowratemustbedoubledtokeeptheequationbalanced;notdoingsoresultedinbarelyanytimesavingsatallwiththeshortercolumn.
Inouralkylphenoneexample[insertequation2a.gif],
flowrate(F2)inmethod2wasincreased,butthegradienttime(tG2)wasnotadjusted,resultinginthesurprisingincreaseinretentionfactors.Whenwedecreasedthegradienttimeby60%tobalancetheequationandkeepk*equivalent,theretentionfactorsmatched.
It’simportanttoremembertoadjustsystemandmethodparameters–flowrate,injectionvolume,gradienttime--whenyouchangeyourcolumndimensions.
TheAgilentLCMethodTranslatorisahelpfultooltouse.Searchforitat
BuffersandMobilePhase
John:
Thenextareawewanttocoveristheimpactofbuffers,ormobilephasemodifiers,onyourcolumn.Everybufferandmobilephasehasanimpactonyourcolumn’sretentiveness.Youmayseeshiftingretentionovertimebasedonthecolumnsinteractionwiththesebuffersespeciallyifyouusemultipledifferentmobilephaseswiththesamecolumn.
Maureen:
Yes.Ifyou’vewatchedmoreofthesegmentsinthisseries,youmayhaveseenthesegmentwhereRonMajorsdiscussestheimportanceofrunningachromatogramwithyournewcolumn,usingastandardthatyou’lluseconsistently,oronyourroutinemethod.Keepingthischromatogramonhandwillbehelpfulforyoutotroubleshoot.
ourtopchromatogramshowstheseparationasitisintended.Overtime,dependingonthemethodconditionssuchaspH,temperature,typeofanalytes,etc.thecolumncanwearout,orage.Asitages,retentioncanshift,asshowninthesecondchromatogram…Inthisexamplepeak2retentionshiftsout,andpeakthreeshiftsin.Astimepasses,thepeakelutionordereventuallychanges,asshowninthebottomchromatogram.Someoneunfamiliarwiththehistoryofthecolumn,ormethodmightassumethatthethreepeaksareinthecorrectorderasthetopchromatogram.
Dependingonthenumberofinjectionsobtained,agoodtoolforextendingcolumnlifewouldbeaguardcolumn.
John:
Anothertrickyissuethatisfairlycommontoseeischangesinretentionthatarecausedbyachangeinmobilephase.Here’sanexampleofananalysisIdidrecentlywithaZORBAXRapidResolutionHDcolumnonthe1290Infinity[insertequilibration_effect.gif]:
Seehowthepeakshapesandretentiondifferinthisanalysisfromruntorun,asthemobilephaseischanged.
[backtoJohn]Sometimes,amobilephasediffers,becauseofsomethingassimpleasthewayitismixedinyourlab.
[demonstratewiththree100mLgraduat
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