retentiontimeshiftswithnotes.docx

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