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LEAPTechnologiesMinimizingandEliminatingCarryoverinPALSystemsAPrimerforReducingCarryoverinPALAutosamplerSystemsAPrimerforReducingCarryoverinPALAutosamplerSystemsRev.
1.
1LEAPTechnologies2005TableofContentsWhatisCarryover3WhatCausesCarryover3WheretheResidualLingers.
3WhatcanbeDonetoMinimizeCarryover4TheBasics4Syringes.
4ValvesandTubing.
5Component&MaterialChoices6StepstoMinimizeorEliminateCarryoverinPALSystems7Hardware7WashProcedures&Solvents.
8Techniques.
9SpecialMacros/Cycles9TroubleshootingSummary.
11ListofReferences11Appendix.
12APrimerforReducingCarryoverinPALAutosamplerSystemsRev.
1.
1LEAPTechnologies20053Twoofthebiggestproblemsinchromatographyarereproducibilityandcarryover.
Thisdocumentisintendedasabriefoverviewoftheissueofcarryover,andtoprovidesomesuggestionsastohowtominimizeoreliminateitasitrelatestoLEAPPALsystems.
WhatisCarryover"Carryover"isadetectorresponsefromapreviouslyinjectedsample,detectablewhenasubsequentinjectionismade.
Theresponseisbestseenwhenthatsubsequentinjectionisa"blank",i.
e.
aninjectionofmobilephasewithnoneofthecompoundintentionallyincluded.
Wheneveraninjectionsystemthatcomesintodirectcontactwithsamplesolutionsgets"re-used"forsubsequentinjections,carryoverwilloccurtoacertainextent.
Itnowbecomesaquestionofdetectorsensitivity.
Theimproveddetectorsensitivitymightbeattheheartofthe"rash"ofcarryoveroccurrencesinrecentyears.
Thefirstquestionauserneedstoansweris:DoestheamountofcarryovermatterHowdoesitcomparetothecompound-specificdetectionlimitsandtoerrorlimitsofthesamplepreparationandLCmethodsExample:Eventhoughvisible,apeakbelowthedetectionlimitforaspecificcompoundis,bydefinition,notdetected.
Intheliterature,carryoverisusuallyquantifiedasapercentagevalueofeitherthe"UpperLevelofQuantitation"(ULOQ)orthe"LowerLevelofQuantitation"(LLOQ).
Thisvalueisalwayscompoundspecificand,therefore,meaninglesseitherbyitselforreferringtoadifferentcompoundthantheoneyouarehavingproblemswith.
Example:UsingaLEAPCTCautosampler,thecarryoverforAcetaminophene(Tylenol)wasdeterminedtobe0.
0004%ULOQ.
Thisisafantasticnumberbutsaysnothingabouttheautosamplerperformanceusingyoursamples.
Duetothesereasonsnogeneralvalueforcarryovercanbegivennorguaranteedforaspecificanalyticalproblem/task.
WhatCausesCarryoverCarryoveriscausedbyafailuretocompletelyeliminateresidualcompoundsfromaprevioussampleinjectionintotheHPLCsystem.
Thelingeringresidualbecomesmixedwithasubsequentinjection.
Thiscanresultfrominadequateorineffectiveflushingafterthepreviousinjection,orbysomeorallofthesamplecomponentsbecomingtemporarilytrappedorboundtothesurfacematerialsomewhereintheHPLCpathway.
WheretheResidualLingersPossiblesitesforresidualaccumulationincludeanywhereandeverythingfromthesyringetothedetector,buttheinjectionvalveandthesyringearethemostlikelyplacesforcompoundstobeunintentionallyretained.
Anysurfacethatisnotcompletelyinerthasthepotentialtobeanadsorptionsite.
Forexample,certaincompoundstendtoadsorbontoactivesitesinStainlessSteel,evenintheflowpath,andrequireenoughoftherightwashsolventintherightconcentrationtobereleased.
ThiscanalsobetrueforPEEKtubing,dependingonthenatureoftheanalytesandthematrix.
Moreinsidiousare"deadvolume"spaces,wheresamplecanremaintrappedafteraninjection,sometimesevenafterroutinerinsing.
TwocommonexamplesofdeadvolumespacesaretheAPrimerforReducingCarryoverinPALAutosamplerSystemsRev.
1.
1LEAPTechnologies20054insideofthesamplesyringeneedleandthevalveinjectionportinletincludingthevalverotorgroove.
Seefigure1.
However,anyspacewheresampleisnotcompletelydisplacedorpurgedbymobilephaseorwashsolventsisapotentialproblem.
Thebiggestsourceofthistypeofcarryoverisfromthefittingsandhowtheyaremade.
Forexample,PEEKfittingswithPEEKferrulescanslipunderhighpressurecreatingadeadvolumeandthusallowingsampletobetrapped.
WhatcanbeDonetoMinimizeCarryoverTheBasicsObviously,flushingthesyringeandvalveisessential.
TheLEAPCTCPALhastwowashstationsincludedasastandardaccessory.
Theselectionofsolventsandflushvolumesareofparamountimportanceandcontrolledbytheuser.
Althoughitmayseemobvious,itisworthstatingthatflushingwithineffectivesolvent(s)isuseless.
Youneedtoconsiderthebasicruleofsolubility:"LikeDissolvesLike".
Therearemanyfactorsthatwillinfluencetheeffectivenessoftherinse,solventstrengthandpolarityamongthem,buttherearenohardandfastrulesthatapplytoeverycase.
Consequently,theend-userchemistmustbesuretochoosesolventsthatwillgetthejobdone.
Often,aone-twopunchofsolvents,e.
g.
anaqueousfollowedbyanorganicwash,canbemoreeffectivethanjustone.
"Cocktails"ofwashsolventmodifierslike0.
1%to1.
0%ofvolatileacid,baseorsaltsmayproducethedesiredresult.
Itisimportanttomakesurethatthechoicesofsolventsarenotimmisciblewitheachother.
SyringesThechoiceofsyringecansignificantlyaffectcarryover.
Needle,plungerandbarrelcharacteristicscanallcontributetoorreduceproblems.
ForLC,theneedleshouldalwaysbea22gauge,pointstyle3,i.
e.
withablunttip,toensureagoodsealatthebottomofthevalveinlet(assumingproperpenetration).
Useofanyothertipwillresultinanunswept"deadvolume"areaaroundthetip,aprimarycauseofcarryover.
Theinnerdiameter(i.
d.
)oftheneedledeterminestheinnersurfaceareaexposedtosampleandtheresidualleftinthedeadvolumeoftheneedleaftertheplungerhasfullyexpelledthecontentsofthebarrel.
Obviously,asmalleri.
d.
reducesboth.
A"gauge22s"needlehasasmalleri.
d.
thana"gauge22"byafactorofapproximately10.
Seefigure2.
Itcanpotentiallyreducecarryoverbyreducingthesurfaceareaandneedlevolume,albeitwithsomesacrificeofinjectionspeedduetothegreaterrestrictionandahigheroccurrenceofneedlecloggingduetothesmallerinnerdiameter.
Figure1:Cutoutofaninjectionvalve;Aninjectionleavestheinjectionportinletfilledwithsample.
Additionallyincaseofaloopoverfillinjection,arotorgroovewillalsocontainsample.
APrimerforReducingCarryoverinPALAutosamplerSystemsRev.
1.
1LEAPTechnologies20055However,additionalrinsescansomewhatreducethepracticaldifferencebetweenthetwo.
Aswiththerestofthesystem,inreducingcarryoverfromthesyringe,theimportanceofdeterminingthemosteffectivesolventsandperformingadequatewashescannotbeoverstated.
Also,thoughmoreexpensive,itispossibletofindneedleswithTeflon(PTFE)orglasscoatingstoreduceadhesion.
Buildupofcompoundsontheinnersurfaceoftheglassbarrelhasbeenclearlyestablishedasaproblem,bothinpotentialleechingintosubsequentsamplesandevenmorenotablyincausingprematurewearofplungertips.
Wornplungertipshavechannelsandpocketswhichareabletoretainsamplesolution.
Bettersyringeshavepolishedand/ortreatedsurfacestoreducethisphenomenon,andtheproperselectionofwashsolventsandtheirapplicationcansubstantiallyreducethisbuildupanditseffects.
Variouscompoundsareusedasplungertipmaterial,thoughmostareeitherPTFEorPolyethylene(PE).
Thereislittleevidencethattheplungertipcontributessignificantlytocarryoverandthedifferenceinthematerialsprimarilyinfluencesdurability.
However,thereisonewayinwhichtheplungercancontributetothecarryoverproblem:iftheplungerfailstoreachthebottomofthebarrel,especiallyduringrinsing,adeadvolumeareawillexistbetweenthetipandthebarrelbottom.
Itisrecommendedthatplungerswithavariableplungerstopareverycarefullyadjusted,oravoidedaltogether.
ValvesandTubingRegularlycheckthatthevalveinletsleevefitssnuglyaroundthesyringeneedlewheninserted.
Otherwise,replacetheinletsleeve.
Thiscaneasilybedeterminedbyinsertingtheneedleintothevalveinletbyhand.
Typicallytheneedlesleeveshouldbechangedevery15to60daysdependingonthenumberofinjectionsmadeandifsaltsarepresentinsampleand/orwashsolutions.
Besurethattheinletsleeveiscorrectlydimensioned.
Thereshouldbeapproximately2.
5mmoftubingbelowtheferrule.
Ifthetubingbelowiseitherlongerorshorter,adeadvolumewillresultinthevalveinjectionport.
Seefigure3.
UseonlyLEAP-approvedinletsleevestoensureconformitytospecifications.
Whenconnectingtubingorloopstothevalve,besurethattheendsarecleanlycutandperpendicular,andtakecaretofullyinsertthetubingendsintoeachportandpushthetubingasyoutightenthefittings(toeliminateanygapbetweentheendofthetubingandthebottomoftheport).
Avoid"Fingertight"fittingsanduserealcrimp-styleferrulefittings.
Fingertightfittingscanloosenenoughunderhighpressuretocauseadeadvolumewithoutleaking.
Figure2:DimensionalcomparisonofcommonlyusedsyringeneedlesizesforLCinjections.
APrimerforReducingCarryoverinPALAutosamplerSystemsRev.
1.
1LEAPTechnologies20056TypicallyPEEKtubingproduceslesscarryoverthanStainlessSteel(SS)tubing,butStainlessSteelferrulesseemtooutperformPEEKferrules.
CaremustbeusedwhenusingaSSferruleonPEEKtubingtopreventclosingoffthei.
d.
byexcesscrimpingand,thus,causingablockageintheflowpath.
InrecentyearsathirdalternativetubingmaterialbecamepracticalforuseinLCapplications.
FusedsilicainformofPEEKsiltubingoffersuniqueanddifferentmaterialpropertiescomparedtoPEEKandSS.
PEEKsilcomesinpre-cuttubinglengthswithpolishedandperpendicularcuttubingendsandshouldbeusedwithoutfurthercuttingorshortening.
CuttingPEEKsilwithoutpropertoolswillresultinajaggededgeofthefusedsilicainnertubingendswhichthenresultsincarryoverandblockagesdownstream.
Seefigure4.
Considerusingavalvethatwillbeinertwithyoursamples.
Valcovalvescomeinseveralmaterialchoices,Nitronic60StainlessSteel,HastelloyC,PAEKpolymeric,andeitherthestandard"rotor"designortheCheminertdesign.
TheValcon"H"rotor,whichisaproprietarypolymerofcarbonfiberreinforcedPTFEisthestandardrotormaterial.
TheValcon"E"materialisamixtureofPAEKandPTFEandismoreinertthanValconH,however,itdoesnotwithstandhigherpressuresverywellandwearsoutmorequicklyasaresult.
The"H"versionisgoodupto10,000PSIandtheEmaterialisgoodupto3,500PSI.
Therotorneedstobecheckedforscratchesorwear.
Replaceascratchedorwornvalverotor.
CheminertvalveswithPAEKstatorandValcon"E"rotoraremoreinertthanstainlesssteelvalves.
(Note:youcanconsulttheVICI(Valco)catalogforadditionalspecificmaterialdescriptionsandcompatibilityissues.
)Component&MaterialChoicesChoosethepropervalvedesignandmaterialbasedonthepotentialforcarryover.
Figure3:Theimportanceofacorrecttubinglengthbelowtheferrule.
Tubingbeingtooshortorlongcanbothproducedeadvolumewhichinturncancausecarryover.
Figure4:CrosssectionofPEEKsiltubing.
Pre-cuttubinglengthscomewithpolishedperpendiculartubingendsandshouldnotbecutorshortenedbytheenduser.
DrawingcourtesyofSGE,Inc.
APrimerforReducingCarryoverinPALAutosamplerSystemsRev.
1.
1LEAPTechnologies20057Choosethepropersyringedesignandsizeforyourapplicationandwashingneeds.
Choosetheproperneedleformat,e.
g.
,"gauge22"vs.
"gauge22s".
Choosethepropertubingmaterialforthesampleloopandothertubingconnections.
Choosetheappropriatesolventsforwashing/rinsingpurposes.
Determineifancillarywashingdeviceswillberequiredi.
e.
LEAPValveSelfWashSystem,ortheDilutormodule.
Orderadditionalcontrolsoftwaretoruncustommacrosorcycleswithspecialwashprocedures.
StepstoMinimizeorEliminateCarryoverinPALSystemsHardware1.
AdjustNeedlepenetrationinthevalveinletto0.
2mmfromthebottomoftheinletport.
Usingthehandheldcontroller,movetheneedleslowlydownuntilaclearaudiblenoise("clunk")occurs.
Movetheneedle0.
2mmupwardsandchecktheposition,again.
2.
Replaceinjectionvalveinletsleevesevery15to60daysorsoonerdependingonthenumberofinjections.
3.
Checktherotorandstatorofthevalveforscratches-theseareconvenientplacesforcompoundstoberetained.
Thestatorcanbecleanedultrasonicallyandtherotorshouldbereplacedasneeded.
4.
Checkthesyringefordiscolorationontheplunger,plungertipwearandtightnessoftheplungerinthesyringebarrel.
5.
Avoidwettingofthelowerneedleguideontheinjectionhead.
Ifthelowerneedleguideisgettingwet,trytofindthesourceoftheliquidandcorrecttheproblem.
ItmaybecomingfromasplashingeffectintheWashStation,fromablockedWashStationdrainlineorfromsamplingoutofopenvials/wells.
6.
RuntheWashStationutilityusingthehandheldcontrollerandverifyactuationoftheWashStationsolenoidvalvesandthegravity-fedflowofwashsolvent.
7.
VerifythatthedrainlineoftheWashStationisnotblockedwhichcanpreventfreshsolventfromgettingintotheWashStation.
8.
BesurethatthecapsonthewashsolventsupplybottlesoftheWashStationareloosetoprovideventilation.
Ifthecapsaretight,arelativevacuummaydevelopinthebottlesasthesolventsareconsumed.
Thisdiminishedpressurewillresultindiminishedsolventflowand,inturn,reducestheefficacyofsyringeneedlewashingFigure5:Positioningoftheinjectionheadneedleguiderelativetotheinjectionvalveinlet.
A2mmgappreventsliquidpossiblypresentontheneedleguideundersidefromcontaminatingtheinjectionvalveinletport.
APrimerforReducingCarryoverinPALAutosamplerSystemsRev.
1.
1LEAPTechnologies20058attheWashStation.
(Note:NormallythereisasmallholeinthecapsprovidedfromCTCespeciallyforthisreason.
However,theholeissmallandonthesideofthecapandinsomecasesmaynotprovideadequateventilation.
Thus,aloosenedcapwillhelptoensurethataircanenterthebottle.
)WashProcedures&Solvents9.
Usetwodifferentwashsolvents,e.
g.
,polarvs.
non-polar.
Makesurethatwashsolventsarecompatible/misciblewitheachotherandwiththesamplesolution.
Asolventmiscibilitytableisprovidedintheappendixtoofferhelpinthewashsolventselectionprocess.
10.
Addavolatilesolventmodifiertothewashsolutions,e.
g.
,acids,bases,salts,etc.
Inadditiontowashsolventmiscibility,thewashsolventmodifiersthemselvesneedtobemiscible/solubleinallwashsolventsandsamplesolution.
11.
Usethemostappropriatewashsolvents.
Example:Whenthesamplecontainsproteins,firstrinsewithanacidifiedaqueoussolvent(e.
g.
,90:10water/Acetonitrilewith0.
5%Formicacid),followedbyamoreaggressiveorganicsolvent(e.
g.
,30:70water/Acetonitrilewith0.
5%Formicacid).
Theacidifiedaqueoussolventwillkeeptheproteinssolubleandminimizetheprobabilityofdenaturing,inturnreducingthechancesoftheproteinsstickingtotheinnersurfacesofthevalveorsyringe.
Theorganicwashsolventwillwashawaythesmallmoleculeanalytes.
Thisexampleillustratesthattheorderofwashsolventsisimportant.
Performasyringewashwiththeaqueoussolventpriortothenextinjectiontoremoveanyhighorganicconcentrations.
12.
Increasethenumberofsyringerinsesandthenumberofvalverinses.
Usea2-to-1ratioofvalvewashestosyringewashes.
13.
Lengthenrinses,possiblyincludinga"dwell"timetopromotea"leeching"effect.
14.
UseaValveSelfWashingSystemorthePALDilutormoduletoincreasethevolumeofvalvewashes.
DilutorasWashStation:-AddingaCTCdilutorcanprovidegreatlyenhancedflushingofthesyringeandvalve.
Alargevolumeofsolventispasseddirectlyintoandthroughthesyringeandhasbeenshowntosignificantlyimproverinsingresults.
(Note:Thisislimitedtoeithera20uLor80uLsamplesyringesize.
)Thisaccessorypermitstheflushingofthevalvewhiletheneedleremainsinthevalveimmediatelyafterinjection.
Youwillhavetodoatleastonesyringeneedlewashinwashsolvent1and/or2priortothenextinjectiontoremoveanyanalytesfromtheneedle'sexternalsurface.
ValveSelf-WashingSystem:-MacroshavebeendevelopedtocontroltheLEAPValveSelfWashingSystemtoprovidespecificwashtimesforthevalve.
Highervolumesofsolventcanbeusedforcleaningthevalvewhilethesyringeisbeingwashedseparately.
Syringelifeisgreatlyenhancedwiththistechnique.
Thesyringeneedleshouldbewashedatleastonceinwashsolvent1and/or2priortothenextinjection.
APrimerforReducingCarryoverinPALAutosamplerSystemsRev.
1.
1LEAPTechnologies20059Techniques15.
Partialloopfillinjectionsisolatethesamplesolutionwithavolumeofsolventthatwasleftintheloop.
Thisprocedurecanminimizeconcentratedsamplecomingintocontactwithvalverotorgrooves,thewasteportoutletandfittings.
Makesurethatthevalveisplumbedinsuchawaythatthesampleleavestheloopthesamewayitcamein,e.
g.
,"lastinfirstout".
Seefigure6.
Theuppersamplevolumelimitforpartialloopfillinjectionsis1/3ofthesampleloopvolume.
(Note:Incaseofloopoverfillinjections,theinjectionvolumeneedstobeatleast3timestheloopvolume.
)SpecialMacros/CyclesCreatemacros/cyclesforthefollowingspecialmethodstohelpminimize/eliminatecarryover.
ThiswillrequiretheuseofCycleComposersoftwaremacrosorCycleEditorsoftwarecycles.
Macro/cycleoperationrequireseitherthenativesoftwarerunningconcurrentlyorLCdatasystemsoftwarecontrollingthePALbasedonmacros/cycles.
Examplesinclude:NeedleDip:-Dipthesyringetipintowashsolvent(s),aftersamplehasbeenaspiratedintothesyringe,justbeforetheinjectiontocleantheoutsideoftheneedlebeforeitisinsertedintotheinjectionport.
Thisprecludesmatrixand/oranalytesfromcontaminatingtheneedleinletsleeveandinjectionport.
ValveToggleandLoopRinse:-Whenoverfillingtheloop,boththeinletandoutletoftheloopmaycontaminatetherespectivecrossoverports.
Togglingthevalveaftertheinjectionsubjectsbothcrossoverportstocleanmobilephase,whichcanreducetheconcentrationofanalytesinthevalve.
Asyringewashoftheinjectionloopcanbeaccomplishedaswell.
Thetogglingofthevalveshouldbedonewhenthegradientstrengthisatitshighestpointtodissolveanyremaininganalytesinthecrossoverport.
Caremustbetakennottoleaveastrongsolventintheloopsothatthenextinjectionisnotsubjectedtothehighersolventstrength.
Theloopmaybewashedwithlowerstrengthsolventsothatthechromatographyisnotcompromised.
DispenseWashSolutionstoWaste:-WashsolventcanbedrawnfromthecapillarytubeoftheFastWashStationandejectedintothewasteposition,Figure6:Loopoverfillvs.
partialloopfillinjections;Duringpartialloopfillinjections,thenumberofrotorandvalvesurfacesincludingfittingsthatcomeincontactwithconcentratedsampleisreduced.
APrimerforReducingCarryoverinPALAutosamplerSystemsRev.
1.
1LEAPTechnologies200510insteadofejectingthecontaminatedsolutionbackintothecapillarytube.
Notethatthisprocedureshouldbefollowedbyatleastonemoreregularwashstepincludingwashsolventejectionintothecapillarytubetorinsetheoutsideoftheneedle.
Undernormaloperation,thesolenoidvalvesclosebeforeawashsolventejection,thus,forcingtheexpelledsolventaroundtheoutsideoftheneedle.
)SandwichInjection:-Asolventplugmaybepickedupbeforethesampleisaspiratedinorderto"isolate"thesyringeplungertipfromcontactwithanalytes.
Thesamesolventplugwillalsocometorestintheinjectionportinletandadjacentrotorgrooveafterthesampleinjectionismade.
Notethatthisprocedureissuccessfulonlyifmixingstrokesarenotrequiredandduringpartialloopfillinjections.
Itcanbedonewithorwithoutanairgapbetweenthesolventandsample.
Thetechniqueminimizesthecontactmadebetweentheconcentratedanalytesandthecrossoverportonthevalve.
Figure7:Sandwichedsamplepickupandinjection;Thesamplesolutionis"embedded"inasandwichofsolventandseparatedbysmallairgaps.
Afterinjection,thesamplewillendupinthemiddleoftheinjectionloop.
Onlysolventswithalowerelutionstrengththanthestartinggradientmobilephasecompositionshouldbeusedtobracketthesample.
APrimerforReducingCarryoverinPALAutosamplerSystemsRev.
1.
1LEAPTechnologies200511TroubleshootingSummaryChecktheeasiestthingsfirst.
WashthesyringeandvalvewiththeproperchoiceofsolventsCheckneedlepenetrationintheinjectionvalveCheckNeedleinletsleeveforwearCheckRotorforscratchesandreplaceasneededChecksyringetobesuretheplungerisgoingallthewaytothebottomofthebarrelChangeyourinjectionproceduregoingfromlowconcentrationstohigherconcentrationsifpossibleUseapartialLoopfillingtechniqueUseValconEinsteadofValconHrotormaterialCheckfittingsbetweenthesampleloopandvalveforproperinstallationUsecustomMacrosorcyclestoenhancerinsingListofReferencesDolan,J.
W.
"AutosamplerCarryover"LCGC19(2)February2001,164-168Dolan,J.
W.
"AttackingCarryoverProblems"LCGC19(10)October2001,1050-1054APrimerforReducingCarryoverinPALAutosamplerSystemsRev.
1.
1LEAPTechnologies200512AppendixWashSolventMiscibilityTable

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