controlling contamination in lc/ms - waters corporation · controlling contamination in lc/ms...
TRANSCRIPT
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715001307REV.G 1OF33
ControllingContaminationinLC/MSSystemsBestPractices
ThisdocumentoutlinesbestpracticesforcontrollingcontaminationinUPLC/MSandHPLC/MSsystems(bothclinicalandnonclinical).Agoodcontaminationcontrolplanincludesmeasuresforpreventing,troubleshooting,andcleaningcontamination.
Note:Tounderstandwhatcausescontamination,seeMajorcontaminantsandtheirsources,page31.
Note:Ifyouhavefollowedtherecommendationsinthisdocumentandstillneedhelpwithcontamination,contactyourWatersrepresentative.
Important:Thisdocumentupdates,supersedes,andsupplementsallotherWatersdocumentsonpreventing,troubleshooting,andcleaningcontamination.
2016 WATERS CORPORATION. ALL RIGHTS RESERVED.
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715001307REV.G 2OF33
TableofContents
Preventingcontamination ....................................................... 3
Select,prepare,andhandlesolventscorrectly......................................3
Cleanlaboratoryglasswareproperly ..............................................6
Prepareandhandlesamplescorrectly ............................................. 7
Usecleanfittingsandtubing..................................................... 8
Weargloves ..................................................................8
Usecleancolumns............................................................. 9
Checklaboratoryair ..........................................................10
TroubleshootingContamination.................................................11
IsolatetheproblemtotheLCorMSsystem.......................................12
TroubleshoottheLCsystem ....................................................12
TroubleshoottheMSsystem ...................................................15
CleaningtoEliminateContamination .............................................16
Generalguidelines............................................................16
CleaningMSSystems .........................................................21
ReferenceInformation .........................................................23
Solventconsiderations.........................................................23
Recommendedwaterpurificationprocess ........................................29
Gloves......................................................................30
Majorcontaminantsandtheirsources ...........................................31
Contaminantdatabase........................................................33
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Preventingcontamination
Preventionisthemostimportantfactorincontrollingcontamination.Itismucheasiertopreventcontaminationthanitistotroubleshootoreliminateit.Topreventcontamination,followthestepsinthissection.
Select,prepare,andhandlesolventscorrectly
Closeattentiontotheselectionanduseofsolvents(ormobilephases)isacriticalsafeguardagainstcontamination(seePossibleeffectsoflowqualitysolvents,page23).Watersrecommendsthefollowingprocedureswhenusingsolvents.1
Note:Formoreinformationonsolventrecommendationsandcautions,seeyoursystemuserguide.
Useclean,particlefreesolvents
Whenpreparingmobilephase,alwaysusechemicallycleanandparticlefreesolvents(Table 3)andreagents.SolventmustbeUPLCgradeoraqualitylevelsuitabletotheapplicationbeingrun.(SeeSolventconsiderations,page23.)
UsefreshsolventsUsethefreshestsolventsavailable;donotstoresolventswithouttakingmeasurestopreventmicrobialgrowth(seeStoresolventsincleanglassreservoirswithcovers,page6).Whensolventsareexposedtotheatmosphere,theybecomevulnerabletoairbornecontamination.
1. TherecommendationsarebasedonexperiencesinWaterslaboratories.
Caution:Failuretousethepropergradeofsolventforyourapplicationcausesexcessivebackgroundnoiseandlossofsensitivity.
Caution:Ifusingorganicsolvents,payattentiontotheexpirationdatesuggestedbythemanufacturer.Openbottlesofsolventcanbecomecontaminated.
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UseultrapurewaterUseultrapurewater(definedaswaterthathasbeenpurifiedthroughasystemthattargetscontaminantsdetrimentaltoUPLC/MSandHPLC/MSsystems).
Note:SeeRecommendedwaterpurificationprocess,page29.
Ultrapurewaterissterileandcontainsnoparticlesgreaterthan0.2microns,nodetectableionizablecompounds(>18MOhm.cmresistivity),andlowUVabsorbanceatthemonitoringwavelengths.Useofultrapurewaterreducesthenumberofimpuritiesinthewaterthatcancollectonthecolumnduringequilibrationwiththeweaksolvent.
PreventmicrobialgrowthAqueousmobilephasesandwateraresusceptibletomicrobialgrowth,whichcancausepeakstoappearduringgradientoperationandincreasebackgroundabsorbanceduringisocraticoperation.Microbialgrowthcanalsoblockfilters,frits,andcolumns,andcancausecheckvalvestomalfunction.Suchproblemscancausehighcolumnorpumpbackpressureandultimatelyleadtoprematurecolumnfailureandsystemshutdown.
Topreventmicrobialgrowthinmobilephase,prepare,filter,anddegasaqueousmobilephasedaily.Duringshutdownoroveralongperiod(suchasaweekend),flushthesystemcompletelywithwater.Thenflushwith10%(minimum)ofanappropriateorganicsolvent(suchasacetonitrileormethanol).
Caution:Topreventmicrobialcontamination,donotstorethesysteminwateror>90%aqueousmobilephase.
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Minimizetheuseofadditives
Toreducebackground,usethelowestconcentrationofmobilephaseadditive(forexample,acid,base,orbufferthatiscompatiblewithgood,stablechromatography).Themoreadditiveyouuse,themorecontaminationcanbeintroducedintothemobilephase.
Whendevelopinganewmethodortransferringanoldonetoanewcolumn,eliminateanyadditivesthatnolongeraffectthechromatography.Forexample,TEAisnolongernecessaryonmanycolumnstoobtaingoodpeakshapeforbasiccompounds.
Usethehighestqualityadditivesavailable(atminimum,LC/MSgradeadditives) Useadditives(forexample,formicacid)thathavelowconcentrationsofironandother
metalions.Aceticacidcancontainasignificantamountofironandothermetalions. Topreventprecipitation,avoidusinginorganicsaltsoradditivesinhighorganiceluents.
Suchsaltsoradditivescanprecipitateatthehighorganicendofthegradient. Useadditivesthatarevolatileandcompatiblewithmassspectrometers.
Volatileadditivescontainingammonium(NH4+),acetate,formate,orcarbonateare
recommended.
Toflushthesystemafterusingmobilephasecontainingadditives,flushthesystemforatleast5minutes(atleast50mLofwater).Followwith10%(minimum)ofanappropriateorganicsolvent(suchasacetonitrileormethanol).
UsemisciblesolventsMakesureallsolventsandsamples,includingadditives,aremiscible.Proteins(fromtissues,blood,orserumsamples)canprecipitateinhigh(>40%)organicsolvents.Theprecipitatedproteinscancloginjectorsandtubing,oradsorbtheanalyteorcontaminants.
Caution:Topreventmicrobialgrowth,donotaddadditivestostockbottles.
Caution:Ifyouareusingamassspectrometer,avoidusingnonvolatileadditivessuchassodium(Na+),potassium(K+),orphosphate(PO4
3).
Caution:Someadditivescanbeincompatiblewithmassspectrometers.Consultthedocumentationshippedwithyoursystemforcompatibleadditives.
Caution:SolventswithapHgreaterthan10dissolvesilica.Ifyoursystemcontainsfusedsilicaandglasscomponents(forexample,ifyouhaveananoACQUITYorMClassUPLCsystem),avoidusingsolventswithapHgreaterthan10.AvoidstoringthesysteminhighpH.Flushandstoreinmethanoloraminimumof10%organicwithnoadditivesorbuffers.
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Storesolventsincleanglassreservoirswithcovers Storemobilephasesincleanamberorbrownstainedborosilicateglassreservoirs(see
Cleanlaboratoryglasswareproperly,page6).Borosilicateglassmustbetype1,classA,ortype3.3.
Coverthereservoirtopreventairbornecontaminantsfromenteringthesolvent. Tocoverthereservoir,usealuminumfoilorcapssuppliedwiththesystem.
Usethesmallestsolventreservoirappropriateforyouranalysis(itdependsonyourflowrateandthelengthofyourruns).
Donottopoffsolvents.Instead,discardoldsolvent,rinsethebottlesandsolventinletfilterswiththesolventthatisused,andthenrefillwithfreshsolvent.Finally,primethesystem.
Cleanlaboratoryglasswareproperly
Anyglasscontainerusedtoprepareorstoremobilephasemustbethoroughlycleanedbeforeuse.
Standardcleaningprocess
Rinseglasswarewithorganicsolventandthenwater.
AggressivecleaningprocessIfmoreaggressivecleaningisrequired(forexample,whenthecontainershistoryisunknown),usethefollowingprocedure:
1. Sonicatewith10%formicornitricacid,thenwater,thenmethanoloracetonitrile,thenwater.
2. Repeattwomoretimes.
Caution:Thebrownbottlesinwhichthemanufacturershipssolventsarenotborosilicateandmustnotbeusedtostoreaqueoussolutions.
Caution:Neverstoreliquidsinplastic,whichcancontainplasticizers(forexample,phthalates)andthuspromoteorganiccontamination.
Caution:DonotuseParafilmorotherplasticfilmstocoversolventreservoirs.
Caution:Neverwashglasswarewithdetergentsorinadishwasher.
Caution:Alwayscleanglasswarewiththesamemobilephase/solventyouuseforthechromatography.
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3. Storeglasswareusedtoprepareorstoremobilephaseseparatelyfromothercommonuseglassware.
4. Ifglasswareorsolventreservoirsbecomecontaminatedwithmicrobialgrowth,treattheminanautoclave:
a.Removeandreplaceallfiltersandtubingbetweenthemobilephasereservoirandtheinstrument.
b.Purgethesystemwithacetonitrileormethanolandletitsitovernight.
Prepareandhandlesamplescorrectly
Makesurethatyoursamplesareparticlefree.Atthesametime,youmusttakecarenottointroducecontaminantsduringtheprocessofpreparingandhandlingsamples.
UseefficientcoldtrapsUseanefficientcoldtrapwhenconcentrating,lyophilizing,ordistillingyoursample.Otherwise,vacuumpumpoilcanbackstreamandcausecontamination.
Usecleanvials,caps,andplates
1. UseWatersbrandvials;theyhavebeencertifiedforcertainlevelsofcleanliness.(Selectthevialappropriateforyourapplication.)Othervialscancontaincontaminantsthatinterferewiththeapplicationandcontaminatethesystem.
2. Makesurethelineronyourvialandbottlecapsdoesnotcontaincontaminants(checkthemanufacturersdescription):
Do not use vials or bottle caps lined with paper. Paper can be a source of contaminants.
Self-sealing septa contain silicone that can interfere with LC/MS applications. Single-layered septa are acceptable if they do not contain plastics or adhesives
that can contaminate the sample manager (PTFE is recommended).
3. UseWatersbrandwellplates.Otherbrandscanleachplasticizers(forexample,diisooctylphthalates).
4. Foillinedplatecoversareacceptableaslongasthealuminumdoesnottouchthesolvent(possiblycausingapossiblereaction).
5. Donotuseselfsealingoradhesivecapmats;theycancontaincontaminantsfromtheadhesives.
6. Donotusecapmatsthatarenotcompatiblewiththesolventsusedforsamplediluents,mobilephase,andwashsolvents.
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Usecleanfittingsandtubing
Useclean,inertmaterialsforconnections
1. Connectionsthatcomeintocontactwithsolventsorsampleincludestoppers,Orings,checkvalves,andsolventinletfilters(sinkers).
2. Labelsoninlettubingcancontaincontaminants.Theymustnotcomeintocontactwithsolventsormobilephases.
3. Sometypesoftubing(suchaspolyvinylchloride,orPVC)containplasticizersorothercontaminants.
Weargloves
Wearparticulatefree,powderfree,nonlatexUseWaterssterilenitrilegloves(seeTable 4)when:
HandlingpartsoftheUPLCorHPLCsystemthatcomeintocontactwithmobilephaseorsample
ReplacingoldpartswithpartsthathavethelabelCriticalClean.
Replaceglovesiftheybecomewet
PuttingonglovesForinstructionsonputtingonglovestopreventcontamination,seeHowtoputongloves,page30.
Caution:Donottouchorbringsurfacesintocontactwithanythingcontainingcontaminants.
Caution:Toavoidincidentalskincontact,donotwearfingercotsasasubstituteforgloves.
Caution:Usecautionwhentouchingsurfaces,evenwhenwearinggloves.Keepglovesdry,andreplaceglovesiftheybecomewet.Wetglovescanintroducecontamination.Changeglovesfrequently.
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Usecleancolumns
UsecleancolumnsAUPLCorHPLCcolumncantrapparticles,precipitatedproteins,andotherorganiccontaminantsattheheadofthecolumn.Thesecontaminantscanadverselyaffectcolumnlifetimebyincreasingoperatingpressureorbyalteringchromatographicselectivity.Inaddition,theycanslowlybleedoff,increasingcarryoverandbackgroundnoise.
Thecolumncanalsoadsorbimpuritiesfromthesolvents.Thisadsorptioncanoccurwhenyou:
Equilibrateareversedphasecolumn(forexample,C18)forlongperiodsoftimeinhighaqueousconditions
RunareversedphasecolumnisocraticallyatlowerorganicconcentrationsTheadsorbedcompoundscaneluteasdistinctpeaksorasasmearacrossthechromatogram.ThetraceenrichingeffectamplifiestheamountofcontaminationpresentinthesolventsortheUPLCorHPLCsystem.
Note:Forguidelinesoncleaningcolumns,seeCleaningcolumns,page17.
StoringcolumnsStorecolumnsinthesolventtheyoriginallycamewith(forexample,100%acetonitrile).Flushcolumnstoremovebuffersandadditivesbeforestoringthem.
Note:Fordetailedinstructions,refertothesolventrecommendationsprovidedbythecolumnmanufacturer.
Important:Fordetailedinstructionsonusingandcaringforcolumns,refertothecareanduseinstructionsprovidedbythecolumnmanufacturer.(AnexampleistheACQUITYUPLCBEHColumnCareandUseManual,715001371.)
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Checklaboratoryair
KeeplaboratoryaircleanCompoundspresentinlaboratoryaircancontaminatetheLC/MSsystem:
Siloxanesareoftenpresentinlaboratoryair.Thesecompounds,whichexistindeodorantandothercosmeticproducts,cancausecontaminationundercertainconditions,suchasNanoFlow(Figure 1).
Figure1ESI+spectrumshowingsiloxanecontamination
Phthalatesarealsopresentinlaboratoryair.Airbornephthalatescomefromairconditioningfiltersandcancontaminateanysolventsorsolidsthatcomeintocontactwiththeair.
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TroubleshootingContamination
ThissectionoutlinesacommonsenseapproachtotroubleshootingcontaminationinanLC/MSsystem.Usetheflowcharttoproceed.
Isitcontinuousbackgroundnoiseor
chromatographicpeaks?
Continuousbackground
Peaks
Removethecolumnandrunablank.Peaksfound?
Runagradientblankwithoutinjecting.Peaks
found?
No
Gradient
Gradientorisocratic?
Isocratic
Yes
Isolatetheproblemtothe
LCorMS
Makeazerovolumeinjection
No
Troubleshootingcontamination
CheckthesolventmanagerYes
Checkthesamplemanager
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IsolatetheproblemtotheLCorMSsystem
Note:Completetheflowchartstepsonpage 11beforebeginningthissection.
1. FlushtheESIprobeandinfusionmechanismwithcleansolventotherthanmobilephaseandconnectasyringeinfusionkitdirectlytotheESIprobe.
If contamination levels decrease, contaminants are probably located primarily in the LC system. Troubleshoot the LC system, page 12.
If contamination spectra do not decrease in intensity, the source of contamination is probably in the MS system (source, inlet tubing, fluidics unit, and so on). Troubleshoot the MS system, page 15.
TroubleshoottheLCsystem
Tryanewcolumn
Note: Contaminantscancollectandconcentrateonacolumn(traceenrichment)whenyourunlowconcentrationorganicmobilephases(forexample,initialconditions)foralongtime.Thecontaminantselutefromthecolumnwhenagradientisrun.
CheckthemobilephaseMix1mLofmobilephaseAwith1mLofmobilephaseBinacleanvial.Infusethemixtureintothemassspectrometer.
Ifcontaminationexists,theproblemisinthebottlesormobilephase.SeetheguidelinesonusingcleansolventsandcontainersinPreventingcontamination,page3.
Ifthereisnocontaminationoninfusion,pumpthe50:50A/Bmixturethroughacleanprobeintothemassspectrometer.Ifyouseecontamination,itislocatedinthesolventmanager/chromatographicpumporsamplemanager/autosampler(orboth).Todeterminewhichcomponentisthesource,Makeazerovolumeinjection.
Makeazerovolumeinjection
Ifyouseecontaminants,Checkthesolventmanager/chromatographicpump. Ifnocontaminantsarepresent,Checkthesamplemanager/autosampler.
Important:InfuseintotheMSthemobilephasethatyouareusinginthesystem(forexample,a50:50A/Bmixtureat0.3mL/min).MakesuretobypasstheentireLCandsolventmanagementsystem.
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Checkthesolventmanager/chromatographicpumpDisconnectthesolventmanager/chromatographicpumpfromthesamplemanager/autosamplerandpumpdirectlyintothemassspectrometer.
Ifcontaminationexists,thesolventmanager/chromatographicpumpisthesourceofthecontamination.Reconnectthesamplemanager/autosamplerandfollowGeneralguidelines,page16.
Ifcontaminationdoesnotexist,Checkthesamplemanager/autosampler.
Checkthesamplemanager/autosampler
1. Removethecolumnifithasnotalreadybeenremoved.2. Pumpawashsolutionthroughthesamplemanager/autosamplertowaste.
Toflushtheneedlewashflowpath,usethesamesolutionyouusedtoflushthepumps.Alsoinjectlargevolumes(forexample,fullloopwith3Xoverfills)ofthecleaningsolution.Ifthewashsolutionisanacid,flushwithcopiousamountsofwater.Thenreturntomobilephaseandflushthoroughly.Ifcontaminationexists,determinewhetheritisinthesample,thediluent,theinfusiondevice,orthesamplecontainer.
3. Checkthesolvent,water,andacidusedfordilution.Infusethesamplediluentforexample,amixtureofequalpartswaterandeitheracetonitrileormethanolplus0.1%formicacidintothemassspectrometertocheckforcontamination.Ifthereisnocontaminationinthisblank,thenthecontaminationcamewiththeoriginalsample.
Ifthecontaminationpersists,Checkthesample.
4. Checkthesample.Infuseadilutedsampleintothemassspectrometer.
Note:PEGcancomefromdetergentsusedforsamplepreparationorPEGylatedpharmaceuticalcompounds.
Ifthecontaminationpersists,Checktheinfusiondeviceandsamplecontainer.5. Checktheinfusiondeviceandsamplecontainer.
Cleanorreplaceeachcomponent.Thenrepeattheinfusiontest.Iftheinfusiondeviceandcontainerareclean,Checktheneedlewashsolutions.
6. Changetheinjectionvolume.Ifreplacingthesamplediluentdidnotsolvetheproblem,tryadjustingtheinjectionvolumebyafactorof2ormore.Ifthecontaminationincreasesordecreasesinproportiontotheinjectionvolumechange,itisprobablethatthesampleiscontaminated.Newsampleorfurthersamplecleanupmayberequired.
Ifthesamplevolumechangehasnoeffectonthesizeofthecarryoverpeak,Changetheinjectionmode.
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7. Changetheinjectionmode.Partialloopneedleoverfill(PLNO)modecancausemorecarryoverthanpartiallooporfullloopmode.ThatisbecausethesampleispulledthroughtheVDD,whichisnotflushedwithstrongneedlewash.
Ifthecontaminationpersists,Checktheneedlewashsolutions.
8. Checktheneedlewashsolutions.Arethewashsolventscorrect?Ifnot,usethecorrectwashsolutions.Makeanotherinjectionandcheckforcontamination.Ifitstillexists,Checkthetubingandfittingsontheinjector.
9. Checkthetubingandfittingsontheinjector.Inparticular,checktheinjectoroutlettocolumninlet.Ifthereisdeadvolume,contaminationcanaccumulateinthosespaces.Replacethetubingandfittings.Makeanotherinjectionandcheckforcontamination.Ifitstillexists,Replacetheneedle.
10.Replacetheneedle.Replacetheneedle,thenmakeanotherinjection.SomehydrophobiccompoundsadsorbtoPEEKneedles.Ifcontaminationpersists,tryreplacingitwithastainlesssteelneedle.Ifcontaminationstillpersists,Replacetheotherinjectorparts.
11.Replacetheotherinjectorparts.Replacetheotherinjectorparts(forexample,needlewashport,injectorvalvepod).Refertotheoperatorsmanualforspecificinjectorpartsthatcanbereplaced.Flushthesystemwithmobilephase.Makeanotherinjectionandthencheckforcontamination.
a.Ifcontaminationstillexists,TroubleshoottheMSsystem,page15.b.Ifcontaminationdoesnotexist,Reinstallthecolumn.
12.Reinstallthecolumn.Reinstallthecolumn,andthencheckforcontamination.Ifcontaminationappears,repeatthefollowingstepswiththecolumninstalled(butomittheinfusionsteps):
Make a zero-volume injection, page 12 Check the solvent manager/chromatographic pump, page 13 Check the sample manager/autosampler, page 13
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TroubleshoottheMSsystem
Iftroubleshootingtheliquidchromatographysystemdoesnotyieldthelocationofcontamination,thelikelysourceisthemassspectrometrysystem.
CheckthefrontendcomponentsLikelylocationsforMScontaminationarefrontendcomponents:
ESIprobe(probetip,capillary,unions) Samplecone LockSpraybaffle Ionsourceblock Sourceenclosure PEEKtubingconnectingcolumnoutlettoAPIsource Componentsoftheintegralflowdivert/injectionvalve(iffitted) Throttlevalve(iffitted) PEEKsupportblock LCtubing Nitrogengastubing Nitrogengassource(forexample,generator)
Remove,cleanorreplace,andtestthecomponentsRemove,cleanorreplace,andtesteachofthesecomponentsoneatatime.Ifcontaminationstillexists,theMScomponentspossiblyhavebecomerecontaminatedaftercleaning.Toavoidthisproblem,cleanandreplaceallsuspectedpartssimultaneously.
CleanorreplacethecontaminatedcomponentIfbackgroundnoiseishighafteranytest,cleanorreplacethelastcomponentadded(seeCleaningtoEliminateContamination,page16).
Important:Donotwastetimeandresourcesattemptingtoremovetypicalbackgroundnoise.ThesensitivenatureofMSsystemsmeansthatsomedegreeofchemicalbackgroundisaconstant.Inaddition,differenttypesofMSsystemshavedifferentdegreesofsensitivity.Forexample,youseeahigherbackgroundinamoresensitiveinstrument.Sensitivityissignaltonoiseratio,notjustabsolutecounts.
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CleaningtoEliminateContamination
IfyoumustcleantheLC/MSsystem,anunderstandingofcontaminantsandtheirsourcesisessential.Forinformation,seeMajorcontaminantsandtheirsources,page31.
Generalguidelines
TocleancontaminationinWaterssystems,useonlymobilephase(highest)qualitysolventsandacidorbase.1Removethecolumnanddetector(anydetector,includingthemassspectrometer).Ifyouknowwhatthecontaminantis,usethemixtureinwhichitismostsoluble.
Flushthesystemcomponentwithahighorganicsolventsuchas80%organicsolventand20%water,andthentestforcontamination.Repeattheprocedureuntilthebackgroundisreducedtoanacceptablelevel.
Afterusinganywash,rinsewith50%acetonitrileormobilephasetoremovethecleaningsolution.Ifyouuseaphosphoricacidwash,pumpultrapurewater(seeUseultrapurewater,page4)throughthesystemuntilthepHisclosetoneutral(pH=7).
Warning:Topreventinjury,alwaysusesafelaboratorypracticeswhenworkingwithsolventsandwashsolutions.Knowthechemicalandphysicalpropertiesofthesolventsandsolutions.Refertothesafetydatasheetsforeachsolventandsolutioninuse.Alwaysuseeyeprotectionandgloveswhenhandlingsolventsorcleaningmixtures.
Caution:Donotattempttocleanthesystemuntilyouhavefoundandeliminatedthesourceofcontamination.Ifyoucleanwithoutfindingthesource,thecontaminationwillsoonreturnandthesystemwillrequirecleaningagain.DisconnecttheLCsystemfromthecolumnanddetectorbeforecleaning.Solventsandacidorbasemustbeofthehighestpurityforcleaninganduse.
1. Thesecleaningguidelinesarebasedontraditionaltechniquesusingmaterialsthatarereadilyavailableinthelaboratory.TheyapplyprimarilytoreversedphaseLC/MS.
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Cleaningcolumns
Tocleanacontaminatedcolumn,washthecolumnwithsolventsthatremovethecontaminantsanddonotdamagethecolumn.Itisgoodpracticetocleanareversedphasecolumnattheendofarunbyflushingitwithahighpercentage(forexample,80%)organicmobilephase.
Note:Forfullinstructionsoncleaningacolumn,refertothecareanduseinstructionsprovidedbythecolumnmanufacturer.
RecommendedcleaningproceduresforMSsystemsTocleanastandaloneMSsystem,usethecleaningprocedure(availableintheWatersSupportLibrary)appropriatetoyoursystem:
Massspectrometryuserguides
RecommendedcleaningmixturesforLCinletcomponents
TocleantheLCinlet,usetherecommendedcleaningmixturesinTable 1.
Forothercleaningproceduresthatmaybeappropriateforyoursystem,refertotheWatersSupportLibrary:
Routinecleaningproceduresforseparationssystems UPLC/UHPLCuserguides AllianceHPLCuserguides
Important:Ifwashedinsolventscontainingadditivessuchasammoniumhydroxideome,silicapackingmaterialsdissolveatpH>8.IfyourmobilephasepHexceeds8,useacolumnthatismorestabletohighpH,suchastheWatersACQUITYUPLCBEHorXBridgecolumn.
Fordetailedguidelinesonwashingsilicapackingmaterials,refertothecareanduseinstructionsprovidedbythecolumnmanufacturer.
Caution:Topreventcrosscontamination,removetheconnectiontothecolumnandmassspectrometerbeforecleaningtheLCinlet.
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FinalrinseAftercleaningwithanyofthemixtures,rinsetheLCinletwithoneofthefollowing:
Ultrapurewater(requiredforphosphoricacidwash) 50:50mixtureofwater/organicsolvent 50:50mixtureofthechromatographicmethodsmobilephases
Important:Alwaysleavethesysteminthewashsolventthatismostsimilartoyourmobilephase(pH)conditions.
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ions Comments
etentiontimeushwithatofasolventwiththeution.formicacidis
washrnightwhen00%organicacid.
Standard,oracidwashCleanstheLCinletforusewithacidicmobilephases.
Formicacidisvolatile.
ACQUITYMCQUITY,orontainingrytubingthatH10.etentiontimeushwithatofsolventwiththeution.washrnightwhen00%organicacid.
BasicwashCleanstheLCinletforusewithbasicmobilephases.
Ammoniumhydroxideisvolatile.
Table1:RecommendedcleaningmixturesforLCinlets1
Cleaningconditions Solventmixtures(v/v) Stockconcentration Description Caut
1 80%2isopropanol(IPA)10%water10%formicacid
~99%formicacid Anaggressivecleaningmixtureforcompoundsmoresolubleinacids
Topreventrproblems,flleast50mLcompatiblecleaningsol
Highpurityexpensive.
Donotleavesolutionovenotflowing.
Donotmix1withstrong
Finalrinse:Primewithultrapurewaterthroughthelinesusedforcleaningforseveralminutes.
2 80%2isopropanol(IPA)10%water10%ammoniumhydroxide(concentrated)
~29%ammonium~56.6%ammoniumhydroxide
Anaggressivecleaningmixtureforcompoundsmoresolubleinbaseforexample,phthalatesandPEGcontainingcompounds,includingdetergents(TritonX100,TritonX114,Brij35).
NeveruseinClass,nanoAanysystemcsilicacapilladissolves>p
Topreventrproblems,flleast50mLcompatiblecleaningsol
Donotleavesolutionovenotflowing.
Donotmix1withstrong
Finalrinse:Primewithultrapurewaterthroughthelinesusedforcleaningforseveralminutes.
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urnsfromolvent,heacidtostirring.iblewithometers.etentiontimeushuntilyoulpH(pH=7)tcompatibleningsolution,100%
washrnightwhensinsolubleinns.Alwaysatewithtiesofwater.00%organicacid.
PhosphoricacidwashPhosphoricacidworksasbothastrongacidandanoxidizingagent,destroyingcompounds.
Notvolatileandnotcompatiblewithmassspectrometers.
Lossinsensitivity.Youmightseeclustersat98.
00%organicacid.ltscontainingassium,recipitates.
N/A
(seeTroubleshootingContamination,pagecolumnmanufacturer.
ions Comments
3 30%phosphoricacid(concentrated)
70%water
~85%Phosphoricacid Anaggressivemixtureforremovingorganiccontaminants.
Topreventbsplatteringsslowlyaddtwaterwhile
Notcompatmassspectr
Topreventrproblems,flreachneutrawithasolvenwiththeclea(forexamplewater).
Donotleavesolutionovenotflowing.
Phosphateihighorganicconcentratioflushphosphlargequanti
Donotmix1withstrong
Finalrinse:Primewithultrapurewaterthroughthelinesusedforcleaningforseveralminutes.
4 100%IPA N/A Neutral,hydrophobiccompounds
Donotmix1withstrong
Inorganicsasodium,potphosphatep
1. UsethesecleaningsolutionsonlytocleanLCinletcomponentsthathavebeenidentifiedasthesourceofcontamination11).Donotuseanyofthesecleaningsolutionstocleanacolumn.Refertothecareanduseinstructionsprovidedbythe
Table1:RecommendedcleaningmixturesforLCinlets1
Cleaningconditions Solventmixtures(v/v) Stockconcentration Description Caut
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Cleaningthesolventmanager/chromatographicpumpPumpthecleaningsolutionthroughthesolventmanager/chromatographicpump(orsolventmanager/chromatographicpumpandsamplemanager/autosampler)towaste.Thenflushthoroughlywithmobilephases.
Cleaningthesamplemanager/autosamplerPumpthecleaningsolutionthroughthesamplemanager/autosamplertowaste.Usethesamesolutiontoflushtheneedlewashflowpath.Alsoinjectlargevolumes(fullloopforthefixedloopsamplemanager,orthelargestvolumeavailablefortheFTNsamplemanager)ofthecleaningsolution.Thenreturntothemobilephaseandwashsolutionsrequiredforanalysisandflushthoroughly.
CleaningthecolumnSeeCleaningcolumns,page17.
CleaningMSSystems
RemoveandcleanorreplacesuspectedMScomponentsRemoveandcleanorreplaceMScomponentssuspectedofcausingcontamination.Usethehighestpuritysolvents.TheobjectiveistodissolvecontaminationfromthesurfaceoftheMScomponent.Toavoidrecontaminatingcleanornewcomponents,useamethodicalcleaningprocess.Sometimesyoucleanorreplaceonecomponentatatime,workingfromthefirstcomponenttocomeintocontactwithsolventtothelastcomponent.Inothercases,itisbettertocleanallsuspectedcomponentssimultaneously.Besuretouseultrapuresolventsandcleanglassware(seePreventingcontamination,page3).
1. Carefullywipethecomponentwithacleanswaborlintfreewipe.
2. Sonicatecomponentsinsolventforbetween15minutesto1hour.SeeTable 1forrecommendedMScleaningsolutions.
Note: Effectiveremovalofcontaminationcanrequireseveralsonicationsteps,usingfreshcleaningsolventateachstep.Eachsonicationstepshouldlastbetween15minutesto1hour.Longertimesmayberequirediftheflowratesarelow(forexample,MClass).
Caution:Ifyouhaveflushedwithphosphoricacid,youmustflushwithwaterbeforemobilephase.
Caution:Toavoidcontactwithtoxiccontaminants,wearglovesandeyeprotection.
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3. Ifthesesolutionsfailtoreducecontaminationlevels,replacetheparts.
4. Besuretorinsetheglasswarethoroughlyandusefresh,cleansolventbetweeneachstep.
5. Afterfinalsonication,removetheMScomponentfromthecleaningsolution.Quicklydrythecomponentwithastrongstreamofclean,drynitrogen.
FluidicsAfterdecontaminatingtherestoftheplumbing,stripandcleanthefluidicsaccordingtomanufacturerinstructions.Payparticularattentiontotherotorseal,onwhichmechanicalwear(observableascirculargrooves)canserveasasiteofcontamination.Replacethesealifnecessary.
APIsourceBecauseitcanbeexposedtoalargequantityofsamplematerialduringnormaloperation,theatmosphericpressureionization(API)sourceisthemostcommonlocationofMScontamination.Disassembleandcleanthesourceusingnormalmaintenanceprocedures.SonicateAPIsourcecomponentsinsolventforbetween15minutesto1hour.
APIprobesReplaceorrebuildcontaminatedAPIprobesratherthancleaningorflushingit.
LCtubingReplacecontaminatedLCtubingratherthancleaningitthroughrepeatedflushing.
Caution:ToavoiddamagingPEEKcomponentsandTWaveassemblies,donotsonicateinchlorinatedsolvents,hexane,acetone,oracids.
Caution:Fast,thoroughdryingisnecessarytopreventsolventspots,whichcanaffectMSperformance.
Important:Ensurethatsolventqualityandglasswarecleanliness(Select,prepare,andhandlesolventscorrectly,page3).
Notice:IonopticsfromtheiontransferregionforwardareanunlikelylocationofcontaminationdetectablebyMSanalyses.
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ReferenceInformation
Solventconsiderations
PossibleeffectsoflowqualitysolventsNotallassaysrequireahighgradesolvent.Whenselectingasolvent,choosethehighestpuritysolventappropriateforyourapplication.Considerthesolventcontaminanttypesandconsequences.Table 2showsthepossibleeffectsoflowqualitysolventsonyourassay.
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Preventiveactions
Useonlyhighpuritysolventsthatarefilteredbythevendortoremoveparticulatesgreaterthan0.2microns.
Wearglovestoeliminateoilsfromskinandhandcreams.
Donotrefiltervendorfilteredsolvents.
Usefreshaqueousmobilephases.
Wearglovestoeliminateoilsfromskinandhandcreams.
Usefresh,highpurity,filteredwater(
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Donotuseplasticbottlesassolventormobilephasereservoirs.
Wearglovestoeliminateoilsfromskinandhandcreams.
Usevials,wellplates,andcapsappropriateforLCorganicsolvents.
NeverwashLCglassware(bottles,funnels,graduatedcylinders,etc.)withdetergents.
HavededicatedLCglassware.Donotrefiltervendorfilteredsolvents.Filtersorfilteringapparatuscanaddcontaminants.
Preventiveactions
UVabsorbingorganiccompounds(detectableinTUV,PDA)
Noisyorchanging(sloping)baseline
GhostpeaksLossofsensitivityLossoflinearityShiftingretentiontimesAbnormalpeakshapes
Forhighsensitivityanddetectionoflowconcentrationsofanalytes,asmooth,flatbaselineisdesiredforthehighestsignaltonoiseratio.Ahighsignaltonoiseratioimprovesthereproducibilityofquantitation.
HighUVbackgroundcandecreasethelinearityofthemethodathighanalyteconcentrations.
Table2:Possibleeffectsoflowqualitysolvents
Solventcontaminants Possibleproblems Analyticalconsiderations
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Donotuseplasticbottlesassolventormobilephasereservoirs.
Plasticsarethesourceofphthalatesandarefoundeverywhereincludinginair,water,andsolvents.
WearglovestoprotectskinfromoilsandhandcreamscontainingPEG.
Usevials,wellplates,andcapsappropriateforLCorganicsolvents.
NeverwashLCglassware(includingbottles,funnels,graduatedcylinders)withdetergentsorinadishwasher.
HavededicatedLCglassware.Donotrefiltervendorfilteredsolvents.Filtersorfilteringapparatuscanaddcontaminants.
Preventiveactions
Ionizableorganiccompoundsdetectableinthemassspectrometer
NoisyorhighbaselineLossofsensitivityGhostpeaksShiftingretentiontimesAbnormalpeakshapes
Contaminationfromlowmassions,PEGcompounds,andphthalates(plasticizers)cancause:
Lossofsensitivity,lowersignaltonoiseratioduetoionsuppression
Spectralpeaksatthesamemassesastheanalytes
Table2:Possibleeffectsoflowqualitysolvents
Solventcontaminants Possibleproblems Analyticalconsiderations
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Usefresh,highpurity,filtered(
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SolventpurityrecommendationsAhighpuritysolventmeetsthecriteriashowninTable 3.
Note:Table 3valueswereobtainedfromknownusableLC/MSsolvents.Somebrandshavecertificatesofanalysisindicatinghigherpuritythanthesevalues.
Table3:LC/MSsolventpurityrecommendations
ParameterSuggestedlevel
Acetonitrile Methanol Isopropanol Water
Assay 99.9% 99.9% 99.9% 99.9%
Filtered 0.2microns 0.2microns 0.2microns 0.2microns
Aluminum(Al) 25ppb 50ppb 20ppb 50ppb
Calcium(Ca) 50ppb 50ppb 50ppb 50ppb
Iron(Fe) 20ppb 20ppb 20ppb 10ppb
Lead(Pb) 20ppb 20ppb 20ppb 10ppb
Magnesium(Pb) 20ppb 20ppb 20ppb 10ppb
Potassium(K) 50ppb 50ppb 50ppb 50ppb
Sodium(Na) 50ppb 50ppb 50ppb 50ppb
Residueafterevaporation
25ppb 25ppb 25ppb 25ppb
Water 0.01% 0.05% 0.05% N/A
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Recommendedwaterpurificationprocess
Ultrapurewaterissterileandcontainsnoparticulates,nodetectableionizablecompounds,andnoUVabsorbingcompounds.Thepurificationprocessmustincludeallthefollowingsteps:
a.Reverseosmosis(toremovemostcontaminants)b.UVsterilization(tokillbacteria)c.Ionexchange(toremoveanyremainingions)d.Carbonfiltration(toremoveanyremainingionizablecompounds)e.Apharmaceuticalgrade0.2mmembranefilter(toremoveanyremainingparticu
lates)
Otherconsiderations
Ifoutletlineshavebeenwithoutflowformorethan24hours,flushthemtoeliminatebacterialgrowth.
Oncethewaterhasbeenpurified,donotstoreitforlongerthan24hourswithouttakingmeasuretopreventthegrowthofmicroorganisms.
Ifusingbottledwater,payattentiontotheexpirationdatesuggestedbythemanufacturer,anddiscardthewaterafterthatdate.Openbottlesofwatercanbecomecontaminated.
Caution:Housedeionized(DI),reverseosmosis(RO),anddistilledwaterdonotmeetthesecriteria.
Caution:Ifusingapurificationsystem,youmustperformregularmaintenanceonit.
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Gloves
Ordergloves
HowtoputonglovesOpentheglovesbyunfoldingthepackagingleavesuntilthecuffsareexposed(Figure 2).
Figure2Removingglovesfrompackage
Graspthecuffofonegloveandpullitoveryourhand,leavingthecuffturnedup.Repeatwiththeotherglove.Thenturndownthecuffsofbothgloves.
Table4:Nitrilegloves
PartNumber Description Qty
700002964 SterileNitrileGloves,Size7 3pairs
700002965 SterileNitrileGloves,Size9 3pairs
Caution:Whenputtingongloves,donottouchtheglovefingerswithyourbarehand.Oncetheglovesareon,donottouchanythingotherthanthecriticalcleanpartsbeinghandledorserviced.
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Majorcontaminantsandtheirsources
ThissectionlistssomemajorcontaminantsinLC/MSsystems,alongwiththeirsourcesandspectra.
Polyethyleneglycol(PEG)orPEGlikematerialsPEG(Figure 3)isasyntheticpolymerproducedinarangeofmolecularweights.CommonsourcesofPEGcontaminationinclude:
Organicsolvents(methanol,2propanol,acetonitrile,water) Massspectrometercalibrationsolution Handcreamandotherpersonalcareproducts Detergent(TritonX100,glasswaredetergents,andsoon) Cuttingsolutionsinmachining Columnmanufacturing
Figure3PEGcontaminationspectra(seriesofmasspeaksseparatedby44Da)
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MetalionsMetalionssuchaslithium(Li),sodium(Na),potassium(K),copper(Cu),platinum(Pt),andiron(Fe)canbesourcesofcontamination.
Forexample,ironformsadductswithvaryingnumbersofacetateinaceticacidoracetatemobilephases.IroncancontaminateanLC/MSsystemthroughthefollowingsources:
Solventssuchaswaterandacetonitrile Aceticacid(lowerinformicacid) Formicacid Nonpassivatedstainlesssteelparts Titaniumorinertmetalpartsfabricatedwithsteeltools
Figure 4showsthetypicalpatternofFeacetateclusterspectra.Thestrongestion(basepeakintensity,orBPI)masscandiffer,dependingonthenumberofacetatesinthecluster.TheupperspectraarebasedontheMassLynxisotopemodel.
Figure4Fecontaminationspectra
PhthalatesPhthalatesarechemicalcompoundsusedchieflyasplasticizers,andcancausecontamination.Thecompoundscanbedetectedonawiderangeoflaboratorymaterials,includingwaterandothersolvents,laboratoryair,andplasticmaterialssuchastubingandwaterstoragecontainers.Commonphthalatesincludedi2ethylhexylphthalate(DEHP),diisodecylphthalate(DIDP),diisononylphthalate(DINP),anddiisooctylphthalate(DIOP).
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Diisooctylphthalatescanformthefollowingadducts:
[M+H]+=391 [M+Na]+=413 [M+K]+=429 [2M+NH4]
+=798 [2M+Na]+=803
Slipagents(amides)Avoidusingcomponentspackedinplasticbagscontainingslipagents,oramides(Figure 5).Thethreemostcommonlyusedamidesare:
Oleamide([M+H]+=282) Stearamide([M+H]+=284) Erucamide([M+H]+=338)
Figure5Contaminationspectrumofoleamideanderucamideinzipbag
Contaminantdatabase
Foracomprehensivelistofmajorcontaminants,seePEGmasterlistandBackgroundIonMasterList.
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http://www.waters.com/webassets/cms/support/docs/peg_table_3_15_2010.pdfhttp://www.waters.com/webassets/cms/support/docs/bckgrnd_ion_mstr_lst_4_13_2010.pdfhttp://www.waters.com/webassets/cms/support/docs/bckgrnd_ion_mstr_lst_4_13_2010.pdf
Controlling Contamination in LC/MS SystemsTable of ContentsPreventing contaminationSelect, prepare, and handle solvents correctlyUse clean, particle-free solventsUse fresh solventsUse ultra-pure waterPrevent microbial growthMinimize the use of additivesUse miscible solventsStore solvents in clean glass reservoirs with covers
Clean laboratory glassware properlyStandard cleaning processAggressive cleaning process
Prepare and handle samples correctlyUse efficient cold trapsUse clean vials, caps, and plates
Use clean fittings and tubingUse clean, inert materials for connections
Wear glovesWear particulate-free, powder-free, non-latexReplace gloves if they become wetPutting on gloves
Use clean columnsUse clean columnsStoring columns
Check laboratory airKeep laboratory air clean
Troubleshooting ContaminationIsolate the problem to the LC or MS systemTroubleshoot the LC systemTry a new columnCheck the mobile phaseMake a zero-volume injectionCheck the solvent manager/chromatographic pumpCheck the sample manager/autosampler
Troubleshoot the MS systemCheck the front-end componentsRemove, clean or replace, and test the componentsClean or replace the contaminated component
Cleaning to Eliminate ContaminationGeneral guidelinesCleaning columnsRecommended cleaning procedures for MS systemsRecommended cleaning mixtures for LC inlet componentsFinal rinseCleaning the solvent manager/chromatographic pumpCleaning the sample manager/autosamplerCleaning the column
Cleaning MS SystemsRemove and clean or replace suspected MS componentsFluidicsAPI sourceAPI probesLC tubing
Reference InformationSolvent considerationsPossible effects of low-quality solventsSolvent purity recommendations
Recommended water purification processOther considerations
GlovesOrder glovesHow to put on gloves
Major contaminants and their sourcesPolyethylene glycol (PEG) or PEG-like materialsMetal ionsPhthalatesSlip agents (amides)
Contaminant database