Controlling Contamination in LC/MS - Waters Corporation · CONTROLLING CONTAMINATION IN LC/MS SYSTEMS 715001307 REV.. G 4 OF 33 Use ultra‐pure water Use ultra‐pure water (defined

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.

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

CONTROLLINGCONTAMINATIONINLC/MSSYSTEMS

715001307REV.G 3OF33

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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715001307REV.G 4OF33

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.


715001307REV.G 5OF33

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.


715001307REV.G 6OF33

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.


715001307REV.G 7OF33

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.


http://www.waters.com/waters/en_US/Sample-Vials/nav.htm?cid=10000711http://www.waters.com/waters/en_US/Sample-Vials/nav.htm?cid=10000711

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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.


715001307REV.G 9OF33

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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715001307REV.G 10OF33

Checklaboratoryair

KeeplaboratoryaircleanCompoundspresentinlaboratoryaircancontaminatetheLC/MSsystem:

Siloxanesareoftenpresentinlaboratoryair.Thesecompounds,whichexistindeodorantandothercosmeticproducts,cancausecontaminationundercertainconditions,suchasNanoFlow(Figure 1).

Figure1ESI+spectrumshowingsiloxanecontamination

Phthalatesarealsopresentinlaboratoryair.Airbornephthalatescomefromairconditioningfiltersandcancontaminateanysolventsorsolidsthatcomeintocontactwiththeair.


715001307REV.G 11OF33

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


715001307REV.G 12OF33

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.


715001307REV.G 13OF33

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.


715001307REV.G 14OF33

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


715001307REV.G 15OF33

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.


715001307REV.G 16OF33

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.


715001307REV.G 17OF33

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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715001307REV.G 18OF33

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


Donotmix1withstrong


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715001307 REV. G

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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).


Phosphateihighorganicconcentratioflushphosphlargequanti

Donotmix1withstrong


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.


Usefresh,highpurity,filteredwater(

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Donotuseplasticbottlesassolventormobilephasereservoirs.


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.


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

Documents

Controlling Contamination in LC/MS - Waters Corporation · CONTROLLING CONTAMINATION IN LC/MS SYSTEMS 715001307 REV.. G 4 OF 33 Use ultra‐pure water Use ultra‐pure water (defined