HYPHENATIONSOF
CAPILLARYCHROMATOGRAPHY WITHMASSSPECTROMETRY
Editedby
PETERQ.TRANCHIDA
LUIGIMONDELLO
Elsevier
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Contributors
PaolaArena
DepartmentofChemical,Biological,PharmaceuticalandEnvironmentalSciences, UniversityofMessina,Messina,Italy
HenrietteEngenBerg
DepartmentofChemistry,UniversityofOslo,Oslo,Norway
FrancescoCacciola
DepartmentofBiomedical,Dental,MorphologicalandFunctionalImagingSciences, UniversityofMessina,Messina,Italy
GiovanniD’Orazio
IstitutoperiSistemiBiologici,ConsiglioNazionaledelleRicerche,Monterotondo,Italy
FrankDavid
ResearchInstituteforChromatography,Kortrijk,Belgium
ChrisG.deKoster
SwammerdamInstituteforLifeSciences,Amsterdam,theNetherlands
PaolaDonato
DepartmentofBiomedical,Dental,MorphologicalandFunctionalImagingSciences, UniversityofMessina,Messina,Italy
PaolaDugo
DepartmentofChemical,Biological,PharmaceuticalandEnvironmentalSciences, UniversityofMessina,Messina,Italy;UnitofFoodScienceandNutrition,Department ofMedicine,UniversityCampusBio-MedicoofRome,Rome,Italy;Chromaleonts.r.l., c/oDepartmentofChemical,Biological,PharmaceuticalandEnvironmentalSciences, UniversityofMessina,Messina,Italy
ChiaraFanali
CentroIntegratodiRicerca,CampusBio-MedicoUniversity,Roma,Italy
SalvatoreFanali
IstitutoperiSistemiBiologici,ConsiglioNazionaledelleRicerche,Monterotondo,Italy; TeachingCommitteeofPh.D.SchoolinNaturalScienceandEngineering,Universityof Verona,Verona,Italy
Hans-GerdJanssen
UniversityofAmsterdam,Van ‘tHoffInstituteforMolecularSciences,Analytical Chemistry-Group,Amsterdam,theNetherlands;UnileverResearchandDevelopment, Vlaardingen,theNetherlands
ArendH.J.Kolk
UniversityofAmsterdam,Van ‘tHoffInstituteforMolecularSciences,Analytical Chemistry-Group,Amsterdam,theNetherlands
ElsaLundanes
DepartmentofChemistry,UniversityofOslo,Oslo,Norway
DomenicaMangraviti
DepartmentofChemical,Biological,PharmaceuticalandEnvironmentalSciences, UniversityofMessina,Messina,Italy
LuigiMondello
DepartmentofChemical,Biological,PharmaceuticalandEnvironmentalSciences, UniversityofMessina,Messina,Italy;Chromaleonts.r.l.,c/oDepartmentofChemical, Biological,PharmaceuticalandEnvironmentalSciences,UniversityofMessina,Messina, Italy;UnitofFoodScienceandNutrition,DepartmentofMedicine,UniversityCampus Bio-MedicoofRome,Rome,Italy;BeSeps.r.l.,c/oDepartmentofChemical,Biological, PharmaceuticalandEnvironmentalSciences,UniversityofMessina,Messina,Italy
MartaP.B.Mourão
UniversityofAmsterdam,Van ‘tHoffInstituteforMolecularSciences,Analytical Chemistry-Group,Amsterdam,theNetherlands
DavidK.Pinkerton
DepartmentofChemistry,UniversityofWashington,Seattle,WA,UnitedStates
SarahE.Prebihalo
DepartmentofChemistry,UniversityofWashington,Seattle,WA,UnitedStates
GiorgiaPurcaro
GemblouxAgro-BioTech,UniversityofLiège,Gembloux,Belgium
BrookeC.Reaser DepartmentofChemistry,UniversityofWashington,Seattle,WA,UnitedStates
HanneRoberg-Larsen
DepartmentofChemistry,UniversityofOslo,Oslo,Norway
PeterJ.Schoenmakers
Van ‘tHoffInstituteforMolecularSciences,UniversityofAmsterdam,Amsterdam, theNetherlands
KristenJ.Skogerboe DepartmentofChemistry,SeattleUniversity,Seattle,WA,UnitedStates
RobertE.Synovec DepartmentofChemistry,UniversityofWashington,Seattle,WA,UnitedStates
PeterQ.Tranchida DepartmentofChemical,Biological,PharmaceuticalandEnvironmentalSciences, UniversityofMessina,Messina,Italy
NathanialE.Watson DepartmentofChemistry,UniversityofWashington,Seattle,WA,UnitedStates
StevenRayWilson
DepartmentofChemistry,UniversityofOslo,Oslo,Norway
MariosimoneZoccali
DepartmentofChemical,Biological,PharmaceuticalandEnvironmentalSciences, UniversityofMessina,Messina,Italy
Preface
Thepresentbook,composedof fivechapters,providesup-to-dateand detailedinformationonthehyphenatedtechnique,capillary chromatography-massspectrometry(CC-MS),initswidevarietyof combinations.Oneofthemainobjectivesofthecontributionistogivethe ReadersufficientinformationtopinpointthemostappropriateCC-MS approachforher/hisanalyticalnecessity,whateveritmaybe.Hopefully, thebookwillalsocontributeinmovingtheviewofthosewhooperatein the fieldofCC-MSandwho:(I)consider,amongchromatographers,the massspectrometeronlyasaspectra-generatingdetector;(II)devote,among massspectrometrists,littleattentiontothechromatographyprocess.
TheheterogeneityofCC-MStechnologiesderivesfromthecurrently availabletypesofchromatographic(gaschromatography GC,liquid chromatography LC,capillaryelectrophoresis CE,etc.)andmass analyzing(singlequadrupole Q,time-of-flight ToF,etc.)systems.Both chromatographyandmassspectrometrycanexistasstandalonetechnologies,andprovidecomplementaryanalyteinformation,whichareideallya retentiontime,amassspectral fingerprint,andthemolecularweight (comprisedinthemassspectra);hence,CC-MSisatwo-dimensional(2D) methodology,withitsanalyticalpotentialdependingtoagreatextenton theMScapabilities(e.g.,lowresolution vs. highresolution,single- vs. dualstageMS).
Two(sometimesmore)separationstepscanbecombinedinamultidimensionalcapillarychromatographyconfiguration(MDCC-MS:e.g., MDGC-ToFMS,MDLC-QMS,etc.),greatlyincreasingtheprobabilityof subjectingtotallyresolvedcompoundstomassspectrometry,thusreducing the “workload” ofthelastanalyticaldimension.Multidimensionalcapillary chromatography,whichisenabledbyusingdedicatedtransferdevices,can beclassifiedintotwogreatgroups:(I)heart-cutting,meaningthatonlya specificnumberof first-dimensioneffluentbandsaretransferredtothe secondcolumn(e.g.,CE-CE,GC-GC);(II)comprehensive,meaningthat theentiresampleisanalyzedonthetwoanalyticalcolumns(e.g.,LC LC, GC GC).SimilartotheconceptofMDCC,massanalyzerscanbe combinedinsequence(CC-MS-MS:e.g.,LC-Q-ToF,GC-triplequadrupoleMS,etc.),greatlyenhancinganalyticalspecificity,thusreducingin manycasestheneedforhigh-resolutionchromatography.Inbothinstances,
eitherMDCC-MSorCC-MS-MS,theanalyticaldimensionsbecomeat leastthree.
Theuseofmultidimensionalapproaches,atboththecapillarychromatographyandmassspectrometrylevels,generatesfourorevenmore analyticaldimensions(e.g.,LC-GC GC-MS-MS).Forsuchpowerful instrumentation,oneofthemaindifficultiesisrelatedtotheselectionofa sufficientlycomplexsample!
AttheendofanyCC-MSprocess,whatevernumberofdimensions,a greatdealofinformationisgenerated,withdifferentdegreesofusefulness. Furthermore,theusefulinformationmustbe,inmanyinstances,isolated fromtheuselessinformation.Sucheventscallforthedevelopmentof suitabledatamanagementandinterpretationstrategies.
Thetopicscontainedinthepresentbookareallconfinedwithinthe conceptsreportedintheaboveparagraphs:
Chapter1 isdedicatedtoconventionalandhigh-speedGC-MS,andis dividedintothreesub-chapters:insub-chapter1.1,thebasicprinciplesof GCanditshyphenationtomassspectrometryaredescribed,alsoinhistoricalterms.Focusisdevotedtoboththemainionizationapproachesand massanalyzersutilized.State-of-the-artapplicationsfollowinsub-chapter 1.2,withadivisonmadeonthebasisoftheMSanalyzerused(lowresolution,highresolution,multipleanalyzers).Awidevarietyofapplications aredescribed:foodaroma,foodcontamination(pesticides,dioxins, flame retardants,etc.),petrochemicals,environmentalcontamination,andhuman fluidmetabolomics.GreaterfocusisdevotedtomorerecentformsofMS, ratherthantomoreclassicalinstrumentation.Fourofthemostpopular approachestoreduceGCseparationtimesarereportedinsub-chapter1.3: capillarycolumnswithareducedinternaldiameterand filmthickness (micro-borecolumns),conditionsofvacuumatthecolumnoutlet(lowpressureGC),veryrapid(resistive)columnheating,and(mostlyshort) narrow-borecapillarycolumns.Basicprinciplesandtheory,aswellas practicalaspects,inrelationtoeachhigh-speedtechnologyaredescribed, alongwithadvantagesanddrawbacks.Aseriesofapplicationsinvolvingthe useofsuchhigh-speedapproaches,withvariousformsofmassspectrometry,aredescribedanddiscussed.
Chapter2 isfocusedon2DGC-basedcapillarychromatography combinedwithmassspectrometry,inasequenceofthreesub-chapters:in sub-chapter2.1,thebasicprinciplesofGC-GCanditshyphenationtomass spectrometryaredescribed.Anoverviewisgivenoftheinstrumental evolutionfromtheoriginalDeansswitchsystem,tostate-of-the-art
Preface xiii
capillary flowtechnology-basedsystems.Thepotentialandadvantagesof heart-cutting2DGC-MSareillustratedwithseveralexamples,including fragrance,foodaroma,petrochemical,andenvironmentalanalysis,andthe determinationofgenotoxicimpuritiesinpharmaceuticals.Insub-chapter 2.2,the firstpartcontainsinformationrelatedtothehistoryanddevelopmentofGC GC(bothwithandwithoutMShyphenation),alongwith generalprinciples,practicalissues(i.e.,modulationprocesses,method optimization,etc.),mainformsofdetection(non-MSdevices),andsome basictheory.Thesecondpartofthesub-chapterisbasedonstate-of-the-art applicationsinvolvingvariousformsofmassspectrometry,frommore popular(lowresolutionToF,singlequadrupole)tolesscommon(high resolutionToF,triplequadrupole)MSinstrumentation.Insub-chapter2.3, themostpopularinterfacesfortheheart-cuttingandcomprehensiveMD combinationofLCandGCarediscussedindetail(LC-GCandLC GC). Furthermore,anoverviewoftheapplication fieldswheresuchapproaches havebeenlargelyappliedisreported,withparticularattentiontothe couplingwithmassspectrometry,eventhoughMShasnotbeenaswidely usedaswithotherGC-basedtechniques.
Chapter3 reportsinformationonone-andtwo-dimensionalcapillary LC,combinedwithMS,infoursub-chapters:sub-chapter3.1,inparticular,isfocusedonthehistoricaldevelopmentandprinciplesofLC-MS ionization,whilesub-chapter3.2providestheoreticalandpracticalguidanceontheanalyticalcapabilitiesofmostcommontypesofMSanalyzers. State-of-the-artcapillaryapplicationsaredescribedanddiscussedinsubchapter3.3,withparticularattentiondevotedtothebioanalyticalresearch field,alongwithinformationrelatedtodrugsofabuse,doping,pharmaceutics,environmentalchemistry,andfoods.Generalprinciplesandtheoretical/practicalaspectsofbothheart-cuttingandcomprehensive2Dliquid chromatographycoupledtomassspectrometrymethodsaredescribedin sub-chapter3.4.Significantapplicationsinthe fieldofdownscaledminiaturizedsystems, viz.,capillaryliquidchromatography,arealsoreportedand aremainlyfocusedonthedevelopmentofinnovativestrategiesinproteomicsstudies.
ThemainfeaturesofCE-MS,alongwithitsadvantagesoverCE techniquesemployingconventionaldetectors,suchasultravioletanddiode arraydetectors,arereportedin Chapter4.Specialattentionisdevotedto thelatestdevelopmentsrelatedtoinstrumentationandmethodologies.The interfacescurrentlyused,i.e., sheath-liquid,sheathless,andliquid-junction, arealsoillustrated.Finally,aseriesofselectedapplicationsinthe fieldsof
proteomics,pharmaceutical,environmental,food,andforensicanalysisare presentedanddiscussed.
Thechallengesofdatamanagementandinterpretationforpurveyorsof capillarychromatographyinstrumentationwithmassspectrometricdetectionarereportedin Chapter5.Techniquestosucceedaredetailedwithin thecontextofstandard,analyticaltargeted,nontargeted,andhybridtargeted/nontargetedwork flows.Theprocessesaredividedintothecommon elementsofpreprocessing,comparativeanalysis,deconvolution(i.e., mathematicalresolution),resultanalysis,andstatisticalpresentation.Timehonoredtechniquesarediscussedthroughoutwithafocusontheadded complicationscreatedbythedatasizeanddensityinmodernexperiments wherethesamplesareanalyzedbychromatographicinstrumentswithmass spectrometricdetection.Multidimensionalandmultivariateoptionsare includedthroughoutandthefocusconstantlyreturnstoharvestinguseful informationfromthewindfallofdatacreatedbythesetechnologies.
Wewouldliketoconcludethisintroductiontothebookbythanking theAuthorsfortheirvaluablesupportandpatience,andtheReviewersfor thenever-easytaskofevaluation.
PeterQ.Tranchida DepartmentofChemical,Biological,Pharmaceuticaland EnvironmentalSciences,UniversityofMessina,Messina,Italy
LuigiMondello
DepartmentofChemical,Biological,Pharmaceuticaland EnvironmentalSciences,UniversityofMessina,Messina,Italy
Chromaleonts.r.l.,c/oDepartmentofChemical,Biological, PharmaceuticalandEnvironmentalSciences,UniversityofMessina, Messina,Italy
UnitofFoodScienceandNutrition,DepartmentofMedicine, UniversityCampusBio-MedicoofRome,Rome,Italy
BeSeps.r.l.,c/oDepartmentofChemical,Biological,Pharmaceuticaland EnvironmentalSciences,UniversityofMessina,Messina,Italy
Generalprinciplesandhistory
MartaP.B.Mourão1,ArendH.J.Kolk1,Hans-GerdJanssen1, 2 1UniversityofAmsterdam,Van ‘tHoffInstituteforMolecularSciences,AnalyticalChemistry-Group, Amsterdam,theNetherlands; 2UnileverResearchandDevelopment,Vlaardingen,theNetherlands
1.1.1Introduction
Detailedinformationonthecompositionofmixturesiscrucialinmany areasofmodernlifeincludingindustry,lifesciences,healthcare,safety, trade,etc.Foranalyticallaboratoriesansweringquestionson what isina mixtureand howmuch ofeachconstituentispresent,isthecoreactivity. Chromatographyisoneofthekeytechniquesappliedtoanswersuch questions.
Chromatographyisaverypowerfulseparationmethodthatcanseparate highlycomplexmixturesintoitsconstituents,butunambiguousinformationontheidentityofthecompoundsisdifficulttoobtainjustfromthe chromatograms.Coupledwithsensitiveandquantitativedetectors,chromatographicseparationscanprovideinformationonhowmuchofaspecies ispresent.Foridentification,spectroscopicdetectorsareusedtoobtain structuralinformation.Severalspectroscopicdetectorshavebeenappliedin thepast,includinginfrared(IR)spectroscopy,ultraviolet/visible(UV/vis) spectroscopy,vacuumultraviolet(VUV),andnuclearmagneticresonance (NMR)spectroscopy.Massspectrometry(MS)usesmolecularfragmentationtoprovideinformationontheidentityofmolecules.Inrecentyears, thecombinationofchromatographywithMShasbecomethekeymethod for(organic)compositionalanalysis.Thisholdsgoodforbothformsof chromatography,liquidchromatography(LC)andgaschromatography (GC).Anin-depthdiscussionontheadvantagesanddisadvantagesofLC andGC,aswellasthediscussionofstrategiesfortheselectionofthemost appropriatechromatographicseparationmodeareoutofscopeofthe presentchapter.WebelievethatGCshouldbethe firstmethodtoconsider, butothersmightdisagreewiththat.Anyway,combiningGCwithMSis mucheasierthanLC-MScouplings,andspectralinterpretationismuch easierinGC-MSthaninLC-MS.Forthisreasonwewillhereexclusively focusonGC-MScouplings.
HyphenationsofCapillaryChromatographywithMassSpectrometry
ISBN978-0-12-809638-3
https://doi.org/10.1016/B978-0-12-809638-3.00001-6
Combinationsofgaschromatographyandmassspectrometryare nowadaysmoreorlessexclusivelyperformedasdirect,on-linecombinations.ThisisreflectedinthehyphenbetweenGCandMS,resultinginthe abbreviationGC-MS,asadirectlycoupledorhyphenatedmethod.InGCMSthecarriergas flowsfreelyfromtheexitoftheGCcolumnintothe ionizationregionoftheMSinstrument,withnocompoundlosses,no changesinthemoleculesorinthecompositionofthesample,noother compoundsbeingadded(exceptmaybeforthetuningstandard),andno analystinvolvement.Althoughoriginallytheword “hyphenated” only meant “directlycoupled,” inrecentyearsitsmeaninghasbroadened dramatically.Hyphenationhasbecomesynonymfor “complementarity,” increasedcertainty,bettereaseofuse,improvedspeed,sensitivity,etc. IndeedwithproperlyinterfacedGCandMSthesebetterperformance characteristicscanbeachieved,butonlyifthetwotechniquesaremutually matchedratherthanmutuallycompromised.Theimportanceofproper mutualmatchingoftheGCandMSperformancesshouldnotbeunderestimated.If,forexample,theMSforsensitivityreasonsrequiresasample amountthatwouldoverloadtheGCcolumn,thiswouldnotbeahappy marriage.OriftheGCstationaryphasehasexcessivebleedingthatrapidly foulstheMSionsource,thiswillalsonullifythebenefitsofhyphenated systems.OrimaginethataGCcolumnthatelutesthreenicelyseparated peaksin1secondiscombinedwithanMSthatrequires1secondtorecord aspectrum.Inthiscouplingthereisaclearmismatch.TheMSspectrum recordedatthetimethatthecompoundselutewouldcontainmixedinformationonthreeanalytesandwillbeuseless.Clearlythebestofboth worldsisonlyobtainedifthehyphenationtrulycombinesthestrengthsof bothtechniquesandthepresenceofonetechniquedoesnotdeterioratethe performanceoftheotherinanunacceptablemanner.Importantareas whereamatchisneededincludetherelativespeedofthetwomethods, theirmolecularcoverageintermsofsizeandpolarityofthemolecules,their sensitivitiesandworkingranges,carriergascompatibilities,etc.Understandingtheseprinciplesiscrucialforsuccessfuluseofthehyphenated methodGC-MS.
Gaschromatography-massspectrometryisattheheartofmany importantanalyticalmethods.Focusingonthisstepoftheanalytical methodalone,however,neglectsthefactthatcompositionalanalysisof complexsamplesisatrulyintegratedchainofsamplepreparation,separation,detection,anddatainterpretation.Thisanalyticalchainisonlyas strongasitsweakestlink.Thiscanbeexemplified,forexample,fromthe
perspectiveofdataanalysis.Formaximumreliabilityofautomateddata interpretationstrategies,threefactorsaregenerallycrucial:stableretention times,purespectra,andthepresenceofbaselinearoundthepeaks.These arebestobtainedifthesamplesarecleanandthepeaksarewellseparated andideallyabitover-resolved.Samplepreparationisindispensableto achievethis.Itisthe firststepintheanalyticalprotocoland,wheremany peoplethoughtsamplepreparationwouldbecomelessimportantduetothe ever-increasingperformanceofGC-MS,theoppositeisprobablytrue. Samplepreparationbecomesevermoreimportanttoallowtoobtainthe muchimprovedperformanceofGC-MS.ThemoresensitivetheGC-MS, themorevulnerableitbecomestothematrix.Repeatedinjectionsof contaminatedsampleswillrapidlydeterioratesensitivity,causinghigh backgroundnoisethatobscurescompoundspresentatlowlevels, complicatingMSdatabasesearchingandadverselyaffectingtheperformanceofautomateddataevaluationprotocols.
Understandinghowsamplepreparation,GCseparation,andMS detectionaffectandcomplementeachotheriscrucialforallapplicationsof GC-MS.Formaximumperformancecarefuloptimizationofeverystepin theanalyticalprotocolisessential,bearinginmindtheperformancecharacteristicsoftheothersteps.Itcanbearguedthattherequirementsonthe GCseparationcanbeabitrelievedwhenMSinstrumentsthatprovidehigh (mass)resolutionareused.Indeedeverystepoftheanalyticalprotocol contributestoselectivity(i.e.,theabilitytodistinguishtheindividual compoundsinacomplexmixturefromeachotherandfromthebackground).Soitisfairtosaythatalltheresolutionprovidedbythedetector doesnothavetobedeliveredbysamplepreparationorseparation.Could onebecomelazybecausethedetectornowdoesallthedirtywork? Generallynot,sinceimpropersamplecleanupwillcontaminatethe expensivedetectorrapidly.Moreover,manylegislations, e.g.,toxicology andsafety,arebasedon “aslowastechnicallypossible,” whichwillrequire deploymentofthelatest,optimizedmethodsinallstagesoftheanalytical protocol.Gaschromatography-massspectrometryexpertsshouldhencealso beexpertsinsamplepreparation,sampleintroduction,andseparation.Users ofhyphenatedGC-MSmustunderstandthepossibilitiesandlimitationsof allstepsoftheanalyticalprotocol.TheymustbeawareofrecentdevelopmentsinGC,MS,andinthecouplingofthetwomethodsandinall pre-GCoperationsandpost-MSsteps.HyphenatedGC-MSisapowerful tool,buttheoperatorneedstobearmedwithlotsofknowledgeand experiencetoreallyexploitthemethodtoitsfullpotential.Thisshouldbe
donebearinginmindtheholisticcombination,andnottheisolation,ofthe performancetobeprovidedbytheGCseparation,bythesampleinjection andthesamplepreparation.
Toemphasizeoncemore,samplepreparation,separationanddetection shouldnotbeseenasseparatestepsthatcanbeoptimizedinisolation. Optimizationofonestephastobeperformedbearinginmind,and exploiting,thepropertiesoftheothertwo.Optimizedseparations,for example,canrelievetherequirementsimposedontothedetectionstepand canproduceamoreefficientutilizationoflaboratoryequipment,higher samplethroughput,betteraccuracyandrepeatability,longercolumnlifetimesandcanreducedetectormaintenance.Foraproperunderstandingof hyphenatedGC-MSandsystemdevelopments,therequirementsand performancespecificationsofbothtechniquesneedtobeconsideredand properlybalanced.Wewilldefinethevariousparametersthatarerelevant forthecouplingofGCandMSandthesuccessfuluseofGC-MSin Sections1.1.2and1.1.3,respectively.
1.1.2GCfundamentalsintheperspectiveofhyphenation andspeedofanalysis
Inthelastdecades,GChasseenanumberofveryrelevantdevelopments. Theimplicationsofthesedevelopmentshaverecentlybeenaddressedbya panelofGC(-MS)expertsinaroundtablediscussion [1].Thedevelopmentsarenumerous.Largevolumeinjection(LVI)methodshave improvedthesensitivityuptohundredsoftimes;solid-phasemicroextraction(SPME)asasamplepreparationtechniquehassimplifiedsample preparation;QuEChERS(quick,easy,cheap,effective,ruggedandsafe) didthesameforcomplexextracts;comprehensivetwo-dimensionalGC resultsin10timesmorepeakcapacity;newstationaryphasesallow obtaininguniqueselectivities;novelMSmethodsimprovetheaccuracy, reliabilityandspeed,etc.Andthesedevelopmentscontinue.ForUV (VUV)detection,forexample,itprovidesawholenewdetectionselectivityingaschromatography.Whenlookingtothesedevelopmentsitis clearthatmanyofthemrefertocouplingsofsamplepreparationand separation,orseparationanddetection,i.e.,tohyphenation.Animportant driverforhyphenationisautomation,sinceautomationeliminatesthe humanfactorandassociatederrors,leadingtomorereliableanalyticalresults.Retentiontimesareimportantforidentification,buttheycannever providepositiveidentificationwhendifferentcompoundseluteatthesame
retentiontime.Foratruepositiveidentification,hyphenatedsystemsusing massspectrometricidentificationanddetectionareclearlyverypowerful. Ofcoursemassspectraarenotalwaysfullyunique:isomerscanhavevery similarifnotidenticalspectra,butthe combination ofspectrumandretention timeshouldbeusedasitcomesveryclosetobeingunique.Ignoringthe retentiontimeinGC-MSismakinguseofonlypartoftheinformation. Thisretentioninformationcannowbeusedconvenientlyandinafully automatedmannerwiththeaidofmodernretentiontimeorretention indexMSdatabases [2].
ForaproperunderstandingofGC-MSanddevelopmentsofnewhyphenatedsystems,therequirementsandperformance/specificationsofboth techniquesneedtobeconsideredandwellbalanced.Forinstance, combiningamediocreGCseparationwithanexcellentMSdimensionwill notdeliveragood finalperformance.Herewewilldefinethevarious parametersthatarerelevantforthetwodimensionsandthatneedtobe properlymatched,suchastheMSspectralacquisitionspeedversuschromatographicpeakwidth,MSsensitivityversusoptimumcolumnloading, temperatures,massrangeversuselutionwindow,stabilityversusanalysis time,etc.Tofurthercomplicatethetaskoftheanalyst,alsosample introductionmethods(discussedin Section1.1.2.2)andsamplepreparation characteristics(in Section1.1.4)havetobeconsidered.Additionally,the typeofcolumnsisalsoimportant,forexample,thin film,short,narrow columnsaremuchlesstoleranttodirtthanwiderborecolumns,ornonpolarpolydimethylsiloxanecolumnsdeterioratefasterwithwaterthan polarphasesasinCarbowax-coatedcolumns.
Thecolumnistheheartofthesystem.Itishencelogicaltoputthe columninacentralpositionduringmethoddevelopment.Theaimofthe columnistoprovidesufficientresolutiontoseparatethecompoundsof interestfromeachother,iftheycannotbedistinguishedbasedonmass spectralinformation,andfrominterferingmatrixspecies.Belowsomebasic conceptsofchromatographyaresummarizedin Table1.1.1.Atthispoint weonlydiscusstheverystronginter-relationshipbetweentheparametersin thetablenexttothepossibilitiesandproblemscausedbytheuseofMS hyphenation.
TheultimategoalofaGC-MSanalysisistoobtaininformationon identityandamountofthecompoundspresentinasample.Forthisresolutionisneeded.Inchromatography,resolutionreferstoseparation:peaks eluteatdifferentretentiontimesandtheyarenarrowenoughtoallowthe determinationoftheirpeakarea.InGC-MSresolutioncanberedefinedas
Table1.1.1 Basicchromatographicconceptsandtheirimportanceinhyphenated systems.
ConceptsDefinitionRemarksregardinghyphenation
Resolution (Rs)
Selectivity (a)
Theseparationoftwo componentsina chromatogram.See Fig.1.1.1
Representstherelative interactionofthesolutes (relativeretentiontimes) withthestationaryphase given.
Peakwidth (Wi)
Peakwidthatbaseline level(Wb),athalf height(Wh),orasthe standarddeviation(s). See Fig.1.1.2.
EfficiencyNumberofplatesor platespermeter.The theoryofband broadeningwas first describedbyGolay [3]
Fullyautomateddeconvolution methodsusingMSspectracan distinguishtwopoorlyseparated compounds.
When a ¼ 1thereisnodifference inaffi nityofthetwoanalytesfor thestationaryphaseandhenceno chromatographicseparation. Throughdifferencesinspectrathey canstillbedistinguished.
Highlyrelevantincoupledsystems asitdeterminesthetimeavailable forspectrumacquisitionbytheMS.
1) Thick filmcolumnshavea lowerefficiencyduetoband broadeninginthestationary phase.Theyshouldonlybeused toretainhighlyvolatilespecies ortoincreasesampleloadability withaninsensitivedetector.
2) Columnlength:thedetector responsesmaybeinsufficientto transducetheaccurateshapesof narrowpeaks.
3) Carriergas:hydrogenisnot oftenusedforsafetyconcerns andcomplicationswhenusing MSdetection.Heliumismost commonandisfasterthan nitrogen.
4) Flowrates:higher flowratesare bettertoavoidbandbroadening, butmightcausepoorvacuumin theMSinterface.
System performance Informationprovided bythechromatogram and/ortheMS backgroundsignal.Key indicators:retention time,sizeandshapeof chromatographicpeaks.
Lossesandincompletedetectionof compoundsduetoadsorption, surface-catalyzedthermal degradation,leaks,andcoldspots.
Table1.1.1 Basicchromatographicconceptsandtheirimportanceinhyphenated systems. cont'd
ConceptsDefinitionRemarksregardinghyphenation
PeakshapeTheideal chromatographicpeakis Gaussian.
Peakasymmetry(Fig.1.1.3)usually causedbyinstrumentalfactors:dead volumesininterfaces,dirt,cold spots,adsorptivesites,lossofthe deactivationlayerinthecolumnor inthetransferlines.
“theabilitytodistinguishcompounds.” Thiscaneitherbefromtheirdifferencesinretentiontimes,orfromdifferencesinMSspectra.Compounds arechromatographicallyseparatediftheyhavedifferentretentiontimes. Thisdifferenceinretentiontimeistheconsequenceofthe selectivity ofthe stationaryphasethatretainsoneanalytemorethantheother.FromtheMS perspectiveselectivitywouldrefertodifferencesinMSspectra.Ifspectraof twoco-elutingcompoundsaredifferent,MScanprovideselectivityaswell. Afurtherparameterthatisimportantisthe efficiency ofthechromatographic system.Basicallythisisameasureforthezonebroadeningthatoccurs duringchromatography.Narrowpeaksareadvantageousintermsofseparationquality,buthavethedisadvantagethatcouplingtoMSbecomes moredifficult.Highermassspectralacquisitionratesareneededanddead
Figure1.1.1 TwonearlyresolvedpeaksA,Bandtheparameterstocalculatethe resolution.Thesymbol “d” representsthedifferenceinretentiontimesofthecompoundsAandB.(Wb)A and(Wb)B arethepeakwidthsatbaselinelevelofeach compound.
Figure1.1.2 IdealGaussianpeak.Thepeakwidthatbaselinelevelis Wb ¼ 4s.Peak widthathalfofthepeakheightis Wh ¼ 2.354s.
volumesbecomemorecritical.Thewidthofachromatographicpeakalso determineshowmanypeaks fitinacertaintime,orintheentirechromatographicrun.Theterm peakcapacity isoftenusedtodescribehowmany peakscanbeseparatedwithagivensystem.Peakcapacityisalsousedto estimatethelikelihoodofbeingabletoseparateallcompoundspresentina
Figure1.1.3 Thetailingfactorisdefinedasb/a(incaseoftailing)ora/b(incaseof fronting).
givensample.TheadditionofMSdetectionstrictlyspeakingdoesnot increasethepeakcapacity,butitcansignificantlyincreasethenumberof compoundsthatcanbedistinguishedinonerun.Fromthisshortsectionit isclearthatthetheoreticalconceptsareveryimportant.Forfurtherreading, extensivereviewsareavailableinliterature [3 5].
1.1.2.1Columntechnologyovertheyears
Nowadays,evenwiththeintroductionofselectivemethodsforsample preparationanddetection,columnquality,inallitsfacets,isstillextremely important.Columnsshouldbehighlyinertandefficienttogenerate symmetricalpeaksfortheminuteamountsofmaterialthatsensitivedetectorscandetect.Additionally,thestationaryphasesshouldbeselectivefor thesolutestobeseparated,stableoverawidetemperaturerange,andhavea lowvaporpressureathightemperatures.Finally,forseparationsnear ambienttemperature,thephasemuststillbealiquidandnotsolidor crystallinetoavoidexcessivestationaryphasebandbroadening.
Despitelotsofresearchintoothercolumnformats,open-tubular,fusedsilicacapillarycolumnsarestillbyfarthemostwidelyusedcolumnsingas chromatography.Packedcolumnshavelargelybeenreplacedbycapillary columns,exceptinpermanentgasanalysisandindustrialanalyzerswhere maximumruggednessiscrucial.Wall-coatedopen-tubularcolumnsarethe mostwidelyusedcapillarycolumns,butporouslayer,open-tubular (PLOT)columnsarefrequentlyusedaswell.Beforewediscussdevelopmentsinthepreparationofthesecolumnsinmoredetail,itis interestingtodiscussothercolumntypesandformatsthathavenot(yet) beenwidelyadoptedinpractice. Table1.1.2 summarizesthecharacteristics andlimitationsofthecolumntypesdescribedinthissection.
Theetchingofcapillarycolumnsinsilicawaferswas firstevaluatedby Terryin1979 [6].Initially,homogeneouschannelswithasquarecrosssectioncouldbeetchedina5cmdiameterwaferwithacolumnlength of1.5m [7].Inthefollowingdecades,numerousattemptswithnovel etchingtechnologiesandmicromachiningprocessesweremade;butinall fairness,theefficiencyandstabilityofetchedcolumnsarestillnotasgoodas thoseoffused-silica,open-tubularcolumns.Inthelateninetiesofthe previouscentury,multicapillarycolumnswerepopularforashorttime. Thesecolumnsconsistedofover900capillarieswithaninnerdiameterof 40 mmandprovidedaveryhighspeed,withouttheassociateddisadvantages ofnarrowcolumns [8].However,duetodifficultiesinhandlingthe
Table1.1.2 Characteristicsandlimitationsofthedifferentcolumntypesincapillary GC.
ColumntypeAdvantagesDisadvantagesReferences
Wafer-etchedSmallvolumeLowstabilityand insuf ficienteffi ciency [6,7]
Multi-capillary fusedsilica
HighspeedFragile,shortlength, differencesbetween thecapillaries [8,9]
MonolithicUnique selectivity,high speed,stability, loadability
Carbon nanotubes (CNTs)
Fusedsilica, open-tubular (siloxane-based phases)
HT-GC (>350 C)
Room temperature ionicliquid (RTIL)
Thermal stability,strong retention
Thermal stability, effi ciency, selectivity
Analysisofhigh molecularweight compounds
Thermal stability, selectivity,low vaporpressure
Loweffi ciency [10,11,14,15]
Mechanicalshocks, conductingmaterials [17,18]
Columnbleeding [20 24]
Leaks,MS contamination, limitedrangeof columnselectivities [25 28]
Longtermstability yettobe demonstrated [29-32,37]
extremelyfragilemulticapillarycolumns,andthedetrimentaleffectoftiny length,diameter,and film-thicknessdifferencesbetweentheindividual capillaries,ontheefficiency [9],thecolumnwastakenfromthemarketa fewyearslater.
Anotherinnovativecolumnformat,introducedmorerecently,isthe monolithiccolumn [10,11].Monolithsareporoussinteredbedsofpolymersorsilicathathaveaspongelikestructure [12].Monoliths,accordingto theory,shouldprovidehighplatenumbersandahighspeedbecausethe characteristicdiffusiondistancesareverysmall.Moreover,thedimensions thatdeterminespeedandefficiency, i.e.,thedimensionsofthethroughchannelsarenotlinkedtothecolumndiameter,whileloadabilityisnot anissueasitwouldbeinnarrow-borecolumns.Thecombinationofhigh speedandgoodloadabilityofmonolithshasbeenusedbyPeroni etal. to
solveproblemswithsampleloadingintheseconddimensionincomprehensivetwo-dimensionalGC [13].TheperformanceofmonolithsinonedimensionalGChasbeenreviewedbyKurganovandSvec etal. [14,15].So far,theefficiencyandspeedofmonolithiccolumnsarepoorerthanexpected,butthephasesprovideauniqueselectivity.
Carbonmaterialsareveryinterestingandvaluablefornanotechnology, electronics,andoptics,butalsoforuseasadsorbents.Inchromatography, thesematerialshavebeeninuseasGCstationaryphases,inparticularfor theanalysisofhighlyvolatilecompounds,sincetheearlydaysofGC. Inspiredbyadvancesinnanotechnology,newcarbonnanoparticle-based materials, i.e.,carbonnanotubes(CNT),anotherformofcarbonwitha characteristiccylindricalnanostructure,weredeveloped.Dependingonthe methodofpreparation,CNTscanexistinvariousopen-orclose-ended structuresandinteractionofsamplemoleculeswiththenanotubearchitecturescanoccurontheoutsidesurface,onthecurvedgrapheneplanes betweenparticles,intheintersticesbetweentubesandinsidethetubes whentheseareopen-ended.Additionally,sievingeffectscanoccurdueto theinterstitialspaceswherelargermoleculescanbeexcluded [16].The combinationofthesevariousretentionmechanismswiththeirexcellent thermalstabilitypropertiesmakescarbonnanotubesaninterestingmaterial foruseasaGCstationaryphase.Additionally,thepolarityofthematerialis verylowandthecapacityfactorsandtheplatenumbersobtainedinrecent studiesarecomparabletothoseobservedonclassicalcarbonpackedGC columns.Thestrongretentioncharacteristicsofthecolumnsallowedthe separationofhighlyvolatilespecies,likemethane,ethane,andacetylene,at aboveambienttemperature [17],whiletheirhighthermalstabilitypermits separationofhighermolecularweightspeciesathighertemperatures [18]. Foruseinhyphenatedsystemstheextraordinarymechanicalstrengthof carbonnanotubematerialsisanadvantage,butmechanicalshockscausedby switchingvalvesorpressurepulsesmightresultinparticleloss.Therefore, morestudiesarenecessaryforuseofsuchmaterialswithmassspectrometric detectors,wherethehighlyconductingcarbonmaterialcouldeasily shortcircuitthehighvoltagesintheionizationsource.Potentially,CNTs couldalsobeusedtostabilizeormodifyotherstationaryphasessimilarto theuseofCNTstostabilizepolymersinautomotivepartsorboathulls [19].
Fusedsilica,open-tubularcolumnscoatedwith filmsofhighviscosity liquidsarebyfarthedominantcolumntypeinGCandGC-MStodate. Selectionofthestationaryphaseshasbecomesomewhateasierbecauseof theveryhighefficiencyofmodernGCcolumns:alowerselectivitycan,to
someextent,becompensatedbyahigherefficiency.Moreover,modern samplepreparationmethodsandmostimportantlyadvancedMSdetection strategiesprovideagreatdealofadditionalselectivity.
StilltodaymostoftheliquidstationaryphasesemployedinGCare polysiloxane-basedpolymers,agroupofpolymerswithabackboneof alternatingsiliconandoxygenatomswhereeachsiliconhastwosubstituent groupsthatdeterminethepolarityofthephase.If,forexample,allsubstituentsaremethylgroups,ahighlynon-polardimethylsiliconephaseis obtained,whichisarealworkhorseincapillaryGC.Furthermore,ifthe methylgroupsarereplacedbyothermorepolarsubstituents,polaror polarizablephasescanbeprepared.ResearchintothesepolarandpolarizablephaseswasstartedbyLee etal. intheearlyeightiesoftheprevious centuryandhasresultedinawiderangeofGCstationaryphaseswitha broadrangeofselectivities [20].Particularlyinhyphenatedsystems,thelow bleedingcharacteristicsofthephenyl-ordiphenyl-substitutedphasesand theirpropertiesareattractive [21].Numerouscombinationsofphenyland otherpolargroupshavebeenevaluatedforuseasselectivephasesinGC,in whichthe fluorophenyl(methyl)substitutedphases [22] andthecyanopropylphases [23] arehighlighted.Thelattergrouppresentsthemostpolar phasesavailabletodateandiswidelyusedin(cis/trans)doublebondseparationsrelevantforthepetrochemicalindustryandinparticularalsothe foodindustry,especiallyfortheanalysisoftransfattyacids,presentindairy fatsorpartiallyhardenedvegetableoils [24].
Animportantparameterinthedevelopmentofstationaryphasesis columnbleeding,inparticular,ifhighmolecularweightbleedcompounds couldcontaminatethedetectorsused.Hence,columnbleedisalsoan importantissueincolumnselectionforGC-MS.Lowbleed,highefficiency,andhighinertnessarethekeycriteriaintheselectionofthecolumn.Thechemistryofcolumnbleedingisnotperfectlyunderstood,butit isclearthatthemainmechanismisbasedontheso-called “back-biting” : reactivegroupsattheendofthepolymerchainbendbackandattacktheir ownchain,inthatwayformingshort,cyclicoligomersthateventuallyelute fromthecolumn.Thesolutiontothisproblemisstiffeningthepolymer backbonethroughtheinclusionofbulkysidegroupssuchasphenylor diphenylgroups.Alternatively,thechainitselfcanbemadeless flexibleby includingrigid,bulkygroupsinthebackbone.Examplesofsuchphasesare thesil-arylene-phasesthatcontainphenylinthebackbone [25] orcarboranephases [26].Withthesestationaryphases finalcolumntemperatures ashighas480 Carepossible [27].Onthisnote,couplingMStohigh-
temperatureGC(HT-GC),with finaltemperaturesinexcessof350 C,is certainlynottrivialsincetherearesignificantinstrumentaldifficulties, especiallyinhyphenatedsystems.InHT-GC-MSfragmentationofthe compoundsisstrongerduetothehigherionsourcetemperatures,thereis highernoiseathigherMStemperatures,anddetectorcontaminationdueto bleedingofthestationaryphaseoccursfrequently.Despitethesepractical problems,HT-GC-MShasbeenwidelyappliedfortheanalysisoffossil fuels,triacylglyceridesandotherlipids,miscellaneousmaterials,polymers andoligosaccharidesinfoods [28].AdrawbackofHT-GCisthevery limitedrangeofcolumnselectivitiesathand.Onlynon-polartomediumpolarcolumnsareavailable.
Afteralmosttwodecadesofhardlyanyprogressinthedevelopmentof GCphases,thecommercialintroductionofroomtemperatureionicliquid (RTILorIL)columnscreatedagreatdealofexcitement.TheRTIL technologyprovidesmoreselectivestationaryphasesthatcanbeusedat hightemperatureswithlowbleedlevels [29].Roomtemperatureionic liquids,ormoltensalts,arenon-molecularionicsolventswithlowmelting points,usuallycomposedofanorganiccationcontainingNorP(i.e.,alkyl imidazolium,phosphonium)withinorganicororganicanions.Propertiesof thesematerialsthatmakethemattractiveforuseasstationaryphasesinGC includetheirverylowvaporpressure,highthermalstability,excellentand uniqueselectivitytowardspecificclassesofcompounds,aswellasthegood wettabilityofthematerialsonfusedsilica.Thesephasesarecharacterizedby anexcellentcomplementaritytoconventionalorganicpolarstationary phases.The firstuseofILsasstationaryphases,atthattimeinpacked columnGC,wasreportedalmost60yearsago [30].Arevivaloccurred around2004withaseriesofpublicationsbyArmstrong etal. whereseveral newionicliquidswereevaluatedintermsoftheirelutionordersand comparedtostandardGCphases [31].Animportantadvantageofionic liquidphaseswastheirabilitytoseparatebothpolarandnon-polarmoleculeswithgoodloadabilitiesforbothclassesofanalytes [32].Thisdualnaturebehaviorbroadensthescopeofthephasesandinthatway minimizestheneedforaspecificphasededicatedforonlyonesingle application.SeveralattemptshavebeenmadetounderstandtheGCrelevantpropertiesofILphases,suchasthermalstability [33,34],viscosity, andrapiddiffusion [35,36],fromthenatureoftheanionandthecation.A detaileddiscussionofthesestudiesisbeyondthescopeofthepresentchapter. Reviewsdescribingtheevaluationandsummarizingapplications,inparticularalsoinmultidimensionalsystems,areavailableintheliterature [37].
1.1.2.2Sampleintroductionandlargevolumeinjection Sampleintroductionisaveryimportantstepinthechromatographic process.Whenchoosingthebestinjectiontechnique,severalsampleparametersneedtobeconsidered,especiallyforGC-MSinstruments,suchas concentrationofthesamplecompounds,theirboilingpoint,andthermal stabilities.Inaddition,thischoiceshouldbeincombinationwiththetype ofcolumnused, e.g.,practicaldifficultiesoccurinon-columninjectionon narrowcolumnsversuswiderborecolumns.
Themostcommonlyappliedinjectionmethodssuchassplit,splitless, andon-columninjectionaresummarizedin Table1.1.3.Additionally,the choiceofthecorrectlinerforeachtypeofsampleintroductionisrelevant, sinceitpreventsdirtfromenteringtheMSdetector.Theseinjectionmodes andthepossibilitiestoalleviatesomeoftheirdrawbacksbyusingprogrammedtemperaturevaporizing(PTV)injectionhavebeenextensively reviewedovertheyears [38 46].Insplit-andsplitlessinjectionsamplesare rapidlyevaporatedinahotliner.Thiscausesthetwomaindisadvantagesof themethods:discriminationanddegradation.ThePTVinjectorreduces thethermalstressappliedtothemoleculesandishenceanattractive replacementfor(hot)splitandsplitlessinjection,nexttobeinggooddevices forlargevolumeinjectionaswillbediscussedbelow.
LargevolumesamplingtechniquesholdaspecialpositionamidtheGC injectiontechniques,andcanbeusedifmaximumsensitivityisneeded, whichdespiteenormousimprovementsinthesensitivityofGC-MSstill occursfrequently.Insuchsituations,LVIeliminatestheneedforapreconcentrationstepinthesamplepreparationprocedure.Foron-linesystemswithcoupledsamplepreparationandGCanalysis, e.g.,solid-phase extraction(SPE)-GC,aswellasintwo-dimensionalLC-GCsystems, LVIisakeyenabler [43].Itensurescompatibilitybetweentheliquid flows andvolumesofthe firstSPEorLCstep,andthetolerablevolumesofthe GCsystem.On-linesystemsbringaseriesofadvantagesforseparation sciencesintermsofautomation,sensitivity,robustness,reliability,and reductionofcosts.Allofthesefeatureswillbediscussedinmoredetailin Section1.1.4.1.
TherearethreetypesofLVIinjectiontechniquesthatwillbediscussed inthischapter:loop-typeinterface,on-column,andprogrammedtemperaturevaporization.Clearly,forPTVLVIthereareseveraldifferenttypes ofinjectionmethodsthatusethePTVinjectorasacommoninterface.This
Table1.1.3 Application,advantages/disadvantagesandlinerselectionofsampleinjectiontechniquesforliquidinjectionsincapillary GC-MS.
TechniqueApplicationAdvantagesDisadvantagesLinerselection
HotsplitMajor compounds
Splitratioprevents overloading;nofocusingon columnneeded/freedomin temperatureselection
Hot splitless Trace analysis
Oncolumn Trace analysis
PTVcold splitand splitless Major compounds (split),trace analysis (splitless)
Sampleloss;sample discrimination
Directquantifi cationLimitedchoiceofapplicable solvents;thermal degradation;discrimination
Nosamplediscrimination; nothermaldegradationupon injection
Reducedthermalstress; flexible;largevolume injection;lesssample discrimination
Deactivatedandpackedliners withglasswoolorfusedsilica beads.
Linerswithagooseneckshape,to focusanalytes.Packedand deactivatedlinerswithglasswool andfusedsilicabeadsalsoused.
Experimentallydifficult;risk ofcolumncontamination; limitedchoiceofsolvents; useofretentiongap Notapplicable
Requiresoptimization; samplediscrimination
AdaptedfromW.M.A.Niessen,CurrentPracticeofGasChromatography-MassSpectrometry,CRCPress,2001.
Lowervolumelinersallowfaster heating.Coldinjectionis preferredtoavoidlinerover flow withevaporatedsample.
willbediscussedinmoredetail.Additionally,othernovelLVItechniques willbebrieflyaddressedaswell [43].
Theloop-typeLVIisaverysimpletechniqueoriginallydesignedfor LC-GCcoupling [47].Inthismethod,thesampleisintroducedintoaloop andthentransferredtoaretentiongapbythecarriergas.Forthisreason,its mainapplicationsarelimitedtosemivolatilesandheavyanalytes.Volatile speciesarelostwiththesolvent.Onepopularapplicationofloop-typeLVI istheanalysisofmineraloilsaturatedandaromatichydrocarbons(MOSH andMOAH)infoodsandcosmeticproductsusinganon-lineLC-GC system [48].
Inon-columnLVI,aretentiongapisplacedbeforetheanalyticalcolumn.Sincetheliquidsampleisdirectlyintroducedintotheretentiongapat atemperaturebelowthesolventboilingpoint,discriminationordegradationofcompoundsisabsent [43,49].Theretentiongapusedhastobe abletoaccommodatetheentireliquidsample,henceitshouldbesuf ficientlylong.Inordertosolvethisproblem, i.e.,shortentheretentiongap,a techniquecalledpartiallyconcurrentsolventevaporation(PCSE)was developed [43].Inthisapproach,around90%ofthesolventisevaporated duringtheintroductionstepoftheliquidsampleinthatwayenabling samplevolumesupto800 mL [50].Adisadvantageoftheearlierversionsof on-columnLVIwasthatthesolventpeakwasverybroadandallsolvent vaporhadtobedischarged via thedetector.Manydetectors,inparticular alsothemassspectrometer,donottoleratetheintroductionoflargevolumesofsolvent.Thisdrawbackwaseliminatedbytheincorporationofan earlysolventvaporexit(SVE) [43].Thisadditionalgasexitisinstalled betweentheretentiongapandtheGCcolumn,acceleratingtheevaporationrateandprotectingthedetectorfromexcessiveamountsofsolvent vapors.Insomecases,anextraso-calledretainingprecolumnneedstobe installedbetweentheretentiongapandtheSVEoutletinordertoavoid lossesofvolatilecompounds [51].Nonetheless,acarefuloptimizationof twoimportanton-columnparametersisnecessarytoachieveoptimum conditions.Specifically,theSVEclosuretimeandtheinjectionrateare critical.Thesetwoparametersshouldbeinbalancewitheachother,where theSVEshouldnotbeclosedtoolateandtheinjectionrateshouldbefaster thantheevaporationrateofthesolvent.Adetailedoptimizationwas described [43].Eventhoughon-columnLVIallowsthedirectintroduction ofthesampleintotheGCcolumn,thistechniquehasanumberofdisadvantagesrelatedtotheretentiongap(contamination,frequentreplacementoftheretentiongap,andre-optimizationoftheSVEclosuretime).
ProgrammedtemperaturevaporizingLVIusesa flexibleinjectorthatis similartoaconventionalsplit/splitlessinjectorbuthasaverysophisticated temperaturecontrolfunctiontoallowfast,programmedheatingand coolingofthesetupduringanalysis.Thisinjectorcanbeusedwithawide rangeofsampleintroductionmethods,includingsplitlessinjection,vapor overflow,solvent-splitandPTVon-columninjection [52].Solvent-split hasbecomethemostwidelyusedPTVinjectiontechnique.Itinvolves sampleintroductionatatemperaturebelowthesolventboilingpoint, solventelimination via thesplitexit,rapidheatingoftheinjectorwith splitlesstransferoftheanalytesfromthelinertothecapillarycolumnand residualsolvent/matrixcomponentselimination via thesplitpurge [53]
Oncemore,carefuloptimizationofthePTVparameters(vent flow, temperature,pressure,andtime)isnecessarytoachievereliableand reproducibleresults.SeveralvendorsofPTVinstrumentsoffercalculators foroptimizationoftheinjectionparameters,specificallysoftwarethatis basedonthepioneeringworkofRijksandStaniewski [54].Strategiesfor systematicoptimizationhavebeendescribedbasedon “onefactoratatime” orusingstatisticaldesignofexperiments(DOE)methods [55 59].Solventsplitlargevolumeinjectioncanbeperformedinthreedifferentways,as director “at-once” injection,speedcontrolledsampleintroduction,and multiple “at-once” injection [43].Director “at-once” injectionismore commonandpreferabletoothermethodsduetoitssimplicity.However, thechoiceoflinersandpackingmaterialsisofcrucialimportanceinorder tomaximizethesampleinjectionvolume,sincethelinershavetoretainthe entireliquidsample.Thepackingmaterialshouldmeetanumberof criteria,suchastheabilitytoretainlargevolumesofsolvent,shouldbe sufficientlyinertandthermallystable.Someexamplesareglasswool,quartz wool,glassbeads,andTenaxTA [43].Otherparametersfordirector “atonce ” injectionareequallyimportanttooptimize, i.e.,thesplitlesstransfer conditions,samplevolume,andsolvent-eliminationtime.Incontrast,speed controlledsampleintroductionisslightlymorecomplicatedtooptimize, sinceitinvolvesamorecontrolledandconstantintroductionofthesample intothelinerandhasnorestrictionsintermsofmaximumsamplevolume.
OtherinterestingdevelopmentsinLVIapproachesthatincludedirect sampleintroductionwithmicrovial-insert(DSI/DMI),splitlessoverflow (CRS-LV),atcolumn,andthroughoventransferadsorption-desorption (TOTAD)werefullyreviewed [43,60 65]
