GC-MS, amp, lcms

Gas Chromatography- Mass Spectrometry

1. 01-Jul-20
1
GC-MS and LC-MS
DrHamayun Khan
GC-MS
Gaschromatography–massspectrometry(GC-MS)isananalyticalmethodthatcombines
thefeaturesofgas-chromatographyandmassspectrometrytoseparateandidentify
differentsubstanceswithinatestsample.
OR/
GC-MSisaninstrumentaltechnique,comprisingofagaschromatographcoupledtoamass
spectrometerbywhichcomplexmixturesofchemicalsmaybeseparated,identified&
quantified.
InordertoacompoundtobeanalyzedbyGC-MSitmustbesufficientlyvolatile&
thermallystable.
Principle
ThesamplesolutionisinjectedintotheGCinletwhereitisvaporized&sweptontoa
chromatographiccolumnbythecarriergas(usuallyhelium).Thesampleflowsthroughthe
column&compoundscomprisingthemixtureofinterestareseparatedbyvirtueoftheir
relativeinteractionwiththecoatingofthecolumn(stationeryphase)&thecarriergas
(mobilephase).
Thedifferenceinthechemicalandphysicalpropertiesofdifferentmoleculesinamixture
willseparatethemoleculesasthesampletravelsthelengthofthecolumn.Themolecules
takedifferentamountsoftime(calledtheretentiontime)tocomeoutof(elutefrom)the
gaschromatograph,andthisallowsthemassspectrometerdownstreamtocapture,ionize,
accelerate,deflect,anddetecttheionizedmoleculesseparately.Themassspectrometer
doesthisbybreakingeachmoleculeintoionizedfragmentsanddetectingthesefragments
usingtheirmasstochargeratio.
INSTRUMENTATION
GC-MS comprise following major blocks
a)theGas chromatograph
b)Interface
c)theMass spectrometer
d)A data system is necessary to handle results obtained during a sample run
A) THE GAS CHROMATOGRAPH
Carriergas

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Split/SplitlessGC-MSinlets
Samplesareintroducedtothecolumnviaaninlet.Thisinletistypicallyinjectionthrough
aseptum.Onceintheinlet,theheatedchamberactstovolatilizethesample.Inasplit
system,aconstantflowofcarriergasmovesthroughtheinlet.Aportionofthecarriergas
flowactstotransportthesampleintothecolumn.Anotherportionofthecarriergasflow
getsdirectedtopurgetheinletofanysamplefollowinginjection(septumpurge).Yet
anotherportionoftheflowisdirectedthroughthesplitventinasetratioknownasthe
splitratio.
Inasplitlesssystem,theadvantageisthatalargeramountofsampleisintroducedtothe
column.However,asplitsystemispreferredwhenthedetectorissensitivetotrace
amountsofanalyteandthereisconcernaboutoverloadingthecolumn.
Oven
TheouterpartoftheGCisaveryspecializedoven.Thecolumnisheatedtomovethe
moleculesthroughthecolumn.Typicaloventemperaturesrangefrom40°Cto320°C.
Column
ThegaschromatographinGC-MSutilizesacapillarycolumn(30meterthintube)which
mostwidelyarethoseinwhichthestationaryphasehasbeenchemicallybondedtothe
fusedsilica.
B)INTERFACE
InitialconcernsforGC-MSwerethesignificantdifferencesinpressure-theGCgasexiting
thesystemisaroundoneatmosphere(760torr)whereastheMSoperatesatavacuumof
around10
-5
–10
-6
torr.ThepressureincompatibilityproblembetweenGCandMSwassolved
byinsertinganInterface.TheGC/MSinterfaceisthesectionoftheinstrumentstartingatthe
columnexitinthegaschromatographandextendingtotheentrancetotheionsourceofthe
massspectrometer.Therearemanyinterfaceslikejet,Electrospray,thermospray,direct
electricalionization,movingwireorbeltinterface.
JetInterface
Theoperationofthisseparatorisbasedona
diffusionprinciple.Thesejetseparatorswork
wellatthehighercarriergasflowrates(10to
40mL/min)TheeluatefromtheGCissprayed
throughanozzleatpositionA,andshootstowardanorificeinthewallofanadjoining
chamberonitswaytotheionsource.AcrossthegapbetweenpointsAandB,thereis
atremendousexpansionofthegases.Compoundsthathavehighdiffusivitywilldiffuseat
rightangles(aprocessknownaseffusion)muchmorethanthosehavingalowerdiffusivity.
Thecarriergasisalmostalwaysasmallmoleculewithahighdiffusioncoefficient,
whereastheorganicmoleculeshavemuchlowerdiffusioncoefficients.
C)MASSSPECTROMETER
Ingeneralamassspectrometerconsistsof:
anionsource,
High-vacuumsystem
amass-selectiveanalyzer,and
anioncollector
IonizationTechniques
Electronimpact(El)
Chemicalionization(CI)
MassAnalyzer:
Themostcommontypeofmassanalyzerassociatedwithagaschromatograph(GC)is:
thequadrupolemassanalyzer,othersinclude:
TimeofFlightAnalyzer
IonTrapAnalyzer
D)ComputerSystem
Thedatafromthemassspectrometerissenttoacomputerandplottedonagraphcalled
amassspectrum.
APPLICATIONS
Itisused:
1.Formetaboliteprofiling
2.Intoxicityassessmentortoxicologye.g.Aspecificlesioninliverorkidneycanbe
profiled.
3.Inhumandosimetry.
4.Fordetectionofillegaldrugslikenarcotics-marijuana,cocaine,opioids,oxycodone
andoxymorphone.
5.Insportsantidopinglaboratoriestotestathletesurinesamplesforprohibited
performanceenhancingdrugs.e.g:anabolicsteroids.
6.Indetectionoflipophiliccompoundsindiverseplanttissues
7.Inanalysisofbiologicallyimportantaromaticamines.
8.Forthequantitativeanalysisofacidicphytohormonesandrelatedcompounds

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9.Inidentificationofvolatilecomponentsinsamples
10.Inanalysisofpesticidesinfoodstuffs,forexamplethedeterminationofpyrethroid
residuesinvegetablesamples
11.Fortrackingorganicpollutantsintheenvironment.
12.Incriminalforensicstohelplinkacriminaltoacrime.Accelerantissignificant
evidenceinafireinvestigationbecauseitsuggeststhatthefirewassetintentionally.
LC-MS
LC-MSisahyphenatedtechnique,whichcombinestheseparatingpowerofHigh
PerformanceLiquidChromatography(HPLC),withthedetectionpowerofmass
spectrometry.
Hyphenatedtechnique:
Thetechniquedevelopedfromthecouplingofaseparationtechniqueandanon-line
spectroscopicdetectiontechnologyisknownashyphenatedtechnique.
PrincipleofLC-MS
TypicalLC-MSsystemiscombinationofHPLCwithMSusinginterface(ionization
source).ThesampleisseparatedbyLC,andtheseparatedsamplespeciesaresprayedinto
atmosphericpressureionsource,wheretheyareconvertedintoionsinthegasphase.The
massanalyzeristhenusedtosortionsaccordingtotheirmasstochargeratioanddetector
countstheionsemergingfromthemassanalyzerandmayalsoamplifythesignal
generatedfromeachion.Asaresult,massspectrum(aplotoftheionsignalasafunction
ofthemass-to-chargeratio)iscreated,whichisusedtodeterminethemassesofparticles
andofmolecules,andtoelucidatethechemicalstructuresofmolecules.
INSTRUMENTATION :
1)HPLCConstitutestheLCPart:
a)SolventSystem(mobilePhase)
b)Pumps
c)Mixeranddegassers
d)Injector
e)Column
2)MassSpectrometer
a)IonSources(functionasinterface)
i)Electrosprayionization.
ii)AtmosphericPressureChemicalIonization.
iii)Atmosphericpressurephotoionization
b)MassAnalyzers
i)Quadrupole
ii)Time-of-flight
iii)Iontrap
iv)Fouriertransform-ioncyclotronresonance(FT-ICR)

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InterfacingLCandMS
oItwasdifficulttocoupleliquidchromatographytoamassspectrometerbecauseofthe
necessitytoremovethesolvent.However,itwasmadepossiblewiththeuseofinterface
whichconnectedtheLCwithMS.
oEarlierLC/MSsystemsusedinterfacesthatinvolvedtechniquesforevaporatingsolvent
andsplittingtheflowfromLCcolumnstoadmitelutedcompoundsintothehighvacuum
ofthespectrometer.Theseapproachesweresuccessfulonlyforaverylimitednumberof
compounds.Theintroductionofatmosphericpressureionization(API)techniques
greatlyexpandedthenumberofcompoundsthatcanbesuccessfullyanalyzedby
LC/MS.Interfacesinvolvingsourcesatvacuum(suchasthermospray,particle-beam
andcontinuous-flowfastatombombardment)arestillinuse,butAPIinterfacesareby
farthemostwidelyused,beingthemostsuitableforLC/MScoupling.
oThecommonlyusedinterfacesare:-
Electrosprayionization(ESI)
Atmosphericpressurechemicalionization(APCI)
Atmosphericpressurephotoionization(APPI)
Thermosprayionization(TSI)
1.Electrospray ionization (ESI):
InESI,theLCeluentissprayed(nebulized)
intoachamberatatmosphericpressureinthe
presenceofastrongelectrostaticfieldand
heateddryinggas.Thecapillarythroughwhich
theeluentpasseshasahighvoltagepotential
acrossitssurface,andsmall,chargeddroplets
areexpelledintotheionizationchamber.The
chargeddropletsaresubjectedtoacounterflowofadryinggas(usuallynitrogen)that
evaporatessolventmoleculesfromthedroplets.Thus,thechargedensityofeachdroplet
increasesuntiltheelectrostaticrepulsiveforcesexceedthesurfacetensionofthedroplet
(theRayleighlimit),atwhichpointthedropletsbreakapartintosmallerdroplets.This
processcontinuesuntilsolvent-freesampleionsareleftinthegasphase.Theseionsare
attractedtoandpassthroughacapillarysamplingorificeintothemassanalyzer.
Electrosprayisespeciallyusefulforanalyzinglargebiomoleculessuchasproteins,
peptidesandoligonucleotides,butcanalsoanalyzesmallermoleculeslikebenzo-
diazepines.
2.Atmosphericpressurechemicalionization(APCI):
InAPCI,theLCeluentissprayedthroughaheated
(typically250°C–400°C)vaporizeratatmosphericpressure.
Theheatvaporizestheliquid.Theresultinggas-phase
solventmoleculesareionizedbyelectronsdischargedfrom
acoronaneedle.Thesolventionsthentransferchargetothe
analytemoleculesthroughchemicalreactions(chemical
ionization).Theanalyteionspassthroughacapillary
samplingorificeintothemassanalyzer.
APCIisapplicabletoawiderangeofpolarandnonpolar
molecules.Sinceitinvolveshightemperatures,APCIis
lesswell-suitedthanelectrosprayforanalysisoflargebiomoleculesthatmaybethermally
unstable.APCIisusedwithnormal-phasechromatographymoreoftenthanelectrosprayis
becausetheanalytesareusuallynonpolar.
3.Atmosphericpressurephotoionization
Atmosphericpressurephotoionization(APPI)forLC/MSisarelativelynewtechnique.As
inAPCI,avaporizerconvertstheLCeluenttothegasphase.Adischargelampgenerates
photonsinanarrowrangeofionizationenergies.Therangeofenergiesiscarefullychosen
toionizeasmanyanalytemoleculesaspossiblewhile
minimizingtheionizationofsolventmolecules.Theresulting
ionspassthroughacapillarysamplingorificeintothemass
analyzer.
MassAnalyzers
Althoughintheoryanytypeofmassanalyzercouldbeused
forLC/MS.Thefollowingfourtypesareusedmostofteneach
hasadvantagesanddisadvantagesdependingonthe
requirementsofaparticularanalysis.
Quadrupole
Time-of-flight
Iontrap
Fouriertransform-ioncyclotronresonance(FT-ICRorFT-MS)

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Detectors:
Onceanionisseparated,isolated,orfragmentedbythemassspectrometer,itisnecessary
toconverttheabundanceofthationintoanelectricsignal,whichcanbereadbyadata
station.Mostmodern-dayinstrumentsrelyon:
Electronmultiplierdetectors.
Microchannelplatedetectors(MCP)
ApplicationsofLC-MS
LC-MSismostwidelyusedinfood,pharmaceuticalandchemicalindustriesfor
quantitativeandqualitativeanalysis
Molecularweightdetermination:
Itisusedtodeterminethemoleculeweightofchemicalsubstance,pharmaceutical
substances,proteins,etc.
Structuraldetermination/elucidation:
Usedforstructuraldeterminatione.g.structuraldeterminationofginsenosides
(sapnins).
Pharmaceuticalapplications:
Itisusedtodeterminethepharmacokineticprofileofdrug,drugmetabolites/
degradationproduct,impuritiesandchiralimpurities.Theseparationanddetectionof
chiralimpuritiesinpharmaceuticalsareofgreatimportancebecausetheD-isomerofa
drugcanhavedifferentpharmacological,metabolicandtoxicologicalactivityfromthe
L-isomer.
UsedinBioequivalenceandbio-avaiabilitystudies.
DetectionofdegradationProductsforcertaindrugswhicharedifficulttobedetectedby
othermethods(UV)e.g.Salbutamol.
Clinicalandbiochemicalapplications:
MALDI-TOFMSisusedquantificationofDNA,geneexpressionanalysis,DNAand
RNAsequencing.
Usedfordetectionoftrimipramineandthioridazine.
ApplicationsinFoodsciencesandpoultry:
Usedtoidentifyaflatoxins(toxicmetabolicproductincertainfungi)infoods.
TodeterminevitaminD
3inpoultryfedsupplements,etc.