mass spectroscopy

Mass spectroscopy
Mass spectroscopy instrumentation

1. ANUBHAV GUPTA
B. PHARM 4
TH
YEAR
Institute of Pharmacy
VBS Purvanchal University

2. 1.INTRODUCTION
▪MassSpectrometry(MS)isatechniquethathelpstoidentifytheamountandtypeofchemicals
presentinasamplebymeasuringthemass-to-chargeratioandabundanceofgas-phaseions.
▪Amassspectrometerworksbygeneratingchargedmoleculesormolecularfragmentseitherina
highvacuumorimmediatelypriortothesampleenteringthehigh-vacuumregion.
▪Massspectrumisaplotofrelativeabundanceagainsttheratioofmass/charge(m/e).
▪Thesespectraareusedtodeterminetheelementalorisotopicsignatureofasample,themassesof
particlesandofmolecules,andtoelucidatethechemicalstructuresofmoleculesandotherchemical
compounds.
▪Massspectrometryprovidesahighlyspecificmethodfordeterminingorconfirmingtheidentityor
structureofdrugsandrawmaterialsusedintheirmanufacture.
▪Massspectrometryinconjunctionwitheithergaschromatography(GC–MS)orliquid
chromatography(LC–MS)providesamethodforcharacterisingimpuritiesindrugsandformulation
excipients.
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3. 2.PRINCIPLE
▪Themassspectroscopyworksonprinciplethatwhenchargedmoleculesormolecularfragmentsare
generatedinahigh-vacuumregion,orimmediatelypriortoasampleenteringahigh-vacuum
region,usingavarietyofmethodsforionproduction.
▪Theionsaregeneratedinthegasphasesothattheycanthenbemanipulatedbytheapplicationof
eitherelectricormagneticfieldstoenablethedeterminationoftheirmolecularweights.
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4. 3.Working of Mass Spectrometry
▪Inatypicalprocedure,asample,whichmaybesolid,liquid,orgas,isionized,forexampleby
bombardingitwithelectrons.
▪Thismaycausesomeofthesample’smoleculestobreakintochargedfragments.Theseionsare
thenseparatedaccordingtotheirmass-to-chargeratio,typicallybyacceleratingthemand
subjectingthemtoanelectricormagneticfield:
▪Ionsofthesamemass-to-chargeratiowillundergothesameamountofdeflection.
▪Theionsaredetectedbyamechanismcapableofdetectingchargedparticles,suchasanelectron
multiplier.
▪Resultsaredisplayedasspectraoftherelativeabundanceofdetectedionsasafunctionofthe
mass-to-chargeratio.
▪Theatomsormoleculesinthesamplecanbeidentifiedbycorrelatingknownmasses(e.g.an
entiremolecule)totheidentifiedmassesorthroughacharacteristicfragmentationpattern.
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5. 4.FRAGMENTATION PATTERN
▪Fragmentationisthedissociationofenergeticallyunstablemolecularionsformedfrompassing
themoleculesintheionizationchamberofamassspectrometer.
▪Fragmentationofthemolecularionstakesplaceinfollowingforms:
1.Homolyticcleavage.Thistypeoffragmentationispromotedbythepresenceofaheteroatom
suchasoxygen,nitrogenorsulphur,andinmoleculescontainingaheteroatomitoftengivesrise
tothemostabundantioninthemassspectrum
2.Heterolyticcleavage.Heterolyticcleavageoccurslessoftenasapredominantfragmentation
mechanismindrugmoleculessincetheyusuallycontainalotofheteroatomstodirectthe
cleavage.Insuchcleavage,thepositivechargeiscarriedbythecarbonatomnotbythe
heteroatom.
3.RetroDiels-Aldersreaction.Itoccursincompoundswithringsystems.Itinvolvesthe
cleavageoftwobondsofacyclicsystemwhichresultsintheformationoftwostable
unsaturatedfragmentinwhichtwonewbondsareformed.
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6. 4.1.Fragmentation Pattern
ALOCOHOL(3-Pentanol) ALDEHYDE(3-Phenyl 2-Propenal)
ALKANE(Hexane) AMIDE(Methyl butyramide)
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7. 5.McLaffertyRearrangement
▪TheMcLaffertyrearrangementisacharacteristicfragmentationofthemolecularionofacarbonyl
compoundcontainingatleastonegammahydrogen,e.g.,Migrationof-hydrogenfollowedby
-bondcleavageandeliminationofethyleneorsubstitutedethyleneneutralmolecule.
▪TheMcLaffertyrearrangement,isrelativelyuncommon
indrugmolecules;itismostoftenencounteredinfatty
acidesters.
▪Thisrearrangementcanoccurincarbonylcompounds
e.g.amides,ketonesandacids.
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8. 6.IONIZATION TECHNIQUES
▪Therearemanytypesofionizationtechniquesthatareusedinmassspectrometry.Someofthem
are:
1.ElectronImpactionization(EI)
2.FastAtomBombardment(FAB)
3.Electrosprayionization(ESI)
4.AtmosphericPressureChemicalIonization(APCI)
5.MatrixAssistedLaserDesorptionIonization(MALDI)
▪APCIisconsideredbestthanEIbecauseAPCIformsaprotonatedmoleculeandiscompletely
compatiblewithLiquidChromatography(LC),whileEIismorelikelytofragmenttheionleading
toapossiblemoreambiguousidentificationofthemoleculeweightandisincompatiblewithLC.
▪MALDIalongwithESIallowsforionizationandmeasurementoflargemolecularweight.
▪ESIhasanadvantageinitseasycompatibilitywithLC.WhileMALDIhasadvantagesforimaging
massspectrometry.
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6.1. Electron ImpactIonization(EI)
▪EI is still used in conjunction with sample introduction either via a direct heated probe or via gas
chromatography (GC):
(i)Thesampleisintroducedintotheinstrumentsourcebyheatingitontheendofaprobeuntilit
evaporates,assistedbythehighvacuumwithintheinstrumentorviaacapillaryGCcolumn.
(ii)Onceinthevapourphase,theanalyteisbombardedwiththeelectronsproducedbyarheniumor
tungstenfilament,whichareacceleratedtowardsapositivetargetwithanenergyof70eV.Theanalyteis
introducedbetweenthefilamentandthetarget,andtheelectronscause
ionisationasfollows:Mþe!Mþ:þ2e.
(iii)Sincetheelectronsusedareofmuchhigherenergythanthe
strengthofthebondswithintheanalyte(4–7eV),extensive
fragmentationoftheanalyteusuallyoccurs.
(iv)Themoleculeanditsfragmentsarepushedoutofthesource
byarepellerplatewhichhasthesamechargeastheions
generated.
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6.2. Fast Atom Bombardment(FAB)
▪FABisatechniquethatwaspopularinthe80'stoearly90'sbecauseitwasthefirsttechniquethat
allowedionizationofnon-volatilecompoundsthatcouldbedonesimply.
▪Itwasdonebybombardingasampleinavacuumwithabeamofatoms,typicallyArorXe,
acceleratedtoKilovoltenergies.Thesamplewastypicallymixedinamatrix.
▪Thetwomostcommonmatrixeswereglyceroland3Nitro-benzoicacid.Thematrixallowedthe
sampletorefreshitself.
▪TheionsformedbyFABwereadductstothemolecule,wherethe
adductscouldbeprotons,sodiumions,potassiumionsor
ammoniumions.
▪AvariationofFABwasreplacementoftheatombeamwitha
beamofions,typicallycesiumions,whichwascalledsecondary
ionmassspectrometry(SIMS).SIMSspectraweretypically
identicaltoFABspectraandthetermsbecameinterchangeable.
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6.3. ElectronsprayIonization(ESI)
▪Theelectronsprayiscreatedbyputtingahighvoltageonaflowofliquidatatmosphericpressure,
sometimesthisisassistedbyaconcurrentflowofgas.
▪Thecreatedsprayisdirectedtoanopeninginthevacuumsystemofthemassspectrometer,where
thedropletsarede-solvatedbyacombinationofheat,vacuumandaccelerationintogasbyvoltages.
▪Theionsareejectedindropletsandacceleratedinto
themassanalyzerbyvoltages.Forlargermolecules,
theionsmaycontainmultiplecharges,allowingthe
detectionofverylargemoleculesonanalyzersthat
havelimitedmasstocharge(m/Z))ratioranges.
Becauseofthenaturaluseofaflowingliquid,itis
easilyadaptedtoliquidchromatography(LC).
▪Therearemanyothertechniquesthatarevariationsof
electrospray,forexample,nanospray,picospray
desorptionelectrosprayionization(DESI).
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6.4. Atmospheric Pressure Chemical Ionization (APCI)
▪Atmosphericpressurechemicalionisation(APCI)iscloselyrelatedtoESIandinstrumentscarrying
outESIcanbereadilyswitchedtooperateinAPCImode.
▪InAPCImodetheeluentfromtheHPLCdoesnotpassthroughachargedneedlebeforeentering
themassspectrometersourcebutviaaheatedtubesothatitformsanaerosol.Uponexitingthe
heatedtubeanelectricdischargeispassedthroughtheaerosolgeneratingreactivespeciessuchas
H3OþandN2þ,whichpromotetheionisationofthe
analytes.
▪Thetechniquehasneverachievedthelevelof
popularityofESIsincemostdrugmoleculescanbe
ionisedunderESIconditions;however,APCIcanbe
employedfortheanalysisofdrugmoleculesoflow
polaritythatdonotioniseefficientlyunderESI
conditions.
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6.5.Matrix Assisted Laser Desorption Ionization (MALDI)
▪MALDIusesanitrogenlasertopromoteionisationofmoleculespriortoionseparationinamass
spectrometer.Itisusuallycombinedwithtimeofflight(TOF)separationoftheionsgenerated.
▪InorderforthesampletobeionisedithastobedissolvedinamatrixthatabsorbsUVradiationat
aroundthewavelength(337nm)producedbythelaser.Asimpleexampleofamatrixis
dihydroxybenzoicacidandthereareanumberofsimilararomaticcompoundswhichareusedto
promoteionisationofdifferentclassesofmolecules.
▪Thesamplesolutionismixedwithmatrixsolutiononametalplateandallowedtodrypriortobeing
introducedintotheinstrument.Thelaseristhendirectedatthetargetplatetopromoteionisation.
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7. INSTRUMENTATION
Massspectrometerhasfollowingcomponents:
A. Sample Inlet, through HPLC, GC, Syringe etc.
B.Ionization,canbeachievedby:
1.ElectronImpactionization(EI)
2.FastAtomBombardment(FAB)
3.Electrosprayionization(ESI)
4.AtmosphericPressureChemicalIonization(APCI)
5.MatrixAssistedLaserDesorptionIonization(MALDI)
C. Acceleration&Deflection
D. Analyser,canbe achieved by analysers like :
1.Magnetic sector mass analyser
2.Double focussing analyser
3.Quadrupole mass analyser
4.Time of Flight analyser (TOF)
5.Ion trap analyser
6.Ion cyclotron analyser
E. Detector
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7.1. Sample Inlet
▪Sample stored in large reservoir from which molecules reaches ionization chamber at low
pressure in steady stream by a pinhole called “Molecular leak”.
▪Molecular leak–It is pin-hole restriction (0.01 to 0.05mm diameter) and made up of gold foil. It
is used for metering the sample to ionization chamber.
7.2. Ionization
▪Atoms are ionized by knocking one or more electrons off to give positive ions by bombardment
with a stream of electrons. Most of the positive ions formed will carry charge of +1.
7.3.Acceleration & Deflection
▪Ions are accelerated so that they all have same kinetic energy.
▪Positive ions pass through 3 slits with voltage in decreasing order.
▪Middle slit carries intermediate and finals at zero volts.
▪Ions are deflected by a magnetic field due to difference in their masses.
▪The lighter the mass, more they are deflected.
▪It also depends upon the no. of +ve charge an ion is carrying; the more +ve charge, more it will be
deflected.
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7.4. Analyser
▪A mass analyser is a device that can separate atoms and molecules according to their mass.
▪The five main characteristics for measuring the performance of a mass analyser are
1) The mass range limit or dynamic range
2) The analysis speed [u (m)S-1]
3) The transmission = No. of ion reaching the ions/No. of ions entering mass analyzer
4) The mass accuracy
5) The resolution.
▪Massanalysersusedare:
1.Magnetic sector mass analyser
2.Double focussing analyser
3.Quadrupole mass analyser
4.Time of Flight analyser (TOF)
5.Ion trap analyser
6.Ion cyclotron analyser
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7.4.1. Quadrupole Mass Analyzer
▪Acheaperandmoresensitivemassspectrometerthanamagneticsectorinstrumentisbasedonthe
quadrupoleanalyser.
▪Itusestwoelectricfieldsappliedatrightanglestoeachother,ratherthanamagneticfield,to
separateionsaccordingtotheirm/zratios.OneofthefieldsusedisDCandtheotheroscillatesat
radiofrequency.
Disadvantage
▪Limitedresolution
▪Peakheightsvariableasafunctionofmass(mass
discrimination).
▪Peakheightvs.massresponsemustbe'tuned'.
▪Notwellsuitedforpulsedionizationmethods.
▪Low-energycollision-induceddissociation(CID)
MS/MSspectraintriplequadrupoleandhybrid
massspectrometersdependstronglyonenergy,
collisiongas,pressure,andotherfactors.
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7.4.2. Time of flight (TOF) analyser
▪Thebasisoftheseparationisthatsmallerionsmovemorequicklythanlargerionsandthusreachthe
detectorfirst.
▪Thetechniquereliesongatingthesignalfromtheionsource,sothatonepacketofionsisallowed
timetoreachthedetectorbeforethenextsetisejectedfromtheionsource,sothatthereisno
overlapbetweenionpackets.Theionsleavingtheionsourcehavedifferentkineticenergiesandthis
compromisesthemassresolution,indeedearlyinstrumentsgaveverywidepeaksforasinglemass.
▪Theproblemofmassresolutionwasresolvedby
usingadevicecalledareflectron,whichopposes
thedirectionthattheionsaremovingbysending
thembackintheoppositedirection.
▪Thefastermovingionspenetratedeeperintothe
reflectronandthusalageffectisproducedfor
fastermovingionssothattheyarefocusedwith
theslowermovingionswhichdonotpenetrateas
farintothereflectron.Pushingtheionsbackinthe
oppositedirectionalsoincreasesthelengthofthe
flighttube,increasinginstrumentresolution.
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7.4.3. Magnetic Sector Mass Analyzer
▪In a magnetic sector instrument the ions generated are pushed out of the source by a repeller
potential of same charge as the ion itself (most often positive).
▪They are then accelerated in an electric field of ca 3–8 kV and travel through an electrostatic field
region so that they are forced to fall into a narrow range of kinetic energies prior to entering the
field of a circular magnet.
▪They then adopt a flight path through the magnetic field depending on their charge to mass (m/z)
ratio; the large ions are deflected less by the magnetic field:
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=
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where H is the magnetic field strength, r is the
radius of the circular path in which the ion travels,
and V is the accelerating voltage.
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Comparison of various analyzers
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7.5. Detectors
▪Theioncollectionsystemmeasurestherelativeabundanceofionfragmentsofeachmass.
▪Severaltypesofdetectorsareavailableformassspectrometers.Thedetectorusedformostroutine
experimentsistheelectronmultiplier.
▪Anothertypeofdetectorisphotographicplatescoatedwithasilverbromideemulsion,itissensitive
toenergeticions.Aphotographicplatecangiveahigherresolutionthananelectricaldetector.
Faraday Cup:
▪Itconsistsofahollowconductingelectrode
connectedtogroundviaahighresistance.
▪Theionshittingthecollectorcauseaflowof
electronsfromgroundtotheresistor.
▪Theresultingpotentialdropacrosstheresistor
isamplified.Aplateheldatabout-80Vin
frontofthecollector,preventsanyejected
secondaryelectronsfromescapingand
causingananomalousreading.
Photographic Plates:
▪Thisdetectorsystemismostsensitivethanany
otherdetectorbecausethephotoplate
integratestheionsignaloveraperiodoftime.
▪Thephotoplateareprocessedbytheusual
photographictechniquesandreadwiththeaid
ofdensitometer.
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ElectronMultipliers:
▪Itismostcommonmeansofdetectingions.Itismadeupofaseries(12to24)ofaluminumoxide
dynodesmaintainedateverincreasingpotentials.
▪Ionsstrikethefirstdynodesurfacecausinganemissionofelectrons.Theseelectronsarethen
attractedtothenextdynodeheldatahigherpotentialandthereforemoresecondaryelectronsare
generated.
▪Ultimately,asnumerousdynodesareinvolved,acascadeofelectronsisformedthatreultinan
overallcurrentgainontheorderofonemillionorhigher.
▪Thehighenergydynode(HED)usesanelectrostaticfieldtoincreasethevelocityoftheionsand
servestoincreasesignalintensityandthereforesensitivity.
Faraday Cup Photoplate Electron Multiplier
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8.APPLICATIONS
▪Massspectrometryprovidesahighlyspecificmethodfordeterminingorconfirmingtheidentityor
structureofdrugsandrawmaterialsusedintheirmanufacture.
▪Massspectrometryinconjunctionwitheithergaschromatography(GC–MS)orliquid
chromatography(LC–MS)providesamethodforcharacterisingimpuritiesindrugsand
formulationexcipients.
▪PharmaceuticalAnalysis
Bioavailability studies
Drug metabolism studies
Characterization of potential drugs
Drug degradation product analysis
Identifying drug targets
▪Biomolecularcharacterization
Characterization of Proteins an Peptides
Oligonucleotides.
▪Environmental Analysis
▪ForensicAnalysis
Anubhav Gupta