Interfaces in chromatography [LC-MS, GC-MS, HPTLC, LC, GC]

THE INTERFACES OF CHROMATOGRAPHY INCLUDES THE CHROMATOGRAPHY CRITEREA WHERE THE DIFFERENT CHROMATOGRAPHY ARE EXPLAINED IN DETAIL WITH PRACTICAL EXAMPLES AND THEIR IMAGES.

1. Interfaces in Chromatography
SHIKHA D. POPALI
HARSHPAL SINGH WAHI

2. LC-MS interface

3. LC-MS
►LC –Separation of the mixture of analytes
►Interface –Separation of the analyte from the solvent
►MA (mass analyzer) –separation of the analyte
molecular ion and fragments according to their mass
to charge ratio
Interface
Mass
Analyzer
DetectorLC
Extraction of
The analyte from
The solvent
Ion evaporation
or ionization.
Fragmentation

4. PROBLEMS IN COMBINING
HPLC AND MS
HPLC
Liquid phase operation
25 -50°C
No mass range
limitations
Inorganic buffers
1 ml/min eluent flow is
equivalent to 500 ml/min
of gas
MS
Vacuum operation
200 -300°C
Up to 4000 Da for
quadrupole MS
Requires volatile
buffers
Accepts 10 ml/min gas
flow

5. Coupling methods
►Direct liquid introduction (DLI) reduces the flow entering the MS
using some kind of splitting device.
►Particle beam (PB)
►Moving belt/wire rely on removal of the solvent prior to entering
the MS.
►Continuous flow fast atom bombardment (cf-FAB)
►A serious drawback of the first approach is the reduction in
sensitivity caused by the split factor.
►The entire flowrate, about 1 mL/min, used with a classic 4.6 mm
i.d. column is only tolerated by techniques such as thermospray
(TSP) and atmospheric pressure chemical ionization (APCI).
►Electrospray (ES) has a working range from nL/min to 0.2 mL/min,
which can be extended to mL/min flows.

6. Direct Liquid Introduction
Scheme of the DLI interface. 1 connection to LC column, 2 diaphragm 5
μm opening to MS, 3 needle valve, 4 cooling region, 5 to UV detector
or waste.

7. ►ThefirstattemptstointroducealiquidintoanMSusingthe
classicelectronimpactionization(EI)/chemicalionization
(CI)sourcewerebasedonthesimpleprinciplethatby
minimizingtheamountofliquid,
►thevacuumsystemwouldremovethesolventleavingthe
analyteinthegasphaseforionization.
►Byusinglargerpumpsystemsanddifferentialpumping,
maintenanceofthevacuumwasensured.
►TheflowfromtheLCcolumnwasreducedbyusingsmalleri.d.
columnsand/orsplittingtheliquidflow.Inordertoassistthe
evaporationaheatedde-solvationchamber couldbe
introduced

8. Particle beam
Schematic showing the principal components of a particle beam or MAGIC
interface

9. ►IonizationinmostLC–MSinterfacesdependsinsomewayonthe
compositionoftheLCsolvent.
►AinterfacethatprovidesthepossibilityofusingEI/CIwithoutthe
mechanicaltransportwasnamedMAGIC,anacronymformono
disperseaerosolgenerationinterfaceforchromatography.This
systemisnowknownasparticlebeam.
►TheLCeluentisforcedthroughasmallnebulizerusingaHegas
flowtoformastreamofuniformdroplets.Thesedropletsmove
throughade-solvationchamberandevaporatetoasolidparticle.
►Theseparticlesareseparatedfromthegas/solventand
transportedintotheMSsourceusingadifferentiallypumped
momentumseparator.
►ThePBinterfaceallowsflow-ratesfrom0.1–0.5mL/min.Most
analytesthatareamenabletoPBLC–MScanbeanalysedusing
GC–MSaswell.

10. Moving Belt/Wire
Schematicshowingtheprincipalcomponentsofamoving-beltinterface

11. ►Themoving-beltinterfaceseparatesthecondensedliquid-phase
sideoftheLCfromthehighvacuumoftheMSandusesabeltto
transporttheanalytesfromonetotheother.
►ThemobilephaseoftheLCisdepositedonabandand
evaporated.Theanalytesremainonthecontinuouslycyclingbelt
andaretransportedfromatmosphericpressureintothevacuum
oftheionsourcethroughtwodifferentiallypumpedvacuum
locks.
►Aheaterintheionsourceevaporatesthesamplefromthebelt
allowingMSanalysis.
►Mostmoving-beltanalysesdealwithvolatileanalytesusingCI/EI;
however,lessvolatilemoleculessuchasnucleosidesand
nucleotidesareanalysedusingthissystem.

12. Continuous flow fast atom bombardment
(cf-FAB)
Basic elements of a cf-FAB probe

13. ►Continuous-flowordynamicFABisamodificationoftheFAB
techniquethatallowscontinuouson-linerefreshingoftheliquid
ontheFABtarget.
►Thisliquid,ahigh-boilingsolventsuchasglycerol,thioglycerolor
nitrobenzylalcohol,isaddedtotheLCeffluentandtransported
throughacapillarytotheprobetip.
►There,fastatoms(oftenArorXe,keVenergies)bombardthe
sampleandionsaresputteredoutofthesolutionandintothegas
phase,andsampledintheMS.
►Togetherwiththesamplesomeofthematrixisionizedaswellas
yieldingapronouncedchemicalbackgroundatlowm/z;however,
thisphenomenonismorepronouncedinstaticFAB.
►Themostsuccessfulapplicationsofcf-FABarefoundinareasin
whichhighlypolar,thermolabile,ofteninvolatilecompoundsareto
beanalysed(surfactants,DNAadducts,oligonucleotides,
pesticides,pharmaceuticalsetc.)becausehighlypolarand/orionic
speciesaresuccessfullyanalysedusingFAB.

14. Thermospray
Thermospray interface. (a) configuration for ‘real-TSP-ionization’
(filament off) or external ionization (filament on) (b)
configuration with discharge electrode for external ionization and
repeller electrode

15. ►TheTSPinterfacewasdevelopedbyM.Vestalandco–workers.A
majoradvantageofTSPoverotherLC–MSinterfacesisitsabilityto
handlethehighflow-ratesdeliveredbyLC(upto2mL/min).
►Asthenamethermosprayimplies,heatingtheliquidflowleavinganLC
systemcreatesasprayofsuperheatedmistcontainingsmallliquid
droplets.
►Severaltechniquesaredevelopedtoheatandvaporizetheeffluent;
however,themostsuccessfulmethodinvolvesdirectingtheliquidflow
throughanelectricallyheatedcapillary,whichcanbedirectlyintroduced
intotheMSionsource.
►Thedropletsarefurthervaporizedastheycollideagainstthewallsof
theheatedionsource.
►Thisionsourceisequippedwithamechanicalpumplineoppositeto
thesprayinordertoevacuatetheexcesssolventvapour
►Therapidheatingandprotectiveeffectsofthesolventallowthe
analysisofnon-volatilesampleswithoutpyrolysis.

16. ►TheanalyteionsaresampledintotheMSthroughasamplingcone,if
necessaryaidedbyanappliedelectricfield(repelleroraccelerating
electrode).
►IonizationoftheanalytesinTSPoccursbymeansofseveralprocesses
whereintwoclassesofionizationcanbedistinguished:
►onewithoutanexternalionizationsource,called‘realthermospray’
►onewithanexternalionization.
►Therealthermosprayusesavolatilebuffer,oftenammoniumacetate,as
partoftheLCeffluent.ThebufferionsNH
4andCH
3COOactaschemical
ionizationreagentionstoformprotonatedordeprotonatedions,
respectively.
►Thebasicityoracidityoftheanalytesrelativetothebufferdetermines
whichionswillorwillnotbeproduced.Thisionizationtakesplacebothin
thegasphaseafterevaporationoftherespectivereagents,orinthe
droplets.
►Ionsformedintheliquidphasearesubsequentlytransferredtothegas
phaseeitherbyevaporationofthesolventorbyionevaporationinwhich
ionsareliberatedimmediatelyfromthedroplet.

17. ►Whennobufferisused,orifahigherpercentageoforganic
modifierisemployed,anexternalionizationisapplied.
►IntheTSPsource,anelectron-impactfilamentcanbeusedto
induceaplasmaofionsfromthemobilephase.Alternatively,a
dischargeelectrodecanbeusedtogeneratethisplasma.These
modes,filament-onanddischarge-on,yieldsolvent-mediatedCI
spectra.
►Eachionizationmode,positiveornegative,yieldsmolecularions
(protonated/deprotonatedoradductswithotherions).
►Dependingonthenatureoftheanalyteandtheexperimental
settingsofsourcetemperatureandrepellervoltage,some
fragmentationwilloccur.
►Althoughbeingahigh-temperaturetechnique,ithasbeen
successfullyappliedtothermolabilecompoundssuchasDNA
adducts,whichcannotbeanalysedwith,forexample,GC–MS.

18. ►TheearliestLC–MStechniques(DLI,TSP,movingbelt,PB),
althoughcommercialized,wereoftendifficulttouse,had
limitedsensitivityandwerenotrobust;however,theywere
veryusefulforspecificapplications.
►TheoverwhelmingincreaseinLC–MSapplicationsismainly
theresultofthesensitivityandruggednessofatmospheric
pressureionization(API)LC–MStechniques.

19. ►APIisageneralnameforallionizationtechniquesinwhichthe
ionsareformedatatmosphericpressure.
►Thoughverypopulartoday,ionizationprocessesatatmospheric
pressure(flames,dischargesetc.)havebeenstudiedusingmass
spectrometers
►InmodernLC–MSapplications;twomajortechniques:ESand
APCI.
►Electrospraycanbesubdividedintotechniquessuchas
pneumatic-assistedES,ES,multiplesprayerESetc.,thatdiffer
mainlyintheformationofasprayfromtheLCflow.
►However,allESvariantsrelyonthesamemechanism(s)toform
ionsfromthedropletsatatmosphericpressure.
►Theionsformedatatmosphericpressurearetransportedfrom
thesourcetothevacuumoftheanalyserthroughoneormore
differentiallypumpedstagesseparatedbyskimmers.Theions
arefocusedandguidedthroughtheskimmeropeningsintothe
MSbyapplyingappropriateelectricfields.

20. ►WhereEShasitsoptimalperformanceatlowflow-rates(nL/min
range)APCIoperateshappilyusingmL/minflow-rates.
►ESandAPCIperformdifferentlyunderdifferentchromatographic
modes.
►TheadvantagesofAPIweresummarizedbyVoyksnerinfour
points:
1.“APIapproachescanhandlevolumesofliquidtypicallyusedin
LC”
2.“APIissuitablefortheanalysisofnonvolatile,polarandthermally
unstablecompoundstypicallyanalysedbyLC”
3.“API-MSsystemsaresensitive,offeringcomparableorbetter
detectionlimitsthanachievedbyGC–MS”
4.“APIsystemsareveryruggedandrelativelyeasytouse.”

21. Electrospray
Basic elements of an electrospray and the layout of a commercial
(Micromass) ES-source and probe.

22. Droplet and ion production under ES conditions.

23. ►InMSresearch,ESisalsodescribedasasampleapplication
methodforplasmadesorption
►TheESsourcecomprisestwoelectrodes;thatis,theEScapillary
andtheatmosphericpressureapertureplateofthemass
spectrometerasthecounterelectrode.
►Ahigh-voltagesupplymaintainsapotentialdifferenceofabout3
kVbetweenbothelectrodes.Underinfluenceoftheappliedelectric
field,ionsofthesamepolaritymigratetowardtheliquidatthe
capillarytip,wheretheliquidsurfaceisdrawnoutofthecapillary
forminga‘Taylorcone’.
►Whenthebuild-upofanexcessofionsofonepolarityatthe
surfaceoftheliquidreachesthepointthatcoulombicforcesare
sufficienttoovercomethesurfacetensionoftheliquid,droplets(1
μm)enrichedinoneionpolarityareemittedfromthecapillary.
►Thesedropletsshrinkbysolventevaporationandrepeated
disintegration,leadingtoverysmall(3–10nm)chargeddroplets
capableofproducinggas-phaseions,yieldingaverysoft
ionizationtechnique.

24. ►Theionsaresampledthroughasetofskimmerelectrodesandfinally
analysedintheMSanalyser.
►Inordertoreducedetrimentaleffectsofdepositsonelectrodesand
skimmers,andtoavoidneutralsenteringtheMSvacuum,agascurtain
isappliedeffectivelyblowingneutralsandlargeparticlesawayfrom
theMSentranceand/orsprayer,andelectrodesaresetupsuchthat
theionsmustroundoneormoreanglesguidedbytheelectricfields.
►ESionizationcanbeusedforsmallmoleculesandlow-polarity
compoundsprovidingthereissomewayto(de)protonateorforma
combinationwithacationoranion.
►However,themoststrikingapplicationisthatofhighmolecularweight,
thermolabile,polarbiomoleculessuchaspeptides,proteins,
oligonucleotidesetc.,
►AnotherhighlyuncommoncharacteristicofESisits‘softness’;thatis,
verylabilestructurescanbecarriedasionsintothegasphasewithout
disruptingtheirstructures.EScanbeusedtostudyproteinfolding
status,non-covalentbonding,DNAduplexesetc.
►ForthesamereasonESspectracontainlittleornostructural
informationbecauseoftheabsenceoffragmentation.

25. Atmospheric Pressure Ionization
Overview of a differentially pumped API source coupled to a mass spectrometer

26. Atmospheric Pressure Chemical Ionization
►InAPCItheliquidflowfromtheLCissprayedandrapidly
evaporatedbyacoaxialnitrogenstreamandheatingthenebulizer
tohightemperature(350–500°C).
►Althoughthesetemperaturesmaydegradetheanalytes,thehigh
flow-ratesandcoaxialN2-flowpreventbreakdownofthe
molecules.
►Ionsalreadypresentinsolutioncanbecarriedintothegasphase,
however,additionalionizationisachievedusingacorona
discharge(3–6kV)inthespray.
►Thisdischargecanionizenotonlytheanalytemolecules,butalso
thesolventmolecules.Thesesolventionscanreactwiththe
analytesinthegasphaseinthesamewaysamplesareionizedin
aCIsourcebythereagentgas.
►Inpositiveionmodeprotonatedmoleculesandadductsare
formed;innegativeionmodeionsareformedbydeprotonation,
combinationswithanionsorelectron-capture.

27. ►UnlikeinES,thesolvent-evaporationandion-formationprocessesare
separatedinAPCI.Thisallowstheuseofsolventsthatare
unfavourableforionformation.
►Theselow-polaritysolventsarecommonlyusedinnormal-phase
chromatographywithlowpolaritysamplesthatcangenerallybe
evaporatedforAPCIionization.
►AnothermajordifferencebetweenAPCIandEScanbefoundintheLC
flow-ratesthatareused.APCIisatechniquewithoptimalperformance
athighflow-rates(1mL/minandhigher).
►Lowerflow-ratescanbeused;however,whenflow-ratesaretoolow
thestabilityofthecoronadischargemaybecomeproblematic.
WhereasESisideallysuitedtominiaturization,reducingtheflowsand
LCdimensionsusingAPCI
►APCIfindsmostofitsapplicationsinmolecularweightsbelow1000Da
formedium-tolow-polaritymolecules.Theanalyteswillneedsome
degreeofvolatilityandshouldnotbetoothermolabile.Typical
moleculesarepesticides,drugs,steroids,PAHsetc.
►Theapplicationscanbefoundinavarietyofplaces,fromquality
control,environmentalmonitoring(water,air,soil),geologicalstudies,
metabolitestudiesetc.

28. ATMOSPHERIC PRESSURE PHOTOSPRAY
IONISATION SOURCE
Uses UV lamp for ionisation (10eV)
Uses toluene as “dopant” (ionised by UV to form photo ions), co-vaporized. Other
dopant acetone, anisole and chlorobenzene, isoprene
Evaporation in a heated probe, similar to APCI
Ion-molecule reactions initiated by the photoions
Proton transfer analytesionised as [M+H]
+
Charge transfer analytesionised as [M]
+•

29. GC-MS interface

30. Mass spectrometer components

31. An ideal interface should:
►Quantitatively transfer all analyte
►Reduce pressure/flow from chromatograph to level
that MS can handle
►No interface meets all requirements.
The interface

32. Interfacing

33. ►The major goal of the interface is to remove most of the
carrier gas -the majority of the effluent.
►Classical or Molecular jet separator
►Permeation separator
►Open split
►Capillary direct

34. Molecular (Ryhage) jet Separator
The classical jet separator or molecular jet interface for
GC/MS was developed from the original Becker jet separator.

35. Intheseseparators,theGCflowisintroducedintoanevacuatedchamber
througharestrictedcapillary.Atthecapillarytipasupersonicexpanding
jetofanalyteandcarriermoleculesisformedanditscoreareasampled
intothemassspectrometer.
Inanexpandingjet,highmolecularmasscompoundsareconcentratedin
thecoreflowwhereasthelighterandmorediffusivecarriermoleculesare
dispersedaway,inpartthroughcollisions.
Thus,samplingofthecoreflowproducesanenrichmentoftheanalyte.
Thejetinterfaceisveryversatile,inertandefficient,despite
disadvantagesofreducedefficiencywithmorevolatilecompoundsand
potentialpluggingproblemsatthecapillaryrestrictor.
Advantage:Relativesimpleandinexpensiveapproach.
Disadvantages:RateofdiffusionisMWdependent
selectivitybasedonMW

36. Permeation interface: Llwellyn Littlejohn separator
Thepermselectivemembraneinterface,developedbyLlewellynand
Littlejohnismadeofasilicone-rubbermembranethattransmits
organicnon-polarmoleculesandactsasabarrierfor(non-organic)
carriergases.
Despitebeingaveryeffectiveenrichmentprocedure,italsosuffers
fromdiscriminationeffectswithmorepolaranalytesandproduces
significantbandbroadeningoftheirchromatographicpeaks.

37. Major problems with this approach
1. Membrane selectivity based on polarity and MW
2. Slow to respond
3. Only a small fraction of analyte actually permeates
through membrane
Watson-Biemann effusion separator

38. Themoleculareffusion(orWatsonBiemann)interfaceisbasedon
themolecularfilteringofthegaseffluentbymeansofaporousglass
frit.Thecolumneffluentpassesthroughafrittedtubesituatedina
vacuumchamber.
Smallmoleculestraversethemicroscopicporesinthetubewallsand
areevacuatedwhereashighmolecularmassmoleculesare
transferredtotheionsource.
Amongtheprincipaldrawbacksofthisinterfacearethehighdead
volumeaddedanditshighsurfacearea.Also,asinthecaseofthejet
separator,thisinterfaceshowsdiscriminationeffectsinthecaseof
smallermolecules.
Thethreemethodspresentedabovearebasedontheenrichmentofthe
analyteinthecarriergasbyeliminatingcarriermolecules.Inthisway,
enoughsamplecanbeintroducedintotheionsourcewithtotalgas
flowscompatiblewiththepumpingcapacityofthesystem.
Amongthem,thejetseparatorhasbeenthe
mostextensivelyusedandisperhapsthemostsuccessfulinterface.

39. Thesimplealternativetoreducedtotalgasflowisflow
splitting.
Inthiscase,nosampleenrichmenttakesplaceandtheseprocedures
aremostusefulwheresensitivityisnotacriticalfactor.
Flowsplittingcanbeperformedattheexitofthegaschromatograph,
allowingthedivertedgastobedirectedtoaparalleldetector,oratthe
interfaceitselfsuchasintheopensplitinterface.
Thelatterisbasedonacapillaryrestrictorthatlimitstheflowentering
theionsourcetoamanageable,constantvalue.TheGCcolumnexitis
situatedclosetotherestrictorentranceinanopenconnector.
TherestrictorsamplestheeffluentfromtheGCcolumnexitandthe
excesscolumnflowisremovedfromtheconnectorbyhelium.
Theopensplitinterfaceisaversatiledevicethatallowsonetowork
withawiderangeofcolumnflowswithoutanyinterfacemodification.

40. Somewhat similar to a jet separator.
The MS pulls in about 1 ml/min through the flow restrictor.
If column flow is above that -excess is vented.
If flow is below that, He from external source is pulled in.
Best for sources that have flows close to 1 ml/min like capillary columns.
Advantages
1. Any gas producing source can be used.
2. Relative low cost and easy to use.
Disadvantages
1. Sample leaves column in split.
2. Split changes as flow changes.
Open split interface

41. Capillary direct interface
Advantages
1 Low cost, simple device
2 No dead volume
3 No selectivity
Disadvantages
1 Limits flow range that column can use
2 Limits the ID of column that can be used
3 Part of column is ‘lost’ -serves as a flow
restrictor.

42. HPTLC-MS interface
Interface
Mass
Analyzer
TLC

43. Advantages of TLC-MS system
1.Direct coupling of TLC plate with MS
without any pretreatment of the
separated chromatographic bands
2.A possibility of coupling a TLC plate
with a variety of MS types
3.A possibility of selecting individual
bands of interest for MS analysis
4.Identification of individual bands with
MS

44. TLC MS interface

45. The interface has the advantage that without
modification it can be integrated into any HPLC-MS
system featuring atmospheric pressure chemical
ionization (APCI), atmospheric pressure photo
ionisation (APPI), or electro spray ionization (ESI).
With two fittings the interface is connected to the
HPLC pump and the mass spectrometer. The
substance of interest is eluted directly from the
TLC/HPTLC plate and is transferred online into the
mass spectrometer.
Within a minute the mass spectrum is obtained.

46. Principle
►The versatile instrument is used to isolate unknown
compounds from a TLC/HPTLC plate and transfer them into
a mass spectrometer foridentification or structure
elucidation. CAMAG TLC-MS Interface can be connected to
any brand of LC-coupled mass spectrometer.
►Plug & play installation by two HPLC fittings at a given
HPLC-MS system
►Semi-automatic instrument involving automatic piston
movement for pressure seal of the TLC/HPTLC zone on both
glass plates and aluminum foils
►Extraction directly from the plate using a suitable solvent
delivered by the HPLC pump
►Online transfer into the mass spectrometer
►Automatic cleaning of the piston between the extractions

47. The extraction principle
►Component mixtures, even with heavy matrix
load, can be separated cost efficiently on
TLC/HPTLC plates or aluminium foils.
►If the target zone is not visible, it can be
marked either under UV 254 nm or UV 366 nm,
by extrapolation of the adjacent zone made
visible by derivatization, or by using the hRF-
value obtained by TLC Scanner 3.
►By means of a laser crosshairs the zone to be
extracted is positioned exactly under the
extraction piston of the interface.
►The TLC-MS Interface is operated in semi-
automatic mode, which means that after
manual positioning of the zone the piston is
lowered at the push of a button.
►Moving a lever starts the solvent flow through
the layer and extracts the zone.

49. TLC-ESI-MS

51. TLC-Fast atom/ion bombardment-MS
TLC-APCI-MS

52. Example
►For identification of the zone at hRF 15 in a standard mixture of
caffeine, paracetamol and acetylsalicylic acid the mass spectrum of the
zone is recorded.
►At the same position a background spectrum of the plate is recorded
and subtracted from the substance spectrum.
►This leads to a mass spectrum free from system peaks showing mainly
substance signals -here the mass signal m/z 195 [M+H]+ for caffeine.
Left: Chromatogram with 4
mm bands, middle:
Same plate after extraction of
zone at hRF 15,
right: Extracted zone identified
as caffeine based on the mass
signal at m/z 195