Liquid chromatography-mass spectrometry (LC-MS) is a technique that combines the separation capabilities of liquid chromatography (HPLC) with the detection capabilities of mass spectrometry, allowing for the identification and structural elucidation of compounds. Key components include interfaces for ionizing samples and various ion sources like electrospray and atmospheric pressure chemical ionization. LC-MS has diverse applications in pharmaceuticals, biochemistry, food safety, environmental monitoring, and forensics.

1. LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
(LC/MS)

2. Principle:
• LC/MS is a technique that combines physical separation
capabilities of liquid chromatography with mass analysis
capabilityofMassspectrometry.
• ItisamethodthatcombinesseparationpowerofHPLCwith
detectionpowerofMassspectrometry.
• InLC-MSweremovethedetectorfromthecolumnofLCandfit the
columntointerfaceofMS.
• InthemostofthecasestheinterfaceusedinLC-MSareionization source.
INTRODUCTION

3. • HPLCisamethod for separatingacomplexmixture in to its
components.
• Highsensitivityof massspectroscopy provides theinformation
for identification of compoundsor structural elucidation of
compounds.
• Combination of thesetwotechniques isLC-MS.
• Asthe metabolitesappearfrom the endof the column they
enter the massdetector, where the solvent isremovedand the
metabolitesareionized.
Theory of LC/MS

4. LC-MS System Components
• Mass spectrometers work by ionizing molecules and then
sorting and identifying the ions according to their mass-
to-charge (m/z) ratios.

5. HPLC
• Liquidphaseoperation
• 25- 50deg.C
• Nomassrange
limitations
• Inorganicbuffers
• 1ml/min eluent flowis
equivalent to 500 ml/min
ofgas
MS
• Vacuumoperation
200- 300deg.C
• Upto 4000Dafor
quadrupole MS
• Requiresvolatile
buffers
• Accepts10ml/min gas
flow
PROBLEMS IN COMBINING HPLC AND MS

6. Themobile phaseisthe solvent that movesthe solute throughout
column.
Generalrequirements:-
(1) Lowcost,UVtransparency,high purity.
(2) Lowviscosity,low toxicity, nonflammability.
(3) Noncorrosiveto LCsystemcomponent.
Solventstrength andselectivity:-
It isthe abilityof solvent to elute solutesfromacolumn.
MOBILE PHASE

7. • The use of di-functional or tri-functional silanes to create bonded
groups with two or three attachment points leading to phases
with higher stability in low or higher pHandlower bleedfor LC-MS
• Most widely usedcolumnsfor LC-MSare :-
(1) fast LC column :-
the useof short column.(15-50mm)
(2) Micro LC column :-
the useof largecolumn. (20-150mm)
COLUMN

8. • Samplepreparation generally consists of concentrating the analyte
and removing compounds that can cause background ion or
suppressionization.
• Exampleof samplepreparationinclude:-
• OnColumnconcentration -to increaseanalyteconcentration.
• Desalting - to reduce the sodium and potassium adduct
formation that commonlyoccursin electrospray.
• Filtration- to separate a low molecular-weight drug from
proteins in plasma,milk, ortissue.
Sample preparation

9. • LC-MSsystemsinclude adevicefor introducing samples(suchas an
HPLC)aninterface for connectingsuchdevice,anion source that
ionizessamples,anelectrostatic lensthat efficiently introduces
the generatedions, amassanalyzerunit that separatesions based
on their mass-to-charge(m/z) ratio, anda detector unit that
detectstheseparatedions.
• In anLC-MSsystem,however, if the LCunit issimplyconnected
directlyto the MSunit, the liquid mobile phasewould vaporize,
resulting in large amountsof gasbeingintroduced into the MS
unit.
• Thiswould decreasethe vacuumlevel andprevent the targetions
from reachingthe detector. Sointerfaces are tobeused.
INTERFACES

10. • It isdifficult to interface aliquid chromatographyto amass-
spectrometercauseof the necessityto removethe solvent.
• Thecommonlyusedinterfacesare:-
(1) Electrosprayionization(ESI)
(2) Thermosprayionization(TSI)
(3) Atmosphericpressurechemicalionization(APCI)
(4) Atmosphericpressurephotoionization(APPI)
TYPES OF INTERFACES

11. • ESIdrawssamplesolutions to the tip of acapillarytube,
whereit appliesahighvoltage of about 3to 5kV.
• A nebulizer gas flows from outside the capillary to spray the
sample.Thiscreatesafine mist of chargeddroplets with the same
polarity asthe appliedvoltage.
• Asthischargedparticles move,the solvent continuesto evaporate,
thereby increasingthe electricfield onthe droplet surface.When
the mutual repulsive force of the charges exceedsthe liquid
surfacetension, then fissionoccurs.
• Asthis evaporation andfissioncycleisrepeated, thedroplets
eventually becomesmallenoughthat the sampleionsare
liberated into the gasphase.
ElectroSprayIonization(ESI)

12. • ESIprovides the softest ionization method available, which
meansit canbeusedfor highly polar, least volatile, or
thermally unstablecompounds.
ElectroSprayIonization(ESI)

13. • APCIvaporizessolvent andsamplemoleculesbysprayingthe
samplesolution into aheater (heatedtoabout 400C)usinga gas,
suchasN2.
• Solventmoleculesareionizedbycoronadischargetogenerate stable
reactionions.
Atmospheric pressurechemical ionization
(APCI)

14. • Theyareof 2type:
• a)Real-TSPionization
• b) Dischargeelectrode for external ionizationandrepeller
electrode
Thermospray ionization (TSI)

15. • TheLC eluent isvaporizedusingaheater at atmospheric pressure.
Theresulting gas ismadeto pass through a beamof photons
generated byadischargelamp(UVlamp) which ionizesthe gas
molecules.
Atmospheric pressurephotoionization(APPI)

16. • Theydeflect ionsdown acurvedtubes in amagnetic fields based
on theirkinetic energydetermined bythe mass,charge and
velocity.
• Themagnetic field isscannedto measuredifferentions.
• Typesof massanalyzer:-
(1) Quadrapolemassfilter.
(2) Timeof flight
(3) Iontrap
(4) Fourier transform ion cyclotron resonance(FT-ICR)
Mass Analyser

17. • A Quadrupolemassfilter consistsof four parallelmetal rodswith different
charges
• Twooppositerodshaveanapplied +potential andthe othertwo
rodshavea-potential
• Theappliedvoltages affect the trajectory of ionstravelingdown the flight
path
• ForgivenDC andAC voltages,only ionsof acertain mass-to-charge ratio pass
through the quadrupole filter and all other ionsare thrownout of their
originalpath.
QuadrupoleMassAnalyzer

18. • TOFAnalyzersseparateionsbytime without the useofan
electric or magneticfield.
• In acrude sense,TOF issimilar to chromatography, except there is
no stationary/ mobile phase,insteadtheseparationis basedon
the kineticenergyandvelocityof the ions.
TOF (Time of Flight)Mass Analyzer

19. • It usesanelectricfield for the separationof the ionbymass to
chargeratios.
• Theelectricfield in the cavitydueto the electrodescauses the
ionsof certainm/z valuesto orbit in the space.
Ion TrapMass Analyzer

20. • Usesamagneticfield in orderto trap ionsinto anor bit inside of it.
• Inthisanalyzerthereisnoseparationthat occursrather all the
ionsof aparticular rangearetrapped inside,andan applied
external electric field helpstogenerate asignal.
Fourier Transform ion cyclotron
resonance (FT-ICR)

21.  PharmaceuticalApplications:
 Rapidchromatographyofbenzodiazepines
 Identificationofbileacidmetabolite
 BiochemicalApplications:
 Rapid protein identification using capillary LC/MS/MS and
databasesearching.
 ClinicalApplications:
 High-sensitivitydetectionoftrimipramineandthioridazine
Applications of LC-MS

22.  Food Applications:
 Identificationofaflatoxinsinfood
 DeterminationofvitaminD3inpoultryfeedsupplements
 EnvironmentalApplications:
 Detectionofphenylureaherbicides
 Detectionoflowlevelsofcarbarylin food
 Forensic Applications:
 illegalsubstances,toxicagents
 Explosives
Applications of LC-MS