57891569 Liquid Chromatography Mass Spectro

8/6/2019 57891569 Liquid Chromatography Mass Spectro

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LIQUID CHROMATOGRAPHYLIQUID CHROMATOGRAPHY--MASSMASSSPECTROMETRYSPECTROMETRY--MASSMASS

SPECTROMETRYSPECTROMETRY

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INTRODUCTION :INTRODUCTION :

LC/MS/MS is an analytical chemistryLC/MS/MS is an analytical chemistrytechnique that combines the physicaltechnique that combines the physicalseparation capabilities ofLC with massseparation capabilities ofLC with massanalysis capabilities ofMSanalysis capabilities ofMS

High sensitivity & specificityHigh sensitivity & specificity

Specific detection and potential

Specific detection and potentialidentification of chemicals in presence ofidentification of chemicals in presence of

other chemicalsother chemicals

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LC/LC-MS/MS-Tandem LC, Tandem MS

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IonizerIonizer

Sample

+_

Mass AnalyzerMass Analyzer DetectorDetector

principle

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Mass Spectrometry (MS)Mass Spectrometry (MS)

Introduce sampletotheinstrumentIntroduce sampletotheinstrument Generateions inthe gas phaseGenerateions inthe gas phase

Separateions onthe basis ofdifferences inSeparateions onthe basis ofdifferences inm/zm/zwith a mass analyzerwith a mass analyzer

DetectionsDetections

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INSTRUMENTATIONINSTRUMENTATION

COMPONENTS:COMPONENTS:

Vacuum systemVacuum systemSample inletSample inletIon sourceIon sourceMass analyzerMass analyzerDetectorDetector

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SCHEMATIC

REPRESENTATION OF MASS

SPECTROMETRY

InletIon

Source

Mass

FilterDetector

Data

System

High Vacuum SystemTurbo pumps

Diffusion pumps

Rough pumps

Rotary pumps

Sample Plate

Target

HPLC

GC

Solids probe

MALDI

ESI

IonSpray

FAB

LSIMS

EI/CI

TOF

Quadrupole

Ion Trap

Mag. Sector

FTMS

Microch plate

Electron Mult.

Hybrid Detec.

PCs

UNIX

Mac

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Vacuum systemVacuum system

Ion source, mass analyzer, detector are toIon source, mass analyzer, detector are tobe maintained under vacuumbe maintained under vacuum

Vacuum system makes desirable ions notVacuum system makes desirable ions notto collide with other ions and moleculesto collide with other ions and molecules

High vacuum is achieved by connecting aHigh vacuum is achieved by connecting alow vacuum pump to output of a highlow vacuum pump to output of a highvacuum pumpvacuum pump

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Inlet systemInlet system

Sample is to be in vapour state and it shouldSample is to be in vapour state and it shouldenter the ionization chamber at constant rate toenter the ionization chamber at constant rate to

achieve this system is usually heated.achieve this system is usually heated. Less volatile liquids and solids may be directlyLess volatile liquids and solids may be directly

vapourised within the ionization chamber.vapourised within the ionization chamber.

Liquids are handled by hypodermic needleLiquids are handled by hypodermic needle

injection through a silicone rubber dam.injection through a silicone rubber dam.

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ION SOURCEION SOURCE

Initial step for MS analysis is formation ofInitial step for MS analysis is formation ofgaseous analyte ionsgaseous analyte ions

Scope ofMS method depend uponScope ofMS method depend uponionization processionization process

These are categorised into two typesThese are categorised into two types

1. Gas phase source1. Gas phase source2. Desorption source2. Desorption source

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In gas phase source sample is firstIn gas phase source sample is firstvapourised and then ionised.vapourised and then ionised.

Compounds of gaseous sources haveCompounds of gaseous sources havemolecular masses ranging 10molecular masses ranging 10 daltons. daltons.

In desorption source solid or liquid sampleIn desorption source solid or liquid sampleis converted directly into gaseous ions. It dois converted directly into gaseous ions. It do

not require volatilization.not require volatilization.



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Ion sources are also classified as:Ion sources are also classified as:

1. hard ionization sources (EI, chemical1. hard ionization sources (EI, chemicalionization)ionization)

2. soft ionization sources (MALDI,ESI)2. soft ionization sources (MALDI,ESI)

Hard ionization gives information aboutHard ionization gives information aboutstructural and functional groups of analytestructural and functional groups of analytemoleculemolecule

Soft sources gives information aboutSoft sources gives information about

molecular mass of analyte molecule.molecular mass of analyte molecule.



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Electron Impact IonizationElectron Impact Ionization

Sampleis broughttohightemperaturesSampleis broughttohightemperaturestoproducemolecular vapourtoproducemolecular vapour

Resulting molecules ionised byResulting molecules ionised bybombarding withenergeticmoleculesbombarding withenergeticmoleculesproduced bytungstenfilamentproduced bytungstenfilament

Primaryproductis singly +veionPrimaryproductis singly +veion



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CHEMICAL IONIZATIONCHEMICAL IONIZATION

In these gaseous atoms of sample areIn these gaseous atoms of sample areionised by collision with ions produced byionised by collision with ions produced byelectron bombardment of excess of reagentelectron bombardment of excess of reagentgas.gas.

Usually +ve ions are used.Usually +ve ions are used.

To carryoutCI experiments ionization areaTo carryoutCI experiments ionization areais modified by adding vaccum pump and byis modified by adding vaccum pump and byreducing the width of the slit.reducing the width of the slit.



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MALDIMALDI

It can be used to obtain accurate molecularIt can be used to obtain accurate molecularmass information about polar bio polymers.mass information about polar bio polymers.

ranging in molecular mass about 10ranging in molecular mass about 10daltons.daltons.

Low concentration analyte is dispersed inLow concentration analyte is dispersed insolid or liquid matrix deposited on the end ofsolid or liquid matrix deposited on the end ofmetal platemetal plate

These plate placed in vaccum chamber &

These plate placed in vaccum chamber &laser beam is focused on to the sample.laser beam is focused on to the sample.



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MatrixMatrix--Assisted LaserAssisted Laser

Desorption IonizationDesorption Ionization

337 nm UV laser

MALDI

Nicotinic acid



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MALDI IonizationMALDI Ionization

++

+

+

-

--

++

+

+

-

---++

Analyte

Matrix

Laser

+

++

Laser beam is focused on to theLaser beam is focused on to thesamplesample

Matrix must strongly absorb laserMatrix must strongly absorb laser

radiationradiation

Matrix and analyte are thenMatrix and analyte are thendesorbed and ionised by thedesorbed and ionised by the

proton transfer with protonatedproton transfer with protonatedmatrix ions. Eg of matrix ismatrix ions. Eg of matrix isnicotinic acid.nicotinic acid.

+

+

+



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ELECTROSPRAY IONIZATIONELECTROSPRAY IONIZATION

It is used for analyzing bio moleculesIt is used for analyzing bio moleculeshaving molecular weight 10 lakhs daltonshaving molecular weight 10 lakhs daltonsor moreor more

Liquid sample is made to pass throughLiquid sample is made to pass throughcapillary stainless steel needle and dryingcapillary stainless steel needle and dryinggas is introduced into the apparatus andgas is introduced into the apparatus andby applying vaccum liquid sample turns toby applying vaccum liquid sample turns todropletsdroplets

ions evaporate from the surface of theions evaporate from the surface of thedroplets.droplets.



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Electrospray (Detail)Electrospray (Detail)



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ESI: Droplet sizereduction &ESI: Droplet sizereduction &

fissionfission Droplet size reduction occurs by theDroplet size reduction occurs by the

continual repetition of two processes:continual repetition of two processes:

1.1. Desolvation (evaporation of neutral solventDesolvation (evaporation of neutral solventand volatile buffers)and volatile buffers)

2.2. Droplet fission caused by electric repulsionDroplet fission caused by electric repulsionbetween like charges.between like charges.

++

++

+

++++

++

+

++

+

+++++++++

++++++

+++++++++

++++++

+++++++++

++++++

+++++++++

++++++

+

+

+

+

+



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Electrospray: OverviewElectrospray: Overview

+

+

++++++++

++++

+

+

+ ++

+

+

+

+

+++

+

++

++ + ++

+

++

++

++

+

+

+

+

+

+

+

To MS

Orifice

Plate

Drying Gas

Droplet Formation

Desolvation

& Fission

Gas Phase Ion

Generation

5kV

Nebulizing

Gas



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Positiveor Negative IonPositiveor Negative Ion

Mode?Mode? Ifthe samplehas functional groups thatIfthe samplehas functional groups thatreadily accept H+ (such as amide andreadily accept H+ (such as amide andamino groups foundinpeptides andamino groups foundinpeptides and

proteins) thenpositiveiondetectionisproteins) thenpositiveiondetectionisusedused--PROTEINSPROTEINS

If a samplehas functional groups thatIf a samplehas functional groups that

readilylose a proton (such as carboxylicreadilylose a proton (such as carboxylicacids andhydroxyls as foundinnucleicacids andhydroxyls as foundinnucleicacids and sugars) thennegativeionacids and sugars) thennegativeiondetectionis useddetectionis used--DNADNA



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Different Mass AnalyzersDifferent Mass Analyzers

Magnetic Sector Analyzer (Magnetic Sector Analyzer (MSAMSA))

Quadrupole Analyzer (Quadrupole Analyzer (QQ))

TimeTime--ofof--Flight Analyzer (Flight Analyzer (TOFTOF))



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Magnetic Sector AnalyzerMagnetic Sector Analyzer



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MAGNETIC SECTOR ANALYZERMAGNETIC SECTOR ANALYZER

Permanent magnet or electro magnet toPermanent magnet or electro magnet tocause beam from ion source travel incause beam from ion source travel incurved pathcurved path

Ions enter in to the mass analyzer tubeIons enter in to the mass analyzer tube

Ions of different masses can be scannedIons of different masses can be scanned

Then ions fall on detector

Then ions fall on detector



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TIME OF FLIGHT MASSTIME OF FLIGHT MASS

ANALYZER :ANALYZER :Ions enter into fieldIons enter into field--free driftfree drifttube by an electric field pulse oftube by an electric field pulse of

1000 to 10000 volts1000 to 10000 voltsIons are separated based on theirIons are separated based on theirmassmass

Lighter particles arrive at theLighter particles arrive at thedetector earlier than the heavierdetector earlier than the heavieronesones



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Quadrupole Mass AnalyzerQuadrupole Mass Analyzer



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Quadruple Mass AnalyzerQuadruple Mass Analyzer

A quadrupole mass filter consists of fourA quadrupole mass filter consists of fourparallel metal rods with different chargesparallel metal rods with different charges

Two opposite rods have an appliedTwo opposite rods have an applied ++potential and the other two rods have apotential and the other two rods have a --

potentialpotentialThe applied voltages affect the trajectory ofThe applied voltages affect the trajectory of

ions traveling down the flight pathions traveling down the flight path

For given dc and ac voltagesFor given dc and ac voltages, only ions of a, only ions of acertain masscertain mass--toto--charge ratio pass through thecharge ratio pass through thequadrupole filterquadrupole filter and all other ions areand all other ions arethrown out of their original paththrown out of their original path



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DETECTORSDETECTORS

The detector counts the ions and signal isThe detector counts the ions and signal isgenerated that is proportional to the ionsgenerated that is proportional to the ions

Once an ionized sample has beenOnce an ionized sample has beenanalyzed by m/e ratio in the analyzeranalyzed by m/e ratio in the analyzer

The ions are detected and reported to theThe ions are detected and reported to thedata systemdata system



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Mass DetectorsMass Detectors

Electron Multiplier (Dynode)



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TANDEM MASSTANDEM MASS

SPECTROMETRYSPECTROMETRYIt is also called as massIt is also called as massspectrometryspectrometry--mass spectrometrymass spectrometry

Ions formed enter mass analyzer1Ions formed enter mass analyzer1Precursor ions enter interactionPrecursor ions enter interactioncellcellProduct ions are formedProduct ions are formedThey are analyzed by massThey are analyzed by massanalyzer2analyzer2They are detected by detectorsThey are detected by detectors



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BLOCK DIAGRAM OF TANDEMBLOCK DIAGRAM OF TANDEMSPECTROMETERSPECTROMETER

Ion

source

Mass

Analyzer 1

Interaction

cell

Mass

analyzer 2Detectors

Original ions

ABC+ ,

ABCD+,

ABCDE+

ABCD+

Precursor

ion

Product

ions

A+

AB+

AABC+

Sample



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Tandem MassTandem MassSpectrometrySpectrometry

Purposeis tofragmentions fromparentPurposeis tofragmentions fromparentiontoprovideiontoprovide structuralinformationstructuralinformationabout a moleculeabout a molecule

Also allowsAlso allows mass separationmass separation andandAAAAidentificationidentification ofcompounds incomplexofcompounds incomplexmixturesmixtures

Uses twoormoremass analyzers/filtersUses twoormoremass analyzers/filtersseparated by a collisioncellfilledwithseparated by a collisioncellfilledwith

Argonor XenonArgonor XenonCollisioncellis where selectedions areCollisioncellis where selectedions are

sentforfurtherfragmentationsentforfurtherfragmentation



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LC/MS/MSLC/MS/MS

The spectra are collected for compoundsThe spectra are collected for compoundsas they exit from a chromatographyas they exit from a chromatographycolumncolumn

These spectra are then stored in aThese spectra are then stored in acomputer for subsequent processing andcomputer for subsequent processing andis introduced to ion source from whichis introduced to ion source from which

ions are formedions are formed



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SCHEMATIC REPRESENTATIONSCHEMATIC REPRESENTATIONOF LC/MS/MSOF LC/MS/MS

HPLC

system

Ion

source

Mass

analyzer1

detectors Massanalyzer2

Interaction

cell



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Applications of MSApplications of MSStructural elucidation of organicStructural elucidation of organic& biological molecules& biological moleculesIdentification of components inIdentification of components inTLC&PCTLC&PCDetermination ofAA sequencesDetermination ofAA sequencesin proteinsin proteinsAnalysis of aerosol particlesAnalysis of aerosol particles

Determination of molecular massDetermination of molecular massof peptides, proteinsof peptides, proteins



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Different compounds can be uniquelyDifferent compounds can be uniquely

identified by their massidentified by their mass

CH3CH2OH

N

OH

HO

-CH2-

-CH2CH-NH2

COOH

HO

HO

Butorphanol L-dopa Ethanol

MW = 327.1 MW = 197.2 MW = 46.1



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Analytical method to measure theAnalytical method to measure the

molecular or atomic weight of samplesmolecular or atomic weight of samples



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Pharmacokinetics: It is used inPharmacokinetics: It is used in

pharmacokinetic studies ofpharmacokinetic studies ofpharmaceuticals and employed inpharmaceuticals and employed inbioanalysisbioanalysis

Drug development: impurity identification,Drug development: impurity identification,invivo drug screening, quality controlinvivo drug screening, quality control

Determine isotopic composition ofDetermine isotopic composition ofelements within a sampleelements within a sample



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Peptide Mass FingerprintingPeptide Mass Fingerprinting

(PM

F)(PM

F)

Characteristic pattern of peptides isused for the identification of proteinthe method is called peptide massfinger printing (PMF)



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REFERENCESREFERENCES

Skoog, Principles of Instrumental Analysis.Skoog, Principles of Instrumental Analysis.

B.K.Sharma, Instrumental and Chemical Analysis.B.K.Sharma, Instrumental and Chemical Analysis.

K.A. Connors, ATextbook of pharmaceuticalK.A. Connors, ATextbook of pharmaceuticalanalysis.analysis.

Gurdeep R. Chatwal Sham k. Anand.Gurdeep R. Chatwal Sham k. Anand.

Robert D. Brown, Introduction to InstrumentalRobert D. Brown, Introduction to Instrumental

Analysis.Analysis.Y. Anjaneyulu and Maraiah, Quality Assurance andY. Anjaneyulu and Maraiah, Quality Assurance and

Quality Management in Pharmaceutical Industry.Quality Management in Pharmaceutical Industry.



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