Download - 57891569 Liquid Chromatography Mass Spectro
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
1/43
LIQUID CHROMATOGRAPHYLIQUID CHROMATOGRAPHY--MASSMASSSPECTROMETRYSPECTROMETRY--MASSMASS
SPECTROMETRYSPECTROMETRY
VisitVisitwww.bpharmstuf.comwww.bpharmstuf.com
For moreFor more pptsppts
1www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
2/43
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
2www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
3/43
LC/LC-MS/MS-Tandem LC, Tandem MS
3www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
4/43
IonizerIonizer
Sample
+_
Mass AnalyzerMass Analyzer DetectorDetector
principle
4www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
5/43
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
5www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
6/43
INSTRUMENTATIONINSTRUMENTATION
COMPONENTS:COMPONENTS:
Vacuum systemVacuum systemSample inletSample inletIon sourceIon sourceMass analyzerMass analyzerDetectorDetector
6www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
7/43
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
7www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
8/43
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
8www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
9/43
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.
9www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
10/43
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
10www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
11/43
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.
11www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
12/43
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.
12www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
13/43
13www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
14/43
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
14www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
15/43
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.
15www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
16/43
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.
16www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
17/43
MatrixMatrix--Assisted LaserAssisted Laser
Desorption IonizationDesorption Ionization
337 nm UV laser
MALDI
Nicotinic acid
17www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
18/43
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.
+
+
+
18www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
19/43
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.
19www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
20/43
Electrospray (Detail)Electrospray (Detail)
20www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
21/43
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.
++
++
+
++++
++
+
++
+
+++++++++
++++++
+++++++++
++++++
+++++++++
++++++
+++++++++
++++++
+
+
+
+
+
21www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
22/43
Electrospray: OverviewElectrospray: Overview
+
+
++++++++
++++
+
+
+ ++
+
+
+
+
+++
+
++
++ + ++
+
++
++
++
+
+
+
+
+
+
+
To MS
Orifice
Plate
Drying Gas
Droplet Formation
Desolvation
& Fission
Gas Phase Ion
Generation
5kV
Nebulizing
Gas
22www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
23/43
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
23www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
24/43
Different Mass AnalyzersDifferent Mass Analyzers
Magnetic Sector Analyzer (Magnetic Sector Analyzer (MSAMSA))
Quadrupole Analyzer (Quadrupole Analyzer (QQ))
TimeTime--ofof--Flight Analyzer (Flight Analyzer (TOFTOF))
24www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
25/43
Magnetic Sector AnalyzerMagnetic Sector Analyzer
25www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
26/43
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
26www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
27/43
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
27www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
28/43
Quadrupole Mass AnalyzerQuadrupole Mass Analyzer
28www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
29/43
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
29www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
30/43
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
30www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
31/43
Mass DetectorsMass Detectors
Electron Multiplier (Dynode)
31www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
32/43
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
32www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
33/43
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
33www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
34/43
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
34www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
35/43
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
35www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
36/43
SCHEMATIC REPRESENTATIONSCHEMATIC REPRESENTATIONOF LC/MS/MSOF LC/MS/MS
HPLC
system
Ion
source
Mass
analyzer1
detectors Massanalyzer2
Interaction
cell
36www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
37/43
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
37www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
38/43
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
38www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
39/43
Analytical method to measure theAnalytical method to measure the
molecular or atomic weight of samplesmolecular or atomic weight of samples
39www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
40/43
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
40www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
41/43
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)
41www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
42/43
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.
42www.bpharmstuf.com
-
8/6/2019 57891569 Liquid Chromatography Mass Spectro
43/43
Visitwww.bpharmstuf.com
For more ppts
43www.bpharmstuf.com