Download - Mass part 2 2
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CONTENTSPRINCIPLEINSTRUMENTATION sampling handling system ion source, mass analysers, detectorsTYPES IONSFRAGMENTAION GC/MSLC/MSINTERPRETATIONAPPLICATIONS
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PRINCIPLEIt is also called as positive ion spectra or line spectraSample is bombarded with the high electron beam
produce the positive ions.They travel in straight path When a maganatic field or electric field is applied
then travels in curved path The fragments of different masses are seperated
based on the radius of curvature.m/e α r2
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Instrumentation:
Sample inlet ion source ion separator
detector
read out device
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1. SAMPLE HANDLING SYSTEM: Different types of samples having the
different sample inlet systems Heated inlet system: gases and less volatile liquids, the liquids vaporized externally an then
slowly introduced into the ionization source. Direct inlet system: Solids, nonvolatile liquids, unstable
compounds directly introduced into the ion source.
Non volatile liquids : steroids, carbohydrates polymeric substances etc..
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2) ION SOURCE
TYPES: 1.ELETRON IMPACT TECHNIQUE (EI)
2.CHEMICAL IONIZATION MS(CIMS)
3.FAST ATOM BOMBARDMENT MS (FAB-MS)
4.MATRIX ASSISTED LASER DESORPTION/IONIZATION MS (MALDI-MS)
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1) ELECTRON IMPACT:Electrons are produced from electrically heated
tungsten. These electrons are accelerated by an electric field to an average electron beam energy of about 70ev.
8-12ev is sufficient to the ionisation of the sample. the vapour of the sample anlaysed introduced at
right angles to the electron beam.The sample pressure is about 10-6 – 10-7 torrDrawback: sample need to be vaporised. It may
cause the thermal decomposition of the compound.
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2) CHEMICAL IONISATION:
In this reagent gas is used normally methane
On electron impact gives primary ion like CH4 .+ CH3.+
These react with excess of CH4 to give secondary ions.
CH4+e CH4++2e
CH4+e CH3++H+2e
CH4+ +CH4 CH5++CH3
CH3.+ +CH4 C2H5+ + H2
these secondary ions react with sample(M)
CH5++M CH4+MH+
C2H5++M C2H4+MH+
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3) FAB:
Few µg of sample is dissolved in few µl of glycerol as matrix.
this solution is bombarded by a beam of fast xenon atoms.
These fast atoms are prepared by accelerating xenon ions to an energy of 6-9 keV, these ions are transfered to the xenon gas ,where these ion get the electrons and forms the high energy xe atom.
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After the impact of fast xenon atoms into the solution, the sample is desorbed as ion by momentum transfer.
The beam of sample ion is analyzed in mass spectrometer.
ADVANTAGES:-• High resolution, rapid & simple• Tolerant to variations in samplingDISADVANTAGES:-• matrix also forms ions on bombardment
which complicates the spectrum
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MATRIX ASSISTED LASER DESORPTION It is new ionization method, which shows
accurate molecular weight information of compounds ranging in molecular weight from few thousands to several hundred thousand Daltons
In this technique low concentration of the analyte is uniformly dispersed in a solid or liquid matrix deposited on the metal plate.
The metal plate put in vaccum chamber and laser beam focussed on the sample.
Then martix and the sample strongly absorb the laser radiation. Then the sample gets ionized.
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The most common type of mass analyser used with the is the time of flight analyserVarious types of matrix Nicotinic acid matrix - to analyte the proteins glycoproteins Ferulic acid matrix to analyte the proteins and Caffieic acid matrix oligonucleotidesSuccinic acid – to analyte the proteins.
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ELECTRON SPRAY IONISATION:.A solution of the sample pumped through a
stainless steel capillary neddle. ↓
The resulting charged spray of fine droplets pass through the desolvating capillary,
↓ where evporation of the solvent attaining the
charge to the molecules(desolvation) ↓
desolvation process continues through various pumping stages as the molecular ion travels
towards the mass analyzer .
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It is one of the most important technique for analysing the biomolecules, proteins and oligonucleotides having the molecular weights of 100000 Da or more
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MASS ANALYSERS: ion seperator
SINGLE FOCUSSING ANALYSERDOUBLE FOCUSSING ANALYSERQUADRUPOLE ANALYSERTIME OF FLIGHT ANALYSER
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1. SINGLE FOCUSSING ANALYSER:
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It has horse shoe shaped glass tube which is evacuated, consists of sample inlet, electron bombarding source, accelerating plates on one end,& collector slit at other end.
• At curvature of tube there is provision to apply electric/magnetic field
• Sample in the form vapour is allowed through inlet and bombarded with electron beam at 70eV.
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It knocks off one electron from every molecule then they become +vely charged ion.
as these molecules become +ve charged, they are accelerated by accelerating plates and travel in straight path.
By application of electric or magnetic field they travel in curved path & molecular ions are separated according to their masses and collected
Different fragments fall on detector then mass spectrum is recorded
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DOUBLE FOCUSSING ANALYSER:
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It is used differentiate the small mass differences of the fragment.
These provides the high resolutionTo achieve better focusing, energy has to be
reduced before ions are allowed to enter the magnetic field and increase resolving power can be obtained two mass analysers in series.
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QUADRUPOLE MASS ANALYSER:
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It consists of 4 voltage carrying rods.The ions are pass from one end to another end During this apply the radiofrequency and
voltage complex oscillations will takes place.Here the single positive charge ions shows the
stable oscillation and the remaining the shows the unstable oscillations
Mass scanning is carried out by varying each of the rf and voltage frequencies ratios keeping their ratios constant. Quadrupole ion storage (ion trap)It store the unsorted ions temporarily, they
released to the detector by scanning the electric field.
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TIME OF FLIGHT ANALYSER:In this type of analyser the sorting of ions is
done in absence of magnetic field.The ions produced are acquiring different
velocities depending on their massesHere the particles reach the detector in the
order of the increasing order of their masses Here electron multiplier detector is used.The resolution power of this is 500-600
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MASS DETECTORS: The Faraday cup detector:
the detector is very simple. The basic principle is that the incident ion strikes the
dynode surface. which emits electrons and induces a current which is
amplified and recorded. The dynode electrode is made of a secondary
emitting material like Cs,Sb, or BeO.
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Electron multiplier
The sensitvity of the detector is 1000times greater than the faradaycup detectorTwo types of detectors 1) series of dyanodes are used 2) single horne shaped dyanode. This is similar to the PMT.
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photomultiplier detector:Positive ions ↓Strike dyanode ↓Release electrons ↓Fall on the phosphorent screen ↓ Realease the photons ↓Transfer to PMT ↓amplification
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Types of ions produced:1)Molecular ion or parent ions2)Fragment ion3)Rearrangement ion4)Metastable ions 5)Multiple charged ions6)Isotope ions7)Negative ions
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Molecular ion:If the electron beam energy is excess than
ionisation potential, electrons may be ejected from a lower lying molecular orbital. That type of ions are called molecular ion.
The molecular ions are formed in the ground state, the yield of molecular ions can be increased by increasing the electron beam energy
Fragment ion:CH3-CH2-Cl CH3CH2Cl+ + 2e-CH3-CH2-Cl- CH3 CH2+ + Cl- CH2CH2+ + HCl
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Rearrangement ions:This ions re produced by rearrangement of
hydrogen atoms one part of the ion to another part.
Rearrangement process common in the unsaturated
compoundsEx : Mc Lafferty rearrangement
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Metastable ions: Stable and unstable ion on fragmentation
gives the sharp peaks, but intermediate stability ions gives the broad peaks
Multiplecharged ions:Loss of two or more electrons from a molecule
with out fragmentation produce double and triple charged ions
M + e- M+++ + 3e-
M + e- M++ + 4e-
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Isotope ions:If the molecule having the F, Cl, Br, I, P produce
the isotope peaks.Ex; methyl bromide CH3 Br79 gives one parent peak at m/e 94 CH3 Br81 gives one parent peak at m/e 96 Negative ions:In few cases only negative ions are formed
during the fragmentation.These are formed by capture of the electron by
the molecule during the collission.
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FRAGMENTATIONThe process of breaking molecules/ions into
fragments is known as fragmentation.This can be seen in the form of peaks in mass spectra Methanol can be divided in to 4fragments
CH3OH CH3OH⁺ +e¯
CH3OH CH3⁺ + OH¯
CH3OH CH2OH⁺+ H¯
CH3OH CHO⁺ + H2¯
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Benzamide C6H5CONH2 C6H5CONH2+ +
2e- -C6H5 -NH2 CONH2+ C6H5CO+
C6H5+
-CO
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FRAGMENTATION RULES:1)Straight chain compound – relative height
molecular ion peak great branched chain – height decreases2)Molecular wt increases - height decreases
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3)Cleavage is favoured at branched carbon atoms, more branched more likely the cleavage
4) Cleavage occurs at alkyl substituted carbon atom, the more substituted, more likely is the cleavage.
Consequence of increased stability of 3˚ carbonium ion over a 2˚ which in turn more stable than 1˚.
[R C ]˙+ R˙ + +C
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5)In alkyl substituted aromatic compounds, cleavage occur at bond β to the ring
6)Cleavage of c-x bond is difficult than c-c bond, if occur +ve charge is carried by carbon atom not by the hetero atom
C C X+ C C+ + X˙
CH2
RCH2 CH2
+
-R .+
βα
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7)Saturated ring lose alkyl side chain at α bond. +ve charge tends to stay with ring fragment.
8)Double bond favours allylic cleavage & gives resonance stabilized allylic carbonium ion.
CH2=CH-CH2-R CH3+-CH=CH2
R .+
+
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FRAGMENTATION PATTERNRelative abundance of ions of various
masses is characteristic of particular compound under the specified conditions of excitation, is known as fragmentation pattern
Strong peak of large mass number is taken as parent peak.
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Molecular peak of a compound depends up on:-
stability of molecular ion & stability of radical lost
Stability of ion can be justified by stabilization of charge
Increased order of stability is amines<alcohols<acids<esters<ethers<alkanes<k
etones< cyclo-alkenes<alkenes< conjugated polyenes<aromatic and hetero aromatic compounds
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MCLAFFERTY REARRANGEMENT:-
Rearrangement ions are fragments, they are formed due to the result of intermolecular atomic rearrangement during fragmentation
To undergo this rearrangement the molecule must posses heteroatom, one double bond and hydrogen atom
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NITROGEN RULE:-
It is used for determination of molecular mass of compounds and its elemental composition
Molecules having odd mass number contain odd number of nitrogen atoms.
Molecules having even mass number contain even no of nitrogen atoms.
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1.Hydrocarbons•Hydrocarbons give clusters of peaks.
•Molecular ion peaks of very low abundance are observed for linear hydrocarbons.•For branched hydrocarbons give a low intensity at M+.
•Intensity of (CnH2n+1) peaks decreases with increasing mass.
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C > C
H
> C
H
H
>H
C
H
H
tert. sec.primary methyl
Cleavage at branched carbon is favored due to higher stability at tertiary carbocation.
General rules of Fragmentation
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+
cleavage at 6-1
cleavage at 6-3
cleavage at 6-2
C H
C4H9
C3H7
C H
CH3
C4H9
+
+
C H
CH3
C3H7
+
(F1)
(F2)
(F3)
H3C CH2 CH2 C
CH3
H
CH2 CH2 CH2 CH3
1 2 3 4 5 6 7 8
Eg.
Produces three secondary cations, the most favored fragments at C-4 of
4- methyl octane.Note that C4 is common for fragments (F1)(F2) And (F3). 49
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X C1C2 R X CH
a b
Most important rule covers 70% of mass fragmentation.
Cleavage favored at bond leaving positive charge on C1.
General rules of Fragmentation
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H3C CH2 O CH2 CH3
H3C CH2 O CH2 CH3
CH2 O CH2
m/e = M-15
1.
H3C
2.
H3C CH2 N CH2
CH2 CH2 CH3
NC2H5
C3H7
H2C
m-57m-29
NC2H5
H2C
H2C
NCH2
C3H7
m-15
CH2
tert.amine
B1B2B3
e.g.: A) (x) = O, N, S.
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3.
CH2 S CH2 CH2 CH3
SH2C
CH2
SH2C
C3H7M-71
M-29
B2 B1
B1B2
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R
CH2CH2
+
+
m/e = ( M-R )Stablebenzylic cation
+
m/e = 91
Tropylium cationm/e = 65
cyclopentadienylcation
+
b)
b) Benzylic clevage
-(x)- =
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Very common fragment for ester
M-31 = methyl esterM-45 = ethyl ester
C. Allylic Cleavage
H2C
R
m/e = M-R stable allyliz cation
CH3H3C
O
R CH3
O+
R C O+CO CH3
m/e = M-R m/e = M-15
Simarly for x= N & S
i)
ii)
CR OCH3
O
CR O+
m/e = M-31
+
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4 Rule of elimination of small neutral molecule
C
H
C
OH
C C
+
+ H2O
m/e M - 18
A) - EliminationThe high temperature and high vacuum are quite favourable for elimination reaction
and hencei)Loss of water (H2O) for alcohols (M-18) is a prominent fragment.Tertiary alcohols lose the water so fast that in many cases M.I. Peak is absent.
General rules of Fragmentation
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C C
NH
C C + NH2
M - 46
C2H5
C2H5
ii)Loss of Ammonia (NH3)(M-17) for primary amines and primary and secondary alkyl ammonia derivatives For
C
H
C
NH2
C C +
M - 17
NH3
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iii)Elimination at Hydrogen sulphide (H2S)[M-34] confirms thiols (mercaptons)
C
H
C
SH
C C + H2S
M - 34
iv)Elimination of Hydrogen cyanide (HCN)[M-27] confirms nitriles.
C
H
C
CN
C C + HCN
M - 27
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v)Elimination of Hydrogen halide(HX),
Common for tertiary halides.
C
H
C
X
C C
m/e = M - HX X = F, Cl, Br, I
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High temperature high vacuum highly favorable for(DA) common for all these six membered cyclic mono olefins.
+
O
O
O + O
O
O
diene dienophile
General rules of Fragmentation
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MCLAFFERTY REARRANGEMENT:-
Rearrangement ions are fragments, they are formed
due to the result of intermolecular atomic
rearrangement during fragmentation
To undergo this rearrangement the molecule must
posses heteroatom, one double bond and hydrogen atom
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McLaffertyMcLafferty
x
CH2
CH2
H
CH2
O
CY
Y Y H, R, OH, NR2 H, R, OH, NR2
Ion Stabilized Ion Stabilized by resonanceby resonance
x
CH2
CH2
H
CH2
O
CY
- CH- CH22=CH=CH22
x
CH2
O
CY
H
CH2+
O+
CY
H
x
CH2+
O
C+
Y
H
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It is used for determination of molecular mass of compounds and its elemental composition
Molecules having odd mass number contain odd number of nitrogen atoms.
Molecules having even mass number contain even no of nitrogen atoms.
NITROGEN RULE:-
CH3
CH3 CH3
H
MW = 59 MW = 59 (odd)(odd)
MW = 58 MW = 58 (even)(even)
Ionisation Ionisation [M+H][M+H]
[M+H][M+H]
MW = 60MW = 60
MW = 59MW = 59
CH3
N
CH3 CH3
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63
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Fragmentation PatternsAlkanes:
Fragmentation often splits off simple alkyl groups:Loss of methyl M+ - 15Loss of ethyl M+ - 29Loss of propyl M+ - 43Loss of butyl M+ - 57
Branched alkanes tend to fragment forming the most stable carbocations.
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Fragmentation PatternsMass spectrum of 2-methylpentane
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CH2
CH3 CH3
CH2
CH2
CH2+
CH3 CH3
CH2+
CH2
CH2
CH3 CH3
CH2
CH3 CH3
CH2+
CH2
CH
CH3CH3
Aklenes (olefins)
CH2
CH3 CH3
CH2
CH3CH3
m/z 69 m/z 67 m/z 93
Fragmentation Patterns
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Fragmentation Patterns
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Fragmentation PatternsAromatics may also have a peak at m/z = 77 for the benzene ring.
NO2
77M+ = 123
77
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Fragmentation PatternsAlcohols
Fragment easily resulting in very small or missing parent ion peak
May lose hydroxyl radical or water
M+ - 17 or M+ - 18
Commonly lose an alkyl group attached to the carbinol carbon forming an oxonium ion.
1o alcohol usually has prominent peak at m/z = 31 corresponding to H2C=OH+
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Fragmentation PatternsMS for 1-propanol
M+M+-18
CH3CH2CH2OH
H2C OH
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/28/09)
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Fragmentation PatternsEthers
-cleavage forming oxonium ion
Loss of alkyl group forming oxonium ion
Loss of alkyl group forming a carbocation
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Fragmentation PatternsAldehydes (RCHO)
Fragmentation may form acylium ion
Common fragments:
M+ - 1 for M+ - 29 for
RC O
R (i.e. RCHO - CHO)
RC O
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Fragmentation Patterns
KetonesFragmentation leads to formation of acylium
ion:
Loss of R forming
Loss of R’ forming
RC O
R'C O
RCR'
O
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Fragmentation PatternsMS for 2-pentanoneCH3CCH2CH2CH3
O
M+
CH3CH2CH2C O
CH3C O
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/28/09)
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Fragmentation PatternsEsters (RCO2R’)
Common fragmentation patterns include: Loss of OR’
peak at M+ - OR’
Loss of R’ peak at M+ - R’
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Fragmentation Patterns
M+ = 136
C
O
O CH3
105
77 105
77
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/28/09)
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GC/MSGC is coupled to MS through an interface, in
this complex mixtures of chemicals are separated, identified and quantified
Compound to be analyzed should be volatile & thermally stable
Sample solution is injected in to GC inlet there it is vapourised and swept on chromatographic column by carrier gas
Sample flows through column and compounds in the sample mixture are separated by their interaction with column coating mixture and carrier gas
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That separated components are passed through the MS inlet, into the MS and there the compounds are analysed and detected.
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LC/MSLiquid chromatography-mass spectrometry is
a technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry .
In this Sample solution is injected in to HPLC columns.
These columns comprises of narrow stain less steel tube, packed with chemically modified silica particles.
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Components eluting from the chromatographic column are then introduced to mass spectra via specialized interface.
The most commonly used interfaces are electrospray ionization, atmospheric pressure chemical ionization interfaces.
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INTERPRETATION OF METHANOL
5 10 15 20 25 30 35
120
100
80
60
40
20
0
CHO⁺
CH3OH⁺
CH3⁺
CH2OH⁺
inte
nsi
ty
m/e
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INTERPRETATION OF PENTANE
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?Why it is use Mass Spectroscopy ?
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APPLICATIONSDetermination of molecular mass & ionization
potentialDetermination of elemental compositionTo know the reaction kineticsTo elucidate chemical structure of moleculeDetection of impuritiesUsed in drug metabolism studiesDetermination of bond dissociation energiesDetermination of isotopic composition of
elements in molecule
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REFERENCESSpectrometric identification of organic
compounds by Robert.M, Silverstein.Instrumental methods of chemical analysis by
Gurdeep, R.chatwal Organic spectroscopy by William kemp
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