Mass part 2

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CONTENTSPRINCIPLEINSTRUMENTATION sampling handling system ion source, mass analysers, detectorsTYPES IONSFRAGMENTAION GC/MSLC/MSINTERPRETATIONAPPLICATIONS

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

Instrumentation:

Sample inlet ion source ion separator

detector

read out device

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..

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)

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.

2) CHEMICAL IONISATION: In this reagent gas is used normally methaneOn 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++CH3CH3.+ +CH4 C2H5+ + H2these secondary ions react with sample(M) CH5++M CH4+MH+

C2H5++M C2H4+MH+

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.

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

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.

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.

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 .

It is one of the most important technique for analysing the biomolecules, proteins and oligonucleotides having the molecular weights of 100000 Da or more

MASS ANALYSERS: ion seperator

SINGLE FOCUSSING ANALYSERDOUBLE FOCUSSING ANALYSERQUADRUPOLE ANALYSERTIME OF FLIGHT ANALYSER

1. SINGLE FOCUSSING ANALYSER:

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.

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

DOUBLE FOCUSSING ANALYSER:

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.

QUADRUPOLE MASS ANALYSER:

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.

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

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.

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.

photomultiplier detector:Positive ions ↓Strike dyanode ↓Release electrons ↓Fall on the phosphorent screen ↓ Realease the photons ↓Transfer to PMT ↓amplification

Types of ions produced:1)Molecular ion or parent ions2)Fragment ion3)Rearrangement ion4)Metastable ions 5)Multiple charged ions6)Isotope ions7)Negative ions

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

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

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-

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.

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¯

Benzamide C6H5CONH2 C6H5CONH2+ + 2e- -C6H5 -NH2 CONH2+ C6H5CO+

C6H5+

-CO

FRAGMENTATION RULES:1) Straight chain compound – relative height molecular

ion peak great branched chain – height decreases2)Molecular wt increases - height decreases

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

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 R CH2 CH2+

-R .+

βα

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 .+

+

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.

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<ketones<

cyclo-alkenes<alkenes< conjugated polyenes<aromatic and hetero aromatic compounds

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

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.

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

X C1C2 R X CH

a b

Most important rule covers 70% of mass fragmentation.

Cleavage favored at β bond leaving positive charge on C1.


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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

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

Α) β - 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.


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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


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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

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

x

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

61

62

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

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


Fragmentation PatternsAromatics may also have a peak at m/z = 77 for the benzene ring.

NO2

77M+ = 123

77

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+

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)

Fragmentation PatternsEthers

α-cleavage forming oxonium ion

Loss of alkyl group forming oxonium ion

Loss of alkyl group forming a carbocation

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


KetonesFragmentation leads to formation of acylium ion:

Loss of R forming

Loss of R’ forming RC O

R'C O

RCR'O

Fragmentation PatternsMS for 2-pentanoneCH3CCH2CH2CH3

O

M+

CH3CH2CH2C O

CH3C O


Fragmentation PatternsEsters (RCO2R’)

Common fragmentation patterns include: Loss of OR’

peak at M+ - OR’

Loss of R’ peak at M+ - R’


M+ = 136

CO

O CH3

105

77 105

77


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

That separated components are passed through the MS inlet, into the MS and there the compounds are analysed and detected.

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.

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.

INTERPRETATION OF METHANOL

5 10 15 20 25 30 35

120

100

80

60

40

20

0

CHO⁺

CH3OH⁺

CH3⁺

CH2OH⁺

inte

nsit

y

m/e

INTERPRETATION OF PENTANE

?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

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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Mass part 2