Mass part 2 2

CONTENTS
PRINCIPLE
INSTRUMENTATION
sampling handling system
ion source, mass analysers, detectors
TYPES IONS
FRAGMENTAION
GC/MS
LC/MS
INTERPRETATION
APPLICATIONS

PRINCIPLE
It is also called as positive ion spectra or line spectra
Sample 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 torr
Drawback: sample need to be vaporised. It may cause
the thermal decomposition of the compound.

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+

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 sampling
DISADVANTAGES:-
• 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 analyser
Various types of matrix
Nicotinic acid matrix - to analyte the proteins
glycoproteins
Ferulic acid matrix to analyte the proteins and
Caffieic acid matrix oligonucleotides
Succinic 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 ANALYSER
DOUBLE FOCUSSING ANALYSER
QUADRUPOLE ANALYSER
TIME OF FLIGHT 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

It is used differentiate the small mass differences of
the fragment.
These provides the high resolution
To 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.

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 masses
Here 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 detector
Two 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 ions
2)Fragment ion
3)Rearrangement ion
4)Metastable ions
5)Multiple charged ions
6)Isotope ions
7)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
compounds
Ex : 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.

FRAGMENTATION
The 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 decreases
2)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
CH2 R CH2 CH2+
α β
-R .
+
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˙

7)Saturated ring lose alkyl side chain at α bond. +ve
charge tends to stay with ring fragment.
R .+
+
8)Double bond favours allylic cleavage & gives resonance
stabilized allylic carbonium ion.
CH2=CH-CH2-R CH3+-CH=CH2

FRAGMENTATION PATTERN
Relative 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.
47

General rules of Fragmentation
Cleavage at branched carbon is favored due to higher stability
at tertiary carbocation.
H
C > C
> C
H
H
>
H
C
H
H
tert. sec. primary methyl
48

CH3
1 2 3 4 5 6 7 8
+
cleavage at 6-1
cleavage at 6-2
cleavage at 6-3
C4H9
C H
C3H7
+
CH3
C H
C4H9
+
CH3
C H
C3H7
+
(F1)
(F2)
(F3)
H3C CH2 CH2 C
H
CH2 CH2 CH2 CH3
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

Most important rule covers 70% of mass fragmentation.
X C1 C2 R X CH
a b
Cleavage favored at b bond leaving positive charge on C1.
50

H3C CH2 O CH2 CH3
H3C CH2 O CH2 CH3
CH2 O CH2
m/e = M-15
1.
H3C
2.
CH2 CH2 CH3
H3C CH2 N CH2
N
C2H5
C3H7
H2C
N
C2H5
H2C
H2C
m-57 m-29
N
CH2
C3H7
m-15
CH2
tert.amine
B1
B3 B2
e.g.: A) (x) = O, N, S.
51

3.
CH2 S CH2 CH2 CH3
H2C S
CH2
H2C S
M-71 C3H7
M-29
B2 B1
B1
B2

R
CH2
CH2
+
+
m/e = ( M-R ) Stable
benzylic cation
+
m/e = 91
Tropylium cation
+
b) Benzylic clevage
b)
m/e = 65
cyclopentadienyl
cation
-(x)- =
53

O
+
O C CH + 3
m/e = M-R m/e = M-15
Simarly for x= N & S
+
Very common fragment for ester
C. Allylic Cleavage
M-31 = methyl ester
M-45 = ethyl ester
H2C
R
m/e = M-R stable allyliz cation
O
H3C CH3
R CH3
R C O
i)
ii)
O
R C OCH3
R C O
m/e = M-31
+
54

4 Rule of elimination of small neutral molecule
A) b - Elimination
The high temperature and high vacuum are quite favourable for elimination reaction
H
C
C
OH
C C
+
+ H2O
m/e M - 18
and hence
i)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.
55

ii)Loss of Ammonia (NH3)(M-17) for primary amines and primary
and secondary alkyl ammonia derivatives
For
C C
NH
C C + NH2
M - 46
C2H5
C2H5
H
C
C
NH2
C C +
M - 17
NH3
56

iii)Elimination at Hydrogen sulphide (H2S)[M-34] confirms thiols
(mercaptons)
H
C
C
SH
C C + H2S
M - 34
iv)Elimination of Hydrogen cyanide (HCN)[M-27] confirms nitriles.
H
C
C
CN
C C + HCN
M - 27
57

v)Elimination of Hydrogen halide(HX),
Common for tertiary halides.
H
C
C
X
C C
m/e = M - HX
X = F, Cl, Br, I
58

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
59

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

MMccLLaaffffeerrttyy
x
CH2
CH2
H
CH2
O
C
Y
+
YY  HH,, RR,, OOHH,, NNRR22
IIoonn SSttaabbiilliizzeedd
bbyy rreessoonnaannccee
x
CH2
CH2
H
CH2
O
C
Y
-- CCHH22==CCHH22
x
CH2
O
C
Y
H
x
+
CH2
O+
C
Y
H
x
CH2
O
C+
Y
H
61

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.
H3C
Molecules having even mass number contain even no of
nitrogen H3C atoms.
CH3
H
MMWW == 5599
((oodddd))
MMWW == 5588
((eevveenn))
IIoonniissaattiioonn
[[MM++HH]]
[[MM++HH]]
MMWW == 6600
MMWW == 5599
CH3
N
H3C CH3
62

Fragmentation Patterns
Alkanes:
Fragmentation often splits off simple alkyl groups:
Loss of methyl M+ - 15
Loss of ethyl M+ - 29
Loss of propyl M+ - 43
Loss of butyl M+ - 57
Branched alkanes tend to fragment forming the most stable
carbocations.
64

FMraassg spmectreumn otfa 2t-mioethnyl pPenatatneterns

H3C CH3
H3C CH3
H3C CH3
CH2
H3C CH3
H3C CH3
+
H2C
CH2
H2C
H3C CH3
H3C CH3
+
H2C
+
CH2
H2C
CH2
CH
CH2
CH2
Aklenes (olefins)
CH2
CH2
m/z 69 mm//zz 6 973

FArormaagticms maey nalstoa hatvieo a npea kP ata mt/zt e= 7r7n fosr the benzene
ring.
NO2
77
M+ = 123
77

FArlcaohgolms entation Patterns
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+

MS for 1-propanol
M+-18 M+
CH3CH2CH2OH
H2C OH
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced
Industrial Science and Technology, 11/28/09)

Ethers
a-cleavage forming oxonium ion
Loss of alkyl group forming oxonium ion
Loss of alkyl group forming a carbocation

Aldehydes (RCHO)
Fragmentation may form acylium ion
RC O
Common fragments:
M+ - 1 for
M+ - 29 for
RC O
R (i.e. RCHO - CHO)

Ketones
O
Fragmentation leads to formation of acylium ion:
 Loss of R forming
 Loss of R’ forming
R'C O
RC O
RCR'

FrMaSg fomr 2e-pnenttaantoinoen Patterns
O
CH3CCH2CH2CH3
CH3CH2CH2C O
M+
CH3C O

Esters (RCO2R’)
Common fragmentation patterns include:
 Loss of OR’
 peak at M+ - OR’
 Loss of R’
 peak at M+ - R’

M+ = 136
77 105
O
C
O CH3
105
77

GC/MS
GC 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/MS
Liquid 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⁺
intensity m/e

? Why it is use Mass Spectroscopy ?
85

APPLICATIONS
Determination of molecular mass & ionization
potential
Determination of elemental composition
To know the reaction kinetics
To elucidate chemical structure of molecule
Detection of impurities
Used in drug metabolism studies
Determination of bond dissociation energies
Determination of isotopic composition of elements
in molecule

REFERENCES
Spectrometric identification of organic compounds
by Robert.M, Silverstein.
Instrumental methods of chemical analysis by
Gurdeep, R.chatwal
Organic spectroscopy by William kemp

Mass part 2 2

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