MASS SPECTROSCOPY (MS)

Ricin (toxic protein)Castor seeds

Does not involve absorption of electromagnetic radiation.

It is a ‘spectroscopic technique’, by virtue of its use in structure elucidation.

MS is used to determine:-1) Molecular weight (from Low Resolution Mass

Spectroscopy, LRMS).2) Molecular formula (from High Resolution Mass

Spectroscopy, HRMS).3) To detect within a molecule the places at which

it is preferred to fragment(recognizable groupings can be deduced).4) To identify unknown compounds by comparison

with MS of standards (fingerprint comparison).

INTRODUCTION

• The three essential components of a mass spectrometer are:-

• 1. The Ion Source• A small sample of a compound is ionized

(individual molecules are changed into ions), usually to cations by loss of an electron.

• 2. The Mass Analyzer• The ions are sorted and separated according to

their mass to charge ratio (m/z). • 3. The Detector• The separated ions are then detected and

tallied, and the results are displayed on a chart.

The Mass Spectrometer

• Ions are very reactive and short-lived, • their formation and manipulation is conducted in

a vacuum.• Different types of ionization are known. • The most common method is:-• Electron Ionization Mass Spectroscopy (EIMS)• ionization is effected by a high energy beam of

electrons, and• ion separation is achieved by accelerating and

focusing the ions in a beam, which is then bent (deflected) by an external magnetic field.

• The ions are then detected electronically and the resulting information is stored and analyzed in a computer.

EIMS SPECTROMETER

• A high energy electron collides with a molecule,• it ionizes it by knocking away one of the molecular

electrons (either bonding or non-bonding). • This results in the formation of a molecular ion.• Residual energy from the collision may cause the

molecular ion to fragment,• (into neutral pieces and smaller fragment ions).

IONIZATION PROCESS

molecular ion(Radical cation)

Radical cation

(radical)cation

fragment ions

Only cations are detected in normal MS spectra;the presence of neutral fragments is recognized through inference.

• Vertical bar graph, in which each bar represents an ion having a specific mass-to-charge ratio (m/z) and

• the length of the bar indicates the relative abundance of the ion.

• Modern mass spectrometers easily distinguish (resolve) ions differing by only a single atomic mass unit (amu),

• and thus provide completely accurate values for the molecular mass of a compound.

• and lower-mass ions are fragments from the molecular ion, assuming the sample is a single pure compound.

The Nature of Mass Spectra

mass-to-charge ratio (m/z)The most intense ion is assigned an abundanceof 100, and it is referred to as the ‘base peak’.Most of the ions have a single charge, so the m/z value is equivalent to mass itselfResolution is, at least, to a single atomic mass unit (amu).

The highest-mass ion is normally the molecular ion.

% a

bund

ance

(rel

ativ

e ab

unda

nce) base peak

106Molecular ion

.

The Molecular Ion• 80-90% of organic compounds give rise to

spectrum in which the peak with the highest m/zvalue corresponds to the molecular ion.

• The stability of the molecular ion and consequently the intensity of the molecular ion peak is related to molecular structure.

• Conjugated systems show stable molecular ion.• Cyclic structures give rise to a relatively strong

molecular ion peak, • (two bonds have to be broken before the m/z

ratio change).• molecular ion will be a weak peak, if

fragmentation results in the formation of neutral molecule or stable fragment ion.

• True molecular-ion peaks are of • even m/z ratio,• unless an odd number of nitrogen atoms are

present in the molecule.

ISOTOPESSince a mass spectrometer separates and detectsions of slightly different masses, it easily distinguishes different isotopes of a given element.

+ .[M] + .[M+1] + .[M+2] etc...

The molecular ion is composed of the most abundant isotopes in the molecules.

Molecular ion Due to the presence of isotopes

(m/z) [M+1]+. at 107

106Molecular ion

natural bromine consists of a nearly 50:50 mixture of isotopes having atomic masses of 79 and 81 amu respectively.

Br2 may be composed of two 79Br atoms (mass 158 amu),two 81Br atoms (mass 162 amu)

the more probable combination of 79Br-81Br (mass 160 amu).

Fragmentation of Br2 to a bromine cation then gives rise to equal sized ion peaks at 79 and 81 amu.

Bromine: 50.50% 79Br and 49.50% 81Br

[M]+ at 158

[M+2]+ at 160

[M+4]+ at 162

chlorine is also composed of two isotopes, the more abundant having a mass of 35 amu, and the minor isotope a mass of 37 amu.

Chlorine: 75.77% 35Cl and 24.23% 37Cl

[M]+ at 62[M]+ at 84

[M+2]+ at 64

[M+2]+ at 86 [M+4]+ at 88

From the pattern of the clusters around the molecular ion peak, one can predict the presence ofcertain hetro-atoms in a molecule

FRAGMENTATION PROCESS• In EIMS bombardment by an electron

beam between 10-15 eV results in the formation of molecular ion.

• 50-70 eV molecular ions breakdown into various fragments.

• The fragmentation process is not randomand leads to well-established fragments.

.M+

Similarly, loss of water:- [M-H2O]+. or [M-18]+., loss of carbon mono-oxide:- [M-CO]+. or [M-28]+. etc..

Fragmentation in Alkanes

RCH2-CH2R' RCH2:CH2R'

e 2e

RCH2+.CH2R'.RCH2

+ CH2R' +.RCH2 +CH2R' + AND/OR

.CH3 + [M-15]+[M-CH3]+ OR

Molecular ions are weak, especially for long chain

The driving force is usually the formation of alkenes.

R H R : H=e 2e

R+.H R+ +.

H

Intensity of the fragment ions depends on carbocation stability

R3C+>R2HC+>RH2C+>H3C+

m/z = 43

m/z = 57

[M]+.[M-Me]+[M-43]+

m/z

+ .[M] +[M-1] + [M-2] etc...

+

+

primary secondary

Effect of of Heteroatoms

Cleavage occurs near functional group

R' RH

:X:

H

e 2e

R' RH

:X+.

H

.

R' H

:X+

H

R+

It is easier to ionize non-bonding electrons than s-electrons;because they are in an orbital of higher energy.

Half arrow:one electron movement

Complete arrow:two electrons movement

ALCOHOLSIn 1º and 2º alcohols, the molecular ion is weakIn 3º alcohols, molecular ion is often undetectable

.

H H

:O+

H

R+

1) Cleavage of C-C bond next to the oxygen

1º

H RH

:O+.

H

oxonium ion

.

R' H

:O+

H

R+

2º

.

R' R''

:O+

H

R+

3º

R' RH

:O+.

H

R' RR''

:O+.

Hoxonium ion

oxonium ion

The largest group isexpelled most readily

2) Loss of water is common in alcohols

RR'

H

OH

+ .

RR'

+ .+ H2O

b

g

R

R''

R'

HH:O+.

R

R''

R'

H H:O+ .

R

R''

R'

H H:O: .

+

R

R''

R'

.+

3) Loss of water with alkene

Ethers1) b-cleavage occurs principally

2) Followed by elimination of alkene, if b-H is present

Fragmentation in AlkenesMolecular ion peaks are normally observed

1) The commonest fragmentation in alkenes involvesrupture of the allylic bond (b-to double bond)

R

H

allylic bond

e 2e

R

H+. C

+H

+ R.

C+

H

HH

Loss of one p-electron

Allyl carbocationstabilized by resonance

[M-Me]+69

2) McLafferty rearrangement in alkenesOccurs if the g-carbon has hydrogen on it

H R''

R R'

b

g

e 2e H R''

R R'

+ .

CH2

R

H

R''

R'

+

+ .

The driving force is the formation of the neutral alkene

H R''

R R'

.+

R''

R R'

H

+

.CH2

R

H R''

R'++ .

Fragmentation in Cyclohexenes

RR'

R''

Elimination of neutral molecule by two s-bond rupture;Retro-Diels-Alder (RDA) reaction mechanism followed

e 2e RR'

R''

+ .

RDA

RR'

X

e 2e RR'

X

+ .

RDAR

R'

X +

+ .

diene monoeneThe diene is almost always contains the radical cation;Except when the monoene has a heteroatom/conjugation

RR'

R''+

+ .

Diels-Alder

Radical MechanismR

R'

R''

e 2e RR'

R''

+.

RDA

RR'

R''++

.

RR'

X

e 2eRDA

diene monoeneR

R'

X+.

Carbonyl Compounds1) -cleavage

Y

XO

+ .

O+

X

CY

+O

+

Y

CX+

OCX+ + OCY

+ +

Acylium ion Acylium ion

The driving force is the formation of carbon mono-oxide

2) McLafferty rearrangement in carbonyl compounds (b-cleavage)

Occurs if the g-carbon has hydrogen on it

OH R''

R R'

b

g

e 2eO

H R''

R R'

+ .

O

R

H R''

R'++ .

The driving force is the formation of the neutral alkene

O

RH

+ .

enolketo

Identify the principal fragment ions and propose mechanism for their formation

Aromatic Hydrocarbon GroupsMolecular ion is strong

Benzyl cleavage is the most common fragmentation

+ . CH2

+ . HH

+

H

H+

H

H+

H

H+

Benzyl cationm/z 91

HHH

+ . + .

+m/z 92 through McLafferty rearrangement

Tropylium ion (aromatic)

Aromatic ethersMolecular ion is prominent

Aromatic esters and acids

XHO

O

R

+ .

X

CO

H O R

+ .

+

R = alkyl group or H

Amines

Tropylium ion

Weak molecular ion

[M-Me]+

IONIZATION PROCESSES

EICIFD

FABESI

MALDI