Mass spectrometry

MASS SPECTROMETRY
A.Solairajan,
M.Pharm,1st year,
S.B.C.P.
31-Aug-14 Solairajan 1

What is Mass
Number?
Zoo Zoo

Mass number:-
The Mass number (A), also called atomic mass number or nucleon
number is the total number of protons and neutrons in an atomic
nucleus.
No.of protons
(Z)
No.of
neutrons (N)
Mass Number (A)

Example:-
Carbon atom have 6 protons and 6 neutrons in the centre of
the nucleus, 2 electrons situated in the inner orbital where as
other 4 electrons are outside the orbital.
We can represent carbon atom like
Mass
Number(A)
No. of protons(Z)
Carbon Atom:-

Mass spectrometry is an instrumental
technique in which sample is converted to rapidly moving
positive ions by electron bombardment and charged
particles are separated according to their masses.
Mass spectrum is a plot of relative abundance
against the ratio of mass/charge(m/e).

•To measure relative molecular masses.
• To know the fragmentation of the molecules.
• Comparison of mass spectra with known
compounds.

Organic molecules are bombarded with electron
converted into Highly energetic positively charged ions
(Molecular ions or Parent ions)
Further break up into smaller ions
(Fragment ions or Daughter ions)
The formed ions are separated by Deflection in Magnetic
field according to their Mass and Charge
MASS SPECTRUM

Loss of electron from a molecule leads to radical cation.
e-
Molecular ion
15 eV
70 eV

 Electron removed from molecule orbital having lowest
ionization potential (IP).
 In general n < π < σ
Compounds Ionization potential
CH4 12.6 eV
C2H4 10.52 eV
CH3NH2 10.3 eV
1 eV = 23 Kcal/mol

• Inlet system
• Ion source
Ionisation methods
• Mass Analysers
• Ion Detectors
• Vacuum System

Inlet system
1.Solid
2.Liquid
3.Gas
Mass
spectrometer
Ionisation
ESI,EI,FAB,
CI,FI,FD,
MALDI,PD,
TI
Mass
Analyser
Quadrupole,
TOF,
Ion trap,
FT-ICR
Detector
Photo multiplier,
Electron
multiplier,
Faraday cup,
Microchannel
plate

• SOLIDS SAMPLES with lower vapour pressure
Inlet
system
directly inserted into the ionization chamber and volatilization
is controlled by heating the probe.
• LIQUIDS are handled by hypodermic needles
injection through a silicon rubber dam.
• GASES SAMPLES are leaked into the ionisation chamber
directly by the help of mercury manometer.

Ionisation
 The ion source is the part of the mass spectrometer that ionizes the
material under analysis (the analyte).
 The ions are then transported by magnetic or electric fields to the
mass analyzer.
 Molecular ions are formed when energy of the electron beam
reaches to 10-15 eV.
 Fragmentation of the ion reaches only at higher bombardment
energies at 70 eV.

.
Samples are ionised outside the ion source.
This technique include,
1.Electron impact ionization (EIS)
2.Chemical ionization.(CI)
3.Field ionisation.(FI)
Samples are ionised inside the ion source.
This technique include,
1. Field desorption.(FD)
2. Fast atom bombardment.(FAB)
Ionisation
31-Aug-314. Laser desorption.(LD) Solairajan 19

Ionisation

Electron impact ionisation Thermally volatile and stable 500 Da
Chemical ionisation Thermally volatile and Stable 500 Da
Electro spray ionisation Polar and Basic 70000 Da
Fast atom bombardment Peptides 7000 Da
Field ionisation Thermally volatile 1000 Da
MALDI Large Biomolecules 3,00,000 Da
Plasma desorption Neutral compounds 500 Da
APCI Thermally liable 1000 Da
SIMS Same as FAB 300-13000 Da
Laser desorption Elemental analysis 500 Da

Ionisation
• A beam of electrons passes through a gas-phase sample and collides
with neutral analyte molecules (M) to produce a positively
charged ion or a fragment ion.
• Generally electrons with energies of 70 eV are used to form a
fragment ions.
• The positive ions are collected in focusing plates and passed to mass
analyzer.

Ionisation

• The ESI source consists of a very fine needle and a series of
skimmers.
• A sample solution is sprayed into the source chamber to form
droplets.
Ionisation
•When droplets carry charge exit the capillary end, as the solvent
evaporates, the droplets disappear leaving highly charged
analyte molecules.

Ionisation
• Chemical Impact Ionisation between interactions of sample
with large amount of reagent gas.
• Commonly used reagent gases include
methane,ammonia,isobutane.
• Oxygen and hydrogens are used in Negative ion chemical
ionisation in MS.

Ionisation
The vaporised sample is introduced into the mass spectrometer with
an excess of a reagent gas (methane) at pressure of about 1 torr.
The excess carrier gas is ionized by electron impact to the primary
.+
+
ions CHand CH.
4
3
These may react with the excess methane to give secondary ions.

• In this method the molecule pass through sharp metal anode
carrying an electric field of 1010 v m-1
• Electrons are analysed in primary focusing cathode slit.
• ADV :- abundance of molecular ions.
• DISADV :- lower resolution.
Ionisation

Ionisation
• Useful for nonvolatile and thermolabile compounds.
• Sample is applied to field ion emitter and the solvent allowed to
evaporate.
• Evaporated sample that leads to chemical ionisation or EIS.
• Example:-Nucleotides & Quarternary ammonium compounds.

• Argon gas ionised by hot filament and focused beam that
bombards the sample.
Ionisation
• Beam impinges the sample, a series of molecular reactions occur
and analyse in MS analyser.
• Ex:-Insulin,Amino glycosides,Phospholipids.

Ionisation
• Sample is coated with a high energetic fragment Californium 252.
• This fission fragment desorbs positive,negative, and neutral molecules.
• 252cf generates 1012 power at 10,000k, this may ionise the target
molecule.
• This method involves the interaction of laser beam with sample to
produce both vaporisation and ionisation.
• The vaporised sample passed to mass spectrometers for analysis.
• Appl:-used for elemental analysis. 31-Aug-14 Solairajan 30

Ionisation
 MALDI is a LIMS method of vaporizing and ionizing and sample
molecules are dispersed in a solid matrix such as nicotinic acid.
 A UV laser pulse ablates the matrix which carries some of the
large molecules into the gas phase in an ionized form so they can be
extracted into a mass spectrometer.
MALDI

Atmospheric pressure chemical ionisation (APCI) is an
analogous ionisation method to chemical ionisation(CI).
Corona discharge is used to ionize the analyte in the
atmospheric pressure region.
Ionisation

Ionisation
 Secondary ion mass spectrometry (SIMS) is based on the
observation that charged particles (Secondary Ions) are ejected
from a sample surface when bombarded by a primary beam of
heavy particles.
 Primary beam species useful in SIMS include Cs+, O2+, O ,
Ar+, and Ga+

An ion, after leaving ion source, the ions are separated
according to their m/e ratio.
Mass
Analyzer
In this area, the ions are accelerated by both electrostatic
and magnetically
Types:-
Magnetic sector mass analysers
Double focussing analysers
Quadrupole mass analysers
Time of Flight analysers (TOF)
Ion trap analyser
Ion cyclotron analyser

m/z= H2r2/2V
H Magnetic field
R Radius of the curvature
Mass
Analyzer
31-Aug-1V4 AppliedSo lavirajoan ltage 35

It contains two analysers namely
•Electrostatic analyser
•Magnetic sector analyser.
Mass
Analyzer

Mass
Analyzer
The quadrupole consists of two pairs of parallel rods with applied
DC and RF voltages.
Ions are scanned by varying the DC/Rf quadrupole voltages.

Mass
Analyzer

Mass
Analyzer
TOF analyzer – ions are accelerated through a flight tube and
the time of fight to the detector is measured.
Typical flight times are 1 to 50μs.

The quadrupole ion trap typically consists of a ring
electrode and two hyperbolic end cap electrodes.
Mass
Analyzer
As the radio frequency voltage is increased, the orbits of
heavier ions become stabilised,and passed into the detector.

Mass
Analyzer
 Fourier transform-ICR mass spectrometry, is a type of mass
analyzer for determining the mass-to-charge ratio (m/z) of ions
based on the cyclotron frequency of the ions in a fixed magnetic
field.
The ions are trapped in a Penning trap(a magnetic field with
electric trapping plates) where they are excited to a larger
cyclotron radius by an oscillating electric field perpendicular to the
magnetic field.

The excitation also results in the ions moving in phase (in a packet).
The signal is detected as an image current on a pair of plates which the
packet of ions passes close to as they cyclotron. The resulting signal is
called a free induction decay (FID), transient or interferogram that
converts signal.
The useful signal is extracted from this data by performing a Fourier
transform to give a mass spectrum.

Faraday cup
Electron Multiplier
photomultiplier
Micro Channel Plate
Detector

Detector
 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 CsSb, GaP or BeO.
 It is ideally suited to isotope analysis.
Faradaycup

Detector
 Electron multipliers are the most common especially when
positive and negative ions need to be detected on the same
instrument.
 Dynodes made up of copper-beryllium which transduces the
initial ion current ,and electron emitted by first dynode are focused
magnetically from dynode to the next.
 Final cascade current is amplified more than million times.
Electron
multipliers

Detector
 The dynode consists of a substance( a scintillator) which emits
photons(light).
 The emitted light is detected by photo multiplier tube and is
converted into electric current.
 These detectors are useful in studies on metastable ions

Detector

All mass spectrometers need a vacuum to allow ions to
reach the detector without colliding with other gaseous molecules
or atoms. If such collisions did occur, the instrument would suffer
from reduced resolution and sensitivity.

TYPES OF PEAKS IN MS
•Molecular ion peak
•Fragment ions peak
•Rearrangement ions peak
•Metastable ion peaks
•Multicharged ions
•Base peak
•Negative ion peak
Molecular ion Peak:-
When a sample is bombarded with electrons of 9 to 15 eV
energy, the molecular ion is produced, by loss of single electron.
M e-
M
+
+ 2 e
-

Fragment ions Peak:-
when an energy is given further more upto 70 eV,
fragment ions produced, it have lower mass number.
Rearrangement ion Peak:-
Recombination of fragment ion is known as
Rearrangement Peaks.
Metastable ion Peak:-
The ions resulting from the decomposition between the
source region and magnetic analyser are called as Meta stable
ions.These appear as broad peaks called Metastable ion Peaks.
Multicharged ions:-
Ions may exist with 2 or 3 charges instead of usual
single charge.The peaks due to these charged ions are known as
Multicharged ion peaks.

Base Peak:-
The largest peak in the mass spectrum corresponding
to the most abundant ion or most intense peak in the spectrum is
called as Base Peak.
Negative ion Peak:-
Negative ions are formed from electron bombardment
of sample. These results due to the capture of electron by a molecule
during collision of molecules
Fragment ion
peak

FRAGMENTATION
Fragmentation is a type of chemical dissociation.
Fragmentation takes place by a process of heterolysis or homolysis.
Types of Fragmentation:-
• Collision induced dissociation(CID)
• Electron capture dissociation(ECD)
• Electron transfer dissociation(ETD)
• Electron detachment dissociation(EDD)
• Photo dissociation
Infrared multiphoton dissociation(IRMPD)
Blackbody infrared radiative dissociation(BIRD)
• Surface induced dissociation(SID)
• Charge remote fragmentation
• Higher energy C-trap dissociation(HCD)

Collision Induced Dissociation
 Molecular ions are accelerated by electrical potential to high
kinetic energy and then allowed to collide with neutral molecules like
He,N or Ar.
 Collision between these molecules leads to bond breakage and
formation of fragment ions.
 These fragment ions are analysed by mass spectrometer.
 Example:- Triple quadrupole spectrometer produces CID
fragments.

SORI-CID:-(Sustained Off-Resonance Irradiation Collision-Induced
Dissociation)
 It is one of CID technique used in Fourier transform ion
cyclotron resonance mass spectrometry.
 In this method accelerating ions in cyclotron motion and
increasing the pressure resulting collisions produce CID fragments.

Electron Capture Dissociation:-
 It is a method of fragmenting gas phase ions for tandem mass
spectrometric analysis (structural elucidation).
 ECD involves the direct introduction of low energy electrons to
trapped gas phase ions.
 Electron-capture dissociation typically involves a multiply
protonated molecule M interacting with a free electron to form an odd-electron
ion.

Electron Transfer Dissociation:-
ETD induces fragmentation of cations by transferring electrons to them.
Example:-peptides or proteins.
Electron Detachment Dissociation:-
EDD is a method for fragmenting anionic species in mass spectrometry.

Photo Dissociation:-
Photodissociation is a chemical reaction in which a chemical compound is
broken down by photons.
IRMPD:-
Absorption of multiple infra red photons by a molecule and leads to
dissociation.
BIRD:-
Long interaction of molecule with radiation field like carbon dioxide laser.
Surface-induced dissociation:-(SID)
It is a technique used in mass spectrometry to fragment molecular ions in the
gas phase by collision of an ion with a surface under high vacuum.

Charge Remote Fragmentation:-
It is a type of covalent bond breaking that occurs in a gas
phase ion in which the cleaved bond is not adjacent to the location of the
charge.
This fragmentation can be observed using tandem mass
spectrometry.
Higher-energy C-trap dissociation:-(HCD)
It is a fragmentation technique, used
for peptide modification analysis.
Immonium ions generated via HCD pinpoint modifications such
as phospho tyrosine.
An added octopole collision cell facilitates de novo sequencing.

Fragmentation of the Molecular ion
Fragmentation of a molecular ion, M, produces a
radical and a cation.
-Only the cation is detected by MS.
A-B
A
Radical Cation
+
A
B
B
• +
Molecular ion
(a radical cation)
+ •
+ •
+
Cation Radical

Description of Fragmentation process:-
Fragmentation of the odd electron molecular ion (M
.+
) may
occur by Homolytic or Heterolytic cleavage of a single bond.

Mass interpretation
Fragmentation rules
Mclafferty rearrangement
Alpha cleavage
Beta cleavage
Nitrogen rule
Retro diels alder reaction
IHD

Fragmentation rules:- (9 rules)
Rule:-1
The height of the M
.+
peak decreases with increasing degree
of branching.

Rule:-2
The height of the M
.+
Peak decreases with
increasing molecular weight.
Example:- Fatty molecules, steroids.

Rule:-3
The cleavage is favored at alkyl substituted carbons
leads to formation of a carbocation.

Rule:-4
Double bonds, cyclic structures and aromatic rings
stabilize M
.+
and increase the probability of its appearance.
Molecular ion peak
& Base peak

Rule:-5
Double bonds favor allylic cleavage to
give the resonance stabilized cation.

Rule:-6
Saturated rings tend to lose alkyl side chains at the α bond (see
rule 3)
Unsaturated rings can undergo a Retro-Diels-Alder reaction

Rule:-7
Alkyl substituted aromatic compounds are cleaved
preferably at the β bond to the ring, giving the resonance stabilized
benzyl ion (or) tropyllium ion.

Rule:-8
C-C bonds next to hetero atom are frequently
cleaved, leaving the charge on the hetero atom (resonance
stabilization).

Rule:-9
Cleavage is often associated with elimination of small
stable, neutral molecules, such as
CO,olefins,water,ammonia,H2S,HCN,ketene or alcohols (often with
rearrangements)
Ex:-Mclafferty rearrangement

Mclafferty Rearrangement:-
 Mclafferty arrangement can occur in
ketones,aldehydes,carboxylic acids, and esters.
 In this rearrangement a radical center in molecular ion derived
from a lone pair or pi bond, removes hydrogen from the Gamma
position(γ), a pi bond is formed between the β and γ position, and
the bond between the α and β positions is broken.

α cleavage
 Alpha cleavage in mass spectrometry is a
characteristic fragmentation of the molecular ion derived
from carbonyl compounds, in which the bond linking the carbonyl
carbon to the atom occupying an alpha position breaks.
It is an expected pathway for carbonyl
compounds,ethers,halides,alcohols,and amines.

β cleavage
Beta cleavage in mass spectrometry is
a characteristic fragmentation of the molecular ion derived
from some organic compounds, most
notably alcohols, ethers, and amines, in which the bond
connecting alpha- and beta-carbons break.

Retro-Diels-Alder reactions:-
 Retro Diels-Alder fragmentation occurs in 3-cyano-cyclohexene,
lets first look at the fragmentation of cyclohexene.
 First ionization occurs and electrons from the double bond
transfer to an adjacent carbon and an electron from the bond
between the 3 and 4 carbons transfers to form a second double bond
that is conjugate with the first one.
These rearrangements cleave the molecule between the 3 and 4
carbon and 5 and 6 (where another electron is transferred to form a
double bond between the 4 and 5 carbons). This leaves an olefin and
a diene.

3
6
2
1
4
5
4
5
Diene Olefin

Nitrogen rule:-
The nitrogen rule states, that a molecule that has no or even
number of nitrogen atoms has an even nominal mass, whereas a
molecule that has an odd number of nitrogen atoms has an odd
nominal mass.
Example:-1 Example:-2

Contd….
The molecular ion appears at m/z 121, indicating an odd number
of nitrogen atoms in the structure.
Odd number of
molecular ion

IHD:-
 In a hydrocarbon where all carbon atoms have only single bonds
and no rings are involved, the compound would have maximum
number of H atoms.
 If any of the bonds are replaced with double or triple bonds, there
would be deficiency of H atoms. By calculating the index of
hydrogen deficiency(IHD), we can calculate molecular formula and
how many multiple bonds and rings are involved. IHD is also called
the Degree of Unsaturation.
 A double bond and ring each counts as one IHD.
 A triple bond counts as two IHD.

Example:-1
Example:-2
CH2=CH2

Mass Spectrum of compounds:-
Alkane:-
Base Peak
Molecular ion
peak

Fragmentation of Cyclo Hexane:-
C6H12
+
= 84 (Molecular ion Peak),
C4H8
+
= 56 (Base Peak), (M-28)
C6H9
+
= 69 (Fragment ion Peak), (M-15)
C3H7
+
= 43 (Fragment ion Peak), (M-41)
C2H5
+
= 29 (Daughter ion Peak),
CH3
+
= 15 (Daughter ion Peak).

Alcohol
Possible Fragmentations are:- Mol.wt-46
C2H5OH+ =46 (Molecular ion peak)
+
CHO
= 31 (Base Peak)
3+
CHO
= 27 (Fragment ion Peak)
CH3
+
= 15 (Daughter ion Peak)
Base Peak
Molecular
ion Peak
Fragment
Daughter ion Peak
ion Peak

Aldehyde:-
Molecular formula:-C6H12O
Molecular Weight:-100
+
C6H12O
= 99 (Molecular ion Peak)
C3H8
+
= 44 (Base Peak)
C4H9
+
C2H5
+
Fragment ion
Peak
Base Peak- Mclafferty
rearrangement
Molecular ion
Peak- α cleavage

Amide:-
Molecular wt :- 87,
Molecular formula :- CHNO
49+
CHNO
49= 87 (Molecular ion Peak),
+
C2H5NO
= 59 (Base Peak)
Fragment ion Peak-
α,β cleavage
Base Peak-
Mclafferty
rearrangement
Molecular ion
Peak-β cleavage

Amine:-
Molecular wt:-59
Mol.formula :-C3H9N
Base Peak-β-H
transfer
Molecular ion Peak-
β-H transfer

Ester
Mol.wt:-102,
Mol.formula:-C5H10O2
Base Peak-α
cleavage
Molecular ion Peak
α-cleavage

Ether
Mol.wt:-130
Mol.formula:-C8H18O
Base Peak-ipso
cleavage
α cleavage Molecular ion
Peak-α cleavage

GC-MS
Gas chromatography–mass spectrometry (GC-MS) is a
method that combines the features of gas-liquid chromatography
and mass spectrometry to identify different substances within a test
sample.
Applications of GC-MS include :-
Drug detection,
Fire investigation,
Environmental analysis,
Explosives investigation, and
Identification of unknown samples.

GC-MS

Tandem MS:-
What is Tandem MS:-
-Uses 2 (or more) mass analyzers in a single instrument.
-One purifies the analyte ion from a mixture using a
magnetic field.
-The other analyzes fragments of the analyte ion for
identification and quantification.

Tandem mass spectrometry, also known
as MS/MS or MS2, involves multiple steps of mass spectrometry
selection, with some form of fragmentation occurring in between
the stages.

Components of Tandem Mass
Spectrometer
MS-1 Collision cell MS-2

Applications of Tandem MS
Biotechnology & Pharmaceutical
 To determine chemical structure of drugs and drug metabolites.
 Detection/quantification of impurities, drugs and their metabolites
in biological fluids and tissues.
 Analysis of liquid mixtures
 Fingerprinting
Nutraceuticals/herbal drugs/tracing source of natural products
or drugs
Clinical testing & Toxicology
 Inborn errors of metabolism, cancer, diabetes, various poisons,
drugs of abuse, etc.

MALDI-MS
Matrix-assisted laser desorption/ionization (MALDI) is a
soft ionization technique used in mass spectrometry allowing the
analysis of biomolecules (biopolymers such
as DNA, proteins, peptides and sugars) and
large organic molecules (such as polymers, dendrimers and
other macromolecules).

 MALDI is based on the bombardment of sample molecules
with a laser light to bring about sample ionisation.
 The sample is pre-mixed with a highly
absorbing matrix compound for the most consistent and reliable
results.
 The matrix transforms the laser energy into excitation
energy for the sample, which leads to sputtering of analyte and
matrix ions from the surface of the mixture.
 Most commercially available MALDI mass spectrometers
now have a pulsed nitrogen laser of wavelength 337 nm.

Common matrix in MALDI
Matrix Solvent Applications
2,5-dihydroxy benzoic
acid
Acetonitrile,water,metha
nol,acetone,CHcl3
Peptides,Nucleotides,
oligo nucleotides
3,5-dimethoxy-4-
hydroxycinnamic acid
Acetonitrile,
water,acetone, CHcl3
Peptides,proteins,lipids
4-hydroxy-3-
methoxycinnamic acid
Acetonitrile, water,
propanol
Proteins
Picolinic acid Ethanol Oligo nucleotides

Applications of MS:-
Elucidation of the structure of the organic and biological molecules.
Determination of molecular mass of peptides, proteins, and
Oligonucleotides.
Monitoring gases in patients breath during surgery.
Identification of drugs abuse and metabolites of drugs of abuse in
blood, urine, and saliva.
Analyses of aerosol particles.
Determination of pesticides residues in food.

References:-
Instrumental methods of chemical analysis by willard
Organic spectroscopy by William kemp
Spectroscopic identification of organic compounds by Silverstein
Instrumental analysis by skoog
Wikipedia
ww2.chemistry.gatech.edu/class/4341-6371/fahrni/set02.pdf

By
A.Solairajan

Mass spectrometry

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