Mass Spectrometry

Dr. Mohammad Shariare

 OUTLINE
Introduction to Mass Spectrometry

Ionization Methods

Mass Analyzer

Fragmentation and MS Interpretation

MS Techniques
Definition of Mass Spectrometry
An analytical technique which is used for the
determination of the composition of a sample or
molecule and elucidation of the chemical structures
of molecules, such as peptides and other chemical
compounds.

Mass spectrometry has been described as the

smallest scale in the world, not because of the mass
spectrometer’s size but because of the size of what it
weighs -- molecules.
 Mass Spectrometry
• Analytical method to measure the
molecular or atomic weight of samples
 Timeline for MS Development
1897 Early Mass Spectrometry
1919 The observation of isotopes using mass spectrometry
2000 Orbitrap
2004 Desorption Electrospray Ionization (DESI) Joseph John Thomson

2004 Electron Transfer Dissociation (ETD) "In recognition of the great merits
of his theoretical and experimental
2005 Direct Analysis in Real Time (DART)
investigations on the conduction of
electricity by gases.“
1906 Nobel Prize
Francis William Aston
"For his discovery, by means of his mass
spectrograph, of isotopes, in a large number
of non-radioactive elements, and for his
enunciation of the whole-number rule."

Mass spectrometry of isotopes

1922 Nobel Prize Replica of J.J. Thomson's third mass spectrometer.
 What information can be
determined?
• Molecular weight
• Molecular formula
• Structure (from fragmentation fingerprint)
• Isotopic incorporation / distribution
• Protein sequence
 Mass Spectrometry 
• Basic principle: Mass spectrometry is the most accurate
method for determining the molecular mass of the compound
and its elemental composition. In this technique, molecules
are bombarded with a beam of energetic electrons. The
molecules are ionized and broken up into many fragments,
some of which are positive ions. Each kind of ion has a
particular ratio of mass to charge, i.e. m/e ratio (value). For
most ions, the charge is one and thus, m/e ratio is simply the
molecular mass of the ion.
• The set of ions (fragment ions or daughter ions) are analyzed in such
a way that a signal is obtained for each value of m/e that is
represented. The intensity of each signal represents the relative
abundance of the ion producing the signal. The largest peak in the
structure is called the base peak and the intensity is taken as 100.
The intensities of other peaks are represented relative to the base
peak.
Mass spectrum: Mass spectrum of a compound is a plot, which represents the
intensities of the signals at various m/e values. It is highly characteristic of
a compound. No two compounds can have exactly similar mass spectra.

Utility: Mass spectra is used in two general ways:

(a) To prove the identity of two compounds.

(b) To establish the structure of a new compound.

The mass spectrum of a compound helps to establish the structure of a new

compound in several ways:

(a)  It can give the exact molecular mass.

(b) It can give a molecular formula or it can reveal the presence of certain
structural units in a molecule.
 Theory
A parent ion results when one electron is removed from the parent
molecule of the substance: M (g) + e M+(g) + 2e.

  The m/e value of the parent ion is equal to the molecular mass of
the compound. The mass spectrometer is designed to perform
three basic functions. These are:

(i) To vaporize compounds of varying volatility.

(ii) To produce ions from the neutral compounds in the vapor phase.

(iii) Separate the ions according to m/e value and detect ions

 
 Background
• The impact of a stream of high energy
electrons causes the molecule to lose an
electron forming a radical cation.
– A species with a positive charge and one unpaired
electron

H H
- -
H C H + e H C H + 2e
H H
Molecular ion (M+)
m/z = 16
 Background
• The impact of the stream of high energy electrons
can also break the molecule or the radical cation
into fragments.
H H
+
H C C H molecular ion (M ) m/z = 30
H H

H H H H
-
H C C H + e + H
H C C
H H H H
m/z = 29

H H
H C + C H (not detected by MS)
m/z = 15 H
H
 Background
• Molecular ion (parent ion):
– The radical cation corresponding to the mass of
the original molecule
H H H
H C H H C C H
H H H

• The molecular ion is usually the highest mass

in the spectrum
– Some exceptions w/specific isotopes
– Some molecular ion peaks are absent.
 Background
• Mass spectrum of ethanol (MW = 46)

M+

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Industrial Science and Technology, 11/1/09)
 Background
• Only cations are detected.
– Radicals are “invisible” in MS.

• The amount of deflection observed depends on

the mass to charge ratio (m/e).
– Most cations formed have a charge of +1 so the
amount of deflection observed is usually
dependent on the mass of the ion.
 Background
• The resulting mass spectrum is a graph of the
mass of each cation vs. its relative abundance.

• The peaks are assigned an abundance as a

percentage of the base peak.
– the most intense peak in the spectrum

• The base peak is not necessarily the same as

the parent ion peak.
 Background
The mass spectrum of ethanol
base peak Molecular
M+ ion peak

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Industrial Science and Technology, 11/1/09)
 Instrumentation:
  The instrument needed to produce the mass spectrum of a compound consists
of the following parts: a) Ion source (b) Mass analyzer (c) Ion detector

(a) Ion source: The common technique used for the production of ion in mass
spectrometer is by the bombardment of moleculs by electron beam produced by an
electrically heated tungsten filament. The minimum energy required to ionize an
atom or a molecule is called ionization potential. The bombarding electrons have
energy of about 70 eV whereas a minimum ionization potential is 10 eV. Due to
bombardment, the molecules generally lose one electron to form a parent radical.
The 70 eV energy is sufficient to produce parent ion along with the fragment ions or
daughter ions. The positive ion (fragment) whether even electron or odd electron
species is detected in the mass spectrometer whereas the neutral molecules are left
undetected.
Schematic Mass Spectrometer

Sample

+
_

Ionizer Mass Analyzer Detector

b) Mass analyzer: The positively charged ion (parent or fragment ions)
produced in the ion chamber are accelerated by applying an
acceleration potential which travels through the whole of the analyzer
portion of the mass spectrometer with high velocity and are separated
according to their m/e ratio.

(c) Ion detector: The ions which are separated by the analyzer are
detected and measured electrically or photographically. The ions pass
through the collecting slit one after the other and fall on the detector.
The ion currents are amplified by using a direct current amplifier. The
spectrum is recorded by using a fast scanning oscillograph.
• The cations that are formed are separated by magnetic
deflection.
 How does a mass spectrometer work?

Create ions Separate ions Detect ions

• Ionization • Mass analyzer • Mass

method
– MALDI-TOF spectrum
• MW
– MALDI – Triple Quadrapole • Database
– Electrospray • AA seq analysis
(Proteins must be – MALDI-QqTOF
charged and dry) • AA seq and MW
– QqTOF
• AA seq and
protein modif.
 The molecular ion or parent ion

The electron bombardment with energy 10-15 eV usually removes

electron from the molecule of the organic compound in the vapor
phase. It results in the formation of molecular ion. An electron from
double bond (two p-electrons) or triple bond (four p-electrons) is
usually lost. In alkanes, the ionization of C-C sigma bonds is easier
than that of C-H bonds.
 What’s in a Mass Spectrum
Mass-to-charge ratios of a molecule or its fragment are
graphed or tabulated according to their relative abundance

Fragment Ions

Fragment Ions: derived from molecular ion or higher weight fragments

 Applications
Biomolecule
Pharmaceutical
characterization
analysis
Proteins and peptides
Oligonucleotides

Paleoclimatology and Archeology

http://www.sciencemag.org/products/lst_20060901.dtl
Paleotemperature
Forensic analysis/clinical O16 and O18

Environmental analysis
Pesticides on foods
Soil and groundwater contamination
Important features of the Molecular/parent ion
peak
(a) The molecular ion peak in aromatic compounds is relatively much intense
due to the presence of p-electrons.

(b) Conjugated olefins show more intense molecular ion peak as compared to the
corresponding non-conjugated olefins with the same number of unsaturation.
Conjugated olefins are more stable than the corresponding non-conjugated
olefins.

(c) Unsaturated compounds give more intense peak as compared to the saturated
or the cyclic molecule.
(d) The relative abundance of the saturated hydrocarbon is more than the
corresponding branched chain compound with the same number of carbon
atoms. For example, the molecular ion peak for n-pentane is more intense
than that of neopentane.

(e) The substituent groups like –OH, -OR, -NH2 etc. which lower the
ionization potential increase the relative abundance in case of aromatic
compounds. Also the groups like –NO2, -CN etc. which increase the
ionization potential, decrease the relative abundance of the aromatic
compounds.

(f) Absence of molecular ion peak in the mass spectrum means that the
compound under examination is highly branched of tertiary alcohols.
(g) In case of chloro or bromo compounds, isotope peaks are also formed along with the
molecular ion peak. In case of bromo compounds, M+ and M+ + 2 peaks are formed in the
intensity ratio 1:1. In case of chloro compounds, M+ and M+ + 2 peaks are formed in the
intensity ration 1: 3.

 
 Bromine:
 M+ ~ M++2 (50.5% 79Br/49.5% 81Br)

2-bromopropane

M+ ~ M+2
Easily Recognized Elements in MS
• Chlorine:
– M++2 is ~ 1/3 as large as M+
Cl M+

M+2

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Industrial Science and Technology, 11/2/09)
Relative Abundance of Isotopes
Atomic weight of an element is a weighted average
of the naturally occurring isotopes.
Important: Nitrogen containing compounds with an odd number of nitrogen
atoms in the molecule must have an odd molecular mass. An even number of
nitrogen atoms or absence of nitrogen atoms in the compound show that the
molecular mass of the compound must be even.

• Nitrogen:
– Odd number of N = odd MW
+
M = 41
 Metastable Ions or Peaks
Metastable ion peaks generate if the transition from parent ion
M+ to daughter ion m+ occur after the ion source and before
arrival at the collector.

Metastable peaks can be easily determined in a mass

spectrum because of their characteristics, such as -
• They do not necessarily occur at the integral m/e values

• They are much broader than the normal peaks and

• They are of relatively low abundance

 Ionization Methods
• Electron bomb Ionization (EI)

• Chemical Ionization (CI)

• Field ionization (FI)

• Matrix Assisted Laser Desorption Ionization (MALDI)

• Fast atom bombardment (FAB)

• Electro Spray Ionization (ESI)

 Electron Bomb Ionization ( EI )
Sample is heated and energized by a beam of electrons, usually gives a
molecular ion (M+) and a lot of fragments.

• Hard ionization
• Electron ionization leads to fragmentation of the molecular ion, which
sometimes prevents its detection.
 Chemical ionization (CI):
A technique that produces ions with little excess energy.
Thus this technique presents the advantage of yielding a spectrum with
less fragmentation in which the molecular species is easily recognized.
Consequently, chemical ionization is complementary to electron
ionization.

Disadvantages:
• Need Volatile Sample
• Need Thermal Stability
• Quantitation Difficult
• Low Mass Compounds (<1000 amu)
• Solids Probe Requires
• Skilled Operator
 Field ionization (FI)
Field ionization (FI) is a method that uses very strong electric
fields to produce ions from gas-phase molecules.

+
+ + +
+ + + + +
- - + + +
- + - + +
+ - + - +
- + - ++ + + + +
+ + + +
- - + -+ - + + +
+
- + + + + +
+ +
+ + +
+ +
+
+ +
+
 Matrix Assisted Laser Desorption
Ionization (MALDI)
Sample is co-crystallized with a matrix and then irradiated
with laser.

MALDI is achieved in two steps. In the first step, the

compound to be analyzed is dissolved in a solvent
containing in solution small organic molecules, called
the matrix. The second step occurs under vacuum
conditions inside the source of the mass spectrometer.
 Fast atom bombardment ( FAB)
Softer than EI and CI. Ions are produced by bombardment with
heavy atoms. Gives (M+H)+ ions and little fragmentation.
Good for more polar compounds.

Ar + e Ar+ acceleration (5-15 KeV)

Ar+ + Ar Ar + Ar+

fast slow + 8 KeV

fast slow
ElectroSpray Ionization (ESI)
Electrospray is abbreviated to ESI, sample is sprayed out
of a narrow nozzle in a high potential field. Generates
positive (M+nH)n+ and negative (M - nH)n- ions and
almost no fragmentation. Generates multiple charged ions.
 Types of Mass Analyzers

Magnetic sector analyzer

Time of Flight analyzer (TOF)

Quadrupole analyzers
Fourier Transform Ion-Cyclotron
 Magnetic Sector Analyzer
Magnetic sector analyzer – Uses electric and/or
magnetic fields to separate ions
 Principle of Magnetic
Sector Analyzer
Only ions of mass-to-charge ratio that have equal
centripetal and centrifugal forces pass through the
flight tube.
It shows that the m/e ratio of the ions that reach
the detector can be varied by changing either the
magnetic field or the applied voltage of the ion
optics.
 Time of Flight Analyzer
TOF analyzer – ions are accelerated through a flight tube
and the time of flight to the detector is measured
Quadrupole Analyzers

Quadrupole analyzers – ions

are filtered or trapped in a
device consisting of several
metal rods using specifically
tailored electromagnetic
fields
 Quadrupole Analyzers
• Electric/magnetic fields trap, store, eject ions
• Requires an in-line quadrupole to act as
mass pre-filter
• Varying RF frequency will vary the m/e ratios
that are trapped
• Additional fragmentation can be performed
on ions stored in the ion trap
Fourier Transform Ion Cyclotron
Resonance (FT ICR) analyzers
Most FTICR mass spectrometers use superconducting
magnets, which provide a relatively stable calibration over a
long period of time.
• Advantages
The highest recorded mass resolution of all mass
spectrometers (>500,000)
Very good accuracy (<1ppm)
Well-suited for use with pulsed ionization methods such as
MALDI
Non-destructive ion detection; ion remeasurement
Stable mass calibration in superconducting magnet FTICR
systems
Fragmentation patterns in MS

Fragmentation leads to smaller ions by the cleaving of parts of molecule

The fragmentation and relative abundance of the fragment ion depends upon –
1)The stability of the ion and
2)The stability of the radical lost
The stability of the ion can be judged by stabilization of the charge which depends
upon –
a)Resonance b) inductive effect c) Polarisability

The radical site is reactive and can form a new bond. The formation of a
new bond is powerful driving force for ion decomposition. The energy released
during bond formation is available for the cleavage of some other bonds in the ion.
Some important fragmentation modes are described in next slides
47
1. Simple cleavage

Homolytic cleavage –
Mode I, Mode II, Mode III
Heterolytic cleavage

2. Other Cleavage pattern-

Retro-Diels Alder reaction
Hydrogen transfer rearrangement
Mclafferty rearrangement ion

48
Examples:

71

57
43

29
O

CH3 CH2 CH2 CH2 CH2 C OH

45

59
73

87
49
 Loss of small molecules, such as H2O, CO, C2H4

C6H13 H2O + C6H13

H HO

OH
H
H2C CHCH3 + H2O + CH2=CH2
CH3

50
 Rule of Thirteen
• The “Rule of Thirteen” can be used to
identify possible molecular formulas for an
unknown hydrocarbon, CnHm.

– Step 1: n = M+/13 (integer only, use

remainder in step 2)

– Step 2: m = n + remainder from step 1

 Rule of Thirteen
• Example: The formula for a hydrocarbon
with M+ =106 can be found:

– Step 1: n = 106/13 = 8 (R = 2)

– Step 2: m = 8 + 2 = 10

– Formula: C8H10
 Rule of Thirteen
• If a heteroatom is present,
– Subtract the mass of each heteroatom from
the MW
– Calculate the formula for the corresponding
hydrocarbon
– Add the hetero atoms to the formula
Example: A compound with a molecular ion peak at m/z = 100 has a
strong peak at 1720 cm-1 in its IR spectrum. Determine its molecular
formula.
 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.
 Fragmentation Patterns
• Mass spectrum of 2-methylpentane
 Fragmentation Patterns
• Alkenes:
– Fragmentation typically forms resonance
stabilized allylic carbocations
 Fragmentation Patterns
• Aromatics:
– Fragment at the benzylic carbon, forming a
resonance stabilized benzylic carbocation (which
rearranges to the tropylium ion at m/z = 91)

H H
H
H C Br H C
H C

or

M+
 Fragmentation Patterns
Aromatics may also have a peak at m/z = 77 for the
benzene ring.

77
NO2

M+ = 123
77
 Fragmentation Patterns
• Alcohols
– 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 Patterns
• MS for 1-propanol
CH3CH2CH2OH

H2C OH

M+-18 M+

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Industrial Science and Technology, 11/28/09)
 Fragmentation Patterns
• Amines
– Odd M+ (assuming an odd number of
nitrogens are present)
 -cleavage dominates forming an iminium ion

CH3CH2 CH2 N CH2 CH2CH2CH3 CH3CH2CH2N CH2

H H
m/z =72
iminium ion
Fragmentation Patterns
86

CH3CH2 CH2 N CH2 CH2CH2CH3

H

72
 Fragmentation Patterns
• Aldehydes (RCHO)
– Fragmentation may form acylium ion
RC O

– Common fragments:

• M+ - 1 for RC O

• M+ - 29 for R (i.e. RCHO - CHO)

 Fragmentation Patterns
• MS for hydrocinnamaldehyde
105 91

H H O
C C C H
M+ = 134
H H
133 105
91

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Industrial Science and Technology, 11/28/09)
 Fragmentation Patterns
O
RCR'
• Ketones
– Fragmentation leads to formation of acylium
ion:

• Loss of R forming R'C O

• Loss of R’ forming RC O
 Fragmentation Patterns
O
CH3CCH2CH2CH3
• MS for 2-pentanone

CH3C O

CH3CH2CH2C O

M+

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Industrial Science and Technology, 11/28/09)
 Fragmentation Patterns
• Esters (RCO2R’)
– Common fragmentation patterns include:
• Loss of OR’
– peak at M+ - OR’

• Loss of R’
– peak at M+ - R’
 Frgamentation Patterns
77 105

O
C O CH3
77
M+ = 136
105

SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced

Industrial Science and Technology, 11/28/09)
Carboxylic acid

Ch3(CH2)4CO (small) 99
CH3(CH2)4 71
CH3(CH)3 57

CH3(CH2)2 43
O
CH3CH2 29
CH3 CH2 CH2 CH2 CH2 C OH
45 CO2H

59(small) CH2CO2H

73 (CH2)2CO2H

87 (CH2)3CO2H

69
Mass Techniques

Chromatography: Separation

Mass: Detection

Chromatography-Mass Spectroscopy :
Separation + Detection 43

57
29 71
15 85
99 113 142
GC-MS LC-MS m/z
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.
HEWLETT Mass
5972A Selective
PACKARD Detector

1.0
DEG/MIN

MS
HEWLETT
PACKARD

5890

Sample Gas Chromatograph (GC) Mass Spectrometer

B
D A
A C B
D
A C
B
DB C
A
C D

Sample Separation Identification

GC-MS

GAS CHROMATOGRAPHY MASS SPECTROMETRY

LC-MS
Liquid chromatography-mass spectrometry (LC-MS) is an analytical
chemistry technique that combines the physical separation capabilities of
liquid chromatography with the mass analysis capabilities of mass
spectrometry.
Different compounds exit Identification of each molecule
at different time ion

LC MS

B
Peak A: mass1
A Peak B: mass2
C
Peak C: mass3

t/min
LC-MS

Liquid chromatography-mass spectrometry (Ion trap LCMS system )

 Exercise 1:

Analysis shows exact mass of compound A is 136.0886 and the formula of this
compound is C9H12O, please confirm the structure of compound A.

Answer

107
100
m/z: 136 M-18 M-H2O
% OF BASE PEAK

79 m/z: 118
77 77
51
50
107 M-29 M-C2H5
41 136
39 118
-C2H5

0 20 40 60 80 100 120 140 160 H2

C CH
CH 3

OH

75