Definition of Mass Spectrometry

Mass spectrometry (MS) :

An analytical technique by using mass spectrometry
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
spectrometers size but because of the size of what it
weighs -- molecules.

What information can be determined?

Molecular weight
Molecular formula (HRMS)
Structure (from
fragmentation fingerprint)
Isotopic incorporation /

distribution
Protein sequence (MS-MS)

Rule of Thirteen
Calculating Mass
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

Nitrogen Rule
This rule states that if a compound has an even no.of N
atoms (or no N atom) , its molecular ion will appear at
an even mass value.
On the other hand , a molecule with an odd no.of N
atom will form a molecular ion with an odd mass.
The N rule stems from the fact that N , although it has
an even mass , has an odd numbered valence.
Consequently , an extra hydrogen atom is included as a
part of a molecule , giving it an odd mass.

Nitrogen Rule
To picture this effect, consider Ehtylamine,
C2H5NH2.
This substance has one N atom, and its mass is
an odd number (45), whereas
EhtyleneDiamine H2N-CH2-CH2-NH2, has 2 N
atoms, and its mass is an even number (60).

Basic principle of working

Find a way to charge an atom or molecule
(ionization)
Place charged atom or molecule in a
magnetic field or subject it to an electric
field and measure its speed or radius of
curvature relative to its mass-to-charge ratio
(mass analyzer)
Detect ions using microchannel plate

Mass Spectrometry
An outline of what happens in a mass
spectrometer
Atoms can be deflected by magnetic fields provided the atom is first turned into an ion.
Electrically charged particles are affected by a
magnetic field although electrically neutral
ones aren't.
The sequence is :

Ionization

Mass Spectrometry

Slightly +ve
To repel +ve ions

Usually carry +1 chrge

Difficult to remove
another electron

Mass Spectrometry

Mass Spectrometry
Assume all streams having +1 charge

Lightest m/z ratio

Bulkiest m/z ratio

Optimum m/z ratio

Mass Spectrometry
Detection

Creating electron vacancy

Causing current to flow which is measured

Elements to Mass Spectrometry

(J.J. Thomson ~ 1910)
Gas Phase/
Ionize

Separate Based on
Mass/Charge

Sample

Electron Impact (EI)

Chemical Ionization (CI)
Electrospray (ESI)
Atmospheric Pressure Chemical
Ionization (APCI)
Photo-ionization (APPI)
Matrix Assisted Laser Desorption
and Ionization (MALDI)

Detector

Mass Spectrometry
The Mass Spectrometer
Fundamental operating principle
Determine mass by manipulating flight path of an ion in a magnetic field
Electron gun

Ionization: X + e- X+. + 2 eMagnet

Measure ion
mass-to-charge ratio
(m/z)

Ionization
m/z
m/ztoo
toosmall
large

sample
introduction

m/z just right

+ Accelerator
plates
Detector quiet

Detector fires
Detector quiet

Detector

14

Isotopes
Isotopes: atoms with same number of protons and same number of electrons
but different numbers of neutrons
Aston mass spectrum of neon (1919)
Ne empirical atomic weight = 20.2 amu
Ne mass spectrum: predict single peak at m/z = 20.2
Results

m/z
20.2
20.0
22.0

relative intensity
no peak
90%
10%

Conclusions
Neon is a mixture of isotopes
Weighted average: (90% x 20.0 amu) + (10.0% x 22.0 amu) = 20.2 amu
Nobel Prize in Chemistry 1922 to Aston for discovery of stable element isotopes
15

The Mass Spectrum

Origin of Relative Ion Abundances

M contributors

M+1 contributors

M+2 contributors

Isotope

Natural
Abundance

Isotope

Natural
Abundance

Isotope

Natural
Abundance

1H

99.9855%

2H

0.015%

3H

ppm

12C

98.893

13C

1.107

14C

ppm

14N

99.634

15N

0.366

16O

99.759

17O

0.037

18O

0.204

19F

100.0

32S

95.0

33S

0.76

34S

4.22

35Cl

75.77

37Cl

24.23

79Br

50.69

81Br

49.31

127I

100.0
16

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)

But when to use which ionization technique?

Ion Source Depends on Sample

Solid Sample

Liquid Sample

Make into Solid ?

Gas Sample

Make into Solution ?

Chemical Properties
of analyte in gas
phase ?

Chemical Properties
of analyte in
solution phase ?

MALDI

APCI

APPI

Turn into Gas?

ESI

CI

EI

Electron Impact
e-

e-

e-

M(g) + e- M+(g) + 2eThis reaction creates the molecular ion so is very useful.
However, the excess energy from the electron can cause
the molecular ion to fall apart:

Neutral
Molecule

IP2
Excess Energy get
redistributed
throughout ion
to cause
fragmentation.

s1
IP

s0
s1

s0

Ionized
Molecule

Electron Impact
e-

e-

e-

A+

M(g) + (g) +
M+(g) A+Fragment 1 (g) + BFragment 2 (g)
e-

M+

2e-

Electron energy is chosen by compromise.

Fragment Information is useful. It can help structural
determination. However, many ions produce only fragments
with no molecular ion remaining. Molecular ion are often very
unstable.
70 eV Classical Spectra to be used for comparisons

Properties of EI
Hard ionization

Gas-phase molecules enter source through heated

probe or
GC column
70 eV electrons bombard molecules forming M+*
ions that fragment in unique reproducible way to
form a collection of fragment ions
EI spectra can be matched to library stds CI (soft
ionization)

Chemical Ionization
EI is not appropriate for some molecules (it
causes too much fragmentation)
Instead, ionize a reagent gas (by EI) then react
it with a analyte molecules
Typically use methane or ammonia for reagent
gas

Properties of CI
Advantages

Disadvantages

Parent Ion
Interface to GC
Insoluble Samples
CI is lower energy
process than EI

No Fragment Library
Need Volatile Sample
Need Thermal Stability
Low Mass Compounds
(<1000 amu)

CI: Form Reagent Ions First

For Example - Methane CI

1. electron ionization of CH4:

CH4 + e- CH4+ + 2e Fragmentation forms CH3+, CH2+, CH+

2. ion-molecule reactions create stable reagent

ions:

CH4+ + CH4 CH3 + CH5+

CH3+ + CH4 H2 + C2H5+
CH5+ and C2H5+ are the dominant methane CI reagent ions

Methane CI Reagent Ions

Ions at m/z 17, 29, and 41 are from methane;
H3O+ is also formed from water vapor in the vacuum
system

Field ionization (FI)

In field ionization, a high-potential electric field is applied to an emitter with a sharp surface, such
as a razor blade, or more commonly, a filament from which tiny "whiskers" have formed. This
results in a very high electric field which can result in ionization of gaseous molecules of the
analyte. Mass spectra produced by FI have little or no fragmentation. They are dominated by
molecular radical cations M+. and less often, protonated molecules.

Probe

+
+

Probe

+
+ +
d<1mm

+
+
+
+ + +
+
Ionization

Field ionization (FI)

Application:
FD/FI being used for analysis of polar and nonvolatile analytes such as
polymers and biological molecules.
However, FD/FI remains one of the only ionization techniques that can
produce simple mass spectra with molecular information from
hydrocarbons and other particular analytes.
The most commonly encountered application of FD/FI at the present time
is the analysis of complex mixtures of hydrocarbons such as that found in
petroleum fractions.

Difference
There are three practical differences between CI and FI: there is less
fragmentation in FI
There is no high-resolution FI, and FI is less sensitive.
Sensitivity is not an issue unless there is an extremely small amount of
sample. FI can be performed by direct probe and GC/MS.

Field ionization (FI)

Matrix Assisted Laser Desorption

Ionization (MALDI)
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.
The use of a chemical matrix in the form of small, laser-absorbing organic
molecules in large excess over the analyte is at the core of the MALDI principle.
One important feature is the way in which the matrix and analyte interact in
the MALDI sample. In a typical UV-MALDI sample preparation small volumes of
an analyte is mixed with amount 106 of matrix. Upon solvent evaporation, the
matrix crystallizes to form a bed of small crystals that range in size from a few to
a few hundred micrometers, depending on the matrix and the details of the
preparation.

Properties of MALDI
Good solubility

Vapour pressure must be sufficiently low to maintain vacuum conditions

Viscosity must allow diffusion of the analyte from the bulk to the surface
Lower PRACTICAL detection limits
Easier to interpret spectra (less multiple charges)
Quick and easy
Higher mass detection
Higher Throughput (>1000 samples per hour)
Low levels of some salts, buffers, and detergents can be tolerated as well as less
than 2% of glycerol.

Principle of MALDI

MALDI mass spectrometry has become a powerful analytical

tool for both synthetic polymers and biopolymers.

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.
It is especially useful in producing ions from macromolecules because it
overcomes the propensity of these molecules to fragment when
ionized.

2. Principle

Properties of ESI
Advantages
Electrospray Ionization can be
easily interfaced to LC.
Absolute signals from
Electrospray are more easily
reproduced, therefore, better
quantitation.
Mass Accuracy is considered
better.
Multiple charging is more
common then MALDI.

Disadvantages
No Fragmentation
Need Polar Sample
Need Solubility in Polar
Solvent (MeOH, ACN, H2O,
Acetone are best)
Sensitive to Salts
Suppression

The Mass Spectrum

Example: methane CH4 + e- CH4+. + 2 e-

Relative ion abundance (%)

Base peak: most abundant ion

m/z = (1 x 12) + (4 x 1) = 16
C
H

mass-to-charge ratio (m/z)

36

Tandem Mass Spectrometry

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

Fragmentation Patterns
The impact of the stream of high energy
electrons often breaks the molecule into
fragments, commonly a cation and a radical.
Bonds break to give the most stable cation.
Stability of the radical is less important.

Fragmentation Patterns
Alkanes
Fragmentation often splits off simple alkyl groups:

Loss of methyl
Loss of ethyl
Loss of propyl
Loss of butyl

M+ - 15
M+ - 29
M+ - 43
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)
H
H C Br

H
H C

H
H C
or

M+

Fragmentation Patterns
Alcohols
Fragment easily resulting in very small or missing
parent ion peak
Commonly losses H2O or OH
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

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

Industrial Science and Technology, 11/28/09)

M+

Fragmentation Patterns
Amines
Odd M+ (assuming an odd number of nitrogens
are present)
a-cleavage dominates forming an iminium ion
CH3CH2

CH2

N CH2
H

CH2CH2CH3

CH3CH2CH2N CH2
H
m/z =72

iminium ion

Fragmentation Patterns
86
CH3CH2

CH2

N CH2
H
72

CH2CH2CH3

Fragmentation Patterns
Aromatics may also have a peak at m/z = 77 for the benzene
ring.
NO2
77

77

M+ = 123

Fragmentation Patterns
Aldehydes (RCHO)
Fragmentation may form acylium ion
RC O
Common fragments:
RC O
M+ - 1 for
M+ - 29 for

(i.e. RCHO - CHO)

Fragmentation Patterns
MS for hydrocinnamaldehyde
105

91

H H O
C C C H
H H
133
91

M+ = 134
105

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

Industrial Science and Technology, 11/28/09)

Fragmentation Patterns
O

Ketones

RCR'

Fragmentation leads to formation of acylium ion:

Loss of R forming

R'C O

Loss of R forming

RC O

Fragmentation Patterns
O
MS for 2-pentanone

CH3CCH2CH2CH3

CH3C O

CH3CH2CH2C O
M+

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

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
105

77
O
C O CH3

77

105

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

Industrial Science and Technology, 11/28/09)

M+ = 136

2. Rearrangement
McLafferty rearrangement
Pattern I

H
+
C

D
H2C

D
C

54

55

Retro Diels-Alder rearrangement

R

-e

R
+

R
+

Examples:

CH3

CH3
+

56

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

C6H13

H2O + C6H13
H HO

OH
H

H2C CHCH3 + H2O + CH2=CH2

CH3

O
- CO

- CO

H
O

O
+

H2O

57

Four-member ring rearrangement

CH3 CH2 O
- C2H4

CH2 CH3

- CH3

CH3 CH2 O

CH2 =

H2C

H2C

O CH2

HO CH2

Other rearrangement

X
R +

58

oConcluding remarks
oSoft techniques produces Molecular ion peaks
whereas hard techniques produces Fregmentation.
oBy Soft techniques exact molecular ion peak can be
obtained for large and small molecules.
oBy Fregmentation connection pattern of a molecule
can be concluded, ehich helps in structure
elucidation.