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.
Molecular weight
Molecular formula (HRMS)
Structure (from
fragmentation fingerprint)
Isotopic incorporation /
distribution
Protein sequence (MS-MS)
What information can be determined?
Rule of Thirteen
Calculating Mass
The Rule of Thirteen can be used to identify
possible molecular formulas for an unknown
hydrocarbon, C
n
H
m
.

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: C
8
H
10


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 :
Mass Spectrometry
Ionization
Slightly +ve
To repel +ve ions
Usually carry +1 chrge
Difficult to remove
another electron
Mass Spectrometry
Mass Spectrometry
Lightest m/z ratio
Bulkiest m/z ratio Optimum m/z ratio
Assume all streams having +1 charge
Mass Spectrometry
Detection
Creating electron vacancy
Causing current to flow which is measured
Gas Phase/
Ionize
Detector
Separate Based on
Mass/Charge
Sample
Elements to Mass Spectrometry
(J.J. Thomson ~ 1910)
Electron Impact (EI)
Chemical Ionization (CI)
Electrospray (ESI)
Atmospheric Pressure Chemical
Ionization (APCI)
Photo-ionization (APPI)
Matrix Assisted Laser Desorption
and Ionization (MALDI)
14
Detector quiet
m/z too small
Mass Spectrometry
The Mass Spectrometer
Fundamental operating principle
Determine mass by manipulating flight path of an ion in a magnetic field
sample
introduction
Measure ion
mass-to-charge ratio
(m/z)
Detector
Ionization
Electron gun
+ -
Accelerator
plates
Magnet
m/z just right
Detector fires
Ionization: X + e
-
X
+.
+ 2 e
-

m/z too large
Detector quiet
15
Isotopes
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 relative intensity
20.2 no peak
20.0 90%
22.0 10%
Conclusions
Neon is a mixture of isotopes
Weighted average: (90% x 20.0 amu) + (10.0% x 22.0 amu) = 20.2 amu
Isotopes: atoms with same number of protons and same number of electrons
but different numbers of neutrons
Nobel Prize in Chemistry 1922 to Aston for discovery of stable element isotopes
16
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
1
H 99.9855%
2
H 0.015%
3
H ppm
12
C 98.893
13
C 1.107
14
C ppm
14
N 99.634
15
N 0.366
16
O 99.759
17
O 0.037
18
O 0.204
19
F 100.0
32
S 95.0
33
S 0.76
34
S 4.22
35
Cl 75.77
37
Cl 24.23
79
Br 50.69
81
Br 49.31
127
I 100.0
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 Gas Sample
EI CI APCI MALDI ESI APPI
Make into Solution ? Make into Solid ? Turn into Gas?
Chemical Properties
of analyte in gas
phase ?
Chemical Properties
of analyte in
solution phase ?
Electron Impact
M
e-
e-
e-
M
(g)
+ e
-
M
+
(g)
+ 2e
-

This reaction creates the molecular ion so is very useful.
However, the excess energy from the electron can cause
the molecular ion to fall apart:
s
0

s
1

IP
s
0

s
1

IP
2

Neutral
Molecule
Ionized
Molecule
Excess Energy get
redistributed
throughout ion
to cause
fragmentation.
Electron Impact
A
+
M
e-
e-
e-
M
(g)
+ e
-
M
+
(g)
+ 2e
-
M
+
(g)
A
+
Fragment 1 (g)
+ B
Fragment 2 (g)

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
B B
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)





Properties of EI
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
Advantages

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

Disadvantages

No Fragment Library
Need Volatile Sample
Need Thermal Stability
Low Mass Compounds
(<1000 amu)
Properties of CI
CI: Form Reagent Ions First
For Example - Methane CI
1. electron ionization of CH4:
CH
4
+ e- CH
4
+
+ 2e
-
Fragmentation forms CH
3
+
, CH
2
+
, CH
+

2. ion-molecule reactions create stable reagent
ions:
CH
4
+
+ CH
4
CH
3
+ CH
5
+
CH
3
+
+ CH
4
H
2
+ C
2
H
5
+

CH
5
+
and C
2
H
5
+
are the dominant methane CI reagent ions
Methane CI Reagent Ions
Ions at m/z 17, 29, and 41 are from methane;
H
3
O
+
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
+
+
+
+
+
+
+
+
+
+
+
+
+
Ionization
d<1mm
Probe
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 10
6
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.
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.


Properties of MALDI
MALDI mass spectrometry has become a powerful analytical
tool for both synthetic polymers and biopolymers.


Principle of MALDI
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.


ElectroSpray Ionization (ESI)

2. Principle
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, H
2
O,
Acetone are best)
Sensitive to Salts
Suppression

Properties of ESI

36
The Mass Spectrum
Example: methane CH
4
+ e
-
CH
4
+.
+ 2 e
-

mass-to-charge ratio (m/z)
R
e
l
a
t
i
v
e

i
o
n

a
b
u
n
d
a
n
c
e

(
%
)

Base peak: most abundant ion
m/z = (1 x 12) + (4 x 1) = 16
C H
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 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)
M
+

C H
H
C H Br
H
C
H
H
or
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.
1
o
alcohol usually has prominent peak at m/z = 31
corresponding to H
2
C=OH
+


Fragmentation Patterns
MS for 1-propanol
M
+
M
+
-18
CH
3
CH
2
CH
2
OH
H
2
C OH
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced
Industrial Science and Technology, 11/28/09)
Fragmentation Patterns
Amines
Odd M
+
(assuming an odd number of nitrogens
are present)
a-cleavage dominates forming an iminium ion
CH
3
CH
2
CH
2
N
H
CH
2
CH
2
CH
2
CH
3
CH
3
CH
2
CH
2
N CH
2
H
m/z =72
iminium ion
Fragmentation Patterns
86
CH
3
CH
2
CH
2
N
H
CH
2
CH
2
CH
2
CH
3
72
Fragmentation Patterns
Aromatics may also have a peak at m/z = 77 for the benzene
ring.
NO
2
77
M
+
= 123
77
Fragmentation Patterns
Aldehydes (RCHO)
Fragmentation may form acylium ion


Common fragments:

M
+
- 1 for

M
+
- 29 for
RC O
R (i.e. RCHO - CHO)
RC O
Fragmentation Patterns
MS for hydrocinnamaldehyde
M
+
= 134
C C C H
H
H
H
H
O
133
105
91
105
91
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced
Industrial Science and Technology, 11/28/09)
Fragmentation Patterns

Ketones
Fragmentation leads to formation of acylium ion:

Loss of R forming

Loss of R forming

RC O
R'C O
RCR'
O
Fragmentation Patterns
MS for 2-pentanone

CH
3
CCH
2
CH
2
CH
3
O
M
+

CH
3
CH
2
CH
2
C O
CH
3
C O
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced
Industrial Science and Technology, 11/28/09)
Fragmentation Patterns
Esters (RCO
2
R)
Common fragmentation patterns include:
Loss of OR
peak at M
+
- OR

Loss of R
peak at M
+
- R


Frgamentation Patterns
M
+
= 136
C
O
O CH
3
105
77
105
77
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced
Industrial Science and Technology, 11/28/09)
54
2. Rearrangement
McLafferty rearrangement
Pattern I
+
A
B
C
D
E
H
A
B
C
D
E
H
A
B
H
2
C
D
E
H
+
A
B
C
D
E
H
A
B
C
D
E
H
C
D
E
H
55
56
Retro Diels-Alder rearrangement
R
- e
R
R
R
+
R
R
+
CH
3
CH
3
+
Examples:
57
Loss of small molecules, such as H
2
O, CO, C
2
H
4
H
HO
C
6
H
13
H
2
O + C
6
H
13
- CO
O
O
O
- CO
+
O
H
O
H
O
H
2
O
H
OH
CH
3
H
2
C CHCH
3
CH
2
=CH
2
H
2
O + +
58
Four-member ring rearrangement

Other rearrangement

CH
3
CH
2
O CH
2
CH
3
- C
2
H
4
CH
3
CH
2
O CH
2
H
2
C
H
2
C O
H
=
CH
2
- CH
3
HO CH
2
X
R R +
X
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.