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Topic : Mass

Spectrometry
Presented by : Zeeshan-Ul-Haq
Refrences : Chemguide,
School of Chemical Sciences (SCS) and
Prof. Yongchi
http://www.chemguide.co.uk/analysis
/masspecmenu.html

http://scs.illinois.edu/massSpec
OUTLINE
Introduction to Mass Spectrometry

Ionization Methods

Fragmentation and MS Interpretation

Hyphenated MS Techniques
Spectroscopy
What methods are commonly used?

Mass spectrometry (MS)* Infrared spectroscopy (IR)


molecular formula functional groups

Nuclear magnetic resonance (NMR) X-ray crystallography*


C/H molecular skeleton spatial position of atoms
*Not rigorously a type of spectroscopy
4
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)
Mass Spectrometer
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 :
Ionizati
Mass Spectrometry
on

Slightly +ve
To repel +ve Usually carry +1
ions 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
Detectio
n

Creating electron vacancy


Causing current to flow which is measured
Elements to Mass Spectrometry
(J.J. Thomson ~ 1910)

Gas Phase/ Separate Based on


Ionize Mass/Charge

Sample

Electron Impact (EI)


Chemical Ionization (CI)
Electrospray (ESI)
Atmospheric Pressure Chemical
Ionization (APCI)
Photo-ionization (APPI) Detector
Matrix Assisted Laser Desorption
and Ionization (MALDI)
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 e-
Magnet
Ionization Measure ion
m/z
m/ztoo
toosmall
large mass-to-charge ratio
sample (m/z)
introduction
m/z just right
+ -

Accelerator
plates
Detector fires

Detector quiet Detector quiet Detector

19
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 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
Nobel Prize in Chemistry 1922 to Aston for discovery of stable element isotopes

20
The Mass Spectrum
Origin of Relative Ion Abundances

M contributors M+1 contributors M+2 contributors


Natural Natural Natural
Isotope Isotope Isotope
Abundance Abundance 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

21
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

Make into Solid ? Make into Solution ? Turn into Gas?

Chemical Chemical
Properties Properties
of analyte in of analyte in
solution phase ? gas phase ?

MALDI APCI APPI ESI CI EI


Electron Impact
e-
e-
e-
M

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:
Neutral IP2
Molecule

Excess
s1 Energy get
redistributed
IP throughout
ion to cause
s0 fragmentation
Ionized .
s1 Molecule

s0
Electron Impact
e-
e-
e-
M

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

Parent Ion Disadvantages


Interface to GC
Insoluble Samples
No Fragment Library
CI is lower energy
process than EI 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 Disadvantages


easily interfaced to LC.
Absolute signals from
Electrospray are more easily No Fragmentation
reproduced, therefore, better Need Polar Sample
quantitation.
Mass Accuracy is considered
Need Solubility in Polar
better. Solvent (MeOH, ACN,
Multiple charging is more H2O, Acetone are
common then MALDI. best)
Sensitive to Salts
Suppression
The Mass Spectrum
Example: methane CH4 + e- CH4+. + 2 e-
m/z = (1 x 12) + (4 x 1) = 16
C H
Base peak: most abundant ion
Relative ion abundance (%)

mass-to-charge ratio (m/z)


41
Hyphenated Mass Techniques

Chromatography: Separation

Mass: Detection

Chromatography-Mass Spectroscopy :
Separation + Detection 43

57
29 71
15 85
99 113 142
GC-MS LC-MS CZE-MS m/z
Hyphenated GC-MS
as chromatography-mass spectrometry (GC-MS) is a method that combines
e features of gas-liquid chromatography and mass spectrometry to identify
fferent substances within a test sample.
Mass
HEWLETT 5972A Selective
PACKARD Detector

1.0
DEG/MI
N

MS
HEW LETT
PACKARD

5890

Sampl Gas Chromatograph (GC) Mass


e B Spectrometer
D A
A C B
D
A C
B
DB C
A
C D

Sampl Separatio Identificatio


e n n
Hyphenated GC-MS

GAS CHROMATOGRAPHY MASS SPECTROMETRY


Hyphenated GC-MS
GAS CHROMATOGRAPHY
The sample is injected into the GC inlet where it is
heated and swept onto a chromatographic column by a
carrier gas.

The pure compounds in a mixture are separated by


interacting with the coating or packing of the column
(stationary phase) and the carrier gas (mobile phase).

This separation is often improved by programming


changes in column temperature and pressure.
Hyphenated LC-MS
quid chromatography-mass spectrometry (LC-MS) is an analytical
hemistry technique that combines the physical separation capabilities of
quid chromatography with the mass analysis capabilities of mass
pectrometry.
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
Hyphenated LC-MS

Liquid chromatography-mass spectrometry (Ion trap LCMS system )


Tandem Mass Spectrometry

dem mass spectrometry, also known as MS/MS, involves multiple


s of mass spectrometry selection, with some form of fragmentat
rring in between the stages.
Tandem Mass Spectrometry
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)
H
H H
H C
H C Br 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 M+

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

M+ = 123
77
Fragmentation Patterns
Aldehydes (RCHO)
Fragmentation may form acylium ion
RC O

Common fragments:
RC O
M+ - 1 for
R (i.e. RCHO - CHO)
M+ - 29 for
Fragmentation Patterns
MS for hydrocinnamaldehyde
105 91

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

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


Advanced Industrial Science and Technology, 11/28/09)
Fragmentation Patterns
O
RCR'
Ketones
Fragmentation leads to formation of
acylium ion:
R'C O
Loss of R forming
RC O
Loss of R forming
Fragmentation Patterns
O
CH3CCH2CH2CH3
MS for 2-pentanone

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
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)
2. Rearrangement
McLafferty rearrangement
Pattern I

H H H
A E A E A E
+
B D B D B D
C C H2C

H H H
A E A E E
+
B D B D D
C C C

66
67
Retro Diels-Alder rearrangement

R R R R
-e
+

R R
+

Examples:

CH3 CH3

68
Loss of small molecules, such as H2O, CO, C2H4

C6H13 H2O + C6H13


H HO

OH
H
H2C CHCH3 + H2O + CH2=CH2
CH3

O O
- CO - CO

O O
H
+ H2O
O
H

69
Four-member ring rearrangement

- CH3 H2C H
CH3 CH2 O CH2 CH3 CH3 CH2 O CH2 =
H2C O CH2
- C2H4
HO CH2

Other rearrangement

X X
R R +

70
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.