Académique Documents
Professionnel Documents
Culture Documents
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
Hyphenated MS Techniques
Spectroscopy
What methods are commonly used?
Molecular weight
Molecular formula (HRMS)
Structure (from
fragmentation fingerprint)
Isotopic incorporation /
distribution
Protein sequence (MS-MS)
Mass Spectrometer
Rule of Thirteen
Calculating Mass
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
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
Sample
Accelerator
plates
Detector fires
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
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
21
Ionization Methods
Electron bomb Ionization (EI)
Chemical Ionization (CI)
Field ionization (FI)
Matrix Assisted Laser Desorption Ionization (MALDI)
Chemical Chemical
Properties Properties
of analyte in of analyte in
solution phase ? gas phase ?
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
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
Viscosity must allow diffusion of the analyte from the bulk to the surface
Advantages
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
LC MS
B
Peak A: mass1
A Peak B: mass2
C
Peak C: mass3
t/min
Hyphenated LC-MS
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+
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
CH3C O
CH3CH2CH2C O
M+
Loss of R
peak at M+ - R
Frgamentation Patterns
77 105
O
C O CH3
77
M+ = 136
105
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
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.