Mass Spectrometry

PRIN
PRINCIPLES
P
NCIPLES OF
MASS
S SPECTROMETRY
Prepared by HUYNH KHANH DUY - HCMUT

HCMC 2013
(C) HKD 2013
BACKGROUND
Mass spectrometry (Mass Spec or MS)

uses high energy electrons to break a
molecule into fragments.
Separation and analysis of the fragments
provides information about:
Molecular weight
Structure
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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 C H
H
H C H
+ 2e
H
Molecular ion (M+)
m/z = 16
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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
H H
H H
H C C H
molecular ion (M ) m/z = 30
H H
+ e
H C C
H H
+ H
H H
m/z = 29
H
H C
H
H
C 2013
H
+(C) HKD
H
(not detected by MS)
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
The molecular ion is usually the highest

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

base peak
M+
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BACKGROUND
MASS RESOLUTION
C3H6O and C3H8O have nominal masses of 58 and 60, and
can be distinguished by low-resolution MS.
C3H8O and C2H4O2 both have nominal masses of 60.
Distinguish between them by high-resolution MS.
Molecular Nominal Precise
Formula
Mass
Mass
C3 H8 O
60
60.05754
C2 H4 O2
60.02112
60
) High resolution MS can

replace elemental analysis for
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chemical formula confirmation.
BACKGROUND
Basic components of mass spectrometer
Ionization
Source
Inlet
Mass
Analzyer
Detector
selected
ions
all ions
Vacuum pumps system

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Data
System
BACKGROUND
WAYS TO PRODUCE IONS

Electron impact (EI): vapor of sample is bombarded with
electrons: M + e 2e + M.+ fragments
Chemical ionization (CI): sample M collides with reagent ions
present in excess e.g.
CH4 + e CH4.+ CH5+
M + CH5+ CH4 + MH+
Fast Atom/Ion Bombardment (FAB): Softer than EI. Ions are
produced by bombardment with heavy atoms. Gives (M+H)+ ions
and litle fragmentation. Good for more polar compounds.
Laser Desorption & Matrix-Assisted Laser Desorption
(MALDI): hit the sample with a laser beam.
Electrospray Ionization (ESI): a stream of solution passes
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20136 V/m).
through a strong electric field
(10
BACKGROUND
ESI
EI
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BACKGROUND
FAB
MALDI
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BACKGROUND
ICP
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BACKGROUND
IONIZATION METHODS
1. Electron Ionization (EI): most common
ionization technique, limited to relatively low
MW compounds (<600 amu).
2. Chemical Ionization (CI): ionization with very
little fragmentation, still for low MW
compounds (<800 amu).
3. Desorption Ionization (DI): for higher MW or
very labile compounds.
4. Spray ionization (SI)
for LC-MS, biomolecules, etc.
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BACKGROUND
Electron ionization
vaporized sample is bombarded with high
energy electrons (typically 70 eV).
hard ionization method leads to significant
fragmentation.
ionization is efficient but non-selective.
eM + e-
M+
ABCD+
ABC+
E << 70 eV
E = 70 eV
fragmentation
. + 2e-
M+
ABCD+
+N
ABC+ + C
BC+ + A
M+
e-
Neutral
molecule (C) HKD 2013
Molecular
ion
+ + +
+ +
Fragment
ions
BACKGROUND
Electron ionization
Advantages
inexpensive, versatile and reproducible.
fragmentation gives structural information.
large databases if EI spectra exist and are
searchable.
Disadvantages
fragmentation at expense of molecular ion.
sample must be relatively volatile.
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BACKGROUND
Chemical ionization (CI)

Vaporized sample reacts with pre-ionized
reagent gas via proton transfer, charge
exchange, electron capture, adduct formation,
etc.
Common CI reagents: methane, ammonia,
isobutane, hydrogen, methanol.
soft ionization gives little fragmentation.
or
Selective
ionization-only
exothermic
thermoneutral ion-molecule reactions will occur.
choice of reagent allows tuning of ionization.
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BACKGROUND
Positive Chemical ionization (CI)

CH4
eHigh
energy
electrons
CH4
CH4+
CH4
CH4
CH3+
CH2
CH4
CH5
MH+
Protonation
Reaction
C2H3+
C2H5+ C2H5
Primary
reagent ions
Reagent gas
Secondary
reagent ions
Analytes
C 2H 4
Uses reagent gas (methane, isobutane or ammonia)

Soft ionization method only little fragmentations
Produces positive ions
Detects pseudo molecular ions molecular weight information can be obtained
HKD 2013
Lower ion source temperature will(C) produce
more pseudo molecular ions. Be
careful that lower ion source temperature can cause more contamination.
BACKGROUND
Negative Chemical ionization (CI)

Reagent gas
eHigh energy
electrons
(from the
filament)
CH4
e-
MX
MX
CH4
MX
MX
CH4
H
CH4+
Analytes
Low energy
electrons
CH4
Associative resonance
capture
CH3+
Dissociative
resonance capture
Uses reagent gas (methane, isobutane or ammonia)

Soft ionization method only little fragmentations
Produces negative ions
Higher reagent gas pressure will produce more low energy electrons, and in turn,
more molecular ions.
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Lowering the ion source temperature
can produce more resonance capture
reaction and increase molecular ions.
BACKGROUND
MASS ANALYSER MAGNETIC SECTOR
m/z = B2r2/2V
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BACKGROUND
MASS ANALYSER MAGNETIC SECTOR

9 Classical mass spectra.
9 Very high reproducibility.
9 Best quantitative performance of all MS analyzers.
9 High resolution.
9 High sensitivity.
9 10,000 Mass Range.
9 Requires Skilled Operator
9 Usually larger and higher cost than other mass analyzers.
9 Difficult to interface to ESI.
HKD 2013 analyzers.
9 Low resolution MS/MS without(C)multiple
BACKGROUND
MASS ANALYSER QUADRUPOLE
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BACKGROUND

m1
m2+
m1+
m2
+
m3
+
+
mmass number
z charge
m3
m/z = k.V
1
m1/z=kV1
m2/z=kV2
m3/z=KV3
k constant
V :voltage applied to rods
We can select mass
Two opposite rods will have a potential of

+(U+Vcos(t)) and the other two -(U+Vcos(t))
where U is DC voltage and Vcos(wt) represents a
radio frequency (RF) field of amplitude V and
(C)
HKD 2013 w. In general, for mass section U and V
frequency
are varied keeping the ratio U/V constant.
BACKGROUND
m/z = K.V/(r22)
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BACKGROUND

9 Easy to use ,simple construction, fast.
9 Good reproducibility.
9 Relatively small and low-cost systems.
9 Able to separate ions at lower vacuum levels (10-2 to 10-3 Pa).
9 Quadrupoles are now capable of routinely analyzing up to a m/q ratio
of 3000, which is useful in electrospary ionization of biomolecules.
9 Low resolution(<4000).
9 Slow scanning.
9 Low accuracy (>100ppm).
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BACKGROUND
MASS ANALYSER ION TRAP
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BACKGROUND

9 Cannot perform SIM measurements for transmission type mass
spectrometry.
9 Only a limited quantity of ions can be trapped, resulting in a narrower
dynamic range than quadrupole MS systems.
9 All trapped ions are detected higher sensitivity in scanning analysis
than quadrupole models.
9 Enables trapping specific ions, then fragmenting them and detecting
the resulting fragment ions a mass spectrometer specialized for
qualitative analysis.
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BACKGROUND

9 Poor quantitation.
9 Less sensitive.
9 Non-classical spectrum.
9 Electrodes tend to be contaminated.
9 Limited column flow rate.
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BACKGROUND
MASS ANALYSER Time-Of-Flight
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m/z =
2eUt /L2
BACKGROUND
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resolution
= 2dt/t
BACKGROUND

9 Good for kinetic studies of fast reactions and for use with gas
chromatography to analyze peaks from chromatograph.
9 High ion transmission.

9 Can register molecular ions that decompose in the flight tube.
9 Extremely high mass range (>1MDa).
9 Fastest scanning.
9 Requires pulsed ionization method or ion beam switching (duty cycle is
a factor).
9 Low resolution (4000).
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9 Limited precursor-ion selectivity
for 2013
most MS/MS experiments
BACKGROUND
Fourier Transform Ion Cyclotron Resonance

(FT ICR) analyzers
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BACKGROUND

(FT ICR) analyzers
y
m/z (A) < m/z (B)

If the frequency of the applied field is the
same as the cyclotron frequency of the ions,
the ions absorb energy thus increasing their
velocity (and the orbital radius) but keeping a
constant cyclotron frequency. Ions having a
different cyclotron frequency are not
accelerated.
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BACKGROUND

(FT ICR) analyzers
= qB/m = v/r
The decay over time of the image current resulting after applying a
short radio-frequency sweep is transformed from the time domain into a
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frequency domain signal by a Fourier
transform
BACKGROUND

(FT ICR) analyzers
9 The highest recorded mass resolution of all mass .spectrometers
(>500,000).
9 Very good accuracy (<1ppm).
9 Well-suited for use with pulsed ionization methods such as MALDI.
9 Non-destructive ion detection; ion remeasurement.
9 Stable mass calibration in superconducting magnet FTICR systems.
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BACKGROUND

(FT ICR) analyzers
9 Expensive.
9 Requires superconducting magnet.

9 Subject to space charge effects and ion molecule reactions.
9 Artifacts such as harmonics and sidebands are present in the mass
spectra.
9 Many parameters (excitation, trapping, detection conditions) comprise
the experiment sequence that defines the quality of the mass
spectrum.
9 Generally low-energy CID, spectrum depends on collision energy,
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collision gas, and other parameters.
BACKGROUND
MASS ANALYSER COMPARISION
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BACKGROUND
Only cations are detected.

Radicals are invisible in MS.
The amount of deflection observed
depends on the mass to charge ratio
(m/z).
Most cations formed have a charge of
+1 so the amount of deflection
observed is usually dependent on the
mass of the ion.
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BACKGROUND
Fragment Ions
Derived from
molecular ion
or higher
weight
fragments
[M+H]+(CI)
Or M+ (EI)
molecular ion
Unit mass
spacing
In CI, adduct ions,

[M+reagent gas]+
High
mass
(C)and
HKD 2013
Mass, as m/z. Z is the charge,
for doubly charged ions (often seen in
macromolecules), masses show up at half their proper value
BACKGROUND
M
Intensity (% of Base Peak)
Molecule
e-
High Energy
Electron
M+
2e-
Molecular
Ion
(Radical Cation)
Base Peak
100
90
80
70
60
50
40
30
20
10
0
CH2OH+
M - (H2O and CH2=CH2)
1-Pentanol - MW 88
M - (H2O and CH3)
CH3(CH2)3 CH2OH
M - H2O
Molecular Ion Peak

M+ - 1
20
30
40
50
60
70
80
90
m/z
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BACKGROUND
A partial MS of dopamine showing all peaks

with intensity equal to or greater than
0.5% of base peak.
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BACKGROUND
Most elements occur naturally as a

mixture of isotopes.
The presence of significant amounts of
heavier isotopes leads to small peaks
that have masses that are higher than
the parent ion peak.
M+1 = a peak that is one mass unit
higher than M+
M+2 = a peak that is two mass units
higher than M+
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BACKGROUND
Bromine:
M+ ~ M+2 (50.5%
79Br/49.5% 81Br)
2-bromopropane
M+ ~ M+2
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BACKGROUND
Chlorine:
M+2 is ~ 1/3 as large as M+
M+
Cl
M+2
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BACKGROUND
Sulfur:
M+2 larger than usual (4% of M+)
M+
S
Unusually
large M+2
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BACKGROUND
Iodine
I+ at 127
Large gap
Large gap
ICH2CN
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FRAGMENTATION PATTERNS
There are 3 type of fragmentations
1) Cleavage off s bond
---- C C ---At heteroatom

---- C Z ----
+.
+.
---- C +
. C ----
---- C +
. Z ----
C=Z +
---- C .
Z+.
---- C = C
D to heteroatom
+.
---- C - C Z ----
+.
---- C - C Z ----
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M+
There are 3 type of fragmentations
2) Cleavage of 2 s bond (rearrangements)
---- HC C Z ----
+.
---- C=C + +
Retro Diels-alder
CH2
+.
HZ
CH2
CH2
CH2
McLafferty
H
Z
+.
+.
R
CH2
CH2
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3) Cleavage of Complex rearrangements
RULES
1.
2.
3.
Intensity of M.+ is Larger for linear chain than for branched

compound
Intensity of M.+ decrease with Increasing M.W. (fatty acid is
an exception)
Cleavage is favored at branching
reflecting the Increased stability of the ion
R
Stability order: CH3+ < R-CH2+ < R CH+ < R C+
R
R
R
R CH
R
HKD 2013
Loss(C)of
Largest Subst. Is most favored
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Branched alkanes
H3C
H3C
H3C
H3C
CH3
CH3
H3C
MW=170
M.+ is absent with heavy branching

Fragmentation occur at branching:
largest
fragment loss
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2013
Illustration of first 3 rules

(Linear alkane with Smaller MW)
Molecular ion is stronger than
in previous sample
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Illustration of first 3 rules

(Branched alkane with Smaller MW)
43
Molecular ion smaller than

linear alkane
Cleavage at branching is
favored
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ALKANES
Rule 3
Cleavage Favored at branching

Loss of Largest substituent
Favored
Rule1: intensity of M.+

is smaller with branching
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RULES
4.
5.
Aromatic Rings, Double bond, Cyclic structures stabilize M.+

Double bond favor Allylic Cleavage
Resonance Stabilized Cation
H2C+
CH
CH2
-R
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H2C
H2C
CH
CH
CH2
CH+2
Aromatic ring has stable M.+
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RULES
6. a) Saturated Rings lose a Alkyl Chain (case of branching)
R
+.
+
-R.
b) Unsaturated Rings Retro-Diels-Alder
CH2
+.
CH2
+
CH2
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CH2
Retro Diels-alder
+.
+.
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RULES
7.
Aromatic Compounds Cleave in Resonance Stabilized

Tropylium ion
R
C
CH2
CH2
-R.
CH
CH
Tropylium ion
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+
m/z 91
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RULES
8. C-C next to Heteroatom cleave leaving the charge on the
Heteroatom
- [RCH2]
CH2
CH2
H2C
H2C
R2
C
R1
- [R2]
R1
R1
+
larger
O(C) HKD 2013
RULES
9. Cleavage of small neutral molecules (CO2, CO, olefins, H2O .)
results often from rearrangement.
McLafferty
CH2
CH2
CH2
CH2
CH2
- CH2=CH2
C
Y
CH2
CH2
Ion Stabilized
by resonance
Y as H, R, OH, NR2
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SUMMARY
Alkanes
Fragmentation often
alkyl groups:
Loss of methyl
Loss of ethyl
Loss of propyl
Loss of butyl
splits off simple

M+
M+
M+
M+
15
29
43
57
Branched alkanes tend to fragment

forming the most stable carbocations.
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SUMMARY
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SUMMARY
Alkenes:
Fragmentation typically forms
resonance stabilized allylic carbocations
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Alkenes
Most intense peaks are often:
m/z 41, 55, 69
Rule 4: Double Bond Stabilize M+

Rule 5:: Doubl
Double
le Bond
B
favo
favor
Allylicc cleavage
CH2 + CH
Me
Et
CH
Et
-Et
-29
+CH
CH
CH2
CH
Me
(C) HKD 2013
M+ = 112
CH
Et
Me
m/z = 83
SUMMARY
Aromatics:
Fragment at the benzylic carbon, forming a
resonance stabilized benzylic carbocation
(which rearranges to the tropylium ion)
H
H C Br
H C
H C
or
M+
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CH +
Et
SUMMARY
Aromatics may also have a peak at m/z = 77 for
the benzene ring.
77
NO2
M+ = 123
77
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SUMMARY
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.
9 1o alcohol usually has prominent peak at
m/z = 31 corresponding to H2C=OH+
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SUMMARY
CH3CH2CH2OH
H2C OH
M+-18
M+
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SUMMARY
Amines
Odd M+ (assuming an odd number of
nitrogens are present).
D-cleavage
dominates
forming
an
iminium ion.
CH3CH2
CH2
N CH2 CH2CH2CH3
H
CH3CH2CH2N CH2
H
m/z =72
iminium ion
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SUMMARY
CH3CH2
86
CH2
N CH2 CH2CH2CH3
H
72
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SUMMARY
Ethers
D-cleavage forming oxonium ion
Loss of alkyl group forming oxonium ion
Loss of alkyl
carbocation
group
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forming
SUMMARY
MS of diethylether (CH3CH2OCH2CH3)
H O CH2
CH3CH2O CH2
H O CHCH3
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SUMMARY
Aldehydes (RCHO)
Fragmentation may form acylium ion
RC O
Common fragments:
9 M+ - 1 for RC O
9 M+ - 29 for R
(i.e. RCHO - CHO)
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SUMMARY
105
H H O
C C C H
M+ = 134
H H
133
91
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SUMMARY
Ketones
Fragmentation leads to formation of
acylium ion:
9 Loss of R forming R'C O

9 Loss of R forming RC O
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SUMMARY
CH3C O
O
CH3CCH2CH2CH3
CH3CH2CH2C O
M+
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SUMMARY
Esters (RCO2R)
Common fragmentation patterns include:
9 Loss of OR: peak at M+ - OR
9 Loss of R: peak at M+ - R
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SUMMARY
77
105
O
C O CH3
77
M+ = 136
105
(C) HKD 2013
RULES 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
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RULES 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
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RULES 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 heteroatoms to the formula.
Example: A compound with a molecular ion
peak at m/z = 102 has a strong peak at
1739 cm-1 in its IR spectrum. Determine
its molecular formula.
(C) HKD 2013

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Prepared by HUYNH KHANH DUY - HCMUT

(C) HKD 2013