Mass Spectrometry

(Mass Spec.)
Prof. Yonghai Chai
School of Chemistry & Materials Science
For Bilingual Chemistry Education

OUTLINE
Introduction to Mass Spectrometry
Ionization Methods
Mass Analyzer
Fragmentation and MS Interpretation
Hyphenated MS Techniques

By James Crawford

How do two people with

different languages
communicate with each other?

Then, how can I catch up, Ms.?

Chemical Identification
Comparison of
Physical Properties
Boiling Point
Melting Point

Elemental Analysis
Burn the compound and
measure the amounts of

Density

CO2, H2O and other

Optical rotation

components that are

Appearance

produced to determine the

Odor

empirical formula

Spectroscopic Methods for

Structure Determination
Ultraviolet-Visible (UV/Vis) spectroscopy:
determination of solutions of transition metal ions and highly
conjugated organic compounds

Infrared (IR) spectroscopy:

Functional groups

Mass spectrometry (MS):

Molecular mass and formula and structure information

Nuclear magnetic resonance (NMR) spectroscopy:

Map of carbon-hydrogen framework

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.

Timeline for MS Development

1897 Early Mass Spectrometry
1919 The observation of isotopes using mass spectrometry
1934 Double Focusing Analyzer
1939 Accelerator Mass Spectrometry

Joseph John Thomson

1946 Time-of-Flight Mass Spectrometry

1947 Preparative Mass Spectrometry
1949 Ion Cyclotron Resonance (ICR)
1953 Reverse Geometry Double
focusing MS
1953 Quadrupole Analyzers

Cited from: http://masspec.scripps.edu/mshistory/

"In recognition of the great merits

of his theoretical and experimental
investigations on the conduction of
electricity by gases.
1906 Nobel Prize

"At first there were very few who believed in the

existence of these bodies smaller than atoms. I was
even told long afterwards by a distinguished physicist
who had been present at my [1897] lecture at the
Royal Institution that he thought I had been 'pulling
their legs."
Replica of J.J. Thomson's third mass spectrometer.

Continuation of Timeline
1956 Gas Chromatography Mass Spectrometry (GC/MS)
1956 Identifying Organic Compounds with Mass
Spectrometry
1962 Mass Spectrometry Imaging
1966 Chemical Ionization
1966 Peptide Sequencing
1966 Tandem Mass Spectrometry
1966 Metabolomics
1968 Electrospray Ionization
1968 Collision Induced Dissociation
1969 Field Desorption-MS of Organic Molecule
Cited from: http://masspec.scripps.edu/mshistory/

Francis William Aston

"For his discovery, by means of his mass
spectrograph, of isotopes, in a large number
of non-radioactive elements, and for his
enunciation of the whole-number rule."
Mass spectrometry of isotopes
1922 Nobel Prize

Continuation of Timeline
1974 Fourier Transform Ion Cyclotron Resonance
1974 Extra-Terrestrial Mass Spectrometry
1975 Atmospheric Pressure Chemical Ionization (APCI)
1976 Californium-252 Plasma Desorption MS

Wolfgang Paul

1978 GC-C-IRMS
1978 Triple Quadrupole Mass Analyzer
1980 Inductively Coupled Plasma MS
1981 Matrix-Assisted Desorption Ionization
1984 Quadrupole/Time-Of-Flight Mass Analyzer
1985 Matrix-Assisted Laser Desorption Ionization (MALDI)

Cited from: http://masspec.scripps.edu/mshistory/

Hans Georg Dehmelt

For the development

of the ion trap
technique.
1989 Nobel prize

Continuation of Timeline
ESI

1987 Soft Laser Desorption of Proteins

1989 ESI on Biomolecules
1989 Monitoring Enzyme Reactions with ESI-MS

John B. Fenn

1990 Protein Conformational Changes with ESI-MS

1990 Clinical Mass Spectrometry
1991 MALDI Post-Source Decay

MALDI

1991 Non-covalent Interactions with ESI

1992 Low Level Peptide Analysis
1993 Oligonucleotide Ladder Sequencing
1993 Protein Mass Mapping
1996 Intact Virus Analyses
Cited from: http://masspec.scripps.edu/mshistory/

Koichi Tanaka
"For the development of soft desorption
ionisation methods for mass spectrometric
analyses of biological macromolecules."

Continuation of Timeline
1998 Electron Capture Dissociation (ECD)
1999 Nanostructure Desorption/Ionization
1999 Quantitative Proteomics and Metabolomics with
Isotope Labels

Fred W. McLafferty Alfred O.C. Nier Alan G. Marshall

2000 Orbitrap
2004 Desorption Electrospray Ionization (DESI)
2004 Electron Transfer Dissociation (ETD)
2005 Direct Analysis in Real Time (DART)

Michael Karas

Malcolm Dole

Brian T. Chait

Klaus Biemann

R. Graham Cooks Donald F. Hunt

Catherine Fenselau Franz Hillenkamp Carol V. Robinson

Cited from: http://masspec.scripps.edu/mshistory/

What information can be

determined?
Molecular weight
Molecular formula (HRMS)
Structure (from
fragmentation fingerprint)
Isotopic incorporation /
distribution
Protein sequence (MS-MS)

Schematic Mass Spectrometer

Whats in a Mass Spectrum

Mass-to-charge ratios of a molecule or its fragment are
graphed or tabulated according to their relative abundance

FragmentIons

FragmentIons:derivedfrommolecularionorhigherweightfragments

Applications
Biomolecule
Pharmaceutical
characterization
analysis
Proteins and peptides
Oligonucleotides

Paleoclimatology
and Archeology
http://www.sciencemag.org/products/lst_20060901.dtl
Paleotemperature
Forensic
analysis/clinical
foraminifera

O16 and O18

Environmental analysis
Pesticides on foods
Soil and groundwater contamination

Relative Abundance of Isotopes

Atomic weight of an element is a weighted average
of the naturally occurring isotopes.

Isotopic Ratio from the Spectra

Mass spec. can be used to measure the isotopic ratios

Continuation of Isotopes

Chlorine (35Cl to 37Cl is 3:1, give M + 2)

Fragmentation

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

Electron Bomb Ionization ( EI )

Sample is heated and energized by a beam of electrons, usually gives a
molecular ion (M+) and a lot of fragments
H H
H C C H
H H
e- +

H H

H C C H

H C C+

H H

H H

(M-R2)+
Mass Spectrum (M-R )+
1
+
M+
(M-R3)

H H

H C+
H

H
C H
H

Electron Bomb Ionization ( EI )

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)
Higher pressure of methane leaked into the source (mtorr)
Reagent ions transfer proton to analyte

Chemical Ionization (CI)

Electron ionization leads to fragmentation of the
molecular ion, which sometimes prevents its detection.
Chemical ionization (CI):
A technique that produces ions with little excess energy.
Thus this technique presents the advantage of yielding a
spectrum with less fragmentation in which the molecular
species is easily recognized.
Consequently, chemical ionization is complementary to
electron ionization.

Chemical Ionization (CI)

Properties of CI
Advantages
Parent Ion
Interface to GC
Insoluble Samples

Disadvantages
No Fragment Library
Need Volatile Sample
Need Thermal Stability
Quantitation Difficult
Low Mass Compounds
(<1000 amu)
Solids Probe Requires
Skilled Operator

Field ionization (FI)

Field ionization (FI) is a method that uses very strong electric
fields to produce ions from gas-phase molecules.

+
+

+
+ +
d<1mm

+
+
+
+ + +
+

Field ionization (FI)

+

+
+
- - +
- + - +
+ - + + - ++ +
+ +
+
+
+ -

+
+
+

+ + +
+
+ +
+
+
+
+
+ +
+
+
+ +
+
+ + + +
+
+
+
+
+
+
+
+ +
+

Matrix Assisted Laser Desorption

Ionization (MALDI)
sample is co-crystallized with a matrix and then irradiated
with laser.
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.

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
Polar : to solvate and separate preformed ion
Less Sensitive to Salts
Lower PRACTICAL detection limits
Easier to interpret spectra (less multiple charges)
Quick and easy
Higher mass detection
Higher Throughput (>1000 samples per hour)

Principle of MALDI

MALDI mass spectrometry has become a powerful analytical

tool for both synthetic polymers and biopolymers.

Fast atom bombardment ( FAB)

Softer than EI and CI. Ions are produced by bombardment with
heavy atoms. Gives (M+H)+ ions and litle fragmentation.
Good for more polar compounds.
Ar + e
Ar+ + Ar
fast

slow

Ar+

acceleration (5-15 KeV)

Ar + Ar+

+ 8 KeV
fast
slow

Properties of FAB
Advantages
Parent Ion
High Mass Compounds
(10,000 amu)
Thermally Labile
Compounds (R.T.)

Disadvantages
No Fragment Library
Solubility in Matrix
(MNBA, Glycerol)
Quantitation Difficult
Needs Highly Skilled
Operator
Relatively Low Sensitivity

ElectroSpray Ionization (ESI)

Electrospray is abbreviated to ESI ample 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.

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

Types of Mass Analyzers

Magnetic sector analyzer
Time of Flight analyzer (TOF) (
Quadrupole analyzers (
Fourier Transform Ion-Cyclotron

Magnetic Sector Analyzer

Magnetic sector analyzer Uses electric and/or
magnetic fields to separate ions

Principle of Magnetic
Sector Analyzer
The ion source accelerates ions to a kinetic
energy given by : (1/2)m2= zV
Where m is the mass of the ion ,v is its velocity, z
is the charge on the ion ,and V is the applied
voltage of the ion optics.

Principle of Magnetic
Sector Analyzer
Only ions of mass-to-charge ratio that have equal
centripetal and centrifugal forces pass through the
flight tube: m v 2 / r = Bzv
By rearranging the equation, m/z = B2r2/2V
It shows that the m/q ratio of the ions that reach
the detector can be varied by changing either the
magnetic field or the applied voltage of the ion
optics.

In summary ,by varying the voltage or magnetic

field of the magnetic-sector analyzer ,the individual
ion beams are separated spatially and each has a
unique radius of curvature according to its
mass/charge ratio.

Advantages
Double focusing magnetic sector mass analyzers are the
"classical" model against which other mass analyzers are
compared.
Classical mass spectra
Very high reproducibility
Best quantitative performance of all MS analyzers
High resolution
High sensitivity
10,000 Mass Range
Linked scan MS/MS does not require another analyzer

 Disadvantages
Requires Skilled Operator
Usually larger and higher cost than other mass analyzers
Difficult to interface to ESI
Low resolution MS/MS without multiple analyzers

Applications
All organic MS analysis methods
Accurate mass measurements
Quantitation
Isotope ratio measurements

Time of Flight Analyzer

TOF analyzer ions are accelerated through a flight tube
and the time of light to the detector is measured

Ions are accelerated and their time of flight to the

detector is measured.

Principle of TOF Analyzer

Uses a pulse of ion mixtures, not steady stream
Ions accelerated into drift tube by a pulsed electric
field called the ion-extraction field
Drift Tube is usually 1-2 m long, under vacuum
Ions traverse the drift tube at different speeds
( L / t ) = v = ( 2zV / m )

Advantages of TOF Analyzer

Good for kinetic studies of fast reactions and for
use with gas chromatography to analyze peaks
from chromatograph
High ion transmission
Can register molecular ions that decompose in the
flight tube
Extremely high mass range (>1MDa)
Fastest scanning

Disadvantages
Requires pulsed ionization method or ion beam
switching (duty cycle is a factor)
Low resolution (4000)
Limited precursor-ion selectivity for most MS/MS
experiments

Applications
Almost all MALDI systems
Very fast GC/MS systems

Quadrupole Analyzers

Quadrupole analyzers ions

are filtered or trapped in a
device consisting of several
metal rods using specifically
tailored electromagnetic
fields

Quadrupole Analyzers

Electric/magnetic fields trap, store, eject ions

Requires an in-line quadrupole to act as

mass pre-filter

Contains a single ring electrode and a top

and bottom cap electrode

Varying RF frequency will vary the m/z ratios

that are trapped

Additional fragmentation can be performed

on ions stored in the ion trap

 Advantages
Easy to use ,simple construction,fast
Good reproducibility
Relatively small and low-cost systems
Quadrupoles are now capable of routinely
analyzing up to a m/q ratio of 3000,which is
useful in electrospary ionization of biomolecules,
which commonly produces a charge distribution
below m/z 3000

 Disadvantages
Low resolution(<4000)
Slow scanning
Low accuracy (>100ppm)
Applications
Majority of benchtop GC/MS and LC/MS systems
Separation of proteins and other biomolecules
with electrosprary
Sector / quadrupole hybrid MS/MS systems

Fourier Transform Ion Cyclotron

Resonance (FT ICR) analyzers

Most FTICR mass spectrometers use superconducting

magnets, which provide a relatively stable calibration over a
long period of time.
Although some mass accuracy can be obtained without
internal calibrant, mass accuracy and resolution are inversely
proportional to m/z, and the best accurate mass measurements
require an internal calibrant.
Unlike the quadrupole ion trap, the FTICR mass spectrometer
is not operated as a scanning device.

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

 Disadvantages
Expensive
Requires superconducting magnet
Subject to space charge effects and ion molecule reactions
Artifacts such as harmonics and sidebands are present in the
mass spectra
Many parameters (excitation, trapping, detection conditions)
comprise the experiment sequence that defines the quality of the
mass spectrum
Generally low-energy CID, spectrum depends on collision
energy, collision gas, and other parameters.

 Applications
Ion chemistry
High-resolution MALDI and electrospray
experiments for high-mass analytes
Laser desorption for materials and surface
characterizatio

The Mass Spectrum

A. Presentation of data
1. The mass spectrum is presented in terms of ion abundance
vs. m/e ratio (mass).
2. The most abundant ion formed in ionization gives rise to the
tallest peak on the mass spectrum this is the base peak.
base peak, m/e 43

58

A. Presentation of data
3. All other peak intensities are relative to the base peak as a
percentage.
4. If a molecule loses only one electron in the ionization
process, a molecular ion is observed that gives its molecular
weight this is designated as M+ on the spectrum.

M+, m/e 114

59

A. Presentation of data
5. In most cases, when a molecule loses a valence electron,
bonds are broken, or the ion formed quickly fragment to
lower energy ions
6. The masses of charged ions are recorded as fragment ions
by the spectrometer neutral fragments are not recorded !

fragment ions

60

B. Determination of Molecular Mass

1. When a M+ peak is observed it gives the molecular mass
assuming that every atom is in its most abundant isotopic
form
2. Remember that carbon is a mixture of 98.9% 12C (mass 12),
1.1% 13C (mass 13) and <0.1% 14C (mass 14)
3. We look at a periodic table and see the atomic weight of
carbon as 12.011 an average molecular weight
4. The mass spectrometer, by its very nature would see a peak
at mass 12 for atomic carbon and a M + 1 peak at 13 that
would be 1.1% as high

- We will discuss the effects of this later

61

B. Determination of Molecular Mass

5. The Nitrogen Rule is another means of confirming the
observance of a molecular ion peak
6. If a molecule contains an even number of nitrogen atoms
(only common organic atom with an odd valence) or no
nitrogen atoms the molecular ion will have an even mass
value
7. If a molecule contains an odd number of nitrogen atoms, the
molecular ion will have an odd mass value
8. If the molecule contains chlorine or bromine, each with two
common isotopes, the determination of M+ can be made
much easier, or much more complex as we will see.

62

member and Review

The Rule of Thirteen Molecular Formulas from Molecular
Mass Lecture 1
When a molecular mass, M+, is known, a base formula can
be generated from the following equation:

M / 13 =

( n + r ) / 13

The base formula being:

CnHn + r

For this formula, the HDI can be calculated from the

following formula:

HDI

(nr+2)/2

63

member and Review

The Rule of Thirteen
The following table gives the carbon-hydrogen
equivalents and change in HDI for elements also
commonly found in organic compounds:
Element
added

Subtrac
t:

H12

(U in
text)
7

H12

-7

CH4

N
S

HDI

Element
added

Subtract:

HDI
(U in text)

Cl

C2H11

Br

C6H7

-3

CH7

CH2

1/2

Si

C2H4

C2H8

C2H7

C9H19

35

79

64

C. High Resolution Mass Spectrometry

1. If sufficient resolution (R > 5000) exists, mass numbers can
be recorded to precise values (6 to 8 significant figures)
2. From tables of combinations of formula masses with the
natural isotopic weights of each element, it is often possible
to find an exact molecular formula from HRMS
Example: HRMS gives you a molecular ion of 98.0372; from
mass 98 data:
C3H6N4
98.0594
C4H4NO2

98.0242

C4H6N2O

98.0480

C4H8N3

98.0719

C5H6O2

98.0368 gives us the exact formula

C5H8NO
C5H10N2
CH

98.0606
98.0845
98.1096

65

D. Exact Mass Determination

1. Need Mass Spectrometer with a high mass accuracy 5 ppm
(sector or TOF)
2. C9H15NO4, FM 201.1001 (mono-isotopic)
3. Mass accuracy = {(Mass Error)/FM}*106
4. Mass Error = (5 ppm)(201.1001)/106 = 0.0010 amu

E. Mass accuracy
1. Mass Error = (5 ppm)(201.1001)/106 = 0.0010 amu
2. 201.0991 to 201.1011 (only 1 possibility)
3. Sector instruments, TOF mass analyzers
4. How many possibilities with MA = 50 ppm? with 100 ppm?

66

F. Important fragmentation patterns in EI

Fragmentation leads to smaller ions by the cleaving of parts of molecule

Unreasonable losses from molecular ion:

M [3~ 14] and M [21~26] are unraesonable losses!

Reasonable losses from molecular ion:

Neutral fragments expelled by simple cleavage
OE+ EE+ + OE
Neutral fragments expelled by multi-centered fragments
OE+ EE + OE +

67

1. Simple cleavage
Radical Remote Fragmentation (a-cleavage)
i. Compounds containing saturated heteroatoms

R'

CR2 Y

R''

R' + CR2

YR''

ii. Compounds containing unsaturated heteroatoms

R'

CR2

R' + CR2

iii. Compounds containing unsaturated carbon-carbon bonds

R

CH2

CH

CH2

R + CH3

CH

CH2

-e

R
CH2

CH2
CH

CH

CH2

CH3
68

Charge Remote Fragmentation ( i-cleavage)

OH R'

R
R

+ OR

+ R'

R'

-Cleavage and i-cleavage are competitive reactions.

The sequence of cleavage tendency:
N > S, O, bond, R > Cl > Br > I
The sequence of i cleavage tendency:
halogen > O, S >> N, C
69

Compounds without heteroatoms ( -cleavage)

R

-e

R'

R + R'

R + R'

Examples:
R

CH

OH

R + CHR'

OH

R'

-cleavage

O
C

NH

CH2

R'

O
C

NH

CH2

R'

R + O

O
C

NH

CH3 + R'

NH

CH2 R

i-cleavage

R'

R + SR'
70

Examples:

71
57
43
29
O
CH3

CH2

CH2

CH2

CH2

OH

45
59
73
87
71

2. Rearrangement
McLafferty rearrangement
Pattern I

A
B

A
B

H
+
C

H
+
H2C

E
D

72

E
D

E
D

Pattern II
A
B

BH

+
C

Examples:
CH3

O
nC4H9

H
CH2

C4H9

OH2

C
CH2
C4H9
CH2

CH2

OH

C
C4H9

C4H9

CH2

OH
C

OH

Second McLafferty rearrangement

CH3

CH2

CH2

CH2

CH2

73

CH
CH3

E
D

Retro Diels-Alder rearrangement

R

-e

R
+

R
+

Examples:

CH3

CH3
+

74

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

H
75

Four-member ring rearrangement

CH3 CH2 O
- C2H4

CH2 CH3

- CH3

CH3 CH2 O

CH2 =

HO CH2

Other rearrangement

C3H7

R +

C3H5 + H2
76

H2C

H2C

O CH2

H
(CH3)2N

(CH3)2N

(CH3)2N

(CH3)2N

(CH3)2N

m/z 84

H
(CH3)2N

(CH3)2N

(CH3)2N

(CH3)2N
m/z 110

77

G. Patterns of different organic compounds fragmentation

Saturated hydrocarbons
1. Alkanes
Dodecane

Figure 1 Mass spectrum of dodecane.

78

2. Branched Alkanes
m/z=43
C3

100

5-Methylpentadecane

169

141

% OF BASE PEAK

90

m/z=57
C4

80
70

CH3(CH2)3

60

C6 m/z=85
m/z=71
C5
m/z=99

50
40
30
20

C7

10
0
0

57

113
C8 C9 C10

CH
CH3

(CH2)9CH3

85

C12

M 15

M
C16

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210220 230

Figure 2 Mass spectrum of 5-methylpentadecane.

79

4-methylundecane

Figure 3 Mass spectrum of 4-methylundecane.

Figure 4 Mass spectrum of 2,2,4,6,6-pentamethylheptane.

80

3. Cycloalkanes
56(C4H8+)

% OF BASE PEAK

100
90
80

84(M )

70
60

41(C3H5+)

Cyclohexane

50
40
30
20
10
0

M=84
0

10 20 30 40 50

60 70 80

90 100 110

Figure 5 Mass spectrum of cyclohexane.

81

1-methyl-3-pentylcyclohexane

Figure 6 Mass spectrum of 1-methyl-3-pentylcyclohexane.

82

Aromatic hydrocarbons

Figure 7 Mass spectrum of 1-phenylhexane

83

Process of fragmentations:
CH2

I.
m/z=91

m/z = 162

HC CH

HC CH

m/z=65

m/z=39
H2
C

II.

m/z=91

CH2

CH2

CH
H

m/z = 162

m/z=92
HC CH

C 6H 9

III.
m/z = 162

m/z=77

m/z=51
84

Alcohols, Phenol and Ether

1. Alcohol

Figure 8 Mass spectrum of 1-dodecanol

85

Process of fragmentations:
I.

R1
R2

OH

R1

R3

OH

R2

R3

m/z: 31,59,73,......

H
II. RHC

III.

H OH

OH
CH2

RHC

C
H2 n

CH2

H 2C
H2
C C
H

C
H
H

- H2O

CH2

RHC

C
H2 n

H
O+
H
CH2 - H2C CH2
RHC
CH2
CH2

- H2O

CH2

(CH2)n

or

RHC

(CH2)n

H2C CH R

M - (Alkene + H2O)

- H2C

CH2
R

CH2

CH2
H2
C CH

86

2. Phenol

H2
C
OH
H
C
H2
H2
C
OH
H
O

- H2O

CH2
CH2

H2O

CH2
O

87

3. Ether

Figure 9 Mass spectrum of hexyl ether.

88

Ketone and Aldehyde

Figure 10 Mass spectrum of 2-dodecanone.

89

57
100
90

CH3(CH2)7CHO

% OF BASE PEAK

80
70
60

MW 142

44

50
40

M-44
M-43

30

M-CH2CH2
M-H2O

20
10

M-1
M

0
0

10 20

30

40

50

60

70

80 90 100 110 120 130 140 150

Figure 11 Mass spectrum of octanal .

90

Carboxylic acid
Ch3(CH2)4CO
CH3(CH2)4
CH3(CH)3

(small) 99
71

57

CH3(CH2)2 43
CH3CH2 29
CH3 CH2

O
CH2

CH2

CH2

C
45

59(small)
73
87

OH
CO2H
CH2CO2H
(CH2)2CO2H
(CH2)3CO2H
91

Ester

Figure 12 Mass spectrum of hexyl benzoate.

92

Other compounds

Figure 13 Mass spectrum of 1-chlorododecane.

93

Figure 14 Mass spectrum of 1-bromododecane.

94

CH3

CH2

CH3

CH3

44

100
% OF BASE PEAK

CH

CH2

CH2

CH3

86

90
80
70
60
50

114

58

40
30
20
10
0

129(M )
29
0 10 20 30 40 50

60 70 80

90 100 110 120 130 140

Figure 15 Mass spectrum of N-isopropyl-N-methylbutan-1-amine

95

84

H3C CH2 NH

% OF BASE PEAK

100
90
80
70
60

70

41

M=113

50
40

27

30

56

113(M )

20
10
0

00

10 20

30 40 50 60 70 80 90 100 110 120

Figure 16 Mass spectrum of N-ethylcyclopentamine.

96

% OF BASE PEAK

100
90
80
70
60
50
40
30
20
10
0

OH
C

74

Methyl octanoate

CH3(CH2)6COOCH3
OCH3
H2C
158(M)
O
159(M+1)
CH2CH2OCH3
160(M+2)
O
87
COCH3
M
121[M-31]
59
M+1
M+2
0 10 20 30 40 50 60 70 80 90 100110 120130140150 160

Figure 17 Mass spectrum of methyl octanoate.

97

Exercise 1:
HRMS shows exact mass of compound A is 136.0886 and the formula of this
compound is C9H12O, please confirm the structure of compound A.
Answer

% OF BASE PEAK

DEB: = (2*9+2-12)/2 = 4
m/z: 118 M-18 M-H2O

107

100
79
77

51

50

20

40

39, 51, 77
136

41
39
0

-OH

107

M-29

118

60

80

M-C2H5

-C2H5

100 120 140 160

H2
C CH

CH
OH

98

Exercise 2:
Please confirm the structure of compound A.

100
% OF BASE PEAK

I158 = 13%
I157 = 3.7%
I156 = 41%

94

156
77

50
65
2739

107

51

158
157

20

40

60

80

100 120

140

160

99

Answer
1. I156/I158 = 3/1

Containing one Cl atom

Containing eight C atoms

2. Nc = 3.7/411.1%8
3. m/z: 39, 51, 77
107

; 94

H2 H2
O C C Cl

OH

O CH2

CH2

4. 156-35-77-16-14 = 14
CH2
O

107

77

Cl

Cl
O

100

Exercise 3:
Based on the EI mass spectrum of compound A, please write the
process of fragmentation

121

% OF BASE PEAK

100
93

65

50

76
77

20

40

60

80

104

134

100 120

150

140
101

160

Answer

NO

O
OH

O
N

-H

-OH

-NO
O

NO2

150

NO

OH
121

134

OH

OH

O
H

-CO
121
93

H
65

102

Hyphenated Mass Techniques

Chromatography: Separation
Mass: Detection

Chromatography-Mass Spectroscopy :
Separation + Detection

43
57
29
15

GC-MS

LC-MS

CZE-MS

71

85

99 113

142
m/z

Hyphenated GC-MS
Gas chromatography-mass spectrometry (GC-MS) is a method that combines
the features of gas-liquid chromatography and mass spectrometry to identify
different substances within a test sample.
HEW LETT
PACKARD

5972A

Mass
Selective
Detector

1.0
DEG/MIN

MS
HEW LETT
PACKARD

5890

Gas Chromatograph (GC) Mass

B
Spectrometer

Sampl
e
A
D

B
C

C
A

Sampl
e

DB

Separatio
n

A
B
C
D

Identificatio
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
Liquid chromatography-mass spectrometry (LC-MS) is an analytical
chemistry technique that combines the physical separation capabilities of
liquid chromatography with the mass analysis capabilities of mass
spectrometry.
Different compounds exit
at different time

Identification of each molecule

ion

LC

MS

B
C

t/min

Peak A: mass1
Peak B: mass2
Peak C: mass3

Hyphenated LC-MS

Liquid chromatography-mass spectrometry (Ion trap LCMS system )

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.

Tandem Mass Spectrometry