Spectrometrie de mase

OUTLINE

Introducere în spectrometria de mase

Metode de ionizare
Analizorul de mase
Fragmentarea și interpretarea SM
Tehnici MS combinate
 Identificarea chimică
Comparison of Elemental Analysis
Physical Properties
Burn the compound and
Boiling Point
measure the amounts of
Melting Point
CO2, H2O and other
Density
Optical rotation components that are
Appearance produced to determine the
Odor empirical formula
 Metode spectroscopice pentru
determinarea structurii
Spectroscopie în ultraviolet-vizibil (UV/Vis):
determinarea concentrației soluțiilor ionilor metalici de tranziție și a
compușilor organici foarte conjugați
Spectroscopie în infraroșu (IR):
Spectrometrie de masă (MS):
Informații privind masa moleculară și formula și structura
Spectroscopie de rezonanță magnetică nucleară (RMN):
Harta interacțiunilor carbon-hidrogen

Grupuri funcționale
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
spectrometer’s 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
1946 Time-of-Flight Mass Spectrometry
1947 Preparative Mass Spectrometry
1949 Ion Cyclotron Resonance (ICR)
1953 Reverse Geometry Double
focusing MS
1953 Quadrupole Analyzers
Joseph John Thomson
"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
Francis William Aston
1966 Tandem Mass Spectrometry "For his discovery, by means of his mass
1966 Metabolomics spectrograph, of isotopes, in a large number
of non-radioactive elements, and for his
1968 Electrospray Ionization enunciation of the whole-number rule."

1968 Collision Induced Dissociation Mass spectrometry of isotopes

1969 Field Desorption-MS of Organic Molecule 1922 Nobel Prize

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

 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 Hans Georg Dehmelt
1985 Matrix-Assisted Laser Desorption Ionization (MALDI) “For the development
of the ion trap
Cited from: http://masspec.scripps.edu/mshistory/ 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
Koichi Tanaka
"For the development of soft desorption
ionisation methods for mass spectrometric
analyses of biological macromolecules."

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

 Continuation of Timeline
1998 Electron Capture Dissociation (ECD)
1999 Nanostructure Desorption/Ionization
1999 Quantitative Proteomics and Metabolomics with
Fred W. McLafferty Alfred O.C. Nier Alan G. Marshall
Isotope Labels
2000 Orbitrap
2004 Desorption Electrospray Ionization (DESI)
2004 Electron Transfer Dissociation (ETD)
2005 Direct Analysis in Real Time (DART) Klaus Biemann R. Graham Cooks Donald F. Hunt

Catherine Fenselau Franz Hillenkamp Carol V. Robinson

Michael Karas Malcolm Dole Brian T. Chait

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

Ce informații pot fi determinate?

• Greutatea moleculară
Formula moleculară (HRMS)
Structura moleculară (de la amprenta fragmentării)
Distribuția izotopică
Secvența proteică (MS-MS)
Schema Spectrometrului de mase
 Ce este într-un spectru de masă?
Raporturile masă/sarcină ale unei molecule sau ale fragmentului
acesteia sunt reprezentate sau ”desenate” în funcție de abundența
lor relativă

Fragment Ions

Ioni fragmentari: derivați din ioni moleculari sau fragmente de greutate mai mare
 Aplicaţii
Analiza farmaceutică Caracterizarea biomoleculei
Proteine și peptide
•Oligonucleotide
•

Paleoclimatologie și Arheologie

Paleotemperatura O16 and O18

foraminifera

Analize medico-legale/clinice
Analiza mediului
Pesticide în alimente
Contaminarea solului și a apelor
subterane
 Abundența relativă a izotopilor
Masa atomică a unui element este o medie ponderată a
izotopilor săi naturali.
 Raportul izotopic din spectre
Spec. de masă poate fi utilizat pentru a măsura raporturile
izotopice
 Continuare –despre izotopi

Clor (raportul 35Cl la 37Cl este de 3:1, formează ioni M + 2)

Fragmentarea
 Ionization Methods

• Ionizare cu impact de electroni (EI)

Ionizare chimică (CI)
Ionizare câmp (FI)
Ionizare cu laser asistată de o matrice
(MALDI)
Bombardament atom rapizi (FAB)
Ionizare prin electro-pulverizare (ESI)
Ionizarea cu impact de electroni ( EI )
Proba este încălzită și energizată de un fascicul de electroni, dă de obicei un
ion molecular (M+) și o mulțime de fragmente

• H H
H C C H
H H
H H H H
e- + H C C H H C C+ H
H H H H

H H
(M-R2)+ H C+ C H
Mass Spectrum (M-R )+
1
(M-R3) + M+ H H
Ionizarea cu impact de electroni ( 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 Disadvantages
No Fragment Library
Need Volatile Sample
Parent Ion
Need Thermal Stability
Interface to GC
Quantitation Difficult
Insoluble Samples
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+ acceleration (5-15 KeV)

Ar+ + Ar Ar + Ar+

fast slow + 8 KeV

fast slow
 Properties of FAB
Disadvantages
Advantages
No Fragment Library
Parent Ion
Solubility in Matrix
High Mass Compounds
(10,000 amu) (MNBA, Glycerol)
Thermally Labile Quantitation Difficult
Compounds (R.T.) 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 Disadvantages
Electrospray Ionization can
be
No Fragmentation
easily interfaced to LC.
Need Polar Sample
Absolute signals from
Electrospray are more easily Need Solubility in Polar
Solvent (MeOH, ACN,
reproduced, therefore, better
H2O, Acetone are best)
quantitation.
Mass Accuracy is considered
Sensitive to Salts
better. Suppression
Multiple charging is more
common then MALDI.
 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)m2= 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 )½
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 electrospray 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

57
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

58
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

59
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…

60
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.

61
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 = ( n – r + 2 ) / 2
62
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 Subtrac  HDI Element Subtract:  HDI

added t: (U in added (U in text)
text)
C H12 7 35
Cl C2H11 3
H12 C -7 Br
79
C6H7 -3
O CH4 1 F CH7 2
N CH2 1/2 Si C2H4 1
S C2H8 2 P C2H7 2
I C9H19 0

63
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 98.0606
C5H10N2 98.0845
C7H14 98.1096

64
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?

65
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 +·

66
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 Y R' + CR2 Y

iii. Compounds containing unsaturated carbon-carbon bonds

-e 
R CH2 CH CH2 R CH2 CH CH2

R + CH3 CH CH2 CH2 CH CH3

67
Charge Remote Fragmentation ( i-cleavage)
i
R OH R' R + OR

R i
C O R + R' C O
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

68
Compounds without heteroatoms ( -cleavage)
-e 
R R' R + R' R + R'

Examples:

R CH OH R + CHR' OH
R' -cleavage
O O
R C NH CH2 R' R C NH CH3 + R'

O
R C NH CH2 R' R + O C NH CH2 R
i-cleavage
+
R S R' R + SR'

69
Examples:

71

57
43

29
O

CH3 CH2 CH2 CH2 CH2 C OH

45

59
73

87
70
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

71
Pattern II
H H H H
A E A E A E A E
+
B D B D B D BH D
C C C C

Examples:
CH3 H
O H
CH2 OH2 O OH
nC4H9 C C4H9
C C C
CH2
C4H9 C4H9 CH2 C4H9
CH2 CH2

OH

C
OH CH2 CH2
Second McLafferty rearrangement
H CH2
CH3 CH2
CH
CH3 72
Retro Diels-Alder rearrangement

R R R R
-e
+

R R
+

Examples:

CH3 CH3

73
 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

74
Four-member ring rearrangement

- CH3 H2 C H
CH3 CH2 O CH2 CH3 CH3 CH2 O CH2 =
H2 C O CH2
- C2H4
HO CH2

Other rearrangement

X X
R R +

C3 H 7 C3H5 + H2

75
 H
(CH3)2N (CH3)2N (CH3)2N

H
(CH3)2N
(CH3)2N m/z 84

H
(CH3)2N
(CH3)2N (CH3)2N

(CH3)2N
m/z 110

76
G. Patterns of different organic compounds’ fragmentation
Saturated hydrocarbons
1. Alkanes

Dodecane

Figure 1 Mass spectrum of dodecane.

77
2. Branched Alkanes

m/z=43 5-Methylpentadecane
C3
100 169 141
90
80 m/z=57 CH3(CH2)3 CH (CH2)9CH3
% OF BASE PEAK

70 C4 CH3
60

50 C6 m/z=85 57 85
40 m/z=71
C5
30 m/z=99
M
C7 113
20
10 C8 C9 C10 C12 M 15 C16
0
0 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.

78
 4-methylundecane

Figure 3 Mass spectrum of 4-methylundecane.

Figure 4 Mass spectrum of 2,2,4,6,6-pentamethylheptane. 79

3. Cycloalkanes

56(C4H8+)
100
90
84(M )
% OF BASE PEAK

80
70
60 41(C3H5+)
Cyclohexane
50
40
30
20
M=84
10
0
0 10 20 30 40 50 60 70 80 90 100 110
Figure 5 Mass spectrum of cyclohexane.

80
 1-methyl-3-pentylcyclohexane

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

81
Aromatic hydrocarbons

Figure 7 Mass spectrum of 1-phenylhexane

82
Process of fragmentations:

I. CH2

m/z = 162 m/z=91

HC CH HC CH

m/z=39 m/z=65 m/z=91

H2
C CH2
CH2
II. H
CH
H H
m/z = 162 m/z=92

C 6H 9 HC CH
III.

m/z = 162 m/z=77 m/z=51

83
 Alcohols, Phenol and Ether
1. Alcohol

Figure 8 Mass spectrum of 1-dodecanol

84
Process of fragmentations:

R1  R3 R1
I. R2 C OH C OH
R3 R2
m/z: 31,59,73,......

H OH H OH
- H2O RHC CH2 RHC CH2
II. RHC CH2 RHC CH2 or
C C
H2 n (CH2)n (CH2)n
H2 n

H
O+
H CH2 - H2C CH2
III. H2C CH R
RHC CH2 - H 2O
CH2 M - (Alkene + H2O)

H 2C
H2
C
H
CH2 - H2C CH2 CH2
H2
R C C H R C CH
H
85
2. Phenol

H2
C CH2
OH - H2 O
H
C CH2
H2
H2
C - H2O CH2
OH
H
O O

86
3. Ether

Figure 9 Mass spectrum of hexyl ether.

87
Ketone and Aldehyde

Figure 10 Mass spectrum of 2-dodecanone.

88
 57
100

90
80 CH3(CH2)7CHO
% OF BASE PEAK

70

60 MW 142
44
50

40
M-44
30
M-43
20 M-CH2CH2 M-1
10 M-H2O
M
0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

Figure 11 Mass spectrum of octanal .

89
 Carboxylic acid

Ch3(CH2)4CO (small) 99
CH3(CH2)4 71
CH3(CH)3 57

CH3(CH2)2 43
O
CH3CH2 29
CH3 CH2 CH2 CH2 CH2 C OH
45 CO2H

59(small) CH2CO2H

73 (CH2)2CO2H

87 (CH2)3CO2H

90
Ester

Figure 12 Mass spectrum of hexyl benzoate.

91
Other compounds

Figure 13 Mass spectrum of 1-chlorododecane.

92
Figure 14 Mass spectrum of 1-bromododecane.

93
 CH3 CH N CH2 CH2 CH2 CH3

CH3 CH3
86
100 44
90
% OF BASE PEAK

80
70
60
50
58 114
40
30
20 129(M )
10 29
0
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

94
 H3C CH2 NH
84
100
90
% OF BASE PEAK

80
70
60 41 70 M=113
50
40 56
30 27 113(M )
20
10
0
0 10 20 30 40 50 60 70 80 90 100 110 120
Figure 16 Mass spectrum of N-ethylcyclopentamine.

95
 OH
100 Methyl octanoate
74
90 C CH3(CH2)6COOCH3
% OF BASE PEAK

80 H2C OCH3
158(M)
70 O 159(M+1)
60
CH2CH2OCH3 160(M+2)
50 O
40
87
COCH3
30
M
20
59 121[M-31] M+1
10
0
M+2
0 10 20 30 40 50 60 70 80 90 100110 120 130140150 160

Figure 17 Mass spectrum of methyl octanoate.

96
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

107 DEB: Ω = (2*9+2-12)/2 = 4

100
m/z: 118 M-18 M-H2O
% OF BASE PEAK

79
-OH
77
51 39, 51, 77
50
107 M-29 M-C2H5
41 136
39 118
-C2H5

0 20 40 60 80 100 120 140 160 H2

C CH
CH 3

OH

97
Exercise 2:

Please confirm the structure of compound A.

94 I158 = 13%
100 I157 = 3.7%
I156 = 41%
% OF BASE PEAK

156

50 77
65
51 107 158
2739
157

0 20 40 60 80 100 120 140 160

98
Answer

1. I156/I158 = 3/1 Containing one Cl atom

2. Nc = 3.7/41÷1.1%≈8 Containing eight C atoms

H2 H2
3. m/z: 39, 51, 77 ; 94 OH O C C Cl

107 O CH2

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

CH2
O

107

O Cl 77

Cl

O H O H O
H

99
 Exercise 3:
Based on the EI mass spectrum of compound A, please write the
process of fragmentation

100 121
93
% OF BASE PEAK

50 65
76
77 104
134 150

0 20 40 60 80 100 120 140 160

100
 O
Answer NO O

H OH
重排

O
N
-OH -NO
-H O

O
O NO
OH

121
NO2 O
150 134

OH OH
O
O
H
-CO

121

93

65 101
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
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.
HEWLETT Mass
5972A Selective
PACKARD Detector

1.0
DEG/MIN

MS
HEWLETT
PACKARD

5890

Sample Gas Chromatograph (GC) Mass Spectrometer

B
D A
A C B
D
A C
B
DB C
A
C D

Sample Separation Identification

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 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

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