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

Elemental Analysis
Burn the compound and measure the amounts of CO2, H2O and other components that are

Melting Point
Density Optical rotation Appearance Odor

produced to determine the

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 1946 Time-of-Flight Mass Spectrometry
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

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/

"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 "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."

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/

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 1978 GC-C-IRMS
Wolfgang Paul

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
1987 Soft Laser Desorption of Proteins 1989 ESI on Biomolecules ESI

1989 Monitoring Enzyme Reactions with ESI-MS

1990 Protein Conformational Changes with ESI-MS 1990 Clinical Mass Spectrometry 1991 MALDI Post-Source Decay 1991 Non-covalent Interactions with ESI 1992 Low Level Peptide Analysis 1993 Oligonucleotide Ladder Sequencing

John B. Fenn

MALDI

Koichi Tanaka

1993 Protein Mass Mapping

1996 Intact Virus Analyses
Cited from: http://masspec.scripps.edu/mshistory/

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

Michael Karas

Malcolm Dole

Brian T. Chait

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

Fragment Ions

Fragment Ions: derived from molecular ion or higher weight fragments

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
H H e+ H C C H H H
H
H H H C C+ H H H

H C H H

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

H C+ 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

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 =
The base formula being:

( n + r ) / 13

CnHn + r

For this formula, the HDI can be calculated from the following formula:

HDI = ( n r + 2 ) / 2
63

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 C H12 O N S

Subtrac t: H12 C CH4 CH2 C2H8

D HDI (DU in text) 7 -7 1 1/2 2

Element added
35Cl 79Br

Subtract:

D HDI (DU in text) 3 -3 2 1 2

C2H11 C6H7 CH7 C2H4 C2H7

F Si P

C9H19

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 C4H4NO2 C4H6N2O C4H8N3 C5H6O2 C5H8NO C5H10N2 C7H14 98.0594 98.0242 98.0480 98.0719 98.0368 gives us the exact formula 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 CH CH2 CH2 -e R CH2 CH CH CH3
68

CH2

R + CH3

CH2

Charge Remote Fragmentation ( i-cleavage)

R
R R'

OH R'
i

+ OR

+ R'

-Cleavage and i-cleavage are competitive reactions.

The sequence of -cleavage tendency: N > S, O, p bond, R > Cl > Br > I The sequence of i-cleavage tendency:

halogen > O, S >> N, C

69

Compounds without heteroatoms ( s-cleavage)

R R' -e R + R' s R + R'

Examples:
R CH R' OH R + CHR' OH

-cleavage
NH CH2 R' R O C NH CH3 + R'

O C

O C

NH

CH2

R'

R + O

NH

CH2 R

i-cleavage

R'

R + SR'
70

Examples:

71 57 43 29 O CH3 CH2 CH2 CH2 CH2 C 45 59 73 87

71

OH

2. Rearrangement
McLafferty rearrangement
Pattern I

H A B C E D A B

H E D C

H A + B H2C D E

H A B C E D A + B

H E C D

H E D C

72

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

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

C CH2 C4H9 CH2

OH C OH

Second McLafferty rearrangement

CH3 CH2

CH2 H CH CH3

CH2 CH2

73

Retro Diels-Alder rearrangement

R -e R R R +

R +

Examples:

CH3

CH3 +

74

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

C6H13 H2O + C6H13 H HO

OH H CH3

H2C CHCH3 + H2O + CH2=CH2

O - CO

O - CO

H O

O + H
75

H2O

Four-member ring rearrangement

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

HO CH2

Other rearrangement
X X R +

C3H7

C3H5 + H2
76

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 m/z=57 C4
5-Methylpentadecane

100 90

169 CH3(CH2)3 57 CH CH3

141 (CH2)9CH3

% OF BASE PEAK

80 70 60 50 40 30 20 10 0 0

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

85

C7 113 C8 C9 C10

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+)
100

% OF BASE PEAK

90 80 70 60 50 40 30 20 10 0

84(M ) 41(C3H5+)
Cyclohexane

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:

I. m/z = 162
HC CH

CH2

m/z=91

HC CH

m/z=39
H2 C

m/z=65

m/z=91

II.
H

CH2 CH H

CH2 H

m/z = 162

m/z=92

III. m/z = 162

C 6H 9

HC CH

m/z=77

m/z=51
84

Alcohols, Phenol and Ether

1. Alcohol

Figure 8 Mass spectrum of 1-dodecanol

85

Process of fragmentations:
R1 R2 R3 - R3 R1 C R2 m/z: 31,59,73,...... OH

I.

OH

H II. RHC

C H2 n

OH CH2

H OH RHC C H2 n CH2

- H2O

RHC

CH2

or

RHC

CH2 (CH2)n

(CH2)n

H O+ H CH2 - H2C CH2 III. RHC CH2 CH2

H2C H2 C C H C H H

- H2O

H2C CH R M - (Alkene + H2O)

CH2

- H2C

CH2 R

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 80

CH3(CH2)7CHO 44

% OF BASE PEAK

70 60 50 40 30 20 10 0
0 10 20 30 40 50 60 70

MW 142
M-44 M-43 M-CH2CH2 M-H2O M-1 M

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 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

(small) 99 71

57

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

CH CH3

N CH3

CH2

CH2

CH2

CH3

86

100
% OF BASE PEAK

44

90 80 70 60 50 40 30 20 10 0

58

114 129(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
100

H3C CH2 NH

% OF BASE PEAK

90 80 70 60 50 40 30 20 10 0

41 56

70

M=113

27

113(M )

10 20

30 40 50 60 70 80 90 100 110 120

Figure 16 Mass spectrum of N-ethylcyclopentamine.

96

100

OH C 74

Methyl octanoate

% OF BASE PEAK

90 80 70 60 50 40 30 20 10 0

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 120 130140150 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

100
% OF BASE PEAK

107 79 51 77

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

-OH

50 41 39

39, 51, 77

136 118

107

M-29

M-C2H5

-C2H5
H2 C CH

20

40

60

80

100 120 140 160

CH OH

98

Exercise 2:
Please confirm the structure of compound A.
94
I158 = 13% I157 = 3.7% I156 = 41%

100

% OF BASE PEAK

156 50 65 2739 51 107 158 157 0 20 40 60 80 100 120 140 160 77

99

Answer
1. I156/I158 = 3/1 2. Nc = 3.7/411.1%8 3. m/z: 39, 51, 77 Containing one Cl atom Containing eight C atoms ; 94
O CH2 OH
H2 H2 O C C Cl

107
4. 156-35-77-16-14 = 14

CH2
CH2 O 107

Cl

77

65

51

39

Cl O H O H H O

94

100

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

20

40

60

80

100 120

140

160
101

Answer

NO

O
OH

H O N

-H

-OH

-NO O

NO

OH 121
NO2

150

134

O
H

OH

OH O -CO 121

93

H 65

102

Hyphenated Mass Techniques

Chromatography: Separation Mass: Detection

Chromatography-Mass Spectroscopy : Separation + Detection

29 15

43 57 71 85 99 113 142 m/z

GC-MS

LC-MS

CZE-MS

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 PACKARD 5972A Mass Selective Detector

1.0 DEG/MI N

MS
HEWLETT PACKARD

5890

Sample

Gas Chromatograph (GC)

B A C D

Mass Spectrometer
A B C D

D A A C

C B

B D
Separation

Sample

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 at different time Identification of each molecule ion

LC

MS

B A C Peak A: mass1 Peak B: mass2 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