Mass spectroscopy and fragmentation rule

Introduction to Mass Spectroscopy, instrumentation Process, and Interpretation of Mass Spectrum, fragmentation rules,

1. Identification of Organic Compounds by Mass
Spectrometry
National Centre of Excellence in Analytical Chemistry
Zahid Wajdan
Zahid
wajdan

2. Contents
Introduction of MS
Tools for the Inspection of MS Spectrum
Masses Game
Fragmentation process
Types of Fragmentation
General Guideline for Fragmentation
Compounds under observation
Spectra Analysis
Alkane, Alcohols, Ethers, Ketone
Acknowledgement
2

3. Introduction of MS
It is a technique used for measuring the molecular weight and determining the
molecular formula of an organic molecule.
Creation of ions – the sample molecules are subjected to a high energy
beam of electrons, converting some of them to ions
Separation of ions – as they are accelerated in an electric field, the ions are
separated according to mass-to-charge ratio (m/z)
Detection of ions – as each separated population of ions is generated, the
spectrometer needs to qualify and quantify them
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4. MS Instrumentation
4

5. What information can be determined
Molecular weight
Molecular formula
Structure (fragmentation fingerprint)
Isotopic incorporation / distribution
Protein sequence (MS-MS)
5

6. Tools for the Inspection of MS Spectrum
●
Nitrogen Rule
●
Rule of Thirteen
●
Hydrogen Density Index (HDI)
●
Stevenson's rule
●
Double Bond Equivalent
6

7. Masses game (MOLECULAR SPECIES RECOGNITION)
Average – calculated using the average mass of each element weighted for its
natural isotopic abundance.
Nominal – the mass of the most abundant isotope of each element rounded to
the nearest integer value. The nominal molecular weight represents only the
integer portion of the value.
Monoisotopic – the mass of the most abundant isotope of each element.
Exact – calculated using the exact mass of a single isotope (most frequently the
lightest isotope) of each element present in the molecule.
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8. Example C33 H40 N2 O9
8

9. Mass spectrum
The x-axis, in units of m/z, represents the
formula weight of the detected ions.
The y-axis represents the relative abundance
of each detected ion.
The most abundant ion (tallest peak) is called
the base peak.
The base peak is usually an easily formed
fragment of the original
compound.
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10. Molecular ion: The ion obtained by the loss of an electron from the
molecule.
Base peak: The most intense peak in the MS, assigned 100% intensity M+
Symbol.
Radical cation: +ve charged species with an odd number of electrons
(M•+).
Fragment ions: Lighter cations formed by the decomposition of the
molecular ion. These often correspond to stable carbcations.
10
Terminology

11. Fragmentation
11

12. fragmentatioN
 Fragmentation is a type of chemical dissociation that can take place
by a process of heterolysis or hemolysis and producing lower weight
fragments.
Fragmentation process:
Bombardment of molecules by an electron beam with energy between 10-15eV
usually results in the ionization of molecules by removal of one electron
(Molecular ion formation).
When the energy of electron beam is increased between 50-70eV, these molecular
ions acquire a high excitation resulting in their break down into various fragments.
This process is called “ Fragmentation process”.
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13. 13

14. Types of fragmentation
There are 13 types of fragmentation;
A1-A5, B, C, D, E1, E2, F, G, and H.
Again these are dived into two classes;
1Simple fragmentation
2Rearrangement fragmentation.
MODES of CLEAVAGE
Sigma bond cleavage
Radical site-initiated cleavage
Charge site-initiated cleavage
14

15. Radical-site-initiated:
is one of the most common one-bond cleavages and is more commonly called an
-cleavage.
α
Inductive cleavage:
involves the attraction of an electron pair by an electronegative heteroatom that
ends up as a radical or as a closed-shell neutral molecule.
Two-Bond Cleavage:
In this process, an elimination occurs, and the odd-electron molecular ion yields an
OE + and an even-electron neutral fragment N,usually a stable small molecule of
some type: H 2 O, a hydrogen halide, or an alkene.
15

16. Types of rearangements
McLafferty rearrangement
Heterocyclic ring fission (HRF)
 Benzofuran forming fission (BFF)
 Quinone methide (QM) fission
 Retro Diels-Alder (RDA)
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17. Fragmentation Guidelines
1.The relative height of the M + peak is greatest for straightchain molecules and decreases
as the branching increases.
2. The relative height of the M+ peak decreases with chain length for a homologous series.
3. Cleavage is favoured at alkyl-substituted carbons, with the probability of cleavage
increasing as the substitution increases.
These rules mostly arise from the fact that carbocation and radical
stability show the following trend:
Most Stable Benzylic > Allylic > Tertiary > Secondary >> Primary
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18. 4.Double bonds, cyclic structures, and especially aromatic rings will stabilize
the molecular ion and increase its probability of appearance.
5. Double bonds favor allylic cleavage to give a resonance stabilized allylic
carbocation, especially for cycloalkenes.
6. For saturated rings (like cyclohexanes), the side chains tend
to cleave first leaving the positive charge with the ring.
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19. 7.Unsaturated rings can also undergo retro-Diels-Alder reactions to eliminate a
neutral alkene.
8. Aromatic compounds tend to cleave to give benzylic cations,
or more likely tropylium cations.
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20. •Alkane
•Alkene
•AlkyneSimple
•Aldehyde
•Ketone
•Carboxalic Acid
•Halogenated Compounds
Carbonyl
•Alcohols
Hydroxyl
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21. alkane
21

22. 22

23. 23
Iodomethane
Halide

24. 24
2-chloropropane

25. Carbonyl compounds
Each functional group exhibits characteristic fragmentation patterns
For example, aldehydes and ketones often undergo the process of
α
cleavage, breaking the bond between the carbonyl carbon and the
carbon adjacent to it. Cleavage yields a neutral radical and a
resonance-stabilized acylium ion
25
All carbonyl-containing compounds have some fragmentations in common: -cleavage,
α
inductive ( -) cleavage, and McLafferty rearrangements are ubiquitous.
β

26. ether
26
Haterolytically
Homolytical

27. 27

28. Aromatic ethers
Form C 6 H 5 O + ions. These fragment
ions then lose CO to form
cyclopentadienyl cations (C 5 H 5 + ). In
addition, an aromatic ether may lose the
entire alkoxy group to yield phenyl
cations (C 6 H 5 + ). The mass spectrum
of ethyl 4-methylphenyl ether (p-
methylphenetole) exhibits a strong mol-
ecular ion at m/z = 136 as well as a
fragment at m/z = 107 from loss of an
ethyl radical. The base peak at m/z = 108
arises from loss of ethene via a
McLafferty rearrangement
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29. the M + 2 Peak
Most of the elements found in organic compounds, such as carbon, hydrogen,
oxygen, nitrogen, sulfur, phosphorus, flouorine, and iodine, have one major
isotope.
Chlorine has two common isotopes, 35 Cl and 37 Cl, which occur naturally in a 3:1
Bromine has two common isotopes, 79 Br and 81 Br, which occur naturally in a 1:1
ratio. Thus, there are two peaks in a 1:1 ratio for the molecular ion of an alkyl
bromide.
29

30. 30

31. Alpha cleavage and dehydration. In the a-cleavage pathway, a C-C bond nearest
the hydroxyl group is broken, yielding a neutral radical plus a resonance-
stabilized, oxygen-containing cation.
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Alcohol

32. 32

33. Water formation in alcohol
In all the fragmentations we have seen so far, only one bond is broken.
An important fragmentation occurs in alcohols, however, that involves
breaking two bonds. Two bonds break because the fragmentation
forms a stable water molecule. The water that is eliminated comes
from the OH group of the alcohol and a hydrogen. Thus, alcohols show
a fragmentation peak at because of loss of water.
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34. 34
Ethanol

35. Benzylic alcohol
35
Loss of a hydrogen atom from
the molecular ion leads to a
hydroxytropylium ion (m/z =
107). The hydroxytropylium ion
can lose
carbon monoxide to form a
resonance-delocalized
cyclohexadienyl cation (m/z =
79). This ion can
eliminate molecular hydrogen
to create a phenyl cation, C 6 H
5 + , m/z = 77.

36. Principal modes of fragmentation include -cleavage and -cleavage.
α β
If carbonyl group contains at least three carbons, McLafferty rearrangement.
M – 1 peak due to the loss of one hydrogen. This peak is observed at m/z = 85 in the mass
spectrum of valeraldehyde HCO + m/z = 29;. The second important mode of
fragmentation for aldehydes
is known as -cleavage (inductive cleavage). In the case of valeraldehyde, -cleavage
β β β
creates a
propyl cation (m/z = 43).
36
ketone

37. 37

38. McLafferty Rearrangement
38

39. If one of the alkyl groups attached to the carbonyl carbon has a hydrogen,
acleavage known as a McLafferty rearrangement may occur. In this rearrangement
the bond between the carbon and the carbon breaks homolytically and a
hydrogen atom from the carbon migrates to the oxygen atom. Again,
fragmentation has occurred in a way that produces a cation with a positive charge
shared by two atoms.
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40. cycloalkane
40

41. amines
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42. Benzene
42
loss of a hydrogen atom from the molecular ion
gives a strong peak at m/z = 91. Although it
might be expected that this fragment ion peak
is due to the benzyl carbocation (C 6 H 5 CH 2
+ ), isotope-
labeling experiments suggest that the benzyl
carbocation actually rearranges to form the
aromatic delo-
calized tropylium ion (C 7 H 7 + , Figure 4.25).
When a benzene ring contains larger side
chains, a favored
mode of fragmentation is cleavage of the side
chain to form initially a benzyl cation, which
sponta-
neously rearranges to the tropylium ion. When

43. 43
In the mass spectrum of butylbenzene, a
strong peak due to the tropylium ion
appears at
m/z = 91. When the alkyl group attached to
the benzene ring is a propyl group or larger,
a McLafferty
rearrangement is likely to occur, producing a
peak at m/z = 92. Indeed, all alkylbenzenes
bearing a side
chain of three or more carbons and at least
one hydrogen on the -carbon will exhibit a
γ
peak at

44. tropylium ion
44
The tropylium ion has
characteristic
fragmentations of its own. The
tropylium ion can fragment to
form the aromatic
cyclopentadienyl
cation (m/z = 65) plus ethyne
(acetylene). The
cyclopentadienyl cation in turn
can fragment to form an-
other equivalent of ethyne and
the aromatic cyclopropenyl
cation (m/z = 39)

45. phenol
45
strong molecular ion peaksm/z = 94 is the base
peak Favored modes of fragmentation involve
loss of a hydrogen atom to create an M – 1 peak
(a small peak at m/z = 93), loss (CO) to produce
a peak at M – 28 (m/z = 66), and loss of a formyl
radical (HCO • ) to give a peak at
M – 29. In the case of phenol itself, this creates
the aromatic cyclopentadienyl cation at m/z =
65.
In some cases, the loss of 29 mass units may be
sequential: initial loss of carbon monoxide
followed
by loss of a hydrogen atom. The mass spectrum
of ortho-cresol (2-methylphenol) exhibits a
much
larger peak at M – 1 (Fig. 4.35) than does
unsubstituted phenol. Note also the peaks at
m/z = 80 and
m/z = 79 in the o-cresol spectrum from loss of
CO and formyl radical, respectively.

46. 46

47. references
Interpretation of Mass Spectra Book by Fred McLafferty
Introduction to Spectroscopy Book by Donald L. Pavia, Gary M. Lampman, and George S.
Kriz
Introduction to mass spectrometry Book by J. Throck Watson
Mass Spectrometry: A Foundation Course Textbook by Kevin Downard
Computational Methods for Mass Spectrometry Book by Ingvar Eidhammer, Kristian
Flikka, Lennart Martens, and Svein-Ole Mikalsen
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48. THANK YOU
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