Mass spectrometery

introduction,instrumentation and application

1. Mr.M.RaguM.Sc,SET
AssistantProfessor
VivekanandaCollege
Tiruvedakamwest
Madurai-625234
Mass Spectrometry

2. Definition
Amassspectrometerbombardsasubstanceunder
investigationwithanelectronbeamandquantitatively
recordstheresultasaspectrumofpositiveionfragments.
ThisrecordisaMassSpectrum.
Amassspectrumisapresentationofthemassesofthe
positivelychargedfragmentsversustheirrelative
abundance.
Separationofthepositivechargeionfragmentisonthe
basisofmasstochargeratio.(Mass/Charge)

3. Mass Spectrum
X -axis: m/z
mass -based on
12
C 12.0000
Z –charge on ion
Y -axis: relative abundance
usually normalized with respect to the
highest peak
(base peak)
0 -100 %

4. 20
40
60
80
100
10 20 30 40 50 60 70
43, base peak
57
72, Molecular ion
42
41
39
27
29CH
3CH
2CH
2CH
2CH
3
m/z

5. Relative Abundance is Expressed as percentage of the base peak.% relative abundance =
Peak height
Peak height of base peak
X 100

6. Types of Mass Spectrometry
ESI (Electro spray Ionization)
FAB (Fast Atom Bombardment)
MALDI (Matrix -Assisted Laser Desorption
Ionization)
GC-MS ( Gas Chromatography Mass
Spectrometry)

7. Fast Atom Bombardment
Worksbestfororganiccompounds with
polarityandeitheracidicandorbasic
functionalgroups.
Basicgroupsrunwellinpositiveionization
mode.
Acidiccentersrunwellinnegativeionization
mode.
ClassesthatuseFABare:peptides,proteins,
fattyacids,organometallics,surfactants,
carbohydrates,antibiotics,andgangliosides.

8. Major Steps in Analysis of Mass
Spectral Data
Identification of molecular ion
Base peak
Analysis of fragmentation pattern
Propose possible structures
Compare postulated species to available reference
spectra

9. Type of ions
Molecular ion
Fragment ion
Rearrangement ion
Metastable ion
Multiple charged ion
Isotope ion
Negative ion

10. Molecular Ion
Ion whose mass equals that calculated from
the molecular formula using the masses for
each element which have the highest natural
abundance; often tallest peak in highest m/z
group
Molecular ion represents the molecular
weight of the sample.
Base peak-most intense peak in spectrum;
not necessarily the molecular ion peak!M + e M
+.+ 2e

11. Fragment ion
Fragmentionsareformedbysimplecleavage
andrearrangementprocesses.
e.gCH3CH2
CH3CH2Cl
+
+
.
+.CH3CH2Cl
Cl

12. Fragmentation ion
Themolecularionsareenergetically
unstable,andsomeofthemwillbreakup
intosmallerpieces.Thesimplestcaseisthat
amolecularionbreaksintotwoparts-one
ofwhichisanotherpositiveion,andthe
otherisanunchargedfreeradical.CH2
H
CH2
Cl
CH2CH2
+.
+HCl
O.E E.E

13. The Nitrogen Rule
Molecule of even number MW contain no nitrogen or even number
of nitrogen
An odd number MW contain odd number of nitrogen atom

14. Problem
Which of the following ions will
appear at an even mass number?
NH
3 MW = 17
C
4H
9N MW = 71
C
2H
5NH
2MW = 45
CH
2N
2 MW = 42
ANS: CH
2N
2

15. Satellite peaks : (M+1) and (M+2) peaks
(M+1) peak can arise by the abstraction of a proton from the neutral
molecule by molecular ion (ion molecule reaction)
Examples: ether, amines and nitriles
(M-1) peak is obtained by the loss of hydrogen
Example: aldehyde
Aromaticcompoundsgiveintensive(presenceof-electronsystem)
Conjugatedolefins-intense peakthanthecorrespondingnon-conjugated
olefinswiththesamenumberofunsaturation.
Aliphatic compounds -branching results decrease in the abundance of
peak
Introduction of groups which lowers the I.P (methoxy, hydroxy, amino etc)
increases the molecular ion abundance
Introduction of groups which increases the I.P (cyano, nitro etc) lowers the
molecurlar ion abundance. M
+. M
+. M
+.

16. Rearrangement ion
Intramolecular reorganization processes involving the migration of hydrogen or
other atoms or groups
Example: Mclafferty rearrangement
Electron impact induced hydrogen transfer rearrangement
“-hydrogen is transferred through a six membered transtion state to an electron
deficient centre followed by the cleavage of the bond”
Reactionresultsintheeliminationofneutralmolecule
Charge will be carried by the oxygen containing fragment
Characteristics of ketones, aldehydes, acids, esters, olfins etc.

17. McLafferty Rearrangement

+



+


+



+
O
CCH
3O
CH
2
CH
2
CR
2
H
R
2
CCH
2
O
H
CCH
3O
CH
2
O
H
CCH
3O
CH
2
O
H
CCH
3O
CH
2




18. Molecularionpeakofastraightchainmonocarboxylicacidis
weakbutusuallydiscernible.Themostcharacteristicpeak
(sometimesthebasepeak)isatm/z=60duetoMcLafferty
rearrangement.
CHO
CH2
OH
HOC
O
H
CH2


+
+
O
COH
CH2
CH2
C
HR
H
H
2CCHR




+



m/z 60

19. H
+
CH2
CH2
+. CH
CH2
CH2
C
CH3O
H
CH2
C
CH3OH
+.
+
CH CH2
+.

20. Structural requirements
1.Side chain containing atleast 3 carbon atoms,
the last bearing a hydrogen atom
2.A double bond (carbonyl, olefin or aromatic
system)
3.Djerassi et al. in a detailed study of steriod
ketones showed that the interatomic distance
for McLafferty rearrangement should be less than
1.8 A°

21. Metastable ion
life time few microseconds
appear as a broad and diffuse peak
If ion of mass m
1decomposes to give a daughter ion of mass m
2, then
the metastable peak m* is given by
Meatstable peaks are not symmetrical
Flat top metastable peak shows K.E is released during the process
From the pattern of the metastable peak it is possible to find out
whether the polyisotopic element is present in the neutral or in the
resulting daughter ion.m*
2
m
m
2
1

23. Acetophenoneshowsamolecularionpeakat
m/z=120anditshowsadaughterionpeak
atm/z=105.Calculatethemetastableion?
m*=(105)
2
/120
=91.88
Acetophenoneshowsapeakatm/z=92.
Problem m*
2
m
m
2
1

24. General Fragmentation Modes
Simple cleavage
Retro-Diels-Alder reaction
Hydrogen transfer rearrangement

25. Redro-Diels-Alder reaction
Multicentred fragmentation mode
involving the cleavage of two bonds
of cyclic system resulting in the
formation of two stable unsaturated
fragments in which two new bonds
are formed.

26. Redro-Diels-Alder reaction
Example:e
e
.
+
+
.
CH2
CH2
+
+
CH2
CH2
+
CH
CH
H2C
H2C
CH
CH
H2C
H2C
+

27. In the mass spectrum of 3,5,6-triphenyl-4-nitro-
1
-
cyclohexene the base peak is due to the diene ion (m/z
206)Ph
Ph
Ph
NO2
+.
Ph
Ph
+. +.
+.
Ph
Ph
+
Ph
Ph
Ph
NO2
Ph
Ph
+
NH2
CH
CH
Ph
NH2
CH
CH
Ph
m/z 206 (100%)
In the spectrum of 3,5,6-triphenyl-4-amino-1-cyclohexene,
the diene and dienophile ions are in the ratio 1:4
100%
25%

28. O
+.
O
+.
+ CH2C
CH3
CH3
-ionone
- ionone
+
+.
+
O O
CH3
. -ionone and -ionone can be differentiated by mass
spectrometry
-ionone undergoes RDA fragmentation to give an intense peak
at m/z 122. This peak is insignificant in mass spectrum of -
ionone
In -ionone , loss of one geminyl methyl group takes place
Among natural products, Norborene, flavones, triterpenes, steroids and
alkaloids undergo RDA fragmentation which is a valuable tool for
structural elucidation

29. Simple cleavage
i)Heterolytic cleavage at the cationic site
Example :
Alkyl halidesCH3CH2 CH2CH
ClH
-HCl
+.
CH3CH2CHCH2
+.
m/z 56
m/z 92 C4H9I
+.
C4H9+I
+.
C4H9
+.
C4H9+
+.
Br Br
m/e 57
m/e 57
m/z 184
m/z 136

30. Ethers:
CH3CH2CH2
CH3CH2CH2
+
+
OCH2CH2CH3
OCH2CH2
CH3
+
m/z 43 (50.3%) m/z 59 (1.7%)
+.

31. Homolytic cleavage induced by a radical site
Oddelectronionshaveanunpairedwhichis
capableofnewbondformationwhichis
exothermicandaccompaniedbybondcleavage.
Thesereactionsaremorecommon.R.
+RCR
+.
X
CR
X
+

32. RCH
OH
+.
R.
+CH
R
R
OH
+
Alcohols
Sec
PriRCH2
OH
+.
R
.
+
CH2
OH
+
Ter
R
R.
+RCR
+.
X
R
CR
X
+
R
CR
X
+

33. R
R.
+
+
Sec
Pri
R R
.
+
Ter
R.
+RCR
+.
+.
R
+.
R
CR
+
CH2
NH2
NH2
CH2
CHR
CHR
NH2
+
NH2
NH2
NH2
Amines

34. m/z 94
m/z 66
.
OH O
H
H
-CO
-CHO
H
-H
+.
----
+
m/z 65
+.

35. +
+
+

-H
-CO
-H2
m/z108
m/z107
m/z79
m/z77
5
CH
2OH
OH
HH
H
H
H
H
H

+
Benzyl alcohol
7 memberd ring
Benzonium ion
Phenyl ion+
C4H3
+
m/z 51

36. .
m/z 108 m/z 93
O
-CO-CH3
+
m/e 65
-C2H4
m/e 39
C6H5
+
m/e 77
-OCH3
OCH3
+.
C3H3
+
+
.

37. Ortho effect
Insuitablysubstitutedaromaticcompoundsorcis-olefins
thesubstituentandhydrogenatomcancomeinclose
proximitysoastohelpeliminationofaneutralmolecule.
Thiseffectisknownasorthoeffect.
Example:
Elimination of methanol from cis-methyl crotonateCH
CH
CH2
H
C
O
OCH3
CH
CH
CH2
CO
+.
+ CH3OH

38. Orthoeffectisusefultodistinguishbetweencis-
andtranscompoundsandorthofrommetaand
paraisomers.O
C
OH
X
H
+.
C
O
X
+H2O
X = O, NH

39. EXAMPLES

40. Mass Spectrum of Pentane
20
40
60
80
100
10 20 30 40 50 60 70
43
57
72, M
+
42
41
39
27
29
m/zCH
3CH
2CH
2CH
2CH
3

41. Mass Spectrum of 2-Methylpentane
m/z
20
40
60
80
100
10 20 30 40 50 60 7080 90
43
57
71
86, M
+
CH
3CH
2CH
2CHCH
3
CH
3

42. Mass Spectrum of Pentane
20
40
60
80
100
10 20 30 40 50 60 70
43
57
72, M
+
42
41
39
27
29
m/zCH
3CH
2CH
2CH
2CH
3

43. 20
40
60
80
100
20 40 60 80100120140
94
156
107
160
94
107
m/z
2-Chloroethylphenyl ether
O
ClH

44. Methyl Ester of Fatty Acids

45. Tropylium ion
Mass Spectrum of n-Octylbenzene
Tropylium ion
Tropylium ion

46. Naphthalene

47. Molecules with Heteroatoms
Isotopes: present in their usual abundance.
Hydrocarbons contain 1.1% C-13, so there will be
a small M+1 peak.
If Br is present, M+2 is equal to M
+
.
If Cl is present, M+2 is one-third of M
+
.
If iodine is present, peak at 127, large gap.
If N is present, M
+
will be an odd number.
If S is present, M+2 will be 4% of M
+
. =>

48. Isotopic Abundance
=>
81
Br

49. Mass Spectrum with Sulfur
=>

50. Mass Spectrum with Chlorine
=>

51. Mass Spectrum with Bromine
=>

52. Mass Spectra of Alkanes
More stable carbocations will be more abundant.
=>

53. Mass Spectra of Alkenes
Resonance-stabilized cations favored.
=>

54. Mass Spectra of Alcohols
Alcohols usually lose a water molecule.
M
+
may not be visible.
=>

57. Problem-1
A hydrocarbon with MF C
7H
12(Molecular ion 96 shows a large peak due to
M-15 and at m/z 54. What structure assigned to this compound?
Answer
Theindexofhydrogendeficiencyinthiscompoundistwo(analkane
withsevencarbonatomswouldbeC
7H
16,thedifferenceC
7H
16-C
7H
12
isfourhydrogenatoms,thustheindexofhydrogendeficiencyistwo).
Thelarge(M-15)pointstothepresenceofabranchedmethylgroup.
Thepeakatm/z54isduetoC
4H
6,i.e.butadiene.ARDAreactionwill
eliminatebutadienefragmentm/z54fromacyclicalkaneandthe
presenceofacycloalkeneisinkeepingwiththeindexofhydrogen
deficiency.Thecompoundis4-methylcyclohexene.CH3
+.
+.
+
CH3
m/z 96
4-methylcyclohexene m/z 54

58. Problem-2
How will you differentiate 3-methylcyclohexene from 4-methylcyclohexene
on the basis of mass spectroscopy?
Answer
IncyclicalkenesaRETRO-DIELS-ALDERreactiongives
achargeddienefragmentandanunchargedethylene
fragment.Them/zofthedienefragmentgivesuseful
informationofthepositionofdoublebond.Thusunlike4-
methylcyclohexene,3-methylcyclohexenegivesadiene
fragment,withm/z=68.CH3
+.
CH3
+.+
m/z 96
m/z683-methylcyclohexene

59. Problem 3
A compound C
9H
10O
2(MW 156.0681) shows a strong peak m/z 119, 118,
91 and 65. What is the structure of the compound?
Answer:
Thecompoundhasanalkylaromaticsystem,thusstrongpeaksat
m/z=91and65willappear.Largepeaksatm/z=118and119willbe
duetolossof32and31amu.Them/z=119isthelossofOCH
3andthe
lossofCH
3OHtogivem/z=118canoccuronlyifthecompoundis
orthoester.CH2
H
C
O
CH3
O C
O
+.
CH2
+CH3OH
m/z 150
m/z 118

60. Problem 3
Ethylbutanoate in its mass spectrum shows two charcteristic
peaks due to odd electron ions at m/z 88 and 60 and
abundant ion at m/z 71. Explain the fragmentation.
Theionsatm/z=88and60arisefromtwosuccessiveMcLafferty
rearrangementswhilethe-cleavagegivestheabundantpeakatm/z=71.m/z 71
CH2
CH2
CH2
C
O
CH2
O
CH3
H
+.
H2C
C
+
CH2
CH2
O
H
O
H
+.
H2C
C
CH2
CH2
O
H
m/z 88
C
OH
H2C OH
+.
+
m/z 60