Mass Spectrometry: A Powerful Method for Organic Chemistry Analysis

Mass Spectrometry
Dr. Krishnaswamy. G Page 1
MASS
SPECTROMETRY
Prepared By
Dr. G. Krishnaswamy
Faculty
DOS & R in Organic Chemistry
Tumkur University
Tumakuru

Mass Spectrometry
Mass Spectrometry is an analytic technique that utilizes the degree of deflection of charged
particles by a magnetic field to find the relative masses of molecular ions and fragments. It is a
powerful method because it provides a great deal of information and can be conducted on tiny
samples. Mass spectrometry has a number of applications in organic chemistry, including:
- Determining molecular mass
- Finding out the structure of an unknown substance
- “Verifying the identity and purity of a known substance”
- Providing data on isotopic abundance.
The mass spectrometer performs three essential functions. First, it subjects molecules to
bombardment by a stream of high energy electrons, converting some of the molecules to ions
which are accelerated in an electric field. Second, the accelerated ions are separated according to
their mass to charge ratios in a magnetic or electric field. Finally the ions that have a particular
mass to charge are detected by a device which can count the number of ions striking it.
A graph of the number of particles detected or relative abundance as a function of mass to charge
ratio is-Mass spectrum.
A sample studied by mass spectrometry may be gas, a liquid or a solid can be introduced into the
ionization chamber through sample inlet system which converts sample to vapor state to obtain
stream of molecules that must flow into the ionization chamber. Once the stream of molecules
has entered the ionization chamber, a beam of high energy electron bombards it emitted from a
filament heated to several thousand degrees Celsius. The emitted electrons have energy of about
70 eV which is routinely used method called electron ionization (EI).These high energy
electrons strike the stream of molecules and ionize the molecules in the stream by removing the
electrons from them; the molecules are thus converted to radical cation (M+.).

Mass Spectrometry
The energy requiredtoremove an electron from an atom or molecule depends on its ionization
potential. Most of the organic molecules have ionization potentials ranging from 8 and 15 eV.
However, a beam of electrons does not create ions with high efficiency until it strikes the stream
with a potential of 50 to 70 eV.
A repellar plate which carries a positive electrical potential directs the newly created ions
towards a series of accelerating plates. A large potential difference ranging from 1 to 10 kV
applied across these accelerating plates produces a beam of rapidly traveling ions. One or more
focusing slits direct the ions into a uniform beam.
From the ionization chamber the beam of ions passes through a short field free region. From
there it enters the mass analyser, the region where the ions are separated according to their mass
to charge ratios.
The kinetic energy of an accelerated ion is equal to
Where m = mass of the ion, v = velocity of the ion, e = charge on the ion and V = potential
difference of the ion accelerating plates.
In the presence of a magnetic field, a charged particle describes a curves flight path. The
equation which yields the radius of curvature of this path is
Where r = radius of curvature of the path and H = strength of the magnetic field. If these two
equations are combined to eliminate the velocity term, the result is

Mass Spectrometry
This is the important equation that governs the behavior of an ion in the mass analyzer portion of
the mass spectrometer.
The molecular ion or parent ion:
The simple removal of an electron from a molecule like highest occupied orbital of aromatic
system and non bonding electron oxygen and nitrogen atoms yields an ion whose mass is the
actual molecular weight of the original molecule. This ion is the molecular ion and symbolized
as M+. The intensity of the molecular ion peak depends on the stability of the molecular ion. The
most stable the molecular ions are those of purely aromatic systems. If the substituents that have
favorable modes of cleavage are present, then the molecular ion peak will be less intense and
fragment peaks relatively more intense.
In general the following group of compounds will in order of decreasing ability give
prominent peaks:
Aromatic compounds >conjugated alkenes >cyclic compounds >organic sulfides >short, normal
alkanes > mercaptans.
The molecular ion is frequently not detectable in aliphatic alcohols, nitriles, nitrites, nitrates and
in highly branched compounds.
The most abundant ion formed in the ionization chamber gives rise to tallest peak in the
mass spectrum called the base peak.
Metastable ion peak:
The ions appear at an m/z ratio that depends on its mass as well as the mass of the original ion
from which it formed. Such an ion is called Metastable ion peak. It is formed due to abnormal
flight path on its way to the detector.
m* = apparent mass of the Metastable ion, m1 = mass of the original ion and m2 = mass of the
new fragment ion.
Characteristics of Metastable ion:
 They do not necessarily occur at the integral m/z values

Mass Spectrometry
 These are much broader than the normal peaks and
 These are relatively less abundance.
Example:Consider the formation of Metastable peak in the mass spectrum of toluene. Two
strong peaks at m/z 91 and at m/z 65 are formed. The peak at m/z 91 is due to formation of
tropylium ion which loses a molecule of acetylene to give C5H5
+ of m/z 65.
-H+ Rearrangement
-C2H2
m/z = 91 m/z = 65
(m1) (m2)
Suppose the transition C7H7+ to C5H5+ occurs, then a Metastable peak is formed. The position of
the broad Metastable peak is determined as
= 65 x 65/ 91 = 46.4
A Metastable peak appears at 46.4 in case of toluene mass spectrum.
Ionization techniques
1. Electron Ionization (EI)
2. Chemical ionization (CI)
3. Field Desorption (FD)
4. Fast Atom Bombardment (FAB)
5. Electron sprayIonization (ESI)
6. Matrix Assisted Laser Desorption/ Ionization (MALDI)
 Electron Ionization (EI): Mass spectra are routinely obtained at an electron beam energy of
70 eV. The simplest event that occurs is the removal of single electron from the molecule in the
gas phase by the electron of the electron beam to form the molecular ion, which is a radical
cation.
 Chemical Ionization (CI): The vaporized sample is introduced into the mass spectrometer
with anlarge excess of reagent gas (1000 – 10000 times) is introduced (Methane, ammonia,
isobutane) at a pressure of about 1 torr. The excess carrier gas is ionized by electron impact to
the primary ions CH4+.and.CH3. These react with the excess methane to give secondary ions like
CH5+, C2H5+ and C3H5+. The secondary ions react with the sample.Chemical ionization mass
spectra show peaks one mass unit higher than those expected in electron impact spectra [M+H]+.
The stability of M+ 1 peak is usually greater than that of the molecular ion.
electronswithninteractiouponformedareCHandCH 34


Mass Spectrometry
 Field Desorption (FD): Stable molecular ions are obtained from a sample of low volatility
which is placed on the anode of a pair of electrode between which there is an intense electric
field. Desorption occurs and molecular and quasimolecular ions are produced with insufficient
internal energy for extensive fragmentation. Usually the major peak represents the [M+ H]+
ion.Synthetic polymers with molecular weights on the order of 10, 000 Da have been analyzed.
 Fast Atom Bombardment (FAB): Polar molecules such as peptides with molecular weight
upto 10, 000 Da can be analyzed by a soft ionization technique called Fast atom bombardment.
The bombarding beam consists of xenon (or Argon) atoms of high translational energy.This
beam is produced by first ionizing xenon atoms with electrons to give xenon radical cation.The
compound of interest is dissolved in a high boiling solvent such as glycerol, a drop is placed on a
thin metal sheet and the compound is ionized by the high energy beam of xenon atoms.
Ionization by translational energy minimizes the amount of vibration excitation and this results
in less destruction of the ionization molecule.
 Electron spray Ionization (ESI): Electron sprayinvolves placing an ionizing voltage several
kilovolts across the nebulizer needle attached to the outlet. This technique is widely used on
water soluble biomolecules proteins, peptide and carbohydrates. Electron spray ionization is one
of several variations of atmospheric pressure ionization (API).
 Matrix Assisted Laser Desorption/Ionization (MALDI): In the MALDI procedure is
mainly used for large biomolecules the sample in a matrix is dispersed on a surface and is
desorbed and ionized by the energy of a laser beam. The MALDI procedure is has been used in
several variations to determine the molecular weight of large protein molecule upto several
hundred kDa.
Determination of Molecular weight
The Nitrogen Rule states that if m/z for M is odd, then the molecular formula must have an
oddnumber of nitrogens. If m/z for M is even, then the molecular formula must have an even
number
of nitrogens (this includes 0).
EX: For 1-bromopropane, m/z for M=122. The even number is in accordance with theeven
number of nitrogens in the formula (zero).
The Hydrogen Rule states that the maximum number of hydrogens in the molecular formula is
2C+N+2

 3544 CHCHCHCH
25243 HHCCHCH  
45 CHMHCHM  
4252 HCMHHCM  

Mass Spectrometry
C: No.of carbons, N: No of nitrogens
EX: For CH3CH2CH2Br, there are three carbons, so the max # of hydrogens is 2(3)+2=8
Index of hydrogen deficiency or Double Bond Equivalent
One Double Bond Equivalent (DBE) (also known as a degree of unsaturation) is one pi bondor
one ring. A triple bond counts as 2 DBE. Having 4 DBE indicates the possibility of a
benzenering, since benzene has three pi bonds plus one ring. The formula for DBE is the
following:
Index of hydrogen deficiency
or
DBE = C – (H/2) _ (X/2) + (N/2) + 1
C = No. of carbons, H = No. of Hydrogen, X = No. of Halogens & N = No. Nitrogen
Determination of Molecular Formulas
A. Precise Mass determination:
Most important application of high Resolution mass spectrometer is the Determinationof
very precise molecular weight of substances. We are accustomed to thinking of atoms as
having integral atomic massed for example H = 1, C = 12 and O = 16. However if we
determine the atomicmasses with sufficient precision we find thatthis not true.Depending
upon the atoms in a molecule it is possible for particle of the same nominal mass to have
slightly different measured masses when precise determinations are possible. Consider a
molecule with a molecular weight of 60 could be C3H8O, C2H8N2, C2H4O2 or CH4N2O.
These species have the following precise masses

Mass Spectrometry
B. Isotope Ratio Data:
In this method of determining molecular formulas is to examine the relative intensities of
the peaks due to the molecular ion and related ion that bear one or more heavy isotopes.If
only C, H, N, O, F, p and I are present, the approximate expected percentage (M+1) and
(M+2) intensities can be calculated by use of the following formula.
Fragmentation:
General rules for predicting prominent peaks in EI spectra:
1. The relative height of the molecular ion peak is greatest for the straight chain compound
and decreases as the degree of branching increases.
2. The relative height of the molecular ion peak usually decreases with increasing molecular
weight in a homologous series.
3. Cleavage is favored at alkyl-substituted carbon the more substituted the more likely is
cleavage. This is consequence of the increased stability of a tertiary carbocation over a
secondary carbocation which in turn is more stable than a primary.

Mass Spectrometry
R
C CR +
4. Double bonds, cyclic structures and especially aromatics rings (or heteroatoms) stabilize
the molecular ion and thus increase the probability of its appearance.
5. Double bonds favors allylic cleavage and give resonance stabilized allylic carbocation.
This rule does not hold good for simple alkenes because of ready migration of double
bond but it does hold for cycloalkenes.
6. Saturated rings tend to lose alkyl side chains at α bond.The positive charge tends stay
with ring fragment.
R
-R
Unsaturated rings can undergo a retro-Diels-Alder reaction.
-R
7. In alkyl substituted aromatic compounds cleavage is very probable at the β bond to the
ring, giving the resonance stabilized benzyl cation or the tropylium ion.
R
H
H
1,2-H shift
-R.
8. The C-C bonds next to a hetero atom are frequently cleaved, leaving the charge on the
fragment containing the heteroatom whose nonbonding electrons provide resonance
stabilization.

Mass Spectrometry
9. Cleavage is often associated with elimination of small stable neutral molecule such as
CO, Olefins, water, ammonia, hydrogen sulfide, hydrogen cyanide, mercaptans, ketenes
or alcohols with rearrangement.
McLafferty Rearrangements:
McLafferty rearrangement involves the migration of γ-hydrogen atom followed by the cleavage
of a β-bond. To undergo McLafferty rearrangement a molecule must possess an appropriately
located heteroatom, a π-system (usually double bond) and an abstractable hydrogen atom γ to the
C=O system.
The rearrangement leads to the elimination of neutral molecules and proceeds through sterically
hindered six membered transition state.
Y
O
H
Y
O
H
Y
O
H
+
Y
O
H
Transition State
Y = H, R, OH, OR, NR2
Example: 1-pentene contains γ-hydrogen atom undergoes McLafferty rearrangement
H
+
m/z= 42
Mass Spectra of some chemical classes:
1. (a) Alkanes:
 The relative height of the parent peak decreases as the molecular mass increases
in the homologous series.
 Groups of peaks in the mass spectrum are observed 14 mass units apart. The most
abundant peaks correspond to CnH+
2n+1 ion.

Mass Spectrometry
 The most intense peaks are due to C3 and C4 ions at m/z 43 and 57 respectively.
 We also notice small peaks for CnH+
2n-1 and CnH+
2n
 Example mass spectrum of octane.
(b) Branched chain alkanes:
 Bond cleavage takes place preferably at the site of branching. Due to such
cleavage, a more stable secondary or tertiary carbocation ion results.
 Generally largest substituent at a branch is eliminated readily as a radical.
 With 2,2,4-trimethylpentane, the cleavage shown leads to formation of tert-butyl
carbocation. Since tertiary carbocations are the most stable of the saturated alkyl
carbocations, this cleavage is particularly favorable and accounts for the intense
fragment peak at m/z = 57.
2. Alkenes:
 The molecular io peak in the unsaturated compounds is more intense than the
corresponding saturated analogues. The reason is the better resonance

Mass Spectrometry
stabilization of the charge on the cation formed by the removal of one of the π
electrons.
 The relative abundance of the molecular ion peak decrease with increasing
molecular mass.
 The general mode of fragmentation induced by a double bond is the allylic
cleavage.
 The CnH2n ions formed by McLafferty rearrangement are more intense.
H
+
m/z= 42
3. Alkynes:
 Themass spectra of alkynes are very similar to those of alkenes. The molecular
ion peaks tend to be rather intense and fragmentation patterns parallel those of the
alkenes.
4. Cycloalkanes:
 The relative abundance of the molecular ion of Cycloalkanes is more as compared
to the corresponding alkanes.

Mass Spectrometry
 It favors cleavage at the bond connecting the ring to the rest of the molecule.
 The stability of the fragment ion depends upon the size of the ring.
+
+
M+
= 126 m/z = 83
m/z = 55
5. Cycloalkenes:
 Cyclic alkenesusually show a distinct molecular ion peak involving retro Diels-
Alder reaction.
-R
6. Aromatic compounds:
 The mass spectra of most aromatic hydrocarbons show very intense molecular ion
peaks.
 If the aromatic ring is substituted by an alkyl group, a prominent peak is formed at
m/z 91, here benzyl cation formed spontaneously rearranges to tropylium cation.

Mass Spectrometry
R
H
H
1,2-H shift
-R.
 When the side chain attached to benzene ring contains three or more carbons ions
formed by a McLafferty rearrangement can be observed.
Example: Fragmentation pattern of n-propyl benzene

Mass Spectrometry
CH2
-(CH3CH2)
M+
m/z = 120
m/z = 91
m/z = 65
HC CH-
7. Alcohols and Phenols:
 The molecular ion peak of primary and secondary alcohol is usually low abundance. It is
not detected in tertiary alcohols.
 The fragmentation of carbon-carbon bond adjacent to oxygen atom (α-bond) is the
preferred.
 Fragmentation involves the loss of an alkyl group or the loss of a molecule of water
(M-18).
 Alcohols containingfour or more carbons may undergo the simultaneous loss of both
water ad ethylene.
 Cyclic alcohols may undergo fragmentation by at least three different pathways.

Mass Spectrometry

Mass Spectrometry
 Phenols typically loss the element of carbon monoxide to give strong peaks at m/z
values that are 28 mass units below the value for the molecular ion (M-28).
 Phenols also lose the elements of the formyl radical (HCO.) to give strong M-29
peaks.M-29 is less intense than M-28
OH
H H
-H.
m/z = 108 m/z = 79
HO
-CO
Tropylium hydroxide
 Benzyl alcohols exhibit intense molecular ion peaks. A favored mode of
fragmentation involves loss of a hydrogen atom, loss of a molecular carbon
monoxide and loss of a formyl radical.
8. Ethers:
 The molecular ion peals of ether are rather weak. Principal modes of
fragmentation include α-cleavage, formation of carbocation fragment and loss of
an alkoxy group.

Mass Spectrometry
 Consider the mass spectrum of diisopropyl ether, loss of alkyl group gives rise a
peak at m/z = 87.
O O
-CH3
.
m/z = 87m/z = 102
 A second mode of fragmentation involves cleavage of the carbon-oxygen bond of
ether to yield carbocation. Cleavage of this type in diisopropyl ether is responsible
for the C3H7+ fragment at m/z = 43.
O OH
+
m/z = 43
 A third type of fragmentation occurs as a rearrangement reaction taking place on
one of the fragmentation ions rather than on the molecular ion itself.This type of
rearrangement is particularly favored when the carbon of the ether is branched. In
the case of diisopropyl ether this rearrangement gives rise to a C2H5O+ fragment
(m/z = 45)

Mass Spectrometry
9. Aldehydes:
 The molecular ion peak of an aliphatic aldehydes is usually observable may be
fairly weak.
 The major fragmentation processes are α- and β- cleavages.
 α- cleavages
 β- cleavages
 If the carbon attached to the carbonyl group contains at least three carbons,
McLafferty rearrangement is also observed.
 Aromatic aldehydes exhibit intense molecular peaks. Consider the mass spectrum
of Benzaldehyde, the M-1 peak appears at m/z = 105. A peak at m/z = 77 which is
due to loss of the –CHO group to give C6H5+.
H O O+
-H. -CO
(M-1) m/z = 77 m/z = 51
+

Mass Spectrometry
10. Ketones:
 The mass spectra of ketones show intense molecular ion peak.
 Loss of the alkyl groups attached to the carbonyl group is one of the most
important fragmentation processes. The pattern of fragmentation is similar to that
of aldehydes.
 The larger of the two alkyl groups attached to the carbonyl group appears more
likely to be lost. Consider the mass spectrum of 2-butanone.
 The peak at m/z = 43 is due to loss of the ethyl group is more intense than the peak
at m/z = 57 due to loss of methyl group.
 When the carbonyl of a ketones attached to alkyl group that is three or more
carbon atom length, McLafferty rearrangement id possible.

Mass Spectrometry
R O O+
-R. -CO
+
 Cyclic ketones may undergo a variety of fragmentation and rearrangement processes.
 Aromatic ketones undergo cleavage to lose alkyl group and form the C6H5CO+ ion
(m/z = 105). This ion loses the carbon monoxide to form the C6H5+ ion (m/z = 77).

Mass Spectrometry
 When larger alkyl group attached to the carbonyl group, McLafferty rearrangement
is possible.
11. Esters:
 The most important of the cleavage reaction involves the loss of the alkoxy group
from an ester to form the corresponding acylium ion RCO+.
 Benzyl esters undergo rearrangement to eliminate the neutral ketene molecule.

Mass Spectrometry
12. Carboxylic acids:
 Aliphatic acids generally show weak but observable molecular ion peaks.
Aromatic acids on the other hand show strong molecular ion peaks.
 The principal mode of fragmentation resembles those of the methyl esters.
 With short chain acids the loss of OH and COOH through α-cleavages on either
side of the C=O group may be observed.
13. Amines:
 Aliphatic amines, the molecular ion peak may be very weak or even absent.
 The most intense peak in the mass spectrum of a aliphatic amine arises from α
cleavage.
 When there is a choice of R group to be lost through this process, the largest
group is lost preferentially.
 Aromatic amines show intense molecular ion peaks. A moderately intense peak
appears at m/z value one mass unit less than that of the molecular ion, due to loss
of a hydrogen atom.
14. Amides:

Mass Spectrometry
 The mass spectra of amides usually show observable molecular ion peaks. The
fragmentation pattern of amides is quite to those of corresponding esters and acids.
 The presence of the strong fragment ion at m/z = 44 is usually indicative of a primary
amide. The peak arises from α-cleavage.

Mass Spectrometry: A Powerful Method for Organic Chemistry Analysis

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