PRINCIPLES OF MASS SPECTROMETRY notes -1.pdf

1
Mass Spectrometry
Introduction:
All chemical substances are combinations of atoms. Atoms of different elements have
different masses (H = 1, C = 12, O = 16, S = 32, etc.). An element is a substance that cannot
be broken down into a simpler species by chemical means - has a unique atomic number
corresponding to the number of protons in the nucleus. Different atoms combine in different
ways to form molecular sub-units called functional groups.
Mass of each group is the combined mass of the atoms forming the group (often unique)
e.g. phenyl (C6H5) mass = 77,
methyl (CH3) mass = 15, etc.
So if you break up a molecule into constituent groups and measure the mass of the individual
fragments (using MS) we can determine what groups are present in the original molecule and
how they are combined together.
What is Mass Spectrometry?
Mass spectrometry is a powerful technique for chemical analysis that is used to
identify unknown compounds, to quantify known compounds, and to elucidate molecular
structure.
A Mass spectrometer is a “Molecule Smasher”, which measures molecular and atomic
masses of whole molecules, molecular fragments and atoms by generation and detection of
the corresponding gas phase ions, separated according to their mass-to-charge ratio (m/z).
Why is mass spectrometry not called spectroscopy?
The reason mass spectrometry is called a spectrometry method and not a spectroscopy
method is because it is an analytical technique where the fragmentation pattern is used to
analyze the molecule, rather than a direct measurement of the interaction of the molecule
with electromagnetic radiation
Principles of mass spectrometry
Mass spectroscopy is the most accurate method for determining the molecular mass of
the compound & its elemental composition. In this the compound under investigation is
bombarded with a beam of energetic electrons. The molecules are ionised & dissociate into
several fragments. Each kind of ion has a particular ratio of mass to charge i.e., m/e ratio. The
charge can normally be taken as one. Thus for most ions, m/e ratio is simply the molecular
mass of the ion. Hence for neopentane m/e ratio is 72.
e- C5H12
+
m/e = 72

2
Molecular ion(C5H12)+
C4H9
+
C3H5+ C2H5
+
C2H3
+
m/e 57 41 29 27
Relative
intensity 100 41.5 38.5 15.7
fragmentation
Thus, for Neopentane,
The molecular ion (here C5H12
+
) is called parent ion and is designated as M+
. is positively
charged molecule with an unpaired electron.
The set of ions (fragment ions or daughter ions) are analysed in such a way that a signal is
obtained for each value of m/e that is represented. The intensity of each signal represents the
relative abundance of the ion producing the signal. The largest peak in the structure is called
the base peak and its intensity is taken as 100. The intensities of other peaks are represented
relative to the base peak.
INSTRUMENTATION
SAMPLE INJECTOR
ION SOURCE MASS ANALYSER
ION
DETECTIR
SIGNAL SENSOR
RECORDER
Mass Spectrometer

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Common mass spectrometer consist of:
1. Sample introducing system
2. Ion source
3. Mass analyser
4. Ion collector & amplifier
5. Recorder
1). SAMPLE INTRODUCING SYSTEM :-
A. Internal sample introducing system:- The sample is placed within the ionisation
chamber either as part of the ion source or coated on a filament.
i) Spark electrode: if the sample is an ionic compound then it can be anlysed by fabricating
spark gas electrodes from the sample itself(10mg). non-conducting samples can be mixed
with some conducting material an converted into an electrode. The method is mainly used for
checking purity of the sample.
ii) Filament Coating: A little amount of the solid sample can be coated on the filament,
which, when heated, yields positive ions directly. The method is useful in the studies of
isotopic abundance.
B. External sample introducing system:-
i) Direct introduction: The sample ranging from 10-4
to 10-9
g is directly introduced into the
ion beam source. The method is applicable to those compounds which are sufficiently volatile
at 373-873K
ii) Glass or Metal inlet: Glass is used when catalytic decomposition of the sample on the
metal surface is anticipated. Introduction of gases involves merely transfer of the
sample(0.1g) from a gas bulb into the metering volume(3cm3
) connected to a mercury
manometer from which it passes and extends into a reservoir volume(expansion volume,
about 5000cm3). For introducing liquid sample, the micropipette filled with the liquid sample
is touched to a sintered glass disc under gallium or mercury.
2). ION SOURCE:- Ion source converts molecules into gaseous ions . The most common
way of producing ions involves bombarding the sample with a beam of energetic electrons
from an ion gun.
3). MASS ANALYSER (separation of ions):- The positively charged ions (parent or
fragment ions) produced in the ion chamber are accelerated by applying an acceleration
potential. These ions then enter the mass analyser. Here the fragment ions are differentiates
on the basis of their m/e ratio. Dempster’s mass spectrometer is used for this purpose. The
positive ions accelerated by electric field travel in a circular path through 180o
under
magnetic field H & fall on a collector. Suppose an ion having a charge which is accelerated
through a voltage V. Then the kinetic energy of the ion is expressed as:
½ mv2
= eV ------ (1)

4
From Newton’s second law of thermodynamics, HeV = mv2
/r ------ (2)
Squaring on both side,
H2
eV2
= m2
v2
/r2
or H2
e2
= m2
v2
/r2
--------- (3)
But, ½ mv2
= eV----- (from equation 1)
mv2
= 2ev putting the value of mv2
H2
e2
= m.2eV/r2
or H2
e = 2mV/r2
or m/e = H2
r2
/2V.............. (4)
The above equation indicates that at a given magnetic field strength & accelerating
voltage, the ions of m/e value will follow a Circular path of radius r. the ions of various m/e
values reach the collector, amplified & recorded. The mass spectrum can be obtained either
by changing H at constant v or by changing v at constant H. when the magnetic field is
varied, the method is called magnetic scanning. It is known as electric voltage scanning when
potential is varied at constant field strength H.
4). ION DECTOR:- The ions which are separated by the analyser are detected & measured
electrically & photographically. The ion beam currents are of the order of 10-15
to 10-5
amps.
The ions pass through the collecting slits one after the other & fall on the detector. The
collector electrode is well shielded from stray ions.
5). RECORDER:- The recorder records peaks of all sizes once the mass spectrum is
scanned by going up the scale. The spectrum is recorded by using fast Scanning
oscillograph. In this type of recording 3 to 5 records of the same peak are made with
galvanometers having different sensitivities.
Different Methods of ionization: (EI, CI, FD and FAB)
Method for thermally volatile materials:
1. Electron Impact (EI),
2. Chemical Ionisation (CI) (Hard method); (for small molecules, 1-1000 Daltons)
Method for thermally non-volatile materials:
3. Field Desorption (FD),
4. Fast Atom Bombardment (FAB) (Semi-hard); peptides, sugars, up to 6000 Daltons
5. Electrospray Ionization (ESI - Soft); peptides, proteins, up to 200,000 Daltons
6. Matrix Assisted Laser Desorption (MALDI-Soft); peptides, proteins, DNA, up to 500 kD
1). Electron Impact (EI) ionisation:
Sample is volatilised in a separate chamber and the vapour is allowed to leak into the
ion source where the ionisation is brought about by bombarding the sample molecules with
high energy electrons. The sample is bombarded with electrons coming from electrically
heated rhenium or tungsten filament. The accelerated electrons have high energy (70 eV) and
on impact with molecules produce positive ions, along with small amounts of negative ions
and neutral particles.

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e-
+ M --> 2e-
+M+.
Fragments sent to mass analyzer
Electron Ionization (EI)
Also referred to as electron impact ionization, this is the oldest and best-characterized
of all the ionization methods. A beam of electrons passes through the gas-phase sample. An
electron that collides with a neutral analyte molecule can knock off another electron, resulting
in a positively charged ion. The ionization process can either produce a molecular ion which
will have the samemolecular weight and elemental composition of the starting analyte, or it
can produce a fragment ion which corresponds to a smaller piece of the analyte molecule.
The ionization potential is the electron energy that will produce a molecular ion. The
appearance potential for a given fragment ion is the electron energy that will produce that
fragment ion. Most mass spectrometers use electrons with an energy of 70 electron volts (eV)
for EI. Decreasing the electron energy can reduce fragmentation, but it also reduces the
number of ions formed
Problems with EI ionisation
(i) Requires sample be in the gas phase before ionisation limits samples to those already
existing in the gas phase or thermally stable samples that are easily volatised (for probe
introduction)
(ii) High Energy (Hard) Ionisation – lots of excess energy given to target – causes
fragmentation to lose energy and become stable – resulting in lots of characteristic fragments
ions, but little parent ion (useful for structural characterisation).
EI mass spectrum of methane.

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2). Chemical Ionisation (CI):
Uses bath gas (CH3/NH4/CH3(CH2)2CH3) to protonate sample often forms MH+
Still only applicable to volatile or thermally stable samples.
The advantage over EI is that it is softer so intact molecular ions are easier to obtain.
A disadvantage with CI compared to EI is that the ion source requires cleaning more often.
In the chemical ionisation method, a reagent such as methane, isobutane or ammonia,
is introduced into a high pressure source (0.1 to 1 torr) and ionised by electron bombardment.
The primary ions produced, undergo ion-molecule collisions to form a stable population of
secondary reagent ions. For example, the methane is ionised by electron impact forming the
primary molecular ion in the usual way as per equation.
CH4 + e --> CH4+ 2e
3). Field Desorption (FD):
Field desorption (FD) was introduced by Beckey in 1969.
FD was the ﬁrst “soft” ionization method that could generate intact ions from
nonvolatile compounds, such as small peptides.
The analytes are placed onto the emitter and desorbed from its surface. Application of
the analyte onto the emitter can be performed by just dipping the activated emitter in a
solution. The emitter is then introduced into the ion source of the spectrometer.
The positioning of the emitter is crucial for a successful experiment, and so is the
temperature setting.
4). Fast Atom Bombardment (FAB):
A technique in which liquid sample is bombarded with energetic atoms typically Ar or Xe
atoms of ~10 keV kinetic energy.
The analyte is dissolved in a viscous liquid, typically glycerol and ionization is
achieved by a beam of fast moving neutral atoms. The bombarding atoms are usually rare
gases, either xenon or argon. After acceleration, the fast moving ions enter into a chamber
containing further gas atoms and collision of ions and atoms leads to charge exchange.
Xe+
. (fast) + Xe (thermal) ---> Xe (fast) + Xe+
. (thermal)
The fast atoms formed in this process retain the original kinetic energy of the fast ions
and proceed towards the analyser. The remaining fast ions and ions with thermal energies can
be removed before sample bombardment by means of a deflector plate with a negative
potential.

7
Advantage and disadvantages of FAB:
Advantage:
1. FAB has been successfully applied to the ionization of high molecular weight samples of
biological origin, and compounds with molecular weights well in excess of 5000 Daltons may
be routinely determined.
2. FAB has been used extensively for obtaining mass spectra of salts. The exact form of the
spectrum obtained is very much dependent on the nature of cation and anion.
3. It provides relatively abundant molecular or quasi-molecular ions and also show some
structurally important fragment ions.
Disdvantages:
1. The disadvantages of FAB is that the matrix also forms on bombardment in addition
to those formed by the sample. This obviously complicates the spectrum.
2. A major drawback of conventional FAB ionization is the problem of quantitative
measurement because the FAB samples the surface rather than the bulk concentration
of the solute present.
Ion separators:-
1. Single focusing separator with magnetic diffraction:
2. Double focusing analyzer:
3. Time-of-fight separator:
4. Quadrupole analyzer:
1. Single focusing with Magnetic sector:
Electrostatic analyser acts as an energy dispersive device. Ions with different kinetic
energy will be focused to different positions. The magnetic sector then disperses the ions
according to m/z. Both sectors create dispersions of the ions according to kinetic energy but
in opposite directions.
It means that only those ions which have the requisite velocity, in accordance with the
above relationship for a given electric field, can pass through the slit kept at the end of the
electrostatic sector and m/z can be measured very accurately.

8
The magnetic sector is a momentum analyzer rather than a direct mass analyzer. An
ion of mass m and charge q traveling at a velocity υ in a direction perpendicular to an
homogeneous magnetic field will follow a circular path of radius rm
This shows the working principle of a magnetic sector where the radius rm depends on
the momentum mυ of an ion
The limitation arises from the fact that ions emerging from the ion source are not
really monoenergetic. Ions of different m/z can obtain the same momentum and thus cause
overlap of adjacent ion beams at the detector.
2. Double focusing analyzer:
In double focusing spectrometer, if all the ions with the same m/e have the same
velocity, a very high resolution is possible. High resolution is achieved by passing the ion
beam through an electrostatic analyser before it enters the magnetic sector. With mass
measurement of 1 ppm accuracy, the atomic composition of the molecular ions can be
determined. However, precise mass measurement requires the use of narrow source exit and
collector slit. This type of spectrometer is generally used for the separation and analysis of
ions and determination of molecular weight. If a mixture of three compounds of molecular
weights M1, M2 and M3 is ionized in the source, then the molecular ions of these can be
separated in the magnetic analyser of this spectrometer. The magnetic field is set to pass only
M2
+
through a slit placed between the magnetic and electric sectors. A collision chamber
behind the slit contains an inert gas at 10-3
to 10-2
, so that when M2
+
has a grazing collision
with an inert gas atom, a tiny proportion of its translational kinetic energy is converted into
internal energy of vibration. Thus fragments are produced from M2
+
.
Limitations:
1. The molecular ions produced from the mixture must be abundant, in order to give
good sensitivity.
2. The fragmentation of the molecular ions in the ion source should be limited so that
the fragment ions may not have the same mass as molecular ions from other
components of the mixture.
By using FAB or SIMS or CI these problems can be eliminated.
3. The mass resolution of electrostatic analyser in this spectrometer is limited to a few
hundred mass units.
4. Structure determinations of novel substances require HPLC or GLC techniques.

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3. Time-of-fight separator:
Velocity separation - E= mv2
Ion packet given constant KE – ions of heavier mass take longer to pass down drift tube and
reach detector
Conceptually easy
Allows very large masses to be measured (500,000Da)
E= 1/2mv2
Time flight of ions through drift tube
Ions of larger mass take longer to reach detector for constant E
In time of flight analysers all the ions leave the accelerating field with same kinetic
energies but with different velocities depending upon their masses. The ion of largest mass
will have the lowest velocities and the longest time of flight over a given distance. This
property is used in this spectrometer, in which pulse on grid A extracts the ions from the
source. The ions are then accelerated by a potential difference between A and B and pass into
the field-free flight tube. They are separated in time, according to their m/z values and
collected at D. since it is common to have differences in arrival times between successive
mass peaks of ≤10-7s, fast electrons are needed to distinguish successive peaks. Fast moving
ions can be produced in pulses lasting about0.25μs at a frequency of 10ooo times per second.

10
This time of flight t is given as t=k√
𝑚
𝑒
. where k is a constant which is dependent on the
length of flight time. The instrument has extremely high scan rate.
4. Quadrupole mass analyser:
Quadrupole mass analyser functions as a “mass filter” and is based on a completely different
principle to magnetic sector instruments. The method employs a radiofrequency electric
quadrupole field to obtain m/z discrimination and consequently no magnet is required.
The analyser consists of four parallel rods, with either circular or hyperbolic cross-
sections, arranged in parallel and are connected to radio frequecy (RF) and direct current
(DC) power supplier Vo Cos ωt and U respectively. Opposing rods are electrically connected
and adjacent rods are oppositely charged. The motion of the ions inside the quadrupole filter
is complex and the solution of rather complex equations of motion of these ions shows that
for paricular values of ω, U and Vo, only ions with one particular m/z value will pass through
the quadrupole analyser without striking one of the electrodes and thereby reach the detector.
The induced oscillations for this particular value of m/z remain quite small whereas the
amplitude of oscillation for other values increases exponentially while traversing the
quadrupole field.
Mass scanning is accomplished by varying U and Vo, so that the ratio U/Vo remains
constant.
Quadrupole instruments are relatively cheaper to construct and fully computer
controlled. They can be made very compact form, as in the “bench top” GC/MS instruments.
Rapid scan rates – enables measurement of transient samples introduced from
chromatographic systems (GC, LC) and at lower resolution – accurate mass NOT possible
Mass spectra: –
The mass spectrum is the plot of mass to charge ratio of positively charged ions
against their relative abundance
Molecular Ion: An ion formed by the removal of one or more electrons to form a positive
ion or the addition of one or more electrons to form a negative ion.

11
Base peak: It is the peak with the greatest intensity, due to its having the greatest relative
abundance. The base peak is assigned a relative intensity of 100%, and the relative intensity
of each of the other peaks is reported as a percentage of the base peak.
Depending on the nature of the compound, it may be either a fragment ion peak or the
parent peak. Of course the molecular ion peak may some times be the base peak. For
example, the molecular ion peak if m/e = 92 and the base peak is m/e = 91.
Metastable Ion: An ion that is formed with internal energy higher than the threshold for
dissociation but with a lifetime long enough to allow it to exit the ion source and enter the
mass spectrometer where it dissociates before detection.
Mass Spectrum with Bromine
Molecular Ion:
The electron bombardment with energy 10-15eV usually removes one electron from
the molecule of the organic compound in the vapor phase. It results in the formation of
molecular ion. The highest occupied orbital of aromatic system and non-bonding electron
orbitals on oxygen and nitrogen atoms readily lose one electron. An electron from double
bond (2-π electrons) or triple bond (4-π electrons) is usually lost. In alkenes, the ionisation of
C – C sigma bonds is easier than that of C – H bonds. Some examples are:
M(g) M+
(g) + 2 e R O R R O R + 2 e
- e
- e
C C C C + 2 e
- e
The mass of the parent ion gives the molecular mass of the sample. It is important to
locate the molecular ion at the high mass region of the spectrum. The stability of the parent
ion decides its relative abundance. In some cases if the rate of decomposition is too high then
parent ion is not formed.
In general, the relative height of the parent peak decreases in the following order.
Aromatica > Conjugated olefins > Alicyclics > unbranched hydrocarbons > Ketones >
Amines > Esters > Ethers > Carboxylic acids > branched hydrocarbons > Alcohols

12
Metastable ions or peaks:
Metastable peaks can be easily determined in a mass spectrum. Some important
characteristics of these peaks are:
(i) They do not necessarily occur at the integral m/e values
(ii) These are much broader than the normal peaks and
(iii) These are of relatively low abundance.
Formation of metastable ions: consider that M1
+
is the parent ion and m1
+
is the daughter
ion. If the reaction M1
+
---> m1
+
takes place in the source, then the daughter ion, m1
+
may
travel the whole analyser region and is recorded as m1+ ion. On the other hand, if the
transition M1
+
to m1
+
occurs after the source exist before arrival at the collector, then m1
+
is
called a metastable ion. We know that in double focusing mass spectrometer, there are two
field free regions. These are called drift regions. The ions pass through these regions after
acceleration. The first field free region refers to the portion of the ion path immediately
before the electrostatic analyser. The second field free region lies between electrostatic
analyser and magnetic analyser.
V
A
B
To collector
Second Field
Free region
First Field
Free Region
Figure showing first and second field free regions.
Electrostatic
Analyser
M
agnetic
Analyser
The position of the metastable peak (m1*) due to the reaction M1
+
---> m1
+
occurring
in the second field free region is such that m* = m1
2
/M1
Note: 1. It is important to remember that for a reaction, M1
+
---> m1
+
, m* (metastable peak)
has a distance below m1 on the mass scale. The distance is approximately similar to the
distance that m1 lies below M1.
2. The relative abundance of the metastable peak (m*) is often of the order of 10-2 or less
compared to the abundance of parent or the daughter ions in 70eV spectrum.
For example: (1) Consider the formation of metastable peaks in the spectrum of p-amino
anisole. The parent (molecular) ion appears at m/e 123. Suppose the fragmentation of parent
ion into daughter ion,(due to the loss of methyl radical, i.e., loss of 15 mass units) takes place
in between the electrostatic and the magnetic analysers, i.e., in second field free region. The
position of the metastable peak can be calculated as follows.

13
The position of the metastable peak, m* =
𝑚1
𝑀1
=
108×108
123
= 94.8
(2) Metastable ion in case of toluene:
-H
-CH3 - CH=CH
m/e=77 m/e=92 m/e=91 m/e=65
CH3 +CH2
m* =
𝑚1
𝑀1
=
65×65
91
= 94.8
Why metastable peaks are broadened?
The b
General rules for fragmentation pattern.
1. Nitrogen rule: “Nitrogen rule states that a molecule of even numbered molecular mass
must contain no nitrogen or an even number of nitrogen atoms. An odd numbered molecular
mass requires an odd number of nitrogen atoms.” This rule holds for all compounds
containing C, H, N, O, S and halogens as well as less usual atoms like P, B, Si, As etc. an
important corollary of this rule states that the fragmentation at a single bond gives an odd
numbered ion fragment from even numbered molecular ion. Similarly, an even numbered ion
fragment results from odd numbered molecular ion. However, the fragment ion must contain
all the nitrogen atoms of the molecular ion.
Consider an example of nitrobenzene (C6H5NO2). The signal for molecular ion
appears at m/e 123 i.e., at odd numbered molecular mass since the compound contains only
one (odd number) nitrogen atoms. Two important ion fragments which are formed in the
mass spectrum of this compound are:
(a) NO2+ at 46 and
(b) NO+ at m/e 30. Both these fragment ions appear at even mass number. Now consider 2,4-
dinitrophenol. This compound contains two (even number) nitrogen atoms. Its signal (M+)
appears at m/e 184. The fragment ion appear at (M+ - H) m/e 183 and (M+ - H - OH) at m/e
155. Thus the fragment ions containing both the N atoms appear at odd mass number. This
proves the validity of the nitrogen rule. The conclusion of any common stable iostope except
O18
alters the use of nitrogen rule.
2. Ortho effect: Elemination reactions: these reactions operate not only from the
molecular ion but also from the fragment ion. The positive charge generally remains on the
carbon containing fragment. Alcohols usually eliminate a molecule of water from the
molecular ion. n-butyl and n-pentyl chlorides undergo hydrogen chloride elimination be the
abstraction of α-hydrogen atom by 1, 3- mechanism. The elimination of ketene (CH2 = C =
O) is a characteristic fragmentation mode of n-alkyl amides and o-acetates of phenols.

14
NH C O
CH2
H
C O
CH2
H
NH3 + CH2 C O
OH2 + CH2 C O
O
Ortho elimination: in ortho substituted aromatic compounds or in cis-olefins, the substituent
and a hydrogen atom can come to close proximity so as to eliminate a neutral molecule.
C
CH2
H
OCH3
O
CH2
CO
+ CH3OH
C
CH2
H
OCH3
O
+ H2O ;
HC
HC
CH2
C
O
H
OCH3
H2C
HC
HC
C
O
+ CH3OH
3. Hydrogen transfer rearrangement: These involve intramolecular hydrogen
rearrangements in aliphatic and aromatic hydrocarbons. In H-transfer rearrangements,
generally a six membered transition state is formed although other transition states are also
common.
H2C
(CH2)n
H2C
X
H
(CH2)n
CH2
CH2
+ HX
4. McLafferty rearrangement:
McLafferty rearrangement involves the cleavage of a β-bond followed by a γ-hydrogen
transfer. The rearrangement leads to the elimination of neutral molecules from amines,
aldehydes, ketones, unsaturated compounds and substituted aromatics. The rearrangemnent
proceeds through a sterically hindered six membered transition stabe. Consider a ketonic
compound,

15
Similarly amines, alcohols, ketones, acids. Esters which conatain a α-hydrogen atom
forms a McLafferty rearrangement ion. Other examples of McLafferty rearrangement are:
The structural requirement for this rearrangement is a side chain containing at least
three carbon atoms., the last bearing a hydrogen atom and a double bond which may be a
carbonyl group, an olefinic double bond or an aromatic system.
Double McLafferty rearrangement:
Double McLafferty rearrangement is reported in certain ketons. The second H-atom
originates exclusively from the γ-position. Secondary hydrogen is freferred to a primary
hydrogen atom in this process. The mechanism involes
(a) Ketonisation of the intermediate enol ion by the hydrogen transfer,
(b) Transfer of hydrogen to enolic oxygen. Consider MR in 4-heptanone.

16
Thus the molecular formula of the unknown compound can be determined from the various
fragment ion and also the parent ion of the mass spectrum.
General rules for fragmentation pattern:
1. Cleavage of a Sigma-Bond
(a)σ-Bond Cleavage in Small Nonfunctionalized Molecules

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(b) σ-Bond Cleavage in Small Functionalized Molecules
2. Even-Electron Rule:
******

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