MASS SPECTROMETRY

Introduction:
Wein showed that deflection of positive ions is possible in presence of electric and
magnetic fields. J J Thomson determined the mass to charge ratio of the electron. These two
phenomena have formed the basis for the development of the technique “Mass Spectrometry”.
This technique is used by organic chemists to characterize organic molecules. The technique is
useful to characterize organic molecules in two principal ways:

1) To measure exact molecular weights as well as exact molecular masses

2) To indicate the exact point at which fragmentation is possible within the molecule and in
turn to identify the presence of certain structural units in the organic compound.

The earliest mass spectrometer was built by A. J. Dempster in 1918. This earliest
spectrometer has undergone tremendous changes in respect to sample introduction, ionization
techniques and ion analysis to become much useful and reliable technique for chemists. This
technique is not only useful for chemists but also for pharmacists, environmental scientists, and
geologists. It is also routinely used in airport security screening and forensic investigations. The
mass spectrometry technique is highly sensitive, reproducible, accurate and can be applied for
samples existing in very low concentrations.

Basic Principle of Mass Spectrometry:

Mass spectrometry is the most accurate method for determining the molecular mass of
the compound and its elemental composition. The compound under investigation is bombarded
with a beam of energetic electrons in this technique. As a result the molecules undergo ionization
and dissociate into fragments. The ions produced have specific ratio of mass to charge i.e., m/z
ratio. The charges can normally be taken as one as multiple charged ions are produced very
rarely relative to singly charged ions.

Initially the process of bombardment of molecules with energetic beam of electrons may
result in the formation of molecular ion (M +). The additional energy of the bombarding electrons
is dissipated in breaking the bonds in the molecular ion, which undergoes fragmentation to yield
several neutral or positively charged species. This fragmentation may result in the formation of
an even- electron ion and a radical

or an odd electron ion and a neutral molecule

The
various ions produced are accelerated and deflected in a magnetic or electrical field. The extent
of deflection of an ion depends on its mass to charge ratio and velocity. To understand the
principle of mass spectrometry, the fragmentation pattern of neopentane has been considered as
an example

m/z
Relative intensity

The molecular ion (here C5H12+) is called parent ion and is designated as M +. The
molecular ion then undergoes fragmentation to form daughter ions or fragment ions and the set
of these ions are analyzed by obtaining a signal for each value of m/z. The intensity of each
signal represents the relative abundance of the ion producing the signal. The largest most intense
peak in the structure is called the base peak and its intensity is arbitrarily assigned a value of
100%. The intensities of other peaks are represented relative to the base peak. The parent peak
may not be confused with the base peak.
 A mass spectrum is a presentation of the masses of positively charged fragments
(including the parent ion) versus their relative concentrations. This graphical representation
consists of intensities of the signals along y-axis and m/z values of the corresponding ions along
x-axis. The mass spectrometry technique is not a true spectroscopic technique but however it
complements information provided by IR, UV and NMR and hence included in spectroscopy
techniques.

Mass
Spectrum of Neopentane

Theory of Mass Spectrometry:

A mass spectrum consists of a display of peaks of different heights. The nature of the
spectrum is dependent on properties of the molecule, ionization potential, sample pressure and
the instrument design. A molecule M is bombarded with high energy electrons in the mass
spectrometer i.e.,

M (g) + e- → M+ (g) + 2e-

Energy of the order of 70eV is required for this ionization. The energy in the form of electrons,
photons, electric fields, heat or electrical discharge is normally deployed for the purpose of
ionization. The energy of 70eV is greater than the typical bond energies encountered in organic
molecules. If the energy of the bombarding electron is equal to the ionization potential then all
the electron’s energy must be used to remove an electron from the molecular orbital of the
molecule M to form the parent ion M+. As the energy of bombarding electron increases, the
probability of the collisions inducing ionization increases. If the energy of the electron is quite
high than M+ then the parent ion may retain such an excess energy in order to rupture the bond
and form a new ion N + and a fragment O. The m/z value of the parent ion is equal to the
molecular mass of the compound. The parent ion has mass equal to that of molecule because the
lost electron has negligible mass. Only the positively charged ions travel towards the detector
and give rise to mass spectrum. Neutral particles, produced in the process of fragmentation (e.g.,
M+ (g) → m1+ + m2 or m1+ m2+ ) cannot be detected in the mass spectrometer. Negative ion
spectra can also be obtained although less commonly used than positive ion spectra.

Instrumentation:

The important basic functions of mass spectrometer are

 To vapourise compounds of varying volatility.
 To generate ions from the neutral compounds in the gas phase.
 To segregate ions according to their mass to charge ratio.
 To sense the ions and produce a resultant signal.
These functions are well performed by mass spectrometers.

The main components of common mass spectrometer are

1. Sample Introduction system
2. The Ion Source
3. Mass Analyser
4. Ion Detector

Schematic Diagram of Mass Spectrometer

1. Sample Introduction system
The sample introduction system is of two types
A) Internal Sample Introducing system: The sample is placed within the ionization
chamber either as a part of the ion source or coated on a filament.
Spark Electrode: If the sample is an ionic compound then it can be analysed by fabricating
the sample itself as spark gas electrode. Non- conducting samples can be mixed with some
conducting material and converted into an electrode.
 Filament Coating: Sometimes sample can be coated on the filament. If the filament is
heated then the sample yields positive ions directly.
B) External Sample Introducing System:
Direct Introduction: The direct introduction method is applicable when the compounds are
sufficiently volatile.
Glass or Metal Inlet: If there is scope for the decomposition of sample on metal surface then
glass inlet system will be used for sample introduction. A syringe with hypodermic needle or
a micropipette can be used as a glass inlet system.

Sample Introduction system

Knudsen Cell: This sample inlet system is used to introduce high melting materials into a
furnace.

Ionization Chamber or Ion Source:

The main purpose of this chamber is to convert molecules into gaseous ions. The most
common way to generate ions involves bombardment of the sample with a beam of energetic
electrons. Important types of ionization chambers are
1. Electron Impact (EI)
2. Chemical Ionization (CI)
3. Electrospray Ionization (ESI)
4. Desorption Ionization Techniques
a. Fast Atom Bombardment (FAB)
b. Matrix Assisted Laser Desorption Ionization (MALDI)
c. Secondary Ionization Mass Spectrometry (SIMS)

1.Electron Impact (EI): In this technique the sample has been introduced using a sample inlet
i.e., metal inlet system. The sample is then interacted with high energy beam of energy 70 ev (1
ev = 23 K Cal/mole or 96 KJ/ mole) and these electrons are accelerated from a tungsten filament.
This electron beam has sufficient energy not only to ionize an organic molecule but also to cause
extensive fragmentation.

Generally the strongest single bond in an organic molecule will have a strength of nearly 4ev.
The electrons in an organic molecule most probably exist in pairs in filled orbitals. Ionisation of
such organic molecule by the removal of electron results in the formation of cation radical.

Electron capture to give an anion radical is less probable, as the bombarding electrons have such
high translational energies that they cannot be captured. The molecular ion produced by the
Electron Impact can fragment either by loss of radical or by loss of a molecule with all its
electrons paired. To understand this, the fragmentation of the molecular ion of butyl acetate is
shown below.

Electron Impact Source

The major advantage with EI technique is extensive fragmentation of sample molecule which
gives rise to a pattern of fragment ions helpful in characterizing the compound. Frequent absence
of molecular ion peak is the major disadvantage with this technique.

2. Chemical Ionization: In this technique a reagent gas like methane, isobutene or ammonia is
sent into ionization chamber initially. The reagent gas undergoes ionization by using electrons
with energies up to 300 ev. The ionization of methane as reagent gas can be given as follows
CH4 + e- → CH4+. + 2e-
Further sequence of ionization can be represented as below
CH4+. → CH3+ + H.
The ions generated in the above manners can collide with the neutral molecules as shown below
CH4+. + CH4 → CH5+ + CH3.
CH3+ + CH4 → C2H5+ + H2
These ions are useful in ionizing the sample molecules when they are introduced into ionization
chamber after reagent gas and the process can be represented as below.
M + CH5+ → MH+ + CH4
Thus in this positive ion CI spectra a protonated species of molecule (MH +) is possible and this
ion will have m/z value one unit more than the true molecular weight. In case of isobutane and
ammonia as reagent gas the possible ions are C 4H9+ and NH4+ in the ionization chamber and
these ions can further ionize the sample molecules. It is very necessary to choose a suitable
reagent gas to best match the proton affinity of the reagent gas with that of the sample, to bring
about efficient ionization of the sample without excessive fragmentation. The greater the
difference between proton affinity of the sample and that of reagent gas, the more will be the
energy that is transferred to the sample during ionization. Therefore, it is necessary to choose a
reagent gas which has closely matched proton affinity with the sample.

Though protonation is the most common method in CI-MS, it is also possible to have reverse
ionization also. If methyl nitrite/ methane mixture is used as reagent gas then ionization of
reagent gas produces CH3O- which abstracts a proton from the sample and leads to the formation
of (M-H)- parent ion. Similarly use of NF3 as reagent gas generates F- which also abstracts a
proton from the sample. This technique is useful in case of samples with low molecular mass.
3.
Electrospray Ionization: This technique is useful in ionizing high molecular weight
biomolecules as well as other labile and volatile molecules. In this technique, a solution of
sample molecules is prepared and is sprayed into a pre heated chamber through a fine capillary
tube. A high voltage potential is applied across the surface of the capillary tube. As a result small
charged droplets of sample solution will be expelled into the chamber. These charged droplets
are subjected to a counter flow of a drying gas (nitrogen gas) to evaporate the solvent molecules
from the droplets. Due to evaporation of solvent molecules, the charge density of the droplets
increases and the electrostatic repulsions exceed the surface tension of the droplet. At this point
the droplets break further into smaller droplets and the process is continued until solvent free
sample ions are left in the gas phase. The sample ions may bear single charge or multiple
charges.

4. Desorption Ionization Techniques (SIMS, FAB, MALDI) : Low molecular weight samples
can be ionized conveniently by EI and CI techniques. Modern ionization techniques like SIMS,
FAB and MALDI can be used to ionize large molecular weight, nonvolatile sample molecules. In
these techniques the sample to be ionized is dispersed or dissolved in a matrix. The matrix
should be nonvolatile, relatively inert, and a reasonable electrolyte to allow ion formation. The
sample molecules in the matrix are then made to interact with high energy beam of ions in case
of SIMS, with neutral atoms in case of FAB and with high intensity photons in case of MALDI.
Beams of Ar+ or Cs+ are often used in SIMS and beams of Ar or Xe atoms are common in FAB.
Most MALDI spectrometers use a nitrogen laser that emit at 337nm, but some applications use
infrared(IR) laser. The collision of these ions /atoms/photons with the sample ionizes some of the
sample molecules and ejects from the surface as shown in the figure. As neutral atoms are used
to ionize the sample molecules, both positive and negative ions detection are possible. (M+H)+
or (M-H)- molecular ions are possible in SIMS and FAB. If the matrix compound is more acidic
than the analyte, then predominantly (M+H)+ ions will be formed, while mostly (M-H)- ions will
result when the matrix is less acidic than the analyte.

Common matricies used for SIMS and FAB mass spectrometry are glycerol, thioglycerol, 3-
nitrobenzyl alcohol, diethanolamine, triethanolamine etc., and common matrices used in MALDI
are nicotinic acid, picolinic acid, 2,5-dihydroxybenzoic acid etc.

Mass Analyzer

The parent and fragment ions produced in the ion chamber are accelerated by applying an
acceleration potential. These ions then enter into mass analyzer. In the mass analyzer the
fragment ions are differentiated on the basis of m/z ratio. The positive ions accelerated by
electric field travel in a circular path through 180 0 under a magnetic field B and fall on a
collector plate. If an ion having charge z is accelerated through a voltage V then the kinetic
energy imparted to the ion can be given as

1/2mv2 = zV …………(1)

Where v is velocity of the ions after acceleration, V is potential applied.

Thus, the different ions move with different velocities depending upon their mass (their charge is
the same). These ions are deflected and forced into circular path, while passing through the
magnetic field, and the radius of which depends upon m/z ratio as per the following equations.

Centripetal force = Bzv

Centrifugal force = mv2 /r

v =Bzr/m

where r is radius of curvature.

Substituting the value of v in equation (1) we get,

m/z = B2r2/2V …………(2)

Equation 2 indicates that at a given magnetic field and accelerating voltage, the ions of m/z value
will follow a circular path of radius r. The radius of curvature of the analyser cannot be varied
and hence either by varying B or V , ions of different m/z are collected successively and hence
resolved according to their m/z value.

The resolving power of Dempster’s mass spectrometer is limited and the resolving power of the
mass analyzer can be increased by placing an electric field prior to magnetic field in mass
analyser. Instruments incorporated with such a system are called double focussing mass
spectrometer.
Ion Detector

The ions which are separated by the analyzer are detected and measured electrically or
photographically. The ions pass through the collecting slits one after the other and fall on the
detector. The collector electrode is well shielded from stray ions. The recorder records the peaks
of different ions with different sizes as per their relative abundance.

Molecular Ion Peak

The bombardment of molecule with electron beam of energy 10-15eV usually removes an
electron and results in the formation of molecular ion peak. The loss of an electron occurs
rapidly from the highest occupied orbital of aromatic system and non bonding electron orbitals
on oxygen and nitrogen atoms. Similarly, the loss of an electron occurs readily from double bond
and triple bond also. In alkanes, the ionization of C-C sigma bond is easier than that of C-H. Few
examples are provided hereunder.

The molecular mass of the compound can be known from the molecular ion peak in the mass
spectrum. This peak always appears at the high mass region of the spectrum. The relative
abundance of the parent ion peak depends on its stability. In some cases, the parent ion may not
appear in the spectrum at all due to decomposition of parent ion at a faster rate. The rate of
decomposition of the parent ion increases with the molecular size in the homologous series. The
relative intensity of the parent peak decreases in the following order.

Aromatics > Conjugated olefins > Alicylics > Unbranched hydrocarbons > Ketones > Amines >
Esters > Ethers > Carboxylic acids > Branched hydrocarbons > Alcohols.

Important features of the parent ion peak

1. In aromatic compounds, the molecular ion peak is relatively much intense due to the
presence of pi- electrons.
2. Much intense molecular ion peak is also possible for unsaturated compounds when
compared to the saturated or the cyclic compounds.
3. The relative abundance of the saturated hydrocarbon is more than the corresponding
branched chain with same number of carbon atoms. For example, the molecular ion peak
of n- pentane is more intense than that of neopentane.
4. In case of highly branched or tertiary alcohol, the molecular ion peak will not appear in
the spectrum.
5. Primary and secondary alcohols give very small molecular ion peaks.
6. Much intense molecular ion peaks are possible in case of conjugated olefins compared to
non conjugated olefins.
7. The presence of substituent’s like -NH 2, -OH, -OR etc., on the aromatic ring, will
increase the relative intensity of the molecular ion peak, whereas the presence of groups
like –NO2, -CN etc., will decrease the intensity of molecular ion peak.
8. Isotope peaks are also possible along with molecular ion peak, in case of chloro or bromo
compounds. The M+ and (M+2)+ ion peaks are possible in the ratio of 1:3 and 1:1
respectively, in case of chloro and bromo compounds.
9. A peak at mass 19 always indicates fluorine, peak at mass 30 is always for primary
amines. The appearance of peaks at 31,45,59 indicates the presence of the oxygen as
alcohol or ether, mass 33 shows thiol, mass 77 corresponds to the presence of benzene
ring, mass 91 indicates a monobasic carboxylic acid or the presence of tolyl group.
10. The peak at mass 29 may be due to C2H5+ which might be a part of C4H10+2. Spectrometers
are unable to distinguish between singly charged ion of a given mass and doubly charged
ion of twice the mass.
11. The peaks due to fragments C 4H10+, C3H7+ and C2H5+ appear sometimes at 59, 44 and 30
respectively. All the three peaks are observed at mass greater than by one unit than the
12
mass of the true fragment due to replacement of one hydrogen by deuterium or C is
replaced by 13C . Mostly due to replacement of 12C by 13C.
12. The molecular mass of an organic compound is odd if it contains odd number of nitrogen
atoms in it or even if it contains (no nitrogen or) even number of nitrogen atoms in it.
Metastable Ions

In the mass spectrum of a molecule normally, peaks appear at integer m/z values
corresponding to parent ion and fragment ions. However, sometimes peaks appear at non-
integer m/z value, such peaks which appear at non-integer m/z are known as metastable
peaks. These metastable peaks are much broader and possess relatively low abundance.

Formation of metastable ions:

Consider M1+ as parent ion and m1+ as daughter ion. If the fragmentation of M 1+ to m1+ takes
place in the ion source, then the daughter ion, may travel the whole analyser region and is
recorded as m1+ ion. If the fragmentation of M1+ to m1+ occurs after the source exit and
before arrival at the collector i.e. in analyser, then m 1+ is called a metastable ion. The ions
coming out of the source are accelerated before entering into mass analyser. Similarly, the
M1+ will also be accelerated before entering into analyser. As fragmentation occurs before
detection, accelerated ion is one (M1+) and resolved as another (m1+). Due to fragmentation
after leaving ion source, the fragment ion travels with different energy than the parent ion as
some of the energy is taken up by the neutral species.

M1+.(accelerated)→ m1+(detected) + R.(neutral)

As m1+ travels with different energy, it undergoes abnormal deflection and is going to be
detected at non integer m/z between m/z of M1+. and m1+. The apparent mass(m*) at which
metastble peak appears at, can be given by

m*= (m1)2/ M1

Importance of metastable peaks: The existence of metastable peaks in the mass spectrum is
helpful in structural elucidation and sometimes helpful in understanding the mechanism of a
reaction.

The mass(m/z) of metastable peak can be known from the position of parent ion peak and
fragment ion peak. The calculation of the metastable peak position can be understood from
the following examples.
Nitrogen rule and extension of nitrogen rule:

Nitrogen rule states that if a compound has even number of nitrogen atoms (or zero nitrogen
atoms also), its molecular ion will appear at an even mass value. On the other hand, a molecule
with an odd number of nitrogen atoms will form a molecular ion with an odd mass. This rule
holds good for all compounds containing C, H, N, O, S and halogens as well as less usual atoms
like P, B, Si, As etc. This rule arises from the fact that nitrogen has odd valence (3) and even
atomic mass (14), where as all other atoms will have odd valence and odd atomic mass (Boron
atom has an atomic mass of 5 and valence is 3) or even valence and even atomic mass (Carbon
atom has a mass of 12 and valence 4).

Examples

Nitrobenzene (C6H5NO2) consists of odd number of nitrogen atoms and has mass of 123(odd),
where 2,4- dinitrophenol consists of two nitrogens and has a mass of 184(even).

This rule is not only applicable for molecular ions but also for fragment ions. The fragment ion
may contain odd number of electrons or even number of electrons. Nitrogen rule can be extended
as follows, if odd electron fragment species contains even number of nitrogens then it will have
even mass. If fragment ion is an even electron species containing even number of nitrogens in it
then it will have odd mass.

Example

If Ethylene diamine (H2N-CH2-CH2-NH2) under goes fragmentation losing a hydrogen radical

(H.) then (M+-H even electron species) peak is possible at 59 (odd mass).

Isotopic Peaks and their importance in Mass spectrum

The mass spectrum of molecules sometimes consists of peaks corresponding to (M+1) + and
(M+2)+ masses with appreciable intensity along with molecular ion peak (M +). The peaks at
(M+1)+ and (M+2)+ m/z values can be attributed to the presence of heavier isotopes. Thus,the M +.
peak is not the peak of highest m/z value. Naturally, several isotopes of most elements are
possible and the lightest of these predominates in abundance. The table of few elements and their
percentage abundance is presented here to understand the importance of isotopes.
For most compounds
the (M+2)+ peak is small, but
for compounds containing
chlorine, bromine and sulphur the (M+2)+ ion peak has substantial intensity. Single bromine
containing compounds exhibit pairs of peaks of roughly equal intensity separated by two mass
units. This is due to almost equal abundance of the two isotopes 79Br and 81Br as shown in the
table (peaks of M+ and (M+2)+ appear in the intensity ratio of 1:1). The existence of such pairs of
peaks can be understood from the mass spectrum of methyl bromide.

Similarly, in case of methyl chloride two peaks are possible corresponding to 35Cl and 37Cl in the
ratio of 3:1 respectively. The presence of such characteristic doublets in the mass spectra of
compounds is helpful in diagnosing the presence of these elements. In the same manner, the
contribution (4.4%) of the 34S to the M+2 peak , is helpful in identifying the presence of sulphur
in the compound.
 Mass Spectrum of methyl chloride.

Index of hydrogen deficiency (or Unsaturation Index or Double bond Equivalence)

The determination of index of hydrogen deficiency is helpful in knowing the number double
bonds or the number of rings possible in an organic compound. The formula of index of
hydrogen deficiency can be derived basing on the following considerations.

1. Alkane CnH2n+2
2. Alkene CnH2n
3. Alkyne CnH2n-2

Alkane differs from alkene by 2 hydrogens and an alkene differs from alkyne by 2
hydrogens. It can be noticed that each time if a ring or pi-bond is introduced into the
molecule, the number of hydrogen atoms in the molecular formula is reduced by 2. For every
triple bond (2 pi bonds), the molecular formula is reduced by four hydrogens.

The hydrogen content in an organic molecule may also vary due to the presence of
noncarbon and nonhydrogen elements. Consequently, the ratio of carbon and hydrogen
content may vary. Three simple rules that may be used to predict how the ratio will change
can be given as follows.

1. If an element of Vth group is present in open chain, saturated hydrocarbon then the
molecule will possess one excess hydrogen than its hydrocarbon analogue.

C2H6 (6 H) , C2H5NH2 (7 H)
2. If an element of VIth group is present in open chain hydrocarbon then the molecule will
possess same number hydrogen atoms as in its hydrocarbon analogue.

C2H6 (6H) , C2H5OH (6H)

3. If an element of VIIth group is present in open chain, saturated hydrocarbon then the
molecule will possess one less hydrogen than its hydrocarbon analogue.

C2H6 (6H), C2H5Cl (5H)

Similarly, the ring closure of hydrocarbon will have hydrogen deficiency of 2 in number
when compared with its open chain compound.

Basing on all the above considerations, the formula to calculate hydrogen deficiency can be
given as follows.

Index of hydrogen deficiency = [2a+2-b+d]/2

Where a is number of tetravalent atoms

b is number of monovalent atoms
d is number of nitrogens or Vth group elements

The rule of thirteen ( Rule-13)

This rule is useful in generating the molecular formula of the compound. Using, this rule it is
possible to generate the base formula of the molecule which contains only carbon and
hydrogen. The mass of carbon and hydrogen put together is 13 and hence the rule is
mentioned as rule-13. The base formula of the molecule is obtained by dividing the mass of
the molecule M by 13. This calculation provides a numerator n and a remainder r.
M/13 = n + r/13
The base formula thus becomes CnHn+r
The index of hydrogen deficiency U can be given as follows
U = (n- r +2)/2
Example
If the molecular mass is 94 then the base formula can be obtained as follows
94/13 = 7 + 3/13
Where n = 7 and r = 3
The base formula is C7H10
The index of hydrogen deficiency is
U = (7 – 3 + 2)/2
i.e. U = 3 Indicates the presence of three double bonds or 1 ring and 2
double bonds etc.