MASS SPECTROSCOPY

Definition:
Mass spectrometry is an instrumental technique in which sample is converted to rapidly
moving positive ions by electron bombardment and charged particles are separated according
to their mass to charge ratio.

Mass spectrum is a plot of relative abundance against the ratio of mass/charge (m/e).

Principle:
Organic molecules are bombarded with electron converted into highly energetic positively
charged ions (Molecular ions or Parent ions) further break up into smaller ions (Fragment
ions or Daughter ions) The formed ions are separated by Deflection in Magnetic field
according to their Mass and Charge and it was represented in the form of mass spectrum.

Components of mass spectrometer:

1. Inlet system
2. Ion source
3. Mass analysers
4. Ion detectors
5. Vaccum system

1. Inlet system:

Solids samples with lower vapour pressure Inlet system directly inserted into the ionization
chamber and volatilization is controlled by heating the probe.

liquids are handled by hypodermic needles injection through a silicon rubber dam.

Gases samples are leaked into the ionization chamber directly by the help of mercury
manometer.

2. Ionisation:
A beam of electrons passes through a gas-phase sample and collides with neutral analyte
molecules (M) to produce a positively charged ion or a fragment ion. Generally electrons
with energies of 70 eV are used to form a fragment ions. The positive ions are collected in
focusing plates and passed to mass analyzer.

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TYPES OF IONS IN MS:

 Molecular ion
 Fragment ion
 Rearrangement ion
 Metastable ion
 Multicharged ion
 Base peak
 Negative ion

Molecular ion:
When a sample is bombarded with electrons of 9 to 15 eV energy, the molecular ion is
produced, by loss of single electron.

Fragment ions:
when an energy is given furthermore up to 70 eV, fragment ions produced, it have lower
mass number.

Rearrangement ion:
Recombination of fragment ion is known as Rearrangement Peaks.

Metastable ion:
The ions resulting from the decomposition between the source region and magnetic analyzer
are called as Meta stable ions. These appear as broad peaks called Metastable ion Peaks.

Multicharged ions:
Ions may exist with 2 or 3 charges instead of usual single charge. The peaks due to these
charged ions are known as Multicharged ion peaks.

Base Peak:
The largest peak in the mass spectrum corresponding to the most abundant ion or most
intense peak in the spectrum is called as Base Peak.

Negative ion:
Negative ions are formed from electron bombardment of sample. These results due to the
capture of electron by a molecule during collision of molecules Fragment ion peak.

Isotope ions:
Most elements are mixture of two or more stable isotopes differing by one or two mass units.
Chlorine and Bromine have two isotopes (35Cl, 37Cl and 79Br, 81Br) in the ratios 3:1 and
1:1 respectively. Thus in the spectrum of methyl bromide the molecular ion peak is the
doublet consisting of two equally intense peaks one at m/e 94 (CH3 79Br) and the other at
m/e 96 (CH3 81Br). These isotopic clusters are referred to as isotopic peaks. They are helpful
in determining the presence of such elements in a molecule.

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Fragmentation:
Fragmentation is a type of chemical dissociation. Fragmentation takes place by a process of
heterolysis or homolysis.

 Homolytic cleavage :

Here fragmentation is due to electron redistribution between bonds.

2. Heterolytic cleavage:

Fragmentation by movement of two electrons: In this type of cleavage both the electrons of
the bond are taken over by one of the atoms; the fragments are an even electron cation and a
radical with the positive charge residing on the alkyl group. It is designated by a conventional
arrow (↶ or ↷) to signify the transfer of a pair of electrons in the direction of the charged
site.

 Retro Diels-Alder reaction:

Elimination by multiple σ bond rupture: cyclohexene is broken down to Diene and

Dienophile. It can be explained by one or two electron mechanism.

One electron mechanism:

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Two electron mechanism:

 Mc Lafferty rearrangement:

Fragmentation due to rearrangement of Molecular or Parent ion: Here cleavage of bonds in

Molecular ion is due to the intramolecular atomic rearrangement. This leads to fragmentation
whose origin cannot be described by simple cleavage of bonds. When fragments are
accompanied by bond formation as well as bond for breaking, a rearrangement process is said
to have occurred. Such rearrangement involves the transfer of hydrogen from one part of the
molecular ion to another via, preferably, a six-membered cyclic transition state. This process
is favored energetically because as many bonds are formed as are broken.

Compounds containing hydrogen atom at position gamma to carbonyl group have been found
to a relative intense peak. This is probably due to rearrangement and fragmentation is
accompanied by the loss of neutral molecule. This rearrangement is known as McLafferty
rearrangement.

The rearrangement results in the formation of charged enols and a neutral olefins. To undergo
McLafferty rearrangement, a molecule must possess

a. An appropriately located heteroatom (ex.oxygen)

b. A double bond

c. An abstractable Hydrogen atom which is γ (gamma) to C= O system.

Ionization techniques:

Electron impact ionization:

In the Electron Impact (EI) process, electrons are emitted from a heated filament (usually
made of tungsten or rhenium) and are accelerated across the source by using an appropriate
potential (5- 100V) to achieve the required electron energy (sufficient to ionize the molecule).

M (g) + e- M+ + 2e-

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Chemical ionization:

Chemical ionization involves the ionization of a reagent gas, such as methane at relatively
high pressure (~1 mbar) in a simple electron impact source. Once produced, the reagent gas
ions collide with the analyte molecules producing ions through gas phase reaction processes
such as proton transfer.

nNH3 + e- NH4+ + 2e-

NH4 + + M NH3 + [M+ H] +

NH4 + + M [M+ NH4] +

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FIELD IONIZATION:

Field ionization (FI) is a method that uses very strong electric fields to produce ions from
gas-phase molecules. It is perfectly suited for the analysis of synthetic polymers or manmade
polymer.

Examples: poly siloxane, poly phosphazene backelite, nylon etc.

In field ionization, electrons are removed from a species by quantum mechanical tunneling in
a high electric field, which results in the formation of molecular ions (M + ̇ in positive ion
mode). This ionization method usually takes places in nonpolar or slightly polar organic
compounds.

FAST ATOM BOMBARDMENT (FAB):

A high-energy beam of netural atoms, typically Xe or Ar, strikes a solid sample causing
desorption and ionization. It is used for large biological molecules that are difficult to get into
the gas phase. FAB causes little fragmentation and usually gives a large molecular ion peak,
making it useful for molecular weight determination. The atomic beam is produced by
accelerating ions from an ion source though a charge-exchange cell. The ions pick up an
electron in collisions with netural atoms to form a beam of high energy atoms.

MALDI:

The analyte is embedded in a very large excess of a matrix compound deposited on a solid
surface called a target, usually made of a conducting metal and having spots for several
different samples to be applied. After a very brief laser pulse, the irradiated spot is rapidly
heated and becomes vibrationally excited. The matrix molecules energetically ablated from

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the surface of the sample, absorb the laser energy and carry the analyte molecules into the gas
phase as well. During the ablation process, the analyte molecules are usually ionized by being
protonated or deprotonated with the nearby matrix molecules. The most common MALDI
ionization format is for analyte molecules to carry a single positive charge.

APCI: Atmospheric Pressure Chemical Ionization

APCI is an ionization method used in mass spectrometry (commonly LC-MS) which utilizes
gas-phase ion-molecule reactions at atmospheric pressure. It is an ionization method that is
similar to chemical ionization (commonly used in GC-MS) where corona discharges on a
solvent spray produce primary ions. APCI is mainly used with polar and relatively non-polar
compounds with a molecular weight of less than 1500 Daltons, generally giving mono-
charged ions.

In APCI, the sample is typically dissolved in a solvent and pumped through a capillary inside
an uncharged quartz tube. At the end of the capillary, but still within the tube, the sample is
converted into an aerosol and then vaporized with the help of nitrogen gas and by heating to
very high temperature (~350- 550 °C). The gaseous solvent (S) and sample (M) are then
ionized by a corona discharge, in which a highly charged electrode creates an electric field
strong enough to ionize nearby molecules. A potential of several kilovolts applied to the

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electrode typically remove an electron from a neutral molecule, without depositing enough
internal energy to cause fragmentation. The corona discharge may directly ionize an analyte
molecule to form a radical cation (M+●):

M + e- → M+● + 2e-

S + e- → S+● + 2e-

Frequent collisions between the ions and molecules can transfer charge from an ion to
another neutral. Collision of an ionized solvent ion with an analyte molecule can create a
direct charge transfer to form a radical cation analyte ion:

S+● + M → M+● + S

Alternatively, collision of solvent ions with a neutral analyte molecule may result in
abstraction of a hydrogen atom from the molecule.

The resulting ionized solvent can then ionize the analyte via proton transfer:

S+● + S → [S+H] + + S [-H] [S+H] + + M → [M+H]+ + S

The resulting analyte ions (M+● or [M+H]+) are then injected into the mass spectrometer for
detection.

APPI:

It has recently become an important ionization source because it generates ions directly from
solution with relatively low background and is capable of analyzing relatively nonpolar
compounds. Similar to APCI, the liquid effluent of APPI is introduced directly into the
ionization source. The primary difference between APCI and APPI is that the APPI vaporized
sample passes through ultra-violet light (a typical krypton light source emits at 10.0 eV and
10.6 eV). Often, APPI is much more sensitive than ESI or APCI and has been shown to have
higher signal-to-noise ratios because of lower background ionization. Lower background
signal is largely due to high ionization potential of standard solvents such as methanol and
water (IP 10.85 and 12.62 eV, respectively) which are not ionized by the krypton lamp.
Disadvantages a. It can generate background ions from solvents. b. It requires vaporization
temperatures ranging from 350-500° C, which can cause thermal degradation. In APPI
technique samples are ionized by using UV light. Molecules interact with photon beam of
UV light with vapors of nebulizer liquid solution. Analyte molecules (A) absorb a photon
(hν) and become an electronically excited molecule. If the ionization energy (IE) of analyte
molecules is lower than the energy of photon, then the analyte molecule releases energetic
electron and become the radical cation.

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ESI:
ESI uses electrical energy to assist the transfer of ions from solution into the gaseous phase.
ESI works on the principle of Soft ionization. Soft ionization is a useful technique when
considering biological molecules of large molecular mass, because in this process
macromolecule is ionized into small droplets, which are then desolvated into even smaller
droplets, which creates molecules with attached protons.

ANALYSERS:

QUADRUPOLE MASS ANALYZER:

Quadrupole mass analyzer is one type of mass analyzer used in mass spectrometry. A typical
quadrupole mass analyzer consists of four rods with a hyperbolic cross section that are
accurately positioned parallel in a radial array. The quadrupole rods are typically constructed
using molybdenum alloys because of their inherent inertness and lack of activity. Very high
degrees of accuracy and precision (in the micrometer region) in rod machining and relative
positioning are required to achieve unit mass accuracy. Quadrupole mass spectrometers
~QMSs’ are widely used in both industry and research for fast accurate analysis of gas and
vapors. The QMS contains basically three elements; i) ion source, ii) mass filter, and iii) ion
detector.

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It consists of 4 voltage carrying rods. The ions are pass from one end to another end. During
this apply the radiofrequency and voltage complex oscillations will takes place. Here the
single positive charge ion shows the stable oscillation and the remaining the shows the
unstable oscillations. Mass scanning is carried out by varying each of the rf and voltage
frequencies ratios keeping their ratios constant. ◦Quadrupole ion storage (ion trap) ◦It store
the unsorted ions temporarily, they released to the detector by scanning the electric field.

TIME OF FLIGHT:
In this type of analyzer the sorting of ions is done in absence of magnetic field. The ions
produced are acquiring different velocities depending on their masses. Here the particles
reach the detector in the order of the increasing order of their masses. Here electron multiplier
detector is used. The resolution power of this is 500-600.
A time-of-flight (TOF) mass spectrometer is a non-scanning mass analyzer that emits pulses
of ions (or transients) from the source. These ions are accelerated so that they have equal
kinetic energy before entering a field free drift region, also known as the flight tube. A time-
of-flight (TOF) instrument consists of a pulsed ion source, an accelerating grid, a field-free
flight tube, and a detector. Different Modes of Time of Flight Analyzers: The mass analyzer
used in TOF-MS can be either be a linear flight tube, applying a bended geometry the flight
path to reduce the influence of neutral particles (Poschenrieder type) or a employ one (V-
type) or more reflectrons (W-type) to enhance the path length within a given flight tube. •
Linear TOF (high mass range but low mass resolution) • Reflectron TOF (lower mass range
but high mass resolution).

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Fragmentation rules:

Nitrogen rule:

The nitrogen rule states that organic molecules that contain hydrogen, carbon, nitrogen,
oxygen, silicon, phosphorus, sulfur, and the halogens have an odd nominal mass if they have
an odd number of nitrogen atoms or an even mass if they have an even number of nitrogen
atoms are present. The nitrogen rule is true for structures in which all of the atoms in the
molecule have a number of covalent bonds equal to their standard valency, counting each
sigma bond and pi bond as a separate covalent bond.

Rings plus double bonds:

From degree of unsaturation principles, molecules containing only carbon, hydrogen,

halogens, nitrogen, and oxygen follow the formula.

𝑅𝑖𝑛𝑔𝑠 + 𝜋 𝑏𝑜𝑛𝑑𝑠 = 𝑢 = 𝐶 – 𝐻/2 – 𝑋/2 + 𝑁/2 + 1

Where, C is the number of carbons, H is the number of hydrogen, X is the number of

halogens, and N is the number of nitrogen.

Even electron rule:

The even electron rule states that ions with an even number of electrons (cations but not
radical ions) tend to form even- electron fragment ions and odd-electron ions (radical ions)
form odd-electron ions or even-electron ions. Even-electron species tend to fragment to
another even-electron cation and a neutral molecule rather than two odd-electron species.

OE+•→EE++ R•, OE+•→OE+•+ N

Stevenson's rules:

The more stable the product cation ion, the more abundant the corresponding decomposition
process. Several theories can be utilized to predict the fragmentation process, such as the
electron octet rule, the resonance stabilization and hyper conjugation.

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Rule of 13:
The Rule of 13 is a simple procedure for tabulating possible chemical formula for a given
molecular mass. The first step in applying the rule is to assume that only carbon and
hydrogen are present in the molecule and that the molecule comprises some number of CH
"units" each of which has a nominal mass of 13.

If the molecular weight of the molecule in question is M, the number of possible CH units is
n and
𝑀/13 = 𝑛 + 𝑟/13
Where r is the remainder.

The base formula for the molecule is 𝐶 𝑛 𝐻 𝑛+𝑟

And the degree of unsaturation is 𝑢 = (𝑛 − 𝑟 + 2)/2.

A negative value of u indicates the presence of heteroatoms in the molecule and a half-integer
value of u indicates the presence of an odd number of nitrogen atoms. On addition of hetero
atoms, the molecular formula is adjusted by the equivalent mass of carbon and hydrogen. For
example, adding N requires removing CH2 and adding O requires removing CH4.

DETECTORS:

Once the ions are separated by the mass analyzer, they reach the ion detector, which
generates a current signal from the incident ions. The most commonly used detectors in MS
are as follows:

 Faraday cup
 Electron multiplier
 Photomultiplier dynode

FARADAY CUP:

A Faraday cup involves an ion striking the dynode (BeO, GaP, or CsSb) surface which causes
secondary electrons to be ejected. This temporary electron emission induces a positive charge
on the detector and therefore a current of electrons flowing toward the detector. This detector
is not particularly sensitive, offering limited amplification of signal, yet it is tolerant of
relatively high pressure.

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ELECTRON MULTIPLIER:

It is the most common means of detecting ions. It is made up of a series (12 to 24) of
aluminum oxide (Al2O3) dynodes maintained at ever increasing potentials. Ions strike the
first dynode surface causing an emission of electrons. These electrons are then attracted to the
next dynode held at a higher potential and therefore more secondary electrons are generated.
Ultimately, as numerous dynodes are involved, a cascade of electrons is formed that results in
an overall current gain on the order of one million or higher. The high energy dynode (HED)
uses an accelerating electrostatic field to increase the velocity of the ions and serves to
increase signal intensity and therefore sensitivity.

PHOTOMULTIPLIER CONVERSION DYNODE:

The photomultiplier conversion dynode detector is not commonly used. It is similar to

electron multiplier in design where the secondary electrons strike a phosphorus screen instead
of a dynode. The phosphorus screen releases photons which are detected by the
photomultiplier and are then amplified using the cascading principle. One advantage of the
conversion dynode is that the photomultiplier tube is sealed in a vacuum, unexposed to the
environment of the mass spectrometer and thus the possibility of contamination is removed.

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 VACUUM SYSTEM:

In order to work in a predictable and efficient way, mass analyzers require high levels of
vacuum so that the analyte ions under investigation must be manageable and sensitive to the
electrostatic components of the instrument. Vacuum technology is used to remove the
majority of background (air) molecules, as the deflection of ions by the spectrometer on a
specific pathway takes place only under the influence of electric, magnetic and/or
radiofrequency fields, which would otherwise result in deviation due to collision. A high
level of vacuum within the instrument prevents deviation of the analyte ion from the specified
path and assists the processes of ion movement and separation within the following ways: By
providing an adequate mean free path for the analyte ions. By facilitating collision free ion
trajectories. By the reduction of ion-molecular reactions. By minimization of background
interference.

Examples of Vacuum Pumps:

 Rotary pumps
 Foreline pumps
 Turbo-molecular pumps
 Diffusion pumps

APPLICATIONS:

Mass spectrometry has both qualitative and quantitative uses.

1. Structure elucidation

2. Detection of impurities

3. Quantitative analysis

4. Drug metabolism studies

5. Clinical, toxicological and forensic applications

6. MS is now in very common use in analytical laboratories that study physical, chemical, or
biological properties of a great variety of compounds.

QUALITATIVE APPLICATIONS:

1. Determination of molecular weight: Mass spectrometry serves as the best possible

technique for the determination or confirmation of molecular weight of compounds that can
be easily volatilized.

2. Determination of molecular formula: For the determination of molecular formula b mass

spectrometry, it is essential to identify the molecular ion peak as well as its exact mass.

3. Determination of partial molecular formula: Generally, atoms are polyisotopic. In mass

spectrometer, the ions are selected according to their actual mass. Exact information about

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 the atomic composition of the selected ions is furnished by the mass distribution of molecular
ions

4. Determination of structure of compounds: Bombardment of vaporized sample

molecules with a high beam of electrons results in their fragmentation producing a large
number of ions with varying masses.

QUANTITATIVE APPLICATIONS

1. Determination of isotope abundance: Although differences in the masses of isotopes of

an element are very small, the isotope abundance i.e., the isotopic composition of molecules
within an easily vaporizable sample can be determined with mass spectrometry.

The information so obtained may be useful for:

(a) Tracer studies with isotopes

(b) Determination of atomic weights of compounds

(c) Determination of age rocks and minerals

(d) Study of origin as well as nature of solar system

2. Determination of isotope ratio: Mass spectroscopy is used to determine isotope ratio

which in turn helps to determine the concentration of individual components present in
complex mixture from which it cannot be separated quantitatively.

3. Differentiation between Cis and Trans isomers: Mass spectrometry may be used to
differentiate between cis and Trans isomers. Both the isomers yield similar spectra but are
differentiated from the intensity of the molecular ion peaks. The molecular ion peak of trans
isomer is more intense than that of cis isomer.

4. Measurement of ionization potential: Ionization potential is the minimum energy

required by the bombarding electrons to produce the molecular ions from a molecule of an
atom.

5. Determination of ion-molecule reactions: Mass spectrometry finds its use in the study of
ion molecule reaction i.e., the reactions in between the fragment ion and the unionized
molecules. The rates of these reactions directly depend on the operating pressure.

6. Detection of impurity: The impurities present sample even in low concentration (parts per
million) can be detected by spectrometry, provide the molecular weights of the impurities
differ considerably from the major components.

7. Identification of unknown compounds: Mass spectrometry can be used to identify the

unknown compounds. This can be achieved by recording the spectrum of the unknown
compounds and comparing it with the spectrum of the standard compound recorded under
identical conditions.

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8. Identification of proteins: Mass spectrometry serves as valuable tool in the study of
structure and functions of proteins (proteomics). Electro spray ionization (ESI) and matrix-
assisted laser desorption/ionization (MALDI) are the widely used ionization methods for this
purpose. Mass spectrometry in the proteomics particularly deals with the analysis of protein
digested by protease like trypsin.

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