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CONTENT Ss:
ae. | INTRODUCTION

4

f DEFINITION
3

OO THEORY
3

did HD
3

“e INSTRUMENTATION

IONIZATION TECHNIQUES

en FRAGMENTATION

APPLICATIONS

INTRODUCTION ~

Mass spectrometry (MS) is an analytical technique that
measures the mass-to-charge particles. It is used for
determining masses of particles, for determining the
elemental composition of a sample or molecule. The MS
principle consists of ionizing chemical compounds to
generate charge molecules or molecule fragments and
measurement of their mass-to-charge ratio by using the
one of a variety of techniques. Mass Spectrometry is a
powerfu l technique for identifying unknowns, studying
molecular structure, and probing the fundamental
principles of chemistry.

 

THEORY:

The relative abundance of positively charged
fragments of various mass-to-charge ratios is the
characteristic feature of the molecule that serve to
identify the substance.

It is determined by
1/2mv2=eV[.’. kinetic energy= 1/2 mv?]  where, m=mass of

ion
v=velocity of ion
V=accelerating

voltage

oi Sys e=charge on ion
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DEFINITION:
Mass spectrometry (MS) is an analytical chemistry
technique that helps identify the amount and type of
chemicals present in a sample by measuring the mass-
to-charge ratio and abundance of gas-phase ions.

PRINCIPLE:
A mass spectrometer generates multiple ions from the
sample under investigation

This molecular ion undergoes fragmentation. Each
primary product ion derived from the molecular ion, in
turn, undergoes fragmentation, and so on.

 

The ions are separated in the mass spectrometer
according to their mass-to-charge ratio, and are
detected in proportion to their abundance. A mass
spectrum of the molecule is thus produced.

lt displays the result in the form of a plot of ion
abundance versus mass-to-charge ratio

 

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OBTAINING MASS SPECTRA CONSIST OF 2 TYPES:

Conversion of neutral molecule into a charged
molecule, preferably to a positively charged
molecule.

Separation of the positively charged fragments
formed, based on their masses, by using
electrical or magnetic field or both.

 

INSTRUMENTATION:

The instrument consists of three major components:

> lon Source: For producing gaseous ions from the
substance being studied.

» Analyzer: For resolving the ions into their characteristics
mass components according to their mass-to-charge ratio.

» Detector System: For detecting the ions and recording
the relative abundance of each of the resolved ionic species.

 

MASS SPECTROMETER

ACCELERATION

IONISATION

 

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DEFLECTION

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DETECTION
amplifier

 

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recorder

 

>» In addition, a sample introduction system is
necessary to admit the samples to be studied to the
ion source while maintaining the high vacuum
requirements (~10-6 to 10-8 mm of mercury) of
the technique; and a computer is required to
control the instrument, acquire and manipulate
data, and compare spectra to reference libraries.

 

To vacuum

pump

Intensity ——>

 

With all the above components, a mass spectrometer should
always perform the following processes:

Y Separate these ions according to their mass-to-charge ratio
in the mass analyzer.

Y Eventually, fragment the selected ions and analyze the
fragments in a second analyzer.

VY Detect the ions emerging from the last analyzer and
measure their abundance with the detector that converts the
ions into electrical signals.

Y Process the signals from the detector that are transmitted to
the computer and control the instrument using feedback.

 

IONIZATION TECHNIQUES:
Electron impact (EI)
Chemical Ionisation (C1)
Fast atom bombardment (FAB)
Field ionization
Plasma desorption

 

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-1.ELECTRON IMPACT (ED: |

In the Electron Impact —_—_—

 

(EI) process, electrons
>.

ws

are emitted from a Transfer line

heated filament (usually
made of tungsten or
rhenium) and = are
accelerated across the > To the mass analyser
source by using an
appropriate potential (5-
100V) to achieve the ] u

required electron energy Hesed Marant
(sufficient to ionize the

Key: Analyte

molecule).

Non lonised

lonised

 

 

2. CHEMICAL IONISATION

(CI)
Chemical ionisation involves : Transfer line

the ionisation 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 Heated filament
through gas phase reaction

Key Analyte Reagent gas
processes such as_ proton

Non lonised & ©
=

nNH, + e& NH,+ + 2e =
lonised ot

NH,* +M NH, + [M+ H]*
NH,* +M _([M+ NH,]*

 

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.

analyte/meatrix

desorbed seconmdary
om beam

probe tip

 

 

 

4.FIELD IONIZATION:
Field ionization (Fl) 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 man made polymer .

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

Positive ion
Rebounding

polarized particle
itical

Critica Field-adsorbed
surface ticl

Impinging te 4 particle

polarized particle a

Ca
* . A field-evaporating

a lattice atom

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Crawling
polarized particle

2

 

-5.Plasma desorption:

Plasma desorption ionization mass spectrometry (PDMS), also called fission
fragment ionization, is a mass spectrometry technique in which ionization of

material in a solid sample by bombarding it with ionic or neutral atoms
formed as a result of the nuclear fission of a suitable nuclide, typically
the californium isotope?Cf.

Sample TOF Mass Spectrometer
pie :

Start : @) Sele
detector detector

_— :
Fragments ,, an al

TDC

Computer

 

 

CHROMATOGRAPHIC TECHNIQUES
COMBINED WITH MASS SPECTROMETRY

» An important enhancement to the mass
resolving and mass determining capabilities of
mass spectrometry is using it in tandem with
chromatographic separation techniques.
> GCMS
>LCMS

 

 

GAS CHROMATOGRAPHY :

» A common combination is gas chromatography-mass
spectrometry (GC/MS or GC-MS). In this technique, a gas
chromatograph is used to separate different compounds.

» This stream of separated compounds is fed online into the
ion source, a metallic filament to which voltage is applied.
This filament emits electrons which ionize the compounds.
» The ions can then further fragment, yielding predictable
patterns.
» Intact ions and fragments pass into the mass
spectrometer’s analyzer and are eventually detected.

 

 

LIQUID CHROMATOGRAPHY

» ndianapolis Museum of Art conservation
scientist performing liquid chromatography-mass
spectrometry.
» Similar to gas chromatography MS (GC/MS),
liquid chromatography-mass spectrometry (LC/MS
or LC-MS) separates compounds

 

» chromatographically before they are introduced to
the ion source and mass spectrometer. It differs from
GC/MS in that the mobile phase is liquid, usually a
mixture of water and organic solvents, instead of gas.

» Most commonly, an electro spray ionization source
is used in LC/MS. Other popular and commercially
available LC/MS ion sources are atmospheric
pressure chemical ionization and atmospheric
pressure photo ionization.

>» There are also some newly developed ionization
techniques like laser spray.

 

 

 

 

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.GC MS
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 the atomic
composition ao 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.Mass spectrometry in thermodynamics:
(a) determination of heat of vapourization

To determine the heat of vapourization of high temperature compounds, data
from the spectrum is collected and a graph is plotted by taking ion intensities on Y-
axis and temperature on X-axis.

(b) Determination of heat of sublimation
To determine the heat of sublimation of a compound, vapours of the sublimed
solids are passed into the ionization chamber of the mass spectrometer. The
spectrum is recorded in which the obtained peak intensities are directly
proportional to the vapour pressure (VP) of the sample in the ionization chamber.
5.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.
6.Detrmination of ion-molecule reactions:
Mass spectrometry finds its use in the study of ion molecule reaction 1.e., the
reactions in between the fragment ion and the unionized molecules. The rate of
these reactions directly depend on the operating pressure.
7.Detction 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.

 

8.ldentification 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.

9.Ildentification 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.

 

ADVANTAGES :
‘Increased sensitivity over most other analytical
techniques because the analyzer, as a mass-charge filter,
reduces background interference
*Excellent specificity from characteristic fragmentation
patterns to identify unknowns or confirm the presence of
suspected compounds, Information about molecular weight.

DISADVANTAGES:
1.Doesn’t directly give structural information

(although owe can often’ ffigure it out).
2.Needs pure compounds .

3.Difficult with non-volatile compounds.

 

CONCLUSIONS AND FUTURE
PROSPECTS:

The inherent analytical advantages of mass
spectrometry, including sensitivity and _ speed,
combined with recent advances promise to make MS
a mainstay of drug design and discovery. The
development of ionization techniques such as ESI
and MALDI now allows almost any compound to be
studied by MS. In addition, MS/MS and MS’ add the
capability for structural analysis of compounds that
are present at low levels and/or are present in
complex mixtures.

 

REFERENCES:
l.e, M. S. & Kerns, E. H. LC/MS applications in drug development. Mass Spectrom. Rev. 18,
187-279 (1999).
2.Cheng, X. & Hochlowski, J. Current application of mass spectrometry to combinatorial
chemistry. Anal. Chem. 74, 2679-2690 (2002).
3.Triolo, A., Altamura, M., Cardinali, F., Sisto, A. & Maggi, C. A. Mass spectrometry and
combinatorial chemistry: a short outline. J. Mass Spectrom. 36, 1249-1259 (2001).
4.Shin, Y. G. & Breemen, R. B. vy. Analysis and screening of combinatorial libraries using
mass spectrometry. Biopharm. Drug Dispos. 22, 353-372 (2001).

5.Kassel, D. B. Combinatorial chemistry and mass spectrometry in the 21%‘ century drug
discovery laboratory. Chem. Rev. 101, 255—267 (2001).
6. Yamashita, M. & Fenn, J. B. Electrospray ion source. another variation of the free-jet
theme. J. Phys. Chem. 88, 4451-4459 (1984).
The initial publication in the development of electrospray as a useful ionization technique
for mass spectrometry.
7.Karas, M., Bachmann, D., Bahr, U. & Hillenkamp, F. Matrix-assisted ultraviolet laser
desorption of non-volatile compounds. Int. J. Mass Spectrom. Ion Processes 78, 53—68
(1987).
8.Seminal publication describing the use of a matrix to enhance the ionization efficiency of
non-volatile compounds — this method of laser/desorption ionization is the most commonly
used today.
9.Horning, E. C., Horning, M. G., Carroll, D. I., Dzidic, I. & Stillwell, R. N. New Picogram
detection system based on a mass spectrometer with an external ionization source at
atmospheric pressure. Anal. Chem. 45, 936—943 (1973