Mass Ionization Techniques

IONIZATION TECHNIQUES IN MASS
SPECTROMETRY
Advanced instrumental analysis
Presented by
B. Likitha
M. Pharmacy I-II
Pharmaceutical analysis

INTRODUCTION
Mass Spectrometry:
Mass spectrometry is an analytical technique that involves the study in the gas phase
of ionized molecules with the aim of one or more of the following:
Molecular weight determination
Structural characterization
Gas phase reactivity study
Qualitative and quantitative analysis of components in a mixture.

Basic Principle:
• Conversion of the sample into rapidly moving gaseous positive ions by electron
bombardment and charged particles are separated according to their masses with
or without fragmentation, which are then characterized by their mass to charge
ratios (m/e) and relative abundances (mass spectrum) is the basic principle of mass
spectrometry.

Working Principle:
In this technique, molecules are bombarded with a beam of energetic electrons.
The molecules are ionized and broken up into many fragments, some of which are
positive ions.
Each kind of ion has a particular ratio of mass to charge, i.e., m/e ratio.
For most ions, the charge is one and thus, m/e ratio is simply the molecular mass
of the ion.
The ion pass through magnetic and electric fields to reach detector where they are
detected and signals are recorded to give a mass spectra.

INSTRUMENTATION OF MASS SPECTROMETRY
MASS SPECTRUM

CATEGORIES OF ION SOURCES
ION SOURCES
GAS PHASE SOURCES DESORPTION SOURCES
 Electron Impact Ionization (EI)
 Chemical Ionization (CI)
 Field Ionization (FI)
 Field Desorption (FD)
 Electrospray Ionization (ESI)
 Matrix Assisted Laser
Desorption/Ionization (MALDI)
 Plasma Desorption (PD)
 Fast Atom Bombardment (FAB)
 Thermo spray Ionization (TI)

IONIZATION TECHNIQUES
1. Electron Spray Ionization(ESI)
2. Atmospheric Pressure Chemical Ionization(APCI)
3. Atmospheric Pressure Photo Ionization(APPI)
4. Matrix Assisted Laser Desorption Ionization Technique(MALDI)
5. Fast Atom Bombardment(FAB)

1. Electrospray ionization
• The method generates ions from solution of a sample by creating fine spray of charged
droplets.
• A solution of sample is pumped through a fine, charged stainless steel capillary needle at
a rate of few micro litres/minute.
• The needle is maintained at a high electric field (several kilovolts) with respect to
cylindrical electrode.
• The liquid phase pushes itself out of the capillary as a mist or aerosol of fine charged
droplets. Set of aerosol droplets is produced by a process involving formation of a
Taylor cone and a jet from the tip of this cone.
• These charged droplets are then passed through desolvating capillary where the solvent
is evaporated in the vacuum and attachment of charge to the analyte molecules takes
place.

• Desolvating capillary uses warm nitrogen as nebulizing gas. The desolvating capillary is
maintained under high pressure.
• As the droplets evaporate the analyte molecules comes closer together.
• These molecules become unstable as the similarly charged molecules comes closer
together and the droplets explode once again.
• This is referred as Coulombic fission. The process repeats itself until the analyte is free
from solvent and is lone ion.
• The ion then moves to the mass analyzer.
• In this method quassi-molecular ions are produced by addition of a proton (hydrogen
ion) to give (M+H) or other cations such as sodium ion (M+Na)+ or removal of
hydrogen ion (M-H).
• Multiply charged ions are often observed and these ions are even electron species
indicating that electrons have neither been added nor removed.

ELECTRON SPRAY IONIZATION
Advantages:
• Used for analysis of high molecular weight
biomolecules such as Polypeptides,
proteins, oligonucleotides and synthetic
polymers.
• Can be used along with LC and capillary
electrophoresis.
• Softest ionization technique.
Disadvantage:
• Multiply charged ions are confusing and
needs careful interpretation.
• Sensitive to contaminants such as alkali
metals or basic compounds.
• Not suitable for low polarity compounds.

2. Atmospheric Pressure Chemical Ionization
• In this method a sample flows through a heated tube where it is volatilized in a
mist and sprayed into a corona discharge with the aid of nitrogen nebulization.
• Corona discharge is used to ionize the analyte in the atmospheric pressure region.
• APCI is best suited to relatively polar or semi-volatile samples.

3. Atmospheric Pressure Photo Ionization
• In APPI, samples are ionized by UV light.
• Molecules with vapors of nebulizer liquid solution, interact with photo beam of
UV light.
• Analyte molecules absorb a photon and become an electronically excited
molecule.
• If the ionization energy of analyte is lower than the energy of photon, then the
analyte molecule releases energetic electron and becomes the radical cation and
moves forward to the MS analyzer.

4. Matrix Assisted Laser Desorption Ionization Technique
• In this method ionization is carried out by bombarding a laser beam of sample dissolved
in a matrix solution.
• Matrix is used in MALDI to
Absorb the laser energy.
Prevent analyte agglomeration.
Protect analyte from being destroyed by direct laser beam.
• Solution of the matrix is made in a mixture of highly purified water and another organic
compound (acetonitrile or ethanol).
• Matrix solution is then mixed with the analyte to be investigated.
• The organic compound acetonitrile dissolves hydrophobic proteins present in the sample
while water dissolves hydrophilic proteins.

• The solution is then spotted in a air tight chamber on the tip of the sample probe.
• With a vacuum pump the air is removed and vacuum is created which leads to
evaporation of the solvent leaving behind a layer of recrystalized matrix
containing analyte molecules.
• Now the laser beam (EMR) is shooted to the sample, the range of UV radiation
used is 360-390nm due to the absorbing substance is present in matrix , it absorbs
radiation or energy and thus it transfers some of its energy to sample molecule
where by the molecular ions are formed and then accelerate to analyzers.

5. Fast Atom Bombardment
• It is an ionization technique in which the analyte and non-volatile liquid matrix
mixture is bombarded by a high energy beam of inert gas such as Argon or Xenon.
• This technique is used for ionization of polar high molecular weight compounds
such as polypeptides. Commonly used matrices include :-
Glycerol
Monothioglycerol
Carbowax
• These solvents easily dissolve organic compounds and do not evaporate in
vacuum.
• The bombarding beam consists of Xenon or Argon atoms of high translational
energy.
• This beam is produced by first ionizing the Xenon (or Argon atoms with electrons
to give Xenon radical cations.
• The radical cations are accelerated and are passed through the chamber containing
xenon atoms.

• During this passage high energy cation obtain electrons from Xenon atoms to
become high energy atoms (Xe). The lower energy ions are removed by
electrostatic deflector.
• The analyte is dissolved in the liquid matrix such as glycerol and applied as a thin
layer on the sample probe shaft.
• The mixture is bombarded with the high energy beam of Xenon atoms. Xenon
ionizes the glycerol molecules to give glycerol ions. These ions react with the
surrounding glycerol molecules to produce (G+H)+ as reactant ions.
• The sample molecules then undergo proton transfer or hydride transfer or ion-pair
interaction with reactant ions to give quassimolecular or psuedomolecular ions
such as (M+H)+, (M-H)- or (M+G+H)+.
• These ions are then extracted from slit lens system designed to collect ions and
directed to mass analyzer.

FAST ATOM BOMBARDMENT
Advantages:
• Used for ionization of polar
high molecular weight
samples.
• Provides rapid heating of
samples and reduces sample
fragmentation.
• Rapid ionization.
Disadvantages:
• Difficult to distinguish
between low molecular
weight compounds.
• Compounds must be soluble
in liquid matrix.
• Not good for multiply
charged compounds.

REFERENCES
1. A. Skoog, F. James Holler and Timothy A. Nieman. Principles of Instrumental
analysis, 5th Edition, 499-511.
2. Willard Meritt. Instrumental Methods Of Analysis, 7th Edition, 468-474.
3. http://www.chem.ox.ac.uk/spectroscopy/massspec/Lecture/oxmain_lectureCI.html
4. http://www.astbury.leeds.ac.uk (A.E. Ashcroft's MS web pages and tutorial)
5. http://en.wikipedia.org/wiki/Atmospheric_pressure_chemical_ionization

Mass Ionization Techniques

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