Ionization Techniques In Mass Spectrometry

1. IONIZATION
TECHNIQUES IN
MASS
SPECTROMETRY
Name – Sayali Pradip Nagrale
Subject – Modern Analytical Technique II
Branch – pharmaceutics
Roll no. - 7
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2. Table of content
 Introduction
 Ionization Methods
 Gas Phase Ionization
 Desorption Ionization
 Evaporation Ionization
 Conclusion
 Reference
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3. Introduction
Component of mass spectrometer
• Analysis by mass spectrometry
begins with the ionization of a
compound into individual, entire or
fragmented species.
• The choice of the ionization
method depends upon the planned
study and the nature of the subject
compound, which could be a small
organic molecule, a metal, a
biological macromolecule and
upon its state(gas, liquid, solid).
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4. Ionization methods
METHODS
Gas Phase
Ionization
Desorption
Ionization
Evaporative
Ionization
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5. Gas phase ionization
• Gas phase ionization is oldest and most popular method and in this method the
sample is vaporized before ionization
Gas Phase Ionization
Electron
Ionization
Chemical
Ionization
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6. Electron ionization
• It is also known as electron bombardment ionization
• Main function of this ionization technique is to convert the gaseous sample into molecular ion
• Electron impact ionization source consists of a ionizing chamber which is maintained at a
pressure of 0.005 torr and temperature of 200 ± 0.25 degrees.
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7. • It is a hard ionization technique as it produces 70eV of potential , ionization potential of
organic compound is approx.8-15 eV and direct bombardment of 70eV is done in this
case , so it will produce large number of fragment ions from molecules.
Advantages
 Gives molecular mass and also the fragmentation pattern of the sample.
 Extensive fragmentation and consequent large number of peaks gives structural
information.
 Gives reproducible mass spectra.
Disadvantages
 Sample must be thermally stable and volatile.
 Unstable molecular ion fragments are formed so readily that are absent from mass
spectrum
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8. Chemical ionization
 Chemical ionization is a gaseous phase ionization technique , it is very important soft
ionization technique .
 Fragmentation is less as compared to EI , so it will give intense peak of molecular ions.
Some molecule like alcohol , amines , esters , ether are highly fragmented so the peaks will
not be clearly detected in EI and to overcome this drawback we are using chemical
ionization .
 In chemical ionization the ionization of the analyte is achieved by interaction of it’s
molecules with ions of a reagent gas in the chamber or source.
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9. Chemical ionization is carried out in an instrument similar to EI with some modifications such
as:-
• Addition of a vacuum pump.
• Narrowing of exit slit to mass analyzer to maintain reagent gas pressure of about 1 torr in
the ionization chamber.
• Providing a gas inlet
It is a two part process.
Step-I Reagent gas is ionized by Electron Impact ionization in the source. The primary ions
of reagent gas react with additional gas to produce stabilized reagent ions.
step-II Reagent ions interact with sample molecules to form molecular ions. In this technique
the sample is diluted with a large excess of reagent gas. Gasses commonly used as reagent
are low molecular weight compounds such as Methane, tertiary Isobutane, Ammonia, Nitrous
oxide, oxygen and hydrogen etc.
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10. Depending upon the type of ions formed CI is categorized as
• Positive Chemical Ionization.
• Negative Chemical Ionization
Positive Chemical Ionization
In this technique positive ions of the sample are produced. In positive chemical ionization gasses such
as Methane, Ammonia, Isobutane etc. are used
For example Ammonia is used as reagent gas.
First ammonia radical cations are generated by electron impact and this react with neutral ammonia to
form ammonium cation (reactive species of ammonia ).
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11. NH4 + reacts with the sample molecules by proton transfer or Adduct formation to produce
sample ions
Negative Chemical Ionization
Negative chemical ionization is counterpart of Positive chemical ionization. Negative ions of
the sample are formed and oxygen and Hydrogen are used as reagent gasses. This method
is used for ionization of highly electronegative samples
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12. Advantages
 Used for high molecular weight compounds.
 Used for samples which undergo rapid fragmentation in EI.
Limitations
 Not suitable for thermally unstable and non-volatile samples.
 Relative less sensitive than EI ionization.
 Samples must be diluted with large excess of reagent gas to prevent primary
interaction between the electrons and sample molecules
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13. Desorption ionization technique
Liquid or solid sample will be converted into gaseous ions and these gaseous ions will be
analyzed by mass analyzer
Desorption Ionization
Technique
Field
Desorption
FAB MALDI
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14. Field desorption
• It is a type of desorption ionization technique which involves direct conversion of solid /
liquid molecules into gaseous ions .
• In this method a multitipped emitter (made up of tungsten wire with carbon or silicon
whiskers grown on its surface).
• The electrode is mounted on a probe that can be removed from the sample
compartment and coated with the solution of the sample. The sample solution is
deposited on the tip of the emitter whiskers either by dipping the emitter into analyte
solution or by using a microsyringe.
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15. • The probe is then reinserted into the sample compartment which is similar to CI or EI unit.
• Then the sample is ionized by applying a high voltage to the emitter. In some cases it is
necessary to heat the emitter by passing a current through the wire to evaporate the
sample.
Advantages
 Works well for small organic molecules, low molecular weight polymers .
Disadvantages
 Sensitive to alkali metal contamination.
 Sample must be soluble in a solvent.
 Not suitable for thermally unstable and non volatile samples.
 Structural information is not obtained as very little fragmentation occurs.
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16. FAB ( Fast Atom Bombardment )
• This technique of ionization is reserved for polar and non-volatile compounds under
vacuum conditions
• Ionization is achieved through the impact of fast and non-ionized heavy atoms (Ar and Xe)
targeting the sample being previously dispersed in a non-volatile liquid matrix (glycerol or
diethanolamine) and deposited into the source at the end of a sample probe.
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• To produce this fast atom bombardment a argon or xenon gas is first ionized by electrons.
These primary ions are then accelerated and directed towards a collision chamber where
they come into contact with slow-moving neutrals atoms of the same gas. During those
collisions, the charge of the fast moving ion is transferred to the slow-moving neutral species
resulting in a fast neutral atom, which is directed at the sample.
• The sample, on the probe, is ionized when bombarded by the fast atom beam.

18. 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.
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19. MALDI
( matrix assisted laser desorption ionization )
• In this method ionization is carried out by bombarding a laser beam on the 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.
Matrix consists of a crystallized molecules of which the
most commonly used are :-
o 3,5 – dimethoxy – 4 – hydroxy cinnamic acid (sinapinic acid).
o 𝛼– cyano – 4 – cinnamic acid (𝛼– cyano or 𝛼 – matrix).
o 2,5 – dihydroxy benzoic acid (DHB)
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20. • Solution of the matrix is made in a mixture of highly purified water and another organic
compound (acetonitrile or ethanol). Triofluoro acetic acid (TFA) is also added.
• If sinapinic acid is used as a matrix the solution is prepared by adding:
 20 mg/ml of sinapinic acid.
• Water: acetonitrile: TFA (50:50:0.1)
• 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.
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21. • 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 recrystallized 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.
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22. Evaporative ionization method
In this technique solvent molecule are removed by evaporation and ions will be produced
Evaporative
Ionization Technique
ESI APCI APPI
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23. ESI ( electro spray 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
microliters/minute.
• The needle is maintained at a high electric field (several
kilovolts) with respect to cylindrical electrode.
• The liquid pushes itself out of the capillary as a mist or
aerosol of fine charged droplets.
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24. Advantages
 Used for analysis of high molecular weight biomolecules such as polypeptides,
proteins, oligonucleotides and synthetic polymers.
 Can be used along with LC .
Disadvantage
 Multiple charged ions are confusing and needs careful interpretation.
 Sensitive to contaminants such as alkali metals or basic compounds.
 Not suitable for low polarity compounds.
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25. APCI
( atmospheric pressure chemical ionization )
• It is a type of soft ionization technique based on mechanism of of evaporation and carried
out at atmospheric pressure
• It is a combination of CI and ESI with some deviation
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26. • Sample will be injected through the
capillary , then it will be converted
into sprayed droplets by nebulizing
gas
• Corona discharge electrode will
ionize the solvent vapor molecule just
like production of primary ions in
chemical ionization
• Sometime analyte vapor may also get
ionized by electrode .
• Polar thermostable substance with
molecular wt. less than 1500 Daltons
can be detected .
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27. APPI
( atmospheric pressure photo ionization )
• It is similar to APCI , in APCI we are using corona discharge electrodes as a ionization
source in APPI we are using Krypton lamp as a ionization source
• Krypton lamp will generate UV light , due to which photons will be generated and that
photons will ionize the analyte
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28. Conclusion
 As mass spectrometer seperates on basis of mass to charge ratio , so ionization of
sample is a prime step .
 we can use different ionization technique and get clear spectra for study of samples
.
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29. References
• Principles of Instrumental analysis. Fifth Edition by Douglas. A. Skoog, F. James Holler and
Timothy A. Nieman. Pg. No. 499 – 511.
• Handbook of pharmaceutical analysis by lena ohannesian and antony J. streeter . Pg.no . 170
-186
• Chemical analysis modern instrumentation and analytical techniques 2nd edition by francis
rouessac and annick rouessac pg.no. 392-404
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