Ionisation Technique of MS

1. Ionisation Method for MS
Presented by Swati Sagar
Roll No.-10831
Division Of Microbiology
IARI-New Delhi
MB 609: RECENT ADVANCES IN MICROBIAL
BIOTECHNOLOGY

2. • Mass spectrometry(MS) is an analytical technique that produces spectra of masses of
molecules constituting a sample.
• MS works by ionizing the chemical compound to generate charged molecules and
measuring their mass-to-charge ratio.
• It identify the molecules based on their molecular weight of fragments formed from the
large biomolecules measured by using electric and magnetic fields.
• It is one of the most sensitive techniques that can be used for identification of
compounds.
Mass Spectrometry
Essential features of mass spectroscopy:-
 Production of ions in gas phase. The sample molecules are break into
charged fragments.
 Acceleration of ion to a specific velocity in an electric field.
 Separation of the ions in mass analyser
 Detection of the fragments on particular m/z ratio

3. Components of MS
Sample Inlet
Many techniques used to ionize the large
molecules into gas phase ions are known
as Ionisation Techniques.
Ion Source
Mass
analyser
Detector
Different types of Ionisation Techniques are:-
1) Electron Impact ionization (EI)
2) Fast Atom Bombardment (FAB)
3) Electrospray ionization (ESI)
4) Chemical Ionization (CI)
5) Matrix Assisted Laser Desorption Ionization
(MALDI)

4. Ionisation techniques
Hard Ionisation
Technique
Soft Ionisation
Technique
EI
CI
FAB
MALDI
ESI
 Large molecules are extensively
fragmented then analysed.
 Analytes are in gas phase.
 Large data is produced.
 Large Molecules are not
extensively fragmented.
 Less data is produced

5. • It is a common method of ionization mostly used for GC-MS.
• Sample is volatilized into gaseous form in a vacuum system which is directly
attached to the analyser.
• The gas phase molecules are bombarded by a beam of electrons formed by
heating a filament.
• The electron beam ejects an ion from the gas phase molecule producing a
radical ion. Hard ionization technique
• Suitable for volatile organic compounds –hydrocarbons, oils, flavours, fragrances.
Electron Impact ionization (EI)

6. • The heated metal filament
produce the stream of
electrons with 70ev
potential energy.
– Ionization only requires
~15 eV
– Bond cleavage requires
~3 – 10 eV
• Produces abundant
fragment ions
Continued..
EI was first described in 1918 by Canadian-American Physicist Sir Arthur
J. Dempster in the article of "A new method of positive ray analysis."

7. Continued..
Advantages Disadvantages
Simple Molecule must be volatile
Sensitive Molecule must be thermally stable
Fragmentation aids Identification of
molecules
Extensive fragmentation- difficult to
interpret data
Library-searchable fingerprint spectra Mass range is low (<1000 Da)
Mechanism

8. • It was the first technique that allowed ionization of non-volatile compounds. Soft
ionisation technique and was popular in the 80's to early 90's.
• This method is known as Liquid Secondary Ion Mass Spectrometry (LSIMS)
• It is done by bombarding a sample in a vacuum with a beam of atoms, typically Ar or Xe,
accelerated to Kilovolt energies.
• The sample was typically mixed in a matrix. The two most common matrixes were glycerol
and 3 Nitro-benzoic acid.
• The ions formed by FAB were adducts to the molecule, where adduct could be protons,
sodium ions, potassium ions or ammonium ions.
• A variation of FAB was replacement of the atom beam with a beam of ions. Typically,
cesium ions is used and is known as Secondary Ion Mass Spectrometry (SIMS). SIMS
spectra were typically identical to FAB spectra.
Fast Atom Bombardment (FAB)

9. Continued..
It was developed by Michael Barber at the University of Manchester
in 1980.

10. Advantages:
• Analyse the mass range upto 7000Da
• Simple and sensitive
• Suitable for polar compounds
Disadvantages:
• Low sensitivity than MALDI
• Little fragmentation
• Not good for non-polar compounds
Continued..

11. • ESI is a most popular ionization technique.
• It is a soft ionisation technique.
• The electrospray is created by putting a high voltage on a flow of liquid at
atmospheric pressure. The charged liquid droplets are produced by atomisation
or nebulization.
• The created spray is directed in the vacuum system of the mass spectrometer,
where these droplets are evaporated by heat in high-vacuum region.
• So, the ions are ejected from the droplets and accelerated into the mass
analyser by voltages. For larger molecules, the ions may contain multiple
charges, allowing the detection of very large molecules on analysers that have
limited mass to charge (m/Z)) ratio ranges.
• It is mostly used with liquid chromatography (LC).
Electrospray ionization (ESI)

12. The ESI technique was developed by John Bennett Fenn in 1984 for the
analysis of biological macromolecules. Nobel prize in chemistry-2002.
Continued..
Mechanism

13. • There are two major theories that explain the final production of gas-phase ions:
• Ion evaporation model (IEM)-The IEM suggests that as the droplet of certain
size has a field strength at its surface and it is large enough to assist the field
desorption of solvated ions
• Charge residue model (CRM)-The CRM suggests that electrospray droplets
undergo evaporation and fission cycles, eventually leading progeny droplets that
contain on average one analyte ion.
Continued..

14. Advantage:-
• Can analyse large biomolecule upto 150-200 KDa
• This technique is more sensitive and can accurately measure the
sample both in term of quality and quantity.
• Suitable for polar compounds.
Disadvantage-
• Not good for complex mixture
• The apparatus is also very difficult to clean
• The multiple charges that are attached to the molecular ions can make
for confusing spectral data
Continued..

15. • Chemical ionization was developed in the 1960s.
• It is a lower energy process as compared to electron ionization (EI).
• Produce less or sometimes no fragmentation, and usually a
simpler spectrum.
Chemical ionization (CI)

16. • Electrons emitted from the source will ionize the reagent gas. The resultant
collisions with other reagent gas molecules will create an ionization plasma.
• Inside the ion source, the reagent gas is present in large excess as compared to
the analyte
• The collision of the analyte with the ions of a reagent gas occur. Common
reagent gases are: methane, ammonia, and iso-butane.
• Positive and negative ions of the analyte are formed by reactions with this
plasma.
Chemical
Ionization
Negative chemical
ionization (NCI)
Atmospheric pressure
chemical ionization
(APCI)
Continued..

17. Advantages
• Large (M+H)+ ion identifies molecular weight (M)
• Soft ionization technique cause simple fragmentation
• Require low energy
Disadvantages
• Simple fragmentation gives little structural information
• Sample must be thermally volatile and stable
• Ion source can easily contaminated.
Continued..

18. • MALDI is a method of ionization in which the sample is bombarded with a
laser.
• The term matrix-assisted laser desorption ionization (MALDI) was coined
in 1985 by Franz Hillenkamp, Michael Karas and their colleagues.
• In 1987, Koichi Tanaka and his co-workers used ultra fine metal plus liquid
matrix method that contain 30 nm cobalt particles in glycerol with a
337 nm nitrogen laser for ionization. Using this laser and matrix
combination, Tanaka was able to ionize biomolecules as large as the
34,472 Da protein carboxypeptidase-A.
• Tanaka received Nobel Prize in Chemistry in 2002 for demonstrating the
proper combination of laser wavelength and matrix for the ionization of
protein.
Matrix Assisted Laser
Desorption Ionization
(MALDI)

19. • The sample is typically mixed with a matrix that absorbs the laser
radiation and transfer a proton to the sample.
• Some small mass samples can be ionized without matrix, but this
is typically called laser desorption.
• Mostly forms singly charged ions and mostly performed on
specially built time-of-flight instruments.
• The matrix consists of crystallized molecules.
• Most commonly used compounds sinapinic acid, α-cyano-4-
hydroxycinnamic acid (alpha-cyano or alpha-matrix) and 2,5-
dihydroxybenzoic acid(DHB).
Continued..

20. Advantage:
 Mostly used to analyse
biomolecules
 High sensitivity
 Simple structure
 Easy operation & maintenance
Disadvantage:
 Delayed extraction
 Different calibration for different mass
range & experimental condition
(laser, matrix)
Continued..