Mass spectrometry is an analytical technique that produces spectra of the masses of atoms and molecules in a sample. It works by ionizing the sample molecules and separating the resulting ions based on their mass-to-charge ratio. Common components include an ion source, mass analyzer, and detector. Common ionization methods are electron impact, chemical ionization, electrospray ionization, and matrix-assisted laser desorption/ionization. Common mass analyzers are the quadrupole and time-of-flight analyzer, which separate ions based on stability in oscillating electric fields or flight time. Mass spectrometry is used to determine molecular structure and composition.

1. MASS SPECTROMETRY
Arushe tickoo
B.Tech Biotech

2. Introduction
 Mass spectrometry (MS) is an analytical technique that produces
spectra (singular spectrum) of the masses of the atoms or
molecules comprising a sample of material.
 It is a technique in which the molecules in the test sample are
converted to gaseous ions that are subsequently separated in a
mass spectrophotometer according to mass to charge(m/z) ratio

3. History
 Mass spectroscopy was first performed at the cambridge
university, in 1912 by J.J Thomson (1856-1940) when he obtained
the mass spectra of O2, N2, CO
 Mass spectroscopy took off in 1930s and advance technology
resulted in the development of double focusing Mass
spectrometers capable of accurate determination.
 The modern techniques of mass spectrometry were devised by
Arthur Jeffrey Dempster and F.W. Aston in 1918 and 1919
respectively. In 1989, half of the Nobel Prize in Physics was
awarded to Hans Dehmelt and Wolfgang Paul for the
development of the ion trap technique in the 1950s and 1960s. In
2002, the Nobel Prize in Chemistry was awarded to John Bennett
Fenn for the development of electrospray ionization (ESI)

4. spectrophotometer

5. (A)

6. (B)

7. Ionization Source Mass Analyser Detector
 Sample under investigation has to be ionized inside the
ionization chamber of the sample.
 These ions are extracted into analyser where they are separated
according to mass to charge (m/z) ratio.
Introduction

8. Components
 A high vacuum system
 An ion source : MALDI(matrix assisted low desorption ionization),
ESI(electrospray ionization), FD(field desorption), EI(electron impact)
 A mass filter/analyser : TOF(time of flight), quadrupole
 A detector : conversion dynode, electron multiplier
INLET
ION
SOURCE
MASS FILTER DETECTOR
DATA
SYSTEM
High vacuum systemHIGH VACUUM SYSTEM

9. Vacuum system
 All mass analysers operate under vacuum in order to minimize collision
between ions and air molecules. Without a high vacuum , the ions
produced in the source will not reach the detector.
 Two vacuum pumps are used :
1. Rotary vane pump : It was invented by Charles C. Barnes of Sackville,
New Brunswick,it is used to provide initial vacuum.
2. Turbomolecular pump : The turbomolecular pump was invented in
1958 by Becke, it provides high vacuum, it is a high speed gas turbine
with moving blades and stators ( fixed blades ) whose rotation forces
the molecules through the blade system.

10. Sample preparation
 Sample can be introduced directly or through sometype of
chromatographic techniques that is HPLC, GC or capillary
electrophoresis.
 Ionization sources :
1. ESI( electrospray ionization )
2. MALDI( matrix assisted laser desorption ionization )
3. FD( field desorption )
4. EI( electron impact )
Method of sample introduction often depends upon the ionization
methods used

11. Hard and Soft ionization techniques
 Field Ionization / Field Desorption
 Possibly the first ‘soft’ ionization method was ‘Field Ionization’,involves desorption of ions
from a surface in a very high electric field.
 The salient feature of soft ionization is that analytes (even large ones) are transferred intact
into the gas phase.
 This includes:
1. Large Polymers
2. Nucleic Acids and non-covalent complexes
3. Proteins and non-covalent complexes
Examples: ESI, MALDI, Chemical ionization
Soft ionization
1. Leave excess energy in molecule – extensive fragmentation(the systematic
rupturing of bonds acts to remove the excess energy, restoring stability to the
resulting ion), resultant ions tend to have m/z lower than the molecular mass
2. Common example of this ionization is electron impact ionization
Hard ionization

12. ESI (electrospray ionization )
 This technique was developed by John Bennett Fenn.
 Sample is dissolved in volatile solvent and pumped through a narrow and
stainless steel capillary. A high voltage of 3-4 kv is applied,as a result of this
strong electric field produced the sample emerging from tip is dispersed into
aerosols of highly charged droplets.
 Electrospray ionization (ESI) is a technique used in mass spectrometry to produce
charged ions. The liquid containing the analyte(s) of interest is dispersed by
electrospray, into a fine aerosol. Because the ion formation involves extensive
solvent evaporation (also termed desolvation), the typical solvents for
electrospray ionization are prepared by mixing water with volatile organic
compounds (e.g. methanol ,acetonitrile).Compounds that increase the
conductivity (e.g. acetic acid) are added to the solution. These species also act to
provide a source of protons to facilitate the ionization process. The aerosol is
sampled into the first vacuum stage of a mass spectrometer through a capillary
carrying a potential difference of approximately 3000V, which can be heated to
aid further solvent evaporation from the charged droplets.

13. Electrospray ionization

14. MALDI( matrix assisted laser desorption ionization)
 Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization
technique used in mass spectrometry, MALDI is a two-step process. First,
desorption is triggered by a UV laser beam. Matrix material heavily absorbs
UV laser light, leading to the adsorption of upper layer of the matrix
material.
 In desorption ionization the sample to be analysed is dissolved in a matrix
and placed in a path of high energy beam of ions or high energy photons
but in case of MALDI high intensity photons are used.
 In this ionization matrix is used to protect the biomolecule from being
destroyed by direct laser beam.
 Solid matrix strongly absorb the laser radiations, with a strong pulse of laser
light the analytes are analysed.

15. MALDI (matrix assisted laser desorption ionization)

16. The matrix consists of crystallized molecules, of which the three most
commonly used are 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), α-
cyano-4-hydroxycinnamic acid (CHCA, alpha-cyano or alpha-matrix) and 2,5-
dihydroxybenzoic acid (DHB). A solution of one of these molecules is made,
often in a mixture of highly purified water and an organic solvent (normally
acetonitrile (ACN) or ethanol). Trifluoroacetic acid (TFA) may also be added.
Matrix
Ionization mechanism
MALDI produces gas phase protonated ions by the excitation of
sample molecules from the energy of the laser transferred via a UV
light absorbing matrix. The sample is mixed with the matrix and dried
on the target plate where they co-crystallize on drying. Pulses of laser
light of a few nano seconds causes rapid excitation and vapourisation
of the crystalline matrix and ejection of analyte ions into the gas
phase. This generates a plume of analyte ions that are analysed in a
mass analyser.

17. Electron ionisation
Electron ionization (EI, formerly known as electron impact) is an
ionization method in which energetic electrons interact with gas
phase atoms or molecules to produce ions. This technique is
widely used in mass spectrometry, particularly for gases and
volatile organic molecules.
In an EI ion source, electrons are produced through thermionic
emission by heating a wire filament that has electric current
running through it. The electrons are accelerated to 70 eV in the
region between the filament and the entrance to the ion source
block. The accelerated electrons are then concentrated into a
beam by being attracted to the trap electrode. The sample under
investigation which contains the neutral molecules is introduced to
the ion source in a perpendicular direction to the electron beam.
Close passage of highly energetic electrons, referred to as a hard
ionization source, causes large fluctuations in the electric field
around the neutral molecules and induces ionization and
fragmentation.[3] The radical cation products are then directed
towards the mass analyzer by a repeller electrode.

18. (A)
(B)

19. Chemical ionization
 In chemical ionization a reagent gas, typically methane or ammonia is
introduced into the mass spectrometer. Depending on the technique
(positive CI or negative CI) chosen, this reagent gas will interact with
the electrons and analyte and cause a 'soft' ionization of the molecule
of interest.One of the main benefits of using chemical ionization is that
a mass fragment closely corresponding to the molecular weight of the
analyte of interest is produced.
 In positive chemical ionization (PCI) the reagent gas interacts with the
target molecule, In negative chemical ionization (NCI) the reagent gas
decreases the impact of the free electrons on the target analyte.

20. Analysers
 Once the sample has been ionized, the beam of ions is accelerated by an
electric field and then passed into mass analyser, the region of mass
spectrophotometer where the ions are separated according to the mass to
charge ratio
 Just like the ionization methods ,there are several types of mass analysers the
most common type of mass analyser is TOF (time of flight)
 once the ions are created and leave the ion source they pass into the mass
analyser, the function of which is to separate the ions and measure their
masses.
 Types of analysers:
1. Quadrupole analyser
2. TOF(time of flight)

21. Quadrupole
 The quadrupole mass analyzer is one type of mass analyzer used in mass
spectrometry. As the name implies, it consists of four cylindrical rods, set
parallel to each other. In a quadrupole mass spectrometer the quadrupole is
the component of the instrument responsible for filtering sample ions, based
on their mass-to-charge ratio (m/z). Ions are separated in a quadrupole based
on the stability of their trajectories in the oscillating electric fields that are
applied to the rods

22. The quadrupole consists of four parallel metal rods. Each opposing rod pair is
connected together electrically, and a radio frequency (RF) voltage is applied
between one pair of rods and the other.
The ions are accelerated down the analyser towards the detector the varying
electric field is precisely controlled so that during each stage , the ions of one
particular mass to charge ratio pass down the length of analyser.

23. TOF (time of flight)
 The TOF mass analyser measures the ion flight time.
 It is based upon the simple idea that velocity of two ions with kinetic energy
will vary depending upon the mass of ion that is lighter ions will have higher
velocity
 Mass spectrometry can be used to measure the mass of biomolecules well
over 200,000 Da to provide sequence information of unknown peptide and
proteins and to detect the non covalent complexes

24. Detector
1. Electron multipliers and conversion dynodes:
Electron multipliers are used as a detector in frequent combination
with conversion dynode which is a device to increase sensitivity.
Conversion dynode is surface that is held at high potential so that ions
striking the surface produce secondary charged particles. The ion beam
from the mass analyser are focused on the conversion diodes.
Positive or negative ions hit the conversion dynode, causing the emission
of secondary particles. The secondary particles are accelerated into the
dynodes of electron multiplier

25. Applications
1. Mass spectrometry is also used to determine the isotopic composition
of elements within a sample. Differences in mass among isotopes of an
element are very small, and the less abundant isotopes of an element are
typically very rare.
2. Mass spectrometry is an important method for the characterization and
sequencing of proteins. The two primary methods for ionization of whole
proteins are electrospray ionization (ESI) and matrix-assisted laser
desorption/ionization (MALDI).
3. Mass spectrometry (MS), with its low sample requirement and high
sensitivity, has been predominantly used in glycobiology for
characterization and elucidation of glycan structures.

26. Disadvantages
 A few of the disadvantages of the method is that often fails to
distinguish between optical and geometrical isomers and the
positions of substituent in o-, m- and p- positions in an aromatic
ring. Also, its scope is limited in identifying hydrocarbons that
produce similar fragmented ions.