Mass spectrometry is a technique used to identify unknown compounds and determine molecular structure. It works by ionizing sample molecules and measuring their mass-to-charge ratios. The document discusses various components of a mass spectrometer including the inlet system, ionization sources, and mass analyzer. Common ionization methods like electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI), fast atom bombardment (FAB), and electron impact (EI) are described in detail, outlining their principles, advantages, and applications. The mass spectrum provides information about molecular weight and structure of compounds.

1. Mass spectroscopy
Assignment1 1
MASS SPECTROSCOPY
Presented by
Kalyani. A
M.Pharmacy (pharmaceutical analysis)
Krupanidhi College of pharmacy
BANGALORE.

2. Mass spectroscopy
Assignment1 2
MASS SPECTROMETRY
INTRODUCTION:
 Mass spectrometry is a powerful analytical technique used to quantify known materials,
to identify unknown compounds within a sample and to elucidate the structure and
chemical properties of different molecules.
 It is a micro analytical technique requiring only a few nanomoles of the sample to obtain
characteristic information pertaining to the structure and molecular weight of analyte.
 This technique basically studies the effect of ionizing energy on molecules.
 Now a day’s mass spectrometry is used in many areas including pharmaceutical, clinical,
geological, biotechnology and environmental.
 It depends upon chemical reactions in the gas phase in which sample molecules are
consumed during the formation of ionic and neutral species.
 This will be happened by converting the material to charged molecules to measure their
mass to charge ratio.
 Mass spectrometry has both qualitative and quantitative uses.
 The mass spectrum of each compound is unique and can be used as a “chemical
fingerprint” to characterize the sample.
MASS SPECTROMETER:
It is an instrument in which the substances in gaseous or vapor state is bombarded with a beam of
electrons, to form positively charged ions (cations) which are further sorted according to their
Mass to charge ratio to record their masses and relative abundances.
MASS SPECTRUM:
 It is a sorted collection of the masses of all the charged molecular fragments produced,
the relative abundance of each is the characteristics of every compound.
 The mass spectrum can give detail information about composition of an organic
compound and the position of functional groups and is also used for the determination of
molecular weight.
PRINCIPLE:
 In mass spectrometry, organic molecules are bombarded with a beam of energetic
electrons (70 eV) in gaseous state under pressure between 10-7 to 10-5 mm of Hg, using
tungsten or rhenium filament. Molecules are broken up into cations and many other
fragments.

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 These cations (molecular or parent ion) are formed due to loss of an electron usually from
n or π orbital from a molecule, which can further break up into smaller ions (fragment
ions or daughter ions).
 All these ions are accelerated by an electric field, sorted out according to their mass to
charge ratio by deflection in variable magnetic field and recorded. The output is known
as mass spectrum.
 Each line upon the mass spectrum indicates the presence of atoms or molecules of a
particular mass.
 The most intense peak in the spectrum is taken as the base peak. Its intensity is taken as
100 and other peaks are compared with it.
BASIC PRINCIPLE OF MASS SPECTROMETRY
10Mass spectra are used in two general ways:
1) To prove the identity of two compounds.
2) To establish the structure of a new a compound.
The mass spectrum of a compound helps to establish the structure of a new compound in
several different ways:
1) It can give the exact molecular mass.
2) It can give a molecular formula or it can reveal the presence of certain structural units in a
molecule.
MASS SPECTROMETER:
A mass spectrometer is an instrument which:
 Generates a beam of positively charged ions from the sample under investigation.
 Produce ions from the sample in the ionization source.
 Separate these ions according to their mass-to-charge ratio in the mass analyzer.
 Eventually, fragment the selected ions and analyze the fragments in a second analyzer.
 Detect the ions emerging from the last analyzer and measure their abundance with the
detector that converts the ions into electrical signals.
 Process the signals from the detector that are transmitted to the computer and control the
instrument using feedback.

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COMPONENTS OF A MASS SPECTROMETER:
The essential components of a mass spectrometer consist of:
 A sample inlet
 An ionization source
 A mass analyzer
 An ion detector
 Vacuum system
FIGURE REPRESENTING INSTRUMENTATION OF A MASS SPECTROMETER
INLET SYSTEM:
 The selection of a sample inlet depends upon the sample and the sample matrix.
 Most ionization techniques are designed for gas phase molecules so the inlet must
transfer the analyte into the source as a gas phase molecule.
 If the analyte is sufficiently volatile and thermally stable, a variety of inlets are available.
 Gases and samples with high vapor pressure are leaked directly into the source region by
the help of mercury manometer.
 Liquids and solids are usually heated to increase the vapor pressure for analysis.
 Liquid samples are handled by hypodermic needles injection through a silicon rubber
dam.
 If the analyte is thermally labile (it decomposes at high temperatures) or if it does not
have a sufficient vapor pressure, the sample must be directly ionized from the condensed
phase.
SAMPLE INTRODUCTION METHODS:
1. Direct Vapor Inlet:
 The simplest sample introduction method.

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 The gas phase analyte is introduced directly into the source region of the mass
spectrometer through a needle valve. Pump out lines are usually included to remove air
from the sample.
 This inlet works well for gases, liquids, or solids with a high vapor pressure.
 It only works for some samples.
2. Gas Chromatography:
 Most common technique for introducing samples into a mass spectrometer.
 Complex mixtures are routinely separated by gas chromatography and mass
spectrometry is used to identify and quantitate the individual components.
 The most significant characteristics of the inlets are the amount of GC carrier gas that
enters the mass spectrometer and the amount of analyte that enters the mass
spectrometer.
 Ideally all the analyte and none of the GC carrier gas would enter the source region.
 The most common GC/MS interface now uses a capillary GC column
3. Liquid Chromatography:
 LC inlets are used to introduce thermally labile compounds not easily separated by gas
chromatography.
 These inlets are used for temperature sensitive compounds.
 The sample is ionized directly from the condensed phase.
4. Direct Insertion Probe:
 The Direct Insertion Probe (DIP) is widely used to introduce low vapor pressure liquids
and solids into the mass spectrometer.
 This is important for analyzing temperature sensitive compounds.
 Although the direct insertion probe is more cumbersome than the direct vapor inlet, it is
useful for a wider range of samples.
5. Direct Ionization of Sample:
 Some compounds either decompose when heated or have no significant vapor pressure
and can be introduced by direct ionization from the condensed phase.
 These are used for LC-MS, glow discharge MS, FAB and laser ablation.
IONIZATION METHODS
Ionization method refers to the mechanism of ionization while the ionization source is the
mechanical device that allows ionization to occur.
The different ionization methods are as follows:
1. Protonation
 Protonation is a method of ionization by which a proton is added to a molecule,
producing a net charge of 1+ for every proton added.
 E.g.: More basic residues of the molecule, such as amines; Peptides.
 Can be achieved through MALDI, ESI and APCI.
2. De-protonation
 De-protonation is an ionization method by which the net charge of 1- is achieved
through the removal of a proton from a molecule.
 E.g.: Acidic species including phenols; carboxylic and sulfonic acids.
 Commonly achieved via MALDI, ESI and APCI.

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3. Cationization
 It produces a charged complex by non-covalently adding a positively charged cation
adduct (e.g. alkali, ammonium) to a neutral molecule.
 E.g.: Carbohydrates are best examples, with Na+ as a common cation adduct.
 Mainly achieved by MALDI, ESI and APCI.
4. Transfer of a Charged Molecule to the Gas Phase
 The transfer of compounds already charged in solution is achieved through desorption or
ejection of the charged species from the condensed phase into the gaseous phase.
 Commonly achieved through MALDI or ESI.
5. Electron Ejection
 Ionization is achieved through the ejection of an electron to produce a 1+ net charge,
often forming radical cations. It generates significantly fragmented ions.
 E.g.: Non-polar compounds with low molecular weights.
 Most commonly achieved with electron ionization (EI) sources.
6. Electron Capture
 With the electron capture ionization method, a net charge of 1- is achieved with the
absorption or capture of and electron.
 E.g.: Molecules with high electron affinity, such as halogenated compounds.
ION SOURCE
Ionization of the organic compound is the primary step in obtaining the mass spectrum. The
minimum energy required to ionize the sample or organic molecule is called as its ionization
potential.
 The ion source is the part of the mass spectrometer that ionizes the material under
analysis (the analyte).
 The ions are then transported by magnetic or electric fields to the mass analyzer.
 Molecular ions are formed when energy of the electron beam reaches to 10-15 eV.
 Fragmentation of the ion reaches only at higher bombardment energies at 70 eV.
Function
1. Produces ion without mass discrimination of the sample.
2. Accelerates ions into the mass analyzer.
Classification of Ion sources
1.Desorption Sources a. Electro spray Ionization (ESI).
b. Matrix assisted laser desorption Ionization (MALDI).
c. Fast Atom Bombardment (FAB).
d. Field Desorption (FD).
e. Plasma desorption (PD).
2. Atmospheric pressure
ionization.
a. Atmospheric pressure chemical ionization (APCI)
b. Atmospheric pressure photo ionization (APPI)
3. Gas Phase Sources. a. Electron Impact Ionization (EI).
b. Chemical Ionization (CI).
c. Field Ionizations (FI).

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ESI (Electrospray Ionization)
 It is a soft ionization technique.
 It is typically used to determine the molecular weights of proteins, peptides and other
biological macromolecules.
 It provides a sensitive, robust and reliable tool for studying.
Advantages
 Has ability to handle samples with large masses.
 One of the softest ionization methods available and has the ability to analyze biological
samples with non-covalent interactions.
 Good sensitivity and therefore, useful in accurate quantitative and qualitative
measurements.
Disadvantages
 Cannot analyze mixtures very well & when forced to do so, results are unreliable.
 Apparatus is very difficult to clean and has a tendency to become overly contaminated
with residues from previous experiments.
 The multiple charges that are attached to the molecular ions can make for confusing
spectral data.
 Prior separation by chromatography is required.
ESI - Principle
 ESI uses electrical energy to assist the transfer of ions from solution into the gaseous
phase.
 ESI works on the principle of Soft ionization.
 Soft ionization is a useful technique when considering biological molecules of large
molecular mass, because in this process macromolecule is ionized into small droplets,
which are then de-solvated into even smaller droplets, which creates molecules with
attached protons.
Applications
a. Protein identification and characterization.
b. Studying non covalent interaction.

8. Mass spectroscopy
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c. Probing molecular dynamics
d. Monitoring chemical reactions and studying reactive intermediates.
e. Chemical imaging.
f. Identification and quantification of hemoglobin variants.
g. Screening for inborn errors of metabolism.
MALDI (Matrix AssistedLaser DesorptionIonization)
 MALDI is also based on “soft ionization” methods where ion formation does not lead to
a significant loss of sample integrity.
 Consequently, the high throughput and speed associated with complete automation has
made MALDI-TOF mass spectrometer an obvious choice for proteomics work on large-
scale.
Advantages
a. Gentle Ionization technique
b. High molecular weight analyte can be ionized
c. Molecule need not be volatile
d. Wide array of matrices
e. Produces singly charged ions thus interpretation becomes easy.
f. Prior separation by chromatography is not required.
Disadvantages
a. MALDI matrix cluster ions obscure low m/z species (<600)
b. Analyte must have very low vapor pressure
c. Pulsed nature of source limits compatibility with many mass analyzers
d. Coupling MALDI with chromatography can be difficult
e. Analytes that absorb the laser can be problematic.
MALDI – Principle
The sample for analysis by MALDI MS is prepared by mixing or coating with solution of an
energy-absorbent, organic compound called matrix. When the matrix crystallizes on drying, the
sample entrapped within the matrix also co-crystallizes. The sample within the matrix is ionized
in an automated mode with a laser beam. Desorption and ionization with the laser beam
generates singly protonated ions from analytes in the sample.
Matrix and Sample Preparation
 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- hydroxy
cinnamic acid (CHCA) 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 such as acetonitrile (ACN) or ethanol.
 A counter ion source such as Trifluoroacetic acid (TFA) is usually added to generate the
[M+H] ions.
 A good example of a matrix solution would be 20 mg/ml sinapinic acid in ACN: water:
TFA (50:50:0.1).
The matrix performs two important functions:
1. It absorbs photon energy from the laser beam and transfers it into excitation energy of the
solid system,

9. Mass spectroscopy
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2. It serves as a solvent for the analyte, so that the intermolecular forces are reduced and
aggregation of the analyte molecules is held to a minimum.
Some desirable characteristics of a typical MALDI matrix are:
a. A strong light absorption property at the wavelength of the laser flux.
b. The ability to form micro-crystals with the sample.
c. A low sublimation temperature, which facilitates the formation of an instantaneous high-
pressure plume of matrix-sample material during the laser pulse duration.
d. The participation in some kind of a photochemical reaction so that the sample molecules can
be ionized with high yields
FAB (Fast Atom Bombardment)
 FAB is an ionization technique used in mass spectrometry in which a beam of high
energy atoms strikes a surface to create ions.
 It was developed by Michael Barber at the University of Manchester.
 When a beam of high energy ions is used instead of atoms (as in secondary ion mass
spectrometry), the method is known as liquid secondary ion mass spectrometry (LSIMS).
1. Advantages
a. FAB is extensively used for the ionization of high molecular weight (>5000 D) samples of
biological origin.
b. Extensively used for obtaining mass spectra of salts depending upon the nature of its cation
and anion.
c. The FAB spectra usually provide relatively abundant molecular or quasi molecular ions and
also show some structurally important fragment ions.
2. Disadvantages
a. The matrix also forms ions on bombardment, in addition to those formed by the sample which
complicates the spectrum.
b. FAB samples the surface rather than the bulk concentration of the solute present and hence
limits quantitative measurement.
Principle
 The analyte is dissolved in a viscous liquid, typically glycerol (matrix material) and
ionization is achieved by bombardment of the sample matrix (a metal plate coated with
viscous solution of the sample) by a beam of fast moving neutral atoms.
 The bombarding atoms are usually rare gases, either xenon or argon.

10. Mass spectroscopy
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 Common matrices include glycerol, thioglycerol, 3-nitrobenzyl alcohol (3- NBA),
triethanolamine etc.
 This technique is similar to secondary ion mass spectrometry and plasma desorption mass
spectrometry.
 In order to achieve a very high kinetic energy, the atoms of the gas are first ionized and
these ions are then passed through an electric field.
 After acceleration, the fast moving ions enter into a chamber containing further gas
atoms and collision of ions and atoms leads to charge exchange.
Xe•+ (fast) + Xe (thermal) Xe (fast) + Xe+• (thermal)
 The fast atoms formed in this process retain the original kinetic energy of the fast ions
and proceed towards the analyzer.
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Matrices
 One of the crucial characteristics of FAB is using liquid matrix.
 Due to the high vacuum condition, usual solvent for chemistry laboratory such as water
and other common organic solvent is precluded for FAB.
 Solvent with high boiling point called matrix is necessary to be employed.
Examples of matrix
Glycerol
Thioglycerol
3-Nitrobenzyl alcohol (3-NOBA)
N-Octyl-3-nitrophenylether (NOP)
Triethanolamine
Diethanolamine
Applications
a. Elucidation of the amino acid sequence of the oligopeptide efrapeptin D. This is a potent
inhibitor of mitochondrial ATPase activity.
b. The separation and MS analysis of peptides arising from protein enzymatic digestion.

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Fielddesorption
Introduction
In field desorption method, a multi-tipped emitter (made up of tungsten wire with carbon or
silicon whiskers grown on its surface) similar to that used in FI is used.
Advantages
○ Works well for small organic molecules, low molecular weight polymers and petrochemical
fractions.
Disadvantages
Sensitive to alkali metal contamination.
Sample must be soluble in a solvent.
Not suitable for thermally unstable and nonvolatile samples.
Structural information is not obtained as very little fragmentation occurs.
Construction & Working
 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 using a micro-syringe.
 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.
 NOTE: In some cases it is necessary to heat the emitter by passing a current through the
wire to evaporate the sample.
Ionization takes place by quantum mechanical tunneling mechanism, which involves transfer of
ions from the sample molecule to the anode (emitter). This results in formation of positive ions
which are radical ions (M+) and cations attached species such as (M+Na)+. (M+Na)+ are
produced during desorption by attachment of trace alkali metal ions present in analyte.

12. Mass spectroscopy
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Plasma Desorption
• Plasma desorption produces molecular ions from the samples coated on a thin foil when a
highly energetic fission fragments from the Californium-252 “blast through” from the opposite
side of the foil.
• The fission of Californium-252 nucleus is highly exothermic and the energy released is carried
away by a wide range of fission fragments which are heavy atomic ion pairs.
• Ion pair fission fragments depart in opposite directions.
• Each fission of this radioactive nucleus gives rise to two fragments traveling in opposite
directions (because necessity of momentum conversation).
• A typical pair of fission fragments is 142Ba18+ and 106TC22+, with kinetic energies roughly
79 and 104 MeV respectively.
• When such a high energy fission fragments passes through the sample foil, extremely rapid
localized heating occurs, producing a temperature in the range of 10000K.
• Consequently, the molecules in this plasma zone are desorbed, with the production of both
positive and negative ions.
• These ions are then accelerated out of the source in to the analyzer system.
Atmospheric Pressure Chemical Ionization
(APCI)
 APCI is an ionization method used in mass spectrometry (commonly LC-MS) which
utilizes gas-phase ion-molecule reactions at atmospheric pressure.
 It is an ionization method that is similar to chemical ionization (commonly used in GC-
MS) where corona discharges on a solvent spray produce primary ions.
 APCI is mainly used with polar and relatively non-polar compounds with a molecular
weight of less than 1500 Da, generally giving mono-charged ions.
Advantages
a. Multiple charging is typically not observed as the ionization process is more energetic than
ESI.
b. Electron transfer or proton loss, ([M-H]-) occurs in the negative mode.
c. Proton transfer (for protonation MH+ reactions) occurs in the positive mode
d. At atmospheric pressure analyte molecules collide with the reagent ions frequently and hence
ionization is very efficient.

13. Mass spectroscopy
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Limitations
a. Very sensitive to contaminants such as alkali metals or basic compounds.
b. Relatively low ion currents.
c. Relatively complex hardware compared to other ion sources.
PRINCIPLE
 In APCI, the sample is typically dissolved in a solvent and pumped through a capillary
inside an uncharged quartz tube. At the end of the capillary, but still within the tube, the
sample is converted into an aerosol and then vaporized with the help of nitrogen gas and
by heating to very high temperature (~350- 550 °C).
 The gaseous solvent (S) and sample (M) are then ionized by a corona discharge, in
which a highly charged electrode creates an electric field strong enough to ionize nearby
molecules.
 A potential of several kilovolts applied to the electrode typically remove an electron
from a neutral molecule, without depositing enough internal energy to cause
fragmentation.
 The corona discharge may directly ionize an analyte molecule to form a radical cation
(M+●): M + e- → M+● + 2e-
S + e- → S+● + 2e-
 Frequent collisions between the ions and molecules can transfer charge from an ion to
another neutral. Collision of an ionized solvent ion with an analyte molecule can create a
direct charge transfer to form a radical cation analyte ion:
S+● + M → M+● + S
 Alternatively, collision of solvent ions with a neutral analyte molecule may result in
abstraction of a hydrogen atom from the molecule. The resulting ionized solvent can then
ionize the analyte via proton transfer:
S+● + S → [S+H]+ + S [-H]
[S+H]+ + M → [M+H]+ + S
 The resulting analyte ions (M+● or [M+H]+) are then injected into the mass spectrometer
for detection.
Applications
a. APCI is suitable for the analysis of organic compounds with medium – high polarity.
b. Since positive ionization is dependent on protonation, molecules containing basic functional
groups such as amino, amide esters, aldehyde/ketone and hydroxyl can be analyzed.
c. Negative ionization depends upon deprotonation, molecules containing acidic functional
groups are analyzed by this method.
d. Can be used as LC/MS interface.
e. In the analysis of pesticides.
f. Analysis of triazines, phenylureas, carbamates and organophosphorous compounds.
g. In the determination of Vit. D3 in poultry feed supplements.

14. Mass spectroscopy
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APPI (Atmospheric Pressure Photoionization)
 It has recently become an important ionization source because it generates ions directly
from solution with relatively low background and is capable of analyzing relatively
nonpolar compounds.
 Similar to APCI, the liquid effluent of APPI is introduced directly into the ionization
source.
 The primary difference between APCI and APPI is that the APPI vaporized sample
passes through ultra-violet light (a typical krypton light source emits at 10.0 eV and 10.6
eV).
 Often, APPI is much more sensitive than ESI or APCI and has been shown to have higher
signal-to-noise ratios because of lower background ionization.
 Lower background signal is largely due to high ionization potential of standard solvents
such as methanol and water (IP 10.85 and 12.62 eV, respectively) which are not ionized
by the krypton lamp.
Disadvantages
a. It can generate background ions from solvents.
b. It requires vaporization temperatures ranging from 350-500° C, which can cause thermal
degradation
Principle
 In APPI technique samples are ionized by using UV light.
 Molecules interact with photon beam of UV light with vapors of nebulizer liquid
solution.
 Analyte molecules (A) absorb a photon (hν) and become an electronically excited
molecule.
 If the ionization energy (IE) of analyte molecules is lower than the energy of photon,
then the analyte molecule releases energetic electron and become the radical cation.
Applications
a. It has the capability to ionize compounds with a wide range of polarities while being
remarkably tolerant of matrix components of HPLC additives.

15. Mass spectroscopy
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b. APPI has been proved to be a valuable tool for analytes which are poorly ionized or not
ionized by ESI and APCI. In particular
c. APPI was shown to be able to detect steroid hormones down to several ng/L and had been
proven to have much higher sensitivity than ESI.
d. Results indicate that APPI using toluene as dopant provides exceptional ionization capabilities
for a broad range of compounds, in particular for hormones and sterols compared to APCI and
HESI.
ELECTRON IMPACT IONIZATION:
 It is the most widely used and highly developed method. It is also known as Electron
bombardment or Electron Ionization.
 Electron impact ionization source consists of an ionizing chamber which is maintained at
a pressure of 0.005 torr and temperature of 200 ± 0.25 degrees.
 Electron gun is located perpendicular to chamber.
 Electrons are emitted from a glowing filament (tungsten or rhenium) by thermionic
emission and accelerated by a potential of 70 V applied between the filament and anode.
 These electrons are drawn in the ionization chamber through positively charged slits.
 The number of electrons is controlled by filament temperature and energy.
 The sample is brought to a temperature high enough to produce molecular vapors.
 The gaseous Neutral molecules then pass through the molecular leaks and enter the
ionization chamber.
 The gaseous sample and the electrons collide at right angles in the chamber and ions are
formed by exchange of energy during these collisions between electron beam and sample
molecules.

16. Mass spectroscopy
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Where,
M = Analyte molecule
e- = Electrons
M. + = Molecular ions
 The positive ions formed in the chamber are drawn out by a small potential difference
(usually 5eV) between the large repeller plate (positively charged) and first accelerating
plate (negatively charged).
 Strong electrostatic field (400 – 4000 V) applied between the first and second
accelerating plates accelerates the ions according to their masses (m1, m2, m3 etc.) to
their final velocities.
 The ions emerge from the final accelerating slit as a collimated ribbon of ions. The
energy and velocity of ions are given by :-
zV = ½ (m1v1) = ½ (m2v2) = ½ (m3v3)
Where,
z = charge of the ion
V = accelerating potential
v = velocity of ion
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.
can be used as GC/MS interface.
Disadvantages
Sample must be thermally stable and volatile.
a small amount of sample is ionized (1 in 1000 molecules).
Unstable molecular ion fragments are formed so readily that are absent from mass spectrum.
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Chemical Ionization
 In chemical ionization the ionization of the analyte is achieved by interaction of its
molecules with ions of a reagent gas in the chamber or source.

17. Mass spectroscopy
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 Chemical ionization is carried out in an instrument similar to electron impact ion source
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. Gases commonly used as reagent are low
molecular weight compounds such as Methane, tertiary Isobutane, Ammonia, Nitrous oxide,
oxygen and hydrogen etc.
 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 CI). NH3 → NH3 + + 2 e-
NH3. + → NH4 + + NH2
NH4 + reacts with the sample molecules by proton transfer or Adduct formation to produce
sample ions
M + NH4+ → [M + H] + + NH3 (Proton transfer)
M + NH4+ → [M + NH4]+ (Adduct formation)
When Methane is used as Reagent gas. Methane is ionized by electron impact:
CH4 → CH4+ + 2e-
Primary ions react with additional reagent gas molecules to produce stabilized reagent ions:
CH4+ + CH4 → CH5+ + CH3
CH3+ + CH4 → C2H5+ + H2
The reagent ions then react with the sample molecules to ionize the sample molecules:
CH5+ + MH → CH4 + MH2+ (Proton transfer)
CH3+ + MH → CH4 + M+ (hydride abstraction)
CH4+ + MH → CH4 + MH+ (Charge transfer)
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 gases.
This method is used for ionization of highly electronegative samples. The negative ions are
formed by following reactions:-
Resonance electron capture M + e- → M-
Dissociative electron capture RCl + e- → R + Cl-
H2O + e- → H + OH-

18. Mass spectroscopy
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The ion molecule reaction occurring between negative ion formed in the chamber source and
the sample molecule include:-
Charge transfer.
Hydride transfer.
Anion- Molecule adduct formation.
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 then EI ionization.
Samples must be diluted with large excess of reagent gas to prevent primary interaction
between the electrons and sample molecules.
FIELD IONIZATION
FI is used to produce ions from volatile compounds that do not give molecular ions by EI. It
produces molecular ions with little or no fragmentation.
Application of very strong electric field induces emission of electrons.
Sample molecules in vapor phase is brought between two closely spaced electrodes in the
presence of high electric field (107 - 108 V/cm) it experiences electrostatic force.
If the metal surface (anode) has proper geometry (a sharp tip, cluster of tips or a thin wire) and
is under vacuum (10-6 torr) this force is sufficient to remove electrons from the sample molecule
without imparting much excess energy.
The electric field is produced by applying high voltage (20 KV) to these specially formed
emitters (made up of thin tungsten wire).
In order to achieve high potential gradients necessary to effect ionization, the anode is
activated by growing carbon micro-needles or whiskers.
As concentration of sample molecules is high at the anode ion-molecule reactions often occur
which results in formation of protonated species (M+H)+. Thus both M+ and (M+H) + is
observed in FI spectrum.
Advantages

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As fragmentation is less, abundance of molecular ions (M+) is enhanced, hence this method is
useful for relative molecular mass and empirical formula determination.
Disadvantages
Not suitable for thermally unstable and nonvolatile samples.
Sensitivity is less than EI ion source.
No structural information is produced as very little fragmentation occurs
54
CLASSIFICATION OF ION SOURCESCLASSIFICATION OF ION SOURCES
20MASS ANALYZERS
With the advent of ionization sources that can vaporize and ionize molecules, it has
become necessary to improve mass analyzer performance with respect to speed,
accuracy, and resolution.
More specifically, quadrupoles, quadrupole ion traps, time-of-flight (TOF), time-of-
flight reflectron, and ion cyclotron resonance (ICR) mass analyzers have undergone
numerous modifications/improvements over the past decade in order to be interfaced
with MALDI and ESI.
TYPESOF ANALYZERS:
Analyzers are typically described as either continuous or pulsed.
Continuous analyzers: These analyzers are similar to a filter or monochromator used
for optical spectroscopy. They transmit a single selected m/z to the detector and the mass
spectrum is obtained by scanning the analyzer so that different mass to charge ratio ions
are detected. They include:
 Quadrupole filters and
 Magnetic sectors.
Pulsed mass analyzers: These are the other major class of mass analyzer.
These are less common but they have some distinct advantages. These instruments
collect an entire mass spectrum from a single pulse of ions. This results in a signal to
noise advantage similar to Fourier transform or multichannel spectroscopic techniques.
Pulsed analyzers include:
 Time-of-flight,
 Ion cyclotron resonance,
 Quadrupole ion trap mass spectrometers.
QUADRUPOLE MASS ANALYZER:
 Quadrupole mass analyzer is one type of mass analyzer used in mass spectrometry.

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 A typical quadrupole mass analyzer consists of four rods with a hyperbolic cross section
that are accurately positioned parallel in a radial array.
 The quadrupole rods are typically constructed using molybdenum alloys because of
their inherent inertness and lack of activity.
 Very high degrees of accuracy and precision (in the micrometer region) in rod machining
and relative positioning are required to achieve unit mass accuracy.
 Quadrupole mass spectrometers ~QMSs’ are widely used in both industry and research
for fast accurate analysis of gas and vapors.
 The QMS contains basically three elements;
i) Ion source,
ii) Mass filter, and
iii) Ion detector
Benefits:
Classical mass spectra.
Good repeatability.
relatively small and cost-effective systems.
Low-energy collision-induced dissociation (CID) MS/MS spectra leads to efficient conversion
of precursor to product.
Limitations:
Limited resolution.
Peak heights are variable as a function of mass discrimination.
Peak height vs. mass response should be 'tuned'.
Not compatible for pulsed ionization methods.
Low-energy collision-induced dissociation (CID) MS/MS rely most probably on energy,
collision gas, pressure, and alternative factors.
Applications:
Majority of bench top GC/MS and LC/MS systems.
Triple quadrupole MS/MS systems.
Sector/quadrupole hybrid MS/MS syst ems.

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MAGNETIC SECTOR MASS ANALYZER:
 Sector mass analyzers are the most mature of the MS mass analysis technologies, having
enjoyed widespread use from the 1950s through to the 1980s.
 Magnetic sectors bend the trajectories of ions accelerated from an ion source into
circular paths; for a fixed accelerating potential, typically set between 2 and 10 kV, the
radii of these paths are determined by the momentum-to-charge ratios of the ions. In
such a manner, the ions of differing m/z are dispersed in space.
 Magnetic sector instruments are often used in series with an electric sector, high
resolution and tandem mass spectrometry experiments.
 When utilizing a magnetic sector alone, resolutions of only a few hundred can be
obtained, primarily due to limitations associated with differences in ion velocities. To
correct for this, electric sectors can be placed before or after the magnetic sector and is
thus called as double focusing sector instruments.
68Benefits:
Classical mass spectra.
Very high reproducibility.
High resolution.
High sensitivity.
High dynamic range.
Limitations:
Not well-suited for pulsed ionization methods (e.g. MALDI).
Usually larger and higher cost than other mass analyzers.
Applications:
All organic MS analysis methods.
Accurate mass measurement.
Isotope ratio measurements.
Quantitation.
6 9
TIME OF FLIGHT ANALYZER:
 A time-of-flight (TOF) mass spectrometer is a non-scanning mass analyzer that emits
pulses of ions (or transients) from the source.

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 These ions are accelerated so that they have equal kinetic energy before entering a field
free drift region, also known as the flight tube.
 A time-of-flight (TOF) instrument consists of a pulsed ion source, an accelerating grid, a
field-free flight tube, and a detector.
Different Modes of Time of Flight Analyzers:
 The mass analyzer used in TOF-MS can be either be a linear flight tube, applying a
bended geometry the flight path to reduce the influence of neutral particles
(Poschenrieder type) or a employ one (Vtype) or more reflectrons (W-type) to enhance
the path length within a given flight tube.
• Linear TOF (high mass range but low mass resolution)
• Reflectron TOF (lower mass range but high mass resolution)
Benefits:
Fastest MS analyzer.
Well suited for pulsed ionization methods (method of choice for majority of MALDI mass
spectrometer systems).
High ion transmission.
MS/MS information from post-source decay.
Highest practical mass range of all MS analyzers.
Limitations:
Requires pulsed ionization method or ion beam switching (duty cycle is a factor).
Fast digitizers used in TOF can have limited dynamic range.
Limited precursor-ion selectivity for most MS/MS experiments.
Applications:
Almost all MALDI systems.
Very fast GC/MS systems,
QUADRUPOLE ION TRAP ANALYZER:
 The Quadrupole ion storage trap mass spectrometer (QUISTOR) is a recently developed
mass analyzer with some special capabilities.
 The quadrupole ion trap is an extraordinary device that functions both as an ion store in
which gaseous ions can be confined for a period of time, and as a mass spectrometer of
large mass range variable mass resolution, and high sensitivity.
 As a storage device, the quadrupole ion trap confines gaseous ions, which are either
positively or negatively charged and when required ions of each polarity also.

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 A typical (three-dimensional quadrupole) ion trap consists of a cylindrical ring electrode
and two end-cap electrodes. The end-cap electrodes contain holes for the introduction of
ions from an external ion source and for the ejection of ions toward an external detector.
 A He bath gas (∼1 mbar) is used to stabilize the ion trajectories in the trap. 74
Benefits:
High sensitivity,
Compactness and mechanical simplicity
Ion/molecule reactions can be studied for mass-selected ions,
High resolution
Non-destructive detection is available using Fourier transform techniques.
Multi-stage mass spectrometry (analogous to FTICR experiments)
Limitations:
Poor quantitation.
Very poor dynamic range (which can be compensated for by employing autoranging).
Collision energy not well-defined in Collision Induced Dissociation [CID] MS/MS.
Quality of the mass spectrum is influenced by many parameters such as excitation, trapping,
and detection conditions.
Mass measurement accuracy is relatively poor.
Applications:
Benchtop GC/MS, LC/MS and MS/MS systems.
Target compound screening.
Ion chemistry.
Non-destructive ion detection
FOURIER-TRANSFORM ION CYCLOTRON
RESONANCE MASS ANALYZER {FTICR-MS/FTMS}
 Fourier transform ion cyclotron resonance mass spectrometry is a type of mass analyzer
(or mass spectrometer) for determining the mass-to-charge ratio (m/z) of ions based on
the cyclotron frequency of the ions in a fixed magnetic field.
 FT-ICR is the highest performance MS technique available, offering unrivalled resolution
and mass accuracy.

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 A FTMS can be considered an ion-trap system, where the ions are trapped in a magnetic
rather than in a quadrupole electric field.
 The Ion Cyclotron Resonance (ICR) mass spectrometer uses a superconducting magnet to
trap ions in a small sample cell. This type of mass analyzer has extremely high mass
resolution and is also useful for tandem mass spectrometry experiments.
 The FTMS consists of an ion source (in this case an Electrospray ion source), some ion
optics to transfer the ions into the magnetic field (in this case an RF-Only Quadrupole
ion guide), and the Ion Cyclotron Resonance (ICR) cell or Penning trap.
Benefits:
Highest mass resolution of all mass spectrometers.
Well-suited for ion chemistry and MS/MS experiments.
Well-suited to be used with pulsed ionization techniques like MALDI,
Non-destructive ion detection; ion re-measurement.
Mass calibration is stable in FTICR systems with superconducting magnet .
Limitations:
Dynamic range is limited.
Strict low-pressure requirements demands a mandatory external source for a number of
analytical applications.
Artifacts like harmonics and sidebands are present in the mass spectra.
Quality of the mass spectrum is influenced by many parameters such as excitation, trapping,
and detection conditions.
Generally low-energy Collision Induced Dissociation, therefore the spectrum depends on
collision energy, collision gas, and other parameters.
Applications:
Ion chemistry.
High-resolution MALDI and electrospray experiments for high-mass analytes.
Laser desorption for materials and surface characterization
DETECTORS
 Once the ions are separated by the mass analyzer, they reach the ion detector, which
generates a current signal from the incident ions.
 The most commonly used detectors in MS are as follows:
Faraday cup

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Electron multiplier
Photomultiplier dynode
Charge (or Inductive) Detector
FARADAY CUP
 A Faraday cup involves an ion striking the dynode (BeO, GaP, or CsSb) surface which
causes secondary electrons to be ejected.
 This temporary electron emission induces a positive charge on the detector and therefore
a current of electrons flowing toward the detector.
 This detector is not particularly sensitive, offering limited amplification of signal, yet it is
tolerant of relatively high pressure.
ELECTRON MULTIPLIER
 It is the most common means of detecting ions. It is made up of a series (12 to 24) of
aluminum oxide (Al2O3) dynodes maintained at ever increasing potentials.
 Ions strike the first dynode surface causing an emission of electrons. These electrons are
then attracted to the next dynode held at a higher potential and therefore more secondary
electrons are generated.
 Ultimately, as numerous dynodes are involved, a cascade of electrons is formed that
results in an overall current gain on the order of one million or higher.
 The high energy dynode (HED) uses an accelerating electrostatic field to increase the
velocity of the ions and serves to increase signal intensity and therefore sensitivity.
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PHOTOMULTIPLIER CONVERSION DYNODE:
 The photomultiplier conversion dynode detector is not commonly used.
 It is similar to electron multiplier in design where the secondary electrons strike a
phosphorus screen instead of a dynode. The phosphorus screen releases photons which
are detected by the photomultiplier and are then amplified using the cascading principle.
 One advantage of the conversion dynode is that the photomultiplier tube is sealed in a
vacuum, unexposed to the environment of the mass spectrometer and thus the possibility
of contamination is removed.
ARRAY DETECTOR
 An array detector is a group of individual detectors aligned in an array format.
 The array detector, which spatially detects ions according to their different m/z, has been
typically used on magnetic sector mass analyzers.
 Spatially differentiated ions can be detected simultaneously by an array detector.
 The primary advantage of this approach is that, over a small mass range, scanning is not
necessary and therefore sensitivity is improved.

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84
CHARGE (OR INDUCTIVE) DETECTOR
 Charge detectors simply recognize a moving charged particle (an ion) through the
induction of a current on the plate as the ion moves past.
 This type of detection is widely used in FTMS to generate an image current of an ion.
 Detection is independent of ion size and therefore has been used on particles such as
whole viruses.
TYPES OF IONS PRODUCED IN MS
1. Molecular ion (Parent ion)
2. Fragment ions
3. Rearrangement ions
4. Metastable ions
5. Multiple charged ions
6. Isotope ions
7. Negative ions
MOLECULAR ION (PARENT ION)
 When a molecule is bombarded with electrons in high vacuum in Mass spectrometer, it is
converted into positive ions by loss of an electron. These ions are called as Molecular or
Parent ions.
M + e → M+° + 2e-
Where, M – represents the Molecule;
M+°– represents the Molecular or Parent ion
 The order of energy required to remove electron is as follows—
σ electrons > non-conjugated π > conjugated π > nonbonding or lone pair of electrons.

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 Most molecules show a peak for the molecular ion, the stability of which is usually in the
order—
Aromatic > Conjugated acyclic polyenes > Alicyclics > n- hydrocarbons > ketones > ethers>
Branched chain hydrocarbons > Alcohols.
Characteristics of Molecular ion
 Molecular peak is observed if molecular ion remains intact long enough (10-6 seconds) to
reach the detector.
 This peak gives the molecular weight of the compound. The molecular ion peak is
usually the peak of the “highest mass number.”
 The molecular ion M+° has mass, corresponding to the molecular weight of the
compound from which it is generated. Thus the mass of a Molecular ion M+° is an
important parameter in the identification of the compound.
Significance of Molecular ion
 Molecular ion peak gives the molecular weight of the compound. i.e. m/z of molecular
ion = molecular weight of the compound.Ex: C2H5+ (m/e=29) gives the molecular
weight of Ethane.
IMPORTANT FEATURES OF PARENT ION PEAK
a) The molecular ion peak in aromatic compounds is relatively much intense due to the presence
of 𝜋- electron system.
b) Unsaturated compounds give more intense peak as compared to saturated or cyclic molecule.
c) Absence of molecular ion peak means that the compound under examination is highly
branched or tertiary alcohol.
d) Primary and secondary alcohol gives very small molecular ion peaks.
e) In case of chloro or bromo compounds isotope peaks are also formed along with the molecular
ion peak.
 For chloro compounds: M+ and (M++2) peaks are formed in the intensity ratio 1:3.
 For bromo compounds: M+ and (M++2) peaks are formed in the intensity ratio 1:1.
FRAGMENT ION
 When the energy is given further more upto 70 eV, fragment ions are produced, which
has smaller masses.
 Formed by both heterolytic and homolytic cleavage of bonds by simple cleavage and
rearrangement process.
 Formation is governed by bond dissociation energy and steric factors. e.g.: ethyl chloride.

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REARRANGEMENT IONS:
 Rearrangement ions are the fragments whose origin cannot be described by simple
cleavage of bonds in the parent ion, but are result of intramolecular atomic
rearrangement during fragmentation.
 These are probably due to recombination of fragment ions and known as rearrangement
peaks.
 Ex: Prominent peak in spectrum of diethyl ether occurs at m/e 31. This is due to the ions
CH3O+, which is formed by rearrangement of C2H5O+ ions.
 The ‘McLafferty rearrangement’ is a common example. In case of carbonyl compound.
93
METASTABLE ION
 Consider that M1 + is the parent ion and m1 + is daughter ion.
 If the reaction M1 + → m1 + takes place in the source, then m1 + may travel the whole
analyzer region and recorded as m1 + ion.
 If the transition M1 +→ m1 + occurs after the source exit and before arrival at the
collector, then m1 + is called a metastable ion.
 The position of metastable ion is given by:
𝑚∗ =𝑚12/𝑀1
 It is important to remember that m* has a distance below m1 on mass scale same as that
of m1 and M1.
 The relative abundance of the metastable peak is often of the order of 10-2 or less
compared to the abundance of parent or daughter ion. 94
CHARACTERISTICS OF METASTABLE IONS:
1) They do not necessarily occur at the integral m/e values.

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2) These are much broader than the normal peaks.
3) These are of relatively low abundance.
4) These have low kinetic energy.
MULTI CHARGED IONS
 Sometimes ions may also exist with two or three charges instead of usual single charge in
the mass spectrum. These are known as doubly or triply charged ions. They are created as
follows:
M+° + e- → M++ + 3e-
M+° + e- → M+++ + 4e-
 But under normal operating conditions, most of the ions produced are single charged. The
doubly or triply charged ions are recorded at a half or one third of the m/e value of the
single charged ions.
 Formation of these multiple charged ions is more common in hetero aromatic
compounds.
 They are also common in inorganic mass spectrum. Gases such as CO, N2, CO2 and O2
have measurable peaks corresponding to CO+2,N+2,and O+2.
ISOTOPE IONS
 Most elements are mixture of two or more stable isotopes differing by one or two mass
units.
 Chlorine and Bromine have two isotopes (35Cl, 37Cl and 79Br, 81Br) in the ratios 3:1
and 1:1 respectively.
 Thus in the spectrum of methyl bromide the molecular ion peak is the doublet consisting
of two equally intense peaks one at m/e 94 (CH3 79Br) and the other at m/e 96 (CH3
81Br).
 These isotopic clusters are referred to as isotopic peaks. They are helpful in determining
the presence of such elements in a molecule. 97
NEGATIVE IONS
 The positive ions predominate in electronic impact ionization because of greater
stability. The Negative ions are not very useful in structural determinations.
 The formation of Negative ions is very rare but these can be produced in three ways:
1. AB + e- → A+ + B-
2. AB + e- → AB-
3. AB + e- → A+ + B- + e-

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