MASS SPECTROSCOPY

PRESENTED BY:
Ajay Kumar Varda
(M.Pharma Pharmaceutics 1st year)
Rayat Institute of Pharmacy, Ropar
 CONTENTS

• Introduction
• Basic principle
• Theory
• Brief outline of instrumentation.
• Ion formation and types
• Fragmentation processes
• Fragmentation patterns
• Fragmentation characteristics in relation to parent structure and functional groups
 INTRODUCTION

• Mass spectrometry (MS) is an analytical technique that measures the mass-to-

charge particles. It is used for determining masses of particles, for determining the
elemental composition of a sample or molecule.
• Mass spectrometry (MS) is an analytical chemistry technique that helps identify the
amount and type of chemicals present in a sample by measuring the mass to charge
ratio and abundance of gas-phase ions.
 INTRODUCTION

• Mass spectrometry is an instrumental technique in which sample is converted to

rapidly moving positive ions by electron bombardment and charged particles are
separated according to their masses.
PRINCIPLE
 PRINCIPLE

• Mass spectroscopy is the most accurate method for determining the molecular mass
of the compound and its elemental composition.
• In this technique, molecules are bombarded with a beam of energetic electrons.
• The molecules are ionised 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(value). For
most ions, the charge is one and thus, m/e ratio is simply the molecular mass of the
ion.
 PRINCIPLE

• Mass spectra is 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.
• Inlet System :
1. SOLID SAMPLES with lower vapour pressure directly inserted to ionization
chamber and volatilization is controlled by heating the probe.
2. LIQUIDS are handled by hypodermic needles injection through a silicon rubber
dam.
3. GASES SAMPLES are leaked into the ionization chamber directly by the help of
mercury manometer.
• Ion Sources:
The ion source is the part of MS that ionizes the material under analysis(the analyte).
Molecular ions are formed when energy of electron beam reaches to 10 – 15 eV.
Fragmentation of the ion occurs only at higher bombardment energies at 70eV.
 TYPES OF IONIZATION METHODS
• Gas Phase Ionisation (gases and vapour) : Samples are ionised outside the ion source.
This technique includes:
1. Electron impact ionization
2. Chemical Ionozation
3. Field Ionization
• Desorption Technique (liquid and solid) : Samples are ionized inside the ion source.
1. Field desorption
2. Fast atom bombardment
3. Laser desorption
 ELECTRON IMPACT
IONISATION
• A beam of electrons(generally have 70 eV
energy) passes through a gas-phase sample
and colloids with neutral analyte molecule
(M) to produce a positively charged ion or a
fragment ion.
• The positive ions are collected in focusing
plate and passed to mass analyzer.
 ELECTROSPRAY IONISATION
• The ESI source consists of a very fine needle and a series of skimmers.
• A sample solution is sprayed into the source chamber to form droplets.
• When Droplets carry charge exit at the capillary end, as the solvent evaporates,
the droplets disappear leaving highly charged analyte molecules.
 CHEMICAL IONISATION
• Chemical impact ionization between
interaction of samples with large
amount of reagent gases.
• Commonly used reagent gases
include methane, ammonia,
isobutane.
• O2 and H2 are used in NEGATIVE
ION CI in MS.
 CHEMICAL IONISATION
The vapourised sample is introduced into the MS with an excess of a reagent gas
(Methane) at a pressure of about 1 torr. The excess carrier gas is ionized by Electron
impact to the primary ions CH4+ and CH3+.
These may react with the excess methane to give secondary ions.
 FIELD IONISATION
• In this method the molecule pass through sharp metal anode carrying an electric
field of 10^10 vm^-1 .
• Electrons are analysed in primary focusing cathode slit.
• ADV : Increase abundance of molecular ions.
• DISADV : lower resolution.
 FIELD DESORPTION

• Useful for nonvolatile and thermolabile

compounds.
• Sample is applied to field ion emitter and the
solvent allowed to evaporate.
• Evaporated sample that leads to chemical
ionization or EIS.
• E.g. Nucleotides & Quarternary ammonium
compounds.
 FAST ATOM BOMBARDMENT
• Argon gas ionized by hot filament and focused beam that bombards the sample.
• Beam impinges the sample, a series of molecular reactions occurs and analyse in MS
analyser.
• E.g. Insulin, Amino glycosides, Phospholipids.
 MALDI
• MALDI is a LIMS method of vapourizing and ionizing and sample molecules are
dispersed in a solid matrix such as nicotinic acid.
• A UV laser pulse ablates the matrix which carries some of the large molecules into
the gas phase in an ionized form so they can be extracted into MS.
 APCI
• Atmospheric pressure chemical ionization (APCI) is an analogus ionization method to
chemical ionization(CI).
• Corona discharge is used to ionize the analyte in the atmospheric region.
 MASS ANALYSERS
An ion, after leaving ion source, the ions are separated according to their m/e ratio.
In this area, the ions are accelerated by both electrostatic and magnetically.
TYPES:
1. Magnetic Sector mass analysers
2. Double focusing analysers
3. Quadrupole MA
4. Time of Flight analysers
5. Ion trap analysers
6. Ion cyclotron analysers
 QUADRUPOLE MASS ANALYSER
• The quadrupole consists of two pairs of parallel rods with applied DC and RF voltages.
• Each pair of Rod have exactly the same voltage as the one directly opposite.
• Depending upon the ratio of DC and AC, ions acquire or affected by magnetic field.
• Ions of correct m/z ratio don’t collide with the rods and travel to detector and other with
laws or high m/z are unstable and doesn’t pass.
• If DC is +ve = high mass ions will have stable path
• If DC is –ve = Low mass ions will have stable path
• Ions are scanned by varying DC/Rf quadrupole voltages.
 TIME OF FLIGHT ANALYSER

• Ions are accelerated through a flight tube and the TOF to the detector is measured.
• Typical flight times are 1 to 50,us.
 DETECTORS

• Faraday cup
• Electron Multiplier
• Photomultiplier
• Micro Channel Plate
 FARADAY CUP
• The basic principle is that the incident ion strikes the dynode surface which emits
electrons and induces a current which is amplified and recorded.
• The dynode electrode is made of a secondary emitting material like CsSb, GaP or
BeO.
• It is ideally suited to isotope analysis.
 ELECTRON MULTIPLERS

• Electron multipliers are the most common especially when +ve or –ve ions need to
be detected on the same instrument.
• Dynodes made up of Cu – beryllium which transduces the initial ion current, and
electron emitted by first dynode are focused magnetically from dynode to the next.
• Final Cascade current is amplified more than million times.
 PHOTOMULTIERS

• The dynode consists of a substance(a scintillator) which emits photons(light).

• The emitted light is detected by photo multiplier tube and is converted into electric
current.
• These detectors are useful in study of metastable ions.
 VACUUM SYSTEM

• All MS need a vacuum to allow ions to reach the detector without colliding with other
gaseous molecules or atoms. If such collisions occur, the instrument would suffer from
reduced resolution and sensitivity 10^-2 to 10^-5 Pa.
 TYPES OF IONS

1. Molecular ion or Parent ion

2. Fragment ions
3. Rearrangement ions
4. Multicharged ions
5. Negative ions
6. Metastable ions
 METASTABLE IONS
Fragment of a parent ion will give rise to a new ion (daughter) plus either a neutral
molecule or a radical.
M1+--------- M2+ + non charged particle
• An intermediate situation is possible; M1+ may decompose to M2+ while being
accelerated. The resultant daughter ion M2+ will not be recorded at either M1 or M2,
but at a position M* as a rather broad, poorly focused peak. Such an ion is called a
metastable ion.
 NATURE OF METASTABLE IONS

Metastable ions have lower kinetic energy than normal ions and metastable peaks are
smaller than the M1 and M2 peaks and also broader. These metastable ions arise
fromfragmentation that takes place during the flight down through ion rather than in the
ionization chamber.
• Molecular ions formed in the ionization chamber do one of the following things:
1. Either they decompose completely and very rapidly in the ion source and never reach
the collector (as in case of highly branched molecular ions with life times less than 10-5
seconds).
2. Or else they survive long enough to reach the collector and be recorded there (life times
longer than 10-5).
 SIGNIFICANCE OF METASTABLE IONS

Metastable ions are useful in helping to establish fragments routes. Metastable ion
peak can also be used to distinguish between fragmentation Processes, which occur in
few microseconds.
 MASS FRAGMENTATION

• Bombardment of molecules by an electron beam with energy between 10-15ev

usually results in the ionization of molecules by removal of one electron (Molecular
ion formation).
• When the energy of electron beam is increased between 50-70ev, these molecular
ions acquire a high excitation resulting in their break down into various fragments.
This process is called “ Fragmentation process”.
 TYPES OF FRAGMENTATION

• Collision induced dissociation(CID)

• Electron capture dissociation(ECD)
• Electron transfer dissociation(ETD)
• Electron Detachment dissociation(EDD)
• Photo dissociation
• Surface induced dissociation(SID)
• Charge remote fragmentation
• Higher energy C-trap dissociation(HCD)
 GENERAL RULES FOR FRAGMENTATION

1. The relative height of the molecular ion peak is greatest for the straight chain
compound and decreases as the degree of branching increases.
 GENERAL RULES FOR FRAGMENTATION
2. The relative height of the Molecular ion peak usually decreases with increasing
molecular weight in a homologous series. For e.g. Fatty molecules , steroids.
3. Cleavage is favoured at alkyl substituted carbon atoms;the more substituted, the
more likely is cleavage. This is a consequence of the increased stability of a tertiary
carbon atom over a secondary, which in turn is more stable than a primary.
CH3+ < RCH2+ < R2CH+ < R3C+
 APPLICATIONS
Mass spectrometry has both qualitative and quantitative uses.
1.Structure elucidation
2.Detection of impurities
3.Quantitative analysis
4.Drug metabolism studies
5.Clinical, toxicological and forensic applications
6.GC MS
MS is now in very common use in analytical laboratories that study physical,
chemical, or biological properties of a great variety of compounds.
 QUALITATIVE APPLICATIONS
1. Determination of molecular weight: Mass spectrometry serves as the best possible
technique for the determination or confirmation of molecular weight of compounds
that can be easily volatilized.
2. Determination of molecular formula :For the determination of molecular
formula b mass spectrometry, it is essential to identify the molecular ion peak as
well as its exact mass.
3. Determination of partial molecular formula: Generally, atoms are polyisotopic. In
mass spectrometer, the ions are selected according to their actual mass. Exact
information about the atomic composition of the selected ions is furnished by the
mass distribution of molecular ions.
 QUALITATIVE APPLICATIONS

4. Determination of structure of compounds: Bombardment of vaporized sample

molecules with a high beam of electrons results in their fragmentation producing a
large number of ions with varying masses.
 QUANTITATIVE APPLICATION
1. Determination of isotope abundance: Although differences in the masses of isotopes of an
element are very small, the isotope abundance i.e., the isotopic composition of molecules
within an easily vaporizable sample can be determined with mass spectrometry. The
information so obtained may be useful for:
(a) tracer studies with isotopes
(b) determination of atomic weights of compounds
(c) determination of age rocks and minerals
(d) study of origin as well as nature of solar system
2. Determination of isotope ratio : Mass spectroscopy is used to determine isotope ratio
which in turn helps to determine the concentration of individual components present in
complex mixture from which it cannot be separated quantitatively.
3. Differentiation between Cis and Trans isomers: Mass spectrometry may be used to
differentiate between cis and trans isomers. Both the isomers yield similar spectra but are
differentiated from the intensity of the molecular ion peaks. The molecular ion peak of trans
isomer is more intense than that of cis isomer.
 QUANTITATIVE APPLICATION
4.Mass spectrometry in thermodynamics:
a) Determination of heat of vapourization : To determine the heat of vapourization of
high temperature compounds, data from the spectrum is collected and a graph is
plotted by taking ion intensities on Yaxis and temperature on X-axis.
b) Determination of heat of sublimation: To determine the heat of sublimation of a
compound, vapours of the sublimed solids are passed into the ionization chamber
of the mass spectrometer. The spectrum is recorded in which the obtained peak
intensities are directly proportional to the vapour pressure (VP) of the sample in
the ionization chamber.
5.Measurement of ionization potential : Ionization potential is the minimum energy
required by the bombarding electrons to produce the molecular ions from a molecule
of an atom.
 QUANTITATIVE APPLICATION
6.Detrmination of ion-molecule reactions: Mass spectrometry finds its use in the
study of ion molecule reaction i.e., the reactions in between the fragment ion and the
unionized molecules. The rate of these reactions directly depend on the operating
pressure.
7.Detction of impurity: The impurities present sample even in low concentration
(parts per million) can be detected by spectrometry, provide the molecular weights of
the impurities differ considerably from the major components.
8.Identification of unknown compounds: Mass spectrometry can be used to identify
the unknown compounds. this can be achieved by recording the spectrum of the
unknown compounds and comparing it with the spectrum of the standard compound
recorded under identical conditions.
9.Identification of proteins: Mass spectrometry serves as valuable tool in the study of
structure and functions of proteins (proteomics). Electro spray ionization (ESI) and
matrix-assisted laser desorption/ionization (MALDI) are the widely used ionization
methods for this purpose. Mass spectrometry in the proteomics particularly deals
with the analysis of protein digested by protease like trypsin.