Presentation on MASS SPECTROSCOPY.pptx

Presentation on
MODERN PHARMACEUTICAL
ANALYTICAL TECHNIQUES
(MPC101T)
TOPIC-MASS SPECTROSCOPY
PRESENTED BY
TAPAS MAJUMDER
M.PHARMA ,1ST SEM
ENROLLMENT NO-2106240007
DEPT.OF PHARMACY
TRIPURA UNIVERSITY(a central university)
PRESENTED TO
MR. RAJAT GHOSH
ASSISTANT PROFESSOR
DEPT.OF PHARMACY
TRIPURA UNIVERSITY(A
CENTRAL UNIVERSITY)

CONTENTS
Introduction
Basic Principle
Brief outline of Instrumentation(Mass spectrometer)
Different sources of ions and types of ionization.
Mass Analyzer
Detectors
Mass fragmentation
Applications
References
2

INTRODUCTION
• Mass spectroscopy is one of the primary spectroscopic methods for molecular analysis available
to organic chemist.
• It is a microanalytical technique requiring only a few nanomoles of the sample to obtain
characteristic information pertaining to the structure and molecular weight of the analyte.
• It involves the production and separation of ionize molecules and theirs ionic decomposition
product and finally the measurement of the relative abundance of different ions produced. It is,
thus a destructive technique in that the sample is consumed during analysis.
3
Replica of J.J Thomson mass spectrometer

BASIC PRINCIPLE
• In this technique ,molecules are bombarded with a beam of energetic electrons(70ev) in
gaseous state using tungsten or rhenium filament. Molecules are broken up into cations
and many other fragments.
• These cations (molecular or parent ion) are formed due to loss of an electron usually from
n or pi 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. Continue…
4

• 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.
5

Ionization
• The atom is ionized by knocking one or more electrons off to give a positive ion.(Mass
spectrometer always work with positive ions).
• The particles in the sample (atoms or molecules) are bombarded with a stream of electrons to
knock one or more electrons out of the sample particles to make positive ions.
7
• Most of the positive ions will carry a charge of +1.
• These positive ions are persuaded out into the rest of the machine by the ion propeller
which is another metal plate carrying a slight positive charge.

ACCLERATION
• The ions are accelerated so that they all have the same kinetic energy.
• The positive ions are repelled away from the positive ionization chamber and pass through 3 slits with
voltage in the decreasing order.
8

DEFLECTION
• The ions are then deflected by a magnetic field according to their masses.
The lighter they are, the more they are deflected.
• The amount of deflection also depends on the number of positive charges on
the ion-the more the ion is charged, the more it gets deflected.
• Different ions are deflected by the magnetic field by different amounts. The
amount of deflection depends on:
• The molecular weight of the ion: lighter ions(ions having lower molecular
wight) are deflected more than heavier ones.
• The charge on the ion: Ions with 2(or more) positive charges are deflected
more than one with only 1 positive charge
9

DETECTION
• The beam of ions passing through the machine is detected electrically.
only ion stream B makes it right through
the machine to the ion detector.
The other ions collide with the walls where they will
pick up electrons and be neutralized.
They get removed from the mass spectrometer by the
vacuum pump.
10
• When an ions hits the metal box, its charge is neutralized by an electron jumping from
the metal on to the ion.
• That leaves a space amongst the electrons in the metal, and the electrons in the wire
shuffle along to fill it.
• A flow of electrons in the wire is detected as an electric current which can be amplified
and recorded. The more ions arriving, the greater the current.

Ionization and sources
• Ionization methods refers to the mechanism of ionization while the ionization source is the mechanical device that allows
ionization to occur.
• ION SOURCE: Ionization of the organic compound is the primary step in obtaining the mass spectrum. 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 ion reaches only at higher bombardment energies at 70 ev.
• TYPES: A)DESORPTION SOURCES
1.Electrospray ionization(ESI) 2. Fast Atom Bombardment (FAB)
3.Matrix-assisted laser desorption ionization(MALDI)
B)ATMOSPHERIC PRESSURE IONIZATION
1.Atmospheric pressure chemical ionization(APCI) 2.Atmospheric pressure
photoionization(APPI)
C)GAS PHASE SOURCES
1.Electron impact ionization(EI) 2.Chemical ionization 3.Field ionization.
11

Electrospray Ionization(ESI)
12
• It is a type of evaporative ionization(soft) technique used to analyte high molecular weight biomolecules,
labile and nonvolatile compound. Due to ESI technique mass spectroscopy become very popular and could
be coupled with chromatography.
• PRINCIPLE:
A solution containing the analyte is sprayed
through the high voltage potential capillary by
the help of Nebulization gas(N2)
Sprayed droplets are being ionized due to
presence of high voltage potential at the tip
of capillary tube.
Heated dissolvation gas(N2) has been entered
into the chamber and they evaporate the
solvent and produce molecular ion(positive
ion) which will further pass through the slit
and entered into ion acceleration chamber .

13
ADVANTAGES:
a) Has the ability to handle the samples with large masses.
b) One of the softest ionization methods available and has the ability to analyze biological
samples with non-covalent interactions.
DISADVANTAGES:
a) Apparatus is very difficult to clean and has a tendency to become overly contaminated withb
residues from previous experiment.
b) The multiple charges that are attached to the molecular ions can make for confusing spectral
data.

MALDI(MATRIX ASSISTED LASER DESORTION
IONIZATION)
 MALDI is also based on “soft ionization” technique m where ion formation does not lead to a
significant loss of sample integrity.
 PRINCIPLE:
14
The sample for analysis by
MALDI is prepared by mixing
or coating with solution of an
energy-adsorbent ,organic
compound called Matrix
When the matrix crystallizes
on drying ,the sample
entrapped within the matrix
also co-crystallizes.
The sample with the matrix is
ionized in an automated mode
with a laser beam. Desorption
and ionization with the beam
generates singly protonated
ions from analytes in the
sample.

15
ROLE OF MATRIX-It absorbs the photon
energy from the laser beam and transfers it into
excitation energy of solid system and it serves as
a solvent for the analyte so that the
intermolecular forces are reduced and
aggregation of the analyte molecule is held to
minimum.
EX-2,5- dihydroxy benzoic acid( at wavelength
337,355,266)
3,5-dimethoxy-4-hydroxycinnamic
acid(337,355,266)
Picolinic acid(266)
ADVANTAGES-
 High molecular weight analyte can be ionized
 Produces singly charged ions thus interpretation becomes
easy.
 Molecules need not to be volatile.
DISADVANTAGES-
 Analyte must have very low vapour pressure.
 Coupling MALDI with chromatography can be difficult.

FAST ATOM
BOMBARDMENT(FAB)
16
 Used for large compounds with low volatility (eg- peptides
,carbohydrates)
 Solid or liquid sample is mixed with a nonvolatile matrix(
glycerol, crown ethers, nitrobenzyl alcohal).
 Immobilized matrix is bombarded with a fast beam of Argon
or xenon atoms
 Charged particles ions are ejected from the matrix and
extracted in the mass analyzers.
 Gives M+H+ ions.
Fast atom bombardment gun

ELECTRON IMPACT
IONIZATION
• Its widely used technique when couples with GC.
• It’s a hard an ionization technique and produces a lots of daughter ion along with molecular ion(M+ ion)
• Suitable for volatile organic compounds like hydrocarbons, oils, flavours, fragrences.
• It produces abundant fragment ions.
• Ionization occurs by removing 1 electron from the analyte molecule thus generating a positively charged ion
with one unpaired electron.
17

CHEMICAL
IONIZATION
It’s a softer ionization technique so that it could be used
instead of EI to prevent excess fragmentation.
It protonates or deprotonates the analyte molecule.
Similar ionization technique to EI except that a reagent
gas is introduced into the chamber in excess of the
sample. Methane, isobutane or ammonia are used as gas.
Ionized reagent gas protonate the sample molecules
leaving a neutral gas species
18
Primary ions:-
CH4
+ e_
CH4
+
+2e_
CH4
+
CH3
+
+H
Secondary ions:-
CH4
+
+CH4
CH5
+
+CH3
CH3
+
+CH4 C2
H5
+
+H2
Proton donation:-
CH5
+
+ M CH4
+
+ MH+

MASS ANALYZER
• Definition-A mass analyzer is a component of the mass spectrometer that takes the ionized masses
and separates them based on charge to mass ratio and outputs them to the detector where they are
detected ad later converted to a digital output.
• TYPES- There are general types of mass analyzer that can be used for the separation of ions in
amass spectrometry.
a. Quadrupole Mass Analyzer
b. Time of flight Mass analyzer
c. Magnetic sector Mass analyzer.
d. Electrostatic sector Mass Analyzer
e. Quadrupole Ion Trap Mass Analyzer
f. Fourier-Transform Ion cyclotron Resonance Mass Analyzer(FTI-MS)
19

QUADRUPOLE MASS ANALYZER
• A typical mass analyzer consists of four rods with a hyperbolic cross section that are accurately positioned
parallel in a radial array.
• Quadrupole rods are typically constructed using Molybdenum alloys because of their inherent inertness and
lack activity.
• The QMS contains basically 3 elements ;
a. Ion source
b. Mass filter
c. Ion detector.
BENEFITS-
a. Good repeatability
b. Relatively small and cost effective system.
LIMITATIONS-
a) Limited resolutions.
b) Not compatible for pulsed ionization mehods.
20

• PRINCIPLE:
21
4 hyperbolic rods are
arranged in parallel to
the direction of ion
beam.
They are connect with
DC voltage and radio
frequency which will
generate an
electrostatic field
between rods.
Ions will enter into the
analyzer and depending
on the ratio of RF
amplitude and DC
voltage ions will
acquire oscillation.
IF RF>DC voltage
larger ion will hit the
detector first
If RF<DC voltage
smaller ion reaches
first
Ions having inappropriate
M/Z ratio undergo unstable
oscillation and collide with
each other or with the wall
and never reach to
detector.

TIME OF FLIGHT(TOF) MASS
ANALYZER
• A time of flight mass spectrometer is a non- scanning mass analyzer that emits pulses of ions (transients)from
the source.
• 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 pulsed ion source ,an accelerating grid, a field free flight tube
and detector.
• BENEFITS:-
• Fastest MS analyzer.
• Well suited for pulsed ionization (method of choice for majority of MALDI mass spectrometer systems).
• High ion transmission.
• LIMITATIONS:-
• Require pulsed ionization method.
• Limited precursor-ion selectivity for most MS/MS experiments.
• APPLICATIONS:- Almost all MALDI systems, Very fast GC/MS systems. 22

• Principle:-TOF mass spectrometry is based on the fact that ions with the same kinetic energy but
different masses travel with different velocities, so that lighter ion have the higher velocities compare go the
heavier ions and will strike the detector faster compare to heavier ions.
• Kinetic energy of an ion accelerated through an electrical potential will be
23
zV=1/2 mv2
(1)
Velocity(v)=L/t (2)
substituting the value of v on
equation 1
zV=1/2 m L2
/t2
(3)
Rearranging the equation the 3
m/z=2V t2
/L2
here V,L are
constant so, k=2V/L2
m/z=kt2
i.e
m/z a t2

DETECTOR
• Once the ions are separated by the mass analyzer, they reach to the ion detector ,which generates a current
signal from the incident ions.
• The most commonly used detectors In MS are as follows:
Faraday cup
Electron multiplier
Photomultiplier dynode.
Ion cyclotron resonance(ICR)/orbitrap.
FARADAY CUP:-
o A Faraday cup is a metal(conductive) cup designed to catch charged particles in vacuum. The resulting
current can be measured and used to determine the number of ions or electrons hitting the cup.
o The Faraday cup was named after Michael Faraday.
24

Principle:
• The ion beam is allowed to collide with the interior walls of an open-ended metal cup all secondary electron
emission is suppressed. The cup is maintained at virtually at ground potential.
• By measuring the electric current (the no of electrons flowing through the circuit per second) in the metal part of
the circuit ,the number of charges being carried by the ions in the vacuum part of the circuit can be determined .
• The total no of ions hitting the cup per unit is
N/t = i/e
N=total no ions hitting
T=time
i=current produced
e=charge on an electron
-minute current is produced
-amplification of current
-Most commonly used detector.
25

Electron multiplier:-
Principle:-
• It is the most common means of detecting ions. It is made up of a series (12-24) of aluminum oxide dynodes
at ever increasing potentials.
• Ions strike the first dynode surface causing an emission of electron. 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 of one million or higher.
26

Mass Fragmentation:-
The process of breaking up of molecular ion into smaller or daughter ion is known as “Fragmentation”.
The molecular ion commonly decomposes to a pair of fragments, which may be either a radical with an ion or
small molecules and a radical cation.
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 50-70 ev, these molecular ions acquire a high
excitation in their breakdown inro various fragments.
Types of ion and peaks in MS:-
Molecular ion/Parent ion:-
Ion formed by the loss of single electron at lowest ionization potential from a molecule.
27

• Fragment ion/ daughter ion:- Generated by the fragmentation of molecular ion in the ionization chamber
• Metastable ion:- some fragmentation may occur during their flight down the ion tube field free region instead
of ionization chamber are known as metastable ions.
• They reach to the detector at masses lower than the actual mass and gives broader peaks.
• Quasi molecular ion:- a protonated molecular ion or an ion formed by removal of 1 hydrogen atom from
molecular ion is known as Quasi molecular ion.
• Multiple charged ion:-some double or triple charged ions are observed .
• Mainly occurs in ESI spectrum and they, different M/Z ratio.
28
M
e-
e-
M
+
M+
2- formed due to energy of
ionization potential
M+
1- Due ro unstability of
molecular ion
M+ H+ MH
+
(M+1)
M-H + H (M-1)
M+

• Base peak:-The most intense/tallest peak in the mass spectrum. It is due to greatest relative abundance.
29
• Isotope peak:-Due to presence of heavier isotope elements ,gives very less intense peak

APPLICATIONS
30
Field of study Applications
Proteomics  Determine protein structure, function, folding and interactions
 Identify a protein from the mass of its peptide fragments.
 Monitor enzyme reactions, chemical modifications and protein digestion.
Drug discovery  Determine structure of drugs and metabolites
 Screen for metabolites in biological systems.
Clinical testing  Perform forensic analyses such as confirmation of drug abuse
 Detect disease biomarkers (newborns screened for metabolic diseases)
Genomics  Sequence oligonucleotides
Environment  Test water quality or food contamination
Geology  Measure petroleum composition
 Perform carbon dating
Military  Mobile mass spectrometers are used to detect liquid chemical warfare agents.

REFERENCES
1) Elementary organic chemistry-principles and chemical applications by Y.R.SHARMA 291-313.
2) https://www.slideshare.net/AmrutaSambrekar/2amruta-mass
3) https://www.slideshare.net/PrincyAgarwal6/mass-spectrometry-81082759
4) https://www.slideshare.net/MuhammadAsif564/mass-spectroscopy-ionization-techniques-and-types-of-mass-analyzers
5) https://www.slideshare.net/Rohitkumar2988/mass-spectroscopy-its-instrumentation
6) https://www.slideshare.net/samiyashaik1/fragmented-techniques
7) https://youtu.be/vxCWBWB20UA
8) https://youtu.be/X9HSc2dMQjc
9) https://www.youtube.com/watch?v=Wc8-GJY42lE
10) https://youtu.be/Hh4ryO4iG1E
11) https://youtu.be/ZCTcKWUudSwhttps://youtu.be/ZCTcKWUudSw
12) https://youtu.be/WzgudLyh0fA
13) https://youtu.be/uOKHufyz_qQ
14) https://youtu.be/DbnNLilCGyk
15) https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules_(Analytical_Chemistry)/Instrumental_Analysis/Mass_Spe
ctrometry/Mass_Spectrometers_(Instrumentation)/Mass_Analyzers_(Mass_Spectrometry)#:~:text=A%20mass%20analyzer%20is%20the,convert
ed%20to%20a%20digital%20output 31

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