sreeprakash.pandey7@gmail.com

1. MASS SPECTROSCOPY
1
Submitted to
Dr. Dhananjay Kumar Singh
Assistance Professor
Department of Pharmacy
School of Health Science
Presented by
Mr. Sree Prakash Pandey
(CUSB2006122010)
M.Pharm 1st year (1st Sem)
Department – Pharmacuetics
School of Health Science
Central University of South Bihar, Gaya

2. CONTENTS
Introduction
Basic Principle and Theory
Different types of ionization
Instrumentation of mass spectrophotometer
Analyzers of Quadrupole
Time of Flight
Mass fragmentation & its rules
Meta stable ion & Isotopic peaks
Application of mass spectroscopy
References
2

3. INTRODUCTION
Mass Spectroscopy is the most appropriate method to
determine the molecular mass of the compound and its
elemental composition.
In this technique, the molecular bombardment with a beam
of energetic electron.
The molecule gets ionized and broken up into many
fragments, some are which positive ions.
And each kind ion has a particular ratio of mass to charge,
i.e. m/e ratio.
This technique is useful for solid, liquid and gas.
Why mass important ?
It can give the exact molecular mass.
It can give a molecular formula.
It also provides the relevant & certain structural units in a
molecule. 3

4. BASIC PRINCIPLE
 A beam of electrons will be bombarded in the
analytical compound & it will leads to removal of one
electron from analyte.
 Due to removal of the electron, molecule will be
positively charged and known as molecular ion and
these molecular ions will be fragmented.
THEORY
 A parent ion results when one electron is removed
from the parent molecule of the substance.
M (g) + e —> M+ (g) + 2e
 The m/e ratio of the parent ion is equal to the
molecular mass of the compound.
4

5. 5
 The kinetic energy of molecular/fragmented ions-
½ mv2 = eV
 Applied centrifugal force to the moving ions is-
mv2 /r = H0eV
 On rearranging above equation we get-
m/e = H0
2r2/2V

6. TYPES OF IONS & PEAKS IN MS
Molecular Ion
 Ion formed by the loss of single electron at lowest
ionization potential from a molecule.
Fragment Ion
 Generated by the fragmentation of molecular ion in
the ionization chamber.
Metastable ion
 Some fragmentation may occurs during their flight
down the ion tube field free region instead of
ionization chamber which known as Metastable ions.
 They reach to the detector at masses lower than the
actual mass and gives broader peaks.
6

7. Quasi Molecular ion
 A protonated molecular ion.
 An ion formed by removal of one H-atom from
molecular ion is known as Quasi molecular ion.
Multiple charge ion
 Some double/triple charged ions are observed .
 Mainly occurs in ESI spectrum.
 Different m/z ratio.
7

8. Base Peak
 The most intense/tallest peak in the mass spectrum.
 It is due to the greatest relative abundance.
Isotope Peaks
 It is due to the presence of heavier isotope element
 It gives less intense peaks.
 Example- 1H1 (99.98 %) & 1H2 (0.015 %)
6C12 (99.89 %) & 6C13 (1.11 %)
8

9. DIFFERENT TYPES OF IONIZATION
9
Classification of Mass Spectroscopy
Desorption source Electrospray Ionization (ESI)
Matrix Assisted Laser Desorption
Ionization (MALDI)
Fast Atom Bombardment (FAB)
Field Desorption(FD)
Plasma Desorption
Gas Phase Source Electron Impact Ionization
Chemical Ionization
Field Ionization
Atomic Pressure
Ionization
Atmospheric Pressure Chemical
Ionization (APCI)
Atmospheric Pressure
Photoionization (APPI)

10. ELECTRON IMPACT IONIZATION
 This technique is used to convert the gaseous sample
into molecular ions.
 It is a hard ionization technique because it will produce
70 eV.
 Ionization potential of organic compounds are
approximately 8-15 eV.
Note: Operating pressure is 10-6 to 10-5 torr.
Potential different between grid 1 (G1) & grid 2
(G2) is 70 V.
Electron beam imparts the Kinetic energy of 70
eV.
Potential different from G3 and G4 is upto
8000V. 10

11.  Here, direct bombardment takes place, so due to
high energy this technique will produce large
number of fragment ions from molecular ions.
11

12. CHEMICAL IONIZATION(CI)
 Chemical ionization is a gaseous phase ionization
method, it is very important soft ionization technique.
 Fragmentation is less & gives intense peak of
molecular ions (more no. of m+).
 A carrier gas/ reagent gas is introduced into the
ionization source at slightly higher pressure. ie, CH4,
NH4 & Isobutane. 12

13. FIELD DESORPTION (FD)
 FD involves direct conversion of solid/liquid molecules
into gaseous ions.
 Here, low volatile samples are loaded on the surface of
the Carbon micro niddles by dipping in the sample.
13

14.  Ion formation takes place mainly by two mechanism-
Field ionization: e- are removed from the
species/analyte in a high electric field.
Cation Attachment: Here, cations will be attached
with the analyte molecule. i.e, H+ or Na+
 The positive ions will be repelled by the anode and
they will go towards the mass analyzer.
14

15. ELECTROSPRAY IONIZATION (ESI)
 ESI is a type of evaporative ionization technique
used to analyze the high molecular bio-molecules
labile & non-volatile compounds.
15

16.  A solution containing the sample molecule is sprayed
through the high voltage potential capillary by the help
of Nebulization gas.
 Sprayed droplets are ionized due to high voltage
potential at capillary.
 Heated disolvation gas will evaporated the solvent and
it will produce the Molecular ions.
 This technique is used to ionizes such as Proteins,
Peptides, Lipids, Oligosaccharides & neucleotides, etc.
16

17. FAST ATOM BOMBARDMENT (FAB)
 FAB is a soft ionization technique, used to determine
the molecular weight of the compound having size from
300 to 6000 Da.
Example- Peptides.
17

18. SAMPLE + MATRIX MATERIAL → SAMPLE - MATRIX MIXTURE
Characteristics of Matrix –
 It should be non volatile.
 It should be low vapour pressure liquids
 Examples- Glycerol, Triglycerol, 3- Nitrobenzyl alcohol,
di & tri-ethaloamine.
 Xe & Ar are accelerated neutral atom to be bombarded
to the sample-matrix mixture and ionize the sample
due to translational energy.
18

19. MATRIX ASSISTED LASER DESORPTION
IONIZATION (MALDI)
 MALDI is a soft ionization technique under
desorption ionization methods, which uses pulsed
LASER (Light Amplification by Stimulated
Emission of Radiation) beam.
19

20.  LASER Beams-
337 nm - Nitrogen LASER of UV range
355 nm – Frequency tripled Nd:YAG
(Neodynium, Ytterium, Aluminium,
Garnet)
326 nm – Frequency Quadrupoled Nd:YAG
294 µm – IR LASER produced by Er: YAG.
 0.5 to 20 nano second explosive time.
 It is used to determine the molecular weight of
peptides, protein, antibodies molecules, etc up to 300
KDa.
20

21. ATMOSPHERIC PRESSURE CHEMICAL
IONIZATION (APCI)
 APCI is a type of soft ionization technique based on the
mechanism of evaporation & atmospheric pressure.
 Actually, APCI is combination of Chemical Ionization
(CI) & Electrospray Ionization (ESI) with some
deviation.
21

22.  The sample is injected through the capillary, then it
will be converted into sprayed droplet & solvent
vapourized due to heating by N2.
 Corona discharged electrode will ionize the solvent
vapour molecule just like production of primary ions in
Chemical ionization.
 Thereby, collision & ion molecule charge transfer
between solvent & analyte .
 So, APCI may produce +ve & -ve ions.
 APCI is used to analyze Polar, thermostable
substance with mol. wt. less then 1500 Da.
22

23. ATMOSPHERIC PRESSURE
PHOTOIONIZATION (APPI)
 APPI is similar to APCI, but ionization is due to Photons
generated by UV light of Krypton Lamp.
 The sample solution will come through the heated
capillary & sprayed droplets will be formed due to N2.
23

24.  Desolvation of gas (heated N2) will be supplied which
will convert the sprayed droplets into the form of
vapours of analyte and solvent.
 The Krypton lamp emitted Photons having 10eV,
which is sufficient to ionized the target molecule.
 Photons will ionized the molecules in 3 mechanism-
i. Direct APPI
ii. Indirect APPI
iii. Dopant assist APPI
 Toluene is used as Dopant agent to increase the
percentage of molecular ion.
24

25. INTRUMENTATION OF MASS SPEC
25

26. 1. SAMPLE INSERTION-INLET SYSTEMS
 Organic compounds that have moderate vapor pressures at
temperatures up to around 300°C (including gases) can be placed in
connected via a reservoir an ampoule to the ionization chamber.
 Samples with lower vapor pressures (for example, solids) are inserted
directly into the ionization chamber on the end of a probe, and their
volatilization is controlled by heating the probe tip.
2. IONIZATION CHAMBER
 The electron bombardment is routinely used.
 Organic molecules react on electron bombardment in two ways:
either an electron is captured by the molecule, giving a radical
anion, or M + e → M+
an electron is removed from the molecule, giving a radical
cation. M → M+ + 2e
 Fragmentation of the molecular ion only reaches at higher bombardment
energies, and 70 eV is used for most organic compound. 26

27. 3. THE ANALYZER
 In a magnetic analyzer ions are separated on the basis of m/z
values,
 When ions are shot into the magnetic field of the analyzer, they
are drawn into circular motion by the field, and at equilibrium the
centrifugal force of the ion (mv2/r) is equalled by the centripetal
force exerted on it by the magnet (zBv), where r is the radius of
the circular motion and B is the field strength.
Types of mass analyzers-
 Magnetic field deflection
 Double focusing
 Quadrupole
 Time of Flight(ToF)
 FT-ICR (Fourier Transform Ion Cyclotron Resonance)
27

28. 4. THE DETECTOR-RECORDER
 The focused ion beam passes through the collector slit to the
detector, which must convert the impact of a stream of positively
charged ions into an electrical current. This must be amplified and
recorded, either graphically or digitally.
 The most common is the electron multiplier, which operates in a
manner similar to the photomultiplier detector.
 Some other detectors such as Faraday cups, Ion-to-Photons
detectors and Microchannel Plate detector.
5. DATA HANDLING
 The analog signal coming from the detector is first converted to
digital form in an analog-to-digital convertor (ADC), and the
digitized data are stored in computer hard disk.
 Computer-controlled instruments produce the mass spectral
data in several forms, either as a list of fragment ions or plotted
directly as a bar diagram.
28

29. QUADRUPOLE MASS ANALYZERS
 It consists of four cylindrical metal rods arranged in a
square parallel to the direction of ion beam.
 Hyperbolic can also be used.
 Radiofrequency (RF) or Direct Current (DC) voltage
is applied.
29

30.  Combination of RF & DC will generate oscillating
electrostatic field between the region of rods.
 Ions will enters into the mass analyzer, depending on the
ratio of RF amplitude & DC voltage oscillating electrostatic
field will be generated for ions.
 Ions will acquire oscillation in two ways-
If RF > DC, then larger ion will hit the detector first:
m+, m1
+, m2
+
If RF < DC, then smaller ion will hit the detector first:
m2
+, m1
+, m+
 When inappropriate m/z ratio of ions (other than 1 to 1000)
is present then they will undergo an unstable oscillation and
hit the rod; and ultimately will not reach to the detector.
 Ions with correct m/z ratio will undergo a stable oscillation
in cork screw trajectory and strike the detector and give
signal.
30

31. TIME OF FLIGHT (TOF )
 The Time of Flight (ToF) is based on the simple
concept that the velocities of two ion varies depending
on the mass of the ions.
 Ions should have created at instant & should have
some Kinetic energy.
 Lighter ions have the higher velocities as compare to
the heavier ion.
31

32.  When ions traveling towards the detector then
lighter ion will be strike the detector first due to
higher velocity.
32

33.  Kinetic energy of an ion accelerated through an electrical
potential will be-
zV = ½ mV2
 The velocity of an ion is path length (L) derived by time (t)-
V = L/t
Put the value of V in above equation we get-
zV= ½ mL2/t2
m/z = 2 V t2/ L2 Where, K=2V/L2
Therefore, m/z ∝ t2
 The m/z of the ions that strike the detector after travelling
the path length (L) is the directly proportional to the square
of the time (t).
 Ions required to ToF must be created in short & well defined
Pulses, so MALDI is used because it follows the condition.
 Note: It gives low resolution peaks.
33

34. METASTABLE IONS & ISOTOPIC
PEAKS
THE NATURE OF METASTABLE ION
 They have lower kinetic energy than have normal
ions, and arise from fragmentations that take place
during the flight down the ion tube rather than in the
ionization chamber.
 The exact position where they are formed in the tube
determines whether or not.
ION TUBE REGIONS
 The metastable ions produced in the ion tube, only a
fraction come to reasonable focus at the collector
34

35.  The first field-free region, in a double-focusing
instrument, lies between the ion source and the
electrostatic analyzer. (This region has no counterpart in
single-focusing instruments.)
 The second field-free region in a double-focusing
instrument lies between the electrostatic and magnetic
analyzers. (In single-focusing spectrometers the
corresponding region is between the ion source and the
magnetic analyzer.)
 Metastables produced in this region will be focused
reasonably sharply by the magnetic analyzer on the bases
of their masses and translational energies,
 The third field-free region lies between the magnetic
analyzer and the collector. Since no focusing takes place
in this region, a parent ion is already immutably on path.
 The metastable ion is detected at the same m/z value
as the parent ion. 35

36. CALCULATION OF METASTABLE ION m/z VALUES
 The apparent mass of a metastable ion A+ (m) can be
calculated fairly accurately from the masses of the parent
ion (m1) and the normal daughter ion A+ (m2) from the
equation
 This equation often gives an apparent mass 0.1- 0.4 mass
units lower than is in fact observed.
SIGNIFICANCE OF METASTABLE IONS
 The presence of a metastable ion in a mass spectrum is
taken as very good evidence that the parent ion undergoes
decomposition in one step to the daughter.
 So that it is of considerable mechanistic importance to
investigate metastable ions. 36

37. FRAGMENTATION PROCESSES & ITS
RULES
37

38. HOMOLYTIC CLEAVAGE (α-Cleavage)
 It is initiated by the radical site.
 Odd electron ions have an unpaired electron which is
having the tendency to create a new bond.
 If any bond is formed then energy will be released
and that energy is used to cleavage of other bond.
38

39. HETEROLYTIC CLEAVAGE
 The cleavage of C-X bond takes place which is
more difficult than C-C bond.
 Here, X= N, O, S, Cl, Br, etc.
 Positive charged carried by carbon atom instead of
Heteroatom.
39

40. MCLAFFERTY REARRANGEMENT
 It involves cleavage of α-β bond followed by transfer
of a γ H-atom.
 This McLafferty rearrangement leads to the
elimination of neutral atoms like amine, aldehyde,
ketone, unsaturated compounds, etc.
40

41. APPLICATION OF MASS SPEC
1. Determination of isotopic composition of element of a
compound.
2. Determination of structure of compound by observing its
fragmentation.
3. Measuring the gases in solution.
4. Characterization & sequencing of Protein.
5. Used in Carbon & Radioactive dating process.
6. Combination of mass spectrophotometer and gas
chromatograph is useful for detection of trace of impurities
in chemical constituents.
7. Application in Forensic analysis.
8. Application in metabolomics such as Cancer screening and
diagnosis.
9. Application in Drinking water testing & Soil contamination
assessment.
10. Application in Preclinical & Clinical Drug Development.
41

42. REFERENCES
 Kemp William; “Organic Spectroscopy”;3rd Edition 1991;
Reprint 2002; Published by- PALGRAVE; Houndmills,
Basingstoke, Hamshite; Fifth Avenue; New York; N.Y.
10010.
 Palvia Donald L.; Lampman Gary M.; Kriz George S.;
Vyvan James R.; “Introduction to Spectroscopy”; 5th Edition
2015; 200 First Stamford Place; 4th Floor; Stamford;
CT06902, USA.
 Hotlas Michael J.; “Mordern Spectroscopy”; 4thEdition
2004; John Wiley & Sons Ltd; The Atrium; Southern Gate;
Chichester; West Sussex; England.
 Sharma Y.R.; “Elementary Organic Spectroscopy”;
Multiedition; S.Chand & Company Ltd; Ram Nagar; New
Delhi-110055.
42

43. 43