Mass spectrometry is an analytical technique that identifies unknown compounds and quantifies known materials by measuring their mass-to-charge ratios. It works by ionizing chemical compounds, generating charged molecule fragments, and measuring their mass-to-charge ratios using techniques like time-of-flight analysis. The document discusses the principles, instrumentation including ion sources, mass analyzers, and detectors, applications in fields like proteomics and metabolomics, and guidelines for interpreting mass spectra.

1. Mass Spectrometry
By – Siddharth Kumar Sahu
M. Pharmacy

2. Content
 Introduction
 Principle
 Theory
 Instrumentation
 Ionisation Technique
 Applications

3. Mass spectrometry (MS) is used for
determining the masses of particles, to
determine the elemental composition of a sample
or molecule..
Mass spectrometry is a powerful analytical
technique that is used to identify unknown
compounds, to quantify known materials, and to
elucidate the structure and chemical properties of
molecules.
Introduction

4. The MS principle consists of ionizing chemical
compounds to generate charge molecules or
molecule fragments and measurement of their
mass-to-charge ratio by using the one of a variety
of techniques.
A mass spectrometer is a instrument that measures
the mass-to-charge (m/z) ratio of ions. (Ionization)
Ionization is a process of charging a molecule.
Molecules must be charged in order to measure
them using a mass spectrometer
Principle

5.  The relative abundance of positively charged
fragments of various mass-to-charge ratios is the
characteristic feature of the molecule that serve to
identify the substance.
 It is determined by
1
2
𝑚𝑣2
= e V
[kinetic energy= 1/2 mv2]
where, m=mass of ion
v=velocity of ion
V=accelerating voltage
e=charge on ion
Theory

6.  A mass spectrometer generates multiple ions from the
sample under investigation
 This molecular ion undergoes fragmentation.
 Each primary product ion derived from the molecular ion,
in turn, undergoes fragmentation, and so on
 The ions are separated in the mass spectrometer according
to their mass-to-charge ratio, and are detected in
proportion to their abundance.
 A mass spectrum of the molecule is thus produced.
 It displays the result in the form of a plot of ion abundance
versus mass-to-charge ratio.

7. Instrumentation
components are :-
Inlet system
Ion source /
Ionisation
chamber
Analyser
Detector
Data system
• Chemical Ionization
• Electro Spray Ionization (ESI)
• MALDI (Matrix Assisted Laser Desorption
Ionisation)
• Atmospheric Pressure Photo Ionization-
(APPI)
• Atmospheric-pressure chemical Ionization
(APCI)
• Quadrupole Mass Analyzer
• Orbitrap Analyzer
• TOF (Time of Flight)
• Electron Multipliers
• Faraday Cup
• Multichannel Plates.
• Array Detector
Vacuum system

8. Schematic diagram -

10.  Mass spectrometers can be divided into three fundamental parts, namely
the Ionization Source, the Analyzer and the Detector.
 Ion Source: For producing gaseous ions from the substance being
studied.
 Analyser: For resolving or separates the ions into their characteristics
mass components according to their mass-to-charge ratio.
 Detector System: For detecting the ions and recording the relative
abundance of each of the resolved ionic species
Instrumentation
Fig:- Representative diagram of Mass Spectrometry

11.  Chemical ionisation involves the ionisation of a reagent gas, such as
methane at relatively high pressure in a simple electron impact
source.
 the reagent gas ions collide with the analyte molecules producing
ions through gas phase reaction
 Reagent gas ionization: CH4 = CH4+ +e– (also CH3+, CH2+)
 ESI -The spray is changed into small droplets, and some of the
solvent is vaporized using a current of heated gas.
 The heated droplets are charged and eliminate most of the solvent.
 Due to ionic repulsion the droplets generate individual ions, most of
them still solvated.
 Multiple charged molecules can also be formed in this process, in
particular for compounds such as peptides.
 Atmospheric Pressure Ionization(API) Source forms gas phase
samples ions from sample molecules that are contained in solution.
 Atmospheric Press. Photoionization technique used to form ions
from the analyte molecule.
Ion Sources-

12. Schematic diagram of an electrospray ionization (ESI)
ESI Source-

13. MALDI (Matrix Assisted
Laser Desorption
Ionisation)
MALDI Technique

14. Mass Analyser
-The mass analyser separates the ions formed in the
ionization source according to their mass-to-charge
(m/z) ratios using some physical property e.g.
electric or magnetic fields.
1. Quadrupole Analyser
2. Orbitrap Analyser
3. TOF (Time Of Flight)

15. 1. Quadrupole type mass spectrometers separate the ions by
passing them along the central axis of four parallel equidistant
rods that have a fixed voltage (DC) and an alternating (RF) voltage
applied to them.
 The field strengths (voltage) can be set such that only ions of one
selected mass can pass through the Quadrupole, while all other
ions are deflected to strike the rods.

16. 2. Orbitrap Analyzer
The Orbitrap analyzer consists of a small electrostatic device into which
ion packets are injected at high energies to orbit around a central, spindle-
shaped electrode.
The image current of the axial motion of the ions is picked up by the
detector and this signal is Fourier transformed (FT) to yield high-
resolution mass spectra.
It capable of MS/MS using a Quadrupole for precursor-ion selection.
Fig. Orbitrap
Analyzer

17. 3. TOF Analysers separate ions by time without the use of an
electric or magnetic field. In a crude sense, TOF is similar to
chromatography, except there is no stationary/ mobile phase,
instead the separation is based on the kinetic energy and velocity
of the ions.
 Ions of the same charges have equal kinetic energies; kinetic
energy of the ion in the flight tube is equal to the kinetic energy
of the ion as it leaves the ion source.

18.  A tiny current is produced when the ion reaches the detector.
 The detector amplifies the signals which are then transmitted to
the data system to be represented as peaks on a mass spectrum
(Mass spectrum: graphic representation of ions separated
according to their m/z ratio)
Some detectors are-
 Electron Multiplier
 Faraday Cups
 Micro channel Plate Detectors
 Array Detector
Mass Detectors

19.  Continuous dynode electron multiplier
 An electron multiplier (continuous dynode electron
multiplier) is a vacuum-tube structure that multiplies incident
charges.
 In a process called secondary emission, a single electron can,
when bombarded on secondary emissive material, induce
emission of roughly 1 to 3 electrons.
 If an electric potential is applied between this metal plate and
yet another, the emitted electrons will accelerate to the next
metal plate and induce secondary emission of still more
electrons.
 This can be repeated a number of times, resulting in a large
shower of electrons all collected by a metal anode, all having
been triggered by just one
1. Electron Multiplier

20.  Faraday cup is part of a circuit where ions are the charge
carriers in vacuum and the faraday cup is the interface to the
solid metal where electrons act as the charge carriers (as in
most circuits).
 By measuring the electrical current (the number 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
2. Faraday Cup

21.  In order to interpret the mass spectrum, one should attain an
understanding of the ionisation process.
 To observe fragmentation pattern.
 1.The exact molecular weight: The molecular weight of a pure
compound from the identification of the parent peak. The
molecular weight one can determine molecular formula.
 2.The isotope effects : Heavy isotopes will exhibit peaks in a mass
spectrum at m/e one or more units higher than normal.
i.e., there will be small peaks at M+1 and M+2.
GENERAL RULES FOR INTERPRETATION
OF MASS SPECTRA

22.  3. The Nitrogen Rule
• In organic compounds , is a relationship between the valance and the mass
of the most common isotope for most elements.
• Even elements have an even valance.
• Odd elements have an odd valance.
• This leads to the ‘nitrogen rule.’ It assumes that we are limiting our elements
to C, H, halogens, O and N.
 4. Molecular formula
 The molecular formula may often be obtained by high-resolution
spectrometer measurements, because atomic weights are not integers. For
example, a distinction among CO, N2, CH2N and C2H4 is possible for
nominal weight.

23.  Mass spectrometry has both qualitative and quantitative uses.
“To identify unknown compound and to Quantify known compound”
QUALITATIVE APPLICATIONS
1. Determination of Molecular Weight.
2. Determination of Molecular Formula.
3. Determination of Partial Molecular Formula.
4. Determination of Structure of Compounds.
QUANTITATIVE APPLICATIONS
1. Determination of Isotope Abundance.
2. Determination of Isotope Ratio .
3. Differentiation between Cis and Trans Isomers.
4. Determination of Ion-molecule Reactions.
5. Identification of Proteins, Peptides, Polymers.
6. Detection of Impurity.
Applications

24. 1. Thermo Fisher Scientific User Guide & Thermo Orbitrap
Scigelova.
2. Some Research Papers on mass spectrometry & Metabolism.
3. A Textbook of
i. “Instrumental Method of Chemical Analysis”.
ii. B.K. Sharma, Instrumental Methods of Chemical Analysis, Goel
Publishing House.
iii. Beckett & Stenlake, Practical Pharmaceutical Chemistry, Vol.-II,
CBS Publishers & Distribution.
4. www.Slideshare.com
i. Rania Mohamed El—Sharkawy, ‘Advances In Medical Research `
From Molecular Medicine-to Clinical Application ` Mass
Spectrometric Techniques.
ii. K . Rakesh Gupta, Mass Spectrometry.
iii. Kommineni, Vidyachowdhary, Basic Principles In Interpretation Of
Mass Spectra.
Reference’s