The given presentation is about mass spectrometry which is an analytical tool used to study molecular mass, structure etc.

1. MASS SPECTROSCOPY
ALIYA FIRDOUS
M.Sc. BIOTECHNOLOGY
III SEMESTER

2. BRIEF INTRODUCTION
 Spectroscopy is the study of interaction of matter with the electromagnetic radiation.
 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.
 The complete process involves the conversion of the sample into gaseous ions, with or
without fragmentation, which are then characterized by their mass to charge ratios
(m/z) and relative abundance.
 This technique basically studies the effect of ionizing energy on molecules. It depends
upon chemical reactions in the gas phase in which sample molecules are consumed
during the formation of ionic and neutral species.

3. Mass spectrometer

4. BASIC PRINCIPLE
 A mass spectrometer generates multiple ions from the sample under investigation, it then
separates them according to their specific mass-to-charge ratio (m/z), and records the relative
abundance of each ion type.
Mass spectra is used in two general ways:
 To prove the identity of two compounds.
 To establish the structure of a new compound.
The mass spectrum of a compound helps to establish the structure of a new compound in
several different ways:
 It can give the exact molecular mass.
 It can give a molecular formula or it can the presence of certain structural units in a
molecule.

5. Organic molecules are bombarded with electrons
Molecules converted into highly energetic
positively charged ions (molecular/ parent ions)
Further breakup of ions into smaller fragments
(fragment/ daughter ions)
Formed ions separated by deflection in magnetic
field, according to their mass to charge ratio
Mass spectrum: m/z vs intensity

6. Four key stages in the process of mass spectroscopy:
 Ionization
 Acceleration
 Deflection
 Detection
WORKING

7. IONIZATION
 The initial sample is first vaporized into gas and then ionized by ion source.
 Most common type of ionization: electro ionization.
 The sample is bombarded with electrons from a heated filament.
 When sample passes through electron stream, the high energy electrons in the stream knocks
out electrons from the sample to form ions.
 Ionization chamber is kept in a vacuum chamber so that the ions produced can progress
through the instrument without colliding with air molecules.
ACCELERATION
 The purpose of acceleration is to give all the ions same kinetic energy.
 It is a simple step in which the ions are placed between charged parallel plates.
 The ions then will be repelled by one plate and attracted by the other.
 There is a slit cut in the plate, the force of attraction and repulsion forces the ions through the
slit at an accelerated speed.
 The speed of acceleration can be adjusted by changing the charge on plates.

8. DEFLECTION
 The ions are deflected by the magnetic field surrounding the instrument.
 Amount of deflection is directly dependent upon mass and charge of the ions.
 Lighter ions or ions with more charge will deflect more than the heavier and less charged ones.
 The mass to charge ratio (m/z) is determined from the ions that hits the detector.
DETECTION
 When ion stream reaches the detector, it hits a metal wire. On hitting the wire, ions becomes
neutralized as electrons jumps from metal wire to the ion.
 The amplifier pucks up on this current being generated between the wire and the ion and
amplifies the signal being detected.
 Computer then picks up this and converts it to mass/charge ratio and a spectrum is produced

9. IONIZATION SEPARATION DETECTION
Detectors:
• Faraday cup
• Electron multiplier
• Photomultiplier conversion
dynode
Mass analyzers:
• Magnetic sector
• Quadrupole
• Time of flight
Ionization methods:
• MALDI
• Electrospray
• Electron ionization
DATA HANDLING
SAMPLE INLET
INSTRUMENTATION
Vacuum system

10. INSTRUMENTATION
1. SAMPLE INSERTION-INLET SYSTEM
 The sample must be introduced into vacuum.
 The sample must be vaporized prior to ionization so that proper ionization can take place.
 One approach to introducing sample is by placing a sample on a probe, which is then inserted
through a vacuum lock into the ionization region of the mass spectrometer. The sample is then
vaporized using one of the several desorption processes available.
 Another method of introducing the sample is capillary infusion. This delivers small quantities of
sample to the ionization chamber without disturbing the vacuum. The capillary can be a column
from a gas chromatograph or a liquid chromatograph.

11. 2.IONIZATION CHAMBER
 A charge is placed on the otherwise neutral molecule.
 The bombarding of the sample is done by the electrons. These electrons move between cathode and
anode. When the sample passes through the electron stream between the electrodes, electrons with
high energy knock electrons out of the sample and form ions.
 Organic molecules react on electron bombardment in two ways:
• Either an electron is captured by the molecule, M + e M+
• An electron is removed from the molecule, M M+ + 2e
 Several different methods are available for ionization:
1. Electron ionization
2. Electrospray
3. MALDI (Matrix-Assisted Laser Desorption/Ionization)
4. Fast atom bombardment
5. Atmospheric pressure chemical ionization
6. Chemical ionization and
7. Inductively coupled plasma.

12. 3. ANALYZER
 After ionization, ions that are in gaseous phase enter the mass analyzer.
 The analyzer separate ions within a selected range of mass-to-charge (m/z) ratios.
 To separate ions, different mass analyzers use magnetic fields, electric fields or the time taken by an
ion to move over a fixed distance.
 Types of mass analyzers:
1. Magnetic field deflection
2. Double focusing
3. Quadrupole
4. Time of flight (ToF)
5. FT-ICR (Fourier Transform Cyclotron Resonance)

13. DETECTOR
 The final element of the mass spectrometer is the detector.
 It generates a signal current from incident ions by generating secondary electrons which are further
amplified.
 The most commonly used detectors are the electron multiplier and scintillation counter. Both these
detectors convert the kinetic energy of incident ions into a cascade of secondary electrons.
 Some of the mostly used detectors:
1. Faraday cup
2. Electron multiplier
3. Photomultiplier conversion dynode
4. Micro channel plate detector

14. DATA HANDLING
 The analog signal coming from the detector is first converted to digital form in an ADC (analog to
digital converter) and the digitalized 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 spectrum.

15. APPLICATIONS
 Pharmaceutical analysis
 Bioavailability studies
 Drug metabolism studies, pharmacokinetics
 Characterization of potential drugs
 Drug degradation product analysis
 Screening of drug candidates
 Identifying drug targets
 Biomolecule characterization
 Proteins and peptides
 Oligonucleotides
 Environmental anlysis
 Pesticides on foods
 Soil and groundwater contamination
 Forensic analysis

16. REFERENCES
 Principles and techniques- Biophysical Chemistry- Avinash-Kakoli-Upadhyay
 Mass spectroscope for analysis in low mass range: Review of scientific
instruments
 Principles and practice of biological mass spectrometry. John Wiley
 https://www.technologynetworks.com/analysis/articles/how-a-mass-
spectrometer-works-types-of-instrumentation-and-interpreting-mass-spectral-
data
 http://www.premierbiosoft.com/mass-spectrometry.html

17. THANK YOU