Mass spectrometry is an extremely valuable analytical technique in which the molecules in a test sample are converted into gaseous ions that are subsequently separated in a mass spectrometer according to their mass-to-charge ratio (m/z) and detected .

1. MASS
SPECTROMETRY

2. MASS SPECTROMETRY
Mass spectrometry is an extremely valuable
analytical technique in which the molecules
in a test sample are converted into gaseous
ions that are subsequently separated in a mass
spectrometer according to their mass-to-charge
ratio (m/z) and detected .

3. Technique can be used for …….
 Qualitative and quantitative analysis
 Providing information about the mass of atoms and molecules
 Molecular structure determination (organic & inorganic)
 Identification and characterization of materials

4. COMPONENTS OF A MASS SPECTROMETER
 - Sample input system
 - Ionization source
 - Mass analyzer
 - Detector
 - Vacuum pumps
 - Computer based data
acquisition and processing
system

5. MASS SPECTRUM
The mass spectrum is a plot of a relative abundance of the
ions at each m/z ratio
In most cases , the nascent molecular ions of the analyte
produce fragment ions by cleavage of the bond and the
resulting fragmentation pattern constitute the mass spectrum

6. PRINCIPLE AND INSTRUMENTATION
1.) IONISATION
 Ionisation is a process of charging a molecule .
 The sample molecule must be charged in order to measure
them using a mass spectrometer
 The atom is ionised by knocking one or more electrons off to
give a positive ion .
 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. PRINCIPLE AND INSTRUMENTATION
2.) ACCELERATION
The ions are accelerated so that they all have the same kinetic
energy
Ionisation
chamber at +1000v
The positive ions are repelled away from the positive
Ionisation chamber and pass through 3 slits with voltage in
the decreasing order
 All the ions are accelerated into a freely focused beam
Ion beam

8. PRINCIPLE AND INSTRUMENTATION
3.) DEFLECTION
The ions in the deflected by
a magnetic field according
to their masses
 Different ions are deflected
by the magnetic field by
different amounts .
Amount of deflection depends
upon :
1) The mass of the ion
2) The charge on the ion

9. PRINCIPLE AND INSTRUMENTATION
4.) DETECTION
 The beam of ions passing through
the machine is detected electrically .
 Ions leaves space in metal by
neutralizing it , & the electrons in
the wire shuffle along to fill it .
The flow of electrons in the wire
is detected as an electric current
which can be amplified & recorded

12. MASS SPECTROMETRY FOR PROTEIN ANALYSIS

13. THE TWO MOST COMMON MEATHOD OF MASS
SPECTROMRTRY FOR PROTEIN ANALYSIS

15. MALDI-TOF MS
The protein sample is mixed with a chemical matrix that
includes a light absorbing substance excitable by a laser .
The laser pulse is used to excite the chemical matrix , creating
a microplasma that transfers a energy to protein molecule
substance excitable by a laser . molecule in the sample
ionizing them and ejecting them into the gas phase .

16. ELECTROSPARY IONISATION MASS SPECTROMETRY
Electrospray is an especially soft ionisation method , capable of generating molecular ions
(without fragmentation) from biological macromolecules present in aqueous solution

17. esI-MS
A solution of macromolecules is sprayed in the form of fine
droplets from a glass capillary under the influence of a strong
electric field
The droplets pick up the positive charge as they exit the
capillary
 Evaporation of the solvent leaves multiply charged molecules

18. Analysis and Separation of Sample Ions
The mass analyzer separates the ions formed in the ionization source according to their
mass-to-charge ratios using some physical property e.g. Electric or magnetic fields .
Time Of Flight Mass Analyzer
In MALDI-TOF the ions produced by a
shorter laser pulse travel across a flight
tube , arriving at different time at the
detector .

19.  - Employs two or more stages of mass analyzers
 - Example is two quadrupoles coupled in series
 - First analyzer selects ion (precursor ion) and second analyzer
selects the fragments of the precursor ion
 - Used to obtain more information about the structure of
fragment ions
 - Fragment ions may be dissociated into lighter fragment ions or
converted into heavier ions by reaction with neutral molecule
MS – MS (TANDEM MS) INSTRUMENTS

20. Applications of mass spectrometry to study proteins
Identifying unknown proteins
 Protein sequencing (peptides are sequenced by generating
multiple sets of proteins
Identification of chemical modification (post translational
modification in proteins after synthesis)
 Identification of organisms ( identifying bacteria by finger
printing proteins )