Mass spectroscopy

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

6. 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.

7. 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

8. 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

9. 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.

10. 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: Electron ionization Electrospray MALDI (Matrix-Assisted Laser Desorption/Ionization) Fast atom bombardment Atmospheric pressure chemical ionization Chemical ionization and Inductively coupled plasma.

11. 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: Magnetic field deflection Double focusing Quadrupole Time of flight ( ToF ) FT-ICR (Fourier Transform Cyclotron Resonance)

12. 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: Faraday cup Electron multiplier Photomultiplier conversion dynode Micro channel plate detector

13. 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.

14. 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

15. 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

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