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It is advantageous to other methods of analysis in its ability to separate and analyze mixtures of compounds. It is an extremely sensitive technique with current levels of accuracy within the range of one mass unit. Slide 3: In MS, the compound in gaseous state, is bombarded with a beam of electron to produce an ionic molecule (Molecular ion) of the original species. The molecular ion has the same MWt as of the original species, which further fragments. The resulting ions formed are then separated according to their masses (m/z). The spectrum produced is known as mass spectrum. This mass spectrum is a record of information regarding various masses produced and their relative abundances. m/e=H²r²/2V Different Components of a Mass spectrometer : Different Components of a Mass spectrometer The three processes are Ionisation Sorting of ions Detection Parts of a Mass Spectrometer : Parts of a Mass Spectrometer Inlet Source Analyser Detector Slide 6: The Sequence is Ionisation Acceleration Deflection Detection Instrumentation of Mass Spectrometer : Instrumentation of Mass Spectrometer The Essential Components are Sample Inlet System Ion Source (Ionisation chamber) Electrostatic accelerating system Magnetic field Ion separator (Analyzer) Ion Collecting System Vacuum System Data Handling Sample Inlet System : Sample Inlet System Handling Gas samples :- Involves transfer of the sample from small containers of known volume coupled to a mercury manometer. The sample is then expanded into a reservoir immediately ahead of the sample “leak”. Introduction of liquids :- Introduced in various ways :- 1) by break-off devices, 2) by touching a micropipet to a sintered glass disk under a layer of molten gallium, or 3) by hypodermic needle injection through a silicone rubber septum. Slide 9: Solids :- solids with a very low pressure can be introduced directly into an entrance to the ion chamber on a silica or platinum volatilized by gently heating until sufficient vapour pressure is indicated by the total ion current indicator. Gas ionization sources techniques : Gas ionization sources techniques are given below: Electron Impact (EI) Chemical Ionization (CI) Field Desorption (FD) Fast Atom Bombardment (FAB) Electrospray (ES) Laser Desorption (LD) Matrix Assisted Laser Desorption/ Ionization (MALDI) Slide 11: Electron Impact (EI) (1920) The original mass spectrometry (MS) ionization method and is still probably the most widely used. In EI, the sample is vaporized into the ion source, where it is impacted by a beam of electrons with sufficient energy to ionize the molecule. M + e → M + + 2e Radical cation EI is only appropriate for molecules that are volatile under the conditions of the ion source. Slide 12: Chemical Ionization (CI) (1965) The sample is combined with an unstable electron-poor species which has been created by electron bombardment. The electron-poor species stabilizes itself by donating a hydrogen ion to the species under study. H2 + electron bombardment → H2+ H2+ + H2 → H3+ + H CH4 + H3+ → CH5+ + H2 CH3CH2OH + CH5+ → CH3CH2OH2++ CH4 Slide 13: Electrospray Ionisation (ESI) (1985) -well-suited to the analysis of polar molecules ranging from 100 to 1,000,000 Da in molecular mass. During standard ESI The strong electric field converts the sample into highly charged droplets. Slide 14: In positive ionization mode, a trace of formic acid is often added to aid protonation of the sample molecules In negative ionization mode a trace of ammonia solution or a volatile amine is added to aid deprotonation of the sample molecules. Matrix Assisted Laser Desorption Ionisation (MALDI) deals well with thermolabile, non-volatile organic compounds especially those of high molecular mass used successfully in biochemical areas for the analysis of proteins, peptides, glycoproteins, oligosaccharides, and oligonucleotides Slide 15: MALDI is also a "soft" ionization method Fragmentation of the sample ions does not usually occur. generates singly charged molecular-related ions regardless of the molecular mass, hence the spectra are relatively easy to interpret Slide 16: MALDI is based on the bombardment of sample molecules with a laser (N2 @ 337nm) light to bring about sample ionization. The sample is pre-mixed with a highly absorbing matrix compound (e.g. sinapinic acid is common for protein analysis) . The matrix transforms the laser energy into excitation energy for the sample, which leads to sputtering of analyte and matrix ions. In this way energy transfer is efficient and also the analyte molecules are spared from excessive direct energy that may otherwise cause decomposition. Positive ionization is used in general for protein and peptide analyses. Negative ionization is used for oligonucleotides and oligosaccharides Slide 17: Fast Atom Bombardment (FAB) FAB remains a popular ionization technique for in volatile and/or thermally labile molecules. It works best for polar and higher molecular weight compounds. Generally FAB utilizes a fast moving beam of neutral atoms (typically Argon or Xenon at 8 kV) which bombard a metal target coated with a liquid matrix in which the sample has been dissolved. Typically [M+H]+ pseudo-molecular ions are formed, together with fragment ions at lower mass. The spectra can be complicated by the presence of : (i) [M+ Cat]+ ions (where Cat = Na, Li etc.); (ii) cluster ions Slide 19: Electron Impact Ion Source Electrostatic Accelerating System : Electrostatic Accelerating System The positive ions formed in the ionisation chamber are withdrawn by the electric field which exists between the first accelerator plate and the second repeller plate. A strong electrostatic field between accelerator and repeller plate of 400-4000V accelerates the ions of masses m1 m2 m3,…… to their final velocities. The ions which escape through slit having velocities and kinetic energies given by eV = ½m1v12 = ½m2v22 = ½m3v32………… When ever the mass spectrometer is started to record the spectrum, the second accelerator is charged to an initial potential of 4000V Magnetic field : Magnetic field As the accelerated particles from the electrical field enter magnetic field, the force of magnetic field requires them to move in a curved path. The radius of this curvature , r, is dependent upon the mass, m, the accelerating voltage, V, the electron charge, e, and the strength of the magnetic field, H. It is the two properties m/e and r upon which mass spectroscopy is based. Mass Analysers /Ion Separators : Mass Analysers /Ion Separators (a) Single Focusing Magnetic-Deflection (b) Double Focusing Mass Analyser (c) Quadrupole Mass Analyser (d) Ion trap Mass Analyser (e) Time-of-flight Mass Analyser Single Focusing Magnetic-Deflection : Single Focusing Magnetic-Deflection It is the most common type of separator. At a given accelerating voltage V, all the singly charged ions are given same kinetic energy. Kinetic energy=½mv2= eV In the magnetic field H, the charged particles experience magnetic force F, F = Hev and counterbalancing centrifugal force Hev=mv2/r on solving equation m/e=H2r2/2V Slide 24: Single Focusing Mass Spectrometer Slide 25: Single Focusing 60°- Sector Mass Spectrometer Slide 26: Single Focusing 180° Mass Spectrometer Double Focusing Mass Analyser : Double Focusing Mass Analyser This instrument is used for high resolution. In this type of instruments, two ion beams from independent sources pass side by side through a common mass analyser and detected by separate collectors. This type is used to compare sample with a standard or in different ionising conditions. The resolving power of this is of order of 30000. Such resolving capability enables high molecular weight fragments, which differ by only one mass unit. Slide 28: Double Focusing Mass Spectrometer Slide 29: Mattauch-Herzog Double Focusing Mass Spectrometer This model makes use of an Electric sector with an angle of 31.5˚. At this angle all ions enter the magnetic field at normal incidence which minimizes the edge effect. The magnetic sector has an angle of 135˚, which brings each ionic species to a focus at the far boundary of the field. Slide 30: Nier-Johnson Double Focusing Mass Spectrometer It makes use of 900 sectors with intermediate slit. Advantages : Advantages Very high reproducibility Best quantitative performance of all mass spectrometer analyzers High resolution High sensitivity High dynamic range Linked scan MS/MS does not require another analyzer Limitations : Limitations Not well-suited for pulsed ionization methods (e.g. MALDI) Usually larger and higher cost than other mass analyzers Linked scan MS/MS gives either limited precursor selectivity with unit product-ion resolution, or unit precursor selection with poor product-ion resolution Quadrupole Mass Analyser : Quadrupole Mass Analyser Quadrupole mass spectrometer, initially devised to separate uranium isotopes. Focusing of ions after accelerating from the ion source is affected by a quadrupole mass filter where they are separated according to mass and detected by an electron multiplier. The mass filter consists of a quadrant of four parallel circular tungsten rods which focus ions by means of an oscillating and variable radiofrequency field. Slide 34: Quadrupole Mass Spectrometer Time-of-flight Mass Analyser : Time-of-flight Mass Analyser The ions leave the accelerating field with different velocities depending on masses on magnetic focussing the ions get separated by changing their directions. If these ions are allowed to travel in a straight line through a magnetic field free region they will take different times to travel a given distance. Then the time of flight is measured t=k √m/e Slide 36: Time-of-flight Mass Spectrometer Slide 37: Ion trap Mass Spectrometer Ion collector : Ion collector The ion beam currents are of the order of 10-15 to 10-19 ampere. The generally employed ion collectors are photographic plates, Faraday cylinders, electron multipliers and electrometers. Vacuum system : Vacuum system A high vacuum is to be maintained. The inlet system is generally maintained at 0.015 torr, the ion source at 10-5 torr and analyzer tube at 10-7 torr or as low as possible. Oil diffusion and mercury diffusion pumps are commonly used in different types of combinations. Slide 40: THANK YOU Slide 41: THANK YOU You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.