Tiêu đề 31. MASS SPECTROSCOPY.pdf ✅

PHARMD GURU Page 1 INTRODUCTION:  Mass-spectrometry (MS) has evolved from the experiments and studies early in the 20th century that tried to explain the behavior of charged particles in magnetic and electrostatic force fields.  Well-known names from these early days are J. J. Thompson investigation into the behavior of ionic beams in electrical and magnetic fields (1912), A. J. Dempster directional focusing (1918) and F. W. Aston energy focusing (1919).  In this way a refinement of the technique was achieved that allowed important information concerning the natural abundance of isotopes to be collected.  Mass spectrometry’s characteristics have raised it to an outstanding position among analytical methods: unequalled sensitivity, detection limits, speed and diversity of its applications.  The first reliable commercial mass spectrometers were produced in the early forties for the quantitative determination of the several components in complex mixtures of crude oil.  In the beginning of the sixties the application of mass-spectrometry to the identification and structure elucidation of more complex organic compounds, including polymers and biomolecules, started.  Since then the technique has developed to a powerful and versatile tool for this purpose, which provides information partly complementary to and overlapping with other techniques, such as NMR.  In mass spectrometry, one generates ions from a sample to be analysed. These ions are then separated and quantitatively detected.  Separation is achieved on the basis of different trajectories of moving ions with different mass/charge ratios in electrical and/or magnetic fields.  The mass to charge ratio is symbolized by m/z, where the mass ‘m’ is expressed in unified atomic mass units and ‘z’ is the number of charges on the ion. Mass spectroscopy is useful for characterization of organic compounds in two ways: 1) It can give the exact molecular masses and thus, the exact molecular formulae. 2) It can show the presence of certain structural units and their points of attachment in the molecule, and thus gives idea about the structure of the molecule. MASS SPECTROSCOPY
PHARMD GURU Page 2 A mass spectrometer should always perform the following processes: 1) Produce ions from the sample in the ionization source. 2) Separate these ions according to their mass-to-charge ratio in the mass analyser. 3) Eventually, fragment the selected ions and analyse the fragments in a second analyser. 4) Detect the ions emerging from the last analyser and measure their abundance with the detector that converts the ions into electrical signals. 5) Process the signals from the detector that are transmitted to the computer and control the instrument through feedback. PRINCIPLE: FIG: MASS SPECTROMETER A small amount of a compound, typically one micromole or less, is evaporated. The vapour is leaking into the ionization chamber where a pressure is maintained of about 10—7 mbar. The vapour molecules are now ionized by an electron-beam. A heated cathode, the filament, produces this beam. Ionization is achieved by inductive effects rather than strict collision. By loss of valence electrons, mainly positive ions are produced. The positive ions are forced out of the ionization chamber by a small positive charge (several Volts) applied to the repeller opposing the exit-slit.
PHARMD GURU Page 3 After the ions have left the ionization chamber, they are accelerated by an electrostatic field of several hundreds to thousands of volts before they enter the analyser. The separation of ions takes place in the analyser, in this example a magnetic sector, at a pressure of about 10—7 mbar. A strong magnetic field is applied perpendicular to the motional direction of the ions. The fast moving ions then will follow a circular trajectory, due to the Lorentz acceleration, whose radius is determined by the mass/charge ratio of the ion and the strength of the magnetic field. Ions with different mass/charge ratios are forced through the exit-slit by variation of the accelerating voltage or by changing the magnetic-field force. After the ions have passed the exit-slit, they collide on a collector-electrode. The resulting current is amplified and registered as a function of the magnetic-field force or the accelerating voltage. The applicability of mass-spectrometry to the identification of compounds comes from the fact that after the interaction of electrons with a given molecule an excess of energy results in the formation of a wide range of positive ions. The resulting mass distribution is characteristic (a fingerprint) for that given molecule. FRAGMENTATION: The structural information is obtained from the fragmentation patterns of the mass spectrum. After a molecule is ionized, the molecular ion retains the excess ionization energy. If this excess energy is greater than the energy required to break a chemical bond, the molecule can fragment. The relative abundance of ions of various masses is characteristic of the particular compound under the specified conditions of the excitation and is known as its fragmentation pattern. Strong peak of the largest mass number is usually taken as the parent peak. It identifies the mass number of the compound itself. The fragmentation processes are typically categorized as direct cleavage or rearrangement. Cleavage reactions are simply the breaking of a bond to produce two fragments. These reactions usually produce an even electron ion (AB+ ). The even electron ion is detected at an odd m/z value (assuming no nitrogen) and a neutral odd electron radical. Since the radical is a neutral fragment it is not observed in the mass spectrum.
PHARMD GURU Page 4 Rearrangements are more complex reactions that involve both making and breaking bonds. These reactions are thermodynamically favorable because they require less energy. However, they also require a concerted mechanism that is not as kinetically favorable when compared to a simple cleavage reaction. Rearrangement ions easily identified because they are observed as odd electron ions with an even m/z value. These fragments often provide important clues about the location and identity of functional groups. FRAGMENTATION MODES: Fragmentation of the molecular ion takes place in following modes: 1) Simple cleavage. 2) Rearrangement reactions accompanied by transfer of atoms. SIMPLE CLEAVAGE: This process involves homolytic or heterolytic cleavage of a single bond.