Spectroscopic Techniques PDF

# Spectroscopic Techniques ## UV Visible Spectroscopy (Part I ) * Ultraviolet (UV) --->200-400 nm * Visible (VIS)----> 400-800 nm) * 100-200 nm---> absorbed by atmospheric gasses. Need to perform measurements under vacuum * Absorption of UV or Vis radiation by a molecule leads transition among ele...

# Spectroscopic Techniques ## UV Visible Spectroscopy (Part I ) * Ultraviolet (UV) --->200-400 nm * Visible (VIS)----> 400-800 nm) * 100-200 nm---> absorbed by atmospheric gasses. Need to perform measurements under vacuum * Absorption of UV or Vis radiation by a molecule leads transition among electronic energy levels of the molecule, it is also often called as electronic spectroscopy. ### Molecular orbitals (MO) * Molecular Orbital theory (MO) is the most important quantum mechanical theory for describing bonding in molecules * MO describes the whole molecule as one big system. * The orbitals from MO theory are spread out over the entire molecule rather than being associated with a bond between only two atoms. * Each MO can have a particular shape such that some orbitals have greater electron density in one place or another, but in the end the orbitals now "belong" to the molecule rather than any particular bond. ### Molecular orbitals (MO) * In general, this mixing of n atomic orbitals (AO) always generates n molecular orbitals (MO) * Bonding orbitals---> low energy * Antibonding---> High energy * Non-bonding orbital---> neither bonding nor antibonding * Carriers pair of unpaired electrons ## UV Visible Spectroscopy (part II ) * Since all these transitions require fixed amount of energy (quantized), an ultraviolet or visible spectrum of a compound would consist of one or more well defined peaks, each corresponding to the transfer of an electron from one electronic level to another. * Absorbance usually ranges from 0 (no absorption) to 2 (99% absorption), and is precisely defined in context with spectrometer operation **Amax: The wavelength at which a substance has its strongest photon absorption** ## Infrared (IR) spectroscopy * Measurement of the absorption of different frequencies of IR radiation by foods or other solids, liquids, or gases. * Wavelengths from 0.8 to 100 micrometers (μm) can be used for IR spectroscopy and are divided into * Near-IR (0.8-2.5 µm; 12,500-4000 cm-1) * Mid-IR (2.5–15.4 µm; 4000-650 cm-1) * Far-IR (15.4–100 μm; 650–100 cm-1) regions ## Infrared (IR) spectroscopy * The frequency for a particular bond is more or less independent of other bonds in the compound. * Therefore, determination of the frequencies in the infrared region which are absorbed by a compound gives information about the types of bonds which are present. * An infrared spectrometer analyses a compound by passing infrared radiation, over a range of different frequencies, through a sample and measuring the absorptions made by each type of bond in the compound. This produces a spectrum, normally a 'plot' of % transmittance against wavenumber. ### Different IRbands * IR bands can be classified as strong (s), medium (m), or weak (w), depending on their relative intensities in the infrared spectrum. * strong band ----> covers most of the y-axis * medium band ----> falls to about half of the y-axis * weak band --> falls to about one third or less of the y-axis. ### Infrared active bonds * Not all covalent bonds display bands in the IR spectrum. Only polar bonds show bands----> IR active * The intensity of the bands depends on the magnitude of the dipole moment associated with the bond in question: * Strongly polar bonds such as carbonyl groups (C=O) produce strong bands. * Medium polarity bonds and asymmetric bonds produce medium bands. * Weakly polar bond and symmetric bonds produce weak or non observable bands. ## Nuclear Magnetic Resonance (NMR) Spectroscopy (part 1) * Some atomic nuclei have a nuclear spin (I), and the presence of a spin makes these nuclei behave like a bar magnet. * If the number of neutrons and the number of protons are both even, then the nucleus has NO spin. * If the number of neutrons plus the number of protons is odd, then the nucleus has a half-integer spin (i.e. 1/2, 3/2, 5/2) * If the number of neutrons and the number of protons are both odd, then the nucleus has an integer spin (i.e. 1, 2, 3) *The overall spin, I, is important. A nucleus of spin / will have 2/ + 1 possible orientations. A nucleus with spin 1/2 will have 2 possible orientations. * 1H and 13C are the most important in NMR, both have spins of 1/2 ### Nuclear Energy Levels in Applied Magnetic Fields * Nuclear Magnetic Resonance (NMR) spectroscopy makes use of yet another type of quantized energy level. * In the presence of an applied external magnetic field. * Considering that the nuclei of some atoms behave as tiny bar magnets. Hence, when the atoms are placed in a magnetic field, their nuclear magnetic moment will have a preferred orientation, just as a bar magnet would behave. * The NMR-sensitive nuclei of general relevance to the food analyst have two permissible orientations. The energy difference between these allowed orientations depends on the effective magnetic field strength that the nuclei experience. * The effective magnetic field strength will itself depend on the strength of the applied magnetic field and the chemical environment surrounding the nuclei in question. * The energy spacings between permissible nuclear orientations, under usable external magnetic field strengths, are of the same magnitude as the energy associated with radiation in the radio frequency range. * In the absence of an external magnetic field, these orientations are of equal energy. If a magnetic field is applied, then the energy levels split. Each level is given a magnetic quantum number, m. ### Chemical shift * Chemical shift is a function of the nucleus and its environment. * It is measured relative to a reference compound. For 1H NMR, the reference is usually tetramethylsilane, Si (CH3)4 where the chemical shift is 0 ppm. ## Nuclear Magnetic Resonance (NMR) Spectroscopy (part II) * Electron with-drawing groups can decrease the electron density at the nucleus, deshielding the nucleus and result in a larger chemical shift. ### Integration * The area of a peak is proportional to the number of H that the peak represents * The integral measures the area of the peak * The integral gives the relative ratio of the number of H for each peak * Summing the integrals can give the empirical number of H and can be related to the molecular formula ### Spin-spin coupling * The proximity of other "n" H atoms on neighboring carbon atoms, causes the signals to be split into "n+1" peaks. * This is also known as the multiplicity or splitting of each signal. ## Mass Spectroscopy * The mass spectrometer vaporize the compounds and produce ions and separate those ions according to mass-charge ratio and detect those. * The three essential functions of a mass spectrometer are: * A small sample is ionized, usually to cations by loss of an electron------> The Ion Source * The ions are sorted and separated according to their mass and charge---->The Mass Analyzer * The separated ions are then measured, and the results displayed on a chart----->The Detector ### Ionization * The two methods commonly used to produce ions from thermally volatile material are electron impact (EI) and chemical impact (CI). ### Electron impact * electrons emitted from a hot filament are accelerated across the ionizer, * Energetic collisions between the accelerated electrons and gas phase sample species, M. * The collision results in the loss of one (or more) electrons, often forming radical cations. * Cause extensive fragmentation ### Chemical Impact * Reagent gas molecules are introduced at a concentration about 100X greater than the sample particles. The reagent molecules, R, are ionized using electrons (produced from a hot filament) to form a reagent ion, R+. * The reagent ions react with the sample molecules, S, to produce the ions, S+, to be sent to the mass analyzer ### Fragmentation * The molecular ions are energetically unstable, and some of them will break up into smaller pieces. The simplest case is that a molecular ion breaks into two parts - one of which is another positive ion, and the other is an uncharged free radical. * The uncharged free radical will not produce a line on the mass spectrum. * Only charged particles will produce a peak. ### Mass Spectrum * A plot of relative abundance of different positively charge fragments vs mass/charge ratio * The most intense ion is assigned an abundance of 100, and it is referred to as the base peak. * Most of the ions formed in a mass spectrometer have a single charge, so the m/z value is equivalent to mass itself. * The highest-mass ion in a spectrum is normally considered to be the molecular ion, and lower-mass ions are fragments from the molecular ion, assuming the sample is a single pure compound. ### Interpreting the mass spectra * Peak at 44 m/z----> CO₂ molecule * Peak at 28 m/z----> CO fragment * Peak at 16 m/z---> 0 * Peak at 89- complete molecule * Which fragments give 57 and 29?