高等食品分析 (Advanced Food Analysis) I. SPECTROSCOPIC METHODS *Instrumental methods: 1. Spectroscopic methods (spectroscopy): a) Electromagnetic radiation
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1 *Instrumental methods: 1. Spectroscopic methods (spectroscopy): a) Electromagnetic radiation (EMR): γ-ray emission X-Ray absorption, emission, fluorescence and diffraction Vacuum ultraviolet (UV) absorption Molecular absorption spectroscopy (UV, visible & near IR) Atomic spectroscopy (absorption, emission & fluorescence) Molecular luminescence: Fluorescence, phosphorescence and chemiluminescence. Infrared absorption spectroscopy Raman scattering: Raman spectroscopy Microwave absorption Electron spin resonance (ESR) spectroscopy Nuclear magnetic resonance (NMR) spectroscopy b) Ions: Mass spectrometry c) Electrons: Electron spectroscopy d) Sound waves: Acoustic spectroscopy 2. Electrometric or electrochemical methods: Potentiometric methods ( 電位測定法 ) Coulometric methods ( 電量測定法 ) Voltammetry ( 電壓電流測定法 ) Thermal Methods: Thermogravimetric methods (TG) ( 熱重量分析法 ) Differential thermal analysis (DTA) ( 示差熱分析法 ) Differential scanning calorimetry (DSC) ( 示差掃描熱分析法 ) 2
2 *Instrumental methods: 3. Methods of separation (chromatography): Plane chromatography (PC and TLC) Liquid phase chromatography on open columns Gas chromatography (GC) High performance liquid chromatography (HPLC) Other separation methods: Supercritical fluid chromatography (SFC) Capillary electrophoresis (CE) 3
3 *Electromagnetic radiation (EMR): 1. Duality: i) Waves: In terms of wavelength (λ), frequency (v), velocity (c) and amplitude (A). ii) Photons: A stream of discrete particles or wave packets of energy. The energy of a photon is proportional to the frequency of the radiation. The dual view of radiation as particles and as waves are not mutually exclusive, rather are complementary. 4
4 *Electromagnetic radiation (EMR): 2. Properties: i) Frequency (v): The number of oscillation per second. Hertz (1 Hz = 1 cycle per sec) v = c/ = c v (1) ii) Wavelength (λ): The linear distance between any two equivalent points on successive waves (maxima or minima). λ = c/v (2) Å nm (m ) m m X-ray & short UV Vis & UV IR iii) Wavenumber ( v ) : Used in IR spectroscopy, also called kaiser; the reciprocal of the wavelength in cm. v = 1/λ or λ v = 1 (3) iv) Velocity (c): x 108 m/sec for radiation traveling through a vacuum. v) Amplitude (A): The length of electric vector at a maximum in the wave. 5
5 *Electromagnetic radiation energy: E = hv = hc/ = hc v (4) Where h: Planck's constant, J s. For example: λ= 200 nm, E =? E = ( J s)( m/s)/( m) = J ( cal/j) = cal ( photons/mol) = 143 kcal/mol For nuclear radiations and x-rays, the energy of photons in ev, kev or MeV is used. 1 ev = x J => v = Hz λ = m in vacuo *Electromagnetic spectrum: The electromagnetic spectrum is divided into several regions based on the methods required to generate and detect the various kinds of radiation. The spectrum of radiant energy is attributable to atomic and molecular excitations and transitions are as follows: γ-ray Nuclear transitions X-Ray K-and L-shell electrons Far UV Middle shell electrons Near UV & Vis Valence electrons Near & mid IR Molecular vibrations (stretching & bending) Far IR Molecular rotations & low-lying vibrations Microwave Molecular rotations Radio waves Nuclear magnetic resonance 6
6 7
7 *Interactions of EMR with matter: Absorption, emission, fluorescence and phosphorescence. 1. Absorption: Absorption of radiation promotes particles, from their ground state to one or more higher-energy excited states. i) Atomic absorption: UV and visible wavelengths have sufficient energy to cause transitions of the outer-most or bonding electrons only. X-Ray frequencies are capable of interacting with electrons closest to nuclei of atoms (innermost electrons). ii) Molecular absorption: E = E electronic + E vibrational + E rotational (5) Where: E electronic > E vibrational > E rotational Transitions between electronic levels are found in the UV & vis. regions; those between vibrational levels lie in the near and mid-ir and can also be observed with Raman techniques. Absorption in the far IR and microwave regions corresponds to low-energy rotation. iii) Absorption induced by a magnetic field: Nuclear magnetic resonance (NMR): Absorption of radio waves at 30 to 500 MHz (λ = 1000 to 60 cm) by nuclei in a magnetic field. Electron spin resonance (ESR): Absorption of microwave at ca MHz (λ = 3 cm) by electrons in a magnetic field. iv) Relaxation processes: Non-radiative relaxation involves the loss of the excitation energy by converting to kinetic energy in collision with other molecules. => Resulting a minute temp increase. Relaxation can also occur by the emission of fluorescence or phosphorescence. 8
8 *Interactions of EMR with matter: 2. Emission: EMR is produced when excited particles (ions, atoms and molecules) relax to lower energy levels by giving up their excess energy as photons. Excitation can be brought about by bombardment with electrons or other elementary particles, exposure to a high-potential alternating current spark, heat treatment in an arc or a flame, or absorption of EMR. The resulting spectrum is discontinuous, termed a line spectrum. Continuous spectra result from excitation of: i) solids or liquids, in which the atoms are so closely packed, and ii) complicated molecules possessing many closely related energy states. Continuous spectra are frequently used as radiation sources in spectrophotometry, while line spectra are allowed to identify and determine the emitted species. 3. Fluorescence: The emitted radiation is complete after ca sec and have less energy per photon, and hence a longer wavelength than the exciting radiation. Many compounds, when irradiated with UV radiation, fluoresce in the visible region. Fluorescence also occurs in atomic spectra, in the UV, IR and x-ray regions. Resonance fluorescence describes a process in which the emitted radiation is identical in frequency to the radiation employed for excitation, and is commonly produced by atoms in the gaseous state. Fluorescence and phosphorescence are most easily observed at 90 angle to the excitation beam. 9
9 *Interactions of EMR with matter: 4. Phosphorescence: The emitted radiation takes places over the periods longer than 10-5 sec and may continue for minutes or even hours. In some molecules, a non-radiative transition from an excited singlet state to the corresponding triplet level. Phosphorescence can be radiated when the molecules release the excess energy to revert to the ground state. *Thermal radiation: Any substance at a temperature above absolute zero emits radiation due to the thermal motion of its electrons. The theory is worked out in terms of an ideal emitter called a blackbody. The blackbody radiation is more characteristic of the temp of the emitting surface than the material of which that surface is composed. The blackbody radiation is distributed as a function of wavelength for various temperatures. Wien displacement law: λ max T = (6) Where max in nanometers (nm) and T in kelvins (K). Due to its reciprocal dependence on temp, the incandescence sources are practical only in the visible and infrared regions, while very high temp is needed for UV coverage. Example: Body temperature: 37 C, λ max =? λ max = /( ) = 9348 nm (IR region) = 1070 cm-1 10
10 *Thermal radiation: Example: UV range: nm, T =? /200 = K /400 = 7245 K *Small amount of pure compounds can be isolated from complex mixtures by chromatography for further identification and structure elucidation. *Spectra used for the identification of organic compounds: IR, NMR and mass spectra. *Two characteristics of these techniques: 1. Rapid and most effective in mg and g level. 2. Neither time nor material is enough to employ classical manipulations such as boiling point, refractive index, functional groups tests and derivative preparation. 11
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