高等食品分析 (Advanced Food Analysis) II. INSTRUMENTS FOR OPTICAL SPECTROSCOPY *Instruments for optical spectroscopy: Optical spectroscopic methods are
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1 *Instruments for optical spectroscopy: Optical spectroscopic methods are based on 6 phenomena: 1) Absorption, 2) Fluorescence, 3) Phosphorescence, 4) Scattering, 5) Emission and 6) Chemiluminescence. Typical spectroscopic instruments contain 5 components: 1) A stable source of radiant energy. 2) A transparent container for holding the sample. 3) A device that isolates a restricted region of the spectrum. 4) A radiation detector that converts radiant energy to a usable signal (usually electrical). 5) A signal processor and readout. Instrument configuration for the measurement of absorption, fluorescence, phosphorescence and scattering, requires an external radiation source. For absorption, the beam from the source passes through the sample directly into the wavelength selector, while in some instruments, the position of the sample and selector is reversed. For fluorescence, phosphorescence and scattering, the source induces the sample held in a container to emit characteristic radiation, which is measured at an angle (usually 90 ) with respect to the source. Emission and chemiluminescence spectroscopy do not required external radiation source due to sample itself the emitter. In emission spectroscopy, the sample container is an arc, a spark or a flame, which holds the sample and causes it to emit characteristic radiation. In chemiluminescence spectroscopy, the radiation source is a solution of the analyte held in a glass sample holder. Emission is brought about by a chemical reaction in which the analyte is directly or indirectly involved. 12
2 *Radiation sources: The problem of source stability was solved by double-beam designs, in which the intensities of the two beams are measured simultaneously so that the effect of fluctuations in the source output is largely canceled. 1. Continuous sources: Emit radiation that changes in intensity only slowly as a function of wavelength. Widespread use in absorption & fluorescence spectroscopy. For UV regions, the most common source is the deuterium (D or 2H) lamp, while high-pressure, gas-filled arc lamps containing argon, xenon or mercury serve when a particularly intense source is required. For visible regions, the tungsten filament lamp is used. IR sources are inert solids heated to 1500 to 2000 K, a temperature at which the max. radiant output occurs at 1.5 to 1.9 m. 13
3 *Radiation sources: 2. Line sources: Emit a limited number of bands of radiation; each band spans a very limited range of wavelengths. Wide use in atomic absorption, atomic and molecular fluorescence, and Raman spectroscopy. Refractometry and polarimetry also employ line spectra. Mercury and sodium vapor lamps provide relatively few sharp lines in the UV and visible regions. 14
4 Hollow cathode lamps and electrodeless discharge lamps are the most important line sources for atomic absorption and fluorescence methods. 3. Lasers: An acronym for light amplification by stimulated emission of radiation. Used in the UV, visible and IR regions. Important in Raman, molecular absorption and emission spectroscopy as well as Fourier transform IR spectroscopy. *Wavelength selection: 1) the use of filter, and 2) geometrical dispersion by means of a prism or grating. 1. Filters: Absorption and interference filters. Absorption filters are restricted to visible region. Interference filters, Fabry-Perot filters, are available to UV, visible and IR regions. Interference filters consist of a transparent dielectric (CaF 2 or MgF 2 ) that occupies the space between two semi-transparent metallic films. The array is sandwiched between two plates of glass or other transparent materials. The wavelength of the transmitted radiation is determined by the thickness of the dielectric layer. 2. Monochromators: Designed for spectra scanning. Spectral scanning is the process of varying the wavelength of radiation continuously over a considerable range. Monochromators for UV, visible and IR radiation are similar in mechanical construction that they employ slits, lenses, mirrors, windows, and gratings or prisms. 15
5 Components of a monochromator: i) An entrance slit providing a rectangular optical image. ii) A collimating lens or mirror producing a parallel beam of radiation. iii) A prism or a grating dispersing the radiation into its component wavelengths. iv) A focusing element reforming the image of the slit and focusing it on a planar surface called a focal plane. v) An exit slit in the focal plane isolating the desired spectra bands. In addition, entrance and exit windows for protection. Currently most monochromators are based on reflection gratings due to their cheaper to fabricate and providing better wavelength separation (linear dispersion). 16
6 *Sample containers: Required for all spectroscopic instruments except emission spectroscopy. Cells or cuvettes (or tubes in photometers) must be made of material that passes radiation in the spectra of interest. Quartz or fused silica is required for UV region (< 350 nm); both are transparent in the visible region and up to ca. 3 m in the IR region. Silicate glasses are used in the regions between 350 to 2000 nm. Plastic containers have also applied in the visible region. Crystalline NaCl is the most common substance employed for cell windows in the IR region. 17
7 *Radiation detectors: The ideal detector: A high sensitivity, a high signal-to-noise (S/N) ratio, a fast response time and a constant response over a considerable range of wavelengths. Two general types of radiation transducer: One responds to photons, the other to heat. Photon detectors (photoelectric or quantum detectors) used for UV, visible and NIR radiation have an active surface capable of absorbing radiation. Thermal detectors used for the detection of IR radiation respond to the average power of the incident radiation. The distinction between these two detectors is important because the shot noise limits the behavior of photon detectors, while thermal noise limits thermal detectors. Due to the low energy of IR photons and thus failing to cause photoemission of electrons, thermal detectors and detection based on photoconduction (limited to ca. 2.5 to 3 m) are required to measure IR radiation. 18
8 *Radiation detectors: Photon detectors: 1) Photovoltaic or barrier-layer cells, in which the radiant energy generates a current at the interface of the semiconductor layer and a metal. Used to detect radiation in the visible region A max. sensitivity is at ca. 550 nm; the response falls off to ca. 10% of the maximum at 350 and 750 nm. 2) Vacuum phototubes, in which radiation causes emission of electrons from a photoemissive solid surface (cathode). 3) Photomultiplier tubes containing a photoemissive surface as well as several additional surfaces that emit a cascade of electrons from the photosensitive area. Photomultipliers are highly sensitive to UV and visible radiation with extremely fast time responses. 4) Photoconductivity detectors, in which absorption of radiation by a semiconductor produces electrons and holes, thus leading to enhanced conductivity. The most sensitive detector used to monitoring radiation in the NIR region of ca to 3 m. Important in infrared Fourier transform instrumentation. 5) Silicon photodiodes, in which photons increase the conductance across a reverse-biased pn junction. More sensitive than a simple vacuum phototube but less sensitive than a photomultiplier tube. Photodiodes have spectral range from ca. 190 to 1100 nm. Multichannel photon detectors: Include linear photodiode array detectors, vidicons and charge-transfer detectors and consist of an array of tiny photosensitive detectors arranged in such a pattern that all elements of a beam of radiation dispersed by a grating can be measured simultaneously. 19
9 *Signal processors and readouts: The signal processor is an electronic device that amplifies the electric signal from the detector. It may perform mathematics on the signal such as differentiation, integration or conversion to logarithm. Readout device: Digital meters, the scale of potentiometers, recorders, cathode-ray tubes (CRT) and PC (IBM compatible or Macintosh). *Instrument designs: 3 basic designs and 2 subclasses each. 1) Temporal designs: Operate with a single detector and are termed as single-channel devices. Successive radiation bands are examined sequential in time. i) Nondispersive instruments: A photometer with a series of narrow band filters of appropriate wavelength. Used for quantitative determination of each of the alkali metals by injection of a sample solution into a flame. Elements are determined by selecting the wavelength using interchanging filters and turnable lasers. 20
10 *Instrument designs: ii) Dispersive instruments: Spectra are obtained by rotating the dispersing element mechanically or manually while monitoring the output of the detector. a) Sequential linear scan instruments: Contains a motor-driven grating system that sweeps the spectra at a constant rate, thus proving a wavelength scan based on time. b) Sequential slew scan instruments: Similar to the abovementioned except that it is programmed to recognize and remain at significant spectral features such as peaks until a suitable signal-to-noise ratio is attained. It is more complex and expensive than linear scan counterparts; provide data more rapidly and efficiently. 2) Spatial designs: Based on multiple detectors or channels to obtain information about different parts or elements of the spectrum simultaneously. i) Nondispersive instruments: Three slits arranged at different angles from the source can be used for the measurement of different elements Multiple filters and photomultiplier tubes are used. ii) Dispersive instruments: Spectrograph is the classic dispersive spatial instrument, in which a photographic plate or film, located at the focal plane of a monochromator. Direct-reading spectrometer consists of a monochromator with a series of slits and photomultiplier tubes along the focal plane. A new type of spatial dispersive instrument is based on the silicon diodes and charge-transfer detectors, providing as many as 4000 individual detectors. 21
11 Advantages: The speed of measurement in which spectra can be obtained without degradation in signal-to-noise ratio, and significantly smaller sample consumption. Disadvantages: More complex and more costly. 3) Multiplex designs: Single-channel devices in which all elements of the signal are observed simultaneously. i) Nondispersive instruments: Fourier transform devices for nuclear magnetic resonance, IR absorption and mass spectroscopy. ii) Dispersive instruments: Hadamard transform. *Fourier transform spectroscopy: 22
12 *Fourier transform spectroscopy: Advantages of the use of Fourier transform instruments: a) Through-put advantage or Jaquinot advantage: Due to few optical elements and no slits to attenuate radiation, the power of radiation reaching the detector is much greater than that in the dispersing instruments and much greater signal-tonoise ratios are observed. S/N is improved by [n measurement]1/2. b) Extremely high wavelength accuracy and precision: Leading to improved signal-to-noise ratios. c) Multiplex or Fellgett advantage: Achieved due to all elements the source reach the detector simultaneously. Leading to obtain an entire spectrum in a short period (1 s or less). Conventional spectroscopy can be termed frequency domain spectroscopy in that radiant power data are recorded as a function of frequency (or the inversely related wavelength). Time domain spectroscopy achieved by Fourier transform is concerned with changes in radiant power with time. The interconversion of time and frequency domain spectra becomes complex and mathematically tedious when more than a few lines are involved; the operation is only practical with a high-speed computer. The device for modulating optical radiation to obtain time domain spectra is a Michelson interferometer. The Michelson interferometer is a device that splits a beam of radiation into two beams of nearly equal power and then recombines them in such a way that intensity variations of the combined beam can be measured as a function of differences in the path lengths of the two halves. 23
13 24
14 Monochromator: Consists of a dispersing element (a prism or grating) together with two narrow slits to serve as entrance and exits ports for the radiation. Polychromator: Similar to monochromator but equipped with two or more exit slits, so that a number of wavelengths can be examined simultaneously. Spectroscope ( 分光鏡 ): An optical instrument used for the visual identification of emission lines, consists of a monochromator in which the exit slit is replace by an eyepiece that can be moved along with the focal plane. Colorimeter ( 比色計 ): An instrument for absorption measurements in which the human eye serves as the detector. Photometer ( 光電比色計 ): An instrument consists of a source of radiation, a filter and a photoelectric detector plus a signal processor and readout. Also referred as colorimeter or photoelectric colorimeter. Fluorometer ( 螢光計 ): Photometer designed for fluorescence measurements. Spectrograph ( 分光儀 ): An instrument similar to the monochromator, but with the focal plane is made up of a holder for a photographic film or plate. Spectrometer ( 分光計 ): A monochromator with a fixed slit in the focal plane. A spectrometer equipped with a photoelectric detector is called a spectrophotometer. Spectrophotometer ( 分光光度計 ): An instrument consists of a source of radiation, a monochromator and a photoelectric detector plus a signal processor and readout. Spectrofluorometer ( 分光螢光計 ): An instrument for fluorescence measurements consists of two monochromators, one to select the wavelength of excitation and the other to select the wavelength of fluorescence. 25
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