Fluorescence spectrophotometry instrumentation

We have seen the relationship between absorption spectrophotometry and spectrofluorometry. A similar relationship exists between atomic absorption spectrophotometry and atomic fluorescence spectrophotometry. In atomic fluorescence, the flame retains its role as a source of atoms these atoms, however, are excited by an intense source of radiation and their fluorescent emission is assayed at an angle of 90° in a manner similar to that of spectrofluorimetry. Lack of sufficiently intense source for many elements has been the limitation of this technique, however, with time instrumental developments are overcoming this problem. High intensity hollow-cathode lamps, or xenon or mercury discharge lamps are used. 

In the search for unknown drugs and/or pharmaceuticals in body fluids, a combined instrumental approach is especially helpful. Table III shows our extraction scheme. For drug analysis, steam distillation is usually omitted. Different extraction steps isolate the strongly acidic, weakly acidic, neutral, basic and amphoteric compounds in separate fractions. All are analyzed by UV-spectro-photometry, in organic solution and (with the exception of the neutral extract) also in water at 3 different pH-values. Thin- layer chromatography, color tests, IR- and fluorescence-spectrophotometry may yield additional information. Fractions of interest are analyzed by GC-MS. 

Cadmium in acidified aqueous solution may be analyzed at trace levels by various instrumental techniques such as flame and furnace atomic absorption, and ICP emission spectrophotometry. Cadmium in solid matrices is extracted into aqueous phase by digestion with nitric acid prior to analysis. A much lower detection level may be obtained by ICP-mass spectrometry. Other instrumental techniques to analyze this metal include neutron activation analysis and anodic stripping voltammetry. Cadmium also may be measured in aqueous matrices by colorimetry. Cadmium ions react with dithizone to form a pink-red color that can be extracted with chloroform. The absorbance of the solution is measured by a spectrophotometer and the concentration is determined from a standard calibration curve (APHA, AWWA and WEF. 1999. Standard Methods for the Examination of Water and Wastewater, 20th ed. Washington, DC American Public Health Association). The metal in the solid phase may be determined nondestructively by x-ray fluorescence or diffraction techniques. 

Calcium may be analyzed by several instrumental techniques such as atomic absorption and emission spectrophotometry, ICP—MS, neutron activation, and x-ray fluorescence and diffraction methods. For all these techniques,... 

Chromium metal may be analyzed by various instrumental techniques including flame and furnace AA spectrophotometry (at 357.9 nm) ICP emission spectrometry (at 267.72 or 206.15 nm), x-ray fluorescence and x-ray diffraction techniques, neutron activation analysis, and colorimetry. 

Palladium metal is digested in aqua regia, evaporated to near dryness. This is followed by addition of concentrated HCl and distdled water and the solution is warmed until dissolution is complete. The solution is aspirated directly into an air-acetylene flame. Palladium is detected by flame-AA spectrophotometry. Other instrumental techniques such as ICP/AES, x-ray fluorescence, and neutron activation analysis are used also. 

Numerous methods have been published for the determination of trace amounts of tellurium (33—42). Instrumental analytical methods (qv) used to determine trace amounts of tellurium include atomic absorption spectrometry, flame, graphite furnace, and hydride generation inductively coupled argon plasma optical emission spectrometry inductively coupled plasma mass spectrometry neutron activation analysis and spectrophotometry (see Mass SPECTROMETRY Spectroscopy, OPTICAL). Other instrumental methods include polarography, potentiometry, emission spectroscopy, x-ray diffraction, and x-ray fluorescence. 

Frequently industrial hygiene analyses require the identification of unknown sample components. One of the most widely employed methods for this purpose is coupled gas chromatography/ mass spectrometry (GC/MS). With respect to interface with mass spectrometry, HPLC presently suffers a disadvantage in comparison to GC because instrumentation for routine application of HPLC/MS techniques is not available in many analytical chemistry laboratories (3). It is, however, anticipated that HPLC/MS systems will be more readily available in the future ( 5, 6, 1, 8). HPLC will then become an even more powerful analytical tool for use in occupational health chemistry. It is also important to note that conventional HPLC is presently adaptable to effective compound identification procedures other than direct mass spectrometry interface. These include relatively simple procedures for the recovery of sample components from column eluate as well as stop-flow techniques. Following recovery, a separated sample component may be subjected to, for example, direct probe mass spectrometry infra-red (IR), ultraviolet (UV), and visible spectrophotometry and fluorescence spectroscopy. The stopped flow technique may be used to obtain a fluorescence or a UV absorbance spectrum of a particular component as it elutes from the column. Such spectra can frequently be used to determine specific properties of the component for assistance in compound identification (9). 

Spectrometers that use phototubes or photomultiplier tubes (or diode arrays) as detectors are generally called spectrophotometers, and the corresponding measurement is called spectrophotometry. More strictly speaking, the journal Analytical Chemistry defines a spectrophotometer as a spectrometer that measures the ratio of the radiant power of two beams, that is, PIPq, and so it can record absorbance. The two beams may be measured simultaneously or separately, as in a double-beam or a single-beam instrument—see below. Phototube and photomultiplier instruments in practice are almost always used in this maimer. An exception is when the radiation source is replaced by a radiating sample whose spectrum and intensity are to be measured, as in fluorescence spectrometry—see below. If the prism or grating monochromator in a spectrophotometer is replaced by an optical filter that passes a narrow band of wavelengths, the instrument may be called a photometer. 

Section I covers the more conventional equipment available for analytical scientists. I have used a unified means of illustrating the composition of instruments over the five chapters in this section. This system describes each piece of equipment in terms of five modules - source, sample, discriminator, detector and output device. I believe this system allows for easily comparing and contrasting of instruments across the various categories, as opposed to other texts where different instrument types are represented by different schematic styles. Chapter 2 in this section describes the spectroscopic techniques of visible and ultraviolet spectrophotometry, near infrared, mid-infrared and Raman spectrometry, fluorescence and phosphorescence, nuclear magnetic resonance, mass spectrometry and, finally, a section on atomic spectrometric techniques. I have used the aspirin molecule as an example all the way through this section so that the spectral data obtained from each... 

In contrast to absorption spectrophotometry, fluorescence and phosphorescence spectrometry involve the recording of both an excitation and an emission spectrum the instruments used are called spectrofluorometers or spectrophosphorimeters. 

Wcssman reviewed a number of instruments used for uranium analyses and ranked their relative measurement sensitivities [32]. The methods include atomic absorption spectrophotometry, colorimetry, neutron bombardment, fission etched track detectors, fluorimetry, laser-induced fluorescence spectrometry, a-spectrometry, isotope dilution mass spectrometry, and spark source mass spectrometry. The majority of urinary bioassay measurements have been performed by fluorimetry, while environmental survey and baseline measurements have been performed by fluorimetry, a-spectrometry, and induced coupled plasma source mass spectrometry. 

Analysis of clays and ceramics, using selected reagents and spectrophotometry to complete an analysis in about 8 hours, instead of several days by the standard wet chemical technique. Modern instrumental techniques such as X-ray fluorescence can produce complete analysis in minutes. 

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