Analytical Applications of Fluorescence

With the development of the photomultiplier tube the measurement of very low light intensities has become relatively simple and the photoelectric recording of fluorescence emission spectra can now compete in terms of sensitivity with the less convenient photographic method. During the last decade the development of the experimental technique has gained considerable impetus as a result of the requirements of analytical chemists for methods of extreme sensitivity. A variety of spectro-fluorimeters have been described in the literature and commercial instruments of high sensitivity are also available. Recent reviews1-2 deal with the principles and analytical applications of fluorescence spectrometry and a textbook of biochemical applications has been published.2... 

Molecular fluorescence and, to a lesser extent, phosphorescence have been used for the direct or indirect quantitative analysis of analytes in a variety of matrices. A direct quantitative analysis is feasible when the analyte s quantum yield for fluorescence or phosphorescence is favorable. When the analyte is not fluorescent or phosphorescent or when the quantum yield for fluorescence or phosphorescence is unfavorable, an indirect analysis may be feasible. One approach to an indirect analysis is to react the analyte with a reagent, forming a product with fluorescent properties. Another approach is to measure a decrease in fluorescence when the analyte is added to a solution containing a fluorescent molecule. A decrease in fluorescence is observed when the reaction between the analyte and the fluorescent species enhances radiationless deactivation, or produces a nonfluorescent product. The application of fluorescence and phosphorescence to inorganic and organic analytes is considered in this section. 

Molecular Fluorescence Spectroscopy Photometric Titrations Analytical Applications of Interferometry Vol. 9 Ultraviolet Photoelectron and Photoion Spectroscopy... 

In a later work, Stokes established the relationship between the intensity of fluorescence and the concentration, pointing out that the emission intensity depended on the concentration of the sample (analyte), but that attenuation of the signal occurred at higher concentrations as well as in the presence of foreign substances. He actually was the first to propose, in 1864, the application of fluorescence as an analytical tool, based on its sensitivity, on the occasion of a conference given previously in the Chemical Society and the Royal Institution, and entitled On the Application of the Optical Properties to the Detection and Discrimination of Organic Substances [5],... 

In addition to these research applications of fluorescence, there is a continuing use of fluorescence detection to replace analytical methods based on radioactivity, as can be judged from the recent books and conferences on fluorescence sensing methods. (7 n) These emerging applications of fluorescence can be seen by the growth and introduction of improved methods for immunoassays, enzyme-linked immunoassays... 

Although the majority of analytes do not possess natural fluorescence, the fluorescence detector has gained popularity due to its high sensitivity. The development of derivatization procedures used to label the separated analytes with a fluorescent compound has facilitated the broad application of fluorescence detection. These labeling reactions can be performed either pre- or post-separation, and a variety of these derivatization techniques have been recently reviewed by Fukushima et al. [18]. The usefulness of fluorescence detectors has recently been further demonstrated by the Wainer group, who developed a simple HPLC technique for the determination of all-trani-retinol and tocopherols in human plasma using variable wavelength fluorescence detection [19]. 

An important application of fluorescence is in immunoassays, which employ antibodies to detect analyte. An antibody is a protein produced by the immune system of an animal in response to a foreign molecule called an antigen. The antibody recognizes the antigen that stimulated synthesis of the antibody. The formation constant for the antibody-antigen complex is very large, whereas the binding of the antibody to other molecules is weak. 

C. Zander, J. Enderlein, and R. Keller, eds., Single Molecule Detection in Solution (New York Wiley, 2002) R. A. Keller, W. P. Ambrose, A. A. Arias, H. Cai, S. R. Emory, P. M. Goodwin, and J. H. Jett, Analytical Applications of Single-Molecule Detection, Anal. Chem. 2002, 74, 317A J. Zimmermann, A. van Dorp, and A. Renn, Fluorescence Microscopy of Single Molecules, ... 

Analytical applications of the malonaldehyde and Maillard fluorescent product formation to assessment of abuse status of oxidizing oils, relative effectiveness of antioxidants, and sugar-amine browning potential are available. 

Work conducted by Tiller and Jones (1997) demonstrated that the fluorescence of PAHs decayed over time under both under anoxic and oxic conditions. Typically, however, the presence of dissolved oxygen had a more pronounced influence on baseline fluorescence decay for all the PAHs studied. Moreover, certain PAHs (pyrene and anthracene) were more susceptible to this phenomenon than others. To date a mechanism to explain this phenomenon has not been identified, but it is probably a combination of complex pathways including the reaction of the analyte with reactive oxygen species formed from the excited triplet state DOM and the direct photolysis of the analyte by the excitation light source. Thus, the application of fluorescence quenching for measuring Kdom is probably limited to systems, which can be analyzed under anoxic conditions. 

Another type of analytical application of sulfuric acid oxidation has been developed recently, based on the intense fluorescence which appears when the sulfuric solutions containing oxidized pheno-thiazine derivatives are diluted with dimethyl sulfoxide. It has been shown that concentrations as low as 0.01 /xg/ml may be detected this way and that substituents in position 2 decrease the intensity of the fluorescence to a greater extent than side chains at position 10.1 1... 

Enedithiolates (dithiolenes ) were used originally together with other ligands for production of chelates of transition metals for determination by UVD. After the discovery of photophysical properties of Pt complexes of certain 1,2-enedithiolates, they were used as phosphorescence and fluorescence dual emitters for oxygen and proton detection. The analytical applications of 1,2-enedithiolates have been reviewed. ... 

Spectroscopy, annual reviews of new analytical instrumentation from the Pittsbuigh Conference on Analytical Chemistry and Applied Spectroscopy. Analytical Chemisty, "Fundamental Reviews" (June 1994, June 1996), analytical applications of infrared, ultraviolet, atomic absorption, emission, Raman, fluorescence, phosphorescence, rhemilnminescence, and x-ray spectroscopy. 

Radiation from radioisotope sources can be used to excite characteristic X-rays in samples upon which the beam of radiation is directed. Detection and analysis of these X-rays yield information about the composition of the sample. This opens the field of analytical applications of X-ray fluorescence analysis. The most frequent applications are in the ore processing and the metal coating industries. 

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