Fluorescence spectroscopy 418 Subject

Chong et al. [742] have described a multielement analysis of multicomponent metallic electrode deposits, based on scanning electron microscopy with energy dispersive X-ray fluorescence detection, followed by dissolution and ICP-MS detection. Application of the method is described for determination of trace elements in seawater, including the above elements. These elements are simultaneously electrodeposited onto a niobium-wire working electrode at -1.40 V relative to an Ag/AgCl reference electrode, and subjected to energy dispersive X-ray fluorescence spectroscopy analysis. Internal standardisation... 

Investigation turned then to chemical and spectroscopic means to obtain the needed mechanistic understanding. Stephenson et al. [17] looked at gas evolution versus exposure, while Pacifici and Straley [18] used UV fluorescence spectroscopy to identify a photo-oxidation product which was later isolated by Valk et al. [19]. In addition, Valk and co-workers [19-21] isolated a number of additional photolysis products by a combination of hydrolysis and chromatography, Marcotte et al. [22] used ESR to look at radicals generated during degradation, and Day and Wiles [23-26] carried out extensive IR and fluorescence spectroscopic investigations on this subject. 

The film reacted with adipoyl chloride followed by coupling with 7-hydroxycoumarin was subjected to methanolysis at 1 N HC1 and 60°C. The regenerated coumarin was assayed at pH 10 by fluorescence spectroscopy at an excitation wavelength of 329 nm and an emission wavelength of 455 nm. A Hitachi MPF-4 Fluorescence Spectrophotometer was used for all fluorescence measurements. 

After extraction, the urethanated films were subjected to alkaline hydrolysis of urethanes to liberate the corresponding amines, while the adipoylated films were hydrolyzed after having reacted with 7-hydroxycoumarin. Amounts of the released amines and coumarin were determined by fluorescence spectroscopy as described in the Experimental section. Since aniline as well as butylamine has no appreciable fluorescence by themselves, their fluorescence assay was made after reacting with o-phthalaldehyde in the presence of mercaptoethanol. In Figure 3, where relative fluorescence intensities are plotted as a function of concentrations of amines and hydroxycoumarin, one can see that the fluorescence intensities vary linearly with their concentration to permit us the quantitative determination of extremely small amounts of amines and hydroxycoumarin. 

As the enzyme itself is usually the focus of interest, information on the behavior of that enzyme can be obtained by incubating the enzyme with a suitable substrate under appropriate conditions. A suitable substrate in this context is one which can be quantified by an available detection system (often absorbance or fluorescence spectroscopy, radiometry or electrochemistry), or one which yields a product that is similarly detectable. In addition, if separation of substrate from product is necessary before quantification (for example, in radioisotopic assays), this should be readily achievable. It is preferable, although not always possible, to measure the appearance of product, rather than the disappearance of substrate, because a zero baseline is theoretically possible in the former case, improving sensitivity and resolution. Even if a product (or substrate) is not directly amenable to an available detection method, it maybe possible to derivatize the product with a chemical species to form a detectable adduct, or to subject a product to a second enzymatic step (known as a coupled assay, discussed further later) to yield a detectable product. But, regardless of whether substrate, product, or an adduct of either is measured, the parameter we are interested in determining is the initial rate of change of concentration, which is determined from the initial slope of a concentration versus time plot. 

Time-resolved fluorescence spectroscopy of polar fluorescent probes that have a dipole moment that depends upon electronic state has recently been used extensively to study microscopic solvation dynamics of a broad range of solvents. Section II of this paper deals with the subject in detail. The basic concept is outlined in Figure 1, which shows the dependence of the nonequilibrium free energies (Fg and Fe) of solvated ground state and electronically excited probes, respecitvely, as a function of a generalized solvent coordinate. Optical excitation (vertical) of an equilibrated ground state probe produces a nonequilibrium configuration of the solvent about the excited state of the probe. Subsequent relaxation is accompanied by a time-dependent fluorescence spectral shift toward lower frequencies, which can be monitored and analyzed to quantify the dynamics of solvation via the empirical solvation dynamics function C(t), which is defined by Eq. (1). 

Zsolnay, A., Baigar, E., Jimenez, M., Steinweg, B., and Saccomandi, F. (1999). Differentiating with fluorescence spectroscopy the sources of dissolved organic matter in soils subjected to drying. Chemosphere 38,45-50. 

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). 

Dufour, C., Dangles, O. (2005). Flavonoid-serum albumin complexation determination of binding constants and binding sites by fluorescence spectroscopy. Biochim. Biophys. Acta-General Subjects 1721, 164-173. 

Summary. Methods for determining the aqueous solubilities of PAHs are subject to errors associated with the preparation, extraction, and quantitative analysis of saturated solutions. There is no one method that has addressed the problems associated with each of these processes. Systematic errors associated with quantitative analyses of saturated solutions should be reduced in methods where selective analytical measurement techniques are used. Chromatographic methods allow separation of nonanalyte signals-in-time from those of the analyte. Fluorescence spectroscopy allows greater selectivity than UV spectroscopy, though less than gas or liquid chromatography. 

Off-line systems are typically used for the detection of the metal analytes occurring in the atmosphere as particulates or fumes which, after collection by an appropriate filter, are subjected to dry ashing followed by acid decomposition [63] or, more commonly, wet ashing [63], and quantitation by ICP-AES [64], flame AAS [65], normal or furnace AAS [66,67], or X-ray fluorescence spectroscopy [68]. Oguma and van Loon [69] reported a method for the... 

Another aspect of optical pumping is related to the coherent excitation of two or more molecular levels. This means that the optical excitation produces definite phase relations between the wave functions of these levels. This leads to interference effects, which influence the spatial distribution and the time dependence of the laser-induced fluorescence. This subject of coherent spectroscopy is covered in Chap. 7. 

The related subject of atomic fluorescence spectroscopy (AES), the emission of photons by excited gas-phase atoms following excitation by absorption of photons, is also covered in this chapter. 

An enormous technology base already exists for familiar optical methods of chemical analysis. There is probably no class of chemical analyte that has not at some time been the subject of optical determination through absorption or fluorescence spectroscopy. Methods also exist for labeling a target analyte with an appropriate chromophore or a fluorophore, a common practice in immunoassays. 

Mark Benvenuto is a Professor of Chemistry at the University of Detroit Mercy and a Fellow of the ACS. His research thrusts span a wide array of subjects, but include the use of energy dispersive X-ray fluorescence spectroscopy to determine trace elemerrts in land-based arrd aqrratic plant matter, food additives, and ancient and medieval coins. Benvenuto received a B.S. in cherrristry from the drginia Military Institute, and after several years in the Atrrty, a Ph.D. in inorganic chemistry from the University of Virginia. After a post-doctoral fellowship at the Pennsylvania State Urriversity, he joined the facrrlty at the University of Detroit Mercy in late 1993. 

Spectroscopic techniques are particularly useful for quantitation. They offer speed and great flexibility in instrumentation. UV spectrophotometry is widely used for quantitation using data at the maximum absorbance of a chromophor. Fluorescence spectrometry is also widely used for quantitation as it provides greater selectivity and sensitivity than UV spectrophotometry. The use of FT-IR spectrometry for quantitation is less common. The use of NIR for quantitation is an area of great activity and innovation. In addition, NMR spectroscopy can be used for quantitation. Here the criteria for success are very different from those for absorption or fluorescence spectroscopy. NMR quantitation is the subject of a separate article in this encyclopedia. 

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