Fluorescence spectroscopy, analytical method Applications

Fluorescence spectroscopy and its applications to the physical and life sciences have evolved rapidly during the past decade. The increased interest in fluorescence appears to be due to advances in time resolution, methods of data analysis and improved instrumentation. With these advances, it is now practical to perform time-resolved measurements with enough resolution to compare the results with the structural and dynamic features of macromolecules, to probe the structures of proteins, membranes, and nucleic acids, and to acquire two-dimensional microscopic images of chemical or protein distributions in cell cultures. Advances in laser and detector technology have also resulted in renewed interest in fluorescence for clinical and analytical chemistry. 

The most frequently applied analytical methods used for characterizing bulk and layered systems (wafers and layers for microelectronics see the example in the schematic on the right-hand side) are summarized in Figure 9.4. Besides mass spectrometric techniques there are a multitude of alternative powerful analytical techniques for characterizing such multi-layered systems. The analytical methods used for determining trace and ultratrace elements in, for example, high purity materials for microelectronic applications include AAS (atomic absorption spectrometry), XRF (X-ray fluorescence analysis), ICP-OES (optical emission spectroscopy with inductively coupled plasma), NAA (neutron activation analysis) and others. For the characterization of layered systems or for the determination of surface contamination, XPS (X-ray photon electron spectroscopy), SEM-EDX (secondary electron microscopy combined with energy disperse X-ray analysis) and... 

Due to the complexity of DOM fractionation has revealed more detailed information on the structural subunits prior to the application of advanced analytical methods. Most effective is the combination of different spectroscopic methods using UV-vis absorbance, fluorescence, 1H- and 13C-nuclear magnetic resonance, and Fourier transform-infrared (FT-IR) spectroscopy. In some studies, also electron paramagnetic resonance spectroscopy (EPR) is used (e.g., Chen et al., 2002). 

The application of analytical methods to speciation measurements in complicated systems has remained rather limited, despite the considerable technological progress during the past 25 years. The characterisation methods (e.g. spectroscopy, nuclear magnetic resonance) are often limited to the study of isolated compounds at relatively high concentrations. They, therefore, necessitate the prior employment of sophisticated separation and pre-concentration methods which introduce severe risks of perturbation. The trace analysis methods are often insensitive to the chemical form of the elements measured (e.g. atomic absorption, neutron activation). Those which possess sufficient element specificity (e.g. electron spin resonance, fluorescence, voltammetry) still require significant development before their full potential can be realised. 

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). 

Odell, W.D. Daughaday, W.H. Principles of Competitive Protein Binding Assays Lippincott Philadelphia 1971. Christian, G.D. Clinical chemistry. Analytical Chemistry John Wiley and Sons Inc. New York, 1994 611-628. Ullman, E.E., Langen, J., Clapp, J.J., Eds. Liquid Assay Analysis of International Development on Isotopic and Non-Isotopic Immunoassay Masson New York, 1981 113. Sharma, A. Schulman, S.G. Fluorescence analytical methods and their applications. Introduction to Fluorescence Spectroscopy, WHey-lntexsdeace.l ew York, 1999 123-158. 

A limitation of the application of luminescence spectroscopy to the analysis of real samples is its lack of specificity owing to similarities in spectral bandshapes and spectral positions of the luminescence spectra of many compounds. An obvious solution to this problem is the separation of the analytical sample s interfering constituents from each other before quantitation by fluorescence. High-performance liquid chromatography (HPLC) and related separation methods can be coupled to fluorescence spectroscopy to take advantage of the sensitivity of the spectroscopic method and the specificity of the separation method. 

Tt may be safe to say that the interest of environmental scientists in airborne metals closely parallels our ability to measure these components. Before the advent of atomic absorption spectroscopy, the metal content of environmental samples was analyzed predominantly by wet or classical chemical methods and by optical emission spectroscopy in the larger analytical laboratories. Since the introduction of atomic absorption techniques in the late 1950s and the increased application of x-ray fluorescence analysis, airborne metals have been more easily and more accurately characterized at trace levels than previously possible by the older techniques. These analytical methods along with other modem techniques such as spark source mass spectrometry and activation analysis... 

Time-resolved luminescence spectroscopy complements the steady-state method and can provide essential kinetic information about the decay of excited states. Application of time-resolved fluorescence spectroscopy for analytical chemistry, where low concentrations might require the use of long... 

The simplicity and robustness of the method makes it well suited to a number of practical analytical applications, such as sensitive noninvasive in vivo disease diagnosis, security screening and the quality control of pharmaceutical tablets. The concept is also potentially applicable to fluorescence spectroscopy, NIR tomography of turbid media and other general applications, where the enhanced coupling of laser radiation into a turbid medium is beneficial an example is the case of photodynamic therapy in cancer treatment of subsurface tissues. 

Important applications of Af-PLS are in the area of multivariate calibrations for excitation/emission fluorescence spectrometry, for hyphenated analytical methods, such as HPLC/diode array detection and GC/MS, or for multidimensional separation techniques with or without coupling to spectroscopy. 

A very detailed review of the HPLC methods has been carried out [7]. The author has described the stationary phase, the mobile phase, flow rate, and detector system used by researchers since 1973. We would like to describe the other analytical methods such as the capillary electrophoresis, flow injection analysis and two-dimensional fluorescence spectroscopy which have foimd applications in ergot alkaloid research. 

After the appearance of the first book on fluorescence in 1951 [45], fluorescence spectroscopy became a widely used scientific tool in biochemistry, biophysics, and in material science. In the last few years, however, several new applications based on fluorescence have been developed, promoting fluorescence spectroscopy from a primarily scientific to a more routine method. The phenomena of fluorescence is for example exploited in simple analytical assays in environmental science and clinical chemistry, in cell identification and sorting in flow cytometry, and in imaging of single cells in medicine. The analyte, whose light emission is investi-... 

A systematic description of the scope of fluorescence spectroscopy in clinical and drug analysis is rendered difficult by the very large number of determinations reported so far. For reasons of space, this article can only present an overview of the wide variety of applications of this technique in these analytical fields, such as chromatographic and electrophoretic methods, its use in enzymatic and immunochemical assays and in biosensors, and its usefulness in new research areas such as proteomic studies. 

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