Fluorescent analytical methods

We will begin by a brief review of the concept of the X-ray fluorescence analytical method widely used in the petroleum industry for studying the whole range of products and for analyzing catalysts as well. 

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. 

Chen GZ. In Fluorescence Analytical Methods, Beijing Science Press, 1990 122. 

Chemical Properties. Elemental analysis, impurity content, and stoichiometry are determined by chemical or iastmmental analysis. The use of iastmmental analytical methods (qv) is increasing because these ate usually faster, can be automated, and can be used to determine very small concentrations of elements (see Trace AND RESIDUE ANALYSIS). Atomic absorption spectroscopy and x-ray fluorescence methods are the most useful iastmmental techniques ia determining chemical compositions of inorganic pigments. Chemical analysis of principal components is carried out to determine pigment stoichiometry. Analysis of trace elements is important. The presence of undesirable elements, such as heavy metals, even in small amounts, can make the pigment unusable for environmental reasons. 

Numerous methods have been pubUshed for the determination of trace amounts of tellurium (33—42). Instmmental 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 instmmental methods include polarography, potentiometry, emission spectroscopy, x-ray diffraction, and x-ray fluorescence. 

Instrumental Methods for Bulk Samples. With bulk fiber samples, or samples of materials containing significant amounts of asbestos fibers, a number of other instmmental analytical methods can be used for the identification of asbestos fibers. In principle, any instmmental method that enables the elemental characterization of minerals can be used to identify a particular type of asbestos fiber. Among such methods, x-ray fluorescence (xrf) and x-ray photo-electron spectroscopy (xps) offer convenient identification methods, usually from the ratio of the various metal cations to the siUcon content. The x-ray diffraction technique (xrd) also offers a powerfiil means of identifying the various types of asbestos fibers, as well as the nature of other minerals associated with the fibers (9). 

The use of agarose as an electrophoretic method is widespread (32—35). An example of its use is in the evaluation and typing of DNA both in forensics (see Forensic chemistry) and to study heritable diseases (36). Agarose electrophoresis is combined with other analytical tools such as Southern blotting, polymerase chain reaction, and fluorescence. The advantages of agarose electrophoresis are that it requires no additives or cross-linkers for polymerization, it is not hazardous, low concentration gels are relatively sturdy, it is inexpensive, and it can be combined with many other analytical methods. 

The complex of the following destmctive and nondestmctive analytical methods was used for studying the composition of sponges inductively coupled plasma mass-spectrometry (ICP-MS), X-ray fluorescence (XRF), electron probe microanalysis (EPMA), and atomic absorption spectrometry (AAS). Techniques of sample preparation were developed for each method and their metrological characteristics were defined. Relative standard deviations for all the elements did not exceed 0.25 within detection limit. The accuracy of techniques elaborated was checked with the method of additions and control methods of analysis. 

The analysis was performed by XRF method with SR. SRXRF is an instrumental, multielemental, non-destructive analytical method using synchrotron radiation as primary excitation source. The fluorescence radiation was measured on the XRF beam-line of VEPP-3 (E=2 GeV, 1=100 mA), Institute of Nuclear Physics, Novosibirsk, Russia. For quality control were used international reference standards. 

It is often experimentally convenient to use an analytical method that provides an instrumental signal that is proportional to concentration, rather than providing an absolute concentration, and such methods readily yield the ratio clc°. Solution absorbance, fluorescence intensity, and conductance are examples of this type of instrument response. The requirements are that the reactants and products both give a signal that is directly proportional to their concentrations and that there be an experimentally usable change in the observed property as the reactants are transformed into the products. We take absorption spectroscopy as an example, so that Beer s law is the functional relationship between absorbance and concentration. Let A be the reactant and Z the product. We then require that Ea ez, where e signifies a molar absorptivity. As initial conditions (t = 0) we set Ca = ca and cz = 0. The mass balance relationship Eq. (2-47) relates Ca and cz, where c is the product concentration at infinity time, that is, when the reaction is essentially complete. 

In order to further extend the utility of fluorescence methods the use of time-resolution methods, fluorescence polarization, and laser techniques should be explored. The addition of other dyes with enhanced fluorescence properties on binding and increased selectivity to various types of nucleic acids will be necessary to further develop more useful analytical methods. 

The PSP toxins represent a real challenge to the analytical chemist interested in developing a method for their detection. There are a great variety of closely related toxin structures (Figure 1) and the need exists to determine the level of each individually. They are totally non-volatile and lack any useful UV absorption. These characteristics coupled with the very low levels found in most samples (sub-ppm) eliminates most traditional chromatographic techniques such as GC and HPLC with UVA S detection. However, by the conversion of the toxins to fluorescent derivatives (J), the problem of detection of the toxins is solved. It has been found that the fluorescent technique is highly sensitive and specific for PSP toxins and many of the current analytical methods for the toxins utilize fluorescent detection. With the toxin detection problem solved, the development of a useful HPLC method was possible and somewhat straightforward. 

Detection of the PSP toxins has proven to be one of the largest hurdles in the development of analytical methods. The traditional means, and still in wide use today, is determination of mouse death times for a 1 mL injection of the test solution. There are a variety of drawbacks to utilization of this technique in routine analytical methods, that have prompted the search for replacements. In 1975 Bates and Rapoport (3) reported the development of a fluorescence technique that has proven to be highly selective for the PSP toxins, and very sensitive for many of them. This detection technique has formed the basis for analytical methods involving TLC (77), electrophoresis (72), column chromatography (7J), autoanalyzers (7 ), and HPLC (5,6,7). 

The most significant differences (i.e. independence) in the analytical methods are provided in the final chromatographic separation and detection step using GC/ MS and LC-FL. GC and reversed-phase LG provide significantly different separation mechanisms for PAHs and thus provide the independence required in the separation. The use of mass spectrometry (MS) for the GC detection and fluorescence spectroscopy for the LG detection provide further independence in the methods, e.g. MS can not differentiate among PAH isomers whereas fluorescence spectroscopy often can. For the GC/MS analyses the 5% phenyl methylpolysiloxane phase has been a commonly used phase for the separation of PAHs however, several important PAH isomers are not completely resolved on this phase, i.e. chrysene and triphenylene, benzo[b]fluoranthene and benzofjjfluoranthene, and diben-z[o,h]anthracene and dibenz[a,c]anthracene. To achieve separation of these isomers, GC/MS analyses were also performed using two other phases with different selectivity, a 50% phenyl methylpolysiloxane phase and a smectic liquid crystalline phase. 

The analytical methods summarized in this article are generally multiresidue methods for the determination of oxime carbamates in different sample matrices (crops, animal tissues, soil, and water). These methods include HPLC with fluorescence, MS, and MS/MS detection. 

There are methods available to quantify the total mass of americium in environmental samples. Mass spectrometric methods provide total mass measurements of americium isotopes (Dacheux and Aupiais 1997, 1998 Halverson 1984 Harvey et al. 1993) however, these detection methods have not gained the same popularity as is found for the radiochemical detection methods. This may relate to the higher purchase price of a MS system, the increased knowledge required to operate the equipment, and the selection by EPA of a-spectrometry for use in its standard analytical methods. Fluorimetric methods, which are commonly used to determine the total mass of uranium and curium in environmental samples, have limited utility to quantify americium, due to the low quantum yield of fluorescence for americium (Thouvenout et al. 1993). 

Moens, L., A. Von Bohlen, and P. Vandenabeele (2000), X ray fluorescence, in Cilib-erto, E. and G. Spoto (eds.), Modern Analytical Methods in Art and Archaeology, Chemical Analysis Series, Vol. 155, Wiley, New York. 

FBAs can also be estimated quantitatively by fluorescence spectroscopy, which is much more sensitive than the ultraviolet method but tends to be prone to error and is less convenient to use. Small quantities of impurities may lead to serious distortions of both emission and excitation spectra. Indeed, a comparison of ultraviolet absorption and fluorescence excitation spectra can yield useful information on the purity of an FBA. Different samples of an analytically pure FBA will show identical absorption and excitation spectra. Nevertheless, an on-line fluorescence spectroscopic method of analysis has been developed for the quantitative estimation of FBAs and other fluorescent additives present on a textile substrate. The procedure was demonstrated by measuring the fluorescence intensity at various excitation wavelengths of moving nylon woven fabrics treated with various concentrations of an FBA and an anionic sizing agent. It is possible to detect remarkably small differences in concentrations of the absorbed materials present [67]. 

When cells are suspended in a biological fluid or culture medium, both serum proteins and cells interact with the surface substrate. Serum protein adsorption behavior on SAMs has been examined with various analytical methods, including SPR [58-61], ellipsometry [13, 62, 63], and quartz QCM [64—66]. These methods allow in situ, highly sensitive detection of protein adsorption without any fluorescence or radioisotope labeling. SPR and QCM are compatible with SAMs that comprise alkanethiols. In our laboratory, we employed SPR to monitor protein adsorption on SAMs. 

Also in the literature, there is little discussion of the accuracy or reproducibility of the analytical technique used for determining the corresponding matrix and particle composition [37, 38], Various analytical methods that have been used to determine the particle concentration in the deposit include gravimetric analysis [29, 31, 39], x-ray fluorescence [5], atomic absorption spectroscopy [33, 40, 41-43], and micro-... 

The principal analytical methods for complex samples are those that separate the mixture by differential migration and then detect the separated components. The separation methods are chromatography, electrophoresis, and field flow fractionation the detection methods—which need not be selective but must be sensitive—include absorption, laser-induced fluorescence, electrochemistry, and mass... 

It can be seen from the above that the sample stream emerging from the plasma will be rich in free ions and atoms of the elements from the sample. Thus, the ICP could provide an attractive source for analytical methods other than those based upon straightforward emission. Instruments using the ICP source as a basis for atomic fluorescence have been developed. 

When primary X-rays are directed on to a secondary target, i.e. the sample, a proportion of the incident rays will be absorbed. The absorption process involves the ejection of inner (K or L) electrons from the atoms of the sample. Subsequently the excited atoms relax to the ground state, and in doing so many will lose their excess energy in the form of secondary X-ray photons as electrons from the higher orbitals drop into the hole in the K or L shell. Typical transitions are summarized in Figures 8.35 and 8.36. The reemission of X-rays in this way is known as X-ray fluorescence and the associated analytical method as X-ray fluorescence spectrometry. The relation between the two principal techniques of X-ray emission spectrometry is summarized in Figure 8.37. 

---