Multidimensional fluorescence analysis

Multidimensional Fluorescence Analysis of Cyclodextrin Solvent—Extraction Systems... 

A detailed account of progress in the applications of multidimensional fluorescence analysis in analytical chemistry can be found elsewhere. The purpose of this article is to outline the scope and potential practical applications of multidimensional fluorescence in analytical chemistry and other related research areas. 

An added feature of total fluorescence techniques is found in systems whose emission and/or excitation wavelength maxima change with the formation of a complex. Multidimensional fluorescence analysis allows the analyst to detect the differences in sensitivity arising from changes in excitation and emission maxima of the analyte. In addition, the technique finds wide application in examining the corrected fluorescence spectra of clinical compounds and their derivatives. In studies involving biological... 

Multidimensional fluorescence analysis is well suited for routine qualitative separation and identification of complex mixtures of PAHs in environmental samples. The separation and extraction of the individual component spectra of PAH mixtures affords valuable information about these hazardous compounds. Such information may allow for a detailed study of the means to remove their influence from the environment. The differences in the intensity contributions from a mixture of PAHs in a multicomponent system could be related to the differences in concentrations of the individual components and their respective quantum efficiencies. The problem is exacerbated by the fact that most solutions containing mixtures of PAHs display broad fluorescence spectral bands at room temperature, resulting in significant overlap and limited specificity for multicomponent analysis. This makes the success of multidimensional fluorescence measurements for complete spectral resolution of a complex multicomponent mixture dependent upon the degree of spectral overlap among the individual components and their fluorescence... 

The combination of multidimensional fluorescence analysis and quenching studies has allowed component resolution of PAHs complexed with P-cyclo-dextrin (ff-CD). For example, upon the addition of iodide, the fluorescence intensity of fluanthrene and pyrene in aqueous solution is quenched to less than 10% of its original intensity. The quenching is dramatically reduced upon addition of jS-cyclodextrin to a 1% f-butanol solution containing pyrene-flu-anthrene. Figure 3 illustrates the utility of a multidimensional fluorescence scheme in combination with cyclodextrin complexation to achieve resolution of a two-component mixture of pyrene and fluanthrene. 

Many petroleum-based samples contain complex mixtures of PAHs, which are known or suspected carcinogens and mutagens. Multidimensional fluorescence analysis is suitable for the identification of fluorophores contained in oil samples. The basis of this technique is to employ a unique spectral fingerprint for the selective characterization and mapping of certain PAHs in the oil. Such a procedure is likely to provide a detection scheme for selective analysis of environmental samples for analytically important hazardous compounds. The primary goal will be to expand this technique to create a library containing... 

Utilization of a multidimensional fluorescence data set that includes, for example, a fluorescence spectrum, an excitation spectrum, a time profile, anisotropy, and spatial localization, is a straightforward and effective approach. Obviously, increasing a dimension (experimental variable) of the data improves the selectivity of the measurement and its subsequent analysis. It has been noticed that 3D or higher dimensional data is inherently different from 2D data, because the decomposition of 3D data is often unique while that of 2D data never is [17]. Therefore, 3D or higher dimensional fluorescence data can be decomposed into individual fluorescent components without any prior knowledge of the autofluorescent molecules. 

In this section, based on the methodology presented in the previous section, we describe multidimensional fluorescence imaging and its application to tracking cell responses. We developed the time- and spectrally-resolved fluorescence imaging system based on line illumination, which is capable of rapid acquisition of fluorescence intensities as a function of Em, x, and xy-positions. We applied it to the analysis of an induced plant defense response, that is, the accumulation of antimicrobial compounds or phytoalexins, in oat (Avena sativa). 

Bystol, A. J., Thorstenson, T. and Campiglia, A. D., Laser-induced multidimensional fluorescence spectroscopy in ShpoTskii matrices in liquid helium (4.2 K) for the analysis of polycyclic aromatic hydrocarbons in HPLC fractions and complex environmental matrices. Environ. Sci. Technol., 36, 4424-4429, 2002. 

Oldham, P.B., Wang, J., Conners, TE. and Schultz, T.P. (1993) Rapid analysis of pulp lignin a review of NIR and FTIR and preliminary investigation of multidimensional fluorescence spectroscopy. Pulping Conference 1993, pp. 653. 

Figure 3 Isometric plots of fluorescence showing the EEMs of (A) a pyrene-fluanthrene mixture in 1 % f-butyl alcohol (B) a pyrene-fluanthrene mixture in 1 % f-butyl alcohol and 100 mmol I iodide (C) a pyrene-fluanthrene mixture in 1% f-butyl alcohol, 100 mmol M iodide, and I.Smmoir -CD (D) a pyrene-fluanthrene mixture in 1% f-butyl alcohol, lOOmmoll" iodide and 3.8mmoir y-CD. (Reprinted with permission from Nelson G, Neal SL, and Warner IM (1986) Resolution of mixtures by cyclodextrin complexation and multidimensional data analysis. Spectroscopy 3 24—28.)...

Multidimensional fluorescence measurement is also very useful for applications that involve the separation and structural elucidation of potential therapeutic drugs. The capability for rapid acquisition of spectral data and enhanced detection limits achieved by use of fluorescence measurements is an important advantage for multidimensional fluorescence measurements. This detection is important for the screening of unknown compounds in complex mixtures, such as in the area of Pharmaceuticals. The utility of a video fluorimeter in this regard is demonstrated for the analysis of unused growth medium, typical cancer cell, and typical normal cell samples (Figure 4). 

Investigation of complex biological samples, such as the analysis of bovine serum albumin, could be best achieved by employing the high resolving power of multidimensional fluorescence spectroscopy. An isometric projection will allow for a simultaneous observation of all the data points in each of the scans. In addition to a more pictorial representation of the... 

See also Fluorescence Overview Multidimensional Fluorescence Spectrometry Time-Resolved Fluorescence Derivatization Quantitative Analysis Environmental Applications Food Applications. 

Hall, G.J., Clow, K., and Kenny, J., Estuarial fingerprinting through multidimensional fluorescence and multivariate analysis. Environ. Sci. TechnoL, 39, 7560, 2005. 

In the analysis of complex PAH mixtures obtained from environmental samples, reversed-phase LC-FL typically provides reliable results for only 8-12 major PAHs (Wise et al. 1993a). To increase the number of PAHs determined by LC-FL, a multidimensional LC procedure is used to isolate and enrich specific isomeric PAHs that could not be measured easily in the total PAH fraction because of interferences, low concentrations, and/or low fluorescence sensitivity or selectivity. This multi-dimensional procedure, which has been described previously (Wise et al. 1977 May and Wise 1984 Wise et al. 1993a, 1993b), consists of a normal-phase LC separation of the PAHs based on the number of aromatic carbon atoms in the PAH, thereby providing fractions containing only isomeric PAHs and their alkyl-substituted isomers. These isomeric fractions are then analyzed by reversed-phase LC-FL to separate and quantify the various isomers. 

Several configurations for the sensor are possible. An especially viable alternative would seem to be the competitive displacement of fluorescent label. Since this is an equilibrium, fouling or contamination of the surface should not alter the absolute result. Krull et al (75) have reported the reproducible immobilisation of a stable phospholipid membrane containing fluorophore in this context. Concurrent fluorescence polarisation measurements can offer the possibility of multidimensional analysis (76) and are in any case experiencing a rejuvenation of interest as a highly selective technique, when the effective molecular weight of the antibody is increased relative to the antigen, by immobilisation on a latex or metal particle (77)... 

The data shown below illustrates how the concept of multidimensional analysis might be used to devise a simultaneous fluorescence assay of warfarin and its major metabolite, 7-0H warfarin. 

Warner and co-workers justify the elaborate optical/detection system and the time commitment required per analysis on the basis of the additional sensitivity available using fluorescence detection, and on the multidimensional CD information available. For applications in which two, or more CD active fluorophores may be present, the ability to provide both an excitation and emission FDCD profile for the sample may allow differentiation of the individual components without pre-separation. Replacement of the mechanical mechanism for prism movement with an electro-optical device may improve both the SNR and reduce the time required per sample. These improvements will greatly facilitate general application of this multidimensional approach to FDCD measurements. 

The parallel factor analysis (PARAFAC) model [18-20] is based on a multilinear model, and is one of several decomposition methods for a multidimensional data set. A major advantage of this model is that data can be uniquely decomposed into individual contributions. Because of this, the PARAFAC model has been widely applied to 3D and also higher dimensional data in the field of chemometrics. It is known that fluorescence data is one example that corresponds well with the PARAFAC model [21]. 

Summarizing this section, we developed the time-gated excitation-emission matrix spectroscopic system and applied it to the decomposition of a mixed solution of a number of fluorescent dyes. We demonstrated that our approach, which was based on unique optical configuration, efficient acquisition of a multidimensional data set, and decomposition of unknown fluorescent components by using the PARAFAC model, was effective for the analysis ofunknown multi-component targets. 

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