Molecular Fluorescence, Phosphorescence, and Chemiluminescence

Steven A. Soper is an Assistant Professor of analytical chemistry at ... A computer search of Chemical Abstracts provided most of the references for t...
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Anal. Chem. 1996, 68, 73R-91R

Molecular Fluorescence, Phosphorescence, and Chemiluminescence Spectrometry Isiah M. Warner,*,† Steven A. Soper,† and Linda B. McGown‡

Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, and Department of Chemistry, Duke University, Box 90346, Durham, North Carolina 27708-0346 Review Contents Books, Reviews, and Chapters of General Interest General Instrumentation Laser-Based Techniques Reviews Instrumentation and Applications Fiber-Optic-Based Fluorescence Reviews Instrumentation and Applications Sample Preparation, Quenching, and Related Phenomena Data Reduction Luminescence in Organized Media Low-Temperature Luminescence Total Luminescence and Synchronous Excitation Spectroscopies and Related Techniques Solid-Surface Luminescence Luminescence in Chromatography, Electrophoresis, and Flow Systems Reviews Flow Injection Analysis Thin-Layer Chromatography and Other Miscellaneous Separation Techniques Fluorogenic Labels for Chromatography Instrumentation and Applications in HPLC Instrumentation and Applications in CE Dynamic Measurements of Luminescence Fluorescence Polarization, Molecular Dynamics and Related Phenomena Chemiluminescence Near-Infrared Fluorescence Reviews Fluorogenic Reagents for Near-IR Fluorescence Instrumentation and Applications Luminescence Techniques in Biological and Clinical Analysis Nucleic Acids Proteins Other Techniques and Applications Reagents and Probes Other Techniques and Applications Literature Cited

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This review covers the approximately two-year period since our last review (A1), roughly from November 1993 through October 1995. A computer search of Chemical Abstracts provided most of the references for this review. Coverage is limited to articles that describe new developments in the theory and practice of molecular luminescence for chemical analysis in the ultravioletvisible and near-infrared region. If you feel that we have omitted an important article published during the above referenced time † ‡

Louisiana State University. Duke University.

S0003-2700(96)00004-2 CCC: $25.00

© 1996 American Chemical Society

period, please forward the reference to one of us and we will be certain to consider it for the next review. Citations may be duplicated between sections due to articles with contents that span several topics. In general, citations are limited to journal articles and usually do not include patents, proceedings, reports, and dissertations. We have also continued to make a concerted effort to reduce the number of citations. This reduction was attempted with more of a focus on articles of general interest and relevance to the field of analytical chemistry. Despite this attempt, the overall number of citations has increased since the last review. Although we are not able to provide extensive coverage of developments in broad areas such as chromatography and biological sciences, we have tried to include major review articles and chapters relevant to these topics. BOOKS, REVIEWS, AND CHAPTERS OF GENERAL INTEREST The number of citations in this area has more than doubled since our last review. This continued growth is an obvious reflection of the increasing use of fluorescence in a wider variety of research areas. A review of the development and commercialization of the spectrofluorometer has been provided by Udenfriend (B1). Particular emphasis is placed on the use of the spectrofluorometer for protein structure and analysis. Some more general reviews have been provided by Varley (B2) and Bright (B3). Miller has reviewed the advantages of fluorescence spectroscopy in the nearinfrared region (B4). The mathematical expressions for fluorescence anisotropy decay analysis have been reviewed by Lakowicz (B5). This same research group has also reviewed lifetime-based sensing (B6). Fluorescence continues to be a viable approach toward achieving multicomponent analysis. In that regard, Soutar and coworkers have reviewed the use of fluorescence techniques for characterization of multicomponent systems (B7). An introduction to multidimensional luminescence has also been provided by Patonay and Warner (B8). A number of general applications of fluorescence spectroscopy have been reviewed. These include fluorescence demodulation spectroscopy (B9) and applications of lanthanide-sensitized luminescence to determination of organic analytes (B10). Various approaches to teaching fluorescence spectroscopy have also been reviewed with a particular emphasis on biophysical measurements (B11). The use of laser-induced fluorescence in conjunction with high-resolution Fourier transform interferometry has been reviewed (B12). The growth of fluorescence spectroscopy as a method for single-molecule detection continues such that two reviews can be cited in this area. Eigen and Rigler have reviewed the detection and identification of single molecules in solution based on Analytical Chemistry, Vol. 68, No. 12, June 15, 1996 73R

fluorescence correlation spectroscopy (B13). Ramsey and coworkers have also reviewed single-molecule detection in solution (B14). The applications of fluorescence to food science have also been reviewed. Strasburg and Ludescher have reviewed the theory and applications of fluorescence spectroscopy in food research (B15). The fluorescence properties of carotenoids have also been reviewed (B16). Environmental applications of fluorescence spectroscopy continue to be a major emphasis by many researchers. Makogan and co-workers have reviewed the use of absorption and laser fluorescence spectroscopies for environmental monitoring (B17). Honing and co-workers have discussed sample preparation and determination of carbamate pesticides in various sample matrices (B18). Emphasis was placed on the use of fluorescence spectroscopy and other analytical techniques for detection and measurement. Two reviews of luminescence for measurement of chemical contaminants in the environment can also be cited (B19, B20). Multicomponent analysis of environmental matrices by use of fluorescence spectroscopy and other analytical techniques has also been reviewed (B21). Two reviews on the use of fluorescence spectroscopy for the analysis of polycyclic aromatic hydrocarbons (PAHs) have been published (B22, B23). Two very interesting reviews on the use of fluorescence to probe chiral properties of molecules are also relevant to our discussion. One review involves fluorescent sensors that can discriminate between enantiomers of various chiral molecules such as carbohydrates (B24). The other review involves detection of chiroptical properties of molecules in the excited state (B25). Study of biological systems is perhaps the fasting growing area for applications of fluorescence spectroscopy. Royer has recently reviewed fluorescence spectroscopy for studying protein systems (B26). Fluorescent protein biosensors for measurement of molecular dynamics in living cells have also been reviewed (B27). Other studies cite the use of fluorescence spectroscopy in turbid media and tissues (B28) and the use of lanthanide ions as probes of protein systems (B29). The use of pseudointrinsic probes for study of proteins and nucleic acids has also been discussed (B30). Some general reviews on the use of fluorescence for studies of protein systems have also been published (B31-B33). A general review with 103 references on the development of autofluorescence for medical diagnosis can be cited (B34). Two other interesting reviews focus on the prospects of using fluorescence spectroscopy to study single ion channels (B35) and for in vivo analysis of tissue (B36). Two reviews provide information on the most widely used mathematical models (B37) and instrumental techniques (B38) for application of fluorescence spectroscopy to biological systems. The application of fluorescence sensing to bioreactors has also been reviewed (B39). Fluorescence methods for the analysis of hemoglobin adducts have also been described (B40). GENERAL INSTRUMENTATION Developments in luminescence instrumentation have primarily focused on modifications of existing designs to achieve specific and/or more selective measurements. One very relevant discussion has suggested improvements in the process of fluorescence instrument performance validation (C1). The measurement of sensitivity in fluorescence spectroscopy has also been discussed (C2). 74R

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The development of a novel spectrometer for measurement of luminescence in highly turbid samples, e.g., those of biological origin, has been described (C3). Purcell has discussed the utility of imaging spectrographs for performing multidimensional spectroscopy (C4). Phelan has discussed the multidimensional capabilities of a commercial rapid-scanning fluorometer (C5). Vonarx and co-workers have designed a laser-based fluorometer to measure tissue photosensitizer concentration (C6). Another instrumental development involves an integrated instrument for light-scattering and time-resolved fluorescence measurements (C7). This instrument has the capability of performing both measurements simultaneously in a thermostated cuvette. Time-resolved fluorescence instrumentation has been developed. Novo and co-workers have described computer interfacing and software development for a boxcar-based laser luminescence spectrometer (C8). This instrument allowed accurate acquisition of luminescence decay curves and time-resolved spectra. A new picosecond fluorescence lifetime system has also been developed (C9). An instrument for time-resolved measurement of circular and linear polarization of luminescence with a time resolution of 50 ns has been described (C10). Another development involves the construction and evaluation of an instrument for the simultaneous measurement of fluorescence intensity, time, and wavelength with a single picosecond pulse (C11). Applications of this system for study of charge-transfer fluorescence is discussed. An inexpensive microprocessor-controlled spectrophotometer for time-resolved fluorescence spectroscopic measurements has been described (C12). Other developments include a fiber-optic-based phase-resolved phosphorescence spectrometer (C13) and an apparatus for detection of emissions with quantum yields as low as 1 × 10-7 and lifetimes as short as 10 µs (C14). Yang and co-workers have described a novel dual-channel phosphorimeter for studies of antigen-antibody complexes (C15). The selectivity of this instrument is provided by use of anisotropy measurements. A simple, modular polarization spectrometer for the measurement of the linear or circular polarization of luminescence has been described (C16). A new instrument for acquisition of fluorescence excitationemission matrices (EEMs) has been described (C17). A 51 × 93 EEM can be acquired in 3.2 min, and a detection limit of 500 ng/L is cited for fluorescein in ethanol. A battery-operated portable synchronous spectrofluorometer has been described by Vo-Dinh and co-workers (C18). A large-aperture spectrofluorometer for analyzing small portions of liquid and solid samples has been described by Popov and co-workers (C19). Krimkin and co-workers have described a spectrofluorometer for remote analysis of oils on a water surface (C20). Nabi has developed a continuous flow analyzer for chemi- and bioluminescence detection (C21). Some developments in fluorescence instrumentation have focused on the detector for improvements. For example, Silzel and Obremski have constructed a short-wave near-infrared spectrofluorometer with a diode laser source and charge-coupled device (CCD) detection (C22). Other developments have included a new emitter-detector-cuvette assembly for measurement of chlorophyll (C23) and photon counting with a position-sensitive detector (C24). Finally, subfemtomole room-temperature phosphorescence detection has been achieved with a charge-coupled device (C25).

LASER-BASED TECHNIQUES Reviews. A general review of laser-induced molecular fluorescence in the analytical sciences has appeared which discussed laser fluorescence in both the liquid and solid states (D1). The review contains over 32 references. A review of laser diagnostics of plasmas using fluorescence and a tunable dye laser system was written which discussed the information obtained on the kinetics of neutral particles in plasmas (D2). Instrumentation and Applications. In order to perform synchronous luminescence, in which both the excitation and emission monochromators are scanned simultaneously, a system was developed that utilized a dye laser for the analysis of multicomponent mixtures (D3). The detection limit for some polyaromatic hydrocarbons was determined to be 680 zmol. Lytle and co-workers developed a cavity-dumped, synchronously pumped dye laser system for performing sensitive two-photon fluorescence with detection limits reported to be in the 38 pM range for 9,10diphenylanthracene (D4). A two-dimensional imaging system was described which utilized a streak camera and mode-locked laser for acquiring lifetimes of rhodamine B at the subpicomole level (D5). Detection sensitivity at the single-molecule level using laserinduced fluorescence continues to be an active area of research. A review of single-molecule detection with 35 references was written by Barnes et al. (D6). The applications of single-molecule detection in biotechnology were discussed in a review article by Rigler, who emphasized the use of fluorescence correlation spectroscopy for large-number-screening applications (D7). In order to enhance the SNR in single-molecule analyses, maximum likelihood algorithms were introduced (D8). The detection and photophysics of single rhodamine molecules in microdroplets was discussed by Ramsey and co-workers (D9), who were able to demonstrate a SNR approaching 30 for single molecules. Mahoney and Hieftje presented results on the detection of R6G molecules in nanoliter droplets (D10). Single-molecule detection has also been used in conjunction with capillary electrophoresis to analyze the contents of single cells (D11), identify single DNA molecules stained with intercalating dyes, and measure mobilities using a two-detector arrangement (D12, D13) and a single detector (D14). Single-molecule detection has also been used to interrogate the photophysics of dye molecules partitioned into micelle aggregates (D15). In flowing sample streams, the fluorescence lifetimes of single rhodamine molecules have been determined using time-correlated single-photon counting (D16). The authors concluded in this work that residual scattering and impurity fluorescence limited the accuracy and precision of the measurement. In a subsequent report, the estimation of background on the least-squares estimation of exponential decay parameters in single-molecule experiments was investigated using Monte Carlo simulation methods (D17). A newly developed time-correlated single-photon-counting electronics system was reported which allows for the continuous registration of time-resolved fluorescence signals in millisecond time intervals (D18). The system was used for monitoring the fluorescence lifetime of single chromophoric molecules. Single-molecule fluorescence was also achieved using a laser confocal microscope system (D19, D20). Confocal imaging allowed the effective probe volume to be reduced to less than 1 fL, thereby reducing background interferences and improving the SNR. Near-field microscopy was also used to image and detect

single molecules at room temperature using fluorescence detection (D21). In a subsequent report, near-field microscopy was used to investigate the photobleaching of single R6G molecules adsorbed to silica surfaces (D22). Near-field microscopy was also used to determine fluorescence lifetimes of single molecular events of sulforhodamine 101 on glass (D23) and R6G on silica (D24). The probe tip was found to have a profound impact on the measured lifetime when operated in the near-field regime. Low-temperature single-molecule detection was discussed as a means to investigate the effects of the nanoenvironment on the single molecule and the potential of optical storage on the singlemolecule level (D25). A confocal system was also developed to observe the fluorescence spectra of single pentacene molecules in p-terphenyl at 1.7 K (D26). A study on the maximum emission rate of single terrylene molecules at low temperature was also undertaken (D27). The authors discovered that the emission rate was dependent upon the orientation of the emission dipole. The fluorescence lifetimes of single pentacene molecules at 1.8 K were also determined in the O1 sites of p-terphenyl (D28). Laser-induced fluorescence was used as a detection strategy for the low-level monitoring of several organic pollutants and constituents. Stevenson and Vo-Dinh monitored the distribution of polynuclear aromatics using a laser-excited synchronous luminescence instrument (D29). Chlorinated hydrocarbons in gas mixtures were determined by laser-induced fluorescence (D30). The possibilities of using laser fluorescence spectroscopy to analyze petroleum hydrocarbons in natural water was also discussed (D31). The real-time measurement of dioxins in combustion sources was carried out using laser-induced fluorescence/continuous monitoring (D32). Fluorescence lifetime parameters, measured using a laser fluorosensor with streak camera detection, were evaluated on crude oils for remote identification of oil spills (D33). The identification process for the oil spills was extended by incorporating multicolor (four wavelengths) parameters into the time-dependent ones (D34). An algorithm was developed in conjunction with a remote-sensing laser fluorescence spectrometer to determine levels of organic substances in natural water (D35). A XeCl and KrF excimer laser system was developed to monitor the presence of synthetic oligopeptides and other bioorganic materials in water (D36). Shu and Hurtubise obtained laserexcited fluorescence spectra of the four isomers of tetrol in order to determine the extent of DNA damage produced by these compounds (D37). The quality of crude palm oil was elucidated using laser-induced fluorescence intensities (D38). The fluorescence intensity was found to correlate with the carotene and deterioration of bleachability index of the crude oil. An analysis of the laser-induced line shape of intact leaves was found to give an indication of UV-induced stress on the leaves (D39). Laser-induced fluorescence was used to measure tissue levels of chloroaluminum sulfonated phthalocyanine in vivo in an implanted hamster to check a pouch carcinoma model (D40). The drug was excited at 610 nm via a pulsed N2 laser-pumped dye laser. Research performed on the use of laser-induced fluorescence spectroscopy as a noninvasive tool to identify diseased tissue sites in situ and in real time was documented by Papazoglou (D41). Zharkova and co-workers developed a compact laserexcited fluorescence system for the diagnosis of tumors in clinical applications (D42). A study to investigate the uptake of porphyrins in neoplasias in the female breast tissue and to evaluate the Analytical Chemistry, Vol. 68, No. 12, June 15, 1996

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potential of fluorescence diagnosis for tumor demarcation using laser-induced fluorescence spectroscopy was performed (D43). Andreoni et al. measured the fluorescence spectroscopy of C60 at room temperature obtained under 514.5- and 337.1-nm excitation (D44). The spectra were measured with subnanosecond laser excitation in a 5-ns gate which was synchronous with the excitation. FIBER-OPTIC-BASED FLUORESCENCE Reviews. Narayanaswamy reviewed the use of chemical transducers based on fiber optics for environmental monitoring applications (E1). Techniques using fiber-optic fluorescence remote sensing in selective environmental monitoring applications were emphasized. Work utilizing fluorometric fiber-optic detection for biosensor applications was also reviewed (E2). In this review, fiber strategies implementing immobilized enzymes and antibodies for detection of antigens, sugars, and nucleic acids were extensively covered. Instrumentation and Applications. Victor and Crouch discussed the construction of a fiber-optic-based fluorometer using synchronous fluorescence spectroscopy to resolve multiple components in mixtures (E3). The fluorometer used a bifurcated fiber diode array that simultaneously measured front-surface fluorescence and absorbance. A confocal fluorescence microscope was constructed using fiber optics and possessed depth resolution of tens of micrometers and lateral resolution of several micrometers (E4). Anders and co-workers discussed the construction of a simple fiber-optic fluorometer and its applications in biotechnology (E5). To obtain information about vital biomass concentrations and their metabolic state, a noninvasive spectrofluorometric method was implemented. In order to improve the sensitivity associated with fluoroimmunoassays, a single-mode tapered optical fiber biochemical sensor was developed (E6). Laser light coupled into one end of the taper excited fluorescence of immobilized analytes with the emission coupled into the guided mode of the fiber and collected at the far end of the taper. An instrument to measure the excited-state lifetimes of phosphorescent materials in real time was described which utilized fiber optics and was targeted for oxygen sensing (E7). A fiber-optic-based phaseresolved phosphorescence spectrometer was discussed which could potentially resolve binary and ternary mixtures of several lumiphors (E8). In time-resolved fiber-optic fluorometers, the wavelength dispersion of the collected emission was studied (E9). A simple correction algorithm was proposed, resulting in timeresolved spectra of second-order, bilinear structure. Dubrovsky and co-workers discussed the fluorescence and phosphorescence study of Langmuir-Blodgett antibody films for applications to immunosensors (E10). Specific quartz surface modifications for immobilizing the antibodies were discussed. Horseradish peroxidase was immobilized in polyurethane membranes and employed in conjunction with a fiber-optic probe to establish an enzyme reactor sensor-based rate assay for hydrogen peroxide (E11). A fiber-optic sensor with fiber lengths over 50 m was constructed for the real-time monitoring of PAHs in aquatic environments. Using time-resolved fluorescence, detection limits in the nanogram per liter range were reported (E12). The optimization of a robust fluorescence detection system was described in which both scattered excitation light and fluorescence emission from rock, core, or cutting samples are guided by optical fibers (E13). 76R

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The fermentation of glucose in a bioreactor was followed in situ by measuring the fluorescence signal produced from NAD (NADH) using a remote-sensing fiber-optic device (E14). Baut and co-workers discussed the use of a remote-sensing fluorometer to control fermentors (E15). The specific fluorescence of NADH was found to be linear with the specific rate of butyric acid production. The analysis of urea was carried out using a fiberbased fluorometer by enzymatically hydrolyzing urea to ammonium and carbon dioxide, causing a pH increase and a concomitant increase in the fluorescence intensity of a pH-sensitive lumiphore (E16). Fiber sensors were also developed for the analysis of nitrate and nitrite at the ppm or ppb level (E17). Draxler and Lippitsch discussed the use of optical sensors based on fluorescence decay times to alleviate the poor long-term stability of optical sensors (E18). They used this concept to perform calibration-free chemical sensing. These authors also developed a time-domain chemical sensor for pH measurements (E19). In thin-layer chromatographic applications, a fiber-optic fluorescence scanning device was constructed by Navas and GarciaSanchez (E20). Various optical configurations were investigated in order to improve the sensitivity of the measurement for fluorophores adsorbed to these solid substrates. The sensitivity enhancement of capillary/fiber-optic sensors for flow systems was investigated and it was found that enhancement can be as high as 2 orders of magnitude better than conventional systems (E21). SAMPLE PREPARATION, QUENCHING, AND RELATED PHENOMENA The solvent effects on the fluorescence properties (quantum yields and excited-state lifetimes) of several donor-spaceracceptor systems were studied (F1). Solvent effects on the excited state of several hydroxy and amino compounds (F2) and styrylpyrenes (F3) were also investigated. In supercritical fluids, the fluorescence spectroscopy of pyrene was initiated to determine the solvent parameters affecting solvation (F4). These studies were expanded to include time-resolved results of pyrene dynamics in supercritical fluids (F5). A study of solvent effects, as well as UV photolysis effects, on the fluorescence of several aromatic pesticides found that the polarity of the solvent played a crucial role in determining the limit of detection for these compounds using fluorometric detection (F6). The analysis of PAHs in sediments was initiated using UV fluorescence and a simple hexane extraction method (F7). The accuracy of the method was found to depend upon the knowledge associated with the site where the sample was collected. The solvent-dependent fluorescence properties of PAHs were carefully examined by several groups. Acree et al. investigated the preferential solvation of PAHs dissolved in mixed binary solvent systems (F8). Tucker et al. looked at solvent polarity and nitromethane effects on the emission behavior of select bipolycyclic aromatic hydrocarbons (F9, F10). Tucker et al. also investigated the fluorescence emission and quenching behavior of selected acenaphthylene derivatives in organic nonelectrolyte solvents (F11) as well as the alkylated pyrene and chrysene derivatives (F12). Stern-Volmer plots were constructed for the selective quenching of several PAHs by nitromethane and nitrobenzene (F13). Two spectrofluorometric probe methods were critically evaluated regarding their ability to model solvation around probe

molecules dissolved in binary solvents (F14). Using a picosecond spectrostreak method, the solvation of fluorescence probes was also investigated in binary solvent mixtures (F15). Lakowicz and co-workers reported on the light quenching of fluorescence induced by laser pulses as a new method for controlling the excited-state population and orientation of chromophores which offers new opportunities for biophysics applications of time-resolved fluorescence (F16). Wilson discussed the quenching of phosphorescence by oxygen as a novel method for mapping oxygen contents in tissues with a few micrometers of spatial resolution (F17). Vinogradov and Wilson also developed a quadratic programming algorithm for determining quencher concentration distributions in heterogeneous systems (F18). As an alternative to electrochemical methods for the determination of molecular oxygen, phosphorescence quenching was utilized (F19). The chromophore used was camphorquinone because of its strong absorption and emission bands in the visible, enabling the use of inexpensive silicon dioxide fiber-optic light guides. Cerebral oxygen pressure was also determined optically by the oxygen-dependent quenching of phosphorescence (F20). The excited-state dynamics of C60 films were studied using luminescence quenching (F21). From the drop in the luminescence yield, it was concluded that branching in the excitation or relaxation processes involved intermolecular charge transfer from within the films. DATA REDUCTION The wealth of information in fluorescence data continues to provide multiple approaches to the analysis and quantification of these data. For example, quantification of PAHs in cooking oil fumes has provided evidence of a link between these PAHs and the high incidence of pulmonary adenocarcinoma in Chinese women (G1). Fell and co-workers have demonstrated the utility of expert systems for the speed of data interpretation of multicomponent fluorescence data (G2). Another expert system has been developed as a guide to experimenters who wish to use basic fluorescence spectroscopy (G3). Apparent content curves have been used to resolve binary fluorescence mixtures (G4). A model has been developed for calculating the analytical sensitivity, limit of detection, precision of fluorescence, and other analytical methods (G5). This method employs a statistical analysis of a linear regression of the calibration experiment. Three other approaches have employed multivariate methods for analyzing fluorescence data. One approach employs multivariate curve resolution methods (G6) and the other uses partial least-squares multivariate calibration to determine binary mixtures of sulfonamides (G7). The third approach has been employed in the development of chemical recognition software for use on data from a multispectral laser remote-sensing system (G8). Several algorithms based on factor analysis have also been developed. For example, Xie and co-workers have developed a hybrid method which employs a combination of the generalized standard addition method and constrained background bilinearization to analyze excitation-emission data (G9). Factor analysis has also been used to separate overlapping fluorescence spectra of multiple species of a hematoporphyrin derivative in aqueous solution of sodium dodecyl sulfate (G10). Pullin and Cabaniss have used rank analysis to analyze the pH-dependent fluorescence spectra of six standard humic substances (G11). Enderlein has shown that the maximum likelihood criterion is a powerful method

for analyzing fluorescence data (G12). Kowalski and co-workers have employed chemometric techniques to analyze excitationemission data obtained for dental calculus deposits from several animals (G13). Evolving factor analysis has been employed for analysis of synchronous fluorescence spectra of fulvic acids in the presence of aluminum (G14). Murillo Pulgarin and Alanon Molina have developed a program to process total luminescence data (G15). Chemometrics has also been used to analyze synchronous data of fulvic acids (G16). New and old algorithms continue to be applied to the analysis of time-resolved data. For example, F.-Calleja and co-workers have studied existing time-interval techniques for analysis of fluorescence decay data (G17). They were able to improve some of these techniques by providing more general expressions. Other approaches have used computer simulation of a boxcar integrator and averager system (G18) and the sine transform of the autocorrelation function (G19) to improve fluorescence decay measurements. Ristein has developed a rigorous sum rule for analyzing frequency-resolved fluorescence data (G20). Patterson and Pogue have developed a mathematical model for time-resolved and frequency-domain fluorescence spectroscopy in biological samples (G21). Numerical solutions to the diffusion in light equation describing light propagation have been used to monitor the fluorescence lifetime of a fluorophore uniformly distributed in tissue samples (G22). The Kalman filter has been used to aid in the resolution of time-resolved solid-surface room-temperature phosphorimetry (G23). LUMINESCENCE IN ORGANIZED MEDIA Micellar systems continue to provide general utility for improved luminescence measurements. The obvious utility of stopped-flow mixing techniques for micelle-stabilized room-temperature liquid phosphorimetry has been exploited (H1). McGown has discussed the applications of organized bile salt media for luminescence analysis (H2). Micellar systems have also been applied to the extraction of PAHs (H3). This procedure provides very selective measurement of PAHs when used in combination with synchronous fluorescence spectroscopy. The study of the determination of tetrachlorobiphenyl in the presence of micelles and cyclodextrins has also been reported (H4). The twisted intramolecular charge transfer of p-(N,N-dimethylamino)benzoic acid in aqueous solutions of cetyltrimethylammonium chloride micellar solutions has been studied (H5). Borges and co-workers have reported on a fluorescence study of the charge and pH dependence of the binding sites of dipyridamole in ionic micelles (H6). Khalaf and co-workers have evaluated the experimental conditions required for sensitized luminescence measurements of propoxur in different surfactants (H7). Octadecylrhodamine B has been used as a probe to study the properties of micelles of Pluoronic F68 (H8). These measurements involved a study of the fluorescence intensity, polarization, and lifetime as a function of concentration and temperature. Fluorescence quenching has been used to study the activity of lipases in esterification reactions involving various alcohols in microemulsions containing AOT reversed micelles in isooctane (H9). Tung and Wu have reported on a novel type of molecular assembly involving aggregates formed by molecules with polar chains in nonpolar solvents (H10). Intramolecular energy transfer between phenanthrene and lanthanide ions in aqueous micellar solutions has been described by Darwent and co-workers (H11). Nonradiative energy transfer Analytical Chemistry, Vol. 68, No. 12, June 15, 1996

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has been used to study polystyrene-poly(ethylene oxide) block copolymer micelle formation in organic and aqueous solvents (H12). Heerklotz and co-workers have reported on the membrane-water partition coefficient of the nonionic detergent C12E7 by use of the fluorescence probe laurdan (H13). The interaction of protein kinase C lipid-loaded mixed micelles has been reported (H14). Several other studies have reported on probing lipid bilayers (H15-H19). Fluorescence spectroscopy has also been used to probe the microenvironment of block copolymer micelles (H20). Cyclodextrins continue to be active areas of investigations for fluorescence spectroscopic studies in organized media. Nagata and co-workers have reported the pH-responsive binding of a cyclodextrin with a polypeptide tail (H21). The chiral recognition properties of cyclodextrins continue to be important. Burns has discussed the versatility of the chiral recognition properties of cyclodextrins in a variety of approaches employing optical spectroscopy and separation techniques (H22). Bara and Scaiano have reported on the photochemistry of aromatic ketones in solid cyclodextrin complexes (H23). The ability of cyclodextrins to stabilize and improve the emission properties of guest molecules has been exploited to provide quantitative determination of retinoids (H24). Flamigni has reported a steady-state and picosecond time-resolved study of the inclusion complexes of fluorescein and halogenated derivatives in R-, β-, and γ-cyclodextrins (H25). Several studies have been reported on luminescence as a probe of inclusion complexes with R-cyclodextrin (H26, H27), β-cyclodextrin (H28-H34), and γ-cyclodextrin (H35). Luminescence has also been used to probe the self-assembly and binding properties of a hydrophobilized polysacccharide structure of a hydrogel nanoparticle (H36). The hydrogen-bonding interaction between two different calix[4]arenes has been probed by use of fluorescence spectroscopy (H37). Other studies report on biacetyl binding in hemicarcerand (H38), fluorescence behavior of polychlorinated dibenzofurans (H39), organized monolayer films for biosensor development (H40), Langmuir-Blodgett films (H41, H42), and self-assembly of peptides (H43). LOW-TEMPERATURE LUMINESCENCE The use of cryogenic, high-resolution molecular fluorescence (Shpol’ski spectroscopy) in analytical chemistry was reviewed (I1). The instrumental developments were addressed as well as the low-level detection of PAHs in complex matrices for environmental studies. Conformational dynamics in light-harvesting systems were also investigated using low-temperature luminescence spectroscopy. Koyama and co-workers reported on the fluorescence and fluorescence excitation spectroscopy at 170 K of all-trans-spheroidene (I2). Vacha and co-workers reported on the lowtemperature phosphorescence of photosystem II particles (I3). The fluorescence spectroscopy of oligo(anthrylenevinylenes) in low-temperature MTHF glasses was reported using a site-selection technique (I4). Fluorescence excitation spectra of small and dilute samples at low temperature in solids was also discussed by Orrit et al. (I5). The authors suggested the use of the narrow lines as probes of the low-temperature matrix around the molecule or as probes of intramolecular transitions between electronic levels. 78R

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Low-temperature luminescence spectra have been reported for several systems, such as, ethenoadenosine at 77 K (I6), o-, m-, and p-phenetidines at 77 K (I7) and 4-methyl-2,6-diacetylphenol at room temperature and 77 K (I8). In addition, the wavelengthdependent emission spectra of several compounds were examined in both pure weakly polar aprotic solvents and in the presence of a base at 77 K (I9). A new method to evaluate the applicability for the analysis of nitro and amino PAH isomers in the environment using lowtemperature, high-resolution fluorescence spectroscopy in Shpol’ski solvents was reported (I10). In this method, the PAHs are treated with potassium borohydride to reduced the PAHs into fluorescent amino PAHs. TOTAL LUMINESCENCE AND SYNCHRONOUS EXCITATION SPECTROSCOPIES AND RELATED TECHNIQUES Several applications of total luminescence spectroscopy appeared, including studies of C60 and C70 fullerenes in various organic matrices (J1) and rapid determination of PAH isomers that could not be adequately resolved by HPLC in environmental samples (J2). In the latter application, the total luminescence spectrum was used as a guide to the selection of an optimal ∆λ for subsequent determination by synchronous excitation spectroscopy. Monte Carlo integration was performed on total spectra to improve the sensitivity of determinations of fluorescein and tryptophan relative to conventional, single-point intensity measurements (J3). The same methodology was applied to total, or threedimensional, synchronous excitation spectroscopy for the simultaneous determination of PAHs in a mixture (J4). Enormous activity continues in the development and application of synchronous excitation luminescence. Victor and Crouch described a fiber-optic-based fluorometer for absorbance-corrected synchronous fluorescence (J5) that simultaneously measures frontsurface fluorescence and absorbance in order to correct for primary and secondary inner-filtering effects. A battery-operated, portable instrument was developed for on-site analysis of groundwater or hazardous waste sites (J6). Stevenson and Vo-Dinh reported the first use of a dye laser as an excitation source for synchronous excitation (J7) and were able to detect as little as 680 zmol of tetracene in the volume probed by the laser. An improved version (J8) provided superior scanning precision and extended wavelength scanning range, as well as time-resolved capabilities for greater selectivity. Fluorescence lifetime selectivity was incorporated into synchronous excitation luminescence in the frequency domain as well, in the lifetime synchronous spectrum (LiSS), which plots lifetime as a function of synchronously scanned wavelength (J9). Shaver and McGown used phase resolution to generate the LiSS and demonstrated its application to coal liquid samples (J9, J10). The technique of matrix isopotential synchronous fluorescence was introduced, in which wavelength is synchronously scanned through a trajectory that joins points of equal intensity in a total luminescence spectrum of a sample with unknown background fluorescence (J11). The technique was applied to the determination of gentisic acid in urine (J11), mixtures of salicylic and gentisic acid (J12), and pyridoxamine in urine (J13). Secondderivative synchronous scanning was introduced as an alternative to time resolution for detection of Tb3+ chelates in immunoassays (J14). The technique, which was demonstrated for determination

of R-fetoprotein, takes advantage of the large Stokes shift of the Tb3+ chelates and the spectral band-narrowing effect of synchronous scanning. The application of an external magnetic field to reduce the undulation of scattered light in resonance synchronous scanning was described (J15). Improved detection limits were obtained for a mixture of anthracene and perylene. The combined technique of derivative variable-offset synchronous scanning was applied to simultaneous analysis of 1-naphthol and 2-naphthol in water samples for improved spectral resolution (J16). Among the numerous applications of synchronous and derivative synchronous spectroscopies are environmental and fuelrelated determinations of PAHs in gases from coal liquids (J17), investigations of coal rank (J18, J19), determination of dicoronylene in hydrocracker streams (J20), PAHs (J21), and their metabolites (J22) in fish bile, and analysis of biodegradable and nonbiodegradable aromatic components of wastewater (J23). Examples of pharmaceutical and biological applications include simultaneous determination of warfarin and bromadiolone (J24), determination of boldine in drug formulations (J25), simultaneous determination of pyridoxal and pyridoxamine (J26), determination of aspirin metabolites in urine (J27), and simultaneous determination of three diuretic drugs (J28). In a related application, synchronous excitation was used to determine the ionization constants of several pharmaceutical compounds (J29). Various organized media have been used in synchronous excitation spectroscopic analysis. Micelles have been used for extraction and enrichment of PAHs in aqueous solutions and soil suspensions (J30), fluorescence enhancement of PAHs in seawater (J31), and analysis of binary mixtures (J32). Selective iodide quenching of three-ring aromatic compounds in β-cyclodextrin was applied to the analysis of mixtures (J33). SOLID-SURFACE LUMINESCENCE Hurtubise and Richmond reviewed the analytical characteristics of β-cyclodextrin-salt mixtures in solid-matrix, room-temperature luminescence analysis (K1), and Sanz-Medel reviewed the coupling of solid-surface photoluminescence with flow injection analysis (K2). New studies by Hurtubise and co-workers examined the luminescence lifetimes and quantum yields of the tetrols of benzo[a]pyrene-DNA adducts in a solid, β-cyclodextrin-NaCl matrix at room temperature and low temperature, and in solution (K3), and applied HPLC and solid-matrix room-temperature luminescence to the separation and analysis of four such tetrols (K4). In other applications, synchronous room-temperature phosphorescence was applied to the analysis of mixtures of purines and pyrimidines on filter paper in the presence of heavy ions (K5), and solid-phase extraction was combined with frontsurface, solid-state luminescence detection for monitoring PAHs in water (K6). Solid-surface techniques were used in investigations of adsorbed molecules, including polynuclear aromatic compounds on soil surfaces (K7), evanescent wave-induced luminescence of the dye auramine-O at a solid-solution interface, for which the maximum entropy method was used to analyze the fluorescence decay data (K8), Rhodamine 6G on saponite in an aqueous suspension (K9), and the orientation distribution of adsorbed fluorophores by total internal reflection fluorescence (K10). Steady-state fluorescence polarization was used to study the orientational parameters of uniaxial films (K11), and phosphorescent compounds were used to probe the molecular dynamics of

the glassy state in amorphous sucrose and the glass-to-rubber transition (K12). LUMINESCENCE IN CHROMATOGRAPHY, ELECTROPHORESIS, AND FLOW SYSTEMS Reviews. Several reviews have appeared over the last two years which have focused on the use of luminescence in both liquid chromatography and electrophoretic methods. van de Nesse and co-workers reviewed the use of laser-induced fluorescence detection of analytes that are naturally fluorescent in column liquid chromatography (L1). The authors critically evaluated aspects that need to be considered when applying lasers instead of conventional lamps in fluorescence detection. A review with 23 references has appeared which discussed the analysis of lipids using microcolumn HPLC and laser-induced fluorescence (L2). Hurst and Zagon reviewed the isolation, separation, and detection of enkephalins using HPLC and capillary electrophoresis with various detection strategies, including fluorescence (L3). Imai and Watanabe reviewed the HPLC of biogenic amines and other neurotransmitters using fluorescence and chemiluminescence detection (L4). The analysis of N-methylcarbamates with HPLC and fluorescence detection was reviewed by McGarvey (L5). Juorio and Sloley reviewed the HPLC determination of amines using both electrochemical and fluorometric detection (L6). The utility of laser-induced fluorescence (LIF) detection in applications of capillary electrophoresis was reviewed (L7). In the article, LIF instrumentation, fluor labeling, and different ways to detect analytes by CE-LIF were discussed. This review contained 122 references and dealt specifically with the analysis of nucleic acids, amino acids, peptides, and proteins. Yoa and Li also reviewed the use of fluorescence detection in CE (L8). As part of a critical review on the development of CE instrumentation, Stevenson discussed fluorescence detection in CE applications (L9). Yeung reviewed various optical detection strategies in capillary electrophoresis, including direct and indirect fluorescence detection (L10). Flow Injection Analysis. Smith and co-workers reported on the fluorescence-based flow injection determination of biotin and biotinylated compounds (L11). Analyses were conducted by taking advantage of the enhancement in fluorescence intensity of a conjugate of avidin-fluorescein in the presence of biotin and biotin derivatives. Pollema and Ruzicka discussed a new approach to immunoanalysis with fluorescence detection utilizing a flow injection renewable surface immunoassay (L12). A flow injection analysis system was developed which was coupled to an evanescent wave sensor employing internal reflection of fluorescence radiation for the detection of organophosphorus compounds (L13). Flippov presented a quantitative theory on the kinetic-diffusive behavior in the flow cell for total internal reflection fluorescence spectroscopy used in flow injection analysis (L14). ValenciaGonzalez and Diaz-Garcia developed alternative approaches for glucose biosensing using a room-temperature phosphorescence oxygen transducer in flow injection devices (L15). Different configurations for biosensing were examined, and their respective analytical characteristics, performance, and stability were evaluated. Perez-Ruiz and co-workers reported on a flow injection configuration for the fluorometric determination of thiourea (L16). Thin-Layer Chromatography and Other Miscellaneous Separation Techniques. Jeger and Briellmann presented a report on the quantitative applications of TLC in the forensic area Analytical Chemistry, Vol. 68, No. 12, June 15, 1996

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(L17). The paper dealt with fluorometric detection in the analyses. A parallel-current open-tubular chromatography system was developed which utilized fluorescence detection (L18). Kim and co-workers developed an immunoaffinity chromatographic method for the analysis of pesticides (L19). Three detection systems were incorporated into the device, including fluorescence detection. A size-exclusion chromatographic system was developed for the determination of β-cyclodextrin derivatives based on fluorescence enhancement after inclusion complexation (L20). The lumiphore used in the analysis was 1-naphthol, which was added to the mobile phase. Suortti reported on the determination of β-glucan using size-exclusion chromatography with postcolumn reaction detection based on fluorescence (L21). The fluorescence was produced upon interaction of β-glucan with calcofluor dye. Fluorogenic Labels for Chromatography. The specific labeling of N-terminal tryptophan-containing peptides was discussed by Kai and co-workers (L22). The derivatives were separated isocratically using reversed-phase LC with subsequent fluorescence detection. Zhoa and Bada presented an assay for R-dialkylamino acids in geological samples by HPLC after derivatization with a chiral adduct of o-phthaldialdehyde (L23). The kinetics of the derivatization reaction were studied with the assay optimized for the detection and enantomeric resolution of R-dialkylamino acids. The use of a europium chelate for the fluorescence detection of amino- and thiol-containing compounds for HPLC was presented (L24). The technique used a labeling step with the nonfluorescent europium chelate and a subsequent replacement step in a postcolumn arrangement to produce a more fluorescent chelate complex. Imakyure and co-workers synthesized two Edman-type fluorogenic labels, 3- and 4-(2-phenanthra[9′,10′-d]oxazolyl)phenyl isothiocyanates (L25). The reaction conditions of amino acids with these reagents were studied for the HPLC determination of amino acids. A sensitive fluorometric HPLC method for the determination of aliphatic thiols, following precolumn derivatization with 2-(4N-maleimidophenyl)-6-methylbenzothiazole was developed (L26). The method was found to be very sensitive and easily adapted for the determination of SH-containing drugs and endogenous thiols in biological samples. The use of 2-(4-N-maleimidophenyl)6-methoxybenzofuran as a fluorogenic precolumn derivatizing agent for the sensitive HPLC determination of aliphatic thiols was also described (L27). The conjugates were separated using reversed-phase LC and detected fluorometrically with excitation at 310 nm and emission at 390 nm. For the detection of target DNA base sequences using fluorescent-labeled oligonucleotides as a hybridizing probe, a rapid and sensitive procedure was developed using HPLC and a 5′-fluorescent-labeled oligonucleotide probe (L28). Sonoki and co-workers described a method for the simultaneous detection of mono-, di-, and trinucleotides by HPLC using N-(dansyl)ethylenediamine as a fluorescent derivatizing reagent (L29, L30). A precolumn fluorescence derivatization method was presented for the determination of guanine and its nucleosides and nucleotides that utilized HPLC and converted the analytes into fluorescence derivatives by reaction with phenylglyoxal in phosphate buffer (L31). Extractive derivatization of the 12-lipoxygenase products, hepoxlins and related compounds, into fluorescent anthryl esters for HPLC determination was presented (L32). The method involves the introduction of a fluorescent ester chromophore into 80R

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the carboxylic group of the hepoxilins under conditions that do not require acidification, leading to stabilization of the derivative. Koga and co-workers developed a fluorogenic label, 4-[(Nchloroformyl)methyl-N-methyl)amino]-7-[(N,N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazole for the analysis of the endogenous ligand for the cannabinoid receptor and its analog (L33). Ferrocenylethylamine was synthesized for the determination of retinoic acids by HPLC (L34). The adduct formed was found to be both fluorescent and electroactive. Marr and co-workers developed several derivatization reagents for the conversion of okadaic acid and related DSP toxins to fluorescent derivatives for analysis by HPLC (L35). Precolumn derivatization using leucine-coumarinylamide for the HPLC determination of mono- and dicarboxylic acids in plasma was also presented (L36). Takeuchi and Miwa studied the effects of cyclodextrin as a mobile-phase additive to enhance the fluorescence from dansylamino acids separated by microcolumn LC (L37). Their results indicated that both β- and γ-cyclodextrin enhanced the fluorescence, especially for the neutral dansyl amino acids. 9-Substituted fluorescent quinolizinocoumarin derivatives were extensively studied spectroscopically due to the wide variation in the emissions observed during HPLC detection in different mobile phases (L38). The observed spectroscopic variations associated with the change in solvents were analyzed in terms of modification in the microenvironment of the derivatives. Instrumentation and Applications in HPLC. Different methods of quantitation that use both fluorescence spectral and retention time data were evaluated for accuracy and precision in diode-array fluorescence detection for HPLC (L39). The methods investigated include peak height and peak area methods of quantitation, an adaptive Kalman filter, and the generalized rank annihilation methods. DeSilva et al. developed a dual-wavelength excitation method for the off-line LC analysis of amino acids derivatized with naphthalenedialdehyde (L40). The method involved the deposition of LC effluents onto TLC plates in order to circumvent the fluorescence loss produced by multiderivatized amino acids. An algorithm was developed for the global analysis of the measured emission decay profiles obtained in LC to resolve poorly separated components (L41). The development of an instrument was discussed using frequency-domain fluorescence lifetime detection in HPLC (L42). The instrument utilized a multiharmonic Fourier transform spectrofluorometer to eliminate the need for multiple injections to obtain the lifetimes. Heath and Giordani reported on an instrument that interfaced three detectors to an HPLC system for the analysis of peptides and proteins (L43). The detectors were situated in series and consisted of UV, fluorescence, and electrospray ionization mass spectrometric detectors. In order to enhance the fluorescence observed from dansyl amino acids separated by HPLC, a packed flow cell where the analytes were detected in the presence of the stationary phase was developed (L44, L45). Kiba and co-workers developed an HPLC system with enzyme reactors for the determination of N-acetyl branched-chain amino acids (L46). The enzymatically produced amino acids were allowed to react with NAD+ in the presence of leucine dehydrogenase, and the reduced NAD was monitored fluorometrically. Neuroactive amino acids were analyzed by HPLC with precolumn derivatization using o-phthalaldehyde (L47). The analysis of penicillin-binding proteins was introduced using reversed-phase chromatography and β-lactam fluorescent probes (L48).

Second-derivative fluorescence spectroscopy was implemented as a detection strategy for the HPLC analysis of salicylic acid in aspirin formulations (L49). Anumula reported on a rapid quantitative determination of sialic acids in glycoproteins by HPLC with sensitive fluorescence detection (L50). The sialic acids were labeled with o-phenylenediamine. Sen and co-workers developed an assay to determine the occurrence of various tetrahydro-βcarboline-3-carboxylic acids in foods and alcoholic beverages (L51). Joshua described the analysis of aflatoxins in naturally contaminated corn using HPLC and postcolumn photochemical derivatization and fluorescence detection (L52, L53). The assay was able to achieve detection limits in the sub-ppb range. Franco and Fernandez-Vila discussed the separation of paralytic shellfish toxins by HPLC with postcolumn reaction and fluorometric detection (L54). Janecek and co-workers also discussed the analysis of paralytic shellfish toxins using automated precolumn oxidation, HPLC separation, and fluorescence detection (L55). The toxins were oxidized using periodate, yielding fluorescent purines. To establish the usefulness of fluorescence detection to quantify urinary concentrations of dextromethorphan and dextorphan for oxidative phenotyping, the molar concentrations of these analytes were determined in 38 subjects (L56). Ou and co-workers identified and quantified choline glycerophospholipids, which contain aldehyde residues, using fluorometric detection and HPLC separation (L57). In this study, formation of aldehydic choline glycerophospholipids was demonstrated for the first time in peroxidized red blood cell membranes. Twenty different fractions of hematoporphyrin derivatives and eight fractions of an HpD dimer mixture were isolated using HPLC and an ionpairing agent and finally characterized using fluorescence spectroscopy (L58). Fluorescence quantum yields and photokill efficiency for each fraction in PTK2 epithelial cells were obtained. Meyns et al. addressed the observed differences in the results obtained for the determination of chlorophyll-a in algae using HPLC and fluorescence spectroscopy (L59). As an extension of previous methods for enantiomeric analyses of diacylglycerols, a highly sensitive HPLC method was developed for the determination of the optical purity in monoacylglycerols using a fluorescent chiral derivatization scheme (L60). An ultrasensitive method was developed for the analysis of NADP+, NADPH, NAD+, and NADH (L61). A simple, rapid reaction of the oxidized nucleotides with cyanide in basic solution produced two stable fluorescent products which could be separated using HPLC. Ogura et al. reported on a method for the analysis of brightners in detergents by HPLC and three different detection strategies, including fluorescence (L62). Panfili and co-workers presented an HPLC method using fluorescence detection with a programmable spectrofluorometer for the analyses of tocopherols, carotenes, and retinol and its geometrical isomers in Italian cheeses (L63). The procedures needed for the optimization of programmable fluorescence detectors required for the analysis of polyaromatic hydrocarbons separated by HPLC were studied (L64). Reupert and co-workers presented an HPLC method for the separation of PAHs with fluorescence detection (L65). The detection was based on fluorescence with wavelength programming in selection of substances according to EPA and with a constant wavelength pair in substance selection according to TVO. Smalley and McGown reported on the detection limits and resolution for on-the-fly lifetime determinations of PAHs separated by HPLC (L66). Garcia

and co-workers developed a method for the determination of PAHs using cloud point preconcentration followed by HPLC separation and fluorescence detection (L67). Methods to improve the sensitivity in fluorescence HPLC protocols for detecting benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide-deoxydenosine adducts in enzyme digests of modified DNA were presented (L68). Instrumentation and Applications in CE. Cannon and Schallern discussed the uses of deep-UV laser-induced fluorescence detection in CE for PAHs at the low-zeptomole limit of detection (L69). The use of laser-induced fluorescence for the trace-level detection of such analytes as peptides and neuropeptides separated by CE was also presented (L70). Sweedler and co-workers presented two unique CE detection strategies for the trace-level analysis of peptides (L71). One method utilized online, multichannel CCD laser-induced fluorescence. Reinhound et al. developed an automated on-capillary isotachophoretic reaction cell for the fluorescence derivatization of small sample volumes at low concentrations followed by CE (L72). Beale and Sudmeier constructed a spatial-scanning laser fluorescence detector for CE using epi-illumination and confocal optical detection which was designed to scan the entire length of the separation capillary (L73). A number of papers have appeared which have constructed microchip CE devices utilizing fluorescence detection. Jacobsen and co-workers developed an argon ion laser-induced fluorescence detector for these microchip CE systems (L74). Effenhauser et al. utilized laser-induced fluorescence detection in microchip CE devices for the analysis of FITC-labeled amino acids (L75). Preand postcolumn reactor systems have been integrated onto these microchip devices for the labeling of amino acids with fluorogenic reagents (L76, L77). Woolley and Mathies reported on the sensitive fluorescence detection of DNA restriction fragments and DNA sequencing fragments separated with microchip CE (L78L80). In order to optimize the fluorescence detection in chemical separations, particularly CE, a theory was developed for calculating the signal-to-noise ratio under such conditions (L81). It was found that, using standard detection procedures in CE, the sensitivity can vary by a factor of 5 depending on when the migrating zone arrives at the fluorescence detector. Selective determination of adenine-containing compounds by CE with laser-induced fluorescence detection was reported (L82). The adenine compounds were derivatized with chloroacetaldehyde as a fluorogenic label. Zhoa et al. separated aminated monosaccharides by CE and detected the compounds using laser-induced fluorescence detection (L83). The analysis of mono- and oligosaccharide isomers derivatized with 9-aminopyrene-1,4,6-trisulfonate by CE with laser-induced fluorescence was investigated (L84). Practical aspects and future prospects for sugar determinations in biochemistry by isoindole derivatization were described (L85). For amino acid analyses, the practical and quantitative aspects of assays using CE with LIF detection and labeling incorporating FITC and DTAF derivatizing reagents were discussed (L86). Mattusch et al. presented results on the sensitive laser-induced fluorescence detection of polyamine-FITC derivatives separated via CE (L87). Chang and Yeung presented results on the analysis of catecholamines in single adrenal medullary cells by CE with laserinduced native fluorescence (L88). At low pH, high sensitivity was reported. On-column derivatization of single mammalian cells Analytical Chemistry, Vol. 68, No. 12, June 15, 1996

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with CE and laser-induced fluorescence detection was described (L89). CE coupled to fluorescence detection was reported for the in vivo determination of the release of multiple neuropeptides from the ewe median eminence (L90). The CE separation of double-stranded DNA fragments stained with monomeric and dimeric fluorescent intercalating dyes using a confocal laser fluorescence detection system was reported by Zhu and co-workers (L91). CE separation of DNA sequencing products using linear acrylamides and laser-induced fluorescence detection was presented (L92). Ueno and Yeung constructed an instrument that was capable of simultaneously monitoring the fluorescence produced from DNA fragments in 100 capillaries (L93). The system used a multichannel CCD detector. For multicolor fluorescent DNA detection in sequencing applications, a multiple sheath flow CE array system was developed (L94). The minimum detectable concentration of dye-labeled DNA was found to be 0.1 pM. For high-sensitivity, on-column detection of DNA sequencing products separated in capillary arrays, a laser-excited confocal fluorescence scanner was reported (L95). The four bases were identified via a binary coding system employing only two different fluorescently labeled dye primers. DYNAMIC MEASUREMENTS OF LUMINESCENCE Reviews addressed potential applications of frequency-domain fluorescence lifetime techniques in industrial, on-line bioreactor control and in clinical monitoring (M1) and the use of timeresolved detection of lanthanide chelate luminescence in bioanalysis (M2). Instrumental advances for lifetime-based measurements include the coupling of a grating objective to a streak camera for simultaneous measurement of intensity as a function of time and of wavelength (M3), a low-cost, closed-loop scheme for phasesensitive lifetime measurements using a fiber-optic sensor (M4), and a modulated deuterium arc lamp light source which can be driven at frequencies up to ∼130 MHz for frequency-domain lifetime measurements and lifetime imaging (M5). McGown and co-workers described a total lifetime distribution analysis approach in which the total fluorescence emission is used in the frequency-domain lifetime analysis and the maximum entropy method is used to recover the lifetime distribution (M6). Shaver and McGown described the lifetime synchronous spectrum based on phase-resolved fluorescence spectroscopy (J9) and applied it to the study of complex coal liquid samples (J10). Galla and co-workers used multiplex dyes with different lifetimes to tag antigens for simultaneous antigen detection (M7). Hutchinson et al. used computational methods to show the feasibility of noninvasive diagnostic monitoring through quantitation of fluorescence lifetime if the lifetime is comparable to photon migration times (M8). Other applications of fluorescence lifetime techniques include a nonisotopic, time-resolved fluorometric receptor assay for benzodiazepine drugs (M9), time-resolved monitoring of local and global dynamics during protein folding (M10), rotational dynamics of C60 and C70 (M11), fast reaction dynamics in the excited states of firefly luciferase (M12), field tests of trace analysis of oil pollution in water and in the ground (M13), and a delayed fluorescence technique for determination of bitterness in beer (M14). 82R

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FLUORESCENCE POLARIZATION, MOLECULAR DYNAMICS AND RELATED PHENOMENA A comparison was made between a GC/MS assay and three immunoassays, including an assay based upon the use of fluorescence polarization immunoassay for the analysis of cannabis metabolites in urine (N1). It was found that the immunoassay results agreed favorably with the GC/MS method and the simple immunoassays could be used for screening purposes. For protein dynamics and flexibility studies in glycerol solvents, the intrinsic phosphorescence of tryptophan residues in several model proteins was measured (N2). The authors concluded that the cosolvent-induced structural changes can be important, even for inner cores of the macromolecules. The dynamics of lightharvesting complexes was studied using picosecond fluorescence spectrochronography (N3). From the data obtained, the authors were able to develop a kinetic model for excitation transfer within the complex. Krishman and co-workers used fluorescence polarization studies to study the binding of antitumor antibiotics to DNA (N4). The increase in the fluorescence polarization was determined to result from the reduced rotational motion of the molecule when bound to DNA. Larsson and co-workers reported on the interactions of the fluorescence dyes, YOYO and TO to T2 doublestranded DNA using fluorescence anisotropy (N5). The conformational changes induced by various substrates binding to glutaminyl-tRNA was evaluated using fluorescence spectroscopy (N6). Phillips and Georghiou developed a global analysis method using trilinear curve resolution to study fluorescence data arrays of mixtures (N7). The methodology is able to separate polarization orientation, excitation wavelength, and emission wavelength. Demidov and Andrews developed a theory focused on the polarized fluorescence and absorption in molecular complexes composed of two chromophores with nonparallel absorption and emission transition dipoles (N8). Detection of the dissociation products of R-urease was studied using fluorescence emission and polarization spectroscopy (N9). Fluorescence polarization technology was used to develop an assay for protease activity that was more sensitive than other nonradioactive assays (N10). In the assay, changes in the molecular volume due to cleavage of intact fluorescein thiocarbamoyl-casein molecules to smaller FITC peptides were measured. The photoisomerization dynamics of the fluorescent dye DTCI was reported (N11). The results of this study suggested that internal conversion, without passing through an intermediate twisted state, is the major nonradiative pathway in this dye. Timeresolved chiroptical luminescence spectroscopy was used to measure the chirality dependence of dynamic excited-state quenching processes in solution (N12). Time-resolved fluorescence spectroscopy was used to study the chain-foldability effect on the coaggregation of cholesterol esters (N13). Collins and Davidsen reported on the photodegradation of fluorescent whitening agents using real-time fluorescence spectroscopy (N14). The degradation of the whitening agents is preceded by an induction period which is attributed to the wool protecting the agents by reaction of its cystyl residues with any photogenerated singlet oxygen. The photodegradation of several fluorescent dyes pertaining to sensitive fluorescence measurements was also reported (N15). It was found that the photon yield

per molecule was nearly 100 times greater in ethanol than in water because of the superior photostabilities of these dyes in ethanol. CHEMILUMINESCENCE There are only three citations in this area over this review period. One includes a detailed study of bacterial luciferase, which catalyzes the bioluminescence reaction in luminous bacteria (O1). Another article involves enhanced chemiluminescence detection of fluorescein-labeled nucleic acids (O2). The third article involves development of a chemiluminescence detection system for fluorescent compounds separated with an acidic mobile phase such as trifluoroacetic acid (O3). NEAR-INFRARED FLUORESCENCE Reviews. Thompson reviewed the practical aspects and methods for performing fluorescence measurements in the red and near-IR regions (600-1000 nm) of the spectrum (P1). The author considered various instrumental components associated with near-IR fluorometry, including excitation sources, detectors, and other optical components as well as the major class of compounds fluorescing in the near-IR. Fluorogenic Reagents for Near-IR Fluorescence. Narayanan and co-workers presented work on the synthetic preparation of several near-IR covalent labeling dyes for bioanalytical applications (P2). Shealy and co-workers synthesized near-IR labeling dyes for use as probes of DNA in sequencing applications (P3). The authors also discussed the chromatographic separation and fluorescence properties of these labeling dyes. Karnes et al. reported on the use of Rhodamine 800, a commercial laser dye, as a precolumn additive for indirect fluorescence detection in liquid chromatography (P4). The authors also reported on the use of Nile Blue as a derivatization reagent for carboxyl-containing analytes. Soper and Mattingly used steady-state and picosecond laser studies to investigate the nonradiative pathways in near-IR dyes and their implications on the preparation of new near-IR dyes with high fluorescence efficiencies (P5). Instrumentation and Applications. An instrument to detect near-IR fluorescence in solid-phase immunoassays was reported (P6). The instrument used a diode laser for excitation and could observe fluorescence in both solid and solution phases. For immunoassays, the antibodies were labeled with near-IR polymethine dyes. Thiols were analyzed using liquid chromatography with a newly developed diode laser detection system in the far-red region of the spectrum (P7). 2-Mercaptobenzothiazole was labeled with a dicarbocyanine dye with a detection limit found to be 8 pM. Primary and secondary amines were separated via LC after precolumn derivatization with a near-IR labeling dye and detected using a diode-based laser-induced fluorescence system (P8). Mank and co-workers compared the applicability of two pulsed lasers, XeCl-excimer/dye laser and Nd:YAG/dye laser, as excitation sources for near-IR fluorescence in LC (P9). Using the model compound, aluminum phthalocyanine, the best detection limits were found for the excimer/dye laser system. The use of ultrasensitive near-IR fluorescence in capillary electrophoretic applications was reported (P10, P11). Separation of native near-IR dyes in nonaqueous running buffers was performed with detection limits found to be in the low-zeptomole range. Amino acids and small peptides were analyzed using capillary electrophoresis after precolumn derivatization with di-

carbocyanine probes (P12). A detection limit of 0.1 amol for dyelabeled glycine was reported. Fuchigami and Imasaka used capillary micellar electrokinetic chromatography with indirect near-IR laser diode fluorescence detection for the determination of some model dinucleotides (P13). In DNA sequencing applications using capillary gel electrophoresis and near-IR fluorescence, Williams and Soper reported on the use of polymethine dye-labeled primers for sequencing applications (P14). Their results indicated improved limits of detection in capillary gel electrophoresis in the near-IR compared to visible fluorescence detection. The use of heavy-atom modified near-IR dyes for a single-lane, single-dye DNA sequencing strategy was also reported (P15). LUMINESCENCE TECHNIQUES IN BIOLOGICAL AND CLINICAL ANALYSIS Schenk reviewed clinical applications of luminescence spectroscopy (Q1), Hemmila reviewed applications of luminescent lanthanide chelates as sensitive labels for clinical diagnostics (Q2), and Lachaine et al. reviewed applications of photoacoustic and fluorescence spectroscopy in biological sciences (Q3). Nucleic Acids. There has been explosive growth in the applications of luminescence techniques to nucleic acid detection. Dye-based methods for quantitation of double-stranded DNA and related binding studies have employed ethidium bromide (Q4Q6), Hoechst 33258 (Q7, Q8), and oxazole yellow (YO) and its homodimer YOYO (Q9, Q10). Lanthanides have been used in time-resolved detection methods (Q11, Q12). Other detection methods used elastase with a fluorogenic rhodamine substrate (Q13) and reactions of quanine and adenine moieties in DNA with phenylglyoxal and chloroacetaldehyde, respectively (Q14). Comparison of spectrofluorometric methods for measuring DNA and RNA in a deposit-feeding bivalve pointed to several problems that led to the recommendation of a protocol for the quantification of nucleic acids (Q15). An automated analysis of DNA restriction fragments in an agarose gel matrix based on fluorescence detection was described (Q16). Fluorescence monitoring of nucleic acid processes is gaining attention as well. Ethidium bromide was used to detect hybridization (Q17, Q18). Strand-specific fluorescence in situ hybridization was used for determining the orientation and direction of DNA sequences (Q19). Fluorescence methods were also described for monitoring helicase-mediated unwinding of duplex DNA (Q20, Q21), real-time kinetics of restriction endonuclease cleavage (Q22), and yeast TATA binding protein interaction with DNA (Q23). In other studies, fluorescent labeling of DNA (Q24) and fluorescent postlabeling of modified DNA bases (Q25) were investigated. Proteins. Steady-state and time-resolved fluorescence of proteins was reviewed (Q26). Absorbance and fluorescence of albumin and myoglobin entrapped in bulk sol-gel glasses were investigated (Q27). Lee reviewed fluorescence studies of proteinlipid interactions (Q28) and protein-protein interactions (Q29) in reconstituted membrane vesicles. Fluorescence studies of protein-lipid interactions included protein kinase C with lipidloaded mixed micelles (Q30) and binding and transfer of phospholipids by lipid-transfer proteins (Q31). Other studies investigated binding of halothane to serum albumin (Q32) and the use of pyridoxic acid bound to protein through a stable amide linkage Analytical Chemistry, Vol. 68, No. 12, June 15, 1996

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to study the fast and slow motions of proteins in the nanosecond and millisecond time scales, respectively (Q33). Applications of front-face fluorometry to the study of dynamic protein structure and clinical analysis were investigated (Q34). Fluorescence determinations of plasma proteins, including total plasma proteins, globulins and albumin, were based on quenching of the intrinsic protein and plasma fluorescence by the dye ANS (Q35). Other Techniques and Applications. Automated, bioluminescent determinations of cellular metabolites were described which employ a novel, fluorometric reagent that can be stored frozen and is relatively inexpensive and stable (Q36). Two fluorescent, aromatic boronic acids were identified among several candidates for the measurement of sugars in water (Q37). A method for determination of glucose was reported (Q38) which is based on substrate-induced quenching of indicator fluorescence. Other applications of luminescence to clinical analysis include a continuous fluorescence displacement method for determination of serum triglyceride (Q39), a fluorescent method for in vivo and in vitro determination of fluorescent lipid peroxidation products (Q40), synthesis and fluorescence characterization of a phosphotyrosine peptide substrate for determination of protein tyrosine phosphatases (Q41), and hydrolysis of a tyrosine peptide to increase its intrinsic fluorescence for use in proteinase determinations (Q42). Phosphorescence quenching was used for in vivo measurement of oxygen pressure in the epicardium of the heart (Q43). REAGENTS AND PROBES There is a large increase in the number of references in this category. This growth is very much a reflection of the increasing utility of fluorescence spectroscopy as an analytical tool. In this regard, Soper and co-workers have examined the photophysical constants of several fluorescent dyes that are used as tags for biological species (R1). New fluorescence reagents for amino acids (R2, R3), fatty acids (R4), folic acid (R5), and mono- and dicarboxylic acids (R6) have been reported. Marr and co-workers have investigated fluorescent derivitization reagents for the analysis of shellfish toxins (R7). Maity and Kasturi have investigated the interaction of cationic and anionic dyes using fluorescence spectroscopy (R8). Several reagents have been reported for the study of oligosaccharides (R9) and saccharides (R10, R11). Pfeiffer and Radler have reported a reagent for determining ethanolamine in wines (R12). Reagents for amine functional groups (R13), hexosamines (R14), and hydrazine derivatives (R15) have also been reported. Several probes for DNA have also been reported (R16-R22). Other probes have been cited for the detection of nucleosides (R23, R24). Fluorescent probes have proved useful for protein analysis. Sutherland has compared two luminescence-based methods for the detection of bacterial proteases (R25). Other reagents have been cited for selective measurement of N-terminal tryptophancontaining peptides (R26). The advantages of using multiplex dyes for simultaneous detection of two antibodies have also been reported (R27). Other fluorescent probes for protein studies have been cited (R28-R33). Zellmer and Lasch have reported a novel technique for determination of phosphotidylserine and phosphatidylethanolamine (R34). 84R

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Several membrane/vesicle/lipid sensitive probes have been described (R35-R37). Yoon and Czarnik have developed a fluorescent chemosensor for catechol and catecholamines (R38). Some general fluorescent probes have been described for use with diode lasers (R39), determination of flavonoids (R40), characterization of mineral oil degradation (R41), examination of preferential solvation in binary solvent mixtures (R42), an estrogen receptor (R43), and a barbiturate receptor (R44). A probe for rapid measurement of antimicrobial activity of natural products has been described by Chand and co-workers (R45). The temperature dependence of the luminescence lifetime on the behavior of a luminescence-quenching oxygen sensor has been described (R46). Other probes have been developed for optical thermometry (R47) and measurement of particle temperatures (R48). The cure characteristics of an epoxy resin have been examined by use of a fluorescent probe (R49). Khan and Bhatti have described a low-dose fluorescence dosimeter (R50). Pal and Pal have found that treatment of a cellulose surface with gelatin tends to enhance the fluorescence of organic compounds (R51). The reagent N-acetylcysteine has been used for the determination of aminoglycoside antibiotics (R52). Other studies focus on probes for thromboxane B2 (R53), a novel electrophilic reagent (R54), a new electrophoric derivatizing reagent (R55), a fluorogenic reagent for detection of (2-chloroethyl)ethyl sulfide (R56), and luminescence of europium in mixed ethanol-water solutions (R57). The characteristics of triplet probes have also been discussed (R58-R60). OTHER TECHNIQUES AND APPLICATIONS A general review described the theory, instrumentation, photoreactions, applications, and future prospects of photochemical fluorometry (S1). Vo-Dinh and co-workers described a new method based on photoactivated luminescence for rapid detection of polychlorinated biphenyls (S2), combining UV photoactivation, excitation of the photoproduct complex, and fluorescence detection of the product. Photochemically induced fluorescence was used for the determination of several biomedically important phenothiazines in aqueous solution at room temperature (S3). Fluorometric determination of four nonfluorescent, aromatic insecticides was accomplished by using UV photolysis to generate fluorescent photoproducts (S4). Applications in the area of environmental analysis included studies of corrected excitation, emission, and synchronous spectra of coastal and marine waters (S5), investigation of the effects of biogenic impurities on the fluorometric determination of polycyclic aromatic hydrocarbons in anthropogenic solutions (S6), use of fluorescence analysis to identify movable oil in self-sourcing reservoirs and thereby differentiate between productive zones and exhausted zones in reservoirs (S7), and monitoring of petroleum hydrocarbon pollution in surface waters (S8). Several methods were described for detection of bacteria, including rapid detection of fecal coliform bacteria as an indicator of sewage contamination in marine waters (S9), rapid detection of PAH-mineralizing bacteria from sewage sludge (S10), rapid detection of total and fecal coliforms from surface water (S11), and quantitation of cyanobacteria in mixed phytoplankton assemblages (S12). Several papers described fluorometric methods for determination of pH. In vivo monitoring of tissue pH was accomplished by using a carboxyseminaphthofluorescein (C-SNAFL-1) dye

(S13). The pH of small solution volumes was determined by applying the solution to a hydroxylapatite surface and measuring the variation in excitation spectra of fluorescein or the lifetime of acridine which were immobilized on the surface (S14). The pH dependence of the fluorescence of 1-hydroxypyrene-3,6,8-trisulfonate that was covalently immobilized on cellulose attached to a plastic strip was used for optical pH sensing in the neutral pH region (S15). Plasma pH was monitored in vivo using transcutaneous detection of injected biscarboxyethyl carboxyfluorescein (S16). The same dye was also used in an optical sensor for the measurement of extracellular pH in hairless mice (S17). A universal phosphorescence immunoassay was described which employed monoclonal antibodies to Pd-coproporphyrin (PdCP) and conjugates of various proteins with Pd-CP (S18). Isiah Manuel Warner is Philip W. West Professor of Analytical and Environmental Chemistry at Louisiana State University (LSU). He received his B.S. in chemistry from Southern University in Baton Route, LA in 1968 and his Ph.D. from the University of Washington in 1977. He joined the faculty of Emory University in 1982 as associate professor and became full professor in 1986. From September 1987, he was Samuel Candler Dobbs Professor of Chemistry at Emory University before joining the faculty of LSU in August 1992. During the 1988/89 academic year, he was on leave to the National Science Foundation (NSF) as Program Officer for Analytical and Surface Chemistry. His current research interests include (1) fluorescence spectroscopy, (2) guest/host interactions, (3) studies in organized media, (4) spectroscopic applications of multichannel detectors, (5) chromatography, (6) environmental chemistry, and (7) mathematical analyses and interpretation of chemical data using chemometrics (chemical data analysis techniques). He is coeditor with Professor Linda McGown of Volumes I and II of a series on Multidimensional Luminescence. He is a member of the American Chemical Society, Society for Applied Spectroscopy, American Association for the Advancement of Science, National Organization of Black Chemists and Chemical Engineers, and Sigma Xi. Steven A. Soper is an Assistant Professor of analytical chemistry at Louisiana State University. He received his B.A. in chemistry from the University of Nebraska (Omaha) in 1982 after which he worked as an Analytical Chemist with Colgate-Palmolive. In 1985, he entered graduate school at The University of Kansas and received his Ph.D. in Bioanalytical Chemistry in 1989. After a two-year postdoctoral fellowship at Los Alamos National Laboratory, he joined the faculty at Louisiana State University. His research interests include applications of near-IR fluorescence in DNA sequencing and capillary electrophoresis, single-molecule fluorescence detection, time-resolved fluorescence spectroscopy, and dye photophysics. Linda Baine McGown received her B.S. in chemistry from the State University of New York at Buffalo in1975 and her Ph.D. in chemistry from the University of Washington in 1979. She joined the faculty at Duke University in 1987, where she currently holds the position of Professor of Chemistry. She currently serves on the editorial boards of Analytical Chemistry, Applied Spectroscopy, and the Journal of Fluorescence and Instrumentation Science and Technology and previously served on the editorial advisory board to Chemical and Engineering News. Dr. McGown’s current research interests include the exploration of new fluorescence lifetime techniques for the characterization and analysis of complex samples, on-the-fly fluorescence lifetime detection in chromatography, molecular probe studies of chiral selectivity and molecular recognition, new approaches to the characterization of heterogeneous binding microenvironments in macromolecular structures and molecular assemblies, exploration of novel organized media and nanostructures for chemical analysis, and fluorescence lifetime resolution in chiroptical measurements. LITERATURE CITED (A1) Soper, S. A.; McGown, L. B.; Warner, I. M. Anal. Chem. 1994, 66, 428R-44R. BOOKS, REVIEWS, AND CHAPTERS OF GENERAL INTEREST (B1) (B2) (B3) (B4) (B5) (B6) (B7) (B8) (B9)

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Analytical Chemistry, Vol. 68, No. 12, June 15, 1996