Using fluorescence measurements

(b) Using fluorescence measurements A method for measurement of lipid peroxides in plasma and serum (Yagi, 1984), that overcomes interference from water-soluble substances that may form adducts with TBA involves the use of a spectrofluorimetric technique. 

Thiobarbituric acid (0.67%, w/v) dissolved in 50% (w/v) glacial acetic acid 

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The data were collected using fluorescence measurements, which allow both identification and quantitation of the fluorophore in solvent extraction. Important experimental considerations such as solvent choice, temperature, and concentrations of the modifier and the analytes are discussed. The utility of this method as a means of simplifying complex PAH mixtures is also evaluated. In addition, the coupling of cyclodextrin-modified solvent extraction with luminescence measurements for qualitative evaluation of components in mixtures will be discussed briefly. 

Teramoto A., Watanabe M., Iizuka E., Abe K. Interaction of polyelectrolytes with albumin using fluorescence measurement. J. M. S. Pure Appl. Chem. 1994 A31(l) 53-64. 

Many scientists use spectroscopy on a daily basis to gain insight into the structure of molecules or the concentration of atoms or molecules in a sample. The chemist uses radio waves and infrared radiation to determine the structure of a new molecule. The geologist uses ultraviolet radiation to determine the concentration of a particular element in a rock or mineral. The microbiologist uses fluorescence measurements to determine the concentration of bacteria in solution, see also Rydberg, Johannes. 

Amrani and co-workers [48] have used fluorescence measurements of energy transfer from donor to acceptor for studies of polymer compatibility. They labelled methyl methacrylate-ethyl methacrylate copolymer and/or methyl methacrylate-butyl methacrylate copolymer with donor-naphthalene and polymethyl methacrylate with acceptor-anthracene. The variation in the ratio of donor to acceptor fluorescence was plotted as a function of butyl methacrylate and ethyl methacrylate in the copolymer and gradual increase of the ratio corresponded to gradual transition from two-phase to a one-phase system. The fluorescence technique was found to be more sensitive to small changes of compatibility of the polymers. 

Winnik [49] used fluorescence measurements of transfer of the electronic excitation between donor-naphthalene and acceptor-pyrene chromophores attached to the same polymer chain for studies of thermoreversible phase separation of aqueous solutions of poly(N-isopropylacrylamide) (PNIPAM). Dilute solutions of the doubly labelled polymer PNIPAM were heated from 277 K to 313 K, and the fluorescence emission intensity of pyrene (integrated spectrum) was measured when the system was excited with 290 nm, donor excitation, and when excited with 328 nm, acceptor excitation. Non radiative energy transfer between excited naphthalene and pyrene occurred in aqueous solution of the polymer. The increase in intensity of pyrene fluorescence when the solution was excited at 290 nm, shown in Figure 4.13, is due to a phase separation process at lower critical solution temperature (LCST). When the LCST was reached, the phase separation into polymer-rich and polymer-lean phases occurred. It was concluded that the collapse of the polymer chain leading to densification of polymer phase is followed by domination of intramolecular contributions to the energy transfer process. 

Study of conformational transitions of insoluble proteins using fluorescence measurements... 

The sensitivities of particular spectroscopic teclmiques to specific chemical features are described more fully in tire next section. Perhaps tire most common and versatile probes of reaction dynamics are time-resolved UV-vis absorjDtion and fluorescence measurements. Wlren molecules contain cliromophores which change tlieir stmcture directly or experience a change of environment during a reaction, changes in absorjDtion or fluorescence spectra can be expected and may be used to monitor tire reaction dynamics. Altliough absorjDtion measurements are less sensitive tlian fluorescence measurements, tliey are more versatile in tliat one need not rely on a substantial fluorescence yield for tire reactants, products or intennediates to be studied. 

Better detection limits are obtained using fluorescence, particularly when using a laser as an excitation source. When using fluorescence detection, a small portion of the capillary s protective coating is removed and the laser beam is focused on the inner portion of the capillary tubing. Emission is measured at an angle of 90° to the laser. Because the laser provides an intense source of radiation that can be focused to a narrow spot, detection limits are as low as 10 M. 

Mineral and Chemical Composition. X-ray diffraction is used to determine the mineral composition of an Mg(OH)2 sample. Induced coupled plasma (icp) spectrophotometry is used to measure the atomic concentrations present in a sample. X-ray fluorescence analysis is another comparative instmmental method of determining chemical composition. 

AH of these properties of x-rays are used to measure various properties of materials. X-ray appHcations can be placed into three categories based on which of the above phenomena are exploited. These categories are x-ray radiography, x-ray fluorescence spectrometry, and x-ray diffraction. 

Fluorescence. The fluorescence detection technique is often used in clinical chemistry analyzers for analyte concentrations that are too low for the simpler absorbance method to be appHed. Fluorescence measurements can be categorized into steady-state and dynamic techniques. Included in the former are the conventional simultaneous excitation-emission method and fluorescence polarization. 

X-Ray Fluorescence (XRF) is a nondestructive method used for elemental analysis of materials. An X-ray source is used to irradiate the specimen and to cause the elements in the specimen to emit (or fluoresce) their characteristic X rays. A detector s)rstem is used to measure the positions of the fluorescent X-ray peaks for qualitative identiflcation of the elements present, and to measure the intensities of the peaks for quantitative determination of the composition. All elements but low-Z elements—H, He, and Li—can be routinely analyzed by XRF. 

Although XRF is generally the X-ray spectrometry method of choice for analysis of major and trace elements in bulk specimens, useful PIXE measurements can be made. A detailed review of the main considerations for thick-target PEXE provides guidance for trace analysis with known and unknown matrices and bulk analysis when the constituents are unknown. Campbell and Cookson also discuss the increased importance of secondary fluorescence and geometrical accuracy for bulk measurements. 

Another major second messenger in cells is calcium ion. Virtually any mammalian cell line can be used to measure transient calcium currents in fluorescence assays when cells are preloaded with an indicator dye that allows monitoring of changes in cytosolic calcium concentration. These responses can be observed in real time, but a characteristic of these responses is that they are transient. This may lead to problems with hemi-equilibria in antagonist studies whereby the maximal responses to agonists may be depressed in the presence of antagonists. These effects are discussed more fully in Chapter 6. 

Several other techniques for have evolved for biochemical assays. In chapter 2 of this book, Omann and Sklar report on a method of fluoroimmunoassay where the bound and unbound antigen are separated by the quenching of fluorescence that accompanies antibody binding. Then, in chapter 3, Holl and Webb show how they achieved a sensitive measurement of nucleic acids by the enhancement in fluorescence that accompanies the binding of fluorescent dyes to nucleic acids. Chandler et al, also used fluorescence enhancement to monitor calcium mobility in neutrophil cells. 

Real-time spectroscopic methods can be used to measure the binding, dissociation, and internalization of fluorescent ligands with cell-surface receptors on cells and membranes. The time resolution available in these methods is sufficient to permit a detailed analysis of complex processes involved in cell activation, particularly receptor-G protein dynamics. A description of the kinetics and thermodynamics of these processes will contribute to our understanding of the basis of stimulus potency and efficacy. 

Definition and Uses of Standards. In the context of this paper, the term "standard" denotes a well-characterized material for which a physical parameter or concentration of chemical constituent has been determined with a known precision and accuracy. These standards can be used to check or determine (a) instrumental parameters such as wavelength accuracy, detection-system spectral responsivity, and stability (b) the instrument response to specific fluorescent species and (c) the accuracy of measurements made by specific Instruments or measurement procedures (assess whether the analytical measurement process is in statistical control and whether it exhibits bias). Once the luminescence instrumentation has been calibrated, it can be used to measure the luminescence characteristics of chemical systems, including corrected excitation and emission spectra, quantum yields, decay times, emission anisotropies, energy transfer, and, with appropriate standards, the concentrations of chemical constituents in complex S2unples. 

Reagents. Perylene was obtained from Sigma Chemical Company (St. Louis, Missouri). All other PAHs were supplied by Aldrich Chemical Company (Milwaukee, Wisconsin) and were reported to contain less that 3% impurities. All PAHs were used without further purification. Isopropyl ether (99%) for extraction work was also purchased from Aldrich. Hydroquinone, a fluorescent stabilizer present in the ether, was removed prior to solution preparation by rotary evaporation. Fluorometric-grade 1-butanol was supplied by Fisher Scientific Company (Fair Lawn, New Jersey). All solutions for extractions of PAHs were prepared by evaporating portions of a stock cyclohexane solution and diluting to the appropriate volume with isopropyl ether. Fluorescence measurements were performed on 1 10 dilutions of the stock and final organic phase solutions. The effect of dissolved CDx on the fluorescence intensity of the organic phase PAH was minimized by dilution with isopropyl ether. 

Samples for studies of CDx effects on fluorescence enhancement in organic solution were prepared using pyrene, because pyrene possesses a long lifetime and is very susceptible to quenching and enhancement in solution (23). An aliquot of pyrene stock solution in cyclohexane was placed under a nitrogen purge to evaporate the cyclohexane. Samples were redissolved in a 1 A mixture of Isopropyl ether and 1-butanol, which was saturated with aqueous CDx solution. Pyrene samples were also prepared in which the organic solvent was not saturated with CDx solution. The mixed solvent was used in order to minimize the effects of ether evaporation and thus allow more accurate quantitation. Fluorescence measurements were made on diluted samples of these solutions. The solvent used to make up the... 

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