Spectroscopic fluorescence spectroscopy

The attachment of pyrene or another fluorescent marker to a phospholipid or its addition to an insoluble monolayer facilitates their study via fluorescence spectroscopy [163]. Pyrene is often chosen due to its high quantum yield and spectroscopic sensitivity to the polarity of the local environment. In addition, one of several amphiphilic quenching molecules allows measurement of the pyrene lateral diffusion in the mono-layer via the change in the fluorescence decay due to the bimolecular quenching reaction [164,165]. 

Measuring Protein Sta.bihty, Protein stabihty is usually measured quantitatively as the difference in free energy between the folded and unfolded states of the protein. These states are most commonly measured using spectroscopic techniques, such as circular dichroic spectroscopy, fluorescence (generally tryptophan fluorescence) spectroscopy, nmr spectroscopy, and absorbance spectroscopy (10). For most monomeric proteins, the two-state model of protein folding can be invoked. This model states that under equihbrium conditions, the vast majority of the protein molecules in a solution exist in either the folded (native) or unfolded (denatured) state. Any kinetic intermediates that might exist on the pathway between folded and unfolded states do not accumulate to any significant extent under equihbrium conditions (39). In other words, under any set of solution conditions, at equihbrium the entire population of protein molecules can be accounted for by the mole fraction of denatured protein, and the mole fraction of native protein,, ie. 

Kazarian et al. [281-283] have used various spectroscopic techniques (including FUR, time-resolved ATR-FHR, Raman, UV/VIS and fluorescence spectroscopy) to characterise polymers processed with scC02. FTIR and ATR-FTIR spectroscopy have played an important role in developing the understanding and in situ monitoring of many SCF processes, such as drying, extraction and impregnation of polymeric materials. 

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

Bigelow, D.J., and Inesi, G. (1991) Frequency-domain fluorescence spectroscopy resolves the location of maleimide-directed spectroscopic probes within the tertiary structure of the Ca-ATPase of sarcoplasmic reticulum. Biochemistry 30, 2113-2125. 

Vukjovic et al.199 recently proposed a simple, fast, sensitive, and low-cost procedure based on solid phase spectrophotometric (SPS) and multicomponent analysis by multiple linear regression (MA) to determine traces of heavy metals in pharmaceuticals. Other spectroscopic techniques employed for high-throughput pharmaceutical analysis include laser-induced breakdown spectroscopy (LIBS),200 201 fluorescence spectroscopy,202 204 diffusive reflectance spectroscopy,205 laser-based nephelometry,206 automated polarized light microscopy,207 and laser diffraction and image analysis.208... 

Materials. The nearly monodisperse atactic PMMA, which was used for the electron beam lithography and fluorescence spectroscopy studies, was obtained from Pressure Chemical. It has a weight average molecular weight (Mw) of 188,100 and Mw/Mn< 1.08. Pyrenedodecanoic acid (PDA) used in the fluorescence studies was obtained from Molecular Probes and used as supplied. Spectroscopic grade benzene purchased from J.T. Baker was used as the spreading solvent in the PMMA and PMMA/PDA solutions. 

In the present work, we have examined poly(N-vinylcarbazole) (abbreviated hereafter as PVCz) and pyrene-doped poly(aethyl methacrylate) (PMMA) films by using a tine-resolved fluorescence spectroscopic aethod. Fluorescence spectra and their dynanic behavior of the forner fila were elucidated with a high intensity laser pulse and a streak camera, which nakes it possible to neasure dynaaics just upon laser ablation. This aethod reveals aolecular and electronic aspects of laser ablation phenomena (17). For the latter fila a laser pulse with weak intensity was used for characterizing the ablated and Basked areas. On the basis of these results, we demonstrate a high potential of fluorescence spectroscopy in aolecular studies on laser ablation and consider its mechanism. Experimental... 

Dendrimers have precise compositional and constitutional aspects, but they can exhibit many possible conformations. Thus, they lack long-range order in the condensed phase, which makes it inappropriate to characterize the molecular-level structure of dendrimers by X-ray diffraction analysis. However, there have been many studies performed using indirect spectroscopic methods to characterize dendrimer structures, such as studies using photophysical and photochemical probes by UV-Vis and fluorescence spectroscopy, as well as studies using spin probes by EPR spectroscopy. 

Investigation turned then to chemical and spectroscopic means to obtain the needed mechanistic understanding. Stephenson et al. [17] looked at gas evolution versus exposure, while Pacifici and Straley [18] used UV fluorescence spectroscopy to identify a photo-oxidation product which was later isolated by Valk et al. [19]. In addition, Valk and co-workers [19-21] isolated a number of additional photolysis products by a combination of hydrolysis and chromatography, Marcotte et al. [22] used ESR to look at radicals generated during degradation, and Day and Wiles [23-26] carried out extensive IR and fluorescence spectroscopic investigations on this subject. 

Such ambiguity and also the low structural resolution of the method require that the spectroscopic properties of protein fluorophores and their reactions in electronic excited states be thoroughly studied and characterized in simple model systems. Furthermore, the reliability of the results should increase with the inclusion of this additional information into the analysis and with the comparison of the complementary data. Recently, there has been a tendency not only to study certain fluorescence parameters and to establish their correlation with protein dynamics but also to analyze them jointly, to treat the spectroscopic data multiparametrically, and to construct self-consistent models of the dynamic process which take into account these data as a whole. Fluorescence spectroscopy gives a researcher ample opportunities to combine different parameters determined experimentally and to study their interrelationships (Figure 2.1). This opportunity should be exploited to the fullest. 

Monitoring of the PFR can be made spectroscopically because the photoproducts have well-defined absorbtion bands in the UV-visible and infrared (IR) ranges [232]. Fluorescence spectroscopy allows the early detection of phenyl salicylate-type products in the photolysis of bisphenol A-based polycarbonates due to the characteristic emission of this chromophore around 470 nm [233]. 

Typically, sample detection in electromigration techniques is performed by on-column detection, employing a small part of the capillary as the detection cell where a property of either the analyte, such as UV absorbance, or the solution, such as refractive index or conductivity, is monitored. This section briefly describes the major detection modalities employed in capillary electromigration techniques, which are accomplished using UV-visible absorbance, fluorescence spectroscopy, and electrochemical systems. The hyphenation of capillary electromigration techniques with spectroscopic techniques employed for identification and structural elucidation of the separated compounds is also described. 

More detailed analyses of sapphyrin anion chelation in solution were made using a full range of tricks borrowed from the supramolecular field. Thus both spectroscopic techniques (e.g., NMR, UV-vis, fluorescence spectroscopy) and transport studies (carried out in a model Pressman type U-tube membrane system ) were employed. From these analyses, it became clear that sapphyrin does in fact bind various negatively charged substrates in solution, but does so both with variable affinity and oft-times remarkable selectivity. These findings/conclusions are detailed further in the paragraphs below. 

One can distinguish between methods in which absorption of the evanescent surface wave in different wavelength regions is measured (these are often called attenuated total reflection methods), and methods which use the evanescent wave to excite other, spectroscopic phenomena, like fluorescence and Raman scattering or light scattering. As the methods of conventional fluorescence spectroscopy have been shown to be exceptionally successful in studies of proteins and other biopolymers, their evanescent surface-sensitive counterparts will be reviewed first. 

In both the far- and near-UV regions, CD spectra can be used empirically as fingerprints of a particular protein, with the spectrum resulting from the aromatic residues being rather more specific and hence diagnostic. The far-UV spectra, however, can provide information about the protein conformation in terms of its secondary structure. As for fluorescence spectroscopy or any spectroscopic method, the sample needs to be chemically pure and homogeneous. 

Substantial evidence suggests that in highly asymmetric supercritical mixtures the local and bulk environment of a solute molecule differ appreciably. The concept of a local density enhancement around a solute molecule is supported by spectroscopic, theoretical, and computational investigations of intermolecular interactions in supercritical solutions. Here we make for the first time direct comparison between local density enhancements determined for the system pyrene in CO2 by two very different methods-fluorescence spectroscopy and molecular dynamics simulation. The qualitative agreement is quite satisfactory, and the results show great promise for an improved understanding at a molecular level of supercritical fluid solutions. 

Spectroscopic techniques, such as ultra-violet (9), Infrared (25), Nuclear Magnetic Resonance (24), and Fluorescence spectroscopies (5-8), constitute direct probes of specific events occurring at the molecular scale. When a quantitative interpretation is possible, spectroscopy provides very detailed microscopic information. Unfortunately however, the interpretation of spectra in terms of molecular events is often complex. Yet another approach that probes events at the molecular scale involves the use of tracers, such as chromophores (1-225). Again, the complexity of the tracer imposes limitations on the extent to which the data can be interpreted quantitatively. 

In this work we investigate such interactions by fluorescence spectroscopy. Probe molecules such as 2-naphthol and its 5-cyano-derivative are effective chromophores for studying acid/base interactions since both are relatively strong photo-acids. In addition, 2-naphthol is a common solute for which SCF solubility and physical property data exist. Ultimately, spectroscopic information will be used to develop a clearer picture of the specific interactions which induce large cosolvent effects on solubility in SCF solutions. 

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