Steady-state fluorescence spectroscopy

Photosensitization of diaryliodonium salts by anthracene occurs by a photoredox reaction in which an electron is transferred from an excited singlet or triplet state of the anthracene to the diaryliodonium initiator.13"15,17 The lifetimes of the anthracene singlet and triplet states are on the order of nanoseconds and microseconds respectively, and the bimolecular electron transfer reactions between the anthracene and the initiator are limited by the rate of diffusion of reactants, which in turn depends upon the system viscosity. In this contribution, we have studied the effects of viscosity on the rate of the photosensitization reaction of diaryliodonium salts by anthracene. Using steady-state fluorescence spectroscopy, we have characterized the photosensitization rate in propanol/glycerol solutions of varying viscosities. The results were analyzed using numerical solutions of the photophysical kinetic equations in conjunction with the mathematical relationships provided by the Smoluchowski16 theory for the rate constants of the diffusion-controlled bimolecular reactions. 

Subsequently, fluorescent MIPs for cGMP were fabricated [46 18, 66, 67]. For that, 1,3-diphenyl-6-vinyl-1 //-pyrazolo 3.4-/ quinoline (PAQ) was introduced as the fluorescent indicator to interact with cGMP in a thin-layer fluorescent MIP chemosensor. Both steady-state (Fig. 1) and time-resolved fluorescence spectroscopy were used as two independent analytical techniques for investigation of the chemosensor properties in the presence of cGMP. Steady-state fluorescence spectroscopy is a common technique applied to MIP sensing. Nevertheless, the use of time-resolved fluorescence spectroscopy combined with microscopy was a new approach to MIP sensing. 

After extraction, the fluorescent indicator was in the unbound state and gave input to the radiative relaxation. Therefore, the fluorescence lifetime increased and, consequently, the intensity as well. After MIP contacting with the analyte, the non-radiative processes were again efficient compared to the radiative processes and, subsequently, fluorescence was quenched. With steady-state fluorescence spectroscopy the cross-reactivity test towards structurally similar biomolecules was performed that yielded selectivity factors for guanosine, cAMP and cCMP of 1.5, 2.5 and 5.1, respectively. 

Steady-state fluorescence spectroscopy has also been used to study solvation processes in supercritical fluids. For example, Okada et al. (29) and Kajimoto and co-workers (30) studied intramolecular excited-state complexation (exciplex) and charge-transfer formation, respectively, in supercritical CHF3. In the latter studies, the observed spectral shift was more than expected based on the McRae theory (56,57), this was attributed to cluster formation. In other studies, Brennecke and Eckert (5,31,44,45) examined the fluorescence of pyrene in supercritical CO2, C2HSteady-state emission spectra were used to show density augmentation near the critical point. Additional studies investigated the formation of the pyrene excimer (i.e., the reaction of excited- and ground-state pyrene monomers to form the excited-state dimer). These authors concluded that the observance of the pyrene excimer in the supercritical fluid medium was a consequence of increased solute-solute interactions. 

To this point it is important to recognize that steady-state fluorescence spectroscopy gives one only an average picture of the system under study. In order to determine the details of the photophysics, it is necessary to employ time-resolved methodologies. 

It is well known that both nanometre and nanosecond-picosecond resolutions at an interface can be achieved by total internal reflection (TIR) fluorescence spectroscopy. Unlike steady-state fluorescence spectroscopy, fluorescence dynamics is highly sensitive to microscopic environments, so that time-resolved TIR fluorometry at water/oil interfaces is worth exploring to obtain a clearer picture of the interfacial phenomena [1]. One of the interesting targets to be studied is the characteristics of dynamic motions of a molecule adsorbed on a water/oil interface. Dynamic molecular motions at a liquid/liquid interface are considered to be influenced by subtle changes in the chemical/physical properties of the interface, particularly in a nanosecond-picosecond time regime. Therefore, time-resolved spectroscopy is expected to be useful to study the nature of a water/oil interface. 

Steady-state fluorescence spectroscopy intensity and wavelength... 

The photochemical and photophysical properties of BPHTs and other phenothiazine derivatives have been investigated by means of a combination of time-resolved laser techniques, including laser flash photolysis and steady-state fluorescence spectroscopy [30,96]. The main goal of these studies was to develop novel photosensitizers for photoresist technology in the photoin-duced crosslinking of polymers. 

Figure 5. Hydrolysis of fluorogenic substrate 1 by HTV protease at 37 C as monitored by steady state fluorescence spectroscopy (Xex+340, A m=490). The reaction was carried out with 10 pM substrate at pH 4.7 in a buffer solution containing 0.1 M NaOAc, 1.0 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% DMSO and 1 mg/mL bovine serum albumin. The arrow indicates the point of addition of HTV protease to a final concentration of 35 nM. Product analysis was carried out by HPLC, mass spectrum and fluorescence lifetime. Inset The initial phase of the hychx)lysis reaction used for rate determinations.

Lulka and co-workers have imprinted two fluorescent compounds, fluorescein (Fig. 2, 6) and A-acetyltryptophanamide (NATA) (Fig. 2,7) with organic silanes bis (2-hydroxyethyl) -aminopropryltriethoxysilane (HAPTES) and tetraethoxysilane (TES) [23]. The binding constants were determined with steady-state fluorescence spectroscopy. It was found that high affinity binding sites with Kj s in the range of sub - to low micromolar were achieved. 

It is also possible to perform steady-state fluorescence spectroscopy experiments and to monitor the decrease in fluorescence intensity of the donor, in both the absence and the presence of the acceptor to estimate the transfer efficiency using... 

Steady-state fluorescence spectroscopy has been used by Torkelson et al. [43,44] to determine the components glass transition temperature (Tg) in various blends, including blends of PS with poly(tert-butyl acrylate) (PtBA), PMMA, and poly(w-butyl methacrylate) (PnBMA) [43], as well as in miscible blends of pyrene-labeled PMMA (MPy-labeled PMMA) with poly(ethylene oxide) (PEO) or poly(vinyl chloride) (PVC) over a broad composition range [44]. In the particular case of this latter study [44], the blend Tg-values were measured upon heating by increasing the... 

In a related study [47], the same group used steady-state fluorescence spectroscopy coupled to epifluorescence microscopy (fluorescence microspectroscopy) for studying the diffusion of the lower-component during the heating of a binary... 

In a related study, the use of a steady-state fluorescence spectroscopy for investigation of chiral recognition ability of amino acid-based L-alanine tert butyl ester bis (trifluoromethane) sulfonamide (L-AlaC4NTf2) chiral ionic liquids was recently demonstrated [24], In this study, L-AlaC4NTf2 was used as a solvent and chiral auxiliary for enantiomeric discrimination of warfarin, naproxen, and... 

The first experiments on the diffusion-controlled end-to-end cycli-zation of polymers were reported by Cuniberti and Perico (34) in 1977. These polymers were studied by steady-state fluorescence spectroscopy in air-saturated solution, and relative values were obtained from measurements of Ie/Im. Absolute values of were obtained by Cheung et al. (35), for a single sample of Py-PEO-Py of M = 9000 in a variety of solvents, using a combination of steady-state and fluorescence decay techniques. With their value of in THF solution, the Ie/In results of Cuniberti and Perico (34) can be calibrated. It is curious, figure 10, that the plot of log (Ie/Im) vs log N for these polymers has a slope significantly less steep than -3/2. 

Chakraborty, R. Berglund, K. A. Steady state fluorescence spectroscopy of pyranine as a trace extrinsic probe to study structure in aqueous sugar solutions. J. Cryst. Growth 1992,125, 81-96. 

Steady-state fluorescence spectroscopy refers to the measurement of the fluorescence intensity of a sample under the condition of constant illumination (excitation) of the sample. This results in a constant rate of absorption, and hence a constant rate of formation of the first excited singlet state. Si, as given by Eq. 3. This leads to the establishment of steady-state conditions, in which the rate of relaxation (decay) of the Si population is exactly the same as the rate of its formation. Thus, a constant Si population is established. Under these conditions, the rate of Eq. 4 ( = f[Si]), i.e. the rate of fluorescence emission, is constant. Since intensity (/p) is defined as the rate of photon emission per unit time (usually expressed in counts per second, cps), the measured fluorescence intensity is therefore constant with time. 

The polarity of IL is one of the spedfic properties that distinguishes it from molecular solvents. The variation of the polarity of IL before and after its covalent immobilization on different polymers was semiquantitatively investigated by means of steady-state fluorescence spectroscopy using pyrene as a probe. Scheme 2.15 [69]. It suggested that the static dielectric constant (e) values deduced from the fluorescence experiments revealed an increase in the polarity of the polymers from e < 5 to e > 10-20 after immobilization of IL. Hence, an IL supported covalently on a polymer exhibits similar polarity as that of the bulk room-temperature IL. For example, the e of the IL [EMIm][NTf2 is 15.76. 

---