Steady-rate fluorescence emission

Molecular rotors are useful as reporters of their microenvironment, because their fluorescence emission allows to probe TICT formation and solvent interaction. Measurements are possible through steady-state spectroscopy and time-resolved spectroscopy. Three primary effects were identified in Sect. 2, namely, the solvent-dependent reorientation rate, the solvent-dependent quantum yield (which directly links to the reorientation rate), and the solvatochromic shift. Most commonly, molecular rotors exhibit a change in quantum yield as a consequence of nonradia-tive relaxation. Therefore, the fluorophore s quantum yield needs to be determined as accurately as possible. In steady-state spectroscopy, emission intensity can be calibrated with quantum yield standards. Alternatively, relative changes in emission intensity can be used, because the ratio of two intensities is identical to the ratio of the corresponding quantum yields if the fluid optical properties remain constant. For molecular rotors with nonradiative relaxation, the calibrated measurement of the quantum yield allows to approximately compute the rotational relaxation rate kor from the measured quantum yield [Pg.284]

One may consider the relaxation process to proceed in a similar manner to other reactions in electronic excited states (proton transfer, formation of exciplexes), and it may be described as a reaction between two discrete species initial and relaxed.1-7 90 1 In this case two processes proceeding simultaneously should be considered fluorescence emission with the rate constant kF= l/xF, and transition into the relaxed state with the rate constant kR=l/xR (Figure 2.5). The spectrum of the unrelaxed form can be recorded from solid solutions using steady-state methods, but it may be also observed in the presence of the relaxed form if time-resolved spectra are recorded at very short times. The spectrum of the relaxed form can be recorded using steady-state methods in liquid media (where the relaxation is complete) or using time-resolved methods at very long observation times, even as the relaxation proceeds. 

FRET interactions are typically characterized by either steady-state or transient fluorescence emission signals from the donor or acceptor species. Efficient nonradiative energy transfer results in donor PL loss associated with acceptor gain in photoluminescence intensity (if the acceptor is an emitter). The rate of this energy transfer is related to the intrinsic lifetime of the isolated donor and depends strongly on the donor-acceptor separation distance ... 

Steady-state fluorescence spectra recorded after the addition of the NDI or PM I acceptor to the bisporphyrin tweezer ( rrl = 660 nm), demonstrated substantial quenching (75%) with increasing quantities of the NDI or PM I acceptors. Time-resolved emission spectra recorded in toluene for the complex 26 were biexponential containing a dominant short-lived CS components (80 ps, -95%) attributed to photoinduced ET from donor porphyrin to NDI, and a minor long-lived component (Ins, 5%). The lifetime of the dominant short-lived CS state is increased two- to threefold relative to covalently linked systems under similar conditions of solvent, donor-acceptor distance and thermodynamics [37]. Charge recombination rates from 1.4 to 3.8 x 1()9s 1 were observed, depending on whether the NDI or PM I acceptor was bound within the cavity. 

The effect of salt on the rate of proton dissociation from excited hydroxypyrene trisulfonate is demonstrated in Figure 6. The effect on the steady-state fluorescence is similar to that shown in Figure 3. The emission of the neutral form is intensified while that of the anion decreases. 

Nicotinamide-nucleotide-linked dehydrogenases were among the earliest two-substrate enzymes to be subjected to detailed kinetic study by steady-state 1-3) and rapid reaction techniques (4), and provided much of the original stimulus for the necessary extension of kinetic theory already developed for one-substrate and hydrolytic enzymes S-8). This was partly because of the convenience and precision with which rates can be measured by means of the light absorption or fluorescence emission 9-11) of the reduced coenzymes and because of the changes of these properties which accompany the binding of reduced coenzymes to many dehydrogenases 12,13). 

The Bloch equations (Eq. 5) can be solved under different conditions. The transient solution yields an expression for 0-22 (0> time-dependent population of the excited singlet state S. It will be discussed in detail in Section 1.2.4.3 in connection with the fluorescence intensity autocorrelation function. Here we are interested in the steady state solution (an = 0-22 = < 33 = di2 = 0) which allows to compute the line-shape and saturation effects. A detailed description of the steady state solution for a three level system can be found in [35]. From those the appropriate equations for the intensity dependence of the excitation linewidth Avfwhm (FWHM full width at half maximum) and the fluorescence emission rate R for a single absorber can be easily derived [10] ... 

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. 

In this paper, we present a preliminary analysis of the steady-state and time-resolved fluorescence of pyrene in supercritical C02. In addition, we employ steady-state absorbance spectroscopy to determine pyrene solubility and determine the ground-state interactions. Similarly, the steady-state excitation and emission spectra gives us qualitative insights into the excimer formation process. Finally, time-resolved fluorescence experiments yield the entire ensemble of rate coefficients associated with the observed pyrene emission (Figure 1). From these rates we can then determine if the excimer formation process is diffusion controlled in supercritical C02. 

It is in the nature of steady-state kinetic calculations that ratios of rate constants are obtained for example, the expressions for the intensity in Eq. 25, or the parameters extracted from the Stern-Volmer treatment, involve ratios of rate constants to the Einstein A factor for emission. Individual rate constants can often be determined from a comparison of kinetic data obtained under stationary conditions with those obtained under nonstationary conditions. For the present purposes, the nonstationary experiment often involves determination of fluorescence or phosphorescence lifetimes (tf, rp). If a process follows first-order kinetics described by a rate constant k, the mean lifetime, r (the time taken for the reactant concentration to fall to 1/e of its initial value), is given by... 

B, then the fluorescence of A would be dominated by the acceptor emission spectrum and that of B by the donor spectrum. Thus, the reaction would cause a blue shift, which, if it were big enough, would enable a cross-correlation measurement. The reverse process with FRET large for B and small for A, would work equally well. For the bimolecular reaction A 4- L B, the flux could be measured if the binding caused a large enough shift in the fluorescence spectrum of A. The analysis for the bimolecular reaction is the same as given earlier in a steady state, the concentration of L is constant and so can be absorbed into a pseudo-first-order rate constant. 

The feature of this result is that the steady-state rate k of Eq. (7.32) is obtained only if the time scale of the experiment exceeds which is not the case with many of the transient experiments that have been performed, in which case only the rates k and are probed. The more general result is obtained if s -I- fc, in Eq. (7.35) is replaced by /4(s). The formula for (s) has been extended by incorporation of fluorescence decay from the excimer and monomer emission. In this case there are at least five poles in (s). 

Rotation occurs about the double bond in a manner similar to what occurs in the mal-ononitrile derivatives described by Loutfy (1982, 1986). However, instead of measuring the steady-state emission intensity, the rate coefficient for the formation of the TICT state was obtained by picosecond laser fluorescence lifetime measurements (Strehmel et al, 1992, 1999, Younes et al, 1994). 

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