Fluorescence Lifetime Behaviour

In a comparison of fluorescence spectra between the ester and thioester derivative crystals of PDA, the ester crystal shows a strong emission whereas the thioester crystal fluoresces much more weakly. For example, the intensity of a PDA methyl thioester crystal is about one-thousandth of that of a PDA methyl ester crystal. Furthermore, the fluorescence lifetime of mixed crystals which consist of a large amount of PDA methyl ester and a small amount of the corresponding thioester moiety is much shortened, compared to the lifetime of pure PDA methyl ester crystals. In quenching experiments in solutions of PDA ester, the fluorescence of the PDA ester is dramatically quenched by thioacetate. Similar behaviour has been obtained with several types of diolefin derivatives having a thioester moiety, where crystal structures are isomorphous with the corresponding ester derivatives. 

These discussions provide an explanation for the fact that fluorescence emission is normally observed from the zero vibrational level of the first excited state of a molecule (Kasha s rule). The photochemical behaviour of polyatomic molecules is almost always decided by the chemical properties of their first excited state. Azulenes and substituted azulenes are some important exceptions to this rule observed so far. The fluorescence from azulene originates from S2 state and is the mirror image of S2 S0 transition in absorption. It appears that in this molecule, S1 - S0 absorption energy is lost in a time less than the fluorescence lifetime, whereas certain restrictions are imposed for S2 -> S0 nonradiative transitions. In azulene, the energy gap AE, between S2 and St is large compared with that between S2 and S0. The small value of AE facilitates radiationless conversion from 5, but that from S2 cannot compete with fluorescence emission. Recently, more sensitive measurement techniques such as picosecond flash fluorimetry have led to the observation of S - - S0 fluorescence also. The emission is extremely weak. Higher energy states of some other molecules have been observed to emit very weak fluorescence. The effect is controlled by the relative rate constants of the photophysical processes. 

The pAT-behaviour of 1-, 2- and 9-anthroic acids in the excited state was studied by Vander Donckt and Porter (1968a). Directly determined p/ (T )-values were found to lie nearer to the p/ (S1)-values calculated using the Forster cycle than to pA (S0). In a study of the fluorescence of 1- and 2-anthroic acids over a wide acidity range (Schulman et al., 1973a), it appeared that the deprotonation reactions did not come to equilibrium in the excited state. For protonation of the carboxyl group only 1-anthroic acid showed an excited state reaction and, as expected, it became more basic in the Si state. Fluorescence lifetime measurements on the prototropic species derived from 1- and 2-anthroic acids help in understanding the failure to reach equilibrium ... 

In the case of xanthone at least, this order is not only shown up in the Forster cycle estimates, but has been confirmed by observing the variation with pH of the optical densities of the triplet states of B and BH+ and comparing it with the fluorescence intensity behaviour (see Fig. 6). Confirmation that the pK order obtained using the Forster cycle is reliable in such cases is also found in a direct determination of p/ (Tj) of benzophenone by a laser technique the value derived is consistent with earlier phosphorescence observations (Rayner and Wyatt, 1974). Ledger and Porter (1972) observed a marked decrease in the phosphorescence intensity of benzophenone near pH 5, and the apparent discrepancy between this result and the p/ (Tj )-value of 1-5 is due to the very large difference in lifetimes of BH+(T,) and B(Tj). Since unprotonated benzophenone has a very shortlived St state [1/kj for the intersystem crossing alone in ethanol is 16 5 ps (Hochstrasser et al., 1974)], protonation in this state is unlikely. However, Forster cycle calculations indicate that the singlet state would be a weaker base than the triplet state. The realization that unprotonated benzaldehyde and acetophenone had Tj states of the... 

Figure 2-17. Time-resolved fluorescence. Chelate complexes of many lanthanides show unusual fluorescence behaviour in that there is a large separation between the fluorescence excitation and emission maxima, and also extremely long fluorescence lifetimes. This enables...

The influence of the counter anion on the excited state relaxation time of cationic polymethine dyes has also been reported . The fluorescence lifetime is dependent on the anion in weakly polar media but independent in polar media. The fluorescence behaviour of highly concentrated rhodamine GG solutions in methanol and water can be separated into monomer and dimer contributions2. Absorption emission and excitation spectral data support the view that the dye rose bengal forms H-type aggregates in water and polar protic solvents 1. The spectroscopic behaviour of rhodamine 6G in polar and nonpolar solvents as well as in thin glass and PMMA films shows dimer formation occurs and their stabilities have been compared under different conditions. The equilibrium between the neutral... 

This phenomenon was first identified and explained by Forster.120 The structureless emission is attributed to an excited pyrene dimer (1P - P) that is formed by collisional association of singlet excited pyrene P with a pyrene molecule P in the ground state. It was subsequently found that many aromatic molecules exhibit similar behaviour. The expression excimer (excited dimer) was proposed by Stevens to distinguish such species from the excited state of a ground-state complex. Excimer formation is prominent at relatively low concentrations of pyrene (Figure 2.22, left), because of its unusually long fluorescence lifetime, 1t = 650 ns, which allows for diffusional encounters of P with P even at low concentration. 

The fluorescence behaviour of a fluorophore is also influenced by the solvent, especially the solvent polarity [308]. Moreover, when a molecule is excited the solvent molecules around it rearrange. Consequently, energy is transferred to the solvent, with the result that the emission spectrum is red-shifted. Solvent (or spectral) relaxation in water happens on the time scale of a few ps. However, the relaxation times in viscous solvents and in dye-protein constructs can be of the same order as the fluorescence lifetime. The measurement of the solvent relaxation can therefore be used to obtain information about the local environment of fluorescent molecules [485]. 

The time behaviour of transient spectra is very essential for the assignment of these spectra to energy levels or species. If possible, independent experimental methods should be used to gain data of the time behaviour. Therefore, we performed time resolved fluorescence measurements on the oligothiophenes and obtained the fluorescence lifetimes of T [3]. Today other references are also available [11]. 

Figure 8. Fluorescence decay of Pr phytochrome (124 kDa) excitation at Aexc = 640 nm, emission measured at Aero — 680 nm. The semilogarithmic plots of the measured decay (curve with signal noise) and the decay function calculated from best-fit kinetics parameters obtained by single-decay analysis (thin line superimposed on measured decay) are shown. In the inset the calculated lifetimes xf 3 and relative amplitudes Rf 3 of the decay components are given. On top, a weighted residuals plot (sigma) indicates the deviations of these computer-fitted parameters from the measured decay, with the value of the squared reduced error (y2) in the inset. The fluorescence decay of the red-light adapted Pr + Pfr mixture exhibited a comparable triexponential behaviour. (After Figure 4 in Holzwarth et al. [76].)...

Figure 12. UV (protein) fluorescence decay of the red-light adapted mixture P, + Pfr (124kDa) at 275 K Aelc = 295 nm, = 330 nm. Inset calculated lifetimes t(t,P)i -4 and relative amplitudes Rftrp)1 -4 °f the decay components calculated by single-decay analysis. Top weighted residuals plot and autocorrelation function of the residuals. The fluorescence decay of pure Pr exhibited a comparable tetraexponential behaviour (Holzwarth et al. [108]).

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