Fluorescence spectra interpretation

The concept of polarity covers all types of solute-solvent interactions (including hydrogen bonding). Therefore, polarity cannot be characterized by a single parameter. Erroneous interpretation may arise from misunderstandings of basic phenomena. For example, a polarity-dependent probe does not unequivocally indicate a hydrophobic environment whenever a blue-shift of the fluorescence spectrum is observed. It should be emphasized again that solvent (or microenvironment) relaxation should be completed during the lifetime of the excited state for a correct interpretation of the shift in the fluorescence spectrum in terms of polarity. 

Attention should be paid to specific interactions, which should be taken into account in the interpretation of spectral shifts in relation to the polarity of a medium. Drastic changes in the fluorescence spectrum may indeed be induced by hydrogen bonding. 

B3.6 Determining the Fluorescence Spectrum of a Protein B3.6.1 Strategic Planning B3.6.1 Basic Protocol l Recording a Fluorescence Emission Spectrum B3.6.5 Basic Protocol 2 Determination of Fluorescence Quenching B3.6.9 Support Protocol Basic Theory and Interpretation of Fluorescence Spectra B3.6.12 Commentary B3.6.19... 

Thus, knowledge of the transition moment direction of a phenol band could help in interpreting the fluorescence spectrum of a tyrosine chromophore in a protein in terms of orientation and dynamics. The absorption spectrnm of the first excited state of phenol was observed around 275 nm with a fluorescence peak aronnd 298 nm in water. The tyrosine absorption was reported at 277 nm and the finorescence near 303 nm. Fluorescent efficiency is about 0.21 for both molecules. The fluorescent shift of phenol between protic and aprotic solvents is small, compared to indole, a model for tryptophan-based protein, due to the larger gap between its first and second excited states, which resnlts in negligible coupling . ... 

In order to proceed it is now necessary to consider the nature of the lowest excited state of these polymers. One description which appears to be particularly appropriate to these materials is that given by the molecular exciton theory (37,38). This of course is suggested by the nature of the fluorescence spectrum itself and in addition this approach has proven to be quite successful in the Interpretation of the electronic states of the alkanes, the structural analogs of the poly(organosllylenes) ( 3, 6). The basic assumption... 

Figure 11.1 Representation on the same graph of the absorbance and fluorescence spectra of an ethlyenic compound. The fluorescence spectrum that resembles the mirror image of the absorbance spectrum, as weU as the Stokes shift can be interpreted by considering the energy diagrams (Figure 11.2). In UV/Vis absorption and fluorescence spectra, bandwidths of 25 nm or more are common. This representation is obtained by uniting on the same graph, with a double scale, the spectrum of absorbance with that of emission. Example extracted from Jacobs H. et al, Tetrahedron 1993, 6045.

At this point, it may seem to the reader that the detailed consideration of quantum beat phase distributions is a somewhat abstract exercise bearing little relation to IVR. We would justify our attention to the problem of phases by noting that the proper interpretation of experimental results from picosecond-jet experiments on IVR relies on the ability to determine how closely one s experimental conditions correspond to one s theoretical model of the experiment. A particularly convenient way to do this is by comparing phase characteristics from experiment with those from theory. In addition, phase characteristics are useful in helping one assign the various bands in a fluorescence spectrum to band types. 

Picosecond-beam studies42 confirm the interpretation that IVR is absent in this low-energy regime. As an example, Fig. 5 shows the fluorescence spectrum that arises upon excitation of the S, + 766 cm"1 (122) level of anthracene and a decay of a band in that spectrum (the shift from the excitation wavelength, vd, for the band being vj = 390 cm"1). The spectrum is analyzed in Ref. 61. The decay is clearly unmodulated, is a single exponential with an 18 nsec lifetime, and is the same as the decays of the other bands in the spectrum. Such decay behavior is consistent with nonexistent IVR. 

Acid Denaturation. LADH loses activity and zinc at pH 5 while still in the dimeric state 177,182). At lower pH dissociation occurs into subunits 182-184) and there are drastic changes in the protein fluorescence spectrum 185-187) and the fluorescence polarization spectrum 182). Different time dependences for the changes of the tyrosine and tryptophan difference fluorescence peaks are observed 187), which is consistent with a slower quenching of the buried Trp-314 (Section II,C,3,c) compared to the more exposed tyrosines. This interpretation implies that a partial unfolding of the tertiary structure occurs prior to the dissociation into subunits at acid pH. 

Figure 11.9. Fluorescence spectra of ZnTPPS-ZnTMPyP and the ZnTPPs-ZnTMPyP ion pair in aqueous solution (a). As for the absorption spectra in Figure 11.8, the fluorescence spectrum of the ion pair cannot be interpreted in term of a linear combination of the individual porphyrin spectra (b).

According to the vibronic interpretation of the absorption spectrum at 1.7 K of open R.vi ridis Res /ll/, the shoulder at 1015 nm reflects 0-0 transition, the peak at 1000 nm belongs to 0-1 transition, the shoulder at 985 nm to 0-2 transition, etc. Fig.4 shows that at 1.7 K in R.vi ridis Res with reduced HL the longest component in the absorption spectrum is located at 1011 nm and the shortest component in the fluorescence spectrum is at 1014 nm. It implies the Stokes shift of 30 cm"l for 0-0 transition. This is consistent with the phonon frequency of 30 cm and Pekar-Huang-Rhys factor Sail found from hole-burning experiments. The latter show that in the presence of reduced HL the narrow hole (zero-phonon line, ZPL) is bleached in the P band at wavelength of excitation (1014 nm) at 1.7 K (Fig.3 /lO/). 

The above section has outlined the physical parameters that describe the fluorescence process. One can measure the fluorescence spectrum, P(X), the singlet excited state lifetime, xs, and determine the Tliese parameters can be interpreted in terms of the structure, environment and ( mamics of the molecule of interest. In this section, the different optical and electronic components comprising an instrument that can measure Fl( ) and will be described. This instrument is generally known as a steady state fluorescence spectrometer, since it integrates the fluorescence intensity over a given time period. Time-resolved fluorescence instrumentation that is used to measure the excited singlet state decay times is described in Chapter 3. 

The evidence from photostationary measurements of fluorescence intensity can thus be interpreted in terms of reversible photodimerisation to form an excited dimer which falls apart when it fluoresces. As with static quenching, there is no permanent chemical change on dilution the original fluorescence spectrum is restored. The essential difference is that excimer formation occurs only when one of the molecules is excited in the ground state, the equilibrium A + A A—A is not observed. The equilibrium constant in the excited... 

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