Fluorescence static quenching

Following an external perturbation, the fluorescence quantum yield can remain proportional to the lifetime of the excited state (e.g. in the case of dynamic quenching (see Chapter 4), variation in temperature, etc.). However, such a proportionality may not be valid if de-excitation pathways - different from those described above - result from interactions with other molecules. A typical case where the fluorescence quantum yield is affected without any change in excited-state lifetime is the formation of a ground-state complex that is non-fluorescent (static quenching see Chapter 4). 

The DPA moiety is less active in forming the CT complex with viologens than the pyrene moiety e.g., for PMAvDPA the KCT values with MV2+ and SPV are 1.3 x 103 M 1 and almost zero, respectively, at pH 8-9 [60, 77], whereas for PMAvPY they are 7.8 xlO4 and 6.3 x 102 M, respectively, at pH 11 [77]. Therefore, the polymer-bound pyrene system undergoes much more static quenching than the polymer-bound DPA system. As will be discussed in Chapter 6, it is very important for charge separation whether the fluorescence quenching is static or dynamic. 

Two kinds of quenching are distinguished. In static quenching, interaction between the potentially fluorescent molecule and the quencher takes place in the... 

Fluorescence quenching is described in terms of two mechanisms that show different dependencies on quencher concentration. In dynamic quenching, the quencher can diffuse at least a few nanometers on the time scale of the excited state lifetime (nanoseconds). In static quenching, mass diffusion is suppressed. Only those dye molecules which are accidentally close to a quencher will be affected. Those far from a quencher will fluoresce normally, unaware of the presence of quenchers in the system. These processes are described below for the specific case of PMMA-Phe quenched by MEK. 

The term static quenching implies either the existence of a sphere of effective quenching or the formation of a ground-state non-fluorescent complex (Figure 4.1) (Case A of Section 4.2.1). 

The excited-state lifetime of the uncomplexed fluorophore M is unaffected, in contrast to dynamic quenching. The fluorescence intensity of the solution decreases upon addition of Q, but the fluorescence decay after pulse excitation is unaffected. Quinones, hydroquinones, purines and pyrimidines are well-known examples of molecules responsible for static quenching. 

Let us consider first the case of static quenching by formation of a non-fluorescent complex. The ratio I0/I obtained for dynamic quenching must be multiplied by the fraction of fluorescent molecules (i.e. uncomplexed)... 

The Dp and Dq are the diffusion coefficients of probe and quencher, respectively, N is the number molecules per millimole, andp is a factor that is related to the probability of each collision causing quenching and to the radius of interaction of probe and quencher. A more detailed treatment of fluorescence quenching including multiexponential intensity decays and static quenching is given elsewhere/635 There are many known collisional quenchers (analytes) which alter the fluorescence intensity and decay time. These include O2/19 2( 29 64 66) halides,(67 69) chlorinated hydrocarbons/705 iodide/715 bromate/725 xenon/735 acrylamide/745 succinimide/755 sulfur dioxide/765 and halothane/775 to name a few. 

The importance of comparing time-dependent and steady-state fluorescence measurements is well illustrated by the difficulty of resolving purely static from purely dynamic quenching. In either case, the basic relationship between the steady-state fluorescence intensity and quencher concentration is the same. The Stem-Volmer relationship for static quenching due to formation of an intermolecular complex is i... 

Time-dependent fluorescence measurements have been made on tyrosine in calf thymus nucleosome core particles by Ashikawa et al. S7) Based on the salt dependence of the decay data, the tyrosines were divided into two classes. At 20 to 400 mM salt, about half of the tyrosine residues appear to be partially quenched, possibly by resonance energy transfer to DNA bases. The other half are thought to be statically quenched, possibly by hydrogen bonds this quenching is partially eliminated at about 2 M salt. In view of the number of tyrosines per nucleosome core particle (estimated at 30), it is impossible to make a more detailed analysis of the decay data. 

Haas et al.(m) have examined the fluorescence decay of tyrosine due to different Tyr-Pro conformations in small peptides to elucidate further the nature of the fluorescence change associated with Tyr-92. These peptides have acetyl groups at the amino terminus and /V-mcthylamidc groups at the carboxyl terminus. They found that whereas the dipeptide fluorescence decay requires a double-exponential fit, that of the tripeptide Tyr-Pro-Asn can be fit by a single exponential. By comparison of the average fluorescence decay time and steady-state quantum yield of the peptide to that of A-acetyltyrosine-A-methylamide, they found a relatively greater reduction in the steady-state quantum yield of the peptides. This is attributed to static quenching, which increased from 5 % in the dipeptide to 25 % in the tripeptide. The conformations of these peptides were also examined by NMR, but the results could be interpreted in terms of either cis-trans isomerization or other conformational isomerizations. 

The motions of chromophore groups and of their environment that lead to temperature-dependent fluorescence quenching are those on the nanosecond time scale. Slower motions cannot manifest themselves in effects on the excited-state lifetime (this corresponds to the limit of high viscosity). On the other hand, if the motions are considerably faster (on the picosecond time scale), then they should give rise to static quenching. 

The illustration in Figure 5.3 shows three types of curves which are commonly obtained when fluorescence quenching data are plotted in the manner shown. Curve a would be typical of static quenching, while curve b would be... 

Figure 5.3. Simulated Stern-Volmer plots of the ratio of the initial fluorescence intensity F0 to the intensity Fin the presence of quencher of concentration [Q] showing (a)static quenching, (b) dynamic quenching (linear), and (c) binding and/or inaccessible quenchers.

The fluorescence emission of 7-amino-4-methylcoumarin is quenched by [Ru(bpy)3] ", and data in aqueous solution are consistent with a static quenching mechanism. ... 

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