Time-dependent fluorescence quenching

Sohn J-H, Kim KH, Lee H-Y, No ZS, Ihee H. Initial Catalyst—Substrate Association Step in Enyne Metathesis Catalyzed by Grubbs Ruthenium Complex Probed by Time-Dependent Fluorescence Quenching. J Am Chem Soc. 2008 130(49) 16506-16507. 

Functionality preferences for metathesis of Ru carbenes to alkenes and alkynes with electronic and steric diversity were studied using time-dependent fluorescence quenching (Scheme 116). ... 

Figure 3 Time-dependant fluorescence quenching by (a) DNT and (b) DMNB. Insets (left) corresponding fluorescence spectra before and after exposure to the analyte vapors for 10 s, and (right) three consecutive quench/regeneration cycles. (Reproduced with permission from Ref 25. Wiley-VCH Verlag GmbH Co. KGaA, 2009.)...

To perform structural research on a food stuff into which a colorant is incorporated, special properties of fluorescing molecules are exploited fluorescence efficiency, fluorescence lifetime, fluorescence quenching, radiationless energy (Foerster) transfer, stationary or time-dependent fluorescence polarization and depolarization." Generally, if food colorants fluoresce, they allow very sensitive investigations which in most cases cannot be surpassed by other methods. 

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. 

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. 

Fig. 4 Time-dependent fluorescence intensity of 12 upon exposure to TNT vapor at 0,10,30, 60,120,180,300, and 600 s (top to bottom), and fluorescence quenching (%) as a function of time (inset). (Reprinted with permission from Ref. [18]. Copyright 1998 American Chemical Society)...

Later work by Fischer and Wan > demonstrated that meta (37 and 38) and para (39) hydroxy-substituted styrenes are also active towards photohydration ( = 0.22, 0.24 and 0.1, respectively). Evidence for formation of quinone methide intermediates was provided by nanosecond LFP. Both steady-state and time-resolved fluorescence quenching of 37 by water showed a cubic dependence on water concentration. This was attributed to an initial ESIPT process in which a three-water-molecule bridge mediates a proton transfer from the phenol to the basic alkene. 

The required exposure times are difficult to estimate. They are best found by trial and error. Documentation of fluorescence quenching at A = 254 nm usually only requires one trial. The exposure time found to be adequate here is normally suitable for all following exposures of fluorescence quenching if the exposure conditions are maintained constant (camera type, film type, distance of objective and lamp, aperture etc.). The exposure time required for fluorescent chromatograms is primarily dependent on the intensity of the fluorescence and, therefore, has to be optimized for each chromatogram. It is best to operate with a range of exposure times, e.g. aperture 8 with exposures of 15,30,60,120 and 240 seconds. Experience has shown that one exposure is always optimal. 

The fluorescence quenching depends on the content of the Phen units (the x values) in APh-x. An aqueous solution of APh-9 contained as many charged groups (SOJ) as about 10 times that of APh-50, when compared at the same molar concentration of the Phen residues. When AMPS homopolymer (PAMPS) was added to a solution of APh-50 so that the SOJ residue concentration was equal to that for APh-9, the kq value for the APh-50 quenching by MV2 + decreased from 2.1 x 1012 to 4.2 x 1011 M-1 s 1, which is close to the kq value for APh-9 (Table 2). From these facts the lower kq values for APh-x with lower x (higher... 

Et /ZG AG -0.2 V fluorescence quenching correlates with CT driving force no spectral overlap for energy transfer no Zs in injection site or intervening sequence k = 2 x 1011 s 1 observed up to 20 A in time-resolved and steady-state experiments shallow dependence of CT yield between 6-24 A (y = 0.2-0.3) no dependence of kci between 10-17 A strong efficiency and distance dependence of CT yield vary with Z flanking sequence and intervening mismatches... 

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 fluorescence decay time, calculated for the 400 nm emission of TIN in PMMA films, decreases from 1.3 0.2 ns to 0.20 0.02 ns as the concentration of TIN is increased from 0.07 mole% to 1.1 mole% respectively. This is further evidence that the TIN molecules are involved in a concentration-dependent, self-quenching... 

The choice of method depends on the system to be investigated. The methods of intermolecular quenching and intermolecular excimer formation are not recommended for probing fluidity of microheterogeneous media because of possible perturbation of the translational diffusion process. The methods of intramolecular excimer formation and molecular rotors are convenient and rapid, but the time-resolved fluorescence polarization technique provides much more detailed information, including the order of an anisotropic medium. 

We also have explored the solution of the time-dependent eq 1 to study the plot corresponding to Figure 1 when the observation of fluorescence quenching in reaction 14 is made at short times. In these short-time calculations we have assumed, for... 

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... 

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