Fluorescence depolarisation

So far, attention has been focussed upon moderately small molecules, little more sizeable than a benzene ring. Studies on larger systems have been made and fluorescence depolarisation is the preferred method for molecules which luminesce. Since such molecules may also participate in fast diffusion-limited chemical reactions, they are of considerable interest. 

In these experiments, the molecule of interest is in solution and excited with plane polarised light. The fluorescence emitted perpendicularly to 

Perrin theory [223] and are typically 0.1—10 ns, depending on solute and temperature. 

When the reactants A and B are not spherically symmetric, their mutual reactivity depends on their orientation. Sole and Stockmayer [256] and Schmitz and Schurr [257] have modified the diffusion equation to include rotational diffusion of both reactants. By defining a reference axis in either species, the probability that a B reactant is a distance r away from A at time t, at an orientation (0, 0) with respect to the laboratory axis, and such that A and B are oriented at angles (0A, 0A) and (0B, 0b), respectively, is p(r,0,0 0a,0a 0b,0b)- It satisfies the expression 

To solve eqn. (114) with the boundary condition above, even for the steady state, is a task of considerable complexity and tedium. Sole and Stockmayer [256] considered the case of axially symmetric molecules and fixed the laboratory framework with 0 = 0. Hence, only 0, 0A and 0B appear in eqn. (115) and thence in the diffusion eqn. (114). By analogy to eqn. (19), the rate coefficient is 

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Fluorescence depolarisation by energy transfer (rather than rotational relaxation) between donor molecules of the same type can occur. Eisenthal [174] excited solutions of rhodamine 6G (9 mmol dm-3) in glycerol with 530 nm light from a frequency-doubled neodymium laser. The polarisation... 

Homopolymers and copolymers made from 3-vinylpyridinium salts form compact coils in solution media" while different isomers of dimethylamino-stilbazolium dyes exhibit different emitting states with a temperature dependent population." Using fluorescence depolarisation the relaxation time of poly-(styrene) chains has been found to decrease with expansion of the chain coils." " ... 

Diffusion coefficients and molecular/ionic interactions between polymers and other species appear to have attracted some interest, particularly with regard to polyions. Quantitative information on coil size and associated changes therein together with the degree of aggregation in blends of polymers and copolymers has been shown to be possible using fluorescence depolarisation measurements. Using copolymer... 

Fluorescence depolarisation studies on mixtures of isotactic and syndiotactic polymethylmethacrylate have shown the formation of a 1 2 complex in toluene while fluorescence anisotropy studies on polybutadienes have shown viscous rotational diffusion with slipping boundary conditions OS. Fluorescence studies on quasi-rigid-rod like probes in anisotropic polyethylene have shown... 

Fluorescence depolarisation by energy transfer (rather than rotational relaxation) between donor molecules of the same type can occur. Eisenthal... 

The efficiency of energy transfer in photosynthesis depends on the relative orientations of the emission and absorption transition moments respectively of the donor and acceptor chlorophyll molecules. This is determined by the mutual orientation of the chromophores within the structure of the photosynthetic membranes. In order to gain a better insight into the molecular mechanism of energy transfer, it is necessary to characterize the directions of the transition moments in the frame of the chromophores. This can be achieved by combining the results of angle - resolved linear dichroism and fluorescence depolarisation measurements on macroscopically oriented lipid membranes with the results of emission-resolved fluorescence anisotropy data from isotropic systems (vesicles) (1). 

It is often convenient to express a relation in terms of (= 1/6D ), as in Equation (3.47), although r contains no more information than (it must not be supposed to tell us anything about the motion of an individual molecule). This is because it happens that is also the relaxation time determined by measurements of various phenomena — light-scattering, fluorescence-depolarisation, and pulsed nmr — which are important in the determination of diffusion coefficients (Section S.4.2.2). 

Fluorescence Depolarisation This is based on the use of fluorescence probes such as pyrene, which has been used to study the interior of micelles. The fluorescence spectrum of pyrene shows a significant change on solubilisation in the core of the micelle. 

Although the relation between fluorescence depolarisation and rotational Brownian motion was first identified by Perrin and the development of the theoretical background of the time-resolved fluorescence depolarization experiments was made by Jablonski use of the technique was limited until the advent of improved fluorescence decay time measurements some fifteen years i. An alternative, related technique, involving excitation using a continuous polarised light source, provides only the time average of the correlation function (Eq. 18) and as such, is less useful than the time resolved method. Other disadvantages are that the natural decay time of the chromophore must be determined from a sqrarate experiment and it is necessary to alter the viscosity, and/or temperature of the medium, often withun-... 

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