Abstract

A new analytical approach to multicomponent fluorimetric analysis is presented theoretically and is experimentally demonstrated. The new approach, termed fluorescence anisotropy selective technique (FAST), allows one to <i>directly</i> resolve the individual components in complex mixtures on the basis of differences in the rotational diffusion rates of the components. Importantly, unlike steady-state polarization-based approaches, FAST does not require <i>a priori</i> information regarding the number of individual components. By the use of FAST, the individual emission spectra of the same fluorophore in two different microenvironments can be simultaneously determined even if the fluorescence lifetimes and emission spectra of the two components are extremely similar. To demonstrate the power of FAST, we show results for the simultaneous resolution of dansylamide bound to beta-cyclodextrin and human serum albumin. In this example, the fluorescence lifetimes of the two components are essentially identical.

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