Total internal reflection fluorescence spectroscopy (TIRF) is an established technique for following the course of interfacial reactions. Theoretically, by gathering TIRF data as a function of observation angle, one can obtain the density of fluorophores with respect to distance away from a solid/liquid interface. In order that the practical application of the theory might be explored, variable observation angle data from solutions of fluorescein and from adsorbed layers of fluorescein isothiocyanate labeled immunoglobulin have been analyzed in terms of simple concentration-distance profiles. In all cases the general shape of the data curves was found to conform to the theoretical expectation. Layer thickness determinations varied over a range of 20 to 100 nm, with concentrations in the layer ranging from 12 to 61 mg/mL. The theoretical background, sources of error, and system improvements are also discussed.
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