Variable-Angle Time-Resolved Evanescent Wave-Induced Fluorescence Spectroscopy (VATR-EWIFS):  A Technique for Concentration Profiling Fluorophores at Dielectric Interfaces

The concentration distributions of fluorescein and acridine orange, at a water/silica interface, are determined using the technique variable-angle time-resolved evanescent wave-induced fluorescence spectroscopy. It is demonstrated theoretically that the radiative fluorescence lifetime of a molecule is affected in only a minor way by the presence of a dielectric interface. Consequently, time-resolved evanescent wave-induced fluorescence spectroscopy is used to measure the variations in the fluorescence quantum yield caused by the pressence of the interface. Knowledge of these variations is then used to determine molecular concentration distributions. The concentration distribution of fluorescein is found to be uniform and homogeneous as a function of distance away from the hydrophilic silica surface. However, it is found that a water/silica interface has a definite affect on the concentration distribution of acridine orange. A “surface associated” acridine orange population distinguishable from “bulk” acridine orange is demonstrated by spectral and kinetic analysis of EWIF emission. This information is then used to create concentration profiles of both molecular populations.