We report on the rotational and translational diffusion dynamics of two monovalent fluorescent probe molecules, cationic oxazine 118 and anionic resorufin, in the glycerol–water solvent system using time-resolved fluorescence anisotropy (TRFA) and fluorescence recovery after photobleaching (FRAP) measurements. Experimental measurements of the chromophores are compared to well-established hydrodynamic models of rotational diffusion by the modified Debye–Stokes–Einstein (DSE) equation and of translational diffusion by the Stokes–Einstein–Sutherland (SES) equation. Other quasi-hydrodynamic models by Geirer–Wirtz (GW) and Dote–Kivelson–Schwartz (DKS) are compared to the modified DSE and SES models to better understand their utility to these systems. Deviations from the theoretically predicted diffusion constants are attributed to local solvation differences between the cationic and anionic chromophores and heterogeneity within the glycerol–water solvent system. Direct comparison between the modified DSE and SES models allows for empirical determination of the solvent–solute frictional interaction factor.
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