Lateral diffusion of molecules partitioned into C-18 ligands on silica surfaces

Lateral diffusion rates of a fluorescent probe molecule partitioned into chromatographic C-18 ligands on a planar silica substrate were measured as a function of overlaying solvent composition. Diffusion coefficients of the probe were measured with a fluorescence recovery after patterned photobleaching experiment. A total internal reflection fluorescence flow cell held the derivatized planar substrate in contact with various aqueous solutions and allowed the fluorescence signal from two interfering, total internally reflected laser beams to be imaged onto a detector. Diffusion coefficients for the partitioned probe were approximately 3 orders of magnitude smaller than diffusion in solution and were found to increase with increasing methanol concentration in the overlaying aqueous solvent mixture. Inability of a solution phase ionic quencher to quench the fluorescence of sorbed rubrene confirmed that the probe is fully partitioned into the C-18 chains.