Single-molecule resolution and fluorescence imaging of mixed-mode sorption of a dye at the interface of C18 and acetonitrile/water

Single-molecule imaging is used for the first time to study the cationic dye, 1,1'-dioctadecyl-3,3,3'3'-tetramethyl-indocarbocyanine perchlorate (DiI), at the chromatographic interface consisting of acetonitrile/water and a hydrocarbon monolayer (C-18) covalently bound to silica. Autocorrelations of burst data agree with our previous single-molecule counting results, showing that most dye molecules are diffusing and that there is a rare specific adsorption site associated with a 0.07-s desorption time. These autocorrelations go further in detecting an even rarer specific adsorption event associated with a 2.6-s desorption time. The latter desorption time would contribute much more significantly to peak tailing in chromatography. In water, the populations of DiI at these two specific adsorption sites are shown to be 11% and 4%, respectively, for the weaker and stronger sites, relative to the diffusing population of DiI. In 60% acetonitrile/water, the relative populations of the specific adsorption sites are 11% and 17%, showing that acetonitrile enhances the population of the stronger specific adsorption site. Fluorescence movies of single and multiple molecules link the stronger specific adsorption sites to specific locations on the surface. The imaging makes rare observations frequent by pinpointing where the events occur spatially. This ability to observe rare events by imaging reveals the presence of a third type of specific adsorption site, for which DiI has a desorption time in excess of 20 s.