This article details the contributions of single-molecule spectroscopy to the current understanding of an important problem in chromatography. Tailing is a long-standing problem in pharmaceutical and protein separations, and it is believed to be caused by unwanted strong adsorption sites on the substrate, which is typically silica. We probed the strong adsorption phenomenon directly by using single-molecule spectroscopy. Single molecules of a fluorophor, 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI), were monitored as they were in dynamic equilibrium at a chromatographic interface composed of chemically modified fused silica in contact with water or acetonitrile. The experiments revealed that most molecules laterally diffused at the interface, but occasionally, a molecule would stop diffusing temporarily and then resume. This was interpreted to be the manifestation of strong adsorption. An analysis of large data sets revealed three different strong adsorption processes occurring with desorption times on distinctly different time scales: 70 ms, 7 s, and >2 min. These events exhibited no pH dependence, and the two longer events were directly observed at nanometer-scale topographical indentations. The desorption kinetics from commercial chromatographic silica gel are in agreement with the single-molecule experiments on fused silica, indicating that the phenomena being probed are chromatographically relevant. These single-molecule experiments provide important new information about the so-called "active silanols" that cause tailing to persist at low pH, revealing that these sites are neutral and that surface topography plays a role.