Single-molecule probing of adsorption to heterogeneous interfaces

Single-molecule spectroscopy and fluorescence microscopy was used to probe the heterogeneous adsorption and lateral diffusion of molecules at chemical interfaces. This research is the first to report the direct observation of adsorption of single molecules to disparate types of adsorption sites at a chromatographic interface.; The theory and instrumental design used to observe single molecules, as well as the history and applications of the technique is described. The instrumentation involves interfacing an inverted microscope, electronic devices, sensitive detectors, and additional optics to achieve spectral resolution of single molecules.; The lateral transport of 1,1′ dioctadecyl-3,3,3 ′3′-tetramethylindocarbocyanine perchlorate, (DiI), and DNA molecules at the C₁₈/water interface was studied. The results revealed the presence of two types of adsorption events, distinguishable by the level of fluctuations of their fluorescent signals. The two types of adsorption events included laterally diffusing molecules having strong fluctuations of their fluorescence due to the random walk of the fluorophor about the Gaussian beam profile, and specifically adsorbed molecules having constant fluorescence within the shot noise.; In addition to single-molecule spectroscopy, fluorescence microscopy in conjunction with atomic force microscopy (AFM) was used to image the adsorption of DiI onto a chemically modified silica substrate. The results revealed that strong adsorption of DiI occurred at nanometer indentations on the surface. Strong adsorption at the nanometer indentations was greater for acetonitrile as the solvent than for water, underscoring the importance of topography in HPLC. This is the first time that topography has been identified as a contributing factor in causing peak tailing. The adsorption of DiI at the indentations was found to be pH independent, behaving similarly at pH 7 and pH 2.