Single-molecule probing of mixed-mode adsorption at a chromatographic interface

This paper reports the use of single-molecule spectroscopy to observe direc tly the presence of two disparate types of adsorption sites on a liquid chr omatographic surface. This so-called mixed-mode adsorption is believed to b e the physical origin of tailing of proteins and pharmaceuticals in HPLC. S ingle-molecule spectroscopy was used to probe the organic dye 1,1'-dioctade cyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), which was adso rbed to the interface of water and a silica surface chemically modified wit h dimethyloctadecylsiloxane (Cls). Using low concentrations of interfacial DiI; single dye molecules were isolated by confocal microscopy, with the fl uorescence from the illuminated interface monitored as a function of time. Two types of adsorption events are shown to be distinguishable by the level of fluctuations on their fluorescence signals: (1) laterally diffusing mol ecules having strong fluctuations on their fluorescence due to the random w alk of the fluorophor about the Gaussian beam profile and (2) specifically adsorbed molecules having constant fluorescence within the shot noise. The study of 2048 single molecules entering the beam revealed that 99% of the o bserved molecules underwent diffusion at the water/C-18 interface, and the diffusion coefficient of the ensemble was 1.3 x 10(-6) cm(2)/s. The remaini ng 1% of the molecules became specifically adsorbed during observation, pre sumably to sites on the exposed silica substrate, and they remained specifi cally adsorbed for an average time of nearly 1s. This represents the first direct experimental observation of the phenomenon that underlies tailing in chromatography.