Probing of chromatographic adsorption by single-molecule fluorescence spectroscopy
Chromatographic surfaces are inherently heterogeneous, giving rise to multiple types of
interactions for important systems such as pharmaceuticals and proteins. For these
systems, it is believed that a small fraction of strong adsorption sites causes tailing and
irreversible adsorption. Single-molecule spectroscopy is unique in its ability to probe
multiple types of interactions and to investigate rare events. We have obtained
fluorescence images of organic cations that mimic pharmaceuticals and of rhodaminetagged proteins at chromatographic interfaces. These studies provide direct experimental
confirmation, for the first time, of the presence of strong adsorption sites. Specifically,
time-resolved imaging shows that the adsorbed analytes freely diffuse on the stationary
phase most of the time, but occasionally become immobilized. Further, strong adsorption
is revealed to occur preferentially in nanometer indentations. The significance of this
finding is that commercial silica gel can be improved by minimizing the number of small
pores. The finding has significance beyond chromatography, pointing to an improved
means of probing the polishing of silica photomasks, and establishing that the adsorption
of proteins could ultimately be directed on the nanometer scale for unique biosensors or
immunoassays.