Mobility-Based Wall Adsorption Isotherms for Comparing Capillary Electrophoresis with Single-Molecule Observations

The adsorption properties of R-phycoerythrin (RPE), an autofluorescent protein, on the fused-silica surface were studied in capillary electrophoresis (CE) and in single-molecule experiments. The band shapes and migration times were measured in CE, and adsorption and desorption events were recorded at the single-molecule level by imaging within the evanescent field layer using total internal reflection fluorescence microscopy. The adsorbed RPE molecules on the surface of the fused-silica prism were counted with confidence based on ImageJ software. The capacity factor and desorption rate were estimated from the counting results. The mobility-based adsorption isotherms were constructed from both computer simulations and experiments to determine the capacity factor.