Single-Molecule Insights into Retention at a Reversed-Phase Chromatographic Interface

The efficiency of chromatographic separations decreases markedly when peaks exhibit asymmetry (e.g., “peak tailing”). Theoretically, these effects can arise from heterogeneous adsorption kinetics. To investigate the nature and consequences of such heterogeneity, we used a combination of single-molecule imaging and reversed-phase liquid chromatography (RPLC). In both single-molecule and macroscopic RPLC experiments, the stationary phase was hydrophobic end-capped (trimethylsilyl-functionalized) silica, which we exposed to different methanol/water solutions (50%–62% methanol), containing a fluorescent fatty acid analyte. Super-resolution maps based on single-molecule observations revealed rare, strong adsorption sites with activity that varied significantly with methanol concentration. The adsorption and desorption kinetics on the strong sites were heterogeneous and positively correlated, suggesting a broad underlying distribution of site binding energies. Adsorption equilibrium on the strong sites was more sensitive to solution conditions than overall retention measured in RPLC experiments, suggesting that the effect of strong sites on the overall adsorption kinetics should change with solution conditions. Interestingly, in RPLC experiments, peak tailing had a nonmonotonic dependence on methanol concentration within the range studied. Using the stochastic model of chromatography, we showed quantitatively that our single-molecule kinetic results were consistent with this macroscopic trend. This approach to identifying and quantifying adsorption sites should be useful for designing better chromatographic separations and for identifying the role of heterogeneous surface chemistry in molecular dynamics.