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

Abstract

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.

Single-Molecule Resolution of Protein Dynamics on Polymeric Membrane Surfaces: The Roles of Spatial and Population Heterogeneity

“…Object positions were calculated as the centroid of intensity with a localization precision of ∼50 nm. 22 Molecular trajectories were constructed by linking an object's center of intensity to the nearest center of intensity within a distance (i.e., tracking radius) of 4 pixels (1.08 μm) in subsequent frames. Protein kinetics were found to be insensitive to the tracking radius selected (see Figure S12 in the Supporting Information).…”

“…By creating these maps, we were able to accurately measure adsorption and desorption kinetics for molecular-scale surface sites and, importantly, identify distinct populations of sites. The specific mapping methods were described previously by Mabry et al 22 The initial position of each molecular trajectory was placed on a pseudoimage with 22.7 nm pixels (10× smaller than camera pixels) and blurred such that an adsorption center was represented by Gaussian peak with a standard deviation σ loc and an amplitude of 1 adsorption event. Super-resolution adsorption event maps were constructed by summing all blurred pseudoimage adsorption events.…”

Tuning the Flight Length of Molecules Diffusing on a Hydrophobic Surface

“…28 Consistent with these previous findings, the frequency of strong adsorption (i.e., confinement) reached a minimum at 25% methanol and was correlated with specific regions of the surface. The TMS surface is known to have spatially rare, anomalously strong surface sites, 14,15 and adsorption maps showed that binding onto strong sites correlated with the frequency of confinement (Figures S-7 and S-8, Table S-3).…”

Capturing Conformation-Dependent Molecule–Surface Interactions When Surface Chemistry Is Heterogeneous

“…The channels were aligned and molecules identified as described previously (using an image processing routine where the raw images were convolved with a disk matrix and then thresholded to identify distinct objects). 76,83 Only molecules adsorbed to the interface were localized during image processing because diffusion in solution was too fast to allow localization at the 100 ms frame acquisition times used in these experiments. Previous studies of FS and TMS surfaces have shown that adsorbed molecules are typically confined to regions smaller than the localization precision (∼100 nm).…”