Single-molecule insights into retention at a reversed-phase chromatographic interface

Area of Science:

• Analytical Chemistry
• Surface Science
• Physical Chemistry

Background:

• Chromatographic separation efficiency is reduced by peak asymmetry, often caused by heterogeneous adsorption kinetics.
• Understanding these adsorption heterogeneities is crucial for improving separation techniques.

Purpose of the Study:

• To investigate the nature and consequences of heterogeneous adsorption kinetics on chromatographic stationary phases.
• To correlate single-molecule adsorption behavior with macroscopic chromatographic performance.

Main Methods:

• Combined single-molecule imaging with reversed-phase liquid chromatography (RPLC).
• Utilized a fluorescent fatty acid analyte on trimethylsilyl-functionalized silica in methanol/water solutions.
• Employed super-resolution mapping and a stochastic model of chromatography.

Main Results:

• Single-molecule observations identified rare, strong adsorption sites with concentration-dependent activity.
• Adsorption/desorption kinetics on strong sites were heterogeneous and correlated with binding energies.
• Peak tailing in RPLC showed a non-monotonic dependence on methanol concentration, consistent with single-molecule findings.

Conclusions:

• Heterogeneous adsorption sites significantly influence chromatographic peak shape and separation efficiency.
• Single-molecule imaging provides quantitative insights into macroscopic chromatographic behavior.
• This approach aids in designing improved chromatographic separations and understanding surface chemistry's role in molecular dynamics.