Liquid Chromatography at the Single-Molecule Level

Liquid chromatography is an indispensable technique in the pharmaceutical industry both for purification and validation of products.  The macroscopic movement and development of chromatographic peaks in a column can be described at the molecular level using the stochastic theory of chromatography.  Using this conceptual link, we compared single-molecule dynamics of fatty acid molecules at hydrophobic interfaces to macroscopic results obtained with a typical analytical chromatography system.  We observed that molecules remained bound over a broad distribution of times.  We made super-resolution spatial maps of molecule adsorption and concluded that the heterogeneous kinetics were not due to large-scale surface defects.

In the single-molecule experiments, we varied the mobile phase composition and found large changes in adsorption rate constants, whereas desorption rate constants were relatively insensitive to the overlying mobile phase.  This finding emphasizes the importance of stationary phase chemistry in determining column efficiency. Our work demonstrates the unique potential of single-molecule measurements for connecting molecular dynamics to macroscopic chromatographic phenomena.