High pressure single-molecule investigation of analyte interactions with chiral stationary phase particles

Many molecules are synthesized as 50/50 mixtures of two mirror-image versions, termed chiral enantiomers. Separating enantiomers is essential to drug development, as each enantiomer can have varied health effects. Chiral chromatography is used to separate enantiomers but lacks a molecular-scale understanding. Previous single-molecule studies from our lab used highly inclined and laminated optical sheet (HILO) microscopy to investigate achiral analyte interactions with chiral stationary phase particles (CSPs) at pressures of 600 psi. Results showed that 2 nm analytes reach a depth of only ~20% of the CSP diameter, despite CSP pores being 50 times larger than analytes. It is unknown whether the inability of analyte molecules to penetrate CSP pores is due to low operating pressures, which are below actual column pressures of ~5,000 psi, or physical blockage by CSP polymer coatings. In this study, we build a high-pressure single-molecule setup to determine if pressure changes analyte penetration into CSPs. We pack CSPs and analyte into optically-transparent capillary tubes compatible with fluorescence microscopy and couple the capillary tube to a pressure generator that can achieve 5,000 psi. We image the packed capillary tubes with HILO microscopy and analyze single-molecule locations with super-resolution algorithms. We hypothesize that high pressures will not cause analyte molecules to penetrate the porous particles and that the lack of analyte penetration can be attributed to the polymer coating.