Single-molecule imaging in commercial stationary phase particles using highly inclined and laminated optical sheet microscopy

Chromatographic separations often rely on porous materials coated with covalently bound polymers to bind and separate analytes from mixtures based on specific forces. Two-dimensional single-molecule and super-resolution microscopy has previously quantified causes of peak broadening in separations, but prior studies have focused in model surfaces that have different physical and chemical properties than the three-dimensional materials used in real columns and membranes. In this work we resolve the three-dimensional, nanoscale locations of single-molecule analytes within unpacked commercial chiral stationary phase particles (CSPs) using highly inclined and laminated optical sheet (HILO) microscopy. We show improvements up to 32% in signal-to-background ratio and 118% in the number of single molecules detected within the CSPs when using HILO compared to epifluorescence. We also generate 3D super-resolution maps of the single-molecule sorption within the particles with nanoscale lateral and axial resolutions, clearly visualizing surface heterogeneities and distinct sorption sites. We observe that the number of localized molecules remains constant axially and between different particles, indicating that heterogeneity in a separation would not derive from such affinity differences at microscales, but instead kinetic differences at nanoscales. Further, the polymer-coated CSPs limit analyte diffusion within the particle, suggesting CSPs may be acting as superficially porous particles compared to results with uncoated porous silica stationary phases. While this work focuses on 5 µm CSPs, HILO can be applied to broad classes of real polymeric and soft materials to inform separations from the bottom-up.