| Direct observation of the molecular distribution in separation columns under real separation conditions has long been desired in the field of chemical separations. This would allow examination of the structure and homogeneity of the packing; and on the molecular level, the study of diffusion, partitioning, and sorption processes at the chromatographic interface. Few techniques allow for this, as direct observation requires an imaging technique with high spatial and temporal resolution with high sensitivity. One such technique is confocal fluorescence imaging.; A commercially available laser scanning confocal microscope was used for studies of capillary electrochromatography and retention in reversed-phase chromatographic stationary phases. Static and time series images were used to investigate interactions between solute molecules and the C18 stationary phase, and dynamic processes that occur within the column. Time series images allowed the observation of dynamic processes such as electrokinetic rearrangement of the packing and bubble formation, a common problem encountered in capillary electrochromatography. Static images showed fluorescein molecules were preferentially distributed in the mobile phase under reversed-phase chromatographic conditions while Nile red and rhodamine 6G preferred environments in the porous C18 beads. The structure and heterogeneity of the packing was observed with confocal images. Anomalous pore structures were present in ∼2% of the silica beads.; Wetting phenomena of the nanometer-sized mesopores were observed with sub-micron resolution. The pores did not wet with highly aqueous mobile phases, beginning to wet only after the concentration of acetonitrile reached 27.5%. They wet at different rates, from all directions, both symmetrically and asymmetrically. The direction and symmetry of wetting was not related to the direction of flow.; Autocorrelation analysis of confocal time series images provided indications of adsorption at the chromatographic interface. Adsorption events were rare, with the size of the sites smaller than the optical resolution of the instrument. Limitations in the commercial confocal system made it difficult to completely characterize the adsorption processes because of noise present in the measurement. A stage scanning confocal microscope specifically designed for both imaging and single molecule fluorescence correlation spectroscopy experiments was built, tested, and overcame these limitations making future studies possible. |
