Imaging Solute Distribution in Capillary Electrochromatography with Laser Scanning Confocal Microscopy

Abstract

A method for the direct observation of solute molecules interacting with a C18 stationary phase under real separation conditions in capillary electrochromatography (CEC) is investigated. The experiments were performed in a capillary electrochromatographic mode; however, the method and findings are useful both in CEC and revered-phase liquid chromatography. The distribution of solute molecules in the packed capillary is directly imaged with laser scanning confocal fluorescence microscopy. Conventional imaging techniques produce images where the C18 silica beads cannot be distinctively identified as a result of the deep depth of field. The optical sectioning capability of confocal imaging overcomes this problem to afford clearly defined images of the stationary-phase packing and the surrounding mobile phase. Fluorescein molecules are preferentially distributed in the mobile phase under reversed-phase chromatographic conditions. Nile Red and rhodamine 6G molecules prefer the environments of the porous C18 beads. Intensity distributions over time for areas within the stationary-phase beads differ from distributions of areas outside the beads in the mobile phase. Images taken at different depths into the capillary probe the internal structure of the C18 beads. While the internal structures of most beads are porous, confocal images show a small fraction (2%) of the silica beads have porous shells and nonporous cores. The capability of imaging the stationary phase distinctively from the mobile phase opens the possibilities of studying the quality of stationary phase, the structure of the column packing, and the mechanisms of separation.