Direct observation of frits and dynamic air bubble formation in capillary electrochromatography using confocal fluorescence microscopy

Confocal fluorescence microscopy has been used to study the capillary electrochromatography (CEC) frits and dynamic air bubble formation under real chromatographic conditions. Confocal fluorescence microscopy provides a nondestructive way to view the three-dimensional structure of the frits with high spatial resolution. Frits prepared with four different procedures were studied: (1) sintering bare silica beads with sodium silicate; (2) sintering bare silica beads wetted with water; (3) sintering C18 beads wetted with water; and (4) sintering C18 beads wetted with water and then surfaced-recovered with C18. Frits prepared with sintering silicate-wetted beads have a high degree of heterogeneity, while the other three types of frits have similar, more homogeneous packing structures. Confocal fluorescence microscopy also provides sufficient temporal resolution for in situ observation of the dynamic processes in air bubble formation. In this study, air bubble formation is imaged during the reorganization process of the packing bed and is shown to occur close to the border between the packing bed and the outlet frit. Confocal fluorescence microscopy opens a new avenue in studying dynamic processes in situ in CEC separations.