Axial heterogeneities in capillary ultrahigh pressure liquid chromatography columns: Chromatographic and bed morphological characterization.

We study axial heterogeneities in capillary ultrahigh pressure liquid chromatography (UHPLC) columns through kinetic performance and bed morphological analysis. Two columns are used in this work, a 75 μm i.d. × 100 cm column packed with 1.3 μm C18-silica particles and a 75 μm i.d. × 45 cm column packed with 1.9 μm C18-silica particles. The long column is chromatographically characterized and is afterwards sectioned into three segments, each analyzed individually. The column packed with the 1.9 μm particles is subjected to a bed morphological analysis using confocal laser scanning microscopy near the inlet, center, and outlet of the column. Chromatographic and morphological characterizations reveal highest separation efficiency and most homogeneous bed microstructure towards the column outlet. Kinetic performance data for inlet and central packing segments indicate enhanced contributions from wall effects to a transcolumn flow heterogeneity. Bed morphological data reveal systematic changes in geometrical and frictional wall effects along the bed: from inlet to outlet, bed morphologies increasingly reflect packing microstructures associated with concentrated slurries. Variations in separation efficiency and bed morphology can be related to the constant-pressure packing mode; the decrease in packing rate along the bed leaves fewer chances for particle rearrangement and bed consolidation from inlet to outlet. It explains the relatively uniform bed morphology towards the outlet and also the relatively loose wall region near the inlet. Bed microstructural features are discussed in a context of previous observations made in the characterization of capillary UHPLC columns.