Influence of the Column Inner Diameter on Chromatographic Efficiency in Miniaturized and Conventional Ultra-High-Performance Liquid Chromatography

To reduce solvent consumption and cycle times of analyses, the inner diameters (ID) of separation columns are being reduced continuously. In particular, 1.0 mm inner diameter columns seem to be the next logical step, as they represent a good compromise between sample loading and analysis speed. However, the necessary optimization of the extra-column volume is often not considered. The aim of this study was therefore to investigate the influence of the column inner diameter on the efficiency in the isocratic and gradient elution mode. The efficiency of 0.3, 0.5, 1.0, and 2.1 mm inner diameter columns were compared using a µ-HPLC and UHPLC system. To enable a fair comparison between the different columns, the linear flow velocity was kept constant. In isocratic mode, the influence of the extra-column volume dispersion was assessed by the peak standard deviation and the reduced plate height. In gradient mode, the separation of 7 antineoplastic drugs was compared in terms of the peak capacity and chromatographic resolution. Amongst other things, it was shown that the ratio between extra-column volume and effective-column volume can be a simple parameter for choosing an appropriate column ID /HPLC combination. An unfavorable ratio between extra-column volume and effective-column volume led to a doubling of the minimum plate height.