Measurement of the Band Broadening of UV Detectors used in Ultra-high Performance Liquid Chromatography using an On-tubing Fluorescence Detector

Abstract

In modern ultra-high performance liquid chromatography set-ups, short columns (max. 10 cm) with a narrow ID (2.1 mm) packed with small, (sub-2 µm) fully or superficially porous particles are used. Since the volume and corresponding peak variance arising from these columns are very small, the dispersion contribution from the chromatographic system has a large effect on the overall separation performance. In gradient elution, the on-column focusing of the sample band at the start of the gradient results in the elimination of the pre-column band broadening. However, since gradient elution separations yield very narrow sample peaks at the outlet of the column, any post-column band broadening has a large effect on the obtained separation quality. In this contribution, the main factor of post-column band broadening is investigated, i.e., that from the UV detector, by comparing the peak width measured on capillary directly in front of the UV detector using an LIF detector and the peak widths obtained in the UV detector. These experiments show that there is a clear increase in peak variance with flow rate up to around 0.4–0.6 mL/min (depending on the investigated flow cell). It is found that modern low-dispersion flow cells generate a dispersion contribution around 0.7 µL² at high flow rates, whereas standard flow cells can have a contribution up to 5.8 µL². For the investigated nano-flow cell (80 nL), no significant dispersion was observed.