Insights on further improving fast size exclusion chromatography separations of biopharmaceuticals using 2.1 millimetre column diameters

Abstract

Recent trends in the pharmaceutical and biotechnology industries call for the miniaturization of size exclusion chromatography. The thought of such a future has been tantalizing but there are many practical and theoretical considerations that have impeded progress. Here, the capabilities of a narrow bore 2.1 mm ID SEC column have been studied and compared to reference 150 × 4.6 mm SEC columns when using UV detection. While our study reconfirms the importance of having very low system dispersion for SEC separations, it goes on to show that a 150 × 2.1 mm 1.7 µm particle SEC column can offer a balanced compromise of performance. Despite the fact that the 150 × 2.1 mm ID 1.7 µm column's intrinsic efficiency was not fully utilized, it still performed with an apparent efficiency similar to that of a 150 × 4.6 mm ID 2.5 µm column. Beyond this, our study provides insights on what more will need to be achieved to robustly establish low flow SEC separations. If SEC chromatographers aim to miniaturize sizing separations to 1 mm diameters or below, there is more work to do on chromatographic instruments and flow paths. In order for an instrument to be optimized for 1 mm ID SEC it would need to exhibit a system variance of less than 0.5 µL2.