Robust determination of characteristic chromatographic parameters from columns exhibiting distinctive non-uniform flow behavior.

Very small columns are difficult to pack, especially with rigid chromatography media. We evaluate the performance of commercial and self-packed anion exchange columns ranging from 0.2 to 10.0 mL column volume by pulse response, breakthrough curves (BTC) and linear gradient elution. In this study we prepare self-packed columns that are intentionally packed in a way to exhibit flow nonuniformities and low performance. At very small scale, the response curves are completely dominated by extra-column dispersion effects and no differences regarding packing quality can be assessed. With increasing column size, differences become progressively better detectable. For 0.2- and 1.0-mL columns, pulse response experiments are still rather insensitive but BTCs indicate flow nonuniformities by a shallow increase in front of the steep breakthrough. For larger columns, lower packing quality is equally well detectable by response peaks with significant fronting and pronounced shallow early section of the BTC. For the columns with flow nonuniformities an analysis considering extra-column dispersion shows that the inherent fronting of the peak is counterbalanced by the peak tailing introduced by the extra-column volume of the chromatography workstation yielding an apparent peak symmetry of 1 and erroneously indicating a perfectly packed column. Using a model that accounts for non-uniform flow, relevant chromatographic parameters such as the effective diffusion coefficient and maximum capacity can be derived from columns with low packing quality, yielding the same values as those with high quality. Additionally, we demonstrate that the performance and resolution of two model proteins using linear gradient elution are nearly independent of packing quality, though it decreases significantly with smaller columns.