Relationship between HETP measurements and breakthrough curves in short chromatography columns.

Abstract

An analysis of the relationship between the number of plates measured with a small molecule tracer and the breakthrough curve of a strongly bound protein in short laboratory chromatography columns (1-5 cm) considering flow nonuniformity is presented. For practical conditions, while axial dispersion has only a small impact on the breakthrough curve, radial flow nonuniformity has a profound effect. Radial parabolic velocity profiles lead to tailing tracer peaks and broader breakthrough curves. Profiles where the velocity varies radially only in a thin region near the column wall lead to fronting tracer peaks and early breakthrough when the velocity at the wall is higher than the average and to tailing peaks and tailing breakthrough curves when the velocity at the wall is lower than the average. Experiments conducted in laboratory minicolumns (0.5-1 cm diameter, 0.5-1 ml volume) show tracer peaks and protein breakthrough curves that are consistent with higher velocities at the wall. The model presented in this work provides a tool to model experimental breakthrough data and to assess the degree of flow uniformity required to obtain meaningful dynamic binding capacity measurements using minicolumns in a high-throughput lab setting.