Assessment of Bed and Particle Integrity of Size-Exclusion Columns Packed with Sub-2-µm Particles

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The Column spoke to Jelle De Vos from the Department of Chemical Engineering at the Vrije Universiteit, in Brussels, Belgium, about his work investigating the performance of columns packed with 1.7-μm particles at high-pressure conditions (ΔPmax = 1034 bar) and linking it to particle and column-bed integrity.

Q. How did the idea of this research project arise and what are the aims of this research?

A: Back in 2016 we published a paper on the effects of operating pressure on selectivity and efficiency in aqueous size‑exclusion mode for the separation of intact proteins (1). We used kinetic plots to describe the possibilities of faster methods in size-exclusion chromatography (SEC) mode when applying ultrahigh pressures, which laid the foundation for the work presented in the poster.

Q. What are you doing in this poster that is novel?

A: The poster explores the potential of sub‑2‑µm particulate SEC columns operated under ultrahigh-pressure liquid chromatography (UHPLC) conditions. There are currently no studies that investigate the effects of high operating pressures on packing stability and particle integrity, and, hence, the selectivity of aqueous size‑exclusion columns. These are essential aspects when method speed-up is envisioned.

Q. What were the main analytical challenges you had to overcome?

A: The experiments were performed using SEC columns packed with 3.5-µm and 1.7‑µm particles. These columns were subjected to extreme pressure stress-tests and their degradation was monitored by separating a mixture of protein standards. At first, we were not able to explain the cause of the impaired chromatography when higher backpressures were applied,
as this is usually not the case in reversed‑phase LC mode. Hence, following the stress tests, the columns were opened and the particles were subjected to physical analytical techniques (scanning electron microscopy, argon adsorption measurements, and mercury intrusion porosimetry). The results were verified using columns originating from different batches. All these results were combined to comprehensively describe the issues that arise at higher operating pressures when using columns packed with sub-2-µm particles.

Q. What were your main findings and why are they useful?

A: The improved mass transfer characteristics of sub-2-µm particles were demonstrated by a sub-min separation of intact proteins by applying a flow rate of 1.8 mL/min, corresponding to a column pressure of 530 bar. High pressure operation typically results in bed compression, which impairs chromatography. Improved column packing is essential to obtain robust high performance SEC separations.

Q. How are you planning to develop this research further?

A: This study demonstrates the potential of sub-2-µm particles in aqueous SEC, and can be expanded towards exploring the potential of even smaller particle diameters ranging around 1 µm.

Reference

• J. De Vos, E.R. Kaal, R. Swart, M. Baca, Y. Vander Heyden, and S. Eeltink, J. Sep. Sci. 39, 689–695 (2016).

Jelle De Vos is at the Department of Chemical Engineering, Vrije Universiteit, in Brussels, Belgium.