Equipment and preliminary results for orthogonal high-pressure electrochromatography

Abstract

This study presents a novel two-dimensional separation technique, Orthogonal High-Pressure Electrochromatography (OHPEC), and a prototype device that combines electrophoresis and liquid chromatography within a single orthogonal separation system. A significant advancement is using an actual planar column packed with bulk adsorbent, overcoming challenges associated with chromatography plates used in planar electrochromatography for close system separations. The OHPEC planar column operates under high-pressure conditions, withstanding short-term pressures of up to 350 bars needed for column ’wet packing’ and long-term of up to 150 bars. Fully automated processes, including sample introduction, separation, detection, and fraction collection, enhance reliability and reduce manual intervention. Notably, OHPEC preserves the core benefits of its predecessor, Orthogonal Pressurised Planar Electrochromatography (OPPEC), including the continuous separation of compounds with distinct electrophoretic mobilities and the periodic isolation of those with similar mobilities but differing retention properties. Its functionality was demonstrated by separating a post-reaction mixture from valine diastereomer synthesis in both analytical and preparative modes, using an applied voltage of up to 2 kV. The preparative separations achieved twice the production rate reported for OPPEC, with further improvements anticipated through process optimisation and the development of planar columns packed with smaller adsorbent particles. OHPEC demonstrated good separation reproducibility, with %RSD values below 2 % for retention time, peak width, and peak area under non-voltage conditions and approximately 2–6 % with applied voltage. While challenges remain, such as improving electrode chamber performance, temperature control, and column efficiency, OHPEC shows great promise for a wide range of applications across various fields.