Preparation of continuous beds for electrochromatography and reversed-phase liquid chromatography of low-molecular-mass compounds

Abstract

High-performance capillary columns for electrochromatography and reversed-phase liquid chromatography are often prepared from silica beads. However, the synthesis of the beads involves many expensive and complicated steps and the packing of reproducible, stable and uniform beds requires great experience. In this paper we propose a simpler and more cost-effective approach, not based on preformed beads but on continuous polymer beds synthesized in situ in the chromatographic tube. The continuous bed columns were prepared by polymerizing two different monomer solutions in two steps directly in the capillary, followed by effective derivatization with hydrophobic ligands (C 18). Electroendosmosis was created by embedding a long, charged polymer (dextran sulfate). The continuous beds, as synthesized previously for the separation of proteins by reversed-phase chromatography, showed low resolution for the separation of low-molecular-mass compounds owing to a relatively low ligand density. This disadvantage was overcome by a new method designed to increase the ligand density and, in addition, to achieve a more rigid gel matrix. For rapid removal of the Joule-heat generated in electrochromatography, it is mandatory to employ very narrow columns. Continuous polymer bed columns with an inner diameter of 25 μm or less are easy to prepare. The potential of this type of capillary column is demonstrated by the separation of non-charged polycyclic aromatic hydrocarbons at an efficiency of 120 000 plates/m for a retained solute using electro-driven buffer flow. The performance is thus comparable to that of electrochromatography columns of 40–50 μm I.D. packed with 3–5 μm silica beads. In accordance with theoretical considerations only a somewhat higher plate height was obtained when the same continuous bed column was eluted with a pressure-driven flow. The columns have the distinct advantage over conventional capillary columns packed with beads that air bubbles seldom form and spoil a run, partly due to the absence of a supporting frit.