Christer Ericson

Preparation of Continuous Beds Derivatized with One-Step Alkyl and Sulfonate Groups for Capillary Electrochromatography

A simple one-step procedure for the preparation of columns for capillary electrochromatography is described. The nonpolar compounds (stearyl or butyl methacrylate) used for the introduction of hydrophobic ligands (C18, C4) are rendered water-soluble in the aqueous monomer mixture used for the synthesis of the matrix by the addition of a surfactant. This solution is sucked into a piece of fused silica tubing, the inner walls of which are activated by methacryl groups. Following polymerization, the continuous bed column is ready for use. Since the bed is attached covalently to the tubing wall, no frit to support the bed is required, which simplifies the preparation of the column. In addition, a frit can easily become clogged and is often a site for the generation of air bubbles. Some new approaches to increase the resolution and shorten the analysis times in electrochromatography are suggested and demonstrated experimentally, such as sharpening the starting zone, employing gradient elution, or adding SDS (below cmc) to the mobile phase. Separation of five polycyclic aromatic hydrocarbons can be achieved in 5 min in a 25-μm-i.d. column.

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 (C18). 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.

(Normal-Phase) Capillary Chromatography Using Acrylic Polymer-Based Continuous Beds

Microchromatographic separations of polar aromatic compounds (pyridine, 4-pyridylmethanol, 4-methoxyphenol, 2-naphthol, catechol, hydroquinone, resorcinol, 2,7-dihydroxynaphthalene) using continuous beds are described. The columns were prepared by a simple one-step in situ polymerization procedure: a solution of acrylic monomers, including the cross-linking agent piperazine diacrylamide, was polymerized in a fused-silica capillary pretreated with 3-(trimetoxysilyl) propyl methacrylate. The continuous bed formed contained a network of channels and was attached covalently to the wall of the silica capillary (100 mm I.D.) via its methacrylate groups. Therefore, the frit used in conventional, packed columns could be omitted. The separation mechanism is discussed, particularly with regard to whether the so-called aromatic adsorption to the matrix itself is involved, an interaction first described by Gelotte [1] (the ligands, isopropyl and sulfonate groups, are not required for separation). This discussion is relevant to the question of whether the separation technique described should be classified as normal-phase or adsorption chromatography. The mobile phase from the HPLC pump was split via an open capillary to get a flow rate through the continuous bed of about 100 nl /min. The beds were tested up to a pressure of 150 bar (8.8 bar /cm). A continuous bed synthesized at a relatively low molar fraction of the cross-linker in the monomer mixture (16.5%) and high total concentration of the monomers (31.9% (w/v)) afforded the highest efficiency for the separation of the polar 21 organic compounds. Plate numbers up to 150 000 m were obtained and the run-to-run reproducibility was high. The selectivity of the separations was adjusted by changing the composition of the mobile phase (hexane–ethanol–methanol). The sample was applied by a diffusion-based injection technique. (Less)

Capillary electrochromatography of hydrophobic amines on continuous beds

The capillary electrochromatographic separation performance of hydrophobic amines and a related quaternary ammonium compound on continuous beds based on polymers of acrylamide has been studied. The chromatographic bed is polymerized in situ and the character of the polymers with regard to hydrophobicity and charge has been systematically changed by regulating its content of isopropyl and sulfonate ligands, respectively. The best performance was obtained for columns with a molar ratio of 1:80 for the sulfonate and isopropyl groups, and resulted in efficiencies up to 200 000 plates per meter. The effects on retention, resolution and elution order by ionic strength, pH, and content of acetonitrile in the mobile phase have been investigated. The quaternary ammonium compound was always the least retained irrespective of pH. By increasing the pH, a reversal of the migration order between the tertiary and secondary amine was obtained. The results indicate a complex migration/retention mechanism where ion‐exchange, adsorption and electrophoretic mobilities play a role. The concentration limit of detection could be lowered from 1.3 μg/mL to 50 pg/mL by using a high content of 2‐propanol (96%) in the sample compared to dissolving the analytes in the mobile phase.

Capillary and rotating-tube isoelectric focusing of a transmembrane protein, the human red cell glucose transporter.

The human red cell glucose transporter (Glut1) is a transmembrane protein. Monomeric Glut1 was purified by ion-exchange chromatography in the presence of the non-ionic detergent n-dodecyl octaoxyethylene (C12E8). For focusing, the ionic strength of the solution of C12E8-Glut1 complexes with co-purified lipids was lowered by dialysis, the detergent concentration was increased and carrier ampholytes were added. Focusing was done for 5 min at 3000 V in a methyl cellulose-coated glass capillary (50 microns I.D.). The anolyte H3PO4 was then replaced by NaOH for mobilization towards the anode. Absorbance monitoring at 280 nm showed two groups of zones at pH 6 and 8. Similarly, isoelectric focusing in a rotating quartz tube (3 mm I.D.) gave Glut1 zones at pH 5.5 and 8.0. Phosphorus analysis revealed that the Glut1 zone at pH 8 contained more phospholipids than did the other one. The above results together with previously determined and calculated isoelectric points (pI) of Glut1 indicate that the Glut1 at pH 8 is monomeric and that the zone at pH 5.5-6 represents oligomeric materials. The pI 8.0 at 22 degrees C applies for monomeric Glut1 in the absence of urea. The results exemplify that capillary isoelectric focusing of hydrophobic membrane proteins is possible.