A new affinity-HPLC packing for protein separation: Cibacron blue attached uniform porous poly(HEMA-<Emphasis Type="Italic">co</Emphasis>-EDM) beads |
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Authors: | Ender?Unsal Aysun?Durdu Begum?Elmas Murvet?Tuncel Email author" target="_blank">Ali?TuncelEmail author |
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Institution: | (1) Chemical Engineering Department, Hacettepe University, 06532, Beytepe, Ankara, Turkey;(2) Chemistry Department, Hacettepe University, 06532, Beytepe, Ankara, Turkey;(3) Anatomy Department, Faculty of Medicine, Hacettepe University, 06532, Beytepe, Ankara, Turkey |
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Abstract: | In this study, a new affinity high-performance liquid chromatography (HPLC) stationary phase suitable for protein separation
was synthesized. In the first stage of the synthesis, uniform porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate), poly(HEMA-co-EDM), beads 6.2 μm in size were obtained. Homogeneous distribution of hydroxyl groups in the bead interior was confirmed
by confocal laser scanning microscopy. The plain poly(HEMA-co-EDM) particles gave very low non-specific protein adsorption with albumin. The selected dye ligand Cibacron blue F3G-A (CB
F3G-A) was covalently linked onto the beads via hydroxyl groups. In the batch experiments, albumin adsorption up to 60 mg
BSA/g particles was obtained with the CB F3G-A carrying poly(HEMA-co-EDM) beads. The affinity-HPLC of selected proteins (albumin and lysozyme) was investigated in a 25 mm×4.0-mm inner diameter
column packed with CB F3G-A carrying beads and both proteins were successfully resolved. By a single injection, 200 μg of
protein was loaded and quantitatively eluted from the column. The protein recovery increased with increasing flow rate and
salt concentration of the elution buffer and decreased with the increasing protein feed concentration. During the albumin
elution, theoretical plate numbers up to 30,000 plates/m were achieved by increasing the salt concentration. |
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Keywords: | Hydroxyethyl methacrylate Affinity chromatography High-performance liquid chromatography Cibacron blue Albumin Lysozyme |
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