Microcolumn Chromatofocusing Using Slurry-Packed Mono P Anion-Exchanger Beads as Chromatographic Matrix

Summary

Chromatofocusing of proteins in a microcolumn format (750 μm i.d.) was evaluated with slurry-packed Mono P anion-exchanger beads (10 μm bead size) as chromatographic matrix. Evaluation was performed with three standard proteins in terms of flow rate, Polybuffer 96–acetic acid, pH 6.0, concentration (eluent buffer strength), and detection limit. A column with the dimension 750 μm i.d. × 50 mm length, a flow rate of 20 μL min⁻¹, and a 1:10–1:20 Polybuffer 96-acetic acid aqueous dilution showed the best resolution between the proteins. Detection limits were 3 μg mL⁻¹ for carbonic anhydrase I and 6 μg mL⁻¹ for myoglobin A employing UV-detection at 280 nm.