Chiral separation of β-blockers by high-performance capillary electrophoresis based on non-immobilized cellulase as enantioselective protein

Abstract

Optical isomers of some basic parmaceutical drugs (β-blockers) were separated by means of high-performance capillary electrophoresis in a carrier-free solution, using the chiral recognition properties of a cellulase (cellobiohydrolase I). High resolution of the isomers and peaks with satisfactory symmetry were obtained only when the enzyme was dissolved at a high concentration (40 mg/ml; total 10 μg) in a buffer of high ionic strength (0.4 M sodium phosphate) supplemented with 2-propanol. Surprisingly, the isomer selectivity was lost when the electrophoresis was carried out in the buffers used for chromatographic separation of the isomers on a bed derivatized with cellulase. At pH 5.1 (the experimental pH), the enantiomers are positively charged and the enzyme is negatively charged. With the cathode at the detection end of the capillary the enzyme accordingly migrated away from the detection point and the enantiomers toward it. Disturbances in the UV detection of the enantiomers otherwise caused by the presence of the enzyme were thus avoided. As the runs are performed in the absence of a supporting medium the analyses can be automated easily, which also facilitates the screening of different proteins for their chiral recognition properties and studies to establish the optimum experimental conditions.