CHIRAL-RECOGNITION CHROMATOGRAPHY OF BETA-BLOCKERS ON CONTINUOUS POLYMER BEDS WITH IMMOBILIZED CELLULASE AS ENANTIOSELECTIVE PROTEIN

Columns prepared by coupling cellulase as a chiral selector to silica beads are very efficient for the separation of enantiomers.20 In this paper we show that continuous polymer beds compete favorably with sili ca beads as chromatographic supports for such separations. The chiral stationary phase is prepared either by entrapment in and simultaneous covalent linkage of allyl cellulase to the continuous beds during thei r preparation or by covalent immobilization of cellulase on an epoxy-a ctivated continuous bed. Enantiomers of beta-blockers were separated r apidly and with high resolution. The enantiomers of practolol were thu s baseline resolved within 45 sec. The recognition center-or at least part of it-coincides with the active center of the enzyme, since the e nantiomers could not be separated in the presence of the competitive e nzyme inhibitors cellobiose and D-glucose and the separation was also impaired upon addition of the substrate carboxymethyl cellulose to the eluent. Similar observations have been reported for silica columns de rivatized with cellulase.26 The capacity factor and the separation sel ectivity could be tuned by the pH and the concentration of the mobile phase, a phosphate buffer. No modifier was required, as is sometimes t he case with silica-based supports.13 The continuous beds give faster enantiomer separations than do columns of silica and are more pH-stabl e and cost effective to prepare. (C) 1993 Wiley-Liss, Inc