Chiral capillary liquid chromatography based on penicillin G acylase immobilized on monolithic epoxy silica column

An epoxy derivatized monolithic silica capillary column (100 μm i.d.) was used as a support for immobilization of penicillin G acylase (PGA), an enzyme used in the production of semisynthetic antibiotics. In order to allow for sensitive UV detection, the PGA-based monolithic capillary column was coupled to an open fused-silica capillary via a TFE (Teflon®) shrink tube sleeve (1 cm long, 300 μm i.d.), which proved to be a robust, dead-volume free and easily replaceable connector. This configuration resulted in a duplex fritless column for capillary liquid chromatography (CLC) and electrically assisted CLC (eCLC). In particular, using the driving pressure (2-12 bar) supplied by the commercial CE instruments, CLC separations could be obtained in short time due to the low column backpressure of the monolith. In particular, the developed stationary phase characterized by the chiral recognition ability of PGA, was successfully applied in enantioseparation of arylpropionic acids of pharmaceutical interest (i.e., profens). As an example, by using a 7 cm long monolith capillary column, the enantioresolution (Rs>3.0) of rac-ketoprofen was achieved in less than 2 min (pressure 12 bar) with a minimum plate height in the order of 20 μm and using as a mobile phase a 50 mM phosphate buffer pH 7.0. Validation data such as repeatability of retention time (intraday<0.62, n=6; interday<1.62, n=9; and column-to-column<10.5, n=2), linearity (r²=0.999), and sensitivity (LOQ 0.25% (w/w) of (R)-ketoprofen with respect to (S)-ketoprofen) showed good method performance. The method was successfully applied to the determination of (S)-ketoprofen in pharmaceutical samples (tablets).