Penicillin G acylase as chiral selector in CE using a pullulan-coated capillary

Area of Science:

• Analytical Chemistry
• Separation Science
• Biochemistry

Background:

• Capillary electrophoresis (CE) is a powerful separation technique.
• Chiral selectors are crucial for enantiomeric separation.
• Protein adsorption to capillary walls can hinder CE performance.

Purpose of the Study:

• To investigate penicillin G acylase (PGA) as a chiral selector in CE.
• To develop a stable capillary coating to mitigate protein-adsorption and electroosmotic flow (EOF).
• To optimize the partial filling technique for enantioseparation of acidic compounds.

Main Methods:

• Chemical coating of silica capillaries with pullulan to create an anti-adhesive and hydrophilic surface.
• Silanization with glycidoxypropyltrimethoxysilane followed by pullulan coupling.
• Optimization of partial filling parameters including buffer pH, PGA concentration, and loading duration.
• Enantioseparation of rac-ketoprofen and other acidic compounds.

Main Results:

• A stable, hydrophilic capillary coating significantly suppressed EOF across a wide pH range (3.0-9.0).
• Optimized partial filling conditions (100 mM sodium phosphate buffer pH 5.5, 240 µM PGA, 120 s filling at 50 mbar) enabled enantioseparation.
• Successful enantioseparation of several acidic compounds was achieved within 10 minutes.
• The capillary coating demonstrated long-term stability, with over 100 injections without significant reproducibility loss.

Conclusions:

• Permanently coated capillaries with pullulan offer a robust platform for using enzymes like PGA as chiral selectors in CE.
• The developed method provides efficient EOF suppression and effective enantioseparation of acidic compounds.
• This approach enhances the applicability of CE for chiral analysis.