Preparation of a Monolith with Covalently Bound Bovine Serum Albumin for Capillary Electrochromatography

Abstract

Capillary electrochromatography (CEC) is important for applications in enantiomer separation. The problems associated with column fabrication bring a challenge in developing monoliths with ease of preparation, robustness of separation, enhanced mass transfer, and lower pressure drop. In this research, the covalent binding of proteins on to a monolithic matrix was investigated to overcome the drawback of loss and/or denaturing of the biomolecules from physical adsorption and encapsulation method. A chitosan/silica hybrid monolith was prepared and a protein, bovine serum albumin, was covalently immobilized on the column. The prepared monolith was evaluated using the enantioseparation of D,L-tryptophan by CEC. It was found that separation of tryptophan enantiomers with a resolution of 2.44 was achieved by using 20 mmol L⁻¹ phosphate buffer at pH 7.5. A higher chitosan concentration was also proven to be of possible use in the synthesis with the aid of acetic acid as the solvent. The much shorter retention time and increased separation ability demonstrate the advantages of capillary column under investigation.

Keywords:

• Bovine serum albumin
• Capillary electrochromatography
• Chitosan
• Enantioseparation
• Monolith

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No.20876128) and the Fundamental Research Funds for the Central Universities (No.2010121049).

Notes

^a The amount of CTAB dissolving in monomer solution.

Conditions: column, 35 cm (ca. 5 cm packed) 100 µm ID monolithic capillary; mobile phase, 20 mmol L⁻¹ phosphate buffer, pH 7.5; applied voltage: 3 kV; Injection: 3 kV/3 s; detection, 254 nm.

^a The selectivity was defined as the ratio of retention times, t _L /t _D , the same as in Kato et al. 2004.