Immobilized Metal Affinity Sequential Injection Chromatography for the Separation of Proteins

Abstract

This paper describes for the first time the automation of immobilized metal affinity chromatography by sequential injection analysis. A generic poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith was synthesized by thermal free radical polymerization inside a 2.1 mm inner diameter activated fused silica-lined stainless steel tubing and then modified with iminodiacetate (IDA) onto which Cu(II) was immobilized. The retention mechanism controlled by metal ion affinity was verified for proteins such as ovalbumin and myoglobin. These proteins were unretained in the monolith modified only with IDA using a mobile phase composed of 20 mM phosphate buffer (pH 7.0) and 0.50 M NaCl. On the other hand, ovalbumin and myoglobin were retained in the monolith to which Cu(II) was immobilized and elution was achieved only following the addition of imidazole to the mobile phase. The sequential injection strategy created a fast gradient by mutual dispersion of three mobile phases containing imidazole at concentrations of 10, 20, and 50 mM, which enabled the efficient separation of cytochrome C, ribonuclease A, and myoglobin. The proposed methodology may be useful to isolate proteins from complex matrices as demonstrated by the separation of ovalbumin and lysozyme from unretained compounds in an egg-white sample.

Keywords:

• Complexation
• flow methods
• iminodiacetate
• liquid chromatography
• macromolecules
• monolithic columns

Acknowledgments

F. H. do Nascimento acknowledges Coordination for the Improvement of Higher Education Personnel (CAPES) for a post-doctoral fellowship (Contract 88882.315696/2019-01).

Additional information

Funding

This work was supported by the São Paulo Research Foundation (FAPESP) [grant number 2013/18507-4] and the National Council for Scientific and Technological Development (CNPq) [grant number 303940/2017-4].