Aqueous chromatographic system for separation of biomolecules using thermoresponsive polymer modified stationary phase.

We have investigated a new method for HPLC using packing materials modified with a functional polymer, such as thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm). PNIPAAm-modified silica exhibits temperature-controlled hydrophilic-hydrophobic surface property changes in aqueous systems. Temperature-responsive chromatography is performed with an aqueous mobile phase without using an organic solvent. We designed ternary copolymers of NIPAAm introduced 2-(dimethyl-amino) ethyl methacrylate (DMAEMA) as a cationic monomer and butyl methacrylate (BMA) as a hydrophobic monomer. A cationic thermoresponsive hydrogel grafted surface would produce an alterable stationary phase with both thermally regulated hydrophobicity and charge density for separation of bioactive compounds. In this study, we achieved successful separation of lysozyme without the loss of bioactivity by temperature-responsive chromatography. The electrostatic and hydrophobic interactions could be modulated simultaneously with the temperature in an aqueous mobile phase, thus the separation system would have potential applications in the separation of biomolecules.