Thermo- and pH-responsive polymer brushes-grafted gigaporous polystyrene microspheres as a high-speed protein chromatography matrix

• A high-speed thermo- and pH-responsive matrix was prepared in two steps.
• Proteins separation was achieved by combining a step pH and temperature gradient.
• A clear separation mechanism was proposed.
• It was able to separate proteins at the mobile phase velocity up to 2528 cm h−1.

Dual thermo- and pH-responsive chromatography has been proposed using poly(N-isopropylacrylamide-co-butyl methacrylate-co-N,N-dimethylaminopropyl acrylamide) (P(NIPAM-co-BMA-co-DMAPAAM)) brushes grafted gigaporous polystyrene microspheres (GPM) as matrix. Atom transfer radical polymerization (ATRP) initiator was first coupled onto GPM through Friedel–Crafts acylation with 2-bromoisobutyryl bromide. The dual-responsive polymer brushes were then grafted onto GPM via surface-initiated ATRP. The surface composition, gigaporous structure, protein adsorption and dual-responsive chromatographic properties of the matrix (GPM-P(NIPAM-co-BMA-co-DMAPAAM) were characterized in detail. Results showed that GPM were successfully grafted with thermoresponsive cationic polymer brushes and that the gigaporous structure was well maintained. A column packed with GPM-P(NIPAM-co-BMA-co-DMAPAAM presented low backpressure, good permeability and appreciable thermo-responsibility. By changing pH of the mobile phase and temperature of the column in turn, the column can separate three model proteins at the mobile phase velocity up to 2528 cm h−1. A separation mechanism of this matrix was also proposed. All results indicate that the dual thermo- and pH-responsive chromatography matrix has great potentials in ‘green’ high-speed protein chromatography.