Homogeneous capture and heterogeneous separation of proteins by PEG-functionalized ionic liquid–water systems

Extraction and separation of proteins is important for their interesting applications as continuous enzymatic reaction and/or product separation systems. In this work, a new highly efficient homogeneous capture and heterogeneous liquid–liquid separation strategy is presented for the extraction of proteins from aqueous solution by PEG (poly(ethylene glycol))-functionalized ionic liquids, which have a lower critical solution temperature (LCST) phase behaviour in water. The factors influencing the solvent extraction process such as homogeneous capture time, pH value of aqueous phase and water content in the ionic liquids were investigated systematically. It was found that homogeneous capture of the proteins was quite fast, typically 1 min was enough to achieve equilibrium, and the extraction efficiency was greatly affected by water phase pH value and water content in the ionic liquid phases. Under optimal conditions, the singlestep extraction efficiency by [PEG800(mim)2][NTf2]2 was higher than 95% for most of the studied proteins (cytochrome c, myoglobin, hemoglobin, lysozyme and papain), but it was only 8% for bovine serum and 2% for peroxidase. Based on the significant differences in the extraction efficiencies observed, selective separation of the proteins from bovine serum or/and peroxidase was successfully performed using the IL/water mixture. In addition, circular dichroism and FT-IR spectroscopy were used to probe the structure and conformation change of the proteins, and the extraction mechanism was also discussed from the point of view of the electrostatic and hydrophobic/hydrophilic interactions of proteins with ionic liquids.