Study of the applicability of non-conventional aqueous two-phase systems in counter-current and centrifugal partition chromatography

• Protein partitioning in ionic liquid-based aqueous two-phase systems is evaluated.
• The influence of ionic liquid anion and cation on protein partitioning is studied.
• Proteins partition into the upper phases of the ionic liquid-based biphasic systems.
• Densities and viscosities of the ionic liquid-based biphasic systems are presented.
• Ionic liquids alter protein partitioning in PEG-based biphasic systems.

Aqueous two-phase systems composed of imidazolium-based ionic liquids and phosphate salts were evaluated for their applicability in liquid–liquid chromatography. The influence of the nature of ionic liquid anion and cation on the partitioning of bovine serum albumin, lysozyme and myoglobin was investigated. A mixture of K2HPO4 and KH2PO4 in a ratio of 1.82:1 wt/wt was used in all of the tested biphasic systems to adjust the pH to a range of 7–8. The results show that more hydrophobic cations decrease the partition coefficients of the proteins in the biphasic systems and outweigh the effect of the anion on the distribution of the macromolecules. Viscosities and densities of the biphasic systems were in a suitable range for liquid–liquid chromatography. Even though the partition coefficients were too high for a conventional batch operation mode, these aqueous two-phase systems show favorable properties for protein capturing in liquid–liquid chromatographic columns. Additionally, the possible application of ionic liquids as modifiers in polyethylene glycol (PEG)-based aqueous two-phase systems was investigated. It could be demonstrated that ionic liquids alter the partition coefficients of the proteins.