On the use of ionic liquids as adjuvants in PEG-(NH4)2SO4 aqueous biphasic systems: Phase diagrams behavior and the effect of IL concentration on myoglobin partition

The introduction of ionic liquids (ILs), either as phase forming components or as adjuvants, in the implementation of aqueous biphasic systems (ABS) enabled to finely tune the properties of the two aqueous phases in equilibrium thus providing the opportunity to increase extraction efficiencies and selectivities of biomolecules. Nevertheless, the effect of ILs addition on the phase diagram behavior and partitioning of biomolecules changes depending on the polymer, salt, ILs and biomolecules used and is still not fully understood. In this context, the influence of the addition of 7 different ILs based on imidazolium, pyrrolidinium or pyrydinium cation on PEG 3350 + ammonium sulfate ((NH4)2SO4) ABS at 25 °C is studied in this work. In terms of phase equilibrium, it was observed that the chemical nature of the IL leads to either a decrease or increase of two-phase region, therefore in the latter affording a decrease in concentrations of phase forming compounds needed for the liquid-liquid demixing. It is shown, that the IL anion affinity for water or the anion's hydrogen-bond basicity controls the phase splitting behavior and thus [C4mim][N(CN)2] is able to greatly enhance two-phase region. Moreover, these PEG 3350 + (NH4)2SO4 + IL ABS were tested in the extraction/concentration of myoglobin from aqueous solutions. The obtained results indicate that the myoglobin partition is strongly influenced by its hydrophilicity and pH of the phases in equilibrium. Consequently, it preferentially partitions to the salt-rich phase. However, partitioning studies in presence of ILs as adjuvants show that these compounds can tune the polarity of the PEG-rich phase thus offering a tunable platform for the purification of myoglobin. This ability greatly depends on the chemical structure of the IL used and closely follows the IL hydrogen-bond basicity (β), enabling the most hydrophilic IL - [C4mim][CH3CO2] - to change the myoglobin partitioning behavior from salt-rich to the PEG-rich phase.