A protein extraction and separation has been reported using the ionic liquid (IL)-based aqueous two-phase system (ATPS). Although ILs have many benefits, there was a problem of unfavorable physical properties such as high viscosity, or high-cost production of ILs. Recently protic ionic liquids (PILs) are emerging as an alternative to conventional ionic liquids. PILs are easily produced through a reaction between Brønsted acid and base at a relatively low cost. In this study, PILs (pyrrolidinium formate [Pyrr][HCOO] and propionate [Pyrr][C2H5COO] and inorganic salts (K3PO4, K2HPO4) as phase-forming constituents of ATPS, were used for the protein extraction.
From the binodal experiments, the phase separation abilities are [Pyrr][HCOO] > [Pyrr][C2H5COO] and K3PO4 > K2HPO4. Because proteins precipitated at the interface when using K3PO4, ATPS composed of [Pyrr][HCOO] and K2HPO4 was selected for the protein extraction. The proteins were successfully extracted to PIL-rich phase. The distribution ratio of each protein was in the order hemoglobin < cytochrome C < α-chymotrypsin < albumin. The partition behavior of proteins in PIL-based ATPS was complex and influenced by combined effect of hydrophobic, electrostatic interactions and salting out. FT-IR spectroscopic characterization indicated that secondary structures of hemoglobin and cytochrome C were retained in PIL-rich phase after extraction.
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