The ionic liquid/aqueous two-phase extraction systems (ATPSs) based on imidazolium ionic liquids were used to extract cytochrome c. Effects of the alkyl chain length of the ionic liquid cations, concentration of potassium citrate, temperature and pH on the extraction efficiency have been investigated. The thermodynamic parameters (ΔG(T)°, ΔH(T)° and ΔS(T)°) associated with Cyt-c partitioning in aqueous two phase systems were determined. Thermodynamic studies indicated that the partitioning of Cyt-c was driven by both hydrophobic and electrostatic interactions in the extraction process. Under the optimum conditions, experiment results showed that 94% of the cytochrome c could be extracted into the ionic liquid-rich phase in a one-step extraction. The structural characterization of Cyt-c in the IL ATPS was investigated by UV-vis and circular dichroism (CD) spectra. The results demonstrated that no direct bonding interaction observed between ionic liquid and cytochrome c, while the native properties of the cytochrome c were not altered. Compared with traditional liquid-liquid extractions based on toxic organic solvents, ionic liquid/aqueous two phase extraction offers clear advantages due to no use of volatile organic solvent and low consumption of imidazolium ionic liquids.
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