A series of hydroxyl ammonium ionic liquids (ILs) have been designed and synthesized. An ionic liquid aqueous two-phase system based on N,N-dimethylethanolamine propionate ([DMEAHPr]) ionic liquid was studied for the extraction of proteins. Based on the single-factor experiment, an initial serial investigative test was used to identify the optimal conditions. Five factors and four levels of orthogonal experiments were used to verify the optimum extraction conditions. The results show that under the optimum conditions, the extraction efficiency could reach up to 99.50%. The RSD of extraction efficiencies in precision experiment, repeatability experiment and stability experiment were 0.3% [n = 5), 1.1% (n = 5) and 1.5% (n = 5), respectively. The conformation of the proteins was not affected after extraction into the IL-rich phase in terms of the determination by UV-vis and FT-IR spectra. According to the determination of conductivity, dynamic light scattering, and transmission electron microscope images, the microstructure of the IL-rich phase and the possible mechanism for the extraction are investigated. Hydrogen bonding interaction, salt out effect and the aggregation phenomenon played important roles in the extraction. The circular dichroism spectral experiment analysis indicated that the secondary structures of the protein were unchanged after extract. Based on these findings, it is suggested that the method of hydroxyl ammonium-based ILs-ATPs have the potential to offer new possibility in the extraction of bio-analytes.
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