# Separation of Albumin from Bovine Serum Applying Ionic-Liquid-Based Aqueous Biphasic Systems
## 4 Conclusions  
The aim of this work was the development of novel IL-based ABS as appropriate protein separation platforms and to provide a better understanding of the conditions required to apply these ABS without leading to the protein's loss of stability. The phase diagrams of all ABS investigated, composed of IL + K\({}_{2}\)HPO\({}_{4}\)/KH\({}_{2}\)PO\({}_{4}\) buffer pH 7 + H\({}_{2}\)O, were firstly determined at 25\({}^{\circ}\)C and atmospheric pressure. Then, the extraction efficiency and yield of pure BSA were evaluated for each system under study. It was found that the best ABS is constituted by [N\({}_{4444}\)][Ac], allowing for extraction efficiencies and recovery yields of 100% and 84.9%, respectively. The performance of this ABS was then evaluated using real bovine serum samples, allowing for the separation of the BSA, with 85.6% of extraction yield and 61.2% of purity when a bovine serum 1:15 (_v:v_) dilution was applied. Finally, the purity increase of BSA was proved by SDS-PAGE, and its stability by circular dichroism. Overall, it is here shown that ABS composed of properly designed quaternary ammonium-based ILs should be more deeply investigated in the field of proteins separation since they provide a biocompatible environment and present a lower cost when compared with the widely studied imidazolium-based counterparts. **Supplementary Materials:** The following material are available online at [[https://www.mdpi.com/article/10.3390/app12020707/s1](https://www.mdpi.com/article/10.3390/app12020707/s1)]([https://www.mdpi.com/article/10.3390/app12020707/s1](https://www.mdpi.com/article/10.3390/app12020707/s1)), Figure S1: 1-butyl-3-methylimidazolium 2-(N-morpholino) ethanesulfonate, [C\({}_{4}\)mim][MES], \({}^{1}\)H NMR (D\({}_{2}\)O, 300 MHz, [ppm]): 7.49 (1H,d, J = 2.0 Hz); 7.39 (1H,d, J = 2.0 Hz); 4.16 (2H,t, J = 7.1 Hz); 3.86 (3H, s); 3.73 (4H, m); 3.10 (2H, m); 2.80 (2H, m); 2.57 (4H, m); 1.82 (2H, m); 1.29 (2H, m); 0.89 (3H, t = 7.4 Hz), Figure S2: 1-butyl-3-methylimidazolium 2-(N-morpholino)ethanesulfonate, [C\({}_{4}\)mim][MES], \({}^{13}\)C NMR (D\({}_{2}\)O, 75.47 MHz, [ppm]): 123.33; 122.08; 66.09; 52.51; 52.19; 49.16; 47.25; 35.48; 31.17; 18.79; 12.64, Figure S3: Tetrabutylammonium 2-(N-morpholino)ethanesulfonate, [N\({}_{4444}\)][MES], \({}^{1}\)H NMR (D\({}_{2}\)O, 300 MHz, [ppm]): 3.77 (m, 4H); 3.178 (m, 10H); 2.843 (m, 2H); 2.61 (t, 4H); 1.66 (m, 8H); 1.38 (m, 8H); 0.96 (t, 12H). Figure S4: Tetrabutylammonium 2-(N-morpholino)ethanesulfonate, [N\({}_{4444}\)][MES], \({}^{13}\)C NMR (D\({}_{2}\)O, 75.47 MHz, [ppm]): 66.02; 58.02; 52.51; 52.19; 47.25; 23.07; 19.11; 12.77. Figure S5: Tetrabutylphosphonium 2-(N-morpholino)ethanesulfonate, [P\({}_{4444}\)][MES], \({}^{1}\)H NMR (D\({}_{2}\)O, 300 MHz, [ppm]): 3.77 (m, 4H); 3.12 (m, 2H); 2.83 (m, 2H); 2.61 (t, 4H); 2.18 (m, 8H); 1.54 (m, 16H); 0.93 (t, 12H). Figure S6: Tetrabutylphosphonium 2-(N-morpholino)ethanesulfonate, [P\({}_{4444}\)][MES], \({}^{13}\)C NMR (D\({}_{2}\)O, 75.47 MHz, [ppm]): 66.09; 52.51; 52.19; 47.24; 23.35; 22.62; 17.90; 12.49. Figure S7: Tetrabutylammonium aginate, [N\({}_{4444}\)][Arg], \({}^{1}\)H NMR (D\({}_{2}\)O, 300 MHz, [ppm]): 3.21 (m, 10H); 1.68 (m, 10H); 1.36 (m, 11H); 0.96 (t, 12H). Figure S8: Tetrabutylammonium aginate, [N\({}_{4444}\)][Arg], \({}^{13}\)C NMR (D\({}_{2}\)O, 75.47 MHz, [ppm]): 180.81; 58.07; 55.51; 40.91; 31.60.24.46; 32.05; 19.08; 12.77. Figure S9: Tetrabutylphosphonium aginate, [P\({}_{4444}\)][Arg], \({}^{1}\)H NMR (D\({}_{2}\)O, 300 MHz, [ppm]): 3.24 (m, 3H); 21.72 (t, 8H); 1.59 (m, 20H); 0.93 (t, 12H). Figure S10: Tetrabutylphosphonium aginate, [P\({}_{4444}\)][Arg], \({}^{13}\)C NMR (D\({}_{2}\)O, 75.47 MHz, [ppm]): 183.71; 55.51; 40.99; 31.63.24.50; 23.13; 17.87; 12.48. Figure S11: Tetrabutylammonium acetate, [N\({}_{4444}\)][Ac], \({}^{1}\)H NMR (D\({}_{2}\)O, 300 MHz, [ppm]): 3.21 (t, 8H); 1.91 (s, 3H); 1.67 (m, 8H); 1.38 (m, 8H); 0.95 (t, 12H).