# Separation of Albumin from Bovine Serum Applying Ionic-Liquid-Based Aqueous Biphasic Systems
## 3 Results and Discussion
### Extraction and Purification of BSA  
After the characterization of the investigated ABS, they were applied to extract BSA and then to purify this protein from bovine serum. To this end, a common ABS mixture point was used: 35 wt% of IL + 20 wt% K\({}_{2}\)HPO\({}_{4}\)/KH\({}_{2}\)PO\({}_{4}\) buffer at pH 7 + 45 wt% aqueous solution of BSA/bovine serum. The extraction efficiency (\(EE\%\)), yield and purity of BSA were obtained by the quantification of BSA and remaining proteins by SE-HPLC. Overall, six out of the eight ILs investigated allowed the partition of the protein in a two-phase system, namely, [C\({}_{4}\)mim][MES], [N\({}_{4444}\)][MES], [P\({}_{4444}\)][MES], [C\({}_{4}\)mim][Ac], [N\({}_{4444}\)][Ac], and [P\({}_{4444}\)][Ac]. The arginate-based-ILs, namely, [N\({}_{4444}\)][Arg] and [P\({}_{4444}\)][Arg], lead to the complete precipitation of the protein in the bottom of the system and did not allow the formation of a liquid-liquid system when in presence of the protein solution. This result shows that relevant interactions between the IL arginate anion and the serum constituents occur, not allowing the creation of ABS. In the studied systems that form an ABS in presence of BSA, the protein preferentially partitions to the top phase (IL-rich phase), in agreement with data reported for ABS composed of other ILs and K\({}_{2}\)HPO\({}_{4}\)/KH\({}_{2}\)PO\({}_{4}\) buffer for other enzymes and proteins, as well as for BSA [34; 35; 36; 37]. This behavior is the result of the salting-out effect provided by the phosphate buffer, thus improving the migration of the proteins to the opposite phase, combined with favorable interactions that may be established between the IL and BSA. These could correspond to hydrogen-bonding, or electrostatic and hydrophobic interactions [38; 39; 40; 36]. In the current work, BSA is negatively charged (isoeletric point (pl) = 4.5-4.9) [41; 42] at the pH at which ABSs were formed, and electrostatic interactions may be established. The extraction efficiencies and yield of the assays carried out with pure BSA are given in Figure 3, with the detailed data provided in the Supporting Information (Table S11). Figure 3 allows comparing the performance of the IL-based ABS under study in terms of extraction efficiency and extractionyield of BSA, allowing the identification of the best system to be used in the next step of the work, i.e., in the separation of BSA from bovine serum albumin. In all ABS, the extraction efficiency of BSA to the IL-rich phase is 100%, whereas the extraction yield values range from 43.7% to 84.9%. The systemconstituted by [C\({}_{4}\)mim][Ac] presents the lowest yield (43.7%), whereas those composed of [N\({}_{4444}\)][Ac] and [P\({}_{4444}\)][Ac] lead to the highest yields, 84.9% and 82.9%, respectively. This set of results show that there is a significant loss of protein stability with systems comprising imidazolium-based ILs; although, these are still the most investigated class of ILs in the field of separations. The denaturation of BSA by imidazolium-based ILs has been previously reported, with loss of secondary and tertiary structure, and results from hydrophobic and electrostatic established between BSA and the IL [43]. On the other hand, quaternary ammonium-based ILs have been reported as protein stabilizers [44], supporting the results here observed and reinforcing their study in protein separation studies. ABS comprisingolinium-based ILs have been also reported for the extraction for BSA in an attempt to use more biocompatible ILs and maintain the protein structure [45]. Extraction efficiencies up to 100% for pure BSA with IL-based ABS have been described with systems constituted byolinium-based ILs + polymer (PPG 400) and phosphonium- and ammonium-based ILs + potassium citrate/citric acid at pH 7, being the reported results [18; 46] similar to those obtained in this work.