# Separation of Albumin from Bovine Serum Applying Ionic-Liquid-Based Aqueous Biphasic Systems
## 3 Results and Discussion
### Extraction and Purification of BSA
On the other hand, ABS witholinium-based ILs combined with amino acids and polymers (polyethylene glycol di-methyl ether 250 and/or PPG, respectively) lead to lower extraction efficiencies, namely, 82.75%, 91.6%, and 85.91% under the best conditions [45; 47; 48], reinforcing the relevance of employing inorganic salts when high extraction efficiencies in IL-based ABS are aimed. Additionally, imidazolium-based systems combined with K\({}_{2}\)HPO\({}_{4}\) led to BSA extraction efficiencies of 82.7-100.7%, but with no extraction yields reported [49]. Regarding the different anions effect it is shown that ABS with the ILs based on the acetate anion lead to the maximum and minimum extraction yields, with the ABS comprising [N\({}_{4444}\)]Ac and [P\({}_{4444}\)][Ac] presenting the maximum values, and that constituted by [C\({}_{4}\)mim][Ac] to the minimum extraction yield. Although no differences are shown in the extraction efficiencies, these results clearly show that the cation effect has a higher impact in the BSA extraction yield, which may be related to the fact of having BSA as a negatively charged protein at the studied pH. Furthermore, these results seem to indicate that IL cations are the species that position themselves closer to the proteins, which is explained by the preferential hydrogen-bonded network established between the IL anions and water in the bulk phase [50]. These results, showing a higher effect of the IL cation,  
Figure 3: Extraction efficiencies (_EE_%) () and yield (_Yield_%) () of bovine serum albumin in the ionic-liquid-rich phase of the studied aqueous biphasic systems composed of 35 wt% of ionic liquid + 20 wt% K\({}_{2}\)HPO\({}_{4}\)/KH\({}_{2}\)PO\({}_{4}\) buffer at pH 7.  
are in agreement with the literature, where it has been shown that the concentration of IL cations is higher than that of anions at the protein surface, regardless of the protein charge [51; 52; 53]. Overall, these results reinforce the need of further investigating new ABS with quaternary ammonium- and phosphonium-based ILs, while also reducing their cost and toxicity when compared to imidazolium-based ILs [54; 55; 56]. Due to its higher extraction yield, thus corresponding to lower losses of BSA, the ABS composed of the IL [N\({}_{444}\)][Ac] was chosen to extract BSA from the real sample, i.e., bovine serum. Using the same mixture point (35 wt% of IL + 20 wt% K\({}_{2}\)HPO\({}_{4}\)/KH\({}_{2}\)PO\({}_{4}\) buffer at pH 7 + 45 wt% bovine serum), four dilutions (1:25,1:20,1:15,1:10 _v:v_) of bovine serum were tested, whose results are given in Figure 4. These experiments are relevant to appraise the protein saturation and possible proteins aggregation/precipitation phenomena. The higher the dilution of serum, the higher is the BSA extraction yield, ranging from 61.0 to 100.0%, thus reflecting the relevance of the IL-rich phase saturation. Detailed results are provided in the Supporting Information, Table S12. On the other hand, an opposite, yet slight, trend is observed when addressing the BSA purity. In this case, values between 51.9% and 61.2% are achieved, with the highest value found for the serum dilution corresponding to 1:15 (_v:v_). These results indicate that there is a competition for the proteins dissolution in the IL-rich phase and respective phase saturation, while considering that immunoglobulin G (IgGG) is the second highest abundant protein in bovine serum, which should partition to the opposite phase, or be precipitated to allow higher purity levels of BSA. In summary, the best results were obtained with the ABS composed of [N\({}_{444}\)][Ac], with a serum dilution of 1:15 (_v:v_), in which an extraction yield of BSA of 85.6% and purity of 61.2% were achieved in one step. The first large-scale technique for separating blood components was developed in the 1940-1950s by [PERSON] and co-workers, corresponding to Fractional Extraction [57; 58].