# Separation of Albumin from Bovine Serum Applying Ionic-Liquid-Based Aqueous Biphasic Systems
## 3 Results and Discussion
### Extraction and Purification of BSA
Up to date, several methods have been developed combining this method resorting to fractionation with ethyl alcohol and other techniques, such as ionic exchange and affinity chromatography. This fractionation step coupled to chromatography allows BSA with a purity of 99% to be obtained [59; 60; 61]. In addition to these methods, other combinations, such as precipitation with ammonium sulfate combined with liquid chromatography [62], and the use of ion exchange [63], affinity [64; 65], and simulated moving bed (SMB) [66]  
Figure 4: Extraction yield (_Yield_%) () and purity (_Purity_%) of bovine serum albumin in the ionic-liquid-rich phase extracted from bovine serum with the aqueous biphasic systems comprising [N\({}_{444}\)][Ac] as function of the bovine serum dilution. chromatography without previous precipitation/fractionation steps, have been reported, resulting in BSA with a purity higher than 90%. In addition to these well-developed techniques, steric exclusion chromatography with a cryogel column has been more recently reported, with purity values of 26.9% in the elution fraction and 103% in the breakthrough fraction [67]. Although our IL-based ABSs do not allow purification levels higher than 90% as the ones here described, it should be remarked that the purity of 61.2% of BSA was achieved in a single-step, i.e., directly from bovine serum without previous fractionation steps. On the other hand, no chromatographic steps are applied. If higher purification levels are required, IL-based ABSs need to be further optimized, in terms of composition, or applied under continuous mode to allow several stages of equilibrium [68]. These could also be combined with other strategies such as chromatography, ultrafiltration, or induced precipitation [69]. SE-HPLC was used to quantify BSA; however, this technique also permits obtaining information about the occurrence of protein aggregates or proteins fragmentation, thus allowing the confirmation that BSA stability is maintained throughout the extraction process in the IL-rich phase (an example of a SE-HPLC chromatogram is provided in Figure S16 in the Supporting Information). The protein profile of the IL-rich phase of the ABS comprising [N\({}_{4444}\)][Ac was evaluated by SDS-PAGE, being given in Figure 5A. Results show that bovine serum is mainly composed of IgG and BSA. After the extraction with ABS, a decrease in the IgG content is visible (as appraised by the intensity of the respective heavy and light chain bands), thus leading to an increase in the purity of BSA in the IL-rich phase. Finally, and to ensure the protein stability after the extraction step and in the IL-rich phase, CD spectroscopy was applied, whose spectra are depicted in Figure 5B. CD spectra allow the secondary structure of proteins to be assessed and their conformation in \(\alpha\)-helix, \(\beta\)-sheet, \(\beta\)-turn, and random coils to be analyzed [70]. Below 240 nm peptide bonds can be observed, while aromatic amino acid side chains and disulfide bonds can be analyzed in the range 260-320 nm and close to 260 nm, respectively. In the case of pure BSA, a protein profile with a rich \(\alpha\)-helix structure is presented, with two minimums occurring at approximately 209 nm and 222 nm and with a maximum between 190 and 195 nm [70; 71]. When comparing the spectra of pure BSA and of BSA after the extraction step to the IL-rich phase, the shape of the two spectra is highly similar, suggesting the maintenance of the secondary structure of BSA in the IL-rich phase after the extraction step. The composition of the BSA secondary structure is mostly \(\alpha\)-helix (62%), with a lower content in \(\beta\)-sheets (7%), turns (12%), and random coils (19%) [72; 73].