# Separation of Albumin from Bovine Serum Applying Ionic-Liquid-Based Aqueous Biphasic Systems
## 2 Materials and Methods
### Extraction and Purification of BSA  
Initial studies were carried out with the pure protein (BSA). ABS were prepared with 35 wt% of each IL, 20 wt% of K\({}_{2}\)HPO\({}_{4}\)/KH\({}_{2}\)PO\({}_{4}\) buffer at pH 7, and 45 wt% of BSA aqueous solution at 2 g\(\cdot\)L\({}^{-1}\) in deionized water. This mixture point was selected according to the phase diagrams determination, fitting within the biphasic region of all studied ABSs. The obtained mixtures were stirred and centrifuged for 10 min at 10,000 rpm, and left to equilibrate for 15 min at 25 (\(\pm\)1) \({}^{\circ}\)C. The two phases were carefully separated and used in several analysis to evaluate the ABS performance on the BSA extraction and separation from the serum samples. For the purification studies of BSA from bovine serum, dilutions of 1:25, 1:20, 1:15, and 1:10 (\(v\!:v\)) of bovine serum in distilled water were prepared and added to the ABS preparations (45 wt%). For all systems, the IL-rich phase corresponds to the top phase, while the salt-rich phase corresponds to the bottom phase.  
BSA and the remaining proteins present in serum bovine were quantified in each ABS phase by size-exclusion high-performance liquid chromatography (SE-HPLC). The samples were diluted at a 1:10 (\(v\!:v\)) ratio with phosphate buffer (50 mmol\(\cdot\)L\({}^{-1}\) phosphate buffer pH 7.0 with NaCl 0.3 mol\(\cdot\)L\({}^{-1}\)) used as the mobile phase for quantification. The equipment used was a Chromaster HPLC system (VWR Hitachi) equipped with a binary pump, column oven (40 \({}^{\circ}\)C), temperature controlled auto-sampler (10 \({}^{\circ}\)C), DAD detector and an analytical column Shodex Protein KW- 802.5 (8 mm \(\times\) 300 mm). A 50 mmol\(\cdot\)L\({}^{-1}\) phosphate buffer pH 7.0 with NaCl 0.3 mol\(\cdot\)L\({}^{-1}\) was run isocartically with a flow rate of 0.5 mL\(\cdot\)min\({}^{-1}\) and the injection volume was of 25 mL. The wavelength was set at 280 nm with a total analysis time of 40 min and the retention time of BSA was found to be ca. 16.5 min. The calibration curve was established with BSA from 0.05 to 7.00 g\(\cdot\)L\({}^{-1}\). The ABS performance was evaluated in terms of extraction efficiency, yield, and purity for BSA, using three replicates.  
The extraction efficiency of the studied systems for BSA, _EE_%, was determined according to the following equation:  
\[EE\%=\frac{w_{\text{BSA}}^{IL}}{w_{\text{BSA}}^{salt}+w_{\text{BSA}}^{ IL}}\times 100 \tag{2}\]  
where \(w_{\text{BSA}}^{IL}\), \(w_{\text{BSA}}^{salt}\) represent the BSA total weight in the IL-rich and salt-rich phases, respectively.  
The extraction yield of BSA (_Yield_%) was determined according to the following equation:  
\[Yield\%=\frac{w_{\text{BSA}}^{IL}}{w_{\text{BSA}_{\text{initial/serum}}} \times 100 \tag{3}\]  
where \(w_{\text{BSA}_{\text{initial/serum}}}\) represents the weight of BSA added to the system and \(w_{\text{BSA}}^{IL}\) the weight of BSA in the IL-rich phase.  
The purity of BSA was calculated dividing the HPLC peak area of BSA (\(A_{\text{BSA}}\)) by the total area of the peaks corresponding to all proteins present in the phases of the different systems \(A_{totalprotein}\), according the following equation:  
\[Purity\%=\frac{A_{\text{BSA}}}{A_{totalprotein}}\times 100 \tag{4}\]