# Separation of Albumin from Bovine Serum Applying Ionic-Liquid-Based Aqueous Biphasic Systems
## 1 Introduction  
Proteins play a fundamental role in biological processes and have high value in different industrial applications, e.g., in the food, biopharmaceutical or cosmetic industries [1]. Conventional techniques for proteins separation and purification include chromatography, electrophoresis, precipitation, and filtration-related techniques, among others [2]. However, there is a need of finding more cost-effective techniques to efficiently separate and purify proteins, while being capable of maintaining their native structure and function/activity [3]. Aqueous biphasic systems (ABS) have been proposed as an alternative technique to separate and purify proteins, mainly due to their versatility and biocompatible character if properly designed [4].  
ABS are formed by the dissolution of at least two water-soluble solutes (typically two polymers, a polymer and a salt, or two salts) in aqueous media [5], above which, in certain concentrations, there is phase separation. Conventional polymer-based ABS have been applied in the extraction and purification of several biological compounds and materials, such as cells [6], nucleic acids [7], amino acids [8], proteins, antibodies [9], and enzymes [10; 11; 12]. However, the coexisting phases in typical polymer-based ABS present a limited polarity difference, preventing high extraction efficiencies and selectivities to be achieved in a single-step [13]. Ionic-liquid-based (IL-based) ABS were later proposedas an alternative to polymer-based ABS, with numerous advantages [14]. These ABS are less viscous, show faster phase separation rates, and generally lead to higher extraction efficiencies. ILs are salts with lower melting temperatures than conventional salts, being constituted by an organic cation and an organic or inorganic anion [15]. Most ILs present a negligible volatility at atmospheric conditions, high thermal and chemical stabilities, a strong solvation capability for a large variety of compounds, and present the ability to conjugate ions according to the intended application, being usually described as \"designer solvents\" [16].  
To improve the biocompatible character of ILs to be applied in ABS, the IL anion can be selected according to the desired application. IL anions derived from biological buffers (Good-Buffer's (GB)-ILs) [17; 18; 19], amino acids [20], and carboxylic acids [21] have been applied in the extraction and separation of amino acids and proteins. However, most ILs reported in the literature were combined with imidazolium cations [22; 23], which may compromise their biocompatibility. On the other hand, the use of ammonium- and phosphonium-based ILs have been reported for the extraction of some proteins, such as ovalbumin and lysozyme [24]. Aiming at identifying the best cation-anion combinations of ILs to form ABS, in this work, ILs combining the imidazolium-, phosphonium-, and tetraalkylammonium cations, and the acetate, originate, and Good-Buffer-based anions, were synthesized, characterized, and used in the creation of IL-based ABS. These systems were then studied as separation platforms of bovine serum albumin (BSA). BSA was studied as the target protein due to its relevance in pharmaceutical studies [25] and due to their presence in bovine serum as a biological and complex medium from which it needs to be isolated.