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Aqueous Two-Phase System (ATPS): an Overview and Advances in Its

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Aqueous Two-Phase System (ATPS): an Overview and Advances in Its Iqbal et al. Biological Procedures Online (2016) 18:18 DOI 10.1186/s12575-016-0048-8 REVIEW Open Access Aqueous two-phase system (ATPS): an overview and advances in its applications Mujahid Iqbal1, Yanfei Tao1*, Shuyu Xie1, Yufei Zhu1, Dongmei Chen1, Xu Wang1, Lingli Huang1, Dapeng Peng1, Adeel Sattar1, Muhammad Abu Bakr Shabbir2, Hafiz Iftikhar Hussain2, Saeed Ahmed2 and Zonghui Yuan1,2* Abstract Aqueous two-phase system (ATPS) is a liquid-liquid fractionation technique and has gained an interest because of great potential for the extraction, separation, purification and enrichment of proteins, membranes, viruses, enzymes, nucleic acids and other biomolecules both in industry and academia. Although, the partition behavior involved in the method is complex and difficult to predict. Current research shows that it has also been successfully used in the detection of veterinary drug residues in food, separation of precious metals, sewage treatment and a variety of other purposes. The ATPS is able to give high recovery yield and is easily to scale up. It is also very economic and environment friendly method. The aim of this review is to overview the basics of ATPS, optimization and its applications. Keywords: Aqueous two-phase system (ATPS), Biomolecule separation, Solvent extraction, Veterinary drug residues History and background hydrophobic groups [5] Interested readers about In 1896, Martinus Willem Beijerinck accidently found aqueous two-phase affinity partitioning (ATPAP) are the ATPS while mixing an aqueous solution of starch referred to an excellent review by Ruiz-Ruiz et al. [6]. and gelatin. However, its real application was discovered Water as the main component of both phases in ATPS by Per-Åke Albertsson. Since then, the ATPS has been forms a gentle environment for biomolecules to separ- used for a range of purposes [1–3]. These systems can ate and polymers stabilize their structure and biological be formed by mixing a variety of components in water activities [3, 4, 7–9] while other liquid-liquid extraction [4]. But two-polymer and polymer-salt (e.g., phosphate, (LLE) methods could damage biological products sulfate or citrate) systems have grown rapidly and a because of the process conditions and organic solvents lot of work has been put into studying this technique [1, 7]. The purpose of this review article is to overview using these types of ATPSs [2]. This method has ad- the technique extensively and its applications in detail. vantages over conventional extraction techniques like, environment-friendly, low cost, capable of continuous Types of aqueous two-phase system (ATPS) operation, ease of scaling-up and is efficient for many The most common biphasic systems are formed by two kinds of experiments specially for the concentration polymers (usually polyethylene glycol (PEG) and dextran) and purification of biomolecules [1, 2, 4, 5]. The use or a polymer and a salt (e.g., phosphate, sulfate or citrate). of affinity ligands in ATPS can result in the higher Other types include, ionic liquids and short-chain alcohols recovery yields and higher purification folds of bio- [1, 2, 4, 6, 7, 10, 11]. In addition to this, ionic and/or non- logical products as it is a primary stage recovery tech- ionic surfactants are used for the formation of micellar nique [6]. Affinity ligands can be covalently attached and reverse micellar ATPSs [6, 12, 13]. Polymer – poly- to polymer or polymer can also be modified with mer/salt systems have been studied for more than five de- cades. Polymer – polymer systems are preferably used for the separation, recovery and purification solutes sensitive * Correspondence: [email protected]; [email protected] to the ionic environment as these systems pose low ionic 1National Reference Laboratory of Veterinary Drug Residues (HZAU)/MOA Key Laboratory of Food Safety Evaluation, Huazhong Agricultural University, strength. While, high ionic strength is the only disadvan- Wuhan, Hubei 430070, China tage of polymer – salts system [14]. Alcohol – salt ATPS Full list of author information is available at the end of the article are inexpensive as compared to polymers and copolymers. © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Iqbal et al. Biological Procedures Online (2016) 18:18 Page 2 of 18 These systems are also characterized by low viscosity, to separate between two different phases. In polymer – easy constituent recovery, and reduced settling times, salt ATPS, salt absorbs large amounts of water and a same but a major drawback of using this type of ATPS is exclusion phenomena can be observed [5, 22]. that many proteins are not compatible with alcohol- Phase separation in ATPS is affected by different fac- rich phase [15, 16]. The aqueous micellar two-phase tors like, concentration and molecular weight (MW) of system was first introduced by Bordier for the separ- polymer, concentration and composition of salt [21, 23]. ation of integral membrane proteins [17]. These sys- The presence of salt also influences phase behavior tems are also useful for ionic environment sensitive which also bring changes according to the type and con- solutes as nonionic surfactants can be used for the centration. Although, the mechanism through which salt formation of these systems. Mixed micellar systems influence ATPS in poorly understood [1, 7]. Generally, are becoming popular for showing selectivity features three forces: gravitational, flotation and frictional, act on [18]. Most recently, ionic liquids (ILs) based ATPSs a drop during phase separation and the balance between are being developed [19]. Poly-phase systems (three these forces determine its movement. The gravitational or four polymer phases) also have been constructed force depends on the density of drops while flotation for the separation of biomolecules [7]. One-polymer and frictional forces depend on the flow properties of ATPSs have also been reported, which utilize only phases [10, 24]. Surface properties of materials and com- one polymer for the formation of ATPS in water [20]. ponents of ATPS determine the partitioning between PEGs of different molecular weights are widely used two phases [7]. Poorly understood partition behavior is a polymers in ATPS due to their low toxicity, low price major barrier in widely adaptation of ATPS on commer- and low volatile nature [21]. Table 1 shows different cial levels for the purification of biomolecules [25]. types of ATPS with representative examples. Phase diagram (see Fig. 1) is like a fingerprint to a sys- tem under specific conditions (e.g., temperature and pH) Two-phase formation, thermodynamics and which is unique and shows the potential working area of partitioning ATPS. It provides a set of information like concentration Miscibility of solutions containing polymers is not a com- of components for two phase formation and their con- mon phenomenon, this property of polymers results in centration in the top and bottom phases [4, 26]. The dia- the formation of two phases [3, 4, 7]. Similar incompatibil- gram (see Fig. 1) shows a binodal curve (TCB), which ity can be observed upon mixing a polymer and a high divides the region of component concentrations. This ionic strength salt. Polymer – polymer system forms large curve splits the concentrations which form two immis- aggregates and because of steric exclusion, polymers start cible aqueous phases (above the binodal curve) from Table 1 Types of ATPS with representative examples Types of ATPS Representative examples Reference Composition of ATPS Product Results Polymer – polymer PEG – dextran Chitinase Successful partitioning of chitinase [161] towards bottom phase PEG – dextran Nanospheres, nanowires and Successful In situ binding Au [162] DNA derivatized nanowires nanospheres with Au nanowires Polymer – salt PEG – K2HPO4 B-phycoerythin Recovery yield = 90 % [163] Purification factor = 4 PEG 4000 – sulfate + 8.8 % NaCl α-Amylase Purification = 53 fold [164] Purity = 86 % Alcohol – salt 2-propanol – K2HPO4 Lipase Recovery yield = 99 % Purification [165] factor = 13.5 Ethanol – K2HPO4 2,3-butanediol Recovery yield= >98 % [16] Micellar/reverse n-Decyl tetra (ethylene oxide) Bacteriophages Bacteriophages partitioning [12] micellar ATPS towards micelle poor phase Isooctane/ethylhexanol/methyltrioctyl Plasmid DNA Successful purification of DNA [166] ammoniumchloride and RNA removal Ionic liquids (ILs) – 1-Butyl-3-methylimidazolium Codeine and papaverine Recovery yield= >90 % (codeine), [167] based ATPS chloride – salt >99 % (papaverine) Imidazolium – K2HPO4 Curcuminoids Extraction yield = 96 % [168] Purity= >51 % Iqbal et al. Biological Procedures Online (2016) 18:18 Page 3 of 18 Fig. 1 Schematic representation of phase diagram. Concentrations above binodal curve (TCB) forms aqueous two-phase system those that make one phase (below the binodal curve). ΔY STL ¼ ð3Þ The line (TB) in the diagram (see Fig. 1) is a tie line; it ΔX connects two nodes, which lie on the binodal curve. All the potential systems (e.g., S1, S2, S3) have same top Binodal can be determined by three methods; turbido- phase and bottom phase equilibrium composition be- metric titration method, cloud point method and node de- cause of being on the same tie line. Point C on binodal termination method [4, 26]. The researchers new to is called as a critical point, just above this point the vol- technique are referred to references [3, 4, 8, 27] for prede- ume of both phases is theoretically equal. At point C the termined phase diagrams and methods for construction. value tie line length (TLL) is equal to zero. The tie line The equilibrium relationship between the top and the length and component concentration has same units.
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