Skip to main content


Current applications of different type of aqueous two-phase systems


In recent year, aqueous two-phase system (ATPS) has become a proven tool used in separation and purification technology. The application of ATPSs in clarification, partitioning and partial purification of biomolecules and bioproducts had showed the rapid development. This method is able to give high recovery yield and high purity in a single step. The ATPS shows characteristics of high selectivity and is easily to scale up. Therefore, ATPS offers an attractive alternative that meets the requirements of the high demand in industry processes and it is also beneficial in terms of economic and environmental protection. In the past, a lot of works and researches have been done in order to develop feasible separation processes using different types of ATPSs and their applications in numerous product separations. This paper aims to review on the recent literature works in the development of different type of ATPSs and their applications in novel separations and purifications of biomaterials.

The development of aqueous two-phase flotation from aqueous two-phase system


Aqueous two-phase system (ATPS) is a liquid–liquid fractionation method. The method is based on incompatibility of two aqueous solutions such as a polymer/salt system [e.g. polyethylene glycol (PEG) and potassium phosphate], a polymer/polymer system (e.g. PEG/dextran) (Show et al. 2013; Rosa et al. 2010), an ionic liquid (IL) and a salt system, and a low molecular weight alcohol and a salt system (Albertsson 1987; Ruiz–Ruiz et al. 2012). Furthermore, micellar and reverse micelle ATPS can also be formed with ionic and/or non-ionic surfactants solution (Ruiz–Ruiz et al. 2012). ATPS can be modified with affinity ligands, which is able to increase the recovery yields and purification folds significantly (Ruiz–Ruiz et al. 2012).

An ATPS, which can provide the non-toxic and low operational cost isolation process besides fast and large-scale purification, has become an ideal technology for separation process (Tang et al. 2014). The ATPSs can be used as a one-step process that allows the removal of contaminant components and at the same time, separating out the desired target products. The separation is based on the selective partitioning of the target products between the two phases (Rosa et al. 2010). Basically, the separation is dependent on the parameters which are related to the system properties. The ATPSs have been successfully applied in the downstream recovering of biopharmaceutical products by manipulating the complexity of the physical and chemical interactions involved in the partitioning process (Rosa et al. 2010).

The design of experiment is a very useful method in finding the optimal purification conditions. This method also can be used to analyse the influence of different parameters involved in the purification process and to evaluate the interactions between the parameters (Rosa et al. 2010). Furthermore, an advanced screening technique that allows a fast evaluation of parameters dependencies and robustness can be employed to establish the optimal conditions to recover the target molecules effectively (Rosa et al. 2010). Benavides, Rito-Palomares (2008) and Rito-Palomares (2004) have suggested a useful strategy for the development of aqueous two-phase extraction (ATPE). Generally, this strategy is divided into four main stages. The first stage is initial physicochemical characterisation of the feedstock, followed by second and third stages where the type of ATPS and system parameters are selected, respectively. The final stage is to evaluate the effect of process parameters on the product recovery and purity (Rosa et al. 2010). The applications of ATPS containing different phase combinations have been reported by Chen et al. (Chen et al. 2014), including the separation and purification of various biological products (Chen et al. 1999), metal ions (Rogers and Bauer 1996), antibodies (Azevedo et al. 2009), antibiotics (Jiang et al. 2009), small organic species (Willauer et al. 2002), nano- and micro-solid particulates (Helfrich et al. 2005) and also as green media (Chen et al. 2005). Different type of APTSs that have been applied into novel partitioning and recovery of various products in different fields will be further reviewed and discussed in this paper.

Applications of ATPSs

Application of polymer/polymer ATPS

A new method of in situ product recovery called extractive bioconversion has been introduced. This technique combines the product formation and separation into single-step process and concurrently, removes the products from the biocatalysts (Zijlstra et al. 1998; Ng et al. 2013). These biocatalysts can be reused for the product recovery from the phase separation of ATPS (Zijlstra et al. 1998; Ng et al. 2013). By incorporating ATPS into extractive bioconversion, the yield of recovered product in enzymatic synthesis can be improved. The product degradation and inhibition which normally happen during the conventional product recovery can be eliminated by rapid removal of the product using extractive bioconversion in ATPS (Ng et al. 2013; Daugulis 1988; Freeman et al. 1993). This method has been demonstrated by Ng et al. (2013) in investigating the synthesis and recovery of cyclodextrins (CDs) from Bacillus cereus cyclodextrin glycosyltransferase (CGTase, EC (Lin YK et al. 2015). An extractive bioconversion in ATPS consisted of 7.7 % (w/w) PEG 20,000 and 10.3 % (w/w) dextran T500 with volume ratio (VR) of 4.0 were selected as the optimum recovery system of CDs. The enzymatic conversion of starch is occured in dextran-rich bottom phase of system. The CDs produced from the systhesis were transferred to the PEG-rich top phase. Higher partition coefficient of CDs was achieved by removing the top phase product and adding starch every 8 h to repeat the CDs systhesis process. The experiments showed an average total CDs concentration of 13.7 mg/mL (composed of 4.77 mg/mL of α-CD, 5.02 mg/mL of β-CD and 3.91 mg/mL of γ-CD) was effectively recovered in the top phase (Ng et al. 2013).

In year 2014, a very impressive new finding in an attempt to remove the metallic single-walled carbon nanotubes (SWCNTs) (Tang et al. 2014) from semiconducting SWCNTs by using a polymer/polymer ATPS has been reported Tang et al. (2014). The rolled up graphene sheets of a nanotube structure is strongly affected by its electrical properties in electronic applications. The electrical conductivity of SWCNTs can be metallic or semiconducting. To separate the metallic SWCNTs from semiconducting SWCNTs, the PEG is dissolved in N-methylpyrrolidone (NMP) while dextran is dissolved in water with 1 % cetyltrimethylammonium bromide (CTAB). NMP and cationic surfactant CTAB are added to the system to disperse SWCNTs, meanwhile dextran will wrap around a SWCNT. CTAB is an amine-bearing surfactant. The selective adsorption of amine onto semiconducting SWCNTs is applied based on the affinity of CTAB on semiconducting SWCNTs and reverse micellar effect. The study reveals that dextran-rich bottom phase contains more semiconducting species while metallic SWCNT is more abundant in the PEG-rich top phase. The finding was proven by infrared spectroscopy analysis where CTAB only present in the dextran-rich bottom phase and metallic-SWNCT can be recovered from dextran-rich bottom phase.

Application of polymer/salt ATPS

The improvement of upstream technologies in manufacturing of biopharmaceutical products has raised the demand and interest to develop a cost-effective and efficient downstream separation technology. Large-scale ATPE is becoming an essential tool for purification of biopharmaceutical products such as interleukin, human growth hormone (hGH), insulin-like growth factor and monoclonal antibodies (Rosa et al. 2010).

The polymer/salt ATPS have been used to purify the interleukin-18 binding protein (IL-18BP) from serum-free Chinese hamster ovary (CHO) cells supernatant. IL-18BP is an important regulator of immune system. For instance, the experiment conducted by Kornmann, Baer (2008) shows that the PEG/sulphate ATPS is an ideal system where the IL-18BP was successfully partitioned to the polymer-rich phase at 2 mL scale in the first cycle. The experiment continued with 10 mL scale at the second cycle to find the optimum pH and concentrations. These were important to obtain the highest recovery and purity of IL-18BP from serum-free CHO cells supernatant (Rosa et al. 2010). The results showed that the best performing ATPS was composed of 11.25 % PEG 10,000 (w/w)/11.25 % Na2SO4 (w/w) at pH 5. The IL-18BP was recovered up to 98 % in the PEG-rich phase with a final purity of 92 % (Rosa et al. 2010). At the same time, Kornmann, Baer (2008) also assessed the purification of IL-18BP directly from crude unclarified harvest. The process performance was found to be insignificantly influenced by the presence of cells. The optimised ATPS had been scaled up to 100-fold and showed no significant difference in process performance. The final purity of 86 % of IL-18BP had recovered (Kornmann and Baer 2008).

An ATPS which consisted of PEG 4600/ammonium sulphate has been developed to recover and purify the recombinant growth hormone antagonists (GHA) from Escherichia coli cells homogenate (Rosa et al. 2010). GHA is an antagonist for hGH. Initially, the extraction was performed at 30 g-scale with 8 % (w/w) PEG 4600 and 10 % (w/w) ammonium sulphate. Then, the extraction was followed by adding 40 % (w/w) of PEG solution to bottom phase to extract the remaining GHA. The results showed that 89 % of GHA in the PEG-rich phase was recovered. By applying Alfa Laval LAPX 202 continuous disc stack centrifuge, the process performance was able to scale up to about 1000-folds. All the GHA has been recovered in PEG-rich phase after two stages of extraction process in this modified centrifuge which function as a purifier for continuous processing (Rosa et al. 2010).

In order to supply a safe and biologically active form of therapeutic protein, a selective removal of process-related impurities are required. The development of the cost-effective and non-chromatographic separation technique such as ATPS has been seen as a potential alternative (Rosa et al. 2010; Azevedo et al. 2009; Asenjo and Andrews 2011; Bhambure et al. 2013). A novel aqueous two-phase-assisted platform for the efficient removal of process related to impurities associated with E. coli, namely host cell proteins (HCP) and nucleic acids (DNA) have been conducted by Bhambure et al. (2013). The purified granulocyte colony stimulating factor (GCSF) has chosen as model biotherapeutic protein product in this study. The study observed that HCP which was produced by E. coli DH5 alpha null cells are preferentially partitioned (Lan et al. 2013) in the salt-rich bottom phase in 8 % PEG 6000– 2 % sodium sulphate (system I) and 10 % PEG 6000–11 % potassium phosphate (system II). The same study also observed that nucleic acids (DNA) are preferentially partitioned to the salt-rich bottom phase too (Bhambure et al. 2013; Duarte et al. 2007; Luechau et al. 2009a; 2009b). Meanwhile, the purified GCSF protein was found to partition in the PEG-rich top phase. Product recovery for system I was 25.1 ± 2.3 %, whereas and for system II was 30.6 ± 0.75 %. The results from analysis showed that 99.5 % of product has been recovered with the concentrations of HCP and DNA that less than 100 ppm and 10 mg/mL were obtained, respectively (Bhambure et al. 2013).

A study of the recovery and partial purification of fibrinolytic activities from oven-dried and freeze-dried basidiocarps of Auricularia polytricha has been conducted by Mohamed Ali et al. (2014). Fibrinolytic activity is defined as the ability of proteolytic enzymes such as nattokinase, streptokinase or urokinase to dissolve the fibrin in blood clots. These proteolytic enzymes are often used as pharmaceuticals to treat cardiovascular disease and stroke. Both fibrin plate assay and Folin-spectrophotometric method were used to assess the effect of processing methods on fibrinolytic activity. The fibrinolytic activities of freeze-dried A. polytricha basidiocarps’ extract exhibited a higher fibrinolytic activity of 3.88 U/mL compared to the oven-dried one (2.19 U/mL). The PEG 8000 and phosphate-based ATPS was employed to further concentrate and recover fibrinolytic crude enzyme. All the system parameters including the molecular mass and concentration of PEG, concentration of salt and crude and pH was evaluated in order to maximise the recovery of the fibrinolytic activity of A. polytricha. With a PEG 8000 20.0 % (w/w):phosphate 11.6 % (w/w) VR of 1.5, at pH of 7.0 together with 25 % (w/w) of crude extract from freeze-dried A. polytricha, sporocarps have shown a purification fold of 7.01-fold. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) has been conducted to characterise the fibrinolytic enzymes. In fibrin plate assay, the crude and concentrated fibrinolytic enzyme showed positive result for fibrinolytic activities. The enzyme has a molecular weight of 66 kDa based on SDS-PAGE result (Mohamed Ali et al. 2014).

The use of poly (ethyleneglycol)/poly (acrylate)/salt system has been successfully explored in the purification of protein and plasmid DNA. This application has become the most recent advance and alternative method in isolation of small DNA fragments that generated from polymerase chain reaction (PCR) (Matos et al. 2014). The main problem of PCR is to obtain the high purity of PCR product as contaminants such as enzymes or larger DNA molecules will be present in final product. The use of commercial kits in purification of PCR products are often result in low recovery which will cause the problem on subsequent steps in molecular biology work (Chen and Thomas 1980; Bellot et al. 2011). The research showed that ATPS method can be selected to isolate the DNA molecules larger than 5000–7000 bp. The PCR products showed the strong partition at between the phases in a two-step extraction process. The DNA is partitioned in PEG-rich phase and the impurities will be partitioned to poly (acrylate)-rich phase at primary extraction. The PEG-rich phase from primary extraction will be used to form the PEG/salt two-phase system in secondary (back) extraction. Finally, the purified target DNA will be strongly partitioned to salt-rich phase and the phase is almost free polymer. The system proven the fast and effective tool in purified the small DNA molecules that less than 4000 bp and able to obtain the high yield (Matos et al. 2014).

Application of thermo-separating polymer ATPS

The application of thermo-separating polymers in forming ATPS has deeply been discussed (Ng et al. 2012). Thermo-separating copolymers were firstly discovered in early 1990s which consisted of ethylene oxide (EO) and propylene oxide (PO) that are able to form ATPS with dextran (Berggren et al. 1995; Johansson et al. 1999). By heating up an aqueous solution above the cloud point (lower critical solution temperature, LCST), the linear and non-ionic EOPO random copolymers will be thermo-separated into two phases from aqueous solution (Ariff et al. 2011; Walter 1986; Jönsson and Johansson 2003; Johansson et al. 1997; Pereira et al. 2003). In this system, the desired target protein is separated to the EOPO-rich top phase at the primary ATPS. Once the temperature increases above the LCST, the EOPO-rich top phase will be removed from the primary system to form the secondary ATPS (Show et al. 2012). A target protein will be partitioned to the top phase, leading the formation of concentrated EO/PO solution at the bottom phase after the secondary ATPS is formed (Show et al. 2011). Since the polymers and salt component can be recycled and reused in subsequent or new primary ATPS extraction (Johansson et al. 1999; Jönsson and Johansson 2003; Johansson et al. 1997), the cost of purification process can be significantly reduced, and at the same time, the environmental pollution can be minimised by reducing the use of harmful organic solvent and salts (Chen et al. 2014). A typical example of the application of thermo-separating polymer ATPS is the use of an EOPO/salts ATPS to extract lysozyme from hen egg white (Dembczynski et al. 2010) and to recover CGTase of B. cereus (Ng et al. 2012).

Show et al. (2011) developed an aqueous two-phase flotation (ATPF) (Lee et al. 2015) that formed by EOPO copolymer and ammonium sulphate to recover the Burkholderia cenocepacia strains ST8 lipase directly from fermentation broth (Show et al. 2012). In this study, ATPF combines solvent sublation and ATPE system (Dembczynski et al. 2010; Pei et al. 2009), which is based on the interaction process between the hydrophilic group (hydroxyl or glucosan) in surface-active compounds and the hydrophobic group (phenyl or alkyl) in water that are adsorbed to the bubbles surfaces of an ascending nitrogen gas stream. The nitrogen gas bubbles will then dissolve in the top polymer layer of the solution. The average separation efficiency is 76 % and purification fold is 13 % higher under the optimal conditions. The study also indicated that 75 % of EOPO polymers had been recovered (Show et al. 2011). By using this ATPF, B. cepacia lipase was satisfactorily purified in single downstream processing step. This technique also obtained a high concentration coefficient, easy separation and operation, low usage of organic solvent, high separation efficiency and low impact to environment (Show et al. 2011).

The thermo-separating polymer ATPS study was extended to investigate the feasibility of recovering lipase of B. cenocepacia strain ST8 and recycling the phase components in ATPS. Lipase (Tan et al. 2015) was successfully recovered from the polymer-rich top phase in the system of EOPO 3900 with 48.5 % (w/w) tie-line length, VR of 2.3 and 20 % (w/w) of crude load at pH 7 in primary ATPS (Show et al. 2012). In secondary system, the concentrated solution of EOPO copolymer was recovered in the bottom phase, while lipase was collected at the top phase. The aqueous salt solution from the primary system will then be mixed with the concentrated polymer solution from the secondary system to form a new ATPS system. The result showed an average purification factor of 14 with the yield as high as 99 % (Show et al. 2012).

In another work, EOPO and phosphate is used to develop a new ATPS to separate and recover the ciprofloxacin (CIP), a type of fluoroquinolone antibiotic. The extraction efficiency of CIP is 97.7 % by using 3.0 mL of 80 % (w/w) EOPO and 7.0 mL of 55 % (w/w) K2HPO4 at pH 11 (Chen et al. 2014). Meanwhile, an CIP extraction efficiency of 85.6 % is achieved in the second extraction. The phase-forming components are able to recycle and reuse for not more than two times (Chen et al. 2014). This new method is successful in determining the CIP in real samples analysis of milk, egg and shrimp with 6.8 mg/g as limit of detection (LOD) in HPLC–UV detection system (Chen et al. 2014).

An ATPS with the combination of EO50PO50 containing 50 % (w/w) EO and 50 % (w/w) PO, and K2HPO4 was used to extract lysozyme from hen egg white. The experiment was performed over four successive extractions in two stages by using the recovery and recycling of phase components. The primary two-phase system of 40 % EO50PO50 and 10 % K2HPO4 (w/w) was formed at the first stage. The lysozyme partitioned to the top copolymer-rich phase and the contaminant proteins still remained in the bottom phosphate-rich phase at this stage (Dembczynski et al. 2010). The secondary two-phase system was composed of a concentrated copolymer solution and a lysozyme solution after induced with temperature at second stage. The specific activity of lysozyme obtained from these four successive extractions was within the range of 38,438–42,907 U/mg of protein (Dembczynski et al. 2010).

Application of alcohol/salt ATPS

The alcohol/salt ATPS is formed by mixing two immiscible phases which are composed of alcohol and salt solutions. By removing the alcohol using evaporation, the target protein can be easily extracted and recovered (Guan et al. 1996; Tianwei et al. 2002). The alcohol/salt ATPS has less toxicity impact to the environment and is cheaper (Zhi and Deng 2006) compared to the conventional ATPSs. The alcohol/salt ATPS have been used to purify the intracellular human recombinant interferon-α2b (IFN-α2b) from E. coli. IFN-α2b purification by nine biphasic systems with combination of alcohol-based top phase (ethanol, 1-propanol and 2-propanol) and salt-based bottom phase (ammonium sulphate, dipotassium hydrogen phosphate and monosodium citrate) were studied by Lin et al. (2013). The optimum conditions for the purification of IFN-α2b was achieved in ATPS composed of 18 % (w/w) of 2-propanol and 22 % (w/w) of ammonium sulphate in 1.0 % (w/w) of sodium chloride. The purification factor of IFN-α2b obtained is 16.2 with the yield of 74.6 % (Lin et al. 2013).

An alcohol/salt-based ATPS was also applied to purify serine proteases from mango (Mangifera Indica Cv. Chokanan) peel by Amid et al. (2012). In this experiment, different types and concentrations of alcohol (e.g. 1-propanol, 2-propanol, and ethanol) and different types of salt (e.g. sodium citrate, potassium phosphate, and ammonium sulphate) were used to investigate their effects on the purification and selective separation of serine protease at different pH and different concentration of NaCl. The serine protease purification achieved a partition coefficient of 64.5 and a selectivity of 343.2 by employing an ATPS of 16 % (w/w) of 2-propanol, 19 % (w/w) of potassium phosphate and 5 % (w/v) of NaCl at pH 7.5. The study also reported that 96.7 % of serine protease can be recovered with a purification factor of 11.6 (Amid et al. 2012).

An innovative method named as microwave-assisted aqueous two-phase extraction (MAATPE) has been formed by integrated between ATPE that had a high electric constant and demixing effect with high efficiency of microwave-assisted extraction (MAE). This is a first novel MAATPE technique developed by Zhang et al. (2015) which has been applied in extraction of alkaloids from Sophora flavescens Ait. The ATPS which consists of ethanol/ammonia sulphate was selected as it showed the low viscosity, easy demixing, solvent recycling, environmental-friendly process compared to other phase-forming components and surfactants which are mostly high viscosity and will caused the difficulty to form transparent solutions in the preparation of ATPS. The response surface methodology is used to optimise the experimental conditions. The final optimised conditions were determined at solvent-to-material ratio 60:1, temperature 90 ℃, extraction time 5 min and microwave power 780 W for the ATPS composed of 28 % (w/w) of ethanol and 18 % (w/w) of (NH4)2SO4. The MAATP showed the less impurities with a better yield of 63.78 ± 0.45 mg/g and a higher recovery (92.09 ± 0.14 %) are obtained in the extraction and purification of alkaloids. At the same time, the MAATPE proven can improve the demixing and mass transfer from solid–liquid extraction to liquid–liquid extraction with the interaction of microwave and ATPS (Zhang et al. 2015).

Application of aqueous two-phase affinity partitioning (ATPAP)

Aqueous two-phase affinity partitioning (ATPAP) is formed when at least one phase forms component of the system is chemically modified by attaching an affinity ligand which is specific to the target molecule (Ruiz–Ruiz et al. 2012). In this system, a selective interaction occurs between the specific ligands in one of the polymeric phases of system and the partition of the desired protein such as cellular fragment or biomolecule towards the modified polymer-rich phase can be achieved (Cordes et al. 1987). Alternative, by adding the free ligands to a conventional ATPS, a partition shift of the desired molecule also can be induced without the chemical modification of the phase-forming component in ATPAP (Azevedo et al. 2009). In 1975, the selective partition of S-23 myeloma proteins in a modified dextran–polyethylene oxide (PEO) of ATPAP system has been investigated by Flanagan and Barondes (1975). Four additional parameters in ATPAP, which must be considered to optimise ATPAP partitioning (Ruiz–Ruiz et al. 2012), have been identified including: 1) pH, 2) ionic strength in polymer/polymer or polymer/salt systems, 3) nature and concentration of affinity ligand and 4) concentration of modified polymers.

Rosa et al. (2007) have conducted a study on the partitioning of human immunoglobulin (IgG) in a polymer/polymer and polymer/salt ATPS with the use of functionalised PEGs. The work aims to compare the partitioning of human IgG in polymer/polymer and polymer/salt system using several different ligands, and to investigate the respective selectivity of these ligands to the human IgG. The hydrophobic, electrostatic and biospecific effects through interaction of ligands with human IgG are the basis factors to be considered when selecting the suitable ligands. The different chain lengths of PEGs can be functionalised with either acidic or basic end groups (Rosa et al. 2007). Initially, the selectivity studies were conducted with pure IgG using the non-functionalised systems. The results showed that the target protein is partitioned to bottom phase. Afterwards, the systems containing pure proteins, an artificial mixture of proteins and a CHO cells supernatant were used to perform the selectivity tests with the most suitable ligands. The results showed that PEG/phosphate system was not desirable for the affinity partitioning of IgG. The diglutaric acid functionalised PEGs (PEG–COOH) were chosen in this study and it exhibited great affinity to IgG where all IgG were completely recovered in the top phase by employing 20 % (w/w) of PEG 150–COOH and 40 % (w/w) PEG 3350–COOH in PEG/dextran system. The result showed that with the application of artificial mixture of proteins such as human serum albumin (Chow et al. 2013) and myoglobin, PEG 3350–COOH did not exhibit affinity to IgG. However, IgG was able to recover with a yield of 91 % in PEG 150–COOH system. The best recovery yield of 93 %, a selectivity of 11 and a purification factor of 1.9 of the IgG from CHO cells supernatant were achieved in a PEG/dextran system when PEG 150–COOH was used. Furthermore, a 60-fold increase in selectivity was obtained when this functionalised PEG was added to the ATPS compared to the systems without functionalised PEG. In other work, triazine-based IgG affinity ligands coupled to the PEGs with carboxylic acid end groups were chosen because the ligands are expected to mimic the effect of the Cl ion and are able to selectively extract IgG to the top phase (Rosa et al. 2007). The modified triazines show affinity towards IgG which observed by Teng et al. (1999) have promoted several of PEGs functionalised with triazine ligands (Rosa et al. 2007).

An attempt to optimise the affinity partitioning conditions of papain in ATPS was carried out by Ling et al. (2010). Papain (EC3.4.22.2) is a cysteine from latex Carica papaya and it is widely used as industrial enzyme (Ling et al. 2010; He et al. 1082). In this system, reactive red 120 was added as free affinity dye ligand. Five parameters including pH, concentrations of ammonium sulphate, sodium chloride, dye and PEG, which affect the papain partitioning in ATPS, were tested by using response surface methodology. The results revealed that the optimum condition of the system was determined as PEG of 0.102 g/mL, sodium chloride of 25 mg/mL, ammonium sulphate of 0.17 g/mL, reactive red 120 of 4.5 μg/mL at pH 8.0. A ratio of specific activity of papain partitioning between top and bottom phase, KU, value is 1.92 with a recovery yield of 50.6 %.

Application of ionic liquid-based ATPS

Ionic liquids are potential green solvents to replace the existing volatile organic solvents (VOCs). Typically, ILs and ionic molten salts (melting point below 100 ℃) are carbon compounds containing one nitrogen atom which have positive charge (imidazolium or pyridinium) by which the organic cation is counter-balanced by an organic or inorganic anion (He et al. 1082). Imidazolium- and pyridinium-based ILs were reported to have highest UV–Vis absorption wavelengths at 210 and 264 nm, respectively, and it is an useful information in determining the concentration of ILs (He et al. 1082). ILs exhibit very low vapour pressure, excellent solvation quality, variable viscosity range and thermal stabilities (Anderson et al. 2002). Up to now, ILs have been widely used in the applications of synthesis (Fukushima et al. 2003), catalysis (Welton 1999), chromatographic separation (Ding et al. 2004; Vaher and Koel 2005), extraction processes (Carda-Broch et al. 2003; Liu et al. 2003), and mass spectrometry analysis (Mank et al. 2004) in chemistry, biotechnology and environmental engineering field. Recently, a new IL/salts ATPS system was developed to recycle, metathesis and determine the distribution ratios of short-chain alcohols (He et al. 1082).

The study done by He et al. (1082) was aimed to develop an environmentally friendly extraction method based on IL/salt system as a new pretreatment procedure to couple with a reversed-phase high-performance liquid chromatography (RP-HPLC) for simultaneous concentration and analysis of testosterone (T) and epitestosterone (ET) in human urine (Antov et al. 2006). A hydrophilic IL, 1-butyl-3-methylimidazolium chloride [(C4mim)Cl], has been selected as the phase-forming IL to study the phase characteristics of ATPS such as phase diagrams and effect of salts (He et al. 1082). By using 3 mL of urine, the experiment began with a single hydrolysis, deproteinisation and extraction step. Finally, the experiment was continued by direct injection of the IL-rich upper phase into HPLC system for further analysis. The method has been successfully analyse the T and ET in human urine with a detection limit of 1 ng/mL and linear range of 10–500 ng/mL for both compounds by optimising the extraction conditions (type and amount of salts, concentration of analytes, and temperature). Extraction efficiencies for both analytes were determined as 80–90 % in the integrated extraction at optimal conditions within 67–600 ng/mL of steroid concentration and 10–50 ℃ of temperature (He et al. 1082).

Another application of IL-based ATPS is to remove the textile dyes in wastewater. The dyes are highly stable and resistant to biodegradation because of the complex aromatic structures and synthetic origin of dyes (Kiran et al. 2006; Aksu and Tezer 2005). The release of large amount of dyes through aqueous effluents can cause environmental and economic problems. In this context, environmental regulations have becoming stricter in the concern of discharging the dyes from aqueous effluents (Robinson et al. 2001; Hessel et al. 2007). The work done by Ferreira et al. (2014) has proposed a novel approach to remove dyes from aqueous discharges by using IL-based ATPS. The partition coefficients and extraction efficiencies of a set of textile dyes (chloranilic acid, Indigo Blue and Sudan III) have been carried out by using ATPS composed of hydrophilic ILs (phosphonium- and imidazolium-based) and an inorganic (aluminium sulphate) or organic salt (potassium citrate). An evaluation of the IL structure, the nature of the salt and the pH of the aqueous medium has been assessed from the data of experiments. A selection of IL and salt that leads to the complete extraction of the three dyes to the IL-rich phase in a single-step procedure has been evaluated from the data analysis. The recovery of dyes will allow the reuse of IL-rich phase. All the partition coefficients and/or extraction efficiencies values reported in this research suggested that the same IL-rich phase can be used up to 800 times without reaching saturation. The saturation limit of the IL-rich phase with Sudan III is 2.12 g/kg IL at 25 ℃ (Ferreira et al. 2014).

Besides the application of thermo-separating polymer ATPS, the IL-based ATPS has been successfully tested in purification of lipase in the latest development. The study is done by Souza et al. (2015) to develop the ATPS that formed with cholinium-based ionic liquid– ILs (or salts) and tetrahydrofuran (THF) to purify the lipase from Bacillus sp. ITP-001. The cholinium-based salts that consist of cholinium chloride, cholinium bitartrate and cholinium dihydrogencitrate together with THF were used to form the ATPS. The lipase from Bacillus sp. ITP-001 is produced by submerged fermentation. The dialysed solution containing the lipase was then used to prepare the ATPS after the pre-purification steps. The optimum conditions for this purification were determined to be 40 % (w/w) THF and 30 % (w/w) cholinium bitartrate at 25 ℃. The result showed a lipase yield of 90.0 ± 0.7 % with a purification factor of 130.1 ± 11.7-fold (Souza et al. 2015).

Application of aqueous surfactant two-phase, ASTP

An new aqueous surfactant two-phase (ASTP) is formed with the solution to divide into two immiscible aqueous phases when cationic and anionic surfactants are mixed at a concentration higher than critical micelle concentration (CMC) and at certain molar ratio of cationic and anionic surfactant compositions (Weschayanwiwat et al. 2008). This novel ASTP extraction system has becoming popular since it also presents a phase separation that mimics to the conventional non-ionic surfactants. ASTP can be formed at low surfactant concentrations and compositions (Weschayanwiwat et al. 2008). The molar ratio of surfactants play an important role in ASTP system (Yin et al. 2002). The aggregates in the phases can exist in different forms including spherical micelles, rodlike micelles or vesicles by simply using different surfactant concentrations and compositions (Weschayanwiwat et al. 2008).

In conventional ASTP extraction, when non-ionic surfactant is heated above the cloud point temperature, the dehydration of detergent will cause phase separation to occur. The detergent-based systems or cloud point extraction (CPE) systems have been introduced and used in conventional ASTP extraction process (Selber et al. 2004). One is the surfactant-rich and the other is the surfactant-dilute phase. In the small volume of surfactant-rich phase, the organic contaminants will partition into the surfactant-rich phase, then aggregate and concentrate. At the same time, a small amount of organic contaminants known as remediated water will remain in the surfactant-dilute phase (Weschayanwiwat et al. 2008). By using a vacuum stripper, the surfactant-rich phase can be recycled and reused if the organic contaminants have high enough volatility (Weschayanwiwat et al. 2008). This kind of ATPS system is mostly used to separate the hydrophobic and amphiphilic molecules based on the solubilisation and partitioning of membrane bound substances. Hinze and Pramauro (1993), Sanchez-Ferrer et al. (1994) have listed all the membrane proteins that have been solubilised from cells including plants and neural cells, archaebacteria, eubacteria and yeasts (Selber et al. 2004). The surfactants mixtures always exhibit superior properties compared to the single surfactants (Kang et al. 2001) and the effectiveness of extraction (Khan and Marques 1999) is also improved which is vital for many types of technical applications (Weschayanwiwat et al. 2008).

ASTP system, which consists of a cationic surfactant [e.g. dodecyltrimethylammonium bromide (DTAB)] and an anionic surfactant [e.g. alkyldiphenyloxide disulphonate (DPDS)] has been used to extract benzene from wastewater. The experiment showed that phase separation occured when the DTAB:DPDS molar ratio range from 1.6:1 to 2.4:1 was used. The lowest CMC value with greatest synergism which mean that the highest negative values of the micellar interaction parameter and the highest extraction efficiency were showed with 2:1 molar ratio of DTAB:DPDS at zero charge of the surfactant aggregates. Compared to ASTP extraction using non-ionic surfactants, the partition ratio of benzene is 48 and 72 % of the benzene is extracted into the surfactant-rich phase at a total surfactant concentration of 50 mM phase solution in a single-stage extraction (Weschayanwiwat et al. 2008).

Another example of ASTP system is demonstrated by Selber et al. (Selber et al. 2004) where the target protein consisted of fungal cellulose endoglucanase I (EGI) and amphiphilic protein hydrophobin I (HFBI) from Trichoderma reesei was chosen. A part of cellulose-binding domain at EGI was replaced by HFBI and this genetically modified engineering protein is named as EGIcore-HFBI. This fusion protein is expressed homologously under the command of the cbh1 promoter. The objective of this work was to examine the technical feasibility of purification of this engineered protein in large scale by using detergent-based ATPS. A non-ionic polyoxyethylene Agrimul NRE 1205 was selected as phase-forming polymer. Agrimul BRE 1205, which is derived from plant oil rich in C12 lipids, has a large extent of C12 chains with small components of C14, C16 and C18 chains. The cloud point at 2 % of Agrimul NRE 1205 in water is at 22 ℃ (Selber et al. 2004). A direct scale up was completed in downstream process from 10 mL to 1200 L. The cultivation was scaled up from 7 L to 1500 L.

Application of ATPS with the combination of carbohydrates

A technique to use mono- and disaccharides to prepare novel ATPS has been developed recently. One of these studies was carried out by de Brito Cardoso et al. (2013) where acetonitrile and carbohydrates are used to prepare ATPS. Acetonitrile is a colourless aprotic solvent and it forms homogeneous mixture when it is mixed with water. The acetonitrile molecules do not strongly interact between each others. However, they are likely to form hydrogen bond networks with water molecules (Takamuku et al. 1998). The carbohydrates molecules [general formula of (CH2O)x] are non-charged. They are biodegradable, non-toxic and renewable feedstock and show an inherent salting-out character (de Brito et al. 2013). The acetonitrile/carbohydrate system has been applied to partition the common antioxidant, vanillin. The ternary phase diagrams were examined at 298 K. The effect of the carbohydrate structure on the liquid–liquid or phase separation ability was evaluated. Numerous of carbohydrates were investigated, including monosaccharides (glucose, mannose, galactose, xylose, arabinose, and fructose), disaccharides (sucrose and mannose) and also commercial food-grade of sucrose, fructose and glucose. However, the main weakness of using food-grade sugar is the tendency of phase separation in reducing the two-phase region which is due to the impurities effect. The vanillin extracted is migrated towards the acetonitrile-rich phase with partition coefficients higher than 3.0, and vanillin has been recovered up to 91 % in a single-step ATPS. Carbohydrates are proven to be a potential substitute of inorganic salts and polymers in the formation of new ATPS. For instance, Wang et al. (2008) have used sugars with the combination of acetonitrile in the formation of ATPS. Also, Wu et al. (2008) and Freire et al. (2011) have applied the use of sugars in combination with the ILs.

An ATPS study on the affinity separation in partitioning Cellulomonas fimi β-mannanase (EC containing a mannan-binding module has been investigated by Antov et al. (2006) using polysaccharides from galactomannan and hydroxypropyl starch. Affinity partitioning of β-mannanase was achieved because of the biospecificity of the mannan-binding module towards the top phase which contained galactomannan. A pH of 8 was used in the experiment in order to inhibit enzyme degradation at the phase containing galactomannan. By lowering the pH to 6 (optimal pH) of β-mannanase, the top phase polymer was removed from degradation by the enzyme. Utilisation of alternative polysaccharide polymers in forming ATPS can improve the partitioning of biological compounds with special interest in plant cell wall polysaccharide degrading enzymes that contain a carbohydrate-binding module (CBM). The functions of CBM are to bind an enzyme to its target substrate and to enhance its action (Tunnicliffe et al. 2005). The study has introduced a new method in affinity partitioning of enzymes that consists of CBMs based on the utilisation of the natural affinity in one of the phase-forming components at the polymer two-phase systems (Antov et al. 2006).

Application of triblock copolymers ATPS

The triblock copolymer ATPS has been discovered based on a hydrophobic complexant for metal separation and purification (Rodrigues et al. 2008). Basically, the triblock copolymers are built by two EO blocks and one PO block, and they have a general structure of (EO)x(PO)y(EO)x. When the copolymers are dissolved in an aqueous solution at critical temperatures and concentrations, a micelle is formed where the core is dominated by hydrophobic units [poly (propylene oxide), PO] surrounded by a crown of hydrophilic units (ethylene oxide, EO) (de Lemos et al. 2013). These cores are able to dissolve the water-insoluble complexing agent and hydrophobic metal complex. An ATPS formed by a triblock copolymer of PEO–PPO–PEO [poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide)], L64, with an average molar mass of 2900 g/mol and 40 % EO, which corresponds to the composition (EO)13(PO)30(EO)13, and an electrolyte (MgSO4, Li2SO4 or Na2C4H4O6) in water at 298 K were used to investigate the separation of copper and zinc by de Lemos et al. (2013). The 1-(2-pyridylazo)-2-naphthol (PAN) acted as an extracting agent in this separation. The factors that affect the metal separation including the amount of extractor added of the ATPS electrolyte, the pH and the composition of the system. The optimum separation (SCu,Zn = 204) of Cu and Zn was obtained using the L64 + Na2C4H4O6 ATPS at pH 3.0 with a tie-line length of 41.28 % (m/m) and a PAN/metal molar ratio of 3.0. The copper stripping percentage achieved a maximum value (% S = 83.3 %) by using 0.076 mol/kg of H2SO4 in this ATPS at thermodynamic condition (de Lemos et al. 2013).

ATPSs composed of macromolecule and electrolyte were used to investigate the separation of metallic ions. These works are mainly based on the PEO (2000 g/mol) and inorganic anions (CO3 2−, SO4 2−, OH or PO4 3−) (Rogers and Bauer 1996; Rogers et al. 1993). Most of the ions are separated into the salt-enriched phase (Silva et al. 2006), except pertechnetate anion (TcO4−) and the pentacyanonitrosylferrate anion [Fe(CN)5(NO)]2− or nitroprusside [Fe(CN)5(NO)]2− (da Silva et al. 2008). The pertechnetate anion is partitioned into the polymer-rich phase. This is due to the reason of hydrophobic nature and small value of estimated hydration Gibbs free energy (ΔhydG) in pertechnetate anion (Rogers and Zhang 1996). On the other hand, the [Fe(CN)5(NO)]2− anion is separated and entered to polymer-rich phase due to strong interaction between the anion and the EO segments of the PEO (Silva et al. 2006). Triblock copolymers-based ATPS has been applied in the separation of the hydrophilic and hydrophobic biomolecules (Bolognese et al. 2005; J-h et al. 2007). The behaviour (Chow et al. 2015) of copolymer solution is highly depending on the EO/PO ratio and the molar mass of the macromolecules. Critical micellization temperature (CMT) and the critical micellization concentration (CMC) are the temperature and concentration where the copolymer molecules began to self-associate, respectively (da Silva et al. 2008). The ATPS composed of potassium phosphate, macromolecules—poly (ethylene oxide) (1500 g/mol) and triblock copolymers, PEO–PPO–PEO were used to study the separating behaviour of [Fe(CN)5(NO)] 2− and [Fe(CN)6]3− anions by da Silva et al. (da Silva et al. 2008). Two copolymers with different hydrophobicity consist of L35 (50 % EO and 1900 g/mol) and F68 (80 % EO and 8400 g/mol) were used. The parameters of the temperature, tie-line length and phases hydrophobicity of the partition coefficient for the [Fe(CN)5(NO)]2− and [Fe(CN)6]3− anions in each ATPS were examined. The result found that the partition coefficient of anions follows the order of L35< F68< PEO relatively. The decrease in partition coefficient of both anions when the temperature was increased indicated that an exothermic process occurred in the separating process. The movement of anions to the top phase is an enthalpic process which is proved by the thermodynamic parameters obtained from Van’t Hoff equation and calorimetric measurements (da Silva et al. 2008).

Summary of the application of ATPSs

All types of ATPS that have been reviewed in this paper have been summarised in Table 1 for comparison of recovery yield, purification factor (PF), purity, fold and selectivity or separation efficiency.

Table 1 Summary of the types and comparison results of ATPS from literature reviews


ATPS has been reported as an attactive alternative or platform in downstream processing that can be adopted by industries due to its high capacity and easily scalability. However, the limited knowledge in the partitioning mechanism and poor understanding of the experimental design, installation and operation of ATPSs are the factors that still hindered its application. More efforts are needed to increase the popularity of the application of ATPSs in the downstream purification and recovery processes in order to replace the conventional techniques for economical and environmental sustainability.

In future, the ATPS is expected to rapidly evolve experimentally and theoretically to become a powerful technology in separation science with many useful applications in the industries. The ATPSs will be modified and improved to overcome the disadvantages and challenges facing in this technique. More novel types of ATPS will be discovered and integrated with other technologies as a promising alternative which can be used as commercial applications in recovering the high value products.


  1. Aksu Z, Tezer S (2005) Biosorption of reactive dyes on the green alga Chlorella vulgaris. Process Biochem 40(3):1347–1361

  2. Albertsson PA (1987) Partition of cell particles and macromolecules, Wiley-Interscience, New York, 346 pp. Analytical Biochemistry 161 (1):227

  3. Amid M, Shuhaimi M, Islam Sarker MZ, Abdul Manap MY (2012) Purification of serine protease from mango (Mangifera Indica Cv. Chokanan) peel using an alcohol/salt aqueous two phase system. Food Chem 132(3):1382–1386. doi:10.1016/j.foodchem.2011.11.125

  4. Anderson JL, Ding J, Welton T, Armstrong DW (2002) Characterizing ionic liquids on the basis of multiple solvation interactions. JACS 124(47):14247–14254. doi:10.1021/ja028156h

  5. Antov M, Anderson L, Andersson A, Tjerneld F, Stålbrand H (2006) Affinity partitioning of a Cellulomonas fimi β-mannanase with a mannan-binding module in galactomannan/starch aqueous two-phase system. J Chromatogr A 1123(1):53–59. doi:10.1016/j.chroma.2006.05.021

  6. Ariff A, Anuar MS, Chen SK, Ling TC, Show PL, Tan CP (2011) Extractive fermentation for improved production and recovery of lipase derived from Burkholderia cepacia using a thermoseparating polymer in aqueous two phase systems. Bioresour Technol 10(1):10–16

  7. Asenjo JA, Andrews BA (2011) Aqueous two-phase systems for protein separation: a perspective. J Chromatogr A 1218(49):8826–8835. doi:10.1016/j.chroma.2011.06.051

  8. Azevedo AM, Rosa PAJ, Ferreira IF, de Vries J, Visser TJ, Aires-Barros MR (2009a) Downstream processing of human antibodies integrating an extraction capture step and cation exchange chromatography. J Chromatogr B 877(1–2):50–58. doi:10.1016/j.jchromb.2008.11.014

  9. Azevedo AM, Rosa PAJ, Ferreira IF, Aires-Barros MR (2009b) Chromatography-free recovery of biopharmaceuticals through aqueous two-phase processing. Trends Biotechnol 27(4):240–247. doi:10.1016/j.tibtech.2009.01.004

  10. Azevedo AM, Rosa PAJ, Ferreira IF, Pisco AMMO, de Vries J, Korporaal R, Visser TJ, Aires-Barros MR (2009c) Affinity-enhanced purification of human antibodies by aqueous two-phase extraction. Sep Purif Technol 65(1):31–39. doi:10.1016/j.seppur.2008.03.006

  11. Bellot G, McClintock MA, Lin C, Shih WM (2011) Recovery of intact DNA nanostructures after agarose gel-based separation. Nat Methods 8(3):192–194. doi:10.1038/nmeth0311-192

  12. Benavides J, Rito-Palomares M (2008) Practical experiences from the development of aqueous two-phase processes for the recovery of high value biological products. J Chem Technol Biotechnol 83(2):133–142. doi:10.1002/jctb.1844

  13. Berggren K, Johansson HO, Yjerneld F (1995) Effects of salts and the surface hydrophobicity of proteins on partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide-propylene oxide copolymers. J Chromatogr A 718(1):67–79. doi:10.1016/0021-9673(95)00657-5

  14. Bhambure R, Sharma R, Gupta D, Rathore AS (2013) A novel aqueous two phase assisted platform for efficient removal of process related impurities associated with E. coli based biotherapeutic protein products. J Chromatogr A 1307:49–57. doi:10.1016/j.chroma.2013.07.085

  15. Bolognese B, Nerli B, Pico G (2005) Application of the aqueous two-phase systems of ethylene and propylene oxide copolymer-maltodextrin for protein purification. J Chromatogr B Analyt Technol Biomed Life Sci 814(2):347–353

  16. Carda-Broch S, Berthod A, Armstrong DW (2003) Solvent properties of the 1-butyl-3-methylimidazolium hexafluorophosphate ionic liquid. Anal Bioanal Chem 375(2):191–199

  17. Chen CW, Thomas CA (1980) Recovery of DNA segments from agarose gels. Anal Biochem 101(2):339–341. doi:10.1016/0003-2697(80)90197-9

  18. Chen J, Ma GX, Li DQ (1999) HPCPC separation of proteins using polyethylene glycol-potassium phosphate aqueous two-phase. Prep Biochem Biotechnol 29(4):371–383. doi:10.1080/10826069908544935

  19. Chen J, Spear SK, Huddleston JG, Rogers RD (2005) Polyethylene glycol and solutions of polyethylene glycol as green reaction media. Green Chem 7(2):64–82

  20. Chen B, Han J, Wang Y, Sheng C, Liu Y, Zhang G, Yan Y (2014) Separation, enrichment and determination of ciprofloxacin using thermoseparating polymer aqueous two-phase system combined with high performance liquid chromatography in milk, egg, and shrimp samples. Food Chem 148:105–111. doi:10.1016/j.foodchem.2013.10.011

  21. Chow YH, Yap YJ, Anuar MS, Tejo BA, Ariff A, Show PL, Ng E-P, Ling TC (2013) Interfacial partitioning behaviour of bovine serum albumin in polymer-salt aqueous two-phase system. J Chromatogr B 934:71–78

  22. Chow YH, Yap YJ, Tan CP, Anuar MS, Tejo BA, Show PL, Ariff AB, Ng E-P, Ling TC (2015) Characterization of bovine serum albumin partitioning behaviors in polymer-salt aqueous two-phase systems. J Biosci Bioeng 120(1):85–90. doi:10.1016/j.jbiosc.2014.11.021

  23. Cordes A, Flossdorf J, Kula MR (1987) Affinity partitioning: development of mathematical model describing behavior of biomolecules in aqueous two-phase systems. Biotechnol Bioeng 30(4):514–520. doi:10.1002/bit.260300408

  24. da Silva LHM, da Silva MdCH, Júnior JA, Martins JP, Reis Coimbra JSd, Minim LA (2008) Hydrophobic effect on the partitioning of [Fe(CN)5(NO)]2− and [Fe(CN)6]3− anions in aqueous two-phase systems formed by triblock copolymers and phosphate salts. Sep Purif Technol 60(1):103–112. doi:10.1016/j.seppur.2007.07.048

  25. Daugulis AJ (1988) Integrated reaction and product recovery in bioreactor systems. Biotechnol Progr 4(3):113–122. doi:10.1002/btpr.5420040302

  26. de Brito Cardoso G, Mourão T, Pereira FM, Freire MG, Fricks AT, Soares CMF, Lima ÁS (2013) Aqueous two-phase systems based on acetonitrile and carbohydrates and their application to the extraction of vanillin. Sep Purif Technol 104:106–113. doi:10.1016/j.seppur.2012.11.001

  27. de Lemos LR, Campos RA, Rodrigues GD, da Silva LHM, da Silva MCH (2013) Green separation of copper and zinc using triblock copolymer aqueous two-phase systems. Sep Purif Technol 115:107–113. doi:10.1016/j.seppur.2013.04.048

  28. Dembczynski R, Bialas W, Jankowski T (2010) Recycling of phase components during lysozyme extraction from hen egg white in the EO50PO50/K2HPO4 aqueous two-phase system. Biochem Eng J 51(1–2):24–31. doi:10.1016/j.bej.2010.04.011

  29. Ding J, Welton T, Armstrong DW (2004) Chiral ionic liquids as stationary phases in gas chromatography. Anal Chem 76(22):6819–6822

  30. Duarte SP, Fortes AG, Prazeres DMF, Marcos JC (2007) Preparation of plasmid DNA polyplexes from alkaline lysates by a two-step aqueous two-phase extraction process. J Chromatogr A 1164(1–2):105–112. doi:10.1016/j.chroma.2007.06.061

  31. Ferreira AM, Coutinho JAP, Fernandes AM, Freire MG (2014) Complete removal of textile dyes from aqueous media using ionic-liquid-based aqueous two-phase systems. Sep Purif Technol 128:58–66. doi:10.1016/j.seppur.2014.02.036

  32. Flanagan SD, Barondes SH (1975) Affinity partitioning. A method for purification of proteins using specific polymer-ligands in aqueous polymer two-phase systems. J Biol Chem 250(4):1484–1489

  33. Freeman A, Woodley JM, Lilly MD (1993) In situ product removal as a tool for bioprocessing. Nat Biotechnol 11(9):1007–1012

  34. Freire MG, Louros CLS, Rebelo LPN, Coutinho JAP (2011) Aqueous biphasic systems composed of a water-stable ionic liquid + carbohydrates and their applications. Green Chem 13(6):1536–1545. doi:10.1039/c1gc15110j

  35. Fukushima T, Kosaka A, Ishimura Y, Yamamoto T, Takigawa T, Ishii N, Aida T (2003) Molecular ordering of organic molten salts triggered by single-walled carbon nanotubes. Science 300(5628):2072–2074. doi:10.1126/science.1082289

  36. Guan Y, Lilley TH, Treffry TE, Zhou C-L, Wilkinson PB (1996) Use of aqueous two-phase systems in the purification of human interferon-α1 from recombinant Escherichia coli. Enzyme Microb Technol 19(6):446–455. doi:10.1016/S0141-0229(96)00051-8

  37. Hayenga KJ, Valex PP (2002) Methodes de purification de proteines par extraction biphasee aqueuse. Google Patents. EP1194445 A1, EP20000928866, 10 Apr 2002

  38. He C, Li S, Liu H, Li K, Liu F (2005) Extraction of testosterone and epitestosterone in human urine using aqueous two-phase systems of ionic liquid and salt. J Chromatogr A 1082(2):143–149. doi:10.1016/j.chroma.2005.05.065

  39. Helfrich MR, El-Kouedi M, Etherton MR, Keating CD (2005) Partitioning and assembly of metal particles and their bioconjugates in aqueous two-phase systems. Langmuir 21(18):8478–8486. doi:10.1021/la051220z

  40. Hessel C, Allegre C, Maisseu M, Charbit F, Moulin P (2007) Guidelines and legislation for dye house effluents. J Environ Manage 83(2):171–180. doi:10.1016/j.jenvman.2006.02.012

  41. Hinze WL, Pramauro E (1993) A critical review of surfactant-mediated phase separations (cloud-point extractions): theory and applications. Crit Rev Anal Chem 24(2):133–177. doi:10.1080/10408349308048821

  42. J-h Ma, Guo C, Y-l Tang, Wang J, Zheng L, X-f Liang, Chen S, H-z Liu (2007) Salt-induced micellization of a triblock copolymer in aqueous solution: a 1H nuclear magnetic resonance spectroscopy study. Langmuir 23(6):3075–3083. doi:10.1021/la063203v

  43. Jiang Y, Xia H, Yu J, Guo C, Liu H (2009) Hydrophobic ionic liquids-assisted polymer recovery during penicillin extraction in aqueous two-phase system. Chem Eng J 147(1):22–26. doi:10.1016/j.cej.2008.11.012

  44. Johansson HO, Karlström G, Tjerneld F (1997) Temperature-induced phase partitioning of peptides in water solutions of ethylene oxide and propylene oxide random copolymers. Biochim Biophys Acta 1335(3):315–325. doi:10.1016/S0304-4165(96)00150-X

  45. Johansson HO, Persson J, Tjerneld F (1999) Thermoseparating water/polymer system: a novel one-polymer aqueous two- phase system for protein purification. Biotechnol Bioeng 66(4):247–257

  46. Jönsson M, Johansson H-O (2003) Protein partitioning in thermoseparating systems of a charged hydrophobically modified ethylene oxide polymer. J Chromatogr A 983(1–2):133–144. doi:10.1016/S0021-9673(02)01695-3

  47. Kang KH, Kim HU, Lim KH (2001) Effect of temperature on critical micelle concentration and thermodynamic potentials of micellization of anionic ammonium dodecyl sulfate and cationic octadecyl trimethyl ammonium chloride. Colloids Surf Physicochem Eng Aspects 189(1):113–121

  48. Khan A, Marques EF (1999) Synergism and polymorphism in mixed surfactant systems. Curr Opin Colloid Interface Sci 4(6):402–410. doi:10.1016/S1359-0294(00)00017-0

  49. Kiran I, Akar T, Ozcan AS, Ozcan A, Tunali S (2006) Biosorption kinetics and isotherm studies of acid red 57 by dried Cephalosporium aphidicola cells from aqueous solutions. Biochem Eng J 31(3):197–203. doi:10.1016/j.bej.2006.07.008

  50. Kornmann H, Baer G (2008) Aqueous two-phase partitioning via polyethylene glycol phase and (Na2)SO4. Google Patents. US7439336 B2, US 11/630,845, 21 Oct 2008.

  51. Lan JC-W, Yeh C-Y, Wang C-C, Yang Y-H, Wu H-S (2013) Partition separation and characterization of the polyhydroxyalkanoates synthase produced from recombinant Escherichia coli using an aqueous two-phase system. J Biosci Bioeng 116(4):499–505. doi:10.1016/j.jbiosc.2013.04.010

  52. Lee SY, Khoiroh I, Ling TC, Show PL (2015) Aqueous two-phase flotation for the recovery of biomolecules. Separation and purification reviews: null-null. doi:10.1080/15422119.2015.1007147

  53. Lin YK, Ooi CW, Tan JS, Show PL, Ariff A, Ling TC (2013) Recovery of human interferon α-2b from recombinant Escherichia coli using alcohol/salt-based aqueous two-phase systems. Sep Purif Technol 120:362–366

  54. Lin YK, Show PL, Yap YJ, Tan CP, Ng E-P, Ariff AB, Mohamad Annuar MSB, Ling TC (2015) Direct recovery of cyclodextringlycosyltransferase from Bacillus cereus using aqueous two-phase flotation. J Biosci Bioeng 120(6):684–689. doi:10.1016/j.jbiosc.2015.04.013

  55. Ling YQ, Nie HL, Su SN, Branford-White C, Zhu LM (2010) Optimization of affinity partitioning conditions of papain in aqueous two-phase system using response surface methodology. Sep Purif Technol 73(3):343–348. doi:10.1016/j.seppur.2010.04.020

  56. Liu JF, Jiang GB, Chi YG, Cai YQ, Zhou QX, Hu JT (2003) Use of ionic liquids for liquid-phase microextraction of polycyclic aromatic hydrocarbons. Anal Chem 75(21):5870–5876

  57. Luechau F, Ling TC, Lyddiatt A (2009a) Partition of plasmid DNA in polymer–salt aqueous two-phase systems. Sep Purif Technol 66(2):397–404. doi:10.1016/j.seppur.2008.12.003

  58. Luechau F, Ling TC, Lyddiatt A (2009b) Selective partition of plasmid DNA and RNA in aqueous two-phase systems by the addition of neutral salt. Sep Purif Technol 68(1):114–118. doi:10.1016/j.seppur.2009.04.016

  59. Mank M, Stahl B, Boehm G (2004) 2,5-Dihydroxybenzoic acid butylamine and other ionic liquid matrixes for enhanced MALDI-MS analysis of biomolecules. Anal Chem 76(10):2938–2950

  60. Matos T, Johansson HO, Queiroz JA, Bulow L (2014) Isolation of PCR DNA fragments using aqueous two-phase systems. Sep Purif Technol 122:144–148. doi:10.1016/j.seppur.2013.11.014

  61. Mohamed Ali S, Ling TC, Muniandy S, Tan YS, Raman J, Sabaratnam V (2014) Recovery and partial purification of fibrinolytic enzymes of Auricularia polytricha (Mont.) Sacc by an aqueous two-phase system. Sep Purif Technol 122:359–366. doi:10.1016/j.seppur.2013.11.016

  62. Ng HS, Tan CP, Mokhtar MN, Ibrahim S, Ariff A, Ooi CW, Ling TC (2012) Recovery of Bacillus cereus cyclodextrin glycosyltransferase and recycling of phase components in an aqueous two-phase system using thermo-separating polymer. Sep Purif Technol 89:9–15

  63. Ng HS, Ooi CW, Mokhtar MN, Show PL, Ariff A, Tan JS, Ng E-P, Ling TC (2013) Extractive bioconversion of cyclodextrins by Bacillus cereus cyclodextrin glycosyltransferase in aqueous two-phase system. Bioresour Technol 142:723–726

  64. Pei Y, Wang J, Wu K, Xuan X, Lu X (2009) Ionic liquid-based aqueous two-phase extraction of selected proteins. Sep Purif Technol 64(3):288–295. doi:10.1016/j.seppur.2008.10.010

  65. Pereira M, Wu Y-T, Venâncio A, Teixeira J (2003) Aqueous two-phase extraction using thermoseparating polymer: a new system for the separation of endo-polygalacturonase. Biochem Eng J 15(2):131–138. doi:10.1016/S1369-703X(02)00190-0

  66. Rito-Palomares M (2004) Practical application of aqueous two-phase partition to process development for the recovery of biological products. J Chromatogr B 807(1):3–11. doi:10.1016/j.jchromb.2004.01.008

  67. Robinson T, McMullan G, Marchant R, Nigam P (2001) Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative. Bioresour Technol 77(3):247–255

  68. Rodrigues GD, da Silva MdCH, da Silva LHM, Paggioli FJ, Minim LA, Reis Coimbra JSd (2008) Liquid–liquid extraction of metal ions without use of organic solvent. Sep Purif Technol 62(3):687–693. doi:10.1016/j.seppur.2008.03.032

  69. Rogers RD, Bauer CB (1996) Partitioning behavior of group 1 and 2 cations in poly(ethylene glycol)-based aqueous biphasic systems. J Chromatogr B Biomed Sci Appl 680(1–2):237–241. doi:10.1016/0378-4347(95)00319-3

  70. Rogers RD, Zhang J (1996) Effects of increasing polymer hydrophobicity on distribution ratios of TcO4− in polyethylene/poly(propylene glycol)-based aqueous biphasic systems. J Chromatogr B Biomed Appl 680(1–2):231–236

  71. Rogers RD, Bond AH, Bauer CB (1993) Metal ion separations in polyethylene glycol-based aqueous biphasic systems. Sep Sci Technol 28(5):1091–1126. doi:10.1080/01496399308018023

  72. Rosa PAJ, Azevedo AM, Ferreira IF, de Vries J, Korporaal R, Verhoef HJ, Visser TJ, Aires-Barros MR (2007) Affinity partitioning of human antibodies in aqueous two-phase systems. J Chromatogr A 1162(1):103–113. doi:10.1016/j.chroma.2007.03.067

  73. Rosa PAJ, Ferreira IF, Azevedo AM, Aires-Barros MR (2010) Aqueous two-phase systems: a viable platform in the manufacturing of biopharmaceuticals. J Chromatogr A 1217(16):2296–2305. doi:10.1016/j.chroma.2009.11.034

  74. Ruiz-Ruiz F, Benavides J, Aguilar O, Rito-Palomares M (2012) Aqueous two-phase affinity partitioning systems: current applications and trends. J Chromatogr A 1244:1–13. doi:10.1016/j.chroma.2012.04.077

  75. Sanchez-Ferrer A, Bru R, Garcia-Carmona F (1994) Phase separation of biomolecules in polyoxyethylene glycol nonionic detergents. Crit Rev Biochem Mol Biol 29(4):275–313

  76. Selber K, Tjerneld F, Collén A, Hyytiä T, Nakari-Setälä T, Bailey M, Fagerström R, Kan J, van der Laan J, Penttilä M, Kula M-R (2004) Large-scale separation and production of engineered proteins, designed for facilitated recovery in detergent-based aqueous two-phase extraction systems. Process Biochem 39(7):889–896. doi:10.1016/S0032-9592(03)00198-5

  77. Show PL, Tan CP, Anuar MS, Ariff A, Yusof YA, Chen SK, Ling TC (2011) Direct recovery of lipase derived from Burkholderia cepacia in recycling aqueous two-phase flotation. Sep Purif Technol 80(3):577–584

  78. Show PL, Tan CP, Anuar MS, Ariff A, Yusof YA, Chen SK, Ling TC (2012a) Primary recovery of lipase derived from Burkholderia cenocepacia strain ST8 and recycling of phase components in an aqueous two-phase system. Biochem Eng J 60:74–80. doi:10.1016/j.bej.2011.10.005

  79. Show PL, Tan CP, Shamsul Anuar M, Ariff A, Yusof YA, Chen SK, Ling TC (2012b) Extractive fermentation for improved production and recovery of lipase derived from Burkholderia cepacia using a thermoseparating polymer in aqueous two-phase systems. Bioresour Technol 116:226–233. doi:10.1016/j.biortech.2011.09.131

  80. Show PL, Ooi CW, Anuar MS, Ariff A, Yusof YA, Chen SK, Annuar MSM, Ling TC (2013) Recovery of lipase derived from Burkholderia cenocepacia ST8 using sustainable aqueous two-phase flotation composed of recycling hydrophilic organic solvent and inorganic salt. Sep Purif Technol 110:112–118

  81. Silva MdCHd, Silva LHMd, Paggioli FJ, Coimbra JSR, Minim LA (2006a) Sistema aquoso bifásico: uma alternativa eficiente para extração de íons. Quim Nova 29:1332–1339

  82. Silva LH, Silva MC, Aquino RA, Francisco KR, Cardoso MV, Minim LA, Coimbra JS (2006b) Nitroprusside-PEO enthalpic interaction as a driving force for partitioning of the [Fe(CN)(5)NO](2−) anion in aqueous two-phase systems formed by poly(ethylene oxide) and sulfate salts. J Phys Chem B 110(46):23540–23546

  83. Souza RL, Lima RA, Coutinho JAP, Soares CMF, Lima ÁS (2015) Aqueous two-phase systems based on cholinium salts and tetrahydrofuran and their use for lipase purification. Sep Purif Technol 155:118–126. doi:10.1016/j.seppur.2015.05.021

  84. Takamuku T, Tabata M, Yamaguchi A, Nishimoto J, Kumamoto M, Wakita H, Yamaguchi T (1998) Liquid structure of acetonitrile-water mixtures by x-ray diffraction and infrared spectroscopy. J Phys Chem B 102(44):8880–8888. doi:10.1021/jp9824297

  85. Tan CH, Show PL, Ooi CW, Ng E-P, Lan JC-W, Ling TC (2015) Novel lipase purification methods–a review of the latest developments. Biotechnol J 10(1):31–44. doi:10.1002/biot.201400301

  86. Tang MS, Whitcher TJ, Yeoh KH, Chua CL, Woon KL, Show PL, Lin YK, Ling TC (2014a) The removal of metallic single-walled carbon nanotubes using an aqueous two-phase system. J Nanosci Nanotechnol 14(5):3398–3402

  87. Tang MSY, Show PL, Lin YK, Woon KL, Tan CP, Ling TC (2014b) Separation of single-walled carbon nanotubes using aqueous two-phase system. Sep Purif Technol 125:136–141. doi:10.1016/j.seppur.2014.01.044

  88. Teng SF, Sproule K, Hussain A, Lowe CR (1999) A strategy for the generation of biomimetic ligands for affinity chromatography. Combinatorial synthesis and biological evaluation of an IgG binding ligand. J Mol Recognit 12(1):67–75. doi:10.1002/(SICI)1099-1352(199901/02)12:1<67:AID-JMR443>3.0.CO;2-4

  89. Tianwei T, Qing H, Qiang L (2002) Purification of glycyrrhizin from Glycyrrhiza uralensis Fisch with ethanol/phosphate aqueous two phase system. Biotechnol Lett 24(17):1417–1420. doi:10.1023/A:1019850531640

  90. Tunnicliffe RB, Bolam DN, Pell G, Gilbert HJ, Williamson MP (2005) Structure of a mannan-specific family 35 carbohydrate-binding module: evidence for significant conformational changes upon ligand binding. J Mol Biol 347(2):287–296

  91. Vaher M, Koel M (2005) Specific background electrolytes for nonaqueous capillary electrophoresis. J Chromatogr A 11(1):83–88

  92. Wang B, Ezejias T, Feng H, Blaschek H (2008) Sugaring-out: a novel phase separation and extraction system. Chem Eng Sci 63(9):2595–2600

  93. Welton T (1999) Room-temperature ionic liquids. Solvents for synthesis and catalysis. Chem Rev 99(8):2071–2084

  94. Weschayanwiwat P, Kunanupap O, Scamehorn JF (2008) Benzene removal from waste water using aqueous surfactant two-phase extraction with cationic and anionic surfactant mixtures. Chemosphere 72(7):1043–1048. doi:10.1016/j.chemosphere.2008.03.065

  95. Willauer HD, Huddleston JG, Rogers RD (2002) Solute partitioning in aqueous biphasic systems composed of polyethylene glycol and salt: the partitioning of small neutral organic species. Ind Eng Chem Res 41(7):1892–1904. doi:10.1021/ie010598z

  96. Wu B, Zhang Y, Wang H (2008) Phase behavior for ternary systems composed of ionic liquid + saccharides + water. J Phys Chem B 112(20):6426–6429. doi:10.1021/jp8005684

  97. Walter H (1986) Partitioning in aqueous two–phase system: theory, methods, uses Elsevier science and applications to biotechnology

  98. Yin H, Mao M, Huang J, Fu H (2002) Two-phase region in the DTAB/SL mixed surfactant system. Langmuir 18(24):9198–9203

  99. Zhang W, Zhu D, Fan H, Liu X, Wan Q, Wu X, Liu P, Tang JZ (2015) Simultaneous extraction and purification of alkaloids from Sophora flavescens Ait. by microwave-assisted aqueous two-phase extraction with ethanol/ammonia sulfate system. Sep Purif Technol 141:113–123. doi:10.1016/j.seppur.2014.11.014

  100. Zhi W, Deng Q (2006) Purification of salvianolic acid B from the crude extract of Salvia miltiorrhiza with hydrophilic organic/salt-containing aqueous two-phase system by counter-current chromatography. J Chromatogr A 1116(1–2):149–152. doi:10.1016/j.chroma.2006.03.036

  101. Zijlstra GM, Gooijer CD, Tramper J (1998) Extractive bioconversions in aqueous two-phase systems. Curr Opin Biotechnol 9(2):171–176

Download references

Authors’ contributions

YKY carried out the aqueous two-phase system studies, participated in the sequence alignment and drafted the manuscript. CWO carried out the bioseparation studies. EPN participated in the sequence alignment. JCJ and TCL participated in the design of the study and performed the statistical analysis. PLS conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


The author wishes to thank Leong Yoong Kit from Department of Chemical and Environmental Engineering, Faculty of Engineering, University of Nottingham Malaysia Campus for editing the English of the manuscript. This study was supported by Ministry of Science, Technology and Innovation, Malaysia (02-02-12-SF0256), Fundamental Research Grant Scheme (FRGS/1/2015/SG05/UNIM/03/1)and Prototype Development Research Grant Scheme (PRGS/2/2015/SG05/UNIM/03/1).

Competing interests

The authors declare that they have no competing interests.

Author information

Correspondence to Pau Loke Show.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, 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.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yau, Y.K., Ooi, C.W., Ng, E. et al. Current applications of different type of aqueous two-phase systems. Bioresour. Bioprocess. 2, 49 (2015).

Download citation


  • Aqueous two-phase system
  • Bioproducts recovery
  • Partitioning
  • Purification
  • Recovery yield