Use Of Aqueous Two-phase Systems Research Articles

Aqueous Two‐Phase Enabled Low Viscosity 3D (LoV3D) Bioprinting of Living Matter (Adv. Sci. 8/2023)

Embedded Bioprinting In article number 2204609 by Jeroen Leijten and co-workers, liquid-in-liquid printing of low viscous solutions is enabled by the novel use of aqueous two-phase systems. Use of low viscous solutions allows for cytocompatible biofabrication of embedded sacrificial strands with an exceptionally large resolution range. The low viscous nature of bio-ink also allows for direct printing of intricate, interconnected, and perfusable networks within 3D structures.

Use of aqueous two-phase systems consisting of sugar alcohols and polymer as a new platform for arbutin separation

Use of aqueous two-phase systems consisting of sugar alcohols and polymer as a new platform for arbutin separation

Pre-treatment strategies based on aqueous two-phase systems comprising ionic liquids to improve the adrenal cancer diagnosis

Cancer remains the cause of millions of deaths every year worldwide, and reliable early-stage diagnosis methods are essential to prevent them. Cancer biomarkers quantification in human fluids has gained relevance in detecting cancer in an early-stage. Among these, the levels of the biomarkers epinephrine, norepinephrine and vanillylmandelic acid in urine have been used for the diagnosis of adrenal cancer. However, due to their low levels in human fluids, expensive and multistep pretreatment methods are necessary to increase their concentration to meet the detection limits of the analytical equipments. In this work, we propose the use of aqueous two-phase systems (ATPS) composed of ionic liquids (ILs), K3PO4 and synthetic/natural urine as an alternative pretreatment strategy to improve the concentration of adrenal cancer biomarkers from human fluids. The ATPS ternary phase diagrams, as well as the respective tie-lines and tie-line lengths, were determined at 25 °C. The performance of these IL-based ATPS for the extraction and concentration of epinephrine, norepinephrine and vanillylmandelic acid from human urine was then evaluated. The obtained results show that in the studied systems, with the exception of IL-based ATPS constituted by ILs with longer alkyl side chains at the cation, all the studied cancer biomarkers preferably migrate to the IL-rich phase. The best ATPS investigated is constituted by 1-ethyl-3-methylimidazolium methylsulfate ([C2mim][CH3SO4]) that allows the complete extraction of all cancer biomarkers to the IL-rich phase in a single-step and a concentration factor up to 500-fold. All biomarkers were accurately quantified in the IL-rich phase after the extraction from urine and the concentration step by UHPLC. According to the obtained results, IL-based ATPS could be used as a novel and effective method for the pretreatment and concentration of cancer biomarkers from human fluids to improve cancer diagnosis.

Stimuli-responsive polymer-based aqueous two-phase extraction of analytes in complex matrices

• Stimuli-responsive polymer-based ATPSs are effective green extraction techniques. • Temperature and pH are the common stimuli used in these ATPSs. • These ATPSs use less aggressive conditions during the extraction of analytes. • The stimuli-responsive polymers in the ATPS are recyclable after the extraction process. The use of aqueous two-phase systems (ATPSs) during extraction of analytes from complex matrices is increasingly gaining popularity. This can be attributed to the fact that these systems are generally simple, fast and environmentally friendly. The success of aqueous two-phase extraction largely depends on the good choice of materials to be used. The availability of smart materials such as stimuli-responsive polymers has facilitated the development of effective ATPSs for the extraction of analytes from complex matrices. This paper explores the utilisation of stimuli-responsive polymers during these biphasic systems. The stimuli-responsive polymers that are in common use in ATPSs include thermo-responsive and pH-responsive polymers. Other researchers used dual-stimuli-responsive ATPSs, which involve a combination of a thermo-responsive polymer and a pH-responsive polymer, during extraction of analytes from complex matrices. These biphasic systems composed of thermo-responsive and pH-responsive polymers are discussed in detail in this paper. In addition, this review explores the challenges associated with the use of stimuli-responsive polymers in ATPSs as well as the future prospects.

Liquid-liquid extraction of rare earth elements using systems that are more environmentally friendly: Advances, challenges and perspectives

• Alternative liquid–liquid techniques for rare earth extraction are reviewed. • Properties and green characteristics of alternative methods are assessed. • Comparisons with traditional solvent extraction method are presented. • Cloud point extraction demonstrates analytical pre-concentration method potential. • Aqueous biphasic systems demonstrate potential for large scale implementation. The rare earth elements, a group of metals consisting of the lanthanide series, together with yttrium and scandium, are increasingly important in high technology industries worldwide. Given the need to expand production of these elements, there is a search for processes to obtain them that are less environmentally damaging, since the rare earths production chain is associated with several environmental problems and concerns. Solvent extraction, one of the important steps involved in the obtaining of rare earth elements, is receiving much attention in the scientific and industrial community, with a renewed search for approaches that avoid the use of organic solvents. Among the various options, cloud point extraction and the use of aqueous two-phase systems are alternatives to the use of traditional organic solvents. This review considers these two more environmentally friendly techniques and their use for the extraction and/or separation of rare earth elements. The issues addressed include their potential to replace traditional liquid–liquid extraction, in terms of the efficiency, toxicity, and biodegradability of the components forming the systems, as well as their potential applications and future perspectives.

Use of aqueous two-phase systems formed by Triton X and choline chloride for extraction of organic and inorganic arsenic

The use of aqueous two-phase systems (ATPSs) for the extraction/speciation of arsenic has received attention in recent years. However, only ATPSs formed by polymer and salt have been used, hindering a global understanding of the mechanism of partitioning of the metalloid and limiting the development of strategies for arsenic speciation in these systems. Therefore, it is important to study the partitioning of different arsenic species in new and more environmentally friendly ATPSs, such as those formed by ionic liquids based on the choline cation. In this work, ATPSs formed by Triton X nonionic surfactant, choline chloride ionic liquid, and water were used to study the partitioning of three arsenic species (As(III), As(V), and dimethylarsinic acid (DMA)), in the absence and presence of the extracting agent ammonium pyrrolidine dithiocarbamate (APDC). Evaluation was made of the effects of the parameters extraction time, pH, tie-line length, extractant concentration, and nature of the surfactant on the distribution of the arsenic species in the ATPS. In the absence of the extracting agent, all the arsenic species were mainly transferred to the choline chloride-rich phase, with maximum extraction percentages of 69.02, 92.99, and 90.05% for As(III), As(V), and DMA, respectively. In the presence of APDC, the As(V) and DMA species mainly remained in the choline chloride-rich phase, while the As(III) was transferred to the surfactant-rich phase, with the best removal to this phase achieved using the ATPS formed by TX-165 + choline chloride + water, at pH 1.00 and with an APDC:arsenic molar ratio of 25. Compared to ATPSs formed by polymer and salt, the ATPSs formed by Triton X and choline chloride required up to nineteen times smaller amounts of APDC for quantitative extraction of As(III) to the phase low in electrolyte. Thus, the results demonstrated the excellent potential of the Triton X + choline chloride + water systems for the extraction, preconcentration, and speciation of arsenic, with the advantage that the components used to form the ATPSs make the technique more environmentally friendly.

Aqueous Two-Phase Systems for Cleanup and Recovery of Enzymes from Plants and Plant-Derived Extracts.

The increasing interest of the biopharmaceutical industry to exploit plants as a commercially viable production system is demanding the development of new strategies to maximize product recovery. Aqueous two-phase systems (ATPSs) are a primary recovery technique that has shown great potential for the efficient extraction and purification of biological products, from organelles to proteins and low-molecular-weight compounds. The evaluation of different system parameters upon the partitioning behavior can provide the conditions that favor the concentration of contaminants and the desired target protein in opposite phases. The protocols described here provide the basic strategy to explore the use of ATPSs for the isolation and partial purification of native and recombinant proteins from plants and plant-derived extracts.

Study of the partition of sodium diclofenac and norfloxacin in aqueous two-phase systems based on copolymers and dextran

The partitioning behavior of norfloxacin (more hydrophilic) and sodium diclofenac (more hydrophobic) was studied considering the use of aqueous two-phase systems (ATPSs) based in copolymers and dextran. For this research, the phase behavior of systems composed of water, a copolymer (Pluronic PE6400, Pluronic L35, Pluronic PE6800, PEG-ran-PPG monobutyl ether and PEG-ran-PPG) and dextran (Mw=40000 and 6000 g.mol−1) were investigated. The phase diagrams showed that by increasing the molecular weight and the copolymer PPG/PEG ratio, the biphasic region is enlarged. After defining the bionodal curves, a preliminary screening on the partition of two pharmaceutic ingredients (sodium diclofenac and norfloxacin) was carried. After selected the most appropriate ATPS for the partition of both drugs, namely systems based on diblock copolymers (poly(ethylene glycol)-poly(propylene glycol)) – dextran T6 and one random copolymer (poly(ethylene glycol)-ran-poly(propylene glycol)) – dextran T6, the effect of the copolymer concentration and different mixture points was assessed. In the end, the results showed that by increasing the copolymer concentration and tie-line length, the partition coefficient of the pharmaceutical ingredients increased in the block copolymer-based ATPS and decreased in systems containing the random copolymer.

Economic analysis of the production and recovery of green fluorescent protein using ATPS-based bioprocesses

Green fluorescent protein (GFP) is a useful biomolecule in biotechnology; however, its price makes its widespread application prohibitive. To overcome this issue, recently, the use of aqueous two-phase systems (ATPS) for GFP purification was proposed as an alternative platform to reduce processing costs. Aligned with this goal, this study performed bioprocess modelling coupled with economic analysis using the software Biosolve to evaluate the potential and commercial applicability of ATPS for GFP purification. This work analysed a collection of fourteen ATPS to discriminate through production costs while also incorporating the concept of product purity into the calculations. The two best systems (a PEG-based and an ionic liquid (IL)-based ATPS) were placed in a full bioprocess at different scale models (1 to 100 L) to elucidate the viability of applying ATPS at large scale. Although the results showed that the PEG-based ATPS exhibit the lowest costs (between USD 3.5x103.g−1 at 1 L and USD 0.33x103.g−1 at 100 L), for further developments, the inclusion of an ATPS granting a higher purity is desired for the development of simpler bioprocesses. Therefore, as a third approach in this work, a sensitivity analysis was performed to determine the impact of varying different model parameters (recovery yield, material costs discount and production titre), to elucidate the circumstances under which the IL-based system can overcome the production costs of the traditional PEG-based ATPS. The results indicate that the best cost-effectiveness approach is to improve the production titre (although it can affect all ATPS studied), as an increase from 1.33 to 3.8 g/L is enough for the IL-based ATPS to be less expensive than the traditional system at all analysed scales. This study demonstrates that ATPS can greatly reduce GFP manufacturing costs, which can potentially help to popularize new applications of fluorescent proteins that are currently mostly restricted to research kits due to their high prices.

Complete process for the selective recovery of textile dyes using aqueous two-phase system

Environmental problems arising from the release of dyes through industrial aqueous effluents are an emerging concern. To mitigate the problem, this work provides an experimental study on the use of aqueous two-phase systems (ATPS) for the selective separation and posterior recovery of dyes. Additionally, the complete recycling of the phase forming components is achieved. Azo dyes used in real textile industries, Direct Red 80 - DR80 and Direct Yellow 86 - DY86, were used. An initial screening of the ATPS forming elements was performed and the ATPS composed of 63 wt% acetonitrile (ACN) + 20 wt% cholinium bitartrate ([Ch][Bit]) + water at 25 °C were selected. An extraction efficiency of around 91% of DY86 to the ACN-rich phase and of approximately 99% of DR80 to the [Ch][Bit]-rich phase was obtained. The Kamlet-Taft parameters of the ATPS coexisting phases were measured aiming to understand the partitioning mechanism, especially for the migration of DY86 to the ACN-rich phase. The recovery of the DR80 from [Ch][Bit]-rich phase was of about 93% using a chitosan adsorbent (1:75, m/v) under constant stirring and at 45 °C, while ACN was completely recovered by evaporation. It was herein demonstrated that an ATPS based on a common organic solvent and a cholinium-based ionic liquid has potential for the separation and recovery of textile dyes.

Optimized Isolation of Extracellular Vesicles From Various Organic Sources Using Aqueous Two-Phase System

From biomarkers to drug carriers, Extracellular Vesicles (EVs) are being used successfully in numerous applications. However, while the subject has been steadily rising in popularity, current methods of isolating EVs are lagging behind, incapable of isolating EVs at a high enough quantity or quality while also requiring expensive, specialized equipment. The “isolation problem” is one of the major obstacles in the field of EV research - and even more so for their potential, widespread use for clinical diagnosis and therapeutic applications. Aqueous Two-Phase Systems (ATPS) has been reported previously as a promising method for isolating EVs quickly and efficiently, and with little contaminants - however, this method has not seen widespread use. In this study, an ATPS-based isolation protocol is used to isolate small EVs from plant, cell culture, and parasite culture sources. Isolated EVs were characterized in surface markers, size, and morphological manner. Additionally, the capacity of ATPS-based EV isolation in removing different contaminants was shown by measuring protein, fatty acid, acid, and phenol red levels of the final isolate. In conclusion, we have shown that EVs originating from different biological sources can be isolated successfully in a cost-effective and user-friendly manner with the use of aqueous two-phase systems.

Phase equilibrium and partitioning of cephalosporins (cephalexin, cefazolin, cefixime) in aqueous two-phase systems based on carbohydrates (glucose, fructose, sucrose, maltose) / acetonitrile

In recent decades, the use of aqueous two-phase system (ATPS) for bio-separation engineering has attracted increasing interest. In this work, ATPSs based on carbohydrates and acetonitrile were used to explore the partitioning behavior of cephalosporin antibiotics including cephalexin, cefazolin, and cefixime. For this purpose, four ATPSs containing acetonitrile and glucose, fructose, sucrose, and maltose have been studied. Initially, the phase diagrams were acquired at 300 K and then correlated by the Merchuk equation. The results showed that the ATPS composed of disaccharides have a broader two-phase region than monosaccharides. Furthermore, for the studied systems, tie-lines were obtained and their accuracy was examined by Othmer-Tobias and Bancroft equations. Moreover, tie-lines length and slope of tie-lines were determined. Besides, the partition coefficient of cephalosporins in four different ATPSs and a total of 48 tie-lines were investigated. Based on the results, it can be concluded that by increasing the concentration of carbohydrates, the partition coefficient increases. The maximum partitioning coefficient of cephalexin and cefazolin were obtained to be 19.2 and 17, respectively, with a recovery yield of 93% in the carbohydrate phase, which corresponds to the system (16 wt % glucose + 50 wt % acetonitrile + water). While the highest partitioning coefficient of cefixime was 6.4, with a recovery percentage of 81% for the system (16 wt % sucrose + 50 wt % acetonitrile + water). The results of this work indicate that these systems have a potential for separation and recovery of the understudied antibiotics.

Partitioning Behavior of Lysozyme and α-lactalbumin in Aqueous Two-Phase System Formed by Ionic Liquids and Potassium Phosphate

Abstract Nowadays ionic liquids (ILs), because of their “green” characteristics, have been used for analytical and separation processes. Therefore the partitioning of lysozyme and α-lactalbumin using aqueous two-phase systems (ATPSs) composed of an ionic liquid (chloride 1-ethyl-3-methylimidazolium) and inorganic salts (K2HPO4, KH2PO4) was studied. Phase equilibrium diagrams were obtained to explore the effect of the different temperatures (293.15, 303.15, 313.15, and 323.15) K and pH (7.5, 8.0, and 8.5) used for the partitioning studies. For both proteins, partition coefficients decreased with increasing temperature. The pH influenced the partition coefficients of lysozyme and α-lactalbumin. The thermodynamic parameters (ΔH◦, ΔS◦, ΔG◦) indicate thermodynamic differences between the partitioning of lysozyme and α-lactalbumin in this system. The ΔH◦, ΔS◦, and ΔG◦ values of the process studied showed that this process is spontaneous. This work demonstrates the possible use of ATPSs with ILs and inorganic salts as a methodology for the partitioning of lysozyme and α-lactalbumin.

Purification of clavulanic acid produced by Streptomyces clavuligerus via submerged fermentation using polyethylene glycol/cholinium chloride aqueous two-phase systems

Clavulanic acid (CA) is an important pharmaceutical compound produced by batch fermentation of Streptomyces clavuligerus. Since, CA is chemically unstable, its downstream processing should be studied to develop more efficient and resolute techniques. Herein, the use of aqueous two-phase systems (ATPS) composed of cholinium chloride, [Ch]Cl, was proposed as a novel platform for the recovery and purification of CA. Thus, the stability of CA in presence of different [Ch]Cl concentrations was initially studied, and the high biocompatibility of this salt was demonstrated by the low CA degradation levels. Then, the partitioning of CA using two types of polymeric ATPS has been investigated. Two ATPS formed by the combination of [Ch]Cl and two polyethylene glycol (PEG) polymers, PEG 600 g mol−1 (PEG-600) and polyethylene glycol methyl ether 550 g mol−1 (PEG-500-OMe), were used to assess the influence of the PEG nature, in addition to the concentration of the phase forming agents on the CA partitioning. It has shown that CA is almost equally distributed between the two-phases in equilibrium (0.6 < KCA < 1.6). Nevertheless, the selective extraction of CA for the [Ch]Cl-rich or PEG-rich phase by the proper adjustment of ATPS composition was attained. In the search for higher extraction efficiencies [EE (%)] and partition coefficients (KCA), a second polymeric ATPS platform composed of PEG-600 and sodium polyacrylate 8000 g mol−1 (NaPA-8000), applying [Ch]Cl as adjuvant was tested. The main results suggest the recovery of CA towards the PEG-rich phase (KCA ≥ 5.6 ± 0.6 and EE ≥ 85.5± 1.4%). The higher migration levels of CA have mainly resulted from the electronegative repulsion of NaPA-8000 over CA molecules. The ATPS with best performance for the CA extraction were selected for the recovery of CA directly from fermented broth of Streptomyces clavuligerus. In this set of experiments, the highest values of CA recovery yield and purification factor (respectively, 64.91± 1.99% and 22.70 ± 0.87) were attained for the systems PEG-600/NaPA-8000 and PEG-600/[Ch]Cl, respectively.

Use of aqueous two-phase PEG-salt systems for the removal of anionic surfactant from effluents

Linear alkylbenzene sulfonates (LAS) are synthetic anionic surfactants that are extensively used in many industries. As a result, large volumes of effluents containing high levels of these compounds are discharged into water bodies, causing risks to aquatic flora and fauna. Then, there is a need for environmentally safe and economically viable technologies for the removal of LAS from aqueous matrices. The present work evaluates the use of aqueous two-phase systems (ATPS) composed of PEG and sulfate salts for this purpose, considering the effects of tie line length (TLL), molar mass of polymer, and type of cation-forming salt on the partitioning behavior of LAS. All the LAS partition coefficient (KLAS) values were greater than unity, and the LAS extraction efficiencies (%ELAS) were higher than 97%. The system consisting of PEG 1500 + (NH4)2SO4 + H2O provided the highest KLAS (1083.34) and %ELAS (99.9%), indicating that the method provided good extraction of LAS to the top phase. This system was applied using a real effluent sample in laboratory-scale experiments as well as in bench-scale batch trials. The results obtained at the laboratory scale showed %ELAS values greater than 98%, while the best KLAS value obtained in the batch experiments was 8.50 (±1.75) (%ELAS = 78.17%). These values demonstrated the potential of ATPS for the removal of LAS from industrial effluents.

Recovery of laccase from processed Hericium erinaceus (Bull.:Fr) Pers. fruiting bodies in aqueous two-phase system

The feasible use of aqueous two-phase systems (ATPSs) to establish a viable protocol for the recovery of laccase from processed Hericium erinaceus (Bull.:Fr.) Pers. fruiting bodies was evaluated. Cold-stored (4.00±1.00°C) H.erinaceus recorded the highest laccase activities of 2.02±0.04U/mL among all the processed techniques. The evaluation was carried out in twenty-five ATPSs, which composed of polyethylene glycol (PEG) with various molecular weights and potassium phosphate salt solution to purify the protein from H. erinaceus. Optimum recovery condition was observed in the ATPS which contained 17% (w/w) PEG with a molecular weight of 8000 and 12.2% (w/w) potassium phosphate solution, at a volume ratio (VR) of 1.0. The use of ATPS resulted in one-single primary recovery stage process that produced an overall yield of 99% with a purification factor of 8.03±0.46. The molecular mass of laccases purified from the bottom phase was in the range of 55-66kDa. The purity of the partitioned laccase was confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Application of affinity aqueous two-phase systems for the fractionation of CD133(+) stem cells from human umbilical cord blood.

In a further attempt to establish a novel stem cell primary recovery strategy, the use of aqueous two-phase systems (ATPS) complemented with the use of antibodies (known as immunoaffinity ATPS) is explored in this work. This type of liquid-liquid extraction systems exploits antigen-antibody affinity and represents a novel and selective approach for the purification of stem cells. The proposed bioengineering strategies include the implementation of traditional [polyethylene glycol (PEG), dextran (DEX) and ficoll] and novel (Ucon) immunoaffinity ATPS to prove the viability of cluster of differentiation 133 (CD133(+) ) stem cells from human umbilical cord blood. Furthermore, the addition of the antibody is implemented to identify conditions under which contaminants and stem cells of interest concentrate in opposite phases. The objective of this work is to establish the initial basis for the development of a novel and scalable purification bioprocess for the selective recovery of CD133(+) stem cells employing immunoaffinity ATPS. The reported methodology allows a partitioning of 62% CD133(+) stem cells to the top phase of the ficoll 400,000-DEX 70,000 immunoaffinity ATPS. In PEG 8,000-DEX 500,000 and Ucon-DEX 75,000 systems, no difference was observed when compared with the conventional ATPS (without antibody addition), as the CD133 antibody does not have preference for the desired clean top phase. In all experiments, cell viability was at least 98% after ATPS recovery. This research highlights the challenges that must be addressed to allow the potential establishment of a separation process using immunoaffinity ATPS for the recovery and purification of stem cells.

Aqueous two-phase printing of cell-containing contractile collagen microgels

This work describes the use of aqueous two-phase systems to print cell-containing contractile collagen microdroplets. The fully aqueous conditions enable convenient formation of sub-microliter ‘microgels’ that are much smaller than otherwise possible to fabricate while maintaining high cell viability. The produced microgels contract over several days, mimicking the behavior of macroscale contraction assays, which have been valued as an important biological readout for over three decades. Use of microgels not only reduces reagent consumption and increases throughput of the assay, but also improves transport of molecules into and out of the collagen matrix, thereby enabling efficient and more precise studies of timed stimulation profiles. Utility of the technology is demonstrated by analyzing the effects of TGF-β1 on gel contraction, and we demonstrate that brief ‘burst’ stimulation profiles in microgels prompt contraction of the matrix, a feature not observed in the conventional macroscale assay. The fully aqueous process also enables the integration of contractile collagen microgels within existing cell culture systems, and we demonstrate proof-of-principle experiments in which a contractile collagen droplet is fabricated in situ on an existing epithelial monolayer. The simplicity, versatility and ability to robustly produce collagen microgels should allow effective translation of this microengineering technology into a variety of research environments.

A novel aqueous two phase assisted platform for efficient removal of process related impurities associated with E. coli based biotherapeutic protein products

This article presents a variant of aqueous two phase system (ATPS) as a tool for selective removal of process related impurities associated with Escherichia coli, namely host cell proteins and nucleic acids. Granulocyte colony stimulating factor (GCSF) expressed in E. coli has been selected as a model protein for the study. While achieving effective removal of host cell impurities as per the regulatory requirement for recombinant therapeutics, high product recovery has been achieved by adopting a novel strategy involving resolubilization of interfacial GCSF precipitate. This has been done such that the structural and biological activity of the product is retained. Exhaustive analysis of structural as well as functional integrity of resolubilized GCSF has been carried out using advanced analytical and in vitro bioassay tools. Product recovery of 99.5% has been achieved with the concentration of host cell proteins less than 100ppm and of nucleic acids below 10ng/ml. We think that the proposed platform can enable use of ATPS as a more economical alternative to process chromatography in industrial biopharmaceutical manufacturing processes.

Isolation of natural red colorants from fermented broth using ionic liquid-based aqueous two-phase systems

There is a growing demand for natural colorants. This is prompting the search for new alternative and "benign" separation systems allowing higher recoveries, extraction yields, and selectivities. This work investigates the use of aqueous two-phase systems (ATPS) based on ionic liquids as extraction processes for the recovery of red colorants from the fermented broth of Penicillium purpurogenum DPUA 1275. Several ATPS based on quaternary ammonium and imidazolium were studied in this work aiming at separating the red colorants produced from the remaining colorants and contaminant proteins present in the fermented broth. The results suggest that the red colorants can be isolated by an appropriate manipulation of some of the process conditions, such as the use of quaternary ammonium with short alkyl chains, alkaline media, and short tie-line lengths (extraction point systems with lower concentrations of ionic liquid). These conditions allow large partition coefficients for the red colorants (K red=24.4±2.3), high protein removal (60.7±2.8%) and selectivity parameters (S red/prot=10.05).