Formation Of Aqueous Two-phase Systems Research Articles

Thermodynamic studies of solute–solute and solute–solvent interactions in ternary aqueous systems containing {betaine + PEGDME250} and {betaine + K3PO4 or K2HPO4} at 298.15 K

In this work, to evaluate solute–solute, solute–solvent and phase separation in aqueous systems containing {betaine + poly ethylene glycol dimethyl ether with molar mass 250 g mol−1 (PEGDME250)}, {betaine + K3PO4} and {betaine + K2HPO4}, first water activity measurements were made at 298.15 K and atmospheric pressure using the isopiestic technique. The water iso-activity lines of these three systems were obtained which have positive deviations from the semi-ideal solutions. This suggests that betaine-polymer and betaine-K3PO4 or betaine-K2HPO4 interactions are unfavorable; and these mixtures may form aqueous two-phase systems (ATPSs) at certain concentrations. Indeed the formation of ATPSs was observed experimentally. Then, osmotic coefficient values were calculated using the obtained water activity data; and, using the polynomial method the solute activity coefficients were determined. Using these activity coefficients, the transfer Gibbs energy (Delta {G}_{tr}^{i}) values were calculated for the transfer of betaine from aqueous binary to ternary systems consisting polymer (PEGDME250) or salts (K3PO4 and K2HPO4). The obtained positive Delta {G}_{tr}^{i} values again indicated that there is unfavorable interaction between betaine and these solutes. Finally, the volumetric and ultrasonic studies were made on these systems to examine the evidence for the nature of interactions between betaine and the studied salts or polymer.

Anthocyanin Partition in Aqueous Two‐Phase Systems Based on Isopropanol and Sodium/Ammonium Sulfate

AbstractPartitioning of anthocyanins contained in the crude extract of Syzygium cumini fruit was investigated in aqueous two‐phase systems (ATPS) made up of isopropanol and Na2SO4/(NH4)2SO4. Na2SO4 induced ATPS formation more effectively than (NH4)2SO4. The increase in temperature enhanced phase separation in the (NH4)2SO4 ATPS, while the opposite occurred in the Na2SO4 ATPS. The higher the overall mixture concentration or temperature, the higher the values of tie‐line length and slope. In all systems and conditions, anthocyanins preferentially partitioned to the top phase. The partition coefficient and theoretical recovery yield in the top phase varied in the ranges 1.14–1.77 and 53.31–63.87 % in the (NH4)2SO4 ATPS, and in the ranges 1.94–21.50 and 65.90–95.55 % in the Na2SO4 ATPS.

Ionic liquid-based multi-stage sugaring-out extraction of lactic acid from simulated broth and actual lignocellulosic fermentation broth

In this study, ionic liquid-based sugaring-out extraction was developed to separate lactic acid from the synthetic solution and actual lignocellulosic fermentation broth. Except for [EOHmim]BF4, the ILs with BF4− and OTF− anion can form aqueous two-phase system (ATPS) with the aid of saccharides. With the same kind of saccharides, the ATPS formation ability of ILs could be promoted by increasing the side-chain length of ILs in the order of [Hmim]BF4 ≈ [Bmim]BF4 ˃ [Emim]BF4 due to the decrease in ILs’ kosmotropicity. On the other hand, for the same type of ILs, an ATPS was formed more easily with glucose than with xylose. When IL concentration varied from 35% (w/w) to 40% (w/w) at a low glucose concentration of 15% (w/w), an interesting phase reversal was observed. When lactic acid was undissociated at pH 2.0, 51.8% LA and 92.3% [Bmim]BF4 were partitioned to the top phase, and 97.0% glucose to the bottom phase using an ATPS consisting of 25% (w/w) glucose and 45% (w/w) IL. The total recovery of LA would increase to 89.0% in three-stage sugaring-out extraction from synthetic solution. In three-stage sugaring-out extraction from the filtered and unfiltered fermentation broth obtained via simultaneous saccharification and co-fermentation (SSCF) of acid-pretreated corn stover by the microbial consortium, the total recovery of LA was 89.5% and 89.8%, respectively. Furthermore, the total removal ratio of cells and pigments from the unfiltered broth was 68.4% and 65.4%, respectively. The results support IL-based sugaring-out extraction as a potential method for the recovery of lactic acid from actual fermentation broth.Graphical

Triple soluting-out effect in ionic liquid− based aqueous triphasic systems revealed by vapor pressure osmometry

An experimental study was conducted on aqueous triphasic systems (A3PSs) to increase molecular understanding of the soluting effect phenomenon occurring in these systems. For aqueous quaternary systems 1‑butyl‑3-methylimidazolium bromide ([Bmim][Br]) + trisodium citrate (Na3Cit) + polypropylene glycol 400 (PPG400) and [Bmim][Br] + disodium sulfate (Na2SO4) + PPG400 which were able to form A3PS, the vapor-liquid equilibria measurements were conducted at 318.15 K. For these systems, deviations from the semi-ideal behavior were addressed using the vapor pressure osmometry (VPO) measurements in monophasic (MR), biphasic (BR), and three-phasic (TR) concentration regions. The VPO data revealed that, because of unfavorable IL-salt, IL-polymer, and polymer-salt interactions as well as the preferential hydration of solutes, the amount of free water molecules in the IL + polymer + salt + water solutions is less than that expected based on the semi-ideal behavior. Therefore, under condition that solutes molalities in the quaternary solutions are the same as those in the corresponding binary solutions, (aw+2)−(awIL∘+awP∘+awS∘) and Δp−(ΔpIL∘+ΔpP∘+ΔpS∘) have negative values and become less negative by formation of aqueous two-phase system (A2PS) and become positive with approaching the triphasic region. The triple soluting-out effect resulted from the unfavorable interactions between all pairs of solutes leads to preferential hydration of solutes and thereby creation of three aqueous salt-rich, IL/polymer-rich, and polymer-rich phases in solution for entropy reason.

Pre-purification of genipin from genipap using aqueous-two-phase systems composed of protic ionic liquids + polymers + water at 298 K and atmospheric pressure

The downstream processing still represents the cost bottleneck of obtaining biomolecules. Aqueous two-phase systems (ATPS) composed of alternative constituents have been presented as a cost-effective platform for the concentration, partition and/or purification of biomolecules. In this sense, this work provides a series of new ATPS composed of polymers (PPG-400, PEG-300, -400, -550 and -750 g gmol−1) + protic ionic liquids (PILs) [based on ethylene glycols (mono-, di- - and tri-ethylene glycol) as cation and inorganic anions (H2PO4−, HSO4−, Cl−, NO3−)] + water at 298 K and atmospheric pressure. An initial screening evaluated the effect of the type of polymer, molecular weight of the polymer, type of cation and anion of the ionic liquid on phases’ separation, while the second series of experiments studied their application as effective platforms for the pre-purification of genipin from genipap. The results showed that the hydrophilic/hydrophobic balance between phase forming components is the driving force for the formation of ATPS, as well as for the pre-purification of genipin from genipap. The system constituted of PEG-750 (40 wt%) + [MEA][H2PO4] (30 wt%) + H2O (30 wt%) exhibited the highest genipin pre-purification performance, i.e., purification factor of 6.03, as confirmed chromatographically.

Aqueous two-phase system formed by alkanolammonium-based Protic Ionic Liquids and acetone: Experimental data, thermodynamic modeling, and Kraft lignin partition

Aqueous two-phase systems (ATPS) formed by Protic Ionic Liquids (PILs) are a potential alternative to recovery PILs after their use in pretreatment step and valorization of lignocellulosic biomass. Besides, ATPS based on PILs may be applied to Kraft lignin fractionation toward a more sustainable environment. In this study, nine alkanolammonium-based PILs were synthesized using organic acids (formic, acetic, propionic, lactic, and glycolic) as precursors. These PILs were combined with acetone to form ATPS at low temperatures, and the partition of Kraft lignin was tested using these same systems. The presence of an extra hydroxyl group in the PILs’ anionic part increased their ability to form ATPS, due to the increase in their acidity. Also, increasing the alkyl chain length of the PILs’ anionic part impairs the formation of ATPS. In contrast, the behavior was observed for the cationic part, due to the increase in the PILs’ hydrophobicity nature. Regarding the partition of Kraft lignin applying 2-hydroxyethylammonium-based PILs, the increase in the alkyl chain length of the PILs’ anionic part leads to stronger PILs-lignin interactions, which favored the lignin’s migration to the bottom phase (PILs-rich phase), and lower partition coefficient values, as follows: formate < acetate < propionate anions. The presence of an extra hydroxyl group in the PILs’ anionic part (lactate and glycolate-based PILs) impaired PILs-lignin interactions, and consequently, increased the partition coefficient, compared to propionate and acetate-based PILs. Finally, the increase in the PILs’ cationic part leads to the improvement in PILs-lignin interactions, and lower partition coefficient values were observed, following the sequence for lactate-based PILs: tris(2-hydroxyethyl)ammonium < bis(2-hydroxyethyl)ammonium < 2-hydroxyethylammonium; and the following sequence for propionate-based PILs: tris(2-hydroxyethyl)ammonium ≈ bis(2-hydroxyethyl)ammonium < 2-hydroxyethylammonium. The glycolate-based PIL showed the highest partition coefficient value, which is related to its small alkyl chain length and the presence of an extra hydroxyl group, making PIL-lignin interactions weaker. The results showed that acetone emerges as a phase-forming agent to remove, at least partially, the lignin dissolved in PILs aqueous solutions, allowing their recycle and reuse after the pretreatment step, for example. Finally, the NRTL activity coefficient model was used to correlate tie-line compositions, and the interaction parameters were estimated for each system determined.

Protic ionic liquids as constituent of aqueous two-phase system based on acetonitrile: Synthesis, phase diagrams and genipin pre-purification

The aim of this study was to evaluate the formation of aqueous two-phase systems (ATPS) based on different protic ionic liquids (PIL), synthesized using amines, and organic or inorganic acid, and acetonitrile (ACN). The ATPSs are used to pre-purify genipin from genipap (Genipa americana L.). All PILs (purity above 98%) were able to form ATPSs with ACN, the size of the biphasic region being directly dependent on salting-out aptitude of PIL anions and relative hydrophilicity of the corresponding cations. The highest solid–liquid extraction of genipin from genipap was achieved by using aqueous ACN solution at 75% (16.79 ± 0.06 mg g−1 dry raw material) as extractant. After the extraction stage, the ATPS was used as an integrated platform to pre-purify the genipin present in the extract. Three different overall mixture point compositions were tested, and those formed by [MEA][H2Cit] (25 wt%) + ACN (45 wt%) + water (30 wt%) at 298.15 ± 1.00 K and 0.10 ± 0.01 MPa showed the highest selectivity (S = 4.202 ± 0.009) and recovery of genipin in top phase (RTG = 56.74 ± 0.04%), and the purification factor in top phase was 5.9 fold.

A stepwise mechanism for aqueous two-phase system formation in concentrated antibody solutions

Aqueous two-phase system (ATPS) formation is the macroscopic completion of liquid-liquid phase separation (LLPS), a process by which aqueous solutions demix into 2 distinct phases. We report the temperature-dependent kinetics of ATPS formation for solutions containing a monoclonal antibody and polyethylene glycol. Measurements are made by capturing dark-field images of protein-rich droplet suspensions as a function of time along a linear temperature gradient. The rate constants for ATPS formation fall into 3 kinetically distinct categories that are directly visualized along the temperature gradient. In the metastable region, just below the phase separation temperature, Tph , ATPS formation is slow and has a large negative apparent activation energy. By contrast, ATPS formation proceeds more rapidly in the spinodal region, below the metastable temperature, Tmeta , and a small positive apparent activation energy is observed. These region-specific apparent activation energies suggest that ATPS formation involves 2 steps with opposite temperature dependencies. Droplet growth is the first step, which accelerates with decreasing temperature as the solution becomes increasingly supersaturated. The second step, however, involves droplet coalescence and is proportional to temperature. It becomes the rate-limiting step in the spinodal region. At even colder temperatures, below a gelation temperature, Tgel , the proteins assemble into a kinetically trapped gel state that arrests ATPS formation. The kinetics of ATPS formation near Tgel is associated with a remarkably fragile solid-like gel structure, which can form below either the metastable or the spinodal region of the phase diagram.

Aqueous Two-Phase System Formation in Small Droplets by Shirasu Porous Glass Membrane Emulsification Followed by Water Extraction

By utilizing water transport phenomena between two different water-in-oil (W/O) emulsion droplets through continuous oil phase, we developed a novel method of aqueous two-phase system (ATPS) formation in small droplets prepared by Shirasu porous glass (SPG) membrane emulsification technique. When we mixed W/O emulsion droplets containing poly(ethylene glycol) (PEG) and dextran (DEX) at concentrations below the threshold of the phase separation, with droplets containing other solutes at high concentrations, water extraction from the droplets containing PEG and DEX to those containing the other solutes occurred, owing to the osmotic pressure difference. This effect increased the concentrations of PEG and DEX in the droplets above the phase separation threshold. We demonstrated the feasibility of the preparation method by varying the pore sizes of the SPG membranes, the solutes, and their concentrations. Only when the concentration of the solute was high enough to extract sufficient amounts of water did the homogeneous disperse phase consisting of PEG and DEX in droplets turn into a PEG-rich phase and DEX-rich phase, showing ATPS. This result was irrespective of the solute itself and pore size of the SPG membrane. In particular, we successfully demonstrated monodisperse ATPS droplets with diameters of approximately 10 μm under a certain condition.

Microfluidic Formation of Hydrogel Microcapsules with a Single Aqueous Core by Spontaneous Cross-Linking in Aqueous Two-Phase System Droplets.

We report a simple process to fabricate monodisperse tetra-arm poly(ethylene glycol) (tetra-PEG) hydrogel microcapsules with an aqueous core and a semipermeable hydrogel shell through the formation of aqueous two-phase system (ATPS) droplets consisting of a dextran-rich core and a tetra-PEG macromonomer-rich shell, followed by a spontaneous cross-end coupling reaction of tetra-PEG macromonomers in the shell. Different from conventional techniques, this process enables for the continuous production of hydrogel microcapsules from water-in-oil emulsion droplets under mild conditions in the absence of radical initiators and external stimuli such as heating and ultraviolet light irradiation. We find that rapid cross-end coupling reaction of tetra-PEG macromonomers in ATPS droplets in the range of pH from 7.4 to 7.8 gives hydrogel microcapsules with a kinetically arrested core-shell structure. The diameter and core-shell ratio of the microcapsules can be easily controlled by adjusting flow rates and ATPS compositions. On the other hand, the slow cross-end coupling reaction of tetra-PEG macromonomers in ATPS droplets at pH 7.0 and lower induces structural change from core-shell to Janus during the reaction, which eventually forms hydrogel microparticles with a thermodynamically stable crescent structure. We believe that these hydrogel microparticles with controlled structures can be used in biomedical fields such as cell encapsulation, biosensors, and drug delivery carriers for sensitive biomolecules.

Purification of the Recombinant Green Fluorescent Protein Using Aqueous Two-Phase System Composed of Recyclable CO2-Based Alkyl Carbamate Ionic Liquid.

The formation of aqueous two-phase system (ATPS) with the environmentally friendly and recyclable ionic liquid has been gaining popularity in the field of protein separation. In this study, the ATPSs comprising N,N-dimethylammonium N′,N′-dimethylcarbamate (DIMCARB) and thermo-responsive poly(propylene) glycol (PPG) were applied for the recovery of recombinant green fluorescent protein (GFP) derived from Escherichia coli. The partition behavior of GFP in the PPG + DIMCARB + water system was investigated systematically by varying the molecular weight of PPG and the total composition of ATPS. Overall, GFP was found to be preferentially partitioned to the hydrophilic DIMCARB-rich phase. An ATPS composed of 42% (w/w) PPG 1000 and 4.4% (w/w) DIMCARB gave the optimum performance in terms of GFP selectivity (1,237) and yield (98.8%). The optimal system was also successfully scaled up by 50 times without compromising the purification performance. The bottom phase containing GFP was subjected to rotary evaporation of DIMCARB. The stability of GFP was not affected by the distillation of DIMCARB, and the DIMCARB was successfully recycled in three successive rounds of GFP purification. The potential of PPG + DIMCARB + water system as a sustainable protein purification tool is promising.

Polyethylene glycol acquires certain compensatory solute properties when used to form aqueous two-phase extraction systems

The polyethylene glycols are currently considered an essential class of polymers with several applications in industry, medicine, and biology. The aqueous two-phase (ATP) extraction based on polyethylene glycol (PEG) and certain salts have gained the attention for further usage in protein purification as a simple and cheap technique; however, it is still under development. In this paper, the formation of ATP systems by PEG as a model polymer and sodium sulfate was studied focusing on (1) some of the conditions under which the polymer may have definite compensatory solute properties, and (2) the analysis of the crystallization degree and the conditions for forming di and triphasic systems. The results demonstrate that the polymer in some systems may have inherent compensatory solute-like behavior which becomes evident under certain conditions, including the type of the other solute molecules, solvent type, concentration, and pH of the system.

Effect of electrolytes as adjuvants in GFP and LPS partitioning on aqueous two-phase systems: 1. Polymer-polymer systems

Abstract The production of recombinant biopharmaceuticals is highly dependent of a proper choice of the downstream processing stages. Particularly, the purification that must ensure that all the endotoxins (lipopolysaccharides - LPS) are efficiently removed from the final product. The efficient removal of LPS has a direct impact on the manufacturing of therapeutic biopharmaceuticals, since LPS is naturally presented in Gram-negative bacterial expression systems. In order to provide a more simple and faster technique for the purification of green fluorescent protein (GFP) and LPS removal, aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) and poly(acrylic acid) (NaPA) and electrolytes were studied. Firstly, the binodal curves of PEG/NaPA + salt systems were established using NaCl, Li2SO4, KI, and KNO3 as additives. It was demonstrated that the formation of ATPS is enhanced following the anion tendency: SO42− > Cl− ≫ NO3− > I−. The stability of GFP in the presence of all different phase forming agents (polymers and salts) was evaluated, and the high biocompatibility of these ATPS demonstrated by the maintenance of GFP fluorescence in all conditions under study. Then, the GFP extraction and LPS removal aptitude of each ATPS was investigated. GFP and LPS were preferentially partitioned into the top (PEG-rich) phase (KGFP > 20), but with a removal of 35% of LPS was attained. Hydrophobic and electrostatic interactions were found to be the major driving forces for GFP partitioning and LPS removal. Moreover, a new effect was found, the presence of high loads of LPS can affect (decrease) the KGFP values (KGFP without LPS > KGFP with 104 EU/mL > KGFP with 106 EU/mL). The ATPS with best GFP extraction performance was selected for the recovery directly from the cell lysates of Escherichia coli. In this experiment, the system composed of 12 wt% PEG 1000 g/mol, 12 wt% NaPA 8000 g/mol, and 0.25 M Li2SO4 lead to a LPS removal (REMLPS) of 13%, KGFP of 20.2, and high selectivity relatively to the total proteins (S = 20), since the majority of contaminants proteins were preferentially partitioned into the bottom (NaPA-rich) phase (purification factor of 4-fold). It is here demonstrated that the ATPS composed of PEG/NaPA + salts as additives can be used as a first step for the recovery of GFP and removal of contaminants (LPS in part, and most contaminant proteins) from cell lysates by applying a system with low polymer content and using mild conditions.

Choline derivative ionic liquids-based aqueous two-phase systems: Phase diagrams and partition of purine alkaloids

Abstract Searching for environmentally friendly aqueous two-phase systems is imperative for the sustainable development of separation technologies. In this work, five kinds of environmentally benign choline derivative ionic liquids were synthesized and used for the formation of aqueous two-phase systems with K3PO4, K3C6H5O7 or K2CO3. In order to understand the phase formation processes of these aqueous two-phase systems and their possible application in extraction, phase diagrams and partition coefficients of purine alkaloids were determined at T = 298.2 K, and the effects of structure of the ionic liquids, nature of the purine alkaloids, composition and temperature of the systems on the partitioning behaviour of purine alkaloids were investigated systematically. It was shown that caffeine, theobromine and 2,6-amino purine could be extracted effectively into the ionic liquid-rich phase of the aqueous two-phase systems, and the partition coefficients were as high as 4.5–42 under the optimized conditions. The partitioning was found to decrease with the increase of the ratio of H2O weight fraction (in the ionic liquid-rich phase and in the salt-rich phase) and hydrophobicity of both the purine alkaloids and the ionic liquids, but not sensitive to the change of temperature.

Use of protic ionic liquids as adjuvants in PEG-based ATPS for the purification of radish peroxidase

This work addresses the use of protic ionic liquids (PIL) as adjuvants (5 wt%) in the formation of aqueous two-phase systems (ATPS), based on polyethylene glycol with different molecular weights + ammonium sulfate + water for peroxidase purification from alternatives vegetables as radish peroxidase (Raphanus sativus L.) from horseradish root peroxidase. Protic ionic liquid as a process adjuvant did not extend the biphasic area in the phase diagram, but the phase behavior was modified according to the hydrophobicity of the PIL used and additional interactions between the enzyme and PIL increased the purification factor of the radish peroxidase from ≈2.5 (no ionic liquid) to ≈ 19.2 fold using 2-hydroxyethylammonium propionate as adjuvant in ATPS.

Integrated process for purification of capsaicin using aqueous two-phase systems based on ethanol

Capsaicin is a very powerful alkaloid with important pharmacological effects and pain relief additives. It also aids in cancer prevention and weight reduction, among others benefits. New and promising techniques based on microwave-assisted and aqueous two-phase systems are considered “green techniques” for extraction and purification of bioactive compounds. This work discusses an integrated process of extraction and purification of capsaicin from biomass Capsicum chinense var. cumari-do-Pará by a microwave-assisted method followed by aqueous two-phase systems (ATPS) based on ethanol+sodium salts. The diagram phases of ATPS were complete characterized and the ability of the salts to promote the formation of ATPS with ethanol follows this trend: NaH2PO4<Na2S2O3<Na2SO4<Na2CO3. Using the optimized conditions, the capsaicin from its natural source, was purified around 5.2-fold with an extraction efficiency of ≈85.6% for the ethanol-rich phase, and DPPH radical inhibition capacity of ≈80%.

Extraction and consecutive purification of anthocyanins from grape pomace using ionic liquid solutions

The global wine industry generates approximately 8.5 million tons of waste annually, which contains several biomolecules of high value. The present study focuses on the extraction of anthocyanins from grape pomace via solid/liquid extraction with aqueous solutions of the ionic liquid 1-ethyl-3-methylimidzaolium acetate [(C2mim]OAc) and sequential purification in aqueous two-phase systems (ATPS) with additional salts. The optimum conditions for solid/liquid extraction were obtained using response surface methodology and yielded up to 3.58 mg g−1 anthocyanins. During ATPS formation, the highest purification factor (PF = 16.2 fold) was obtained in the bottom and salt-rich phase using K2CO3 (29.41 wt%) and [C2mim]OAc (29.28 wt%) at 35 °C and atmospheric pressure. The partition process is exothermic, spontaneous and governed by enthalpy forces.

Primary and secondary aqueous two-phase systems composed of thermo switchable polymers and bio-derived ionic liquids

The liquid-liquid equilibrium (LLE) data for aqueous two-phase systems (ATPSs) comprising poly(propylene glycol) 400 (PPG 400) and cholinium-aminoate-based ([Ch][AA]) ionic liquid were determined experimentally at T=(288.15 and 308.15)K, while the LLE data at T=298.15K was adopted from our previous work for comparison. The experimental binodal data were satisfactorily fitted to a temperature-dependent nonlinear empirical expression. The reliability of tie-line data was confirmed by fitting the experimental data with the Othmer-Tobias and Bancroft equations. Furthermore, for the first time, the electrolyte nonrandom two-liquid model (e-NRTL) was used to correlate the tie-line data of PPG 400+[Ch][AA]+water systems. The correlations of LLE data using these models provide a good description of the experimental values. The effect of temperature on the phase-forming capabilities of the corresponding [Ch][AA] was assessed using the experimental binodal data and the salting-out coefficient (k2) derived from the Setschenow-type equation. The values of k2 were well correlated to the phase-forming abilities of [Ch][AA], and were found to increase at higher temperature. Upon heating to 308.15K, the solution of (PPG 400)-rich top phase from the primary PPG 400+[Ch][AA]+water systems formed the secondary ATPSs. The LLE data of the secondary PPG 400+[Ch][AA]+water systems was also determined. The PPG 400 was concentrated in the top phase of the secondary ATPS; this could serve as a means to recover PPG 400 from the primary ATPS via the formation of secondary ATPS.

Phosphoric acid partition in aqueous two phase systems

In this work, a polyethylene glycol (PEG)/Na2SO4 aqueous two phase system (ATPS) was formulated for the extraction of phosphoric acid from an aqueous solution to a polymer rich phase. The optimal conditions to form a biphasic system in the presence of phosphoric acid were determined experimentally. The phase diagram of ATPS composed of water, PEG, Na2SO4 and H3PO4 was obtained. Different components as PEG 1000g∙mol−1, Na2SO4, H3PO4, phosphoric and acid impurities as Al(III), Ca(II), Fe(III), Cd(II), V(IV) were titrated in each phase of the systems showing that it was possible to transfer approximately a 75% of loaded H3PO4 to the polymer rich phase. Moreover, less than 15% of ions were extracted except for V(IV) for which it was 35%. The stripping of H3PO4 has been performed by the formation of Na2HPO4 using NaOH, which led to the formation of a second ATPS allowing the recovery of 87% of phosphate salt in the salt rich phase in only one stage. This work demonstrated for the first time the possible use of ATPS for designing a greener purification process of phosphoric acid.

Recovery of PEGylated and native lysozyme using an in situ aqueous two‐phase system directly from the PEGylation reaction

AbstractBACKGROUNDPurification of PEGylated proteins from reactions is still a challenge due to the formation of isomers. In this work, the recovery of PEGylated lysozyme was studied in a novel integrative approach called in situ aqueous two‐phase systems (ATPS). The excess of PEG in the lysozyme PEGylation reaction (LPR) was used as part of the phase forming chemicals with different polymer (UCON, ficoll, dextran) and salt (sodium phosphate, potassium sulphate, sodium sulphate, ammonium sulphate, sodium carbonate) solutions.RESULTSThe best option for the in situ ATPS formation was the addition of a 4 mol L−1 ammonium sulphate in 20 mmol L−1 Tris–HCl (pH 7.0) solution to the PEGylation reaction. PEGylated conjugates of lysozyme exhibited a preferential partition to the top (PEG‐rich) phase while native protein was partitioned to the bottom (salt‐rich) phase. Di‐PEGylated lysozyme partitioned entirely to the top phase (recovery of 100%), while the mono‐PEGylated protein presented a recovery yield of 56% in the top phase. On its part, 97.7% of the native lysozyme was concentrated in the bottom phase.CONCLUSIONThe proof‐of‐concept presented in this work allows the recovery of PEGylated proteins directly from a PEGylation reaction. It is the first time that PEGylated proteins have been fractionated using a PEG–ammonium sulphate system taking advantage of the unreacted PEG left in a PEGylation reaction. Based on reaction engineering, in situ ATPS are a potential mechanism for PEGylation control, a good alternative for ‘packed‐bed’ or on‐column PEGylation processes and a pioneer strategy in the development of phase transfer catalysis in PEGylation. © 2017 Society of Chemical Industry