Aqueous Biphasic Systems Research Articles

Liquid-liquid equilibria of aqueous biphasic systems containing 1-alkyl-3-methylimidazolium amino acid ionic liquids with different anions (L-Leucine, L-Valine, L-Lysine) and inorganic salt (tripotassium phosphate, potassium carbonate)

Aqueous biphasic systems containing water-miscible ionic liquids (ILs) and inorganic salt are considered as an effective media for liquid-liquid extraction of bioactive materials. One of the important advantages of ILs is the possibility for a modification of their chemical structure and, as a result, their properties, i.e. their multifunctionality. Among the modified ILs, the amino acid ILs (AAILs) attract much attention due to their low toxicity and biodegradability. In the present work, new experimental data on phase behavior (binodal curves and tie lines) were obtained at 298.15 K for the systems containing 1-alkyl-3-methylmidazolium AAILs, [Cnmim]Х, with different hydrocarbon chain length (n = 4, 8), and with the amino acid anions: X = [Leu] (L-Leucine), [Val] (L-Valine), [Lys] (L-Lysine), and inorganic salts (K3PO4, K2CO3). AAILs under investigation were synthesized and characterized by means of thermogravimetric analysis, 1H- and 13C-NMR spectra. The ability of the studied systems to phase separation, the lengths and the slopes of the tie lines were discussed in terms of AAIL's structure. Systems containing [C8mim][Lys] show stronger tendency for phase separation. The slopes of the tie lines increase with the length of alkyl chain of IL's cation. For the aqueous systems [Сnmim]X + inorganic salt, the slopes of the tie lines increase in the sequence: [Lys] < [Val] < [Leu] < Cl < Br. Phase behavior of the investigated ternary systems favors their use for the extraction of bio-solutes.

Alcohol-based aqueous biphasic system applied to partition four different natural bioactive compounds from Garcinia indica Choisy

ABSTRACT An attempt was made to simultaneously partitioning Anthocyanins (ACN), Hydroxycitric acid (HCA), Garcinol (GL), and Isogarcinol (IGL) from fruit rinds of Garcinia indica using the 1-propanol and magnesium sulfate system. The influences of 1-propanol and magnesium sulfate concentration and Tie line Length (TLL) were investigated to obtain the optimal partitioning and recovery of GL and IGL into the top-phase and ACN and HCA to the bottom phase. The 25% w/w 1-propanol and 12% w/w magnesium sulfate system showed the maximum extraction efficiency for all the compounds. The purity and recovery were further improved by employing the second stage ATPS.

Designing novel biocompatible oligopeptide-based ionic liquids for greener downstream processes

In this work, a new family of ILs is here firstly presented, built with the biocompatible and biodegradable choline cation and oligopeptide-based anion. The simplest tripeptide triglycine (Gly-Gly-Gly) was employed for conforming a new choline tripeptide (Ch3Pe) IL, which sets the foundations for novel families of ILs based on peptides. Aiming at exploring the applicability of the novel solvents, we focused our endeavours in developing efficient and green biomolecule extraction processes for biotechnological industries, in particular for microalgae biorefineries. For this purpose, the Ch3Pe IL was evaluated in (i) lysing a model microalga (Tetraselmis suecica), (ii) generating aqueous biphasic systems (ABS) with surfactants and (iii) extracting representative biomolecules in the ABS conformed. The performances of the new IL in such tasks were compared with previous glycine IL analogues (choline amino acid and choline dipeptide) and the ABS configured with ChGly-Gly-Gly seemed to be the most promising one. Finally, this system was employed for simultaneously (i) lysing T. suecica and (ii) recovering β-carotene (20.9 μg/gb), α-tocopherol (141 μg/gb) and chlorophyll A (1392 μg/gb), which demonstrates the applicability of the novel IL in developing greener downstream processes.

The influence of zwitterions on the partition of biomolecules in aqueous biphasic systems

The use of hydrophilic zwitterionic compounds (ZI) to act as phase forming components of aqueous biphasic systems (ABS) has been attracting increased interest. Although previous works studied the phase behavior of ZI, there is still a lack of knowledge on the partition behavior of compounds in ZI-based ABS. This work reports a study on the influence of ZI structure for the extraction of 7 different biomolecules selected as molecular probes – 2 alkaloids (nicotine and caffeine), 2 phenolic compounds (gallic acid and vanillic acid) and 3 amino acids (L-tryptophan, L-tyrosine and L-phenylalanine). The partition and extraction efficiencies of these biomolecules were evaluated in ternary systems composed of hydrophilic ZI constituted by ammonium, imidazolium, pyridinium and piperidinium cationic and SO3− anionic groups, K2CO3 and water. The obtained results show that all the biomolecules preferentially partition to the ZI-rich phase, and the extent of their partition was closely related with the octanol–water partition coefficients of both biomolecules and ZI. It was demonstrated that the partition of biomolecules in ZI-based ABS is firstly ruled by hydrophobic/hydrophilic effects and only in some particular cases specific interactions between the biomolecule and the ZI affect their partition. Furthermore, despite the similarity between ZI and ionic liquids behavior as phase forming compounds of ABS for the extraction of biomolecules, ZI-based ABS presented a better performance in the extraction of phenolic compounds in alkaline environment.

Extraction and ICP-OES determination of heavy metals using tetrabutylammonium bromide aqueous biphasic system and oleophilic collector

Preconcentration of Cd(II), Co(II), Cu(II), Ni(II), Pb(II), and Zn(II) in aqueous biphasic system tetrabutylammonium bromide (TBAB) – H2O – (NH4)2SO4 followed by ICP-OES determination is reported for the first time. All metal ions extracted quantitatively with 4-(2-pyridylazo)-resorcinol. Nonwoven polypropylene collector was used to capture the extract and to assist phase separation. The concentrate was eluted with 0.3 mol L−1 nitric acid and introduced directly into ICP-OES sampling line. Detection limits for cadmium, cobalt, copper, nickel, lead, and zinc were 0.012, 0.2, 0.17, 0.11, 0.2, and 0.4 μg L−1, respectively; preconcentration factor was 150. The method accuracy was verified by the analysis of certified reference materials samples of waste waters and surface water, as well as the real samples of spring and well water. The use of oleophilic polypropylene collector allowed to handle small volume extract, attain high preconcentration factor and significantly decrease detection limits for ICP-OES determination of heavy metals.

A Single-Molecule Surface-Based Platform to Detect the Assembly and Function of the Human RNA Polymerase II Transcription Machinery

Single-molecule detection and manipulation is a powerful tool for unraveling dynamic biological processes. Unfortunately, success in such experiments is often challenged by tethering the biomolecule(s) of interest to a biocompatible surface. Here, we describe a robust surface passivation method by dense polymer brush grafting, based on optimized polyethylene glycol (PEG) deposition conditions, exactly at the lower critical point of an aqueous biphasic PEG-salt system. The increased biocompatibility achieved, compared with PEG deposition in sub-optimal conditions away from the critical point, allowed us to successfully detect the assembly and function of a large macromolecular machine, a fluorescent-labeled multi-subunit, human RNA Polymerase II Transcription Pre-Initiation Complex, on single, promoter-containing, surface-immobilized DNA molecules. This platform will enable probing the complex biochemistry and dynamics of large, multi-subunit macromolecular assemblies, such as during the initiation of human RNA Pol II transcription, at the single-molecule level.

Purification of virus-like particles using aqueous biphasic systems composed of natural deep eutectic solvents

Virus-like particles (VLPs) mimic the structure of viral particles, presenting antigens in a repetitive and ordered structure, which makes them excellent vaccine immunogens. However, a major bottleneck in the VLPs industrial production process is the lack of robust purification methods, which can account for up to 80% of total manufacturing cost.Aqueous biphasic systems (ABS) are portrayed as viable and efficient platform in purification of wide variety of biomolecules, since they can be designed to be versatile, biocompatible and easy to scale-up, depending on the phase forming components. In the last years, natural deep eutectic solvents (NADES) have emerged and gained a lot of attention in several fields and different applications, including the ABS implementation, where they have been proposed as versatile, cost-effective and biocompatible phase forming compounds.In this work, a study of the liquid-liquid equilibria (LLE) of ABS composed of carbohydrates and carbohydrates-based NADES is reported and the mechanisms associated with phase equilibria discussed. The carbohydrates used in the NADES preparation are osmolytes, which can be regarded both as extractants and excipients that stabilize the final formulation of biopharmaceuticals, avoiding the tricky back extraction of VLPs. It is here shown that the use of NADES leads to an increase of two-phase region, when compared to the use of only one of the NADES components, thus offering liquid-liquid demixing at low concentrations of phase forming compounds. Furthermore, the cytotoxicity studies of these NADES and their individual components using two different human cell lines, HEK 293 and Huh-7, was evaluated, in order to conclude about the biocompatibility and suitability of proposed systems in vaccine development. Finally, the potential of the proposed ABS in purification of VLPs from bovine serum albumin (BSA) is demonstrated, while their structural properties are also assessed.

Aqueous two-phase system (polyethylene glycol + ionic liquid) for extraction of α-amylase: phase diagrams, systems characterization and partition study

The increasing demand for safe and effective methods for the separation of biological materials combining the advantageous properties of polyethylene glycol (PEG) (1500 g mol−1 or 4000 g mol−1) and ionic liquid has led to attractive and promising applications of the new aqueous biphasic systems. In this work, the liquid–liquid extraction process with aqueous two-phase systems (ATPS) was studied for α-amylase partitioning. The phase diagrams composed of PEG (1500 or 4000) g mol−1 + 1-ethyl-3-methylimidazolium chloride + water, were determined experimentally by the turbidimetric method at different temperatures T = (293.15, 303.15, 313.15 and 323.15) K. The effect of temperature and polymer molar mass on the phase diagrams, as well as the phase densities, refractive indexes and electrical conductivity were evaluated. Subsequently, the α-amylase partitioning was performed, where the partition coefficient was determined, besides the activity and selectivity. It was found that a temperature decrease and a polymer molar mass increase favored the formation of two phases. It was also found that α-amylase showed a preference for the bottom phase composed of PEG and the systems composed of PEG 1500 presented better results.

Cholinium dipeptide as the cornerstone to build promising separation processes: A simultaneous recovery strategy for microalgae biorefineries

This work bets in Choline (Ch) Amino Acid (AA)-based Ionic Liquids (ILs) to design a biocompatible separation process. This family represents a greener alternative regarding more toxic and recalcitrant analogues, like conventional imidazolium ILs. In this vein, the library of Ch-based ILs has been rationally incremented by utilizing L-Phenylalanyl-L-Phenylalanine (Phe-Phe) as an anionic counterpart. Aqueous Biphasic Systems (ABS) have been recently proposed for the recovery of high value metabolites in microalgae biorefineries. In this study, an Ch-based IL, ChPhe-Phe, was synthesized, and its industrial potential was evaluated for obtaining natural antioxidants from the model microalga Tetraselmis suecica. Therefore, ABS-forming, biomolecule extraction and lytic capacities were investigated separately for the appropriate selection of ILs. The synthesized ChPhe-Phe demonstrated a superior performance than the simpler ChAA when segregated with K3PO4 aqueous solutions in terms of tie line length (TLL) and slope (S), improving the results obtained with 1-ethyl-3-methyl imidazolium methylsulfate (C2C1ImC1SO4). Finally, ChPhe-Phe was employed for setting-up a simultaneous lytic and biomolecule recovery stage from T. suecica, which returned promising results for the development of this biocompatible technology.

Chiral Ionic Liquids: Structural Diversity, Properties and Applications in Selected Separation Techniques.

Ionic liquids (ILs) are chemical compounds composed of ions with melting points below 100 °C exhibiting a design feature. ILs are commonly used as the so-called green solvents, reagents or highly efficient catalysts in varied chemical processes. The huge application potential of ionic liquids (IL) justifies the growing interest in these compounds. In the last decade, increasing attention has been devoted to the development of new methods in the synthesis of stable chiral ionic liquids (CILs) and their application in various separation techniques. The beginnings of the successful use of CILs to separate enantiomers date back to the 1990 s. Most chiral ILs are based on chiral cations or chiral anions. There is also a limited number of CILs possessing both a chiral cation and a chiral anion. Due to the high molecular diversity of both ions, of which at least one has a chiral center, we have the possibility to design a large variety of optically active structures, thus expanding the range of CIL applications. Research utilizing chiral ionic liquids only recently has become more popular. However, it is the area that still has great potential for future development. This review aimed to describe the diversity of structures, properties and examples of applications of chiral ionic liquids as new chiral solid materials and chiral components of the anisotropic environment, providing chiral recognition of enantiomeric analytes, which is useful in liquid chromatography, countercurrent chromatography and other various CIL-based extraction techniques including aqueous biphasic (ABS) extraction systems, solid–liquid two-phase systems, liquid–liquid extraction systems with hydrophilic CILs, liquid–liquid extraction systems with hydrophobic CILs, solid-phase extraction and induced-precipitation techniques developed in the recent years. The growing demand for pure enantiomers in the pharmaceutical and food industries sparks further development in the field of extraction and separation systems modified with CILs highlighting them as affordable and environmentally friendly both chiral selectors and solvents.

Neochloris oleoabundans biorefinery: Integration of cell disruption and purification steps using aqueous biphasic systems-based in surface-active ionic liquids

In this work, an approach to integrate the downstream processing of bioactive compounds present in the microalgae cells by combining the use of tensioactive compounds and aqueous biphasic systems (ABS) is proposed. For this purpose, several aqueous solutions using solvents with and without tensioactive nature were investigated on their capacity to disrupt the microalgae cells as well as to extract the different classes of biomolecules, namely pigments (chlorophylls a and b, and lutein), proteins and carbohydrates. Cationic tensioactive compounds were selected due to their high ability to simultaneously extract the different classes of compounds present in the Neochloris oleoabundans biomass. To fractionate pigments, proteins and carbohydrates extracted from the microalgae, ABS formed by polyethylene glycol (PEG 8000) and sodium polyacrylate (NaPA 8000) were used, with the solvent selected to disrupt the cells acting as electrolyte. This allowed to tune the biomolecule’s partition reaching a selective fractionation. This approach provided the simultaneous extraction of different biomolecules (pigments, protein and carbohydrates) from the cells and, the subsequent origin of two fractions, one rich in proteins (extraction efficiencies of 100%) and carbohydrates (extraction efficiency of 80%) and the second concentrated in pigments (e.g. lutein, extraction efficiency of 98%). The further isolation of the biomolecules from the ABS forming solvents is proposed aiming at the development of an integrated downstream process, including the cell disruption/compounds extraction, the fractionation, and the isolation of the biomolecules.

Phase Diagrams of Aqueous Biphasic Micellar Systems Formed by Polyethylene Glycol (10,000 or 35,000 g mol–1) + Triton X-100 + Water at Different Temperatures: Experimental and Correlation

A wide characterization of the phase equilibrium involved in aqueous biphasic micellar systems (ABMSs) formed by 10,000 or 35,000 g mol–1 polyethylene glycol (PEG) + t-octylphenoxypolyethoxyethanol...

Determination, characterization and modeling of aqueous biphasic systems composed of propylammonium-based ionic liquids and phosphate salts

Ionic liquids (ILs)-based aqueous biphasic systems (ABS) are efficient platforms for the extraction and purification of biomolecules. To find more biocompatible ABS, propylammonium-based ILs, phosphate salts and water were mixed at three temperatures, evaluating the influence of IL and temperature on the liquid-liquid equilibria (LLE). The biphasic region increases with the increase of IL’ anionic/cationic chain and decreases with the increase of temperature. There is good agreement between the IL’ ability to form ABS and the hydrogen bonding interaction predicted by COSMO-RS. The subtle balance of hydrogen bonding between salt anion-water and IL anion-water governs the phase separation.

Tuning the Partition Behavior of PEG-Based Aqueous Biphasic Systems Using Cholinium Chloride

Aqueous biphasic systems (ABS) have positive aspects associated with the low-cost, eco-friendly system, biomolecules separation ability and high extraction yield. The presence of quaternary ammoniu...

Recovery of platinum group metals using magnetic nanoparticles modified with ionic liquids

The development of a principally new approach to preparation of IL-modified magnetic nanoparticles and their application as sorbent materials for recovery of platinum group metals from model technological solutions are described. The formation of Fe3O4 nanoparticles, their simultaneous coating with a polymer shell and an ionic liquid are implemented in aqueous biphasic systems by a single step synthesis. Different ionic liquids have been tested as modifying agents: Cyphos® IL 101, Cyphos® IL 110, 1-butyl-3-methylimidazolium hexafluorophosphate, 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, 1-octyl-3-methylimidazolium hexafluorophosphate. The prepared magnetic nanoparticles have been characterized by scanning and transmission electron microscopy, X-ray diffraction, thermogravimetric analysis and applied as sorbents for magnetic solid-phase extraction of platinum metals from chloride solutions. The results indicate that the recovery of the platinum metals from (0.1 M HCl + 1 M NaCl) solution is 99% for Pd(II) and 98% for Pt(IV) within 30 min and their quantitative stripping can be achieved using 2 M HNO3 solution.

Aqueous biphasic systems comprising copolymers and cholinium-based salts or ionic liquids: Insights on the mechanisms responsible for their creation

Aqueous biphasic systems (ABS) formed by copolymers and ionic liquids (ILs) have demonstrated to be effective separation platforms, but there is still a gap on the complete understanding of the molecular-level mechanisms ruling the two-phase formation for this type of systems. This work addresses the determination of the liquid–liquid equilibrium of ABS composed of cholinium-based salts or cholinium-based ILs and the triblock copolymer Pluronic PE6200 (PL6200). It is demonstrated that PL6200 can form ABS with all investigated cholinium-based salts or ILs, contrarily to most poly(ethylene)glycol polymers, which is due to the presence of hydrophobic propylene oxide (PO) blocks. From the phase diagrams behavior and IL/salt anions properties, it is shown that the formation of ABS with cholinium-based salts is ruled by the anions polar surface and ability to be hydrated, whereas in systems comprising ILs van der Waals interactions between the copolymer and the IL cannot be discarded. The partition of a series of alkaloids in these systems, namely caffeine, nicotine, theophylline, and theobromine, was additionally appraised. It is shown that caffeine, theophylline, and theobromine preferentially migrate to the more hydrophobic PL6200-rich phase, and that their partition depends on the water content in the respective phase, being ruled by the phases’ hydrophobicity. On the other hand, nicotine, with the most prominent hydrophobic character amongst the studied alkaloids, preferentially migrates to the salt- or IL-rich phase, in which interactions occurring between this alkaloid and the IL/salt cannot be discarded. The ABS formed by cholinium dihydrogenphosphate is the most selective system identified to separate nicotine from the remaining alkaloids, giving some insights into their investigation as separation platforms for alkaloids from natural extracts.

Enhanced performance of polymer-polymer aqueous two-phase systems using ionic liquids as adjuvants towards the purification of recombinant proteins

Protein biopharmaceuticals, among which interferon alpha-2b (IFNα-2b) that can be used in the treatment of chronic hepatitis C and hairy cell leukemia, have become an indispensable product of current medicine. However, their current high costs derived from the lack of cost-effective downstream strategies still limits their widespread use. Polymer-based aqueous two-phase systems (ATPS) or aqueous biphasic systems (ABS) can be used in biopharmaceuticals purification. This work investigates the application of ionic liquids (ILs) as adjuvants (at 5 wt%) in ATPS constituted by polyethylene glycol with a molecular weight of 600 g mol−1 (PEG 600) and polypropylene glycol with a molecular weight of 400 g mol−1 (PPG 400) at constant pH (8) to purify the recombinant protein IFNα-2b from Escherichia coli lysates. IFNα-2b was produced from isopropyl β-d-1-thiogalactopyranoside (1 mM)-induced Escherichia coli BL21 (DE3) cultures and recovered from inclusion bodies after mechanical lysis, involving glass beads and a solubilization step with urea (8 M) in alkaline pH (12.5). PEG-PPG-based ATPS involving ILs as adjuvants were subsequently applied for IFNα-2b purification, in which the target protein tends to migrate to the PEG-rich phase (being the phase also enriched in IL) and the remaining proteins tend to precipitate at the interface (fitting within the three-phase partitioning approach). In comparison with the ATPS without adjuvant, most systems comprising ILs as adjuvants lead to enhancements in the purification factors of IFNα-2b, namely from 2.28 ± 0.06 up to 6.77 ± 0.49. The purity of IFNα-2b is maximized using ILs composed of aromatic cations and anions with high hydrogen-bond basicity. The secondary structure of IFNα-2b is preserved during the purification step, as appraised by circular dichroism and western-blot studies. Overall, the obtained results demonstrate the ILs ability to tune the characteristics of the ATPS coexisting phases towards improved purification processes, paving the way for their investigation in the purification of other high-value biological products.

Insights on the laccase extraction and activity in ionic-liquid-based aqueous biphasic systems

Due to their catalytic properties, selectivity, and efficiency, enzymes are excellent biocatalysts. In particular, laccases are versatile multi-copper oxidases with great interest for a wide plethora of biotechnological and environmental applications. Even though several laccase-catalysed processes have been reported at an industrial level, the high costs of their downstream processing required to provide biocatalysts with high purity levels, stability and activity remains one of the main drawbacks when economically evaluating the overall processes. Aqueous biphasic systems based on ionic liquids (ILs) can be foreseen as a promising alternative approach for the extraction and activity maintenance/improvement of enzymes, essentially due to the designer solvents ability of ionic liquids. However, to take advantage of this feature and to use the full potential of IL-based aqueous biphasic systems, it is necessary to understand the effect of ILs as phase-forming constituents and how they affect the enzymes extraction and activity. In order to overcome the lack of information on this topic in the literature, in this work, IL-based aqueous biphasic systems were investigated to extract and enhance the laccase activity, in order to gather evidences that could be used to improve the enzymes downstream processing. To this end, a wide screening of imidazolium-, pyridinium-, pyrrolidinium-, piperidinium-, tetraalkylphosphonium-, and tetraalkylammonium-based ILs as phase-forming components of ABS was carried out. Furthermore, these ILs were used to create ABS combined with salts, polymers and used as adjuvants in polymer-based ABS. Most ABS comprising ILs revealed to be highly efficient extraction platforms, allowing the complete extraction of laccase for all the conditions tested, and with an enzyme activity enhancement by more than 50%. Overall, the obtained results demonstrate that laccase preferentially partitions to the most hydrophilic phase in ABS comprising ILs, both used as adjuvants or as phase-forming components, corresponding to the phase in which the IL is enriched. Furthermore, the IL chemical structure of the IL plays a significant role in the enzyme activity, where ILs with a higher number of hydroxyl groups seem to be relevant to improve the laccase activity.

Primary purification of intracellular Halomonas salina ectoine using ionic liquids-based aqueous biphasic system

Ectoine is a zwitterionic amino acid derivative that can be naturally sourced from halophilic microorganisms. The increasing demands of ectoine in various industries have urged the researches on the cost-effective approaches on production of ectoine. Ionic liquids-based aqueous biphasic system (ILABS) was applied to recover Halomonas salina ectoine from cells hydrolysate. The 1-butyl-3-methylimidazolium tetrafluoroborate (Bmim)BF4 was used in the ILABS and the recovery efficiency of ILABS to recover ectoine from H.salina cells lysate was evaluated by determining the effects of phase composition; pHs; crude loading and additional neutral salt (NaCl). The hydrophilic ectoine was targeted to partition to the hydrophilic salt-rich phase. A total yield (YB) of 96.32% ± 1.08 of ectoine was obtained with ILABS of phase composition of 20% (w/w) (Bmim)BF4 and 30% (w/w) sulfate salts; system pH of 5.5 when the 20% (w/w) of crude feedstock was applied to the ILABS. There was no significant enhancement on the ectoine recovery efficiency using the ILABS when NaCl was added, therefore the ILABS composition without the additional neutral salt was recommended for the primary purification of ectoine. Partition coefficient (KE) of 30.80 ± 0.42, purity (PE) of 95.82% and enrichment factor (Ef) of 1.92 were recorded with the optimum (Bmim)BF4/sulfate ILABS. These findings have provided an insight on the feasibility of recovery of intracellular biomolecules using the green solvent-based aqueous system in one single-step operation.

Inhibition effect of amine compounds derived from hardening agents on the extraction of hydrogen bromide by water from the pyrolysis oil of brominated printed circuit boards

The pyrolysis oil of waste printed circuit boards (PCBs) obtained through industrially feasible methods commonly contains high concentrations of hydrogen bromide (HBr). This HBr must be further eliminated if the oil is intended to be used in commercial applications. For this purpose, a conventional liquid-liquid extraction process was explored using water as the extracting solvent. It was shown that the extraction efficiency decreases with the concentration of HBr in oil, reaching a limiting concentration below which HBr can hardly be removed. This finding was explained by considering the presence of amines in oil that result from the decomposition of amine-hardening agents in epoxy boards. In connection with these results, the distribution equilibria of HBr in a phenolic model oil–aqueous biphasic system was theoretically and experimentally analysed. The results revealed that the presence of amines in concentrations as low as 0.1 wt% of nitrogen was sufficient to inhibit the removal of HBr below the 1000 ppm level. The dissociation constant of HBr in oil and the partition coefficient of bromine ions between the two phases were evaluated, obtaining 0.088 mol dm−3 and 2.6 × 10−4, respectively. The findings of this work are fundamental for the optimal production of bromine-free oils from waste PCBs.