Towards Enhanced Tunability of Aqueous Biphasic Systems: Furthering the Grasp of Fluorinated Ionic Liquids in the Purification of Proteins.

This work unfolds functionalized ABSs composed of FILs ([C2C1Im][C4F9SO3] and [N1112(OH)][C4F9SO3]), mere fluoro-containing ILs ([C2C1Im][CF3SO3] and [C4C1Im][CF3SO3]), known globular protein stabilizers (sucrose and [N1112(OH)][C4F9SO3]), low-molecular-weight carbohydrate (glucose), and even high-charge density salt (K3PO4). The ternary phase diagrams were determined, stressing that FILs highly increased the ability for ABS formation. The functionalized ABSs (FILs vs. mere fluoro-containing ILs) were used to extract lysozyme (Lys). The ABSs' biphasic regions were screened in terms of protein biocompatibility, analyzing the impact of ABS phase-forming components in Lys by UV-VIS spectrophotometry, CD spectroscopy, fluorescence spectroscopy, DSC, and enzyme assay. Lys partition behavior was characterized in terms of extraction efficiency (% EE). The structure, stability, and function of Lys were maintained or improved throughout the extraction step, as evaluated by CD spectroscopy, DSC, enzyme assay, and SDS-PAGE. Overall, FIL-based ABSs are more versatile and amenable to being tuned by the adequate choice of the phase-forming components and selecting the enriched phase. Binding studies between Lys and ABS phase-forming components were attained by MST, demonstrating the strong interaction between Lys and FILs aggregates. Two of the FIL-based ABSs (30 %wt [C2C1Im][C4F9SO3] + 2 %wt K3PO4 and 30 %wt [C2C1Im][C4F9SO3] + 25 %wt sucrose) allowed the simultaneous purification of Lys and BSA in a single ABS extraction step with high yield (extraction efficiency up to 100%) for both proteins. The purity of both recovered proteins was validated by SDS-PAGE analysis. Even with a high-charge density salt, the FIL-based ABSs developed in this work seem more amenable to be tuned. Lys and BSA were purified through selective partition to opposite phases in a single FIL-based ABS extraction step. FIL-based ABSs are proposed as an improved extraction step for proteins, based on their biocompatibility, customizable properties, and selectivity.

Aqueous biphasic systems developed with deep eutectic solvents and polymer for the efficient extraction of pigments from beverages.

Novel aqueous biphasic systems (ABSs) developed with benzyl-based quaternary ammonium salts-deep eutectic solvents (DESs) and polypropylene glycol (PPG) were herein proposed. The liquid-liquid equilibrium and the partitioning behavior of pigments in the systems were addressed. The results suggested that the shorter the carbon chain length of the DES, the easier to form two phases. The analysis of mixed samples showed that the selective separation was achieved in the ABSs, including 99.47% of tartrazine in the DES-rich phase and 98.47% of sudan III in the PPG-rich phase. Additionally, the systems were successfully applied to the extraction of pigments from the actual beverage samples with recoveries ranging from 93.43% to 102.15%. Furthermore, the study on the separation mechanism indicated that the hydrogen bonding played a significant role in the separation process. All the above results highlight the proposed DES/polymer-based ABSs have great advantages in selective and high-performance separation of pigments from beverages.

Simultaneous Purification of Human Interferon Alpha-2b and Serum Albumin Using Bioprivileged Fluorinated Ionic Liquid-Based Aqueous Biphasic Systems.

Interferon alpha-2b (IFN-α2b) is an essential cytokine widely used in the treatment of chronic hepatitis C and hairy cell leukemia, and serum albumin is the most abundant plasma protein with numerous physiological functions. Effective single-step aqueous biphasic system (ABS) extraction for the simultaneous purification of IFN-α2b and BSA (serum albumin protein) was developed in this work. Effects of the ionic liquid (IL)-based ABS functionalization, fluorinated ILs (FILs; [C​2C​1Im][C​4F​9SO​3] and [N​1112(OH)][C​4F​9SO​3]) vs. mere fluoro-containing IL ([C​4C​1Im][CF​3SO​3]), in combination with sucrose or [N​1112(OH)][H​2PO​4] (well-known globular protein stabilizers), or high-charge-density salt K​3PO​4 were investigated. The effects of phase pH, phase water content (%wt), phase composition (%wt), and phase volume ratio were investigated. The phase pH was found to have a significant effect on IFN-α2b and BSA partition. Experimental results show that simultaneous single-step purification was achieved with a high yield (extraction efficiency up to 100%) for both proteins and a purification factor of IFN-α2b high in the enriched IFN-α2b phase (up to 23.22) and low in the BSA-enriched phase (down to 0.00). SDS-PAGE analysis confirmed the purity of both recovered proteins. The stability and structure of IFN-α2b and BSA were preserved or even improved (FIL-rich phase) during the purification step, as evaluated by CD spectroscopy and DSC. Binding studies of IFN-α2b and BSA with the ABS phase-forming components were assessed by MST, showing the strong interaction between FILs aggregates and both proteins. In view of their biocompatibility, customizable properties, and selectivity, FIL-based ABSs are suggested as an improved purification step that could facilitate the development of biologics.

Green Extraction Strategy Using Bio-Based Aqueous Biphasic Systems for Polyphenol Valorization from Grape By-Product.

Polyphenols are natural compounds with enhanced antioxidant properties. They are present in relatively high concentrations in fruit/vegetable by-products. Therefore, there is a need for the development of efficient and cost-effective methods for the separation and purification of these valuable compounds. Traditional extraction with organic solvents needs to be switched to novel methods that are more efficient, with reduced extraction times and low consumption of organic solvents. Aiming at developing sustainable processes for the separation and purification of phenolic compounds, we used three model compounds, namely resveratrol, quercetin, and gallic acid, to investigate ionic liquid-based aqueous biphasic systems (IL-ABSs) formed by cholinium-based IL in combination with polypropylene glycol with a molecular mass of 400 g/mol (PPG400). The ABS composition in the two-phase region was selected according to a previously determined phase diagram. Extraction studies indicated the preferential partition of resveratrol and quercetin toward the hydrophobic PPG-rich phase that is mainly dominated by its hydrophobic nature and the strong salting-out effect of ILs. On the other hand, due to its considerably hydrophilic nature, gallic acid preferentially migrates toward the IL phase. The achieved results from grape stem extract demonstrated high extraction efficiencies of cholinium dihydrogen phosphate (~99% for resveratrol for the PPG phase and 78% for gallic acid for the IL phase), with considerable selectivity, demonstrating promising outcomes for potential applications.

Aqueous two-phase system based on benzethonium chloride and sodium dihexyl sulfosuccinate for extraction and ICP-OES determination of heavy metals.

An aqueous two-phase system (ATPS) based on benzethonium chloride (BztCl) and sodium dihexyl sulfosuccinate (NaDHSS) was proposed for the first time for liquid-liquid microextraction of Cd(II), Co(II), Cu(II), Mn(II), Ni(II), and Pb(II) followed by ICP-OES determination. The mixture of cationic and anionic surfactants, BztCl and NaDHSS, showed liquid-liquid phase separation at the molar ratio of 1:1, and the total surfactant concentration of 0.01-0.2 mol L-1 forming ATPS that was investigated in the extraction process. The extraction efficiency for Cd(II), Co(II), Mn(II), Ni(II), and Pb(II) was nearly 100 %, and for Cu(II) - not lower than 88 % in the presence of 8-hydroxyquinoline as a complexing agent. The surfactant-rich phase containing analytes was subjected to back-extraction with 0.2 M HNO3 before ICP-OES measurements. The preconcentration in the proposed BztCl-NaDHSS-H2O ATPS for 30 s and the high degree of back-extraction, which was achieved in 1 min, significantly reduced the sample preparation time, matrix effects and provided low LODs in the range of 0.04-1.0 µg L-1, the preconcentration factor was 120. The analysis of a certified reference material sample of surface water and the real samples of tap, sea, and waste water verified the method accuracy.

Isocyanide-based Multicomponent Reactions (IMCRs) in Water or Aqueous Biphasic Systems.

BACKGROUND: Isocyanide is an intriguing one-carbon synthon that is frequently employed in a variety of carbon-carbon and carbon-heteroatom bond-forming reactions. Isocyanide-based multicomponent reactions (IMCRs) are effective synthetic tools in organic synthesis for the preparation of complex heterocyclic molecules. The IMCRs in water have become an attractive research direction, enabling simultaneous growth of both IMCRs and green solvents towards ideal organic synthesis. OBJECTIVE: The goal of this review is to provide a general overview of IMCRs in water or biphasic aqueous systems for accessing various organic molecules, as well as an examination of their benefits and mechanistic insights. CONCLUSION: High atom economies, mild reaction conditions, high yields, and catalyst-free processes are crucial features of these IMCRs in water or biphasic aqueous systems.

Combined Use of Ionic Liquid-Based Aqueous Biphasic Systems and Microfluidic Devices for the Detection of Prostate-Specific Antigen.

Prostate cancer (PCa) is one of the cancer types that most affects males worldwide and is among the highest contributors to cancer mortality rates. Therefore, there is an urgent need to find strategies to improve the diagnosis of PCa. Microtechnologies have been gaining ground in biomedical devices, with microfluidics and lab-on-chip systems potentially revolutionizing medical diagnostics. In this paper, it is shown that prostate-specific antigen (PSA) can be detected through an immunoassay performed in a microbead-based microfluidic device after being extracted and purified from a serum sample through an aqueous biphasic system (ABS). Given their well-established status as ABS components for successful bioseparations, ionic liquids (ILs) and polymers were used in combination with buffered salts. Using both IL-based and polymer-based ABS, it was demonstrated that it is possible to detect PSA in non-physiological environments. It was concluded that the ABS that performed better in extracting the PSA from serum were those composed of tetrabutylammonium chloride ([N4444]Cl) and tetrabutylphosphonium bromide ([P4444]Br), both combined with phosphate buffer, and constituted by polyethylene glycol with a molecular weight of 1000 g/mol (PEG1000) with citrate buffer. In comparison with the assay with PSA prepared in phosphate-buffered saline (PBS) or human serum in which no ABS-mediated extraction was applied, assays attained lower limits of detection after IL-based ABS-mediated extraction. These results reinforce the potential of this method in future point-of-care (PoC) measurements.

New liquid supports in the development of integrated platforms for the reuse of oxidative enzymes and polydopamine production.

Polydopamine (PDA), a bioinspired polymer from mussel adhesive proteins, has attracted impressive attention as a novel coating for (nano) materials with an adequate conformal layer and adjustable thickness. Currently, PDA is obtained from dopamine chemical oxidation under alkaline conditions, limiting its use in materials sensible to alkaline environments. Envisaging a widespread use of PDA, the polymerization of dopamine by enzymatic catalysis allows the dopamine polymerization in a large range of pHs, overcoming thus the limitations of conventional chemical oxidation. Moreover, the conventional method of polymerization is a time-consuming process and produces PDA films with poor stability, which restricts its applications. On the other hand, the main bottleneck of enzyme-based biocatalytic processes is the high cost of the single use of the enzyme. In this work, laccase was used to catalyse dopamine polymerization. To improve its performance, a liquid support for integrating the laccase and its reuse together with the PDA production and recovery was developed using aqueous biphasic systems (ABS). Firstly, dopamine polymerization by laccase was optimized in terms of pH, temperature and initial dopamine concentration. It was demonstrated that the highest enzymatic polymerization of dopamine was achieved at pH 5.5, 30°C and 2 mg ml-1 of dopamine. Then, ABS composed of polymers, salts and ionic liquids were evaluated to optimize the laccase confinement in one phase while PDA is recovered in the opposite phase. The most promising ABS allowing the separation of laccase from the reaction product is composed of polypropylene glycol (400 g mol-1) and K2HPO4. The polymerization of dopamine in ABS leads to a remarkable improvement of polymerization of 3.9-fold in comparison to the conventional chemical PDA polymerization. The phase containing the confined laccase was reused for four consecutive reaction cycles, with a relative polymerization of 68.9% in the last cycle. The results of this work proved that ABS are a promising approach to create a liquid support for enzyme reuse allowing the process intensification efforts. The use of biocatalysts in ABS emerges as sustainable and alternative platforms from environmental and techno-economic points of view.

Purified Saponins in Momordica charantia Treated with High Hydrostatic Pressure and Ionic Liquid-Based Aqueous Biphasic Systems.

Momordica charantia L. (Cucurbitaceae) is rich in saponins, which have multiple biological effects. In this study, the total saponins of M. charantia were extracted by high hydrostatic pressure (HHP) technology. The optimal extraction process was determined (ethanol concentration 68%, pressure-holding time 8 min, ratio of material to solvent 1:35 and pressure 510 MPa), and the extraction amount of saponins reached 127.890 mg/g. On this basis, an ionic liquid-based aqueous biphasic system was constructed to purify the total saponins. Under the optimized conditions, the purity of M. charantia saponins was 76.06%. Liquid chromatography-mass spectrometry (LC/MS) was used to characterize the saponins in the purified extract of M. charantia. It was found that there were four kinds of saponins in the extract of M. charantia: kuguaglycoside A, momordicoside L, kuguacin B and kuguacin J, providing a basis for the study of the biological activity of saponins.

Opposite Effects Induced by Cholinium-Based Ionic Liquid Electrolytes in the Formation of Aqueous Biphasic Systems Comprising Polyethylene Glycol and Sodium Polyacrylate.

Cholinium-based ionic liquids ([Ch]-based ILs) were investigated as electrolytes in the formation of aqueous biphasic systems (ABS) composed of polyethylene glycol (PEG) and sodium polyacrylate (NaPA) polymers. Both enhancement and decrease in the liquid-liquid demixing ability induced by electrolytes in PEG-NaPA aqueous biphasic systems were observed. It is shown that the ILs that most extensively partition to the PEG-rich phase tend to act as inorganic salts enhancing the two-phase formation ability, while those that display a more significant partition to the NaPA-rich phase decrease the ABS formation capacity. The gathered results allowed us to confirm the tailoring ability of ILs and to identify, for the first time, opposite effects induced by electrolytes on the PEG-NaPA ABS formation ability. The distribution of the electrolyte ions between the coexisting phases and the polyelectrolyte ion compartmentalization are key factors behind the formation of PEG-NaPA-based ABS.

Advances Brought by Hydrophilic Ionic Liquids in Fields Involving Pharmaceuticals.

The negligible volatility and high tunable nature of ionic liquids (ILs) have been the main drivers of their investigation in a wide diversity of fields, among which is their application in areas involving pharmaceuticals. Although most literature dealing with ILs is still majorly devoted to hydrophobic ILs, evidence on the potential of hydrophilic ILs have been increasingly provided in the past decade, viz., ILs with improved therapeutic efficiency and bioavailability, ILs with the ability to increase drugs' aqueous solubility, ILs with enhanced extraction performance for pharmaceuticals when employed in biphasic systems and other techniques, and ILs displaying low eco/cyto/toxicity and beneficial biological activities. Given their relevance, it is here overviewed the applications of hydrophilic ILs in fields involving pharmaceuticals, particularly focusing on achievements and advances witnessed during the last decade. The application of hydrophilic ILs within fields involving pharmaceuticals is here critically discussed according to four categories: (i) to improve pharmaceuticals solubility, envisioning improved bioavailability; (ii) as IL-based drug delivery systems; (iii) as pretreatment techniques to improve analytical methods performance dealing with pharmaceuticals, and (iv) in the recovery and purification of pharmaceuticals using IL-based systems. Key factors in the selection of appropriate ILs are identified. Insights and perspectives to bring renewed and effective solutions involving ILs able to compete with current commercial technologies are finally provided.

Enantioselective liquid-liquid extraction of tryptophan enantiomers by a recyclable aqueous biphasic system based on stimuli-responsive polymers.

The hydrophobic organic solvents/water biphasic system had been always used in the traditional enantioselective liquid-liquid extraction (ELLE). In recent years, aqueous biphasic systems (ABSs) are considered as a promising method used in the ELLE. In the present work, a recyclable ABS composed of a temperature-responsive polymer poly(MAH-ß-CD-co-NIPAAm) (PN-CD) and a pH-responsive polymer poly(AA-DMAEMA-BMA) (PADB) was employed in the enantioseparation of tryptophan enantiomers. The polymer PN-CD acted as not only the phase-forming component but also the chiral selector, which can be recycled by changing the temperature. The polymer PADB can be used as the phase-forming component, which can also be recycled by adjusting the pH. The phase behaviors of this PN-CD/PADB ABS had been studied. The influencing parameters were studied for this chiral separation process, including the polymer concentration, initial tryptophan concentration, extraction temperature, and system pH. The maximum separation factor (α) of 1.42 was obtained by one-step extraction under the optimal conditions. Meanwhile, the distribution coefficients of L-tryptophan (L-Trp) and D-tryptophan (D-Trp) were 2.79 and 1.96, respectively. This study develops a green and sustainable strategy for enantioseparation by using the ELLE.

Sustainable liquid supports for laccase immobilization and reuse: Degradation of dyes in aqueous biphasic systems.

Novel liquid supports for enzyme immobilization and reuse based on aqueous biphasic systems (ABS) constituted by cholinium-based ionic liquids (ILs) and polymers for the degradation of dyes are here proposed. The biocatalytic reaction for dye decolorization using laccase occured in the biphasic medium, with the enzyme being "supported" in the IL-rich phase and the dye and degradation products being enriched in the polymer-rich phase. An initial screening of the laccase activity in aqueous solutions of ABS constituents, namely cholinium dihydrogen citrate ([Ch][DHC]), cholinium dihydrogen phosphate ([Ch][DHP]), cholinium acetate ([Ch][Acet]), polypropylene glycol 400 (PPG 400), polyethylene glycol 400 (PEG 400) and K2 HPO4 was carried out. Compared to the buffered control, a relative laccase activity of up to 170%, 257%, and 530% was observed with PEG 400, [Ch][DHP], and [Ch][DHC], respectively. These ABS constituents were then investigated for the in situ enzymatic biodegradation of the Remazol Brilliant Blue R (RBBR) dye. At the optimized conditions, the ABS constituted by PPG 400 at 46 wt% and [Ch][DHC] at 16 wt% leads to the complete degradation of the RBBR dye, further maintaining the enzyme activity. This ABS also allows an easy immobilization, recovery, and reuse of the biocatalyst for six consecutive reaction cycles, achieving a degradation yield of the dye of 96% in the last cycle. In summary, if properly designed, high enzymatic activities and reaction yields are obtained with ABS as liquid supports, while simultaneously overcoming the safety and environmental concerns of conventional organic solvents used in liquid-liquid heterogeneous reactions, thus representing more sustainable biocatalytic processes.

Novel aqueous biphasic system based on ionic liquid for the simultaneous extraction of seven active pharmaceutical ingredients in aquatic environment.

Nonsteroidal anti-inflammatory drugs and antibiotics are classes of active pharmaceutical ingredients (APIs), which are continuously contaminating the ecosystem through various anthropogenic activities. Because of their pseudo-persistence in the aquatic environment and their potentially chronic effects on aquatic life, it is important to closely monitor their concentrations in the aquatic environment using a sensitive analytical method. Sustainable aqueous biphasic systems (ABSs) composed of ionic liquids and biodegradable organic salt (sodium malate) were proposed. The phase diagrams of the systems were firstly determined, and [N4444]Cl-based ABS was selected for the simultaneous extraction and preconcentration of seven APIs. With the developed ABS, extraction efficiencies of APIs close to 100% were obtained. For the developed method, limits of detection (LODs) of 45, 65, 76, 14, 60, 48, and 51 ng L-1 were obtained for indomethacin, ibuprofen, diclofenac, naproxen, ketoprofen, flurbiprofen, and chloramphenicol, respectively, providing from 1216- to 1238-fold improvement as compared with the analysis without preconcentration. From an economic and environmental point of view, we can predict the prospects and competitive position of the method developed.

Purification of Pseudomonas sp. proteases through aqueous biphasic systems as an alternative source to obtain bioactive protein hydrolysates.

Aqueous biphasic systems (ABSs) are an interesting alternative for separating industrial enzymes due to easy scale-up and low operational cost. The proteases of Pseudomonas sp. M211 were purified through ABS platforms formed by polyethylene glycol (PEG) and citrate buffer salt. Two experimental designs 23 + 4 were performed to evaluate the following parameters: molar mass of PEG (MPEG ), concentration of PEG (CPEG ), concentration of citrate buffer (CCit ), and pH. The partition coefficient (K), activity yield (Y), and purification factor (PF) were the responses analyzed. The best purification performance was obtained with the system composed of MPEG  = 10,000 g/mol, CPEG  = 22 wt%, CCit  = 12 wt%, pH = 8.0; the responses obtained were K = 4.9, Y = 84.5%, PF = 15.1, and tie-line length = 52.74%. The purified proteases of Pseudomonas sp. (PPP) were used to obtain hydrolysates of Lupinus mutabilis (Peruvian lupin cultivar) seed protein in comparison with the commercial protease Alcalase® 2.4L. A strong correlation between hydrolysis degree and radical scavenging activity was observed, and the highest antioxidant activity was obtained with Alcalase® (1.40 and 3.47 µmol Trolox equivalent/mg protein, for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and oxygen radical absorbance capacity, respectively) compared with PPP (0.55 and 1.03 µmol Trolox/mg protein). Nevertheless, the IC50 values were lower than those often observed for antioxidant hydrolysates from plant proteins. PEG/citrate buffer system is valuable to purify Pseudomonas proteases from the fermented broth, and the purified protease could be promising to produce antioxidant protein hydrolysates.

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.

Insights on the use of alternative solvents and technologies to recover bio-based food pigments.

This review will discuss, under the Circular Economy and Biorefinery concepts, the performance of the alternative solvents in the downstream process to recover natural pigments in a more sustainable way. Conventionally, pigments marketed on an industrial scale are produced through chemical synthesis by using petroleum derivatives as the main raw material. Also, the current production chain of the synthetic dyes is linear, with no solvent recycling and waste generation. Thus, the most promising processes of extraction and purification of natural pigments and strategies on the polishing of the solvents are here reviewed. In this review, the use of alternative solvents, namely, ionic liquids, eutectic solvents, aqueous solutions of surfactants, and edible oils, for recovering natural pigments was reviewed. Works discussing higher extraction yields and selectivity, while maintaining the stability of the target pigments, were reported. Also, a panorama between Sustainability and Circular Economy prospection was discussed for better comprehension of the main advances in the field. Behind the analysis of the works published so far on the theme, the most important lacunas to overcome in the next years on the field were pointed out and discussed. Also, the future trends and new perspectives to achieve the economic viability and sustainability of the processes using alternative solvents will be scrutinized.

Simultaneous Separation of Antioxidants and Carbohydrates From Food Wastes Using Aqueous Biphasic Systems Formed by Cholinium-Derived Ionic Liquids.

The food industry produces significant amounts of waste, many of them rich in valuable compounds that could be recovered and reused in the framework of circular economy. The development of sustainable and cost-effective technologies to recover these value added compounds will contribute to a significant decrease of the environmental footprint and economic burden of this industry sector. Accordingly, in this work, aqueous biphasic systems (ABS) composed of cholinium-derived bistriflimide ionic liquids (ILs) and carbohydrates were investigated as an alternative process to simultaneously separate and recover antioxidants and carbohydrates from food waste. Aiming at improving the biocompatible character of the studied ILs and proposed process, cholinium-derived bistriflimide ILs were chosen, which were properly designed by playing with the cation alkyl side chain and the number of functional groups attached to the cation to be able to create ABS with carbohydrates. These ILs were characterized by cytotoxicity assays toward human intestinal epithelial cells (Caco-2 cell line), demonstrating to have a significantly lower toxicity than other well-known and commonly used fluorinated ILs. The capability of these ILs to form ABS with a series of carbohydrates, namely monosaccharides, disaccharides and polyols, was then appraised by the determination of the respective ternary liquid-liquid phase diagrams at 25°C. The studied ABS were finally used to separate carbohydrates and antioxidants from real food waste samples, using an expired vanilla pudding as an example. With the studied systems, the separation of the two products occurs in one-step, where carbohydrates are enriched in the carbohydrate-rich phase and antioxidants are mainly present in the IL-rich phase. Extraction efficiencies of carbohydrates ranging between 89 and 92% to the carbohydrate-rich phase, and antioxidant relative activities ranging between 65 and 75% in the IL-rich phase were obtained. Furthermore, antioxidants from the IL-rich phase were recovered by solid-phase extraction, and the IL was recycled for two more times with no losses on the ABS separation performance. Overall, the obtained results show that the investigated ABS are promising platforms to simultaneously separate carbohydrates and antioxidants from real food waste samples, and could be used in further related applications foreseeing industrial food waste valorization.

Ionic liquid-based miniaturized aqueous biphasic system to develop an environmental-friendly analytical preconcentration method.

Two ILs containing guanidinium cations (butylguanidinium chloride -C4Gu-Cl- and hexylguanidinium chloride -C6Gu-Cl-) were synthesized and characterized. Their cytotoxicity was also assessed, obtaining adequate CC50 values of 680 ±â€¯99 mg·L-1 for C4Gu-Cl and 135 ±â€¯8 mg·L-1 for C6Gu-Cl. Miniaturized aqueous biphasic systems (µ-ABSs) were developed using amounts lower than 1% (w/w) of these synthesized guanidinium-based ILs, K3PO4 as salting-out agent, and ultrapure water. The phases diagrams of both systems were determined, and the C4Gu-Cl-based µ-ABS was selected for the development of a microextraction method in combination with high performance liquid chromatography (HPLC) with fluorescence detection (FD) for the determination of five polycyclic aromatic hydrocarbons (PAHs) as model analytes. A point of the biphasic region of the C4Gu-Cl-based µ-ABS corresponding to a mixture of 0.75% (w/w) of the IL, 37.7% (w/w) of K3PO4 and 61.55% (w/w) of ultrapure water, and 30 min of equilibrium time, were selected as optimum conditions to obtain high enrichment factors and proper analytical microextraction performance. The C4Gu-Cl-based µ-ABS-HPLC-FD method exhibited low limits of detection, between 0.010 ng·L-1 and 2.0 ng·L-1, average relative recoveries of 96.7%, high enrichment factors ranging from 44.1 to 60.4, average extraction efficiencies of 61.7%, and intermediate precision relative standard deviations lower than 17% for a concentration level of 12 ng·L-1. The developed method was applied successfully in the analysis of different tap water samples.

Development of deep eutectic solvent-based aqueous biphasic system for the extraction of lysozyme.

Aqueous Biphasic Systems (ABSs) based on deep eutectic solvents (DESs) were determined and applied in the extraction of lysozyme from chicken egg white. Tetrabutylammonium bromide (TBAB) and benzyltributylammonium bromide were utilized as hydrogen-bond acceptors to synthesize six kinds of DESs with different carboxylic acids (such as glycolic acid, Gly). The phase-formation ability of these DESs was evaluated by combining several salts. The results revealed that the content of hydrophilic group and the alkyl side chain length of the carboxylic acids played a crucial role in phase separation process, and the introduce of the benzyl group for quaternary ammonium salt had an aptitude to promote two-phase splitting. Then the system comprising [TBAB][Gly] and Na2SO4 was used to appraise the effect of different experimental parameters on the extraction efficiency, including the amount of DES and salt, the temperature, the values of pH and the ionic strength. More than 98% of lysozyme was transferred into the DES-rich phase at the optimum condition. The activity of lysozyme after the process of extraction still retained 91.73% of initial activity, demonstrating high biocompatibility of the studied system. What's more, the proposed method was successfully utilized for the real sample analysis. Finally, UV-vis, FT-IR, circular dichroism spectra, dynamic light scattering and transmission electron microscopy were employed to investigate the extraction mechanism. All of these results verify the excellent feasibility of the proposed system in the analysis of biological samples.