Bottom Phase Research Articles

Experimental and Theoretical Validation of System Variables That Control the Position of Particles at the Interface of Immiscible Liquids.

We construct a mathematical model describing the equilibrium flotation height of a spherical particle at the interface of immiscible liquids. The behavior of such a system depends on several experimentally measurable parameters, which include surface tensions, densities of all phases, and system scale. These parameters can be absorbed into three quantities that entirely determine the equilibrium position of the particle: the contact angle between the interface and particle, the Bond number, and the ratio of particle buoyant density to liquid phase densities-a new, dimensionless number that we introduce here. This experimentally convenient treatment allows us to make predictions that apply generally to the large parameter space of interesting systems. We find the model is in good agreement with experiments for particle size and interfacial tension spanning 3 orders of magnitude. We also consider the low interfacial tension case of aqueous two-phase systems (ATPSs) theoretically and experimentally. Such systems are more sensitive to changes in density than higher-tension aqueous/organic two-phase systems; we experimentally demonstrate that a millimeter-sized bead in an ATPS can be controllably positioned with between 5.9 and 95.1% of its surface area exposed to the bottom phase, whereas the same bead in an aqueous/organic system is limited to a range of 18.2-61.6%. Finally, we discuss the potential for wettability-based control for micron length-scale particles, which are not sensitive to changes in density. Our results can be used to simply define the experimentally controllable parameters that affect the equilibrium position and the length scales of a particle over which such parameters can be effectively tuned. A complete understanding of these properties is important for a number of applications including colloidal self-assembly and chemical patterning (e.g., formation of desymmetrized or Janus particles). By considering ATPSs, we broaden the potential uses to biological applications such as cell separation and interfacial tissue assembly.

Short-Chain n-Alcohol-Induced Changes in Phase Behaviors of Aqueous Mixed Cationic/Anionic Surfactant System.

The short-chain n-alcohol-induced changes in phase behaviors of aqueous mixed 1,3-propanediyl bis(dodecyl dimethylammonium bromide) (12-3-12) and sodium dodecyl sulfonate (AS) system have been investigated. For the 12-3-12/AS/H2O mixed system, there are two kinds of aqueous two-phase systems with excess cationic surfactant (ATPS-C). The molar ratio of 12-3-12 to AS (MR12-3-12/AS) and the total surfactant concentration ( mT) in the top phase are smaller than those in the bottom phase of ATPS-C. It is worth noting that the addition of ethanol or n-propanol leads to different influences on the ATPS-C. Molecular dynamics (MD) simulation results illustrate that the different influences ascribe to the difference in the cosurfactant effect of ethanol and n-propanol. When ethanol is used as additive, the difference in mT leads to the difference in interactions between surfactants and ethanol for the two coexisting phases of ATPS-C, determining the difference in their combination ability with the mixed solvent. It is the main reason for the ethanol-induced phase inversion of the first kind of ATPS-C. When n-propanol is added, in addition to mT, MR12-3-12/AS is also a key factor influencing the interactions between 12-3-12 and AS and between surfactants and n-propanol due to the stronger cosurfactant effect of n-propanol. MD simulations indicate that vesicles with smaller MR12-3-12/AS are easier and faster to form. These vesicles spontaneously accumulate at the top phase accompanied by certain amount of mixed solvent transferred from the bottom phase of ATPS-C. Meanwhile, the competition for the mixed solvent arising from the surfactant-rich bottom phase prevents the transferring. The two factors work together to cause the increase of mT in the top phase of ATPS-C with the addition of n-propanol, leading to n-propanol-induced phase concentration inversion rather than phase inversion of ATPS-C. On the basis of the experimental results and MD simulations, ethanol-induced phase inversion mechanism or n-propanol-induced phase concentration inversion mechanism of ATPS-C has been proposed.

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.

Ionic liquid-based sugaring-out and salting-out extraction of succinic acid

In this study, ionic liquids-based salting-out extraction and sugaring-out extraction were developed to extract succinic acid. Among these five investigated ionic liquids, the ionic liquid with BF4- and OTF− anion showed the ability to form two phases not only with glucose but also with salts. However, the ionic liquid with Br− anion cannot form aqueous two-phase system with glucose, but with salts. Relatively high extractabilities were obtained by ILs–based salting-out extraction systems compared to sugaring-out systems. The extraction efficiency values ranging between 47% and 86% were obtained when succinic acid concentration increased from 5 g/L to 70 g/L in [Bmim]BF4/(NH4)2SO4 systems. It was implied that succinic acid concentration was a key factor in ILs-based salting-out extraction system. Compared to salting-out extraction, sugaring-out extraction was more suitable for succinic acid separation due to the bottom phase reutilization to achieve coupling of fermentation and separation. The distribution behavior of succinic acid in [Bmim]BF4/glucose sugaring-out extraction system depends on not only the concentrations of ionic liquid and glucose, but also the pH. The partition coefficient (2.53) and recovery (75.96%) of succinic acid were obtained by a sugaring-out system consisting of 45% (w/w) [Bmim]BF4 and 30% (w/w) glucose at pH 2.0. Moreover, to support the applicability of ILs-based sugaring-out extraction for SA separation, the most prominent systems were further tested with actual fermentation broth. The extraction efficiencies were 73.71% and 72.53% for filtered and unfiltered fermentation broth, respectively. The remarkable results obtained in this work support the establishment of IL-based sugaring-out extraction as a sound basis of greener cost-effective strategies with a substantial reduction in the environmental footprint and economical issues.

(Invited) New Aqueous Two-Phase Systems for Sorting DNA-Wrapped SWCNTs

Previous studies have shown that polymer-polymer aqueous two-phase systems are very effective for DNA-wrapped SWCNTs sorting. Here we report that polymer-salt systems are also very effective. In some of the new aqueous polymer-salt two-phase systems, we can easily obtain pure SWCNTs of certain chirality from the top phase by just one-step extraction. By continuously changing the composition of the system, certain SWCNTs can also be purified from the bottom phase without using extra modulating agent. Since the polymer-salt system contains only one kind of polymer, it can greatly increase the flexibility in designing sorting processes to meet the application needs of sorted SWCNTs.

Aqueous micellar two-phase system as an alternative method to selectively remove soy antinutritional factors

In this work, different antinutritional factors (trypsin inhibitors, isoflavones and raffinose family oligosaccharides) were selectively removed from soy flour by using aqueous micellar two-phase systems (AMTPS). The effects of independent variables including temperature (30–60 °C), time (10–40 min) and solid to liquid ratio (0.025–0.050 g/L) on the extraction of each antinutritional factor were analyzed using a full factorial design. As general tendency, temperature and time were the most significant parameters (p < 0.05). The best condition for the selective recovery (97% of isoflavones at top phase, and more than 50% of the rest of ANFs at bottom phase) were 5 g/L of Genapol X-080, 0.2 moL/L of sodium citrate pH 5.00, 30 °C, 40 min and 0.050 g/L. Besides, in vitro gastrointestinal digestions assays demonstrated that the treated soy flour improved its protein digestibility. The findings of this work represent the introduction of a novel methodology to selectively remove soy antinutritional factors.

Optimization of aqueous two-phase systems for the production of 6-aminopenicillanic acid in integrated microfluidic reactors-separators

Aqueous two-phase systems (ATPSs) were screened for the production of 6-aminopenicillanic acid (6-APA) catalyzed by penicillin acylase, followed by the extractive separation of 6-APA from the reaction mixture. The key point of this study was to find an ATPS exhibiting a large difference in the partition coefficients of the biocatalyst and reaction products. Several ATPSs based on polyethylene glycol (PEG)/phosphate, PEG/citrate, and PEG/dextran were tested. We found that an ATPS consisting of 15 wt% of PEG 4000, 10 wt% of phosphates, 75 wt% of water (pH value 8.0 after dissolution) provided optimal separation of 6-APA from the enzyme. While the 6-APA was mainly found in the top PEG phase, the free enzyme favored the bottom salt-rich phase. This ATPS also fulfils other important requirements: (i) high buffering capacity, reducing an undesirable pH decrease due to the dissociation of phenylacetic acid (the side product of the reaction), (ii) a relatively low cost of the ATPS components, (iii) the possibility of electrophoretic transport of fine droplets as well as the reaction products for both the acceleration of phase separation and the enhancement of 6-APA concentration in the product stream. Extraction experiments in microcapillary and batch systems showed that the transport of 6-APA formed in the salt-rich phase to the corresponding PEG phase could occur within 30 s. The experimental results described form a base of knowledge for the development of continuously operating integrated microfluidic reactors-separators driven by an electric field for the efficient production of 6-APA.

Optimization of an aqueous two-phase extraction method for the selective separation of sulfated polysaccharides from a crude natural mixture

Abstract In this study, one aqueous two-phase system (ATPS) composed of polymer and salt was employed to extract sulfated polysaccharides (sulfated dextran) to polymer-rich phase from model polysaccharides mixture (dextran and sulfated dextran), and another ATPS was employed to back-extract sulfated polysaccharides to a salt phase. This process was further applied to separate sulfated Enteromorpha polysaccharides (EPs) from crude EPs. For optimal PEG1000/(NH4)2SO4 (TLL45) ATPS, the extraction efficiencies were 93.2 ± 0.1%, 95.4 ± 2.0%, 56.3 ± 4.4% for 5, 50, 500 kDa sulfated dextran in top PEG-rich phase and were 91.9 ± 1.4%, 97.1 ± 2.9%, 95.2 ± 0.9% for 5, 50, 500 kDa dextran in bottom salt-rich phase, respectively. The 500 kDa sulfated dextran presented precipitation at the interface, and the extraction efficiency was 26.9 ± 5.7% for 500 kDa sulfated dextran in the interface. Different ATPSs composed of PEG1000 and another salt (sodium citrate, K2HPO4, sodium tartrate, etc.) were performed to back-extract sulfated polysaccharides of PEG-rich phase to another salt phase. Under the PEG1000/sodium tartrate ATPS, the best back-extraction efficiencies of 5, 50, 500 kDa sulfated dextran were 75.1 ± 4.1%, 86.1 ± 0.2% and 88.3 ± 1.6%, respectively. Crude EPs was extracted by ultrasonic/microwave-assisted extraction from Enteromorpha powder with yield of 9.5% (w/w, dry algae powder). After that, crude EPs was separated by ATPSs, and analyzed using anion exchange chromatography and FT-IR. The results of anion exchange chromatography indicated that crude EPs contained three fractions (EP-1, EP-2, EP-3). The results of crude EPs separating by ATPSs demonstrated that majority of EP-3 concentrated in top PEG-rich phase, and EP-2 in bottom salt-rich phase of PEG1000/(NH4)2SO4 ATPS. FT-IR results showed that EP-3 presented S O and C O S strength vibrations at 1254 and 848 cm−1 (SO42− group), while EP-2 had no correlative strength vibrations. These results revealed that an ATPS method was established preliminarily for sulfated polysaccharides separation from a natural polysaccharides mixture.

A novel dispersive liquid-liquid microextraction method with high performance liquid chromatography for detection of 2,6-dimethylaniline in lidocaine hydrochloride injection

A novel hexafluoroisopropanol (HFIP)-octanol supramolecular solvent (SUPRAS) based dispersive liquid-liquid microextraction (DLLME) method was developed for the determination of 2,6-dimethylaniline (2,6-DMA) in lidocaine hydrochloride injection coupled with high performance liquid chromatography-ultraviolet detection (HPLC-UV). n-Octanol was selected as extraction solvent while HFIP was served as dispersing agent, self-assembly inducer of n-octanol as well as density-regulating agent of n-octanol. The HFIP-octanol SUPRAS displays reverse micellar aggregate structures (2-6 μm) with hydrophilic inner cores and is located in the bottom phase of the system after phase separation, which not only facilitates the efficient extraction and enrichment of polar 2,6-DMA, but also simplifies the extraction process. Several parameters influencing the extraction efficiency of 2,6-DMA were investigated and optimized. Under optimum conditions (0.4%(v/v) n-octanol, 5% (v/v) HFIP, vortex for 3 s at 60 W, standing for 3 min, centrifugation for 3 min at 3000 r/min, sample solution pH 9), the novel DLLME-HPLC method shows good linearity for quantitative detection of 2,6-DMA in the range of 1-100 μg/L (R=0.9989). The limit of detection (LOD) was 0.33 μg/L. The enrichment factor (EF) reached about 63. Intra-day and inter-day precisions (n=3) were all below 2.5%. The recoveries were from 93.9% to 100.8%. The results demonstrate that the novel DLLME-HPLC method is suitable for the accurate quantitative determination of 2,6-DMA in lidocaine hydrochloride injection with advantages of simplicity, rapidness, high efficiency and environmental friendliness, and may own high potential in future prospects.

Effect of hybrane hyperbranched polymer additive on partitioning of cephalexin antibiotic in aqueous biphasic systems

In this research, the partitioning of cephalexin as a broad-spectrum antibiotic has been investigated in an aqueous two-phase system (ATPS) comprised of poly(ethylene glycol) (PEG), trisodium citrate (Na3Cit), as well as a special hyperbranched polymer (i.e. Hybrane®) which has the potential to help the purification process be more efficacious. Adding 4wt% Hybrane® (HB) into the system elevated the maximum antibiotic partition coefficients from 2.40 to 3.07 and from 2.1 to 2.85 for the ATPSs containing PEG 600 and PEG 2000, respectively. The phase diagram results suggested that using PEG 2000 in the ATPS enlarged biphasic area compared to using PEG 600. Also, the introduction of HB into the ATPS decreased the salt concentration in the top phase, and fostered its tendency towards the bottom phase. In general, the values of partition coefficient for the ATPSs containing PEG 600 are higher than those for the (PEG 2000)-based ATPSs. Furthermore, increasing the polymer and salt concentrations had an adverse effect on the partition coefficient. The partitioning behavior of cephalexin demonstrates that our ATPS, featuring HB, can serve as a favorable, benign milieu for the separation and release of this antibiotic.

Sustainable approach in recycling of phase components of large scale aqueous two-phase flotation for lipase recovery

Abstract Recently, aqueous two-phase flotation system has become a powerful method for separation of biomolecules. However, the application of this technique for large scale operation elevates a concern on the environmental issue. This is due to the large amount of chemical consumption for phase components formation. Recycling of hydrophilic organic solvent and inorganic salt in aqueous two-phase flotation is an efficient and green technique for biomolecules recovery. Up to date, there is limited research concerning the recycling of the phase components. In this present work, recycling capability of pilot scale aqueous two-phase flotation composing alcohol/salt was employed to separate lipase from fermentation broth. The drive of this research is to economize the usage of chemicals, to reduce environmental pollution and to establish this approach for sustainable recovery of lipase. The aqueous two-phase flotation system investigated was composed of 1-propanol and ammonium sulphate whereby both phase components went through complete recycling process. Recycling of entire phase components is found to be a sustainable approach as it able to reduce the consumption of chemicals and it is an eco-friendly technique. From the result obtained, it was exhibited that by reusing the bottom phase, separation efficiency was sustained beyond 70%. As for the lipase yield, 80% of yield was achieved from recycling the crystallized salt that was recovered by dilution crystallization with methanol.

Phase equilibrium and protein partitioning in aqueous two-phase systems containing imidazolium ionic liquids and surfactant at low voltage levels

In this paper, the binodal data of aqueous two-phase system (ATPS) containing imidazolium-based ionic liquids (chlorinated 1-ethyl-3-methylimidazole, [Emim]Cl), and a non-ionic surfactant (octylphenol polyethoxylene, Triton X-100) was obtained by means of the cloud point method in the temperature intervals T=(288.15–308.15) K. It was found that the [Emim]Cl/Triton X-100 ATPS was sensitive to temperature and had a long phase segregation time. Screening experiments of various additives showed that adding 7% of sodium dodecyl sulfate (SDS) to the total mass of surfactants (w/w) reduced the temperature sensitivity, and improved the phase separation rate of the system. The liquid-liquid equilibrium (LLE) data of [Emim]Cl/Triton X-100 ATPS without additive and containing 7% SDS at 298.15K was further studied according to the cloud point method. To further shorten the long phase disengagement time, a low-voltage alternating current (AC) electric field was added to the [Emim]Cl/surfactant ATPS. Through the optimization of AC electric field conditions (such as electric field type, frequency and duty ratio), a relatively fast and stable [Emim]Cl/surfactant ATPS was established. In 0.67V/cm field strength, 1000Hz frequency and 50% duty ratio of impulse waveform, the phase splitting time is shortened by 2–3 times. With and without electric field, protein partitioning of cytochrome c (Cyt-c), bovine serum albumin (BSA) and lysozyme (Lys) in [Emim]Cl/surfactant ATPS indicated that Cyt-c and BSA tended to distribute in the bottom phase, while Lys was inclined to distribute in the top phase. The application of the AC electric field can still decrease the phase separation time in the protein distribution process, and has no significant effect on the distribution behavior of proteins. Furthermore, Lys activity in the top phase of ATPS was significantly enhanced, which could be caused by the low concentration of Triton X-100 in the top phase based on the comparison of Lys fluorescence spectra analysis in aqueous solution, in the top phase and in the bottom phase of [Emim]Cl/surfactant ATPS.

Ethanol/salt aqueous two-phase system based ultrasonically assisted extraction of polysaccharides from Lilium davidiivar. unicolor Salisb: Physicochemical characterization and antiglycation properties

An effective method based on ultrasonically assisted aqueous two-phase extraction (UA-ATPE) was developed for extracting polysaccharides from Lilium davidiivar. unicolor Salisb (LPS). Four different types of ethanol/salts of aqueous two-phase system (ATPS) were evaluated. Ethanol/K2HPO4 system exhibited good selective extraction ability. An optimum ATPS of 16.54% (w/w) ethanol and 31.45% (w/w) K2HPO4 was considered to be a satisfactory system for extracting LPS. The optimum ultrasound-assisted conditions were as follows: 25:1 (g/g) of solvent to solid ratio, 10min of extraction time, 60°C of extraction temperature, pH of 11 (ATP system with no pH adjustment), 190W of ultrasound power, and 22KHz of ultrasound frequency. The yield of LPS was 36.58% from the bottom phase, and the purity of LPS was 84.71%. Compared with heat water extraction (HWE) and ultrasonic-assisted extraction (UAE), UA-ATPE improved the yield and purity of LPS. There was no significant difference among LPS prepared by three methods from flourier transformation infrared spectroscopy. Circular dichroism spectra, thermogravimetric analysis, and scanning electron microscope indicated that the chemical and physical properties of LPS were affected by different extraction methods to some extent. Besides, LPS has a certain antiglycation activity. Therefore, as an innovative and promising technique, this approach using ATPS as an extraction system and ultrasound as a source of energy provided better choice for the separation of active components from other natural products.

Three-liquid-phase extraction and separation of V(V) and Cr(VI) from acidic leach solutions of high-chromium vanadium–titanium magnetite

Abstract A new method by liquid–liquid–liquid three phase system, consisting of acidified primary amine N1923 (abbreviated as A-N1923), poly(ethylene glycol) (PEG) and (NH4)2SO4 aqueous solution, was suggested for the separation and simultaneous extraction of V(V) and Cr(VI) from the acidic leach solutions of high-chromium vanadium–titanium magnetite. Experimental results indicated that V(V) and Cr(VI) could be selectively enriched into the A-N1923 organic top phase and PEG-rich middle phase, respectively, while Al(III) and other co-existing impurity ions, such as Si(IV), Fe(III), Ti(IV), Mg(II) and Ca(II) in acidic leach solutions, could be enriched in the (NH4)2SO4 bottom aqueous phase. During the process for extraction and separation of V(V) and Cr(VI), almost all of impurity ions could be removed. The separation factors between V(V) and Cr(VI) could reach 630 and 908, respectively in the organic top phase and PEG middle phase, and yields of recovered V(V) and Cr(VI) in the top phase and middle phase respectively were all above 90%. Various effects including aqueous pH, A-N1923 concentration, PEG added amount and (NH4)2SO4 concentration on three-phase partitioning of V(V) and Cr(VI) were discussed. It was found that the partition of Cr(VI) into the PEG-rich middle phase was driven by hydrophobic interaction, while extraction of V(V) by A-N1923 resulted of anion exchange between NO3− and H2V10O284 −. Stripping of V(V) and Cr(VI) from the top organic phase and the middle PEG-rich phase were achieved by mixing respectively with NaNO3 aqueous solutions and NaOH-(NH4)2SO4 solutions. The present work highlights a new approach for the extraction and purification of V and Cr from the complex multi-metal co-existing acidic leach solutions of high-chromium vanadium–titanium magnetite.

Protic ionic liquids as a constituent of biphasic systems based on acetonitrile: Phase diagram and alkaloid partitioning

Abstract The present work focuses on the use of protic ionic liquids (PILs) in the formation of biphasic phase systems with acetonitrile, followed by a study of the partitioning of alkaloids such as caffeine, theobromine and theophylline between the phases. Initially, new phase diagrams were built at 298.15 K and atmospheric pressure to observe the ease of phase formation. All PILs were able to form biphasic systems with acetonitrile, except for those based on pentanoate as anion. From these studies, the order of ease of formation was estimated using the logarithm of the distribution coefficient of system constituents; acetate was the most apt to form biphasic systems. Furthermore, the replacement of amine hydrogen (ethanolamine) by methyl or 2-hydroxymethyl group did not alter the ease of phase formation. All alkaloids (caffeine – CF, theobromine – TB and theophylline – TF) migrated to the bottom phase (PIL-rich phase) due to their higher hydrophilicity, where good recovery rates were verified (CF: 61%, TB: 71% and TF: 69%).

Equilibrium data and thermodynamic studies of α-amylase partition in aqueous two-phase systems

Liquid-liquid equilibrium data (LLE) were obtained for aqueous two-phase systems (ATPS) composed of polyethylene glycol (average molar mass of 1500 g mol−1), sodium citrate/citric acid and water, at different pH (4.0, 5.0, 6.0 and 7.0) and temperatures T = (303.15 and 313.15) K. The thermodynamic study of the phase equilibrium was performed. The experimental results of the LLE were correlated using the UNIversal Functional Activity Coefficient (UNIFAC) model. The salting-out effect evaluated using the model of the effective excluded volume (EEV). Partitioning of the α-amylase (EC 3.2.1.1) was investigated as a function of pH, temperature, and tie line length (TLL). It was observed that the temperature had no influence on the formation of the biphasic region. Increments in pH value resulted in the raising of biphasic region. Isothermal titration calorimetry (ITC) assays was performed in order to deeply understanding the intermolecular interactions involved in the α-amylase partition and the driving forces that govern this process. It was measured the Gibbs free energy transfer (ΔtrG), transfer enthalpy variation (ΔtrH) and transfer entropy variation (ΔtrS) during the enzyme partition, as a function of the pH, temperature and TLL. For α-amylase partition, it was verified that at pH 4.0 the enzyme had a strong tendency to transfer to the polymer-rich phase. At pH 5.0 α-amylase showed affinity for the salt-rich bottom phase, while at pH 6.0, the partition coefficient of the enzyme increased as temperature was reduced to 303.15 K. The opposite behavior was noted for α-amylase at pH 7.0. Thermodynamic analysis based on isothermal titration microcalorimetry indicated the partition of α-amylase partition in the ATPS at pH 4.0 and 7.0 and 313.15 K was accompanied by endothermic heat and was entropically driven to the upper phase. Results of yield (95.373%) parameter indicated the applicability of ATPS for amylase purification.

Extraction method based on emulsion breaking for the determination of Cu, Fe and Pb in Brazilian automotive gasoline samples by high-resolution continuum source flame atomic absorption spectrometry

This work reports a new method for extraction of Cu, Fe and Pb from Brazilian automotive gasoline and their determination by high-resolution continuous source flame atomic absorption spectrometry (HR-CS FAAS). The method was based on the formation of water-in-oil emulsion by mixing 2.0mL of extraction solution constituted by 12% (w/v) Triton X-100 and 5% (v/v) HNO3 with 10mL of sample. After heating at 90°C for 10min, two well-defined phases were formed. The bottom phase (approximately 3.5mL), composed of acidified water and part of the ethanol originally present in the gasoline sample, containing the extracted analytes was analyzed. The surfactant and HNO3 concentrations and the heating temperature employed in the process were optimized by Doehlert design, using a Brazilian gasoline sample spiked with Cu, Fe and Pb (organometallic compounds). The efficiency of extraction was investigated and it ranged from 80 to 89%. The calibration was accomplished by using matrix matching method. For this, the standards were obtained performing the same extraction procedure used for the sample, using emulsions obtained with a gasoline sample free of analytes and the addition of inorganic standards. Limits of detection obtained were 3.0, 5.0 and 14.0μgL−1 for Cu, Fe and Pb, respectively. These limits were estimated for the original sample taking into account the preconcentration factor obtained. The accuracy of the proposed method was assured by recovery tests spiking the samples with organometallic standards and the obtained values ranged from 98 to 105%. Ten gasoline samples were analyzed and Fe was found in four samples (0.04–0.35mgL−1) while Cu (0.28mgL−1) and Pb (0.60mgL−1) was found in just one sample.

Partition behavior of monocarboxylic acids in salting-out extraction systems of monohydric alcohols and dipotassium phosphate

Abstract Extraction of organic acids has been attracted much attention due to their hydrophilicity, pH-dependence and structural diversity. Partition behavior of seven monocarboxylic acids with different chemical structures, including formic acid, acetic acid, n-propionic acid, n-butyric acid, n-valeric acid, glycollic acid and lactic acid, have been investigated in salting-out extraction systems (SOES) composed of monohydric alcohol (ethanol, isopropanol, n-propanol, or tert-butanol)/K2HPO4. The relative hydrophobicity of the equilibrium phases in the SOES has been characterized by the Gibbs energy of transfer for methylene group from the salt-rich bottom phase to the solvent-rich top phase. The effects of the structures of target products and organic extractants, SOES composition, system pH and temperature on partition behavior were investigated. The results showed that natural logarithm of partition coefficient (ln K) of target products increased linearly with their calculated 1-octanol/water partition coefficient (Clog P), but decreased with the calculated 1-octanol/water partition coefficient of organic extractant. The partition coefficient of target products increased or decreased linearly as tie line length, which depends on their hydrophobicity. The partition coefficient and recovery decreased with the increasing of system pH values. In addition, the increasing of temperature will decreased the extraction efficiency of monocarboxylic acids, because the salting-out extraction is an exothermic process.

Aqueous biphasic systems formed by deep eutectic solvent and new-type salts for the high-performance extraction of pigments

Deep eutectic solvent (DES) composed of polypropylene glycol 400 (PPG 400) and tetrabutylammonium bromide (TBAB) was combined with a series of new-type salts such as quaternary ammonium salts, amino acid and polyols to form Aqueous Biphasic Systems (ABSs). Phase-forming ability of the salts was investigated firstly. The results showed that polyols had a relatively weak power to produce phases within studied scopes. And the shorter of carbon chain length of salts, the easier to obtain phase-splitting. Then partitioning of three pigments in PPG 400/betaine-based ABSs was addressed to investigate the effect of pigments’ hydrophobicity on extraction efficiency. It was found that an increase in hydrophobicity contributed to the migration of pigments in the DES-rich phase. On the other hand, with a decline in phase-forming ability of salts, the extraction efficiency of the whole systems started to go down gradually. Based on the results, selective separation experiment was conducted successfully in the PPG 400/betaine-based systems, including more than 93.00% Sudan Ⅲ in the top phase and about 80.00% sunset yellow FCF/amaranth in the bottom phase. Additionally, ABSs constructed by DES/betaine for partitioning amaranth were further utilized to explore the performances of influence factors and back extraction. It can be concluded that after the optimization above 98.00% amaranth was transferred into the top phase. And 67.98% amaranth can be transferred into the bottom phase in back-extraction experiment. At last, dynamic light scattering (DLS) and transmission electron microscope (TEM) were applied to probe into extraction mechanism. The results demonstrated that hydrophobicity played an important role in the separation process of pigments. Through combining with new-type DES, this work was devoted to introducing plentiful salts as novel compositions of ABSs and providing an eco-friendly extraction way for partitioning pigments, which boosted development of ABSs in the monitoring food safety field.

Physicochemical characterization and in vitro hypoglycemic activities of polysaccharides from Sargassum pallidum by microwave-assisted aqueous two-phase extraction

Microwave-assisted aqueous two-phase extraction (MAATPE) was applied for simultaneous extraction and separation of polysaccharides from Sargassum pallidum (SPPs). The optimal extraction parameters, physicochemical properties, and hypoglycemic activities in vitro of SPPs were investigated. The results revealed that the optimal extraction conditions were as follows: 21.0% ethanol (w/w) and 22.0% ammonium sulfate (w/w) for ATPS, ratio of material to liquid 1:60 (g/mL), extraction time 15 min, microwave power 830 W, and extraction temperature 95 °C. Under the optimal these conditions, the maximum yields of SPPs were 0.75 ± 0.04% of the top phase (SPP-1) and 6.81 ± 0.33% of the bottom phase (SPP-2). SPP-1 and SPP-2 were homogeneous with molecular weights of 1518.6 and 50.6 kDa, respectively. SPP-1 mainly consisted of fucose, galactose, mannose, and glucuronic acid with a molar ratio of 4.97:9.75:6.44:6.07, whereas SPP-2 was mainly composed of fucose, galactose, glucose, and mannose with a molar ratio of 4.20:2.88:18.05:7.83. SPP-1 and SPP-2 exhibited favorable α-amylase and α-glucosidase inhibitory activities, and could remarkably improve glucose consumption in insulin resistance (IR) model cells. Notably, SPP-1 exhibited stronger α-glucosidase inhibitory activity than SPP-2, and even was comparable with acarbose.