Investigation and characterization of liquid two-phase systems for the separation of crystal mixtures by interfacial partitioning

The interfacial partitioning behavior of ampicillin and phenylglycine crystals in different two-phase systems has been investigated. The two-phase systems employed are water/dodecane, water/1-butanol, and water/pentane/methanol. By means of partition experiments and microscopic imaging, it has been shown that the mechanism of separation strongly depends on the choice of the two-phase system. While water/dodecane features a mechanism of sheer competitive adsorption at the interface, separation in water/1-butanol is mainly due to partitioning into both liquid phases, leading to a higher degree of separation. Experiments with water/pentane/methanol have illustrated the large potential of three-component systems, as slight variations in the composition can have large effects on the separation.     

Analysis of solvent-free ethyl oleate enzymatic synthesis at equilibrium conditions

This work reports experimental equilibrium data for the esterification of pure oleic acid and a fatty acid mixture with ethanol, using an immobilized Candida antarctica B lipase as catalyst. Reactions are performed in a solvent-free system, containing a mixture of substrates and different amounts of distilled water. According to the initial amount of water and the extent of the reaction, one or two liquid phases are present. Therefore, when the equilibrium is achieved, the liquid–liquid and chemical reaction equilibria have to be simultaneously satisfied.

Several reports dealing with enzymatic reactions performed in two-phase systems have found that the value of the reaction equilibrium constant calculated from overall experimental concentrations varies not only with temperature but also with substrate ratio and water content. Although this approach is a valuable way to explore equilibrium shifts in biphasic systems, it is limited to ideal systems with constant partition coefficients. The aim of this work is to consider the biphasic nature of the reactive mixture through a computational procedure that simultaneously takes into account liquid–liquid and reaction equilibria. This approach enables the determination of a classical temperature-dependent thermodynamic equilibrium constant, which accurately fits experimental equilibrium conversions over a wide range of operating conditions.     

Imaging of anisotropic cellulose suspensions using environmental scanning electron microscopy

The effect of concentration on anisotropic phase behavior of acid-hydrolyzed cellulose suspensions has been examined using conventional polarizing microscopy and the novel technique of environmental scanning electron microscopy (ESEM). Microcrystalline cellulose dispersed in water formed biphasic suspensions in a narrow concentration range, 4-12 wt % for a suspension pH of 4, where the upper and lower phases were isotropic and anisotropic (chiral nematic), respectively. It is known from previous work that within the biphasic regime total suspension concentration affects only the volume fractions of the two phases, not phase concentration or interfacial packing. As the total suspension concentration surpassed the upper critical limit (c), however, a single anisotropic phase of increasing concentration was observed. It was evident from polarizing microscopy that the chiral nematic pitch of the anisotropic phase decreased with increasing concentration, which has been attributed to a reduction in the electrostatic double layer thickness of the individual rods, thus increasing intermolecular interactions. Chiral nematic textures were also visible using ESEM. This technique has the advantage of studying individual rod orientation within the liquid crystalline phase as it permits the high resolution of electron microscopy to be applied to hydrated samples in their natural state. To our knowledge this is the first time such lyotropic systems have been observed using electron microscopy.     

Aqueous biphasic systems composed of ionic liquids and sodium carbonate as enhanced routes for the extraction of tetracycline

Aqueous biphasic systems (ABS) using ionic liquids (ILs) offer an alternative approach for the extraction, recovery, and purification of biomolecules through their partitioning between two aqueous liquid phases. In this work, the ability of a wide range of ILs to form ABS with aqueous solutions of Na₂CO₃ was evaluated. The ABS formed by IL + water + Na₂CO₃ were determined at 25°C, and the respective solubility curves, tie‐lines, and tie‐line lengths are reported. The studied ILs share the common chloride anion, allowing the IL cation core, the cation isomerism, the presence of functionalized groups, and alkyl side chain length effects to be evaluated. An increase in the cation side alkyl chain length leads to a higher ability for liquid–liquid demixing whereas different positional isomers and the presence of an allyl group have no major influence in the phase diagrams behavior. Quaternary phosphonium‐ and ammonium‐based fluids are more able to form an ABS when compared with imidazolium‐, pyridinium‐, pyrrolidinium‐, and piperidium‐based ILs. Moreover, the presence of an aromatic cation core has no major contribution to the formation of ABS when compared to the respective nonaromatic counterparts. Finally, to appraise on the systems applicability in downstream processing, selected systems were used for the partitioning of tetracyclines (neutral and salt forms) — a class of antibiotics produced by bacteria fermentation. Single‐step extraction efficiencies for the IL‐rich phase were always higher than 99% and confirm the great potential of ILs to be applied in the biotechnological field. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:645–654, 2013     

Cell surface energy,contact angles and phase partition III. Adhesion of bacterial cells to hydrophobic surfaces

The densities of adhesion of Staphylococcus epidermis, Staphylococcus aureus and Serratia marcescens to five types of plastics were studied in relation to interfacial free energies at the aqueous interfaces of both the bacteria and the plastics. The free energy of adhesion of bacteria to plastic in an aqueous medium is a linear function of partition of the bacteria between the solid surface and the liquid phase. These results show that the thermodynamics of the partitioning of a suspended particle between two immiscible liquid phases also apply to partitioning between a liquid and a solid phase.     

Dissipative particle dynamics of non-spherical particles using a Gaussian density model

The Gaussian density molecular model has been adapted for dissipative particle dynamics. The model, when combined with a soft potential, is shown to be a very flexible mesoscale model exhibiting a wide range of phase behaviour. The soft potential allows relatively large time steps to be used and hence a more rapid equilibration. In addition, the model can be used to study both uniaxial and biaxial systems. We have undertaken a number of pilot studies and have demonstrated that the Gaussian model is able to identify nematic–isotropic phase transitions in liquid crystals and the formation of ordered discotic phases.     

Biocatalysis in multi-phase reaction mixtures containing organic liquids

A wide range of enzymes and whole microbial cells will act as catalysts in reaction mixtures that contain 2 or more phases, one of which is an organic liquid (either a reactant or including water-immiscible organic solvents). These "biphasic" systems have a variety of structures, knowledge of which aids predictions about biocatalyst activity and stability. There is often a dilute aqueous solution phase (containing the biocatalyst), which may be emulsified with the organic phase, or "trapped" within catalyst particles; sometimes however there may only be traces of water adsorbed to the enzyme or cells. These reaction systems offer several advantages for industrial applications, notably the higher solubilities of many reactants of interest, and the ability of readily available hydrolytic enzymes to catalyse syntheses. The most non-polar organic liquids are least likely to inactivate biocatalysts, though many do remain active with relatively polar solvents. Modification of the biocatalyst may stabilise against inactivation, especially where this is due to direct contact with the phase interface. The mass transfer processes required in these systems remain poorly understood, particularly because the interfacial area is often unknown. Attractive continuous reactors may be operated using a packed bed of catalyst with a trapped aqueous phase.     

Effects of speciation on partitioning of iodine in aqueous biphasic systems and onto ABEC resins

Polyethylene glycol (PEG)-aqueous biphasic systems (ABS) and PEG-grafted aqueous biphasic extraction chromatographic (ABEC) resins have been shown to remove inorganic species from environmental and nuclear wastes. The partitioning behavior of several iodide species (iodide, iodine, triiodide, iodate, and 4-iodo-2,6-dimethylphenol (I-DMP)) have been studied for PEG (MW 2000)-salt systems and ABEC resins. Iodide partitioning to PEG-rich phases or onto ABEC resins can be enhanced by derivatization with 2,6-dimethylphenol to form 4-iodo-2,6-dimethylphenol or by addition of I(2) to form triiodide. Conversely, iodide partitioning to the PEG-rich phase or onto ABEC resins is reduced by oxidation of iodide to IO(3)(-). Partitioning studies of iodide, iodate, and iodine in a PEG-ABS are compared to results using ABEC resins.     

Evaluation of parallel milliliter-scale stirred-tank bioreactors for the study of biphasic whole-cell biocatalysis with ionic liquids

As clear structure-activity relationships are still rare for ionic liquids, preliminary experiments are necessary for the process development of biphasic whole-cell processes involving these solvents. To reduce the time investment and the material costs, the process development of such biphasic reaction systems would profit from a small-scale high-throughput platform. Exemplarily, the reduction of 2-octanone to (R)-2-octanol by a recombinant Escherichia coli in a biphasic ionic liquid/water system was studied in a miniaturized stirred-tank bioreactor system allowing the parallel operation of up to 48 reactors at the mL-scale. The results were compared to those obtained in a 20-fold larger stirred-tank reactor. The maximum local energy dissipation was evaluated at the larger scale and compared to the data available for the small-scale reactors, to verify if similar mass transfer could be obtained at both scales. Thereafter, the reaction kinetics and final conversions reached in different reactions setups were analysed. The results were in good agreement between both scales for varying ionic liquids and for ionic liquid volume fractions up to 40%. The parallel bioreactor system can thus be used for the process development of the majority of biphasic reaction systems involving ionic liquids, reducing the time and resource investment during the process development of this type of applications.     

Dynamic interactions between enzyme activity and the microstructured environment

A new approach for the study of an enzyme's relationship with its own reaction medium has been developed. One technique of micellar enzymology is the use of pseudohomogeneous systems composed of surfactant/water/organic solvent. In such systems, the physicochemical properties and textures of the medium depend on the relative ratios of the different components. Enzymes are catalytically active in such systems and up to the present have been studied in different microenvironments, such as micelles, microemulsions and lyotropic liquid crystals. Our purpose was to develop a system in which the enzyme could, by its activity, modify one of the components in such a way that the relative ratios among them changed sufficiently to produce a transition from one phase domain to another. The three components, water (or glucose in water), octanol and octyl-beta-D-glucoside, form a classical ternary water/oil/surfactant system. The relevant phase diagram shows different macroheterogeneous phases and microstructured domains. The enzyme beta-D-glucosidase hydrolyses octyl-beta-D-glucoside to form glucose and octanol. The enzyme was found to change the relative ratios of water (or glucose in water), octanol and octyl-beta-D-glucoside in such a manner that the physicochemical structure of the medium was modified. At the beginning of the reaction beta-D-glucosidase was present in a micellar solution of octyl-beta-D-glucoside in water. As the enzymatic reaction proceeded, the medium became biphasic. One of the two phases was the micellar solution of octyl beta-D-glucoside in water, while the other phase was either a microemulsion or a liquid crystalline phase. In addition the enzyme, through its catalytic activity, was able to modify the physiocochemical properties of the reaction medium.     

The opposite effect of temperature on polyethylene glycol-based aqueous biphasic systems versus aqueous biphasic extraction chromatographic resins

Variation in operational temperatures has revealed differences in the partitioning behavior of probe solutes between the phases in aqueous biphasic systems (ABS) and the related aqueous biphasic extraction chromatographic resin (ABEC). This difference has been studied using the hydrophobic anion, 99TcO4-, as a probe and (NH4)2SO4 as the kosmotropic salt. Distribution of the hydrophobic anion 99TcO4- to the PEG-rich phase in a MePEG-5000/(NH4)2SO4 ABS increases with increasing temperature, but decreases are observed in batch uptakes of this anion to ABEC resins from (NH4)2SO4 solutions. Phase diagrams were constructed at five different temperatures from 10 to 50 degreesC using cloud point titration for the ABS and a correlation between the phase divergence, measured in terms of tie line length (TLL), and the temperature of the partitioning system was verified. Thermodynamic parameters (deltaHdegrees,deltaSdegrees, deltaGdegrees, ) as a function of temperature were calculated for the various systems studied and the results imply thermodynamic differences between partitioning in ABS versus ABEC.     

The development of biphasic chiral recognition in chiral separation

In chiral separation, enantioseparation factor is an important parameter which influences the resolution of enantiomers. In this current overview, a biphasic chiral recognition method is introduced to the readers. This method can significantly improve the enantioseparation factor in two‐phase solvent through adding lipophilic and hydrophilic chiral selectors which have opposite chiral recognition ability to organic and aqueous phases, respectively. This overview presents the development and applications of biphasic chiral recognition in liquid‐liquid extraction and counter current chromatography. It mainly focuses on the topics of mechanism, advantages and limitations, applications, and key factors of biphasic chiral recognition. In addition, the future outlook on development of biphasic chiral recognition also has been discussed in this overview.     

Improved dilute bicelle solutions for high-resolution NMR of biological macromolecules

Dissolving biological macromolecules in dilute bicelle solutions, which form oriented liquid crystals in the presence of a magnetic field, permits measurement of anisotropic spin interactions such as dipolar couplings [Tjandra, N. and Bax, A., Science, 278, 1111–1114]. However, the lifetimes and temperature ranges of orientation for these samples are critically dependent on sample composition and experimental conditions. This paper demonstrates that doping dilute bicelle solutions with small amounts of charged amphiphiles substantially improves the stability and degree of alignment, as well as extends the temperature range of orientation for these systems. An explanation of the dependence of bicelle aggregation on sample composition is proposed based on the DLVO theory of colloids.     

Growth models of cultures with two liquid phases. IV. Cell adsorption, drop size distribution, and batch growth

In this work, a mathematical model which can be used to describe butch growth in fermentations with two liquid phases present is developed for systems in which the growth limiting substrate is dissolved in the dispersed phase. The model takes into account the drop size distribution, the rate of adsorption of cells on the drop surface, the rate of desorption of cells from the drop surface, substrate transport between phases, phase equilibrium, and growth kinetics. The model also considers the effect, of coalescence and redispersion of oil drops in the system. It is assumed that the composition of the dispersed phase is such that substrate utilization from it causes little or no change in the interfacial area. A discrete uniform distribution and a discrete normal distribution which is obtained from an experimental distribution curve are used as drop size distributions. Simulation results are obtained for a wide range of parameter values using the IBM S/360 Continuous System Modeling Program.     

A unified partition coefficient theory for chromatography,immobilized enzyme kinetics,and affinity chromatography

A unified treatment of systems containing immobilized biochemical and chromatographic systems was developed from basic thermodynamic considerations of partitioning in biphasic systems. Division of the overall partitions coefficient into electrostatic and nonelectrostatic interactions provided an effective stratagem for analysis of these systems. The properties of both strong and weak ionogenic matrices were explored. It was Found that the matrix charge concentration and the bulk solutions pH and ionic strength completely determine the electrostatic partition coefficient. Hence, the relationships developed allow prediction of partition coefficients from readily obtainable experimental parameters. It was also shown that even at low concentrations, the presence of immobilized protein can alter the properties of the matrix phase. However, a weak ionogenic matrix has an unusual property which allows for a biological switching device. Ina characteristics pH range, such matrices will maintain a constant micro environmental pH while the partitioning of a substrate ion is greatly variable. Finally, the theoretical treatment suggests simple procedures for determination of binding constants from affinity or adsorption chromatography.     

Solute partitioning into lipid bilayer membranes

We have measured the membrane/water partition coefficients of benzene into lipid bilayers as a function of the surface density of the phospholipid chains. A simple 2H NMR method was used for the measurement of surface densities; it is shown to give results similar to those obtained from more demanding X-ray diffraction measurements. We observe that benzene partitioning into the bilayer is dependent not only on the partitioning chemistry, characterized by the oil/water partition coefficient, but also on the surface density of the bilayer chains. Increasing surface density leads to solute exclusion: benzene partitioning decreases by an order of magnitude as the surface density increases from 50% to 90% of its maximum value, a range readily accessible in bilayers and biomembranes under physiological conditions. This effect is independent of the nature of the agent used to alter surface density: temperature, cholesterol, and phospholipid chain length were tested here. These observations support the recent statistical thermodynamic theory of solute partitioning into chain molecule interphases, which predicts that the expulsion of solute is due to entropic effects of the orientational ordering among the phospholipid chains. We conclude that the partitioning of solutes into bilayer membranes, which are interfacial phases, is of a fundamentally different nature than partitioning into bulk oil and octanol phases.     

A determination of liquid–vapour interfacial properties for methanol using a linear-combination-based isotropic periodic sum

We performed molecular dynamics simulations of the methanol liquid–vapour interfacial systems for a wide temperature range of 200–350 K. The linear-combination-based isotropic periodic sum (LIPS) method was used for estimating Coulombic interactions of the systems. The temperature dependence of the liquid and vapour phase densities, surface thickness and surface tension using the LIPS method was almost equal to that of the Ewald sum method. The temperature dependence of the electrostatic potential profile using the LIPS method was very similar to that of the Ewald sum method. We conclude that the LIPS method provides a reasonable accuracy in determining the liquid–vapour interfacial properties of polar molecules for many temperature conditions.     

Invited review liquid crystal models of biological materials and silk spinning

A review of thermodynamic, materials science, and rheological liquid crystal models is presented and applied to a wide range of biological liquid crystals, including helicoidal plywoods, biopolymer solutions, and in vivo liquid crystals. The distinguishing characteristics of liquid crystals (self-assembly, packing, defects, functionalities, processability) are discussed in relation to biological materials and the strong correspondence between different synthetic and biological materials is established. Biological polymer processing based on liquid crystalline precursors includes viscoelastic flow to form and shape fibers. Viscoelastic models for nematic and chiral nematics are reviewed and discussed in terms of key parameters that facilitate understanding and quantitative information from optical textures and rheometers. It is shown that viscoelastic modeling the silk spinning process using liquid crystal theories sheds light on textural transitions in the duct of spiders and silk worms as well as on tactoidal drops and interfacial structures. The range and consistency of the predictions demonstrates that the use of mesoscopic liquid crystal models is another tool to develop the science and biomimetic applications of mesogenic biological soft matter.     

南极假丝酵母脂肪酶B在离子液体/超临界流体体系中的稳定性模拟

脂肪酶在离子液体/超临界流体体系中的结构稳定性是影响其活性的重要因素。本文采用分子动力学方法分别研究了南极假丝酵母脂肪酶B(CALB)在离子液体CYPHOS IL-201/极性超临界流体CHF_3两相体系和离子液体CYPHOS IL-201/非极性超临界流体CO_2两相体系中的结构稳定性,揭示影响CALB结构稳定性的因素。研究结果表明,在超临界CHF_3中,CHF_3破坏蛋白维持α螺旋结构的氢键是蛋白结构不稳定的主要原因;在超临界CO_2中,CALB蛋白的结构紧密性降低,有序二级结构发生了变化,导致稳定性下降。离子液体和两种超临界流体均形成了两相体系,蛋白处于离子液体相中,离子液体不溶于超临界流体,但超临界流体部分进入离子液体相,降低了离子液体相的黏度。其中,相比于CYPHOS IL-201/CO_2体系,CYPHOS IL-201/CHF_3体系的黏度降低多。在离子液体CYPHOS IL-201与超临界流体(CHF_3、CO_2)形成的两相体系中,离子液体CYPHOS IL-201具有保护蛋白结构的作用,使CALB蛋白结构更加稳定。     

Fluorescence probe partitioning between Lo/Ld phases in lipid membranes

Fluorescence microscopy imaging is an important technique for studying lipid membranes and is increasingly being used for examining lipid bilayer membranes, especially those showing macroscopic coexisting domains. Lipid phase coexistence is a phenomenon of potential biological significance. The identification of lipid membrane heterogeneity by fluorescence microscopy relies on membrane markers with well-defined partitioning behavior. While the partitioning of fluorophores between gel and liquid-disordered phases has been extensively characterized, the same is not true for coexisting liquid phases. We have used fluorescence microscopy imaging to examine a large variety of lipid membrane markers for their liquid phase partitioning in membranes with various lipid compositions. Most fluorescent lipid analogs are found to partition strongly into the liquid-disordered (L(d)) phase. In contrast, some fluorescent polycyclic aromatic hydrocarbons with a flat ring system were found to partition equally, but others partition preferentially into liquid-ordered (L(o)) phases. We have found these fluorescent markers effective for identification of coexisting macroscopic membrane phases in ternary lipid systems composed of phospholipids and cholesterol.