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Phase separation in alcohol/liquid carbon dioxide solvent systems facilitates critical point drying.

Carbon dioxide and methanol or ethanol, although miscible, form alcohol/CO2 solutions that do not easily mix with additional pure liquid CO2. If the CO2 inlet is situated at the top of a critical point drying apparatus chamber, pure CO2 will entirely displace the alcohol/CO2 phase (which is more dense) while keeping the chamber filled with liquid. This unexpected phenomenon is invaluable in critical point drying delicate biological tissues which remain continuously immersed, avoiding surface or convection currents. By providing an objective criterion for intermediate solvent displacement, the protocol also eliminates ambiguous 'flushing' steps.

Carbon Dioxide↗

NMR evidence for a two-step phase separation in Nd1.85Ce0.15CuO4-delta.

By Cu NMR we studied the spin and charge structure in Nd(2-x)Ce(x)CuO(4-delta). For x=0.15, starting from a superconducting sample, the low temperature magnetic order in the sample reoxygenated under 1 bar oxygen at 900 degrees C reveals a peculiar modulation of the internal field, indicative of a phase characterized by large charge droplets ("blob" phase). By prolonged reoxygenation at 4 bars the blobs break up and the spin structure changes to that of an ordered antiferromagnet. We conclude that the superconductivity in the n-type systems competes with a genuine type I Mott-insulating state.

Journal Article↗

Two-dimensional and serial column reversed-phase separation of phenolic antioxidants on octadecyl-, polyethyleneglycol-, and pentafluorophenylpropyl-silica columns.

The separation selectivity of octadecyl-silica (C18) and of bonded pentafluorophenylpropyl-silica (F5) and PEG-silica columns was compared for natural phenolic antioxidants. The separation selectivities for phenolic antioxidants on C18 and F5 columns are strongly correlated, but low selectivity correlation indicating strong differences in the retention mechanism was observed between the C18 and PEG columns. Hence, the combination of a C18 and a PEG column is useful for separation of phenolic antioxidants that are not fully separated on single columns. Two-dimensional comprehensive liquid chromatography using a short PEG-silica column in the first dimension and a conventional C18-silica in the second dimension has the advantage of on-column focusing of the fractions transferred onto the C18 column in the second dimension, as a weaker mobile phase is used in the first dimension than in the second dimension. However, a stop-flow set-up in the first dimension system is necessary after the transfer of each fraction to the second dimension. Peak capacity is considerably larger but the separation time is much longer than with serially coupled PEG and C18 columns, which were employed for separation of beer and hop extract samples in connection with coulometric detection.

Antioxidants↗

Detection of phase separation in fluid phosphatidylserine/phosphatidylcholine mixtures.

The nonideal mixing of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine, (16:0, 18:1)PS, and 1,2-didodecenoyl-sn-glycero-3-phosphocholine, (12:1, 12:1)PC, in fluid lamellar model membranes was studied by measuring binding of aqueous Ca2+ ions and by x-ray diffraction. A region of two-phase coexistence was found by invariance of the aqueous concentration and by the appearance of two sets of lamellar spacings. The phases were identified as fluid from the diffuse x-ray diffraction in the wide-angle region. The width of the two-phase coexistence region was greater at higher ionic strength. In 800 mM KCl, the phase boundaries were at PS mole fraction 0.5 and 0.8. In 100 mM KCl, the phase boundaries were at PS mole fraction 0.52 and 0.62. Monte Carlo simulations of the lateral distributions of these PS/PC mixtures show pronounced clustering of the lipids.

Binding Sites↗

Scalable recovery of plasmid DNA based on aqueous two-phase separation.

Future developments in gene therapy and DNA vaccination depend on cost-effective large-scale production of pharmaceutical-grade pDNA (plasmid DNA). Given the large amount of impurities present in the feedstock, purification processes that have high specificity and capacity at a moderate cost are required. In the present study, we describe a non-chromatographic procedure based on aqueous two-phase extraction allowing a fast and simply scalable capture step. PEG [poly(ethylene glycol)] in combination with potassium citrate or potassium phosphate was tested as phase component for extraction. By increasing either PEG or salt concentration, the partitioning of nucleic acids changed from bottom to top phase. Phase systems with a composition of 15% PEG 800 and 20% potassium phosphate at pH 7.0 showed a strong partitioning of pDNA to the bottom phase, linked to a clear decrease in open circular pDNA, while proteins, genomic DNA and RNA remain at the top or at the interphase. A great advantage of the current process is that the complete procedure of lysis, precipitation, clarification and extraction can be performed in a single vessel. The number of denatured and sheared genomic DNAs in a spiking experiment was found to be depleted by more than 99%.

Chemical Fractionation↗

The effects of glycols, aldehydes, and acrylamide on phase separation and opacification in the calf lens.

Glycols, aldehydes, and acrylamide inhibit the in vitro formation of cold cataracts in calf lenses. The inhibition is reversed when the glycols or the acrylamide are diffused out of the lenses. The inhibition by aldehydes or polacrylamide is irreversible, suggesting that the crosslinking reagents may permanently modify lens structure to prevent the development of lens opacities due to a phase transition.

Acrylamides↗

Phase separation of myelin proteins in triton X-114: differential behavior of myelin basic protein in purified myelin and in cultured oligodendrocytes.

Rabbit central (CNS) and peripheral nervous system (PNS) myelin, as well as nonmyelinating pig oligodendrocytes in culture, were extracted at 0-4 degrees C with the nonionic detergent Triton X-114. The solubilized proteins were partitioned into the detergent-rich and detergent-depleted (aqueous) phases that form upon heating to 37 degrees C. The proteolipid protein (PLP), myelin-associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG) and P0 extracted from myelin were found exclusively in the detergent phase which is characteristic of the intrinsic membrane proteins. This was also the case for Wolfgram protein (WP), although this protein lacks transmembrane domains. A small fraction of the MAG and MOG extracted from oligodendrocytes partitioned into the aqueous phase, suggesting an altered conformation outside myelin or a different state of glycosylation. P2 and myelin basic protein (MBP) showed distinct patterns of behavior. P2 was found mainly in the aqueous phase giving strong support to its theoretically predicted conformation. Eighty-nine percent of the MBP extracted from CNS myelin and 81% of the pure MBP partitioned into the detergent phase. Surprisingly, most of the MBP extracted from the oligodendrocytes was recovered in the aqueous phase. We speculate that, in these cells, a hydrophilic protein might bind to the MBP in a specific manner, thereby preventing it from binding inappropriately to cellular components before its insertion into myelin.

Animals↗

Direct electron spin resonance evidence for alpha-tocopherol-induced phase separation in model membranes.

The high resolution (narrow linewidth) amphiphilic spin probe perdeutero di-t-butyl nitroxide (PDDTBN) has been used to investigate the effect of alpha-tocopherol on lecithin liposomes. The electron spin resonance (ESR) results obtained as a function both of alpha-tocopherol concentration and of temperature indicate the presence of two different hydrophobic sites for the spin probe molecules. The presence of two distinct phases, one alpha-tocopherol-poor and the other alpha-tocopherol-rich, is suggested in these phospholipid bilayers.

Electron Spin Resonance Spectroscopy↗

Trehalose maintains phase separation in an air-dried binary lipid mixture.

Mixing and thermal behavior of hydrated and air-dried mixtures of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1,2-distearoyl-d70-sn-glycero-3-phosphocholine (DSPCd-70) in the absence and presence of trehalose were investigated by Fourier transform infrared spectroscopy. Mixtures of DLPC:DSPCd-70 (1:1) that were air-dried at 25 degrees C show multiple phase transitions and mixed phases in the dry state. After annealing at high temperatures, however, only one transition is seen during cooling scans. When dried in the presence of trehalose, the DLPC component shows two phase transitions at -22 degrees C and 75 degrees C and is not fully solidified at -22 degrees C. The DSPCd-70 component, however, shows a single phase transition at 78 degrees C. The temperatures of these transitions are dramatically reduced after annealing at high temperatures with trehalose. The data suggest that the sugar has a fluidizing effect on the DLPC component during drying and that this effect becomes stronger for both components with heating. Examination of infrared bands arising from the lipid phosphate and sugar hydroxyl groups suggests that the strong effect of trehalose results from direct interactions between lipid headgroups and the sugar and that these interactions become stronger after heating. The findings are discussed in terms of the protective effect of trehalose on dry membranes.

Air↗

Nuclear magnetic resonance description of molecular motion and phase separations of cholesterol in lecithin dispersions.

Proton-enhanced 13C nuclear magnetic resonance is used to obtain signals from labeled cholesterols in lecithin dispersions. The [26-(13)C]cholesterol resonance indicates that the aliphatic tail of the molecule undergoes reorientation fast enough to average completely the chemical shift anisotropy. In contrast, [4-(13)C]cholesterol signals are characteristic of limited anisotropic reorientation. The resonances from the 4 position are sensitive to the temperature-concentration phase diagram. A phase boundary is observed at about 20 mole percent cholesterol.

Chemical Phenomena↗

Analysis of diffusion in a solid polymer electrolyte in the context of a phase-separated system.

The salt and ionic diffusion coefficients of the salt LiN(CF3SO2)(2) (LiTFSI) dissolved in high-molecular-weight poly(ethylene oxide) [PEO] have been measured over a broad concentration range, and the interrelationship of the measured values is demonstrated to be in good agreement with basic electrochemical theory. In the light of recently published structural findings, we propose a biphasic model for the conducting amorphous state, consisting of a stoichiometric P(EO)6LiTFSI phase dispersed into a salt-containing disordered phase, and analyze the ionic diffusion data within the framework of the Bruggeman-Landauer theory. The agreement between experimental and fitted data is shown to be excellent. We conclude by pointing out and discussing potential weaknesses of our analysis.

Journal Article↗

Swelling behavior and controlled release of theophylline and sulfamethoxazole drugs in beta-lactoglobulin protein gels obtained by phase separation in water/ethanol mixture.

Physically cross-linked beta-lactoglobulin (BLG) protein gels containing theophylline and sulfamethoxazole low molecular weight drugs were prepared in 50% ethanol solution at pH 8 and two protein concentrations (6 and 7% (w/v)). Swelling behavior of cylindrical gels showed that, irrespective of the hydrated or dehydrated state of the gel, the rate of swelling was the highest in water. When the gels were exposed to water, they first showed a swelling phase in which their weight increased 3 and 30 times for hydrated and dehydrated gels, respectively, due to absorption of water, followed by a dissolution phase. The absorption of solvent was however considerably reduced when the gels were exposed to aqueous buffer solutions. The release behavior of both theophylline and sulfamethoxazole drugs from BLG gels was achieved in a time window ranging from 6 to 24 h. The drug release depended mainly on the solubility of the drugs and the physical state of the gel (hydrated or dry form). Analysis of drug release profiles using the model of Peppas showed that diffusion through hydrated gels was governed by a Fickian process whereas diffusion through dehydrated gels was governed partly by the swelling capacities of the gel but also by the structural rearrangements inside the network occurring during dehydration step. By a judicious selection of protein concentration, hydrated or dehydrated gel state, drug release may be modulated to be engineered suitable for pharmaceutical as well as cosmetics and food applications.

Ethanol↗

Nonideal mixing and phase separation in phosphatidylcholine-phosphatidic acid mixtures as a function of acyl chain length and pH.

The miscibilities of phosphatidic acids (PAs) and phosphatidylcholines (PCs) with different chain lengths (n = 14, 16) at pH 4, pH 7, and pH 12 were examined by differential scanning calorimetry. Simulation of heat capacity curves was performed using a new approach that incorporates changes of cooperativity of the transition in addition to nonideal mixing in the gel and the liquid-crystalline phase as a function of composition. From the simulations of the heat capacity curves, first estimates for the nonideality parameters for nonideal mixing as a function of composition were obtained, and phase diagrams were constructed using temperatures for onset and end of melting, which were corrected for the broadening effect caused by a decrease in cooperativity. In all cases the composition dependence of the nonideality parameters indicated nonsymmetrical mixing behavior. The phase diagrams were therefore further refined by simulations of the coexistence curves using a four-parameter approximation to account for nonideal and nonsymmetrical mixing in the gel and the liquid-crystalline phase. The mixing behavior was studied at three different pH values to investigate how changes in headgroup charge of the PA influences the miscibility. The experiments showed that at pH 7, where the PA component is negatively charged, the nonideality parameters are in most cases negative, indicating that electrostatic effects favor a mixing of the two components. Partial protonation of the PA component at pH 4 leads to strong changes in miscibility; the nonideality parameters for the liquid-crystalline phase are now in most cases positive, indicating clustering of like molecules. The phase diagram for 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid:1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine mixtures at pH 4 indicates that a fluid-fluid immiscibility is likely. The results show that a decrease in ionization of PAs can induce large changes in mixing behavior. This occurs because of a reduction in electrostatic repulsion between PA headgroups and a concomitant increase in attractive hydrogen bonding interactions.

1,2-Dipalmitoylphosphatidylcholine↗

Charge-induced unilamellar vesicle formation and phase separation in solutions of Di-n-decylmethylamine oxide.

A double-tail amine oxide surfactant, di-n-decylmethylamine oxide (2C10MAO), was prepared, and the effects of protonation on aggregate structure were examined by small-angle neutron scattering (SANS), cryo-transmission electron microscopy (cryo-TEM), turbidity, electric conductivity, and solubilization of an oil-soluble dye at various degrees of neutralization, X, defined as the mole ratio of HCl/2C10MAO. The surfactant makes an L(2) phase in the nonprotonated state (X = 0) in water. The L(2) phase is in equilibrium with an aqueous L(1) phase. On protonation, unilamellar vesicles (ULVs) are formed over a wide range of compositions (0.05 < X< 0.4-0.5 at C = 10 mM) as observed by cryo-TEM. At X = 0.2, the ULV is stable over a wide concentration range (3 mM < or = C < 0.1 M), but an L(alpha) phase replaces the vesicle phase at C > 0.1 M. SANS results show that the mean radius of the ULV is about 25 nm and the bilayer thickness is about 2 nm, consistent with the extended configuration of the alkyl chains of the surfactant. An important contribution to the enhanced stability of the bilayer structures over the L(2) phase is suggested to be the translational entropy of the counterions. The enhanced stability of the bilayers diminishes as the counterion concentration increases either by an increase of X or by the addition of a salt. When the counterion concentration exceeds a critical value, the ULV solutions transform into the L(2) phase (or L(2)/L(1) two-phase system at low surfactant concentrations). The critical composition X is about 0.4-0.5 in water, but it is below 0.4 in D(2)O. The critical NaCl concentration is below 5 mM at X = 0.2. The stability of ULVs against multilamellar vesicles is ascribed partly to undulation forces and partly to the adjustable nature of the spontaneous curvature of amine oxide monolayers. The characteristics of the ULV of the surfactant remain the same within a temperature range 25-50 degrees C at X = 0.2. An iridescent lamellar phase and possibly an L(3) phase were observed in a very narrow X range (0 < X < 0.02) prior to the vesicle phase.

Cryoelectron Microscopy↗

Noise probe of the dynamic phase separation in La(2/3)Ca(1/3)MnO3

Giant random telegraph noise (RTN) in the resistance fluctuation of a macroscopic film of perovskite-type manganese oxide La(2/3)Ca(1/3)MnO3 has been observed at various temperatures ranging from 4 to 170 K, well below the Curie temperature ( T(C) approximately 210 K). The amplitudes of the two-level fluctuations vary from 0.01% to 0.2%. We discuss the origin of the RTN to be a dynamic mixed-phase percolative conduction process, where manganese clusters switch back and forth between two phases that differ in their conductivity and magnetization.

Journal Article↗

Beta transition and stress-induced phase separation in the spinning of spider dragline silk.

Spider dragline silk is formed as the result of a remarkable transformation in which an aqueous dope solution is rapidly converted into an insoluble protein filament with outstanding mechanical properties. Microscopy on the spinning duct in Nephila edulis spiders suggests that this transformation involves a stress-induced formation of anti-parallel beta-sheets induced by extensional flow. Measurements of draw stress at different draw rates during silking confirm that a stress-induced phase transition occurs.

Animals↗

Evidence for phase separation in the membrane of an osmotically stabilized fatty acid auxotroph of E. coli and its biological significance.

1. An unsaturated fatty acid auxotroph of Escherichia coli accumulated a high content of saturated fatty acids in its membrane when it was cultured under osmotically stabilized conditions. The physicochemical properties of the phospholipid extracts and of the membrane fraction from the cells were investigated by means of proton magnetic resonance, infrared spectroscopy and differential scanning calorimetry. 2. Physicochemical studies indicate that the phospholipid bilayers in the membranes exhibit at least two phase transitions, a minor one at approx. 19 degrees C and a major one at approx. 43 degrees C. Between the two temperatures, gel and liquid crystalline domains co-exist. Moreover, even in the gel state, phospholipids seem to segregated into domains containing different proportions of unsaturated fatty acids. 3. The Arrhenius plot of beta-galactoside transport rates is biphasic. The inflection point is at 22 degrees C. This means that the appearance of the fluid region in the bilayer at approx. 19 degrees C is important in the activation of membrane transport.

Biological Transport↗

Realistic calculation of the low- and high-density liquid phase separation in a charged colloidal dispersion.

A realistic statistical-mechanics model is applied to describe the repulsive interaction between charged colloids. The latter, in combination with the long-range van der Waals attraction simulated under excess salt environment, gives rise to a total intercolloidal particle potential showing a clear second potential minimum. Differing from the usual Derjaguin-Landau-Verwey-Overbeek (DLVO) model, the present model is valid at any finite concentration of colloids and is thus an appropriate model for investigating the low- and high-density liquid phase transition. Employing this two-body colloid-colloid potential and in conjunction with the Weeks-Chandler-Andersen [J. D. Weeks, D. Chandler, and H. C. Andersen, J. Chem. Phys. 54, 5237 (1971)] thermodynamic perturbation theory, we derive analytical expressions for the pressure, chemical potential, and related thermodynamic functions. These thermodynamic quantities were used to calculate the phase diagrams of charged colloidal dispersions in terms of the critical parameters: temperature, volume fraction, and electrolyte concentration parameter k(D). Compared with the DLVO model, we find the areas enclosed within the spinodal decomposition and also the liquid-liquid coexistence curves broader in the present model for an excess salt condition kappa=k(D)sigma(0)< or similar to 200, sigma(0) being the macroion diameter, in addition to exhibiting a shift in the critical point kappa(c) to lower values; for kappa>300, the disparities between the two models reduce. The same thermodynamic perturbation theory has been employed to study also the weak reversible coagulation whose physical origin is attributed to the presence of the second potential minimum. We examine various colloidal parameters that affect the structure of the latter and deduce from our analysis the conditions of colloidal stability. In comparison with the measured flocculation data for a binary mixture of polystyrene lattices and water, we find that our calculated results are generally reasonable, thus lending great credence to the presently used model.

Journal Article↗