Collapse Surface Pressure Research Articles

Morphology of Mixed Langmuir and Langmuir-Schaefer Monolayers with Covered CdSe/CdS/ZnS Quantum Dots and Arachidic Acid.

The process of formation of a Langmuir-Schaefer (LS) matrix based on a mixed monolayer of arachidic acid (AA) and 8 nm CdSe/CdS/ZnS quantum dots (QDs) stabilized by molecules of trioctylphosphine oxide (TOPO) was investigated. The change in the morphology, monolayer compressibility, and area per elementary cell of the created mixed monolayers, depending on the ratio of the components, was studied. It is shown that the change in the morphology of Langmuir-Blodgett (LB) monolayers begins to occur at a ratio between the number of QDs and AA molecules of 1:24. Dendrimeric structures with a thickness of the order of 30-40 nm appear in the mixed monolayer when LB film deposition was carried out above the collapse surface pressure of a Langmuir film from only TOPO-covered QDs. Information on the dependence of the morphology of such structures on the molar ratio of the components is necessary for the production of ordered 2D nanostructures containing 0D and 1D objects with quantum bonds. Such nanostructures can be used in nanoelectronic and optoelectronic devices as a sensitive sensor element. The obtained results would be relevant for any type of spherical shape nanoparticles.

Capacity of cholesteryl hemisuccinate in ion pair/phospholipid complex to improve drug-loading, stability and antibacterial activity of clarithromycin intravenous lipid microsphere.

Current research was to evaluate the capacity and molecular interaction of ion pair/phospholipid complex during preparation of clarithromycin intravenous lipid microsphere (CLA-LM) for improving drug-loading, stability and antibacterial activity. The optimum pH range for the presence of ion pair formed by CLA and cholesteryl hemisuccinate (CHEMS) was found to be between 6.4 and 8.2. CLA-LM prepared by ion pair/phospholipid complex possessed improved drug-loading (5-10 mg/ml), plasma stability, storage stability (24 months at 4 ± 2℃) and better activity for multi-drug-resistant Mycobacterium tuberculosis (MIC = 0.058 μg/ml). The release profile in vitro was pH-sensitive and greater in more acidic condition, which was effective for pathological targeting. CLA-LM presented controlled release and low drug leakage in plasma. Langmuir monolayer showed that the incorporation of CHEMS obviously improved interfacial molecular interactions of the mixed monolayer. The ordering and condensing effect of CHEMS resulted in higher collapse surface pressure and smaller limiting area, as well as reduced compression modulus. Close aggregate network distribution with particular micro domains in atomic force microscopy images reflected the enhanced miscibility and thermodynamic stability. The change of the spatial location and protonation degree of CHEMS resulted from the aqueous subphase pH value from 6 to 8 was observed. More acidic environment allowed for better molecular interaction and interface behavior. These results indicated that CHEMS enhances the phospholipid packing to achieve stable CLA-encapsulation in phospholipid interface and identify the preference of ion pair/phospholipid complex as a valuable delivery strategy to develop CLA formulations for improving drug-loading, stability and antibacterial activity.

Regioselective Synthesis of Eight-Armed Cyclosiloxane Amphiphile for Functional 2D and 3D Assembly Motifs.

A crystalline tetramethylcyclotetrasiloxane (TMCS)-derived amphiphile was regioselectively synthesized with eight peripheral hydrophilic amide groups and hydrophobic dodecyl chains by Pt(0)-catalyzed hydrosilylation and amidation reactions. The as-synthesized materials showed ordered lamellar structure formation in the powder form. It also exhibits superior two-dimensional (2D) monolayer formation properties at the air-water interface with unexpectedly high collapse surface pressure and elastic modulus. The monolayers act as two-dimensional building blocks with finely controllable thickness on a several nanometer scale irrespective of the substrate type and properties. The amphiphile forms nanofibers spontaneously by good-poor solvent strategies, which contributes to porous three-dimensional (3D) structures possessing superhydrophobic surface wettability.

β-Galactosidase Langmuir Monolayer at Air/X-gal Subphase Interface.

This article investigates the surface chemistry properties of the β-galactosidase monolayer at the air-subphase interface at the vicinity of its substrate, X-gal. We have demonstrated that the β-galactosidase in the monolayer form remained active and performed hydrolysis of the X-gal in the subphase. We investigated the β-galactosidase Langmuir monolayer in absence and presence of X-gal in the subphase of varying concentration of X-gal with the sodium chloride solution. It was found that the limiting molecular area as well as the collapse surface pressure kept on decreasing with the increasing concentration of X-gal. In accordance to the data obtained from the isotherm it was also found that β-galactosidase forms a stable monolayer that does not aggregate at the air-subphase interface. The stability of the monolayer at the air-subphase interface was studied by using compression-decompression cycles with and without X-gal at varying concentration and different surface pressures. The infrared reflection-absorption spectroscopy (IRRAS) and Brewster angle microscopy (BAM) of β-galactosidase Langmuir monolayer was also investigated for pure and mixed β-galactosidase at the air-subphase.

Surface pressure‐induced isothermal 2D‐ to 3D‐transitions in Langmuir films of poly(ε‐caprolactone)s and oligo(ε‐caprolactone) based polyesterurethanes

Surface pressure‐induced isothermal 2D‐ to 3D‐transitions in Langmuir films of biodegradable poly(ε‐caprolactone) (PCL) and oligo(ε‐caprolactone) based polyester‐urethanes P(OCL‐U)s are investigated in order to gain deeper insights into the influence of the linker species on the crystallization and aggregation behavior of macromolecules in a biomimetic aqueous environment. The presence of three urethane linkers derived from 2, 2 (4), 4‐trimethyl‐hexamethylene‐diisocyanate (TMDI), hexamethylene diisocyanate (HDI) and lysine ethylester diisocyanate (LDI) induces remarkable changes in the mesoscopic structure compared to PCL Langmuir films. The pronounced changes in the morphology of the 3D structures co‐existing with the Langmuir film above a collapse surface pressure are visualized by Brewster angle microscopy (BAM). Hysteresis of the compression–expansion isotherm in the surface pressure range of the 2D‐ to 3D‐transition indicates the influence of the urethane linkers on the diffusion‐limited kinetics and on the reversibility of this phase transition. Copyright © 2015 John Wiley & Sons, Ltd.

Calf Lung Surfactant Recovers Surface Functionality After Exposure to Aerosols Containing Polymeric Particles.

Recent studies have shown that colloidal particles can disrupt the interfacial properties of lung surfactant and thus key functional abilities of lung surfactant. However, the mechanisms underlying the interactions between aerosols and surfactant films remain poorly understood, as our ability to expose films to particles via the aerosol route has been limited. The aim of this study was to develop a method to reproducibly apply aerosols with a quantifiable particle dose on lung surfactant films and investigate particle-induced changes to the interfacial properties of the surfactant under conditions that more closely mimic those in vivo. Films of DPPC and Infasurf® were exposed to aerosols containing polystyrene particles generated using a Dry Powder Insufflator™. The dose of particles deposited on surfactant films was determined via light absorbance. The interfacial properties of the surfactant were studied using a Langmuir-Wilhelmy balance during surfactant compression to film collapse and cycles of surface compression and expansion at a fast cycling rate within a small surface area range. Exposure of surfactant films to aerosols led to reproducible dosing of particles on the films. In film collapse experiments, particle deposition led to slight changes in collapse surface pressure and surface area of both surfactants. However, longer interaction times between particles and Infasurf® films resulted in time-dependent inhibition of surfactant function. When limited to lung relevant surface pressures, particles reduced the maximum surface pressure that could be achieved. This inhibitory effect persisted for all compression-expansion cycles in DPPC, but normal surfactant behavior was restored in Infasurf® films after five cycles. The observation that Infasurf® was able to quickly restore its function after exposure to aerosols under conditions that better mimicked those in vivo suggests that particle-induced surfactant inhibition is unlikely to occur in vivo due to an aerosol exposure.

Facile Preparation of Highly Oriented Poly(vinylidene fluoride) Langmuir–Blodgett Nanofilms Assisted by Amphiphilic Polymer Nanosheets

We describe a facile and novel method for preparing highly dense Langmuir–Blodgett (LB) nanofilms of poly(vinylidene fluoride) (PVDF) with precisely adjustable film thickness from several to hundreds of nanometers, assisted by amphiphilic poly(N-dodecylacrylamide) (pDDA) nanosheets. Even at a molar mixing ratio of PVDF:pDDA up to 50:1, the high collapse surface pressure of 44.4 mN/m obtained using this method is a breakthrough for the preparation of PVDF LB nanofilms, which is devoted to the resulting high-density PVDF nanofilms. As shown by FTIR and XRD measurements, the mixed LB nanofilms without any postprocessing comprised dominant ferroelectric β phase of ∼95% and negligible paraelectric α phase. Furthermore, through control of the surface pressure, controllable PVDF crystal morphologies were achieved. Moreover, β phase PVDF dominates in all cases. After applying a dc bias of 5 V through a conductive cantilever, the local polarized pattern on the surface of a nine-layer mixed LB nanofilm observed using Kelvin probe force microscopy indicates that it is possible to induce all dipoles in one direction in the mixed LB nanofilm, which is promising for application in low-voltage nanoelectronics.

Oleamide and Oleamide–Lipid Mixed Monolayers

Oleamide (OA) and its mixtures with other lipids are of interest in some biological systems, such as the tear film or the cerebrospinal fluid. In this work, the behavior of OA, OA–dipalmitoylphospatidylcholine (DPPC), and OA–cholesterol films is studied using surface pressure–area isotherms and atomic force microscopy, and analyzing the collapse pressure vs. composition, the compressibility, and the mean area vs. composition for several surface pressures. It is observed that OA forms homogeneous monolayers in a liquid-expanded state until the collapse surface pressure and the mixing with DPPC and cholesterol. The collapse surface pressure changes with the mixture composition, and in the case of DPPC, a noticeable influence of OA in the liquid-expanded/liquid-condensed phase change of DPPC is observed. The excess area is positive for the OA/DPPC films, but mostly negative for the OA–cholesterol films. These results are of interest for the target of formulation of artificial tears containing lipids.

Bis (monoacylglycero) phosphate interfacial properties and lipolysis by pancreatic lipase-related protein 2, an enzyme present in THP-1 human monocytes

The interfacial physical properties of bis(monoacylglycero)phosphate (BMP) and its derivatives with three oleoyl chains (hemi-BDP) and four oleoyl chains (bis(diacylglycero)phosphate, BDP) were investigated using Langmuir monomolecular films. The mean molecular area of BMP at the collapse surface pressure (45mN m−1) was similar to those measured with other phospholipids bearing two acyl chains (66 and 59.6Å2 molecule−1 at pH 5.5 and 8.0, respectively). In Hemi-BDP and BDP, the mean molecular area increased by 26 and 35Å2 molecule−1 per additional acyl chain at pH 5.5 and 8.0, respectively. When BMP was added to a phospholipid mixture mimicking late endosome membrane composition at pH 8.0, the mean phospholipid molecular area increased by 7% regardless of the surface pressure. In contrast, the variation in molecular area was surface pressure-dependent at pH 5.5, a pH value close to that of intra-endosomal content. BMP and hemi-BDP, but not BDP, were hydrolyzed by pancreatic lipase-related protein 2 (PLRP2), which exhibits phospholipase A1 activity. At pH 5.5, the maximum activities of PLRP2 on BMP were recorded at high surface pressures (25–35mN/m). At pH 8.0, the PLRP2 activity vs. surface pressure showed a bell-shaped curve with maximum activities at 15mN/m for both BMP and hemi-BDP. This is a new activity for this enzyme which could degrade cellular BMP since both human PLRP2 (HPLRP2) and BMP were localized in human monocytic THP-1 cells. This is the first report on the cellular localization of HPLRP2 in human monocytes.

Native Pulmonary Surfactant Membranes in Mice Show Coexistence of Two Different Phases in Bilayers and Monolayers: When the Lipid Composition can Predict the Structural Phase Segregations

Pulmonary surfactant is a surface active material composed both of lipids (aprox. 90% by weight) and proteins (aprox. 10%) produced by type II pneumocyte cells in the alveoli. This tension-active material forms a unique air-liquid interface at the alveolar cell surface that reduces surface tension close to 0 mN/m and maintains lung volumes and alveolar homeostasis at the end of the expiration. There are four pulmonary surfactant proteins (SP-A, -B, -C and -D). SP-A and -D have an important role in the immunological response against pathogens. The particular lipid composition of the lung surfactant suggests that surfactant mono- and bi-layer-based structures could exhibit lateral phase segregation at physiological temperatures. This work shows that in native pulmonary surfactant membranes a close lipid compositional study is crucial to understand the structure and biophysical function of these complex mixtures. Observing Giant Unilamellar Vesicles under conventional and two-photon excitation microscopy allow us to characterize and quantify the coexistence of two fluid-like phases in the wild-type (wt) native pulmonary surfactant membranes and a gel/fluid-like segregation pattern in the Knocked-out protein D (KOD) membranes. The atomic force microscopy studies of supported Langmuir-Blodgett bilayers and monolayers at different surface pressure show the same phase pattern before the collapse surface pressure of native pulmonary surfactant (∼40mN/m). Above this surface pressure different protruded structures can be observed arising from the more fluid phases. A closer look at the lipid composition reveals a higher content of saturated phospholipid species in the KOD native pulmonary surfactant membranes. This last finding explain the coexistence phenomena observed and allow us to conclude that the pulmonary surfactant segregation pattern could be predicted by an accurate lipid compositional study.

Schizophrenic Behavior of a Thermoresponsive Double Hydrophilic Diblock Copolymer at the Air−Water Interface

The thermoresponsive behavior of the rhodamine B end-labeled double hydrophilic block copolymer (DHBC) poly(N,N-dimethylacrylamide)-b-poly(N,N-diethylacrylamide) (RhB-PDMA(207)-b-PDEA(177)) and the 1:1 segmental mixture of PDEA and rhodamine B end-labeled PDMA homopolymers was studied over the range of 10-40 degrees C at the air-water interface. The increase in collapse surface pressure (second plateau regime) of the DHBC with temperature confirms the thermoresponsiveness of PDEA at the interface. The sum of the pi-A isotherms of the two single homopolymers weighted by composition closely follows the pi-A isotherm of the DHBC, suggesting that the behavior of each block of the DHBC is not influenced by the presence of the other block. Langmuir-Blodgett monolayers of DHBC deposited on glass substrates were analyzed by laser scanning confocal fluorescence microscopy (LSCFM), showing schizophrenic behavior: at low temperature, the RhB-PDMA block dominates the inside of bright (core) microdomains, switching to the outside (shell) at temperatures above the lower critical solution temperature (LCST) of PDEA. This core-shell inversion triggered by the temperature increase was not detected in the homopolymer mixture. The present results suggest that both the covalent bond between the two blocks of the DHBC and the tendency of rhodamine B to aggregate play a role in the formation of the bright cores at low temperature whereas PDEA thermoaggregation is responsible for the formation of the dark cores above the LCST of PDEA.

Numerical simulations of non-spherical bubble collapse.

A high-order accurate shock- and interface-capturing scheme is used to simulate the collapse of a gas bubble in water. In order to better understand the damage caused by collapsing bubbles, the dynamics of the shock-induced and Rayleigh collapse of a bubble near a planar rigid surface and in a free field are analysed. Collapse times, bubble displacements, interfacial velocities and surface pressures are quantified as a function of the pressure ratio driving the collapse and of the initial bubble stand-off distance from the wall; these quantities are compared to the available theory and experiments and show good agreement with the data for both the bubble dynamics and the propagation of the shock emitted upon the collapse. Non-spherical collapse involves the formation of a re-entrant jet directed towards the wall or in the direction of propagation of the incoming shock. In shock-induced collapse, very high jet velocities can be achieved, and the finite time for shock propagation through the bubble may be non-negligible compared to the collapse time for the pressure ratios of interest. Several types of shock waves are generated during the collapse, including precursor and water-hammer shocks that arise from the re-entrant jet formation and its impact upon the distal side of the bubble, respectively. The water-hammer shock can generate very high pressures on the wall, far exceeding those from the incident shock. The potential damage to the neighbouring surface is quantified by measuring the wall pressure. The range of stand-off distances and the surface area for which amplification of the incident shock due to bubble collapse occurs is determined.

Fluorescence Microscopy Imaging of Giant Folding in a Catanionic Monolayer

The behavior of the catanionic system of dioctadecyldimethylammonium bromide (DODAB) and sodium dodecyl sulfate (SDS) was investigated at 23 +/- 1 degrees C at the air-water interface using a Langmuir trough. The surface pressure as a function of surface area was measured while monitoring domain structures using epifluorescence microscopy. At high surface densities, the monolayer exhibits collapse through reversible folding at about 47 mN m(-1). This corresponds to the DODAB collapse surface pressure. The number of folds increases with the rate of compression speed and is history-dependent.

Langmuir Monolayer and Langmuir−Blodgett Films Formed by a Melamine-Headed Azobenzene-Derived Amphiphile: Interfacial Assembly Affected by Host−Guest Interaction

A novel azobenzene-derived amphiphile with a melamine head, 2Azo-2C12H2-melamine, has been synthesized. pi-A isotherm measurements displayed that this amphiphile is able to form a stable Langmuir monolayer on both pure water and barbituric acid (BA)- or thymine (T)-containing subphases. The collapse surface pressure and limiting molecular area of its Langmuir monolayer on pure water are 40 mN/m and 0.56 nm2, respectively. However, when barbituric acid or thymine was introduced into the subphase, the corresponding pi-A isotherms of the monolayers exhibited a lower collapse surface pressure (22 mN/m for BA, 21 mN/m for T) and smaller limiting molecular area (0.54 nm2 for BA and 0.52 nm2 for T). UV-vis and FT-IR studies of the LB films formed by 2Azo-2C12H25-melamine have also been carried out. The results indicated that the LB films of 2Azo-2C12H25-melamine deposited from pure water undergo distinct collapse of the H-aggregate upon UV irradiation, while the LB films deposited from a BA- or T-containing subphase retain the H-aggregate. The host-guest-interaction-induced blockage of azobenzene photoisomerization should be responsible for the stabilized H-aggregate. A 1:1 host/guest binding mode to form a linear supramolecular polymeric chain has been proposed in the Langmuir monolayers formed on a BA- or T-containing subphase. The current results suggest that the host-guest interaction should be an effective means to manipulate the interfacial assembly of azobenzene-derived amphiphiles.

Phase behaviour of stearic acid–stearonitrile mixtures: A thermodynamic study in bulk and at the air–water interface

The solid–liquid phase behaviour of stearic acid (SA) and stearonitrile (SN) in binary mixtures was investigated by differential scanning calorimetry (DSC), and the formation of SA–SN mixed monolayers at the air–water interface was followed by surface pressure–area ( π–A) measurements and by Brewster angle microscope (BAM) observation. The solid–liquid phase diagram is a eutectic type phase diagram, with the eutectic composition 0.90 < X SN < 0.95 and T eut = 40.9 °C. The DSC results also suggest that the two components are immiscible in the solid phase but form a liquid mixture with positive deviations to the ideal behaviour. At the air–water interface, the two components form liquid condensed monolayers in the entire range of compositions, at low surface pressures, while solid mixed monolayers only form at high surface pressures for X SN < 0.8. Thermodynamic analysis indicates that SA and SN are miscible in the liquid condensed phase, with negative deviations from the ideal behaviour. The variation of the collapse surface pressure of mixed monolayers also indicates miscibility at the air–water interface.

Influence of the chain length of ubiquinones on their interaction with DPPC in mixed monolayers

The thermodynamic behavior of representative short (UQ2), middle (UQ4 and UQ6) and long-chain (UQ10) ubiquinones (UQ) mixed with dipalmitoyl-phosphatidylcholine (DPPC) was studied in monolayers at the air–water interface. The influence of isoprenoid chain-length of UQ on miscibility of both lipids was investigated by analysis of surface pressure-area isotherms and using fluorescence microscopy. Analysis of excess areas ( A ex) and free energies of mixing (Δ G m), calculated from compression isotherms in the full range of ubiquinones concentrations, has given evidences for UQ-rich constant-size (UQ6, UQ10) or less growth limited (UQ2, UQ4) microdomains formation within mixed films. Fluorescence microscopy observation revealed that ubiquinones are preferentially soluble in the expanded phase. When lateral pressure increased, concomitant evolutions of A ex and Δ G m parameters, and composition dependence of collapse surface pressures, argue for an evolution towards a total segregation, never reached due to expulsion of ubiquinones from the film. The possible significance of these observations is discussed in relation to ubiquinones organization and similar chain length effects in membranes.

Two-Dimensional Miscibility StudiesThe Analysis of Interaction between Long-Chain Alcohols and Semifluorinated Alkanes

This work presents the results of phase behavior studies of two-dimensional (2D) binary systems involving semifluorinated alkanes (SFAs) and fatty alcohols. Four different SFAs were selected for investigations: (i) with a short and branched perfluorinated moiety (iF3H20), (ii) with a short and normal perfluorinated chain (F4H20), (iii) with a long and branched perfluorinated fragment (iF9H20), and (iv) with a long and normal perfluorinated group (F10H20). Two alcohols were selected to mix with the above-mentioned SFAs: tetradecanol and docosanol. The measurements were based on surface pressure/area isotherms in addition to Brewster angle microscope (BAM) imaging. Dependencies of the collapse surface pressure and the compression modulus vs the monolayer composition together with the excess free energy of mixing values, complemented with BAM images, enabled us to draw some general conclusions regarding the phase behavior of the investigated mixed systems. Generally, it has been noticed that the addition of docosanol into an SFA monolayer exerts a condensing effect, contrary to tetradecanol. Moreover, SFAs with a long perfluorinated segment mix to a larger extent with alcohols as compared to their analogues having a short perfluorinated moiety. The resultant phase diagrams for all the investigated eight mixtures are presented and discussed.

Memory effects on the interfacial characteristics of dioctadecyldimethylammonium bromide monolayers at the air–water interface

The structural and dynamic characteristics of dioctadecyldimethylammonium bromide (DODAB) monolayers on a pure water subphase were investigated by surface film balance, Brewster angle microscopy, and relaxation in area and surface pressure at constant surface pressure and area, respectively. The first compression–expansion cycle of the monolayer is not reversible and the second π– A compression isotherm deviates to larger molecular areas relative to the first one. At a microscopic level this hysteresis may be assigned to an irreversible hydration of the ammonium groups of DODAB. The morphology and reflectivity of DODAB monolayers during compression and expansion on the monolayer depend on the monolayer history. Bright domains randomly dispersed were observed during compression before collapse. Surprisingly, this random distribution of domains changes into a fractal-like structure during the monolayer expansion in a narrow range of surface pressures. This morphology does not form when the monolayer is previously compressed above the collapse surface pressure. 2D foam-like structure is often observed when the film is expanded at maximum area. Relaxation phenomena in DODAB monolayers are attributed to monolayer reorganization and nucleation of liquid-condensed domains from the liquid-expanded phase. These time-dependent processes are irreversible.

Molecular Organization in Langmuir Films of a Dichroic Azo Dye-Liquid Crystal Mixture. I. Thermodynamic Data and BAM Observations

1 Two-component films of 4-octyl-4’-cyanobiphenyl (8CB) and a nonamphiphilic azo dye on an airwater interface have been studied by means of surface pressure-area (π-A) isotherm measurement, Brewster angle microscopy (BAM), and absorption spectroscopy. Conventional Langmuir technique was used to form /8CB (guest-host) films during compression and expansion of an area occupied by the molecules. The pure dye, spread on the air-water interface, formed irregular three-dimensional structures (3D), visible by the naked eye. In 1/8CB films the dye was distributed homogeneously only at low mole fraction. At mole fractions higher than 0.3, just after spreading the 1/8CB solution at the interface part of 1 formed 3D structures. On the basis of π-A isotherm, information about the miscibility of the two components in the 1/8CB mixtures was obtained by using the area additivity criterion and surface phase rule. BAM images allowed to draw conclusions on the molecular organization of mixed Langmuir films at the air-water interface. The presence of 1, roughly up to a mole fraction of 0.5, causes the mixed film to be less compressible and more thermodynamically stable (with higher collapse surface pressure). The absorption spectra of the films of 1/8CB mixtures revealed the formation of dye aggregates.

Liquid crystalline binary mixtures at the air–water interface

The interaction behaviour of the binary mixtures composed of the liquid crystals 4-octyl-4′-cyanobiphenyl (8CB) and 4-pentyl-4′-cyanoterphenyl (5CT) as well as biphenyl (BP) and terphenyl (TP) at different molar ratios has been studied using Langmuir technique. The surface pressure-mean molecular area isotherms have been recorded. The miscibility of the components in Langmuir films has been determined by using the additivity rule and collapse surface pressure criterion. It has been found, that 8CB is partially miscible with BP and TP. 5CT is miscible with TP, whereas the mixtures of two liquid crystals, 8CB and 5CT reveal the phase separation over a whole range of composition.