Liquid-liquid Phase Separation Mechanism Research Articles

The liquid-liquid phase separation in programmed cell death

In recent years, the physical phenomenon of liquid-liquid phase separation has been widely introduced into biological research. Membrane-free organelles have been found to exist in cells that were driven by liquid-liquid phase separation. Intermolecular multivalent interactions can drive liquid-liquid phase separation to form condensates that are independent of other substances in the environment and thus can play an effective role in regulating multiple biological processes in the cell. The way of cell death has also long been a focus in multiple research. In the face of various stresses, cell death-related mechanisms are crucial for maintaining cellular homeostasis and regulating cell fate. With the in-depth study of cell death pathways, it has been found that the process of cell death was also accompanied by the regulation of liquid-liquid phase separation and played a key role. Therefore, this review summarized the roles of liquid-liquid phase separation in various cell death pathways, and explored the regulation of cell fate by liquid-liquid phase separation, with the expectation that the exploration of the mechanism of liquid-liquid phase separation would provide new insights into the treatment of diseases caused by regulated cell death.

Phosphorylation regulates tau’s phase separation behavior and interactions with chromatin

Tau is a microtubule-associated protein often found in neurofibrillary tangles (NFTs) in the brains of patients with Alzheimer’s disease. Beyond this context, mounting evidence suggests that tau localizes into the nucleus, where it may play a role in DNA protection and heterochromatin regulation. The molecular mechanisms behind these observations are currently unclear. Using in vitro biophysical experiments, here we demonstrate that tau can undergo liquid-liquid phase separation (LLPS) with DNA, mononucleosomes, and reconstituted nucleosome arrays under low salt conditions. Low concentrations of tau promote chromatin compaction and protect DNA from digestion. While the material state of samples at physiological salt is dominated by chromatin oligomerization, tau can still associate strongly and reversibly with nucleosome arrays. These properties are driven by tau’s strong interactions with linker and nucleosomal DNA. In addition, tau co-localizes into droplets formed by nucleosome arrays and phosphorylated HP1α, a key heterochromatin constituent thought to function through an LLPS mechanism. Importantly, LLPS and chromatin interactions are disrupted by aberrant tau hyperphosphorylation. These biophysical properties suggest that tau may directly impact DNA and chromatin accessibility and that loss of these interactions could contribute to the aberrant nuclear effects seen in tau pathology.

Solute interaction-driven and solvent interaction-driven liquid-liquid phase separation induced by molecular size difference.

We conducted molecular dynamics (MD) simulations in a binary Lennard-Jones system as a model system for molecular solutions and investigated the mechanism of liquid-liquid phase separation (LLPS), which has recently been recognized as a fundamental step in crystallization and organelle formation. Our simulation results showed that LLPS behavior varied drastically with the size ratio of solute to solvent molecules. Interestingly, increasing the size ratio can either facilitate or inhibit LLPS, depending on the combination of interaction strengths. We demonstrated that the unique behavior observed in MD simulation could be reasonably explained by the free energy barrier height calculated using our thermodynamic model based on the classical nucleation theory. Our model proved that the molecular size determines the change in number of interaction pairs through LLPS. Varying the size ratio changes the net number of solute-solvent and solvent-solvent interaction pairs that are either broken or newly generated per solute-solute pair generation, thereby inducing a complicated trend in LLPS depending on the interaction parameters. As smaller molecules have more interaction pairs per unit volume, their contribution is more dominant in the promotion of LLPS. Consequently, as the size ratio of the solute to the solvent increased, the LLPS mode changed from solute-related interaction-driven to solvent-related interaction-driven.

Phase Separation of PML/RARα Microspeckles Governs Transcriptional Dysregulation through Genomic Rewiring of BRD4 in Acute Promyelocytic Leukemia

Introduction: Abnormal nuclear morphologies are frequently observed in cancer cells, which have been implicated in biological reactions such as gene regulation and DNA damage repair. However, the nature of altered nuclear structures, particularly molecular components determining the specificity, regulatory functions and oncogenic activity of nuclear compartments, has remained largely enigmatic and awaits characterization. In acute promyelocytic leukemia (APL), the PML/RARα fusion oncoprotein, which is produced by t(15;17), has been long observed under the microscope for its ability in destroying PML nuclear bodies (NBs) and subsequently forming microspeckles. Restoration of PML NBs has been reported during the treatment with retinoic acid (RA) or arsenic trioxide (ATO). Despite being long observed, how PML/RARα-mediated microspeckles are formed and linked to APL leukemogenesis remains a mystery. To address this knowledge gap, we present a comprehensive analysis of the driving forces and constituent components of PML/RARα-assembled microspeckles and their functional implications in transcriptional dysregulation and leukemogenesis. Methods: High-resolution imaging techniques, multi-omics strategies, integrative bioinformatics analysis, and in-depth functional validation are used in this study. We employ Immunofluorescence, Fluorescence recovery after photobleaching and in vitro droplet formation assays to determine the chemical and physical properties and constituent components of PML/RARα microspeckles. Co-IP and mass spectrometry are performed to identify interactome of PML/RARα. ChIP-seq is utilized to determine the exact genomic regions where PML/RARα microspeckles occur and to evaluate the impact on chromatin occupancy of PML/RARα and its co-factors upon genetic or pharmacological perturbation. RNA-seq is performed to evaluate the transcriptional output of repression of condensates mediated co-factor activity. Functional experiments, including CCK-8 assay and Flow cytometry, are used to assess the pathological functions of PML/RARα assembled and BRD4 recruited microspeckles. Results: We uncover the biophysical mechanism of liquid-liquid phase separation (LLPS) underlying the assembly of PML/RARα microspeckles and elucidate their role in APL leukemogenesis. Our findings reveal that PML/RARα co-assembles with BRD4 to form de novo nuclear phase-separated condensates, which distinguish them from PML nuclear bodies. PML/RARα and BRD4 co-assembled condensates exhibit preferential occupancy on super-enhancers and broad-promoters, targeting genes essential for APL leukemogenesis. Mechanically, PML/RARα incorporates BRD4 into nuclear condensates, thereby facilitating its chromatin binding and redistribution. Importantly, blockage of the condensate-mediated co-activator BRD4 activity suppresses APL cell proliferation and induces apoptosis, thereby impairing PML/RARα-driven leukemogenesis. Finally, perturbation of LLPS depletes the chromatin co-occupancy of PML/RARα and BRD4 and attenuates their target gene activation, reinforcing the importance of LLPS in transcriptional dysregulation. Conclusions: In this study, we have provided a comprehensive analysis of PML/RARα-assembled microspeckles, shedding light on their driving forces, constituent components, and regulatory function. Our findings have resolved the long-standing cognitive stagnation regarding the morphological characteristics of PML/RARα microspeckles. We have demonstrated that PML/RARα forms de novo phase-separated condensates, which selectively recruit the co-activator BRD4 to gene loci with both super enhancers and broad promoters. This aberrant recruitment leads to dysregulated transcriptional programs and the development of malignant phenotypes. Importantly, our present study is the first to reveal the biophysical nature of PML/RARα-mediated microspeckles and elucidate the fundamental regulatory mechanism underlying their formation. We have also highlighted the role of PML/RARα phase separation in the pathogenesis of APL leukemogenesis. Therefore, our study provides valuable biophysical insights into the molecular basis for PML/RARα to exert its oncogenic activity.

Decoding protein methylation function with thermal stability analysis

Protein methylation is an important modification beyond epigenetics. However, systems analyses of protein methylation lag behind compared to other modifications. Recently, thermal stability analyses have been developed which provide a proxy of a protein functional status. Here, we show that molecular and functional events closely linked to protein methylation can be revealed by the analysis of thermal stability. Using mouse embryonic stem cells as a model, we show that Prmt5 regulates mRNA binding proteins that are enriched in intrinsically disordered regions and involved in liquid-liquid phase separation mechanisms, including the formation of stress granules. Moreover, we reveal a non-canonical function of Ezh2 in mitotic chromosomes and the perichromosomal layer, and identify Mki67 as a putative Ezh2 substrate. Our approach provides an opportunity to systematically explore protein methylation function and represents a rich resource for understanding its role in pluripotency.

Enhanced liquidity of p62 droplets mediated by Smurf1 links Nrf2 activation and autophagy

BackgroundMacro-autophagy/Autophagy is an evolutionarily well-conserved recycling process to maintain the balance through precise spatiotemporal regulation. However, the regulatory mechanisms of biomolecular condensates by the key adaptor protein p62 via liquid-liquid phase separation (LLPS) remain obscure.ResultsIn this study, we showed that E3 ligase Smurf1 enhanced Nrf2 activation and promoted autophagy by increasing p62 phase separation capability. Specifically, the Smurf1/p62 interaction improved the formation and material exchange of liquid droplets compared with p62 single puncta. Additionally, Smurf1 promoted the competitive binding of p62 with Keap1 to increase Nrf2 nuclear translocation in p62 Ser349 phosphorylation-dependent manner. Mechanistically, overexpressed Smurf1 increased the activation of mTORC1 (mechanistic target of rapamycin complex 1), in turn leading to p62 Ser349 phosphorylation. Nrf2 activation increased the mRNA levels of Smurf1, p62, and NBR1, further promoting the droplet liquidity to enhance oxidative stress response. Importantly, we showed that Smurf1 maintained cellular homeostasis by promoting cargo degradation through the p62/LC3 autophagic pathway.ConclusionsThese findings revealed the complex interconnected role among Smurf1, p62/Nrf2/NBR1, and p62/LC3 axis in determining Nrf2 activation and subsequent clearance of condensates through LLPS mechanism.

Uncovering the conditions that dictate the liquid-liquid phase separation of the CTD of RNAPII.

The intrinsically disordered carboxy terminal domain (CTD) of RNA polymerase II (RNAPII) may nucleate the formation of transcription factories through a mechanism of liquid-liquid phase separation (LLPS). Transcription factories are densely packed membraneless organelles (MLOs) that contain all the necessary components to perform the transcription cycle. The CTD in model organisms such as S. cerevisiae, S. pombe and C. albicans, is composed of ∼26 copies of consensus repeats (Y1S2P3T4S5P6S7). However, CTD sequences of fungi isolated from geographically distinct locations from temperate to polar regions, show high sequence diversity at positions 1, 4, and 7. The CTD is highly conserved at its Ser-Pro motifs, which are targets of phosphorylation and cis-trans prolyl isomerization by Ess1. Ess1, an essential peptidylprolyl isomerase in yeast, has a highly conserved evolutionary relationship with the CTD. Here, we found that different CTDs isolated from yeasts found in geographically distinct regions ranging from temperate to polar climates undergo LLPS, but do so differently. We hypothesize Ess1 and phosphorylation may modulate the phase behavior of the CTD. The objective of this study is to determine how linear amino acid sequence, phosphorylation, and cis-trans prolyl isomerization influences CTD phase behavior. To do so, we utilize a variety of biophysical characterization assays such as UV-Vis spectroscopy, DIC microscopy, and phase separation prediction algorithms. These results will elucidate what conditions promote the LLPS of the CTD and its orthologs, thereby setting the stage for further investigating the conditions that promote the formation of transcription factories.

DNA damage-induced cellular senescence is regulated by 53BP1 accumulation in the nuclear foci and phase separation.

Cellular senescence is linked to a wide range of age-related diseases and can be triggered by a variety of stresses, including DNA damage. A variety of genotoxic stressors, such as anti-cancer drugs, cause DNA double-strand breaks (DSBs), which trigger the accumulation of the tumour suppressor protein p53 in the nucleus. Cellular stresses stabilize and activate the p53 signalling pathway, which regulates various cellular processes, such as apoptosis, DNA repair, and senescence. Although p53 signalling is a well-known tumour suppressor pathway, it remains unclear how it is regulated during cellular senescence. Here, we show that p53-binding protein 1 (53BP1) accumulation in the nuclear foci is required for DNA damage-induced cellular senescence via p53 activation. In human immortalized fibroblast, shRNA-mediated 53BP1 depletion decreased not only the expression of p53-target genes but also the cellular senescence induced by adriamycin treatment. Furthermore, we confirmed that DSBs trigger the hyperaccumulation of 53BP1 in the nuclear foci, which plays a key role in the regulation of cellular senescence. To prevent the accumulation of 53BP1 in the nuclear foci, we used phase separation inhibitors, and siRNA against RNF168, which accumulates at DSB loci and forms complexes with 53BP1. This blocks the formation of 53BP1 nuclear foci and DNA damage-induced cellular senescence by activating the p53 signaling pathway. In conclusion, we demonstrated that increased accumulation of 53BP1 in the nuclear foci following DNA damage activates p53 and governs cellular senescence via a liquid-liquid phase separation mechanism.

Tau liquid-liquid phase separation in neurodegenerative diseases.

Aggregation of the microtubule-associated protein tau plays a major role in Alzheimer's disease and several other neurodegenerative disorders. An exciting recent development is the finding that, akin to some other proteins associated with neurodegenerative disease, tau has a high propensity to condensate via the mechanism of liquid-liquid phase separation (LLPS). Here, we discuss the evidence for tau LLPS in vitro, the molecular mechanisms of this reaction, and the role of post-translational modifications and pathogenic mutations in tau phase separation. We also discuss recent studies on tau LLPS in cells and the insights these studies provide regarding the link between LLPS and neurodegeneration in tauopathies.

Signalling mechanisms and cellular functions of SUMO.

Sumoylation is an essential post-translational modification that is catalysed by a small number of modifying enzymes but regulates thousands of target proteins in a dynamic manner. Small ubiquitin-like modifiers (SUMOs) can be attached to target proteins as one or more monomers or in the form of polymers of different types. Non-covalent readers recognize SUMO-modified proteins via SUMO interaction motifs. SUMO simultaneously modifies groups of functionally related proteins to regulate predominantly nuclear processes, including gene expression, the DNA damage response, RNA processing, cell cycle progression and proteostasis. Recent progress has increased our understanding of the cellular and pathophysiological roles of SUMO modifications, extending their functions to the regulation of immunity, pluripotency and nuclear body assembly in response to oxidative stress, which partly occurs through the recently characterized mechanism of liquid-liquid phase separation. Such progress in understanding the roles and regulation of sumoylation opens new avenues for the targeting of SUMO to treat disease, and indeed the first drug blocking sumoylation is currently under investigation in clinical trials as a possible anticancer agent.

Microscopic Exploration of Liquid-liquid Phase Separation Mechanism in LCST Solutions

Microscopic Exploration of Liquid-liquid Phase Separation Mechanism in LCST Solutions

RNA polymerase II clusters form in line with surface condensation on regulatory chromatin.

It is essential for cells to control which genes are transcribed into RNA. In eukaryotes, two major control points are recruitment of RNA polymerase II (Pol II) into a paused state, and subsequent pause release toward transcription. Pol II recruitment and pause release occur in association with macromolecular clusters, which were proposed to be formed by a liquid–liquid phase separation mechanism. How such a phase separation mechanism relates to the interaction of Pol II with DNA during recruitment and transcription, however, remains poorly understood. Here, we use live and super‐resolution microscopy in zebrafish embryos to reveal Pol II clusters with a large variety of shapes, which can be explained by a theoretical model in which regulatory chromatin regions provide surfaces for liquid‐phase condensation at concentrations that are too low for canonical liquid–liquid phase separation. Model simulations and chemical perturbation experiments indicate that recruited Pol II contributes to the formation of these surface‐associated condensates, whereas elongating Pol II is excluded from these condensates and thereby drives their unfolding.

Microanalysis of Single Poly(N-isopropylacrylamide) Droplet Produced by an Optical Tweezer in Water: Isotacticity Dependence of Growth and Chemical Structure of the Droplet.

Thermoresponsive phase separation mechanisms of aqueous poly(N-isopropylacrylamide) (PNIPAM) solutions were investigated using an optical tweezer combined with a Raman microspectroscope. A near-infrared laser beam (λ = 1064 nm) was focused into the solution to produce and trap a single polymer microdroplet under an optical microscope. The laser beam played two important roles: The first role is to locally heat the solution to induce phase separation in which numerous polymer microdroplets are generated around the focus, while the second one is to collect these microdroplets. Eventually, a single polymer droplet was stably produced and trapped at the focus. Our method enabled us to perform two types of microanalysis for the droplet. Analysis I is real-time monitoring the growth of the polymer droplets by which we can determine the growth rate of droplets. Analysis II is Raman microspectroscopy to reveal chemical components of the droplets. By means of these two analyses, we revealed important phase separation mechanisms in terms of stereoregularity (isotacticity) dependence. From analysis I, we show that droplet growth is governed by the Ostwald ripening mechanism and the growth is accelerated by increasing the isotacticity. From analysis II, we show that the gelation is promoted in the droplet (physical gel formation) with increasing isotacticity. Our technique should be a versatile tool to explore liquid-liquid phase separation mechanisms for various binary solution systems.

Structural conversion of PLLA/ZnO composites facilitated by interfacial crystallization to potential application in oil-water separation

Focus on the potential application in oil-water separation for porous and biodegradable Poly(L-latic acid) (PLLA) amenable to economically viable water treatment, we designate the controllable pores, fibrillary network and protrusion structure by crystallization induced phase separation in poly(L-latic acid)/Zinc oxid (PLLA/ZnO) materials, ready for their potential application in oil-water separation. It’s worth noting that the interface effect between PLLA and ZnO is strengthened by silane coupling agent KH570. The crystallinity of the PLLA/ZnO@KH570 materials are correspondingly increased from 36.9% to 41.1% as the slightly increasing content of ZnO@KH570 from 1% to 7%, indicating that ZnO@KH570 nanoparticles can act as heterogeneous nucleating agent to promote crystallization of PLLA, which facilitate the conversion of PLLA solution from liquid-liquid phase separation mechanism to solid-liquid phase separation mechanism, resulting in the formation of microstructures. Very interestingly, the porosity of PLLA/ZnO@KH570 composite is highest up to 94.1% compared to its value of 87.8% for pure PLLA. Due to the multiple structures, the water contact angle of composite is significantly improved to 146°, allowing the oil/water mixture solution to be effectively separated. In brief, we have successfully manufactured hydrophobic PLLA/ZnO@KH570 composite with the multi-scale microstructure which may be potentially applied in oil-water separation.

Concentration fluctuations in the early stages of LLPS and partial dissolution of melt-memory in broadly distributed ethylene copolymers

The mechanism of liquid-liquid phase separation (LLPS) is investigated in a random ethylene 1-hexene copolymer (E/H) with a broad comonomer content distribution. SANS measurements were first carried out using the original resin with deuterium added via direct isotope exchange to preserve the original branching distribution. Upon lowering the melt temperature, the upshift of SANS profile observed only below a critical scattering vector is consistent with the signature of nucleation and growth during LLPS in the metastable region. Differences in branching distribution between the original broad resin and a broad multi-components blend of narrow E/H copolymers studied previously, result in slower LLPS dynamics in the former that has a more continuous change of comonomer content between molecules. Time-resolved light scattering and light transmittance measurements in the metastable region of the broad resin, reveal the nucleation and growth stages of LLPS. Dissolution of crystalline melt-memory, associated with an inversion of the crystallization rate with decreasing the temperature of the initial melt, is found to occur mainly within the LLPS induction period and is associated with concentration fluctuations prior to the LLPS nucleation step. The typical sea-island morphology associated with LLPS via nucleation and growth is observed under bright-field optical microscopy after one-day annealing in the metastable region.

Preparation and properties of PVDF/SiO2@GO nanohybrid membranes via thermally induced phase separation method

Graphene oxide (GO), one of the most promising filter materials, is intensively employed to prepare the membranes via non-solvent induced phase separation(NIPS) method, but it has not been found to modify polymeric membranes via thermally induced phase separation (TIPS) method. In this work, the derivative of GO, SiO2@GO nanohybrid, is fabricated and employed to synthesize PVDF/SiO2@GO nanohybrid membranes via TIPS method for the first time. The results indicate that the PVDF/SiO2@GO nanohybrid membranes experience liquid-liquid phase separation mechanism, and exhibit bi-continual and asymmetric structure. The included SiO2@GO nanohybrid is uniformly dispersed in the membrane matrix. With the addition of SiO2@GO, the top surface becomes denser and the pore size decreases; but overhigh SiO2@GO addition for membrane M-5 triggers in the adverse trend. This is caused by the combined actions of nucleation and growth of PVDF and viscosity increase of cast solution due to the addition SiO2@GO nanohybrid. The BSA rejection of membrane gradually increases with the addition of PVDF/SiO2@GO nanohybrid accompanied with the decline of pure water permeation flux. As the SiO2@GO content increases to 0.9wt%, the PVDF/SiO2@GO nanohybrid membrane M-4 presents the highest BSA rejection of 91.7% and the lowest permeation flux of 182.6Lm−2h−1bar−1. However, the overhigh SiO2@GO addition (1.2wt%) leads to the outstanding pure water permeation flux of 679.1Lm−2h−1bar−1 and a much lower BSA rejection. The addition of PVDF/SiO2@GO nanohybrid evidently improves the surface hydrophilicity and antifouling ability of resultant membranes. The XRD patterns and FTIR spectra of membranes verify the exclusive α-phase of PVDF, and the melting temperature(Tm) and crystallinity(xc) evidently increase with the addition of SiO2@GO nanohybrid up to 0.9wt% in the dopes. Overhigh SiO2@GO addition (1.2wt%) leads to small decline for these parameters.

S.12.01 Does more long-term-potentiation mean better memory? Studies of genetically modified mice with altered NMDAR function

Long-term potentiation (LTP) of synaptic transmission is considered to be a cellular counterpart of learning and memory. Activation of postsynaptic NMDA type glutamate receptor (NMDA-R) induces trafficking of AMPA type glutamate receptors (AMPA-R) and other proteins to the synapse in sequential fashion. At the same time, the dendritic spine expands for long-term and modulation of actin underlies this (structural LTP or sLTP). How these changes persist despite constant diffusion and turnover of the component proteins have been the central focus of the current LTP research. Signaling triggered by Ca2+-influx via NMDA-R triggers kinase including Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII can sustain longer-term biochemical signaling by forming a reciprocally-activating kinase-effector complex with its substrate proteins including Tiam1, thereby regulating persistence of the downstream signaling. Furthermore, activated CaMKII can condense at the synapse through the mechanism of liquid-liquid phase separation (LLPS). This increases the binding capacity at the synapse, thereby contributing to the maintenance of enlarged protein complexes. It may also serve as the synapse tag, which captures newly synthesized proteins.

Gelation mechanism of ultra-high-molecular-weight polyethylene (UHMWPE) chains in dispersion solutions containing multiwall carbon nanotubes (MWNTs) analyzed in terms of liquid-liquid phase separation

Abstract Gelation mechanisms of ultra-high-molecular-weight polyethylene (UHMWPE) in dispersion solutions containing multiwall carbon nanotubes (MWNTs) were investigated in terms of liquid-liquid phase separation mechanisms. When an incident beam of He-Ne gas laser was directed to the dispersion solution quenched to a desired temperature, the logarithm of scattered intensity increased linearly with elapsing time and tended to deviate from this linear relationship. The two different increasing behaviors of the scattered intensity were termed as initial and latter stages, respectively, of the phase separation. The linear increase in logarithmic light-scattered intensity was analyzed within the framework of the linear theory of spinodal decomposition in terms of the viewpoint that the quenched solution becomes thermodynamically unstable and tends to incur phase separation to resolve the unstable state. The growth rate of the concentration fluctuation of neat UHMWPE solution (without MWNTs) calculated from the slope of linear increase was faster than that of the dispersion solution. In spite of the rapid growth rate, however, the gelation time of the dispersion solution was much shorter than that of the neat UHMWPE solution and the gelation and quasi-spinodal temperatures were slightly higher. These phenomena were in contradiction with the result of the concentration fluctuation. This contradiction was thought to be due to the fact that diffused UHMWPE chains in the dispersion solution were gelled easily on the MWNT surface rather than the gelation by self-coagulation.

STRUCTURE-PROPERTY RELATIONS IN REACTION-INDUCED, PHASE-SEPARATED POLY(2-PHENOXYETHYL ACRYLATE)/POLYSTYRENE BLENDS

Structure-property relations were studied in reaction-induced, phase-separated polymer blends. An amorphous-amorphous system consisted of polystyrene (PS) dissolved in the monomer 2-phenoxyethyl acrylate (POA). When the POA was polymerized to poly(2-phenoxyethyl acrylate) (PPOA), phase separation and phase inversion were induced, and a polymer blend was formed. The reaction kinetics were measured by monitoring the reduction in the intensity of the C╤C stretching vibration band in the Raman spectrum of POA. The phase separation kinetics were determined using light transmission experiments and were combined with the reaction kinetics so that a ternary phase diagram could be defined for the reactive system. Structure development was monitored using small-angle laser light scattering (SALLS) and optical microscopy, which showed that spinodal decomposition was the mechanism of liquid-liquid phase separation. Plots of the relative invariant with time showed an increase in the degree of phase separation. The Fourier transforms of the microscopy images had peaks in the radial intensity distributions, again implying that spinodal decomposition was the phase separation mechanism. Tensile testing showed that PPOA was soft and rubbery at 20°C. Both PS and PPOA had comparable toughness when tested to failure; however, the blend containing 17 wt% PS had a toughness more than 10 times that of either PS or PPOA in isolation. Both modulus and tensile strength increased with PS content, while the ultimate strain decreased. The Nielsen model best described the tensile modulus data, providing further evidence for co-continuous phase structure.

A phase-separating centrifuge for automated radioanalysis

A simple centrifuge controlled by a suitable laboratory programmer has been designed to perform automatically the purification procedures in radioanalysis which involve precipitation or solvent extraction. The mechanism of liquid-liquid phase separation depends on the intrinsic properties of the liquids; either surface tensions, or, when predictable volumes are used, the densities rather than detection of the interface level as in the manual method. The liquid-liquid phase-separating performance of 3 versions of the centrifuge having different orifice materials has been studied and data are presented which should enable the analyst to select the most appropriate version for a particular solvent extraction method.