Quantification of binary diffusion in protein crystals

The use of confocal laser scanning microscopy for visualization and quantification of binary diffusion within anisotropic porous material is described here for the first time. The dynamics of adsorption profiles of dianionic fluorescein, zwitterionic rhodamine B, and their mixture in the cationic native orthorhombic lysozyme crystal were subsequently analyzed. All data could be described by a classical pore diffusion model. There was no change in the adsorption characteristics, but diffusion decreased with the introduction of a second solute in the solution. It was found that diffusion is determined by the combination of steric and electrostatic interactions,while adsorption is dependent on electrostatic and hydrophobic interactions. Thus, it was established that the outcome of binary transport depends on the solute, protein, and crystal characteristics.     

Morphological changes of ethylene/vinyl acetate-based controlled delivery systems during release of water-soluble solutes

Thin slabs of theophylline and monomer albumin release systems were prepared by dispersing 212-300 μm and 300-25 μm particles respectively, of these bioactive agents in a methylene chloride solution of ethylene/vinyl acetate (EVAc) copolymer (40 wt% vinyl acetate), and evaporating the solvent at low temperatures according to the Langer—Folkman technique. Compositions containing 21.41 wt%, 31.04 wt% and 40.0 wt% albumin, and 19.32 wt% theophylline were prepared. Solute release experiments were performed in deionized water at 37 ± 0.1°C under perfect-sink conditions. The concentration of released solute was determined by measuring the absorbance of the UV spectra at 276 nm for albumin and 272 nm for theophylline. Both solutes could be released for long periods of time at controlled rates. The main mechanism of release was established to be solute dissolution and diffusion through the generated, waterfilled pore structure. Photomicrographs present the main features of this pore network. Mercury porosimetry was used to determine the pore volume and size of pores for freezedried slabs before, during and after the dissolution/diffusion/release process. Considerable pore collapse was observed and pore diameters of 8-650 μm were detected. In addition to solution diffusion through large pores, diffusion might occur through small constrictions between large pores or through a pore network of much smaller pores created in the matrix.     

Molecular simulations of solute transport in xylose isomerase crystals

Cross-linked enzyme crystals (CLECs) enclose an extensive regular matrix of chiral solvent-filled nanopores, via which ions and solutes travel in and out. Several cross-linked enzyme crystals have recently been used for chiral separation and as biocatalysts. We studied the dynamics of solute transport in orthorhombic d-xylose isomerase (XI) crystals by means of Brownian dynamics (BD) and molecular dynamics (MD) simulations, which show how the protein residues influence the dynamics of solute molecules in confined regions inside the lattice. In the BD simulations, coarse-grained beads represent solutes of different sizes. The diffusion of S-phenylglycine molecules inside XI crystals is investigated by long-time MD simulations. The computed diffusion coefficients within a crystal are found to be orders of magnitude lower than in bulk water. The simulation results are compared to the recent experimental studies of diffusion and reaction inside XI crystals. The insights obtained from simulations allow us to understand the nature of solute-protein interactions and transport phenomena in CLECs, which is useful for the design of novel nanoporous biocatalysts and bioseparations based on CLECs.     

Further studies on solvent extraction with immobilized interfaces in a microporous hydrophobic membrane

Investigations on solvent extraction of acetic acid into xylene or methyl isobuty] ketone by using immobilized interfaces in microporous hydrophobic membranes have now been extended to a number of different membranes with a wide variation in pore size and porosity. Measured intrinsic membrane transfer coefficients of the solute are adequately described by the simple model of unhindered diffusion in tortuous pores developed earlier. Applied pressure difference did not influence the overall solute transfer coefficient as long as it was not close to that required for the breakthrough of aqueous phase into organic phase. Aqueous and organic boundary layer mass transfer coefficients in the flow type test cell have been determined with a known membrane and utilized to predict effectively the overall solute transfer coefficient observed with other membranes.     

The sepiolite membrane for ultrafiltration

Novel inorganic membranes were prepared from clay (sepiolite) suspensions, which were formed by dispersing clay particles in water either by applying ultrasonic wave or by magnetic stirring. Films can be formed easily from such suspensions due to fibrous nature of sepiolite. Thus, this offers a method much simpler than the conventional sol–gel method. The membranes were further tested for ultrafiltration of polyethylene glycol and polyethylene oxide solutes of different molecular weights. It was found that the correlation between the separation and the Einstein–Stokes radius of solute fits the log-normal distribution very well. The mean pore size of 23–26 nm and the standard deviation of 1.91–2.04 were obtained from the above correlation. It was also found that the mean pore size and the pore size distribution did not depend very much on the membrane preparation method.     

Local and average diffusion of nanosolutes in agarose gel: the effect of the gel/solution interface structure

Fluorescence correlation spectroscopy (FCS) has been used to study the diffusion of nanometric solutes in agarose gel, at microscopic and macroscopic scales. Agarose gel was prepared and put in contact with aqueous solution. Several factors were studied: (i) the role of gel relaxation after its preparation, (ii) the specific structure of the interfacial zone and its role on the local diffusion coefficient of solutes, and (iii) the comparison between the local diffusion coefficient and the average diffusion coefficient in the gel. Fluorescent dyes and labeled biomolecules were used to cover a size range of solutes of 1.5 to 15 nm. Their transport through the interface from the solution toward the gel was modeled by the first Fick's law based on either average diffusion coefficients or the knowledge of local diffusion coefficients in the system. Experimental results have shown that, at the liquid/gel interface, a gel layer with a thickness of 120 microm is formed with characteristics significantly different from the bulk gel. In particular, in this layer, the porosity of agarose fiber network is significantly lower than in the bulk gel. The diffusion coefficient of solutes in this layer is consequently decreased for steric reasons. Modeling of solute transport shows that, in the bulk gel, macroscopic diffusion satisfactorily follows the classical Fick's diffusion laws. For the tested solutes, the local diffusion coefficients in the bulk gel, measured at microscopic scale by FCS, were equal, within experimental errors, to the average diffusion coefficients applicable at macroscopic scales (>or=mm). This confirms that anomalous diffusion applies only to solutes with sizes close to the gel pore size and at short time (相似文献   

Modelling of the pore size distribution of ultrafiltration membranes

A dilute aqueous solution of polydisperse neutral dextrans was used to determine the sieving properties (flux and rejection) of porous polyacrylonitrile membranes. Gel ermeation chromatography was used to measure the solute mole and concentration in the permeate. From these data, rejection coefficients were calculated as a function of solute molecular size. A mathematical model was then developed to relate the flux and solute rejection to pore size distribution and the total number of pores, based upon the assumption that solute rejection was the result of purely geometric considerations. As a first approximation, a solute molecule was considered either too large to enter a membrane pore, or if it entered, its concentration in the permeate from that pore, as well as the solvent flux through the pore, were not affected. This model also considered the effects of steric hindrance and hydrodynamic lag on the convection of solute through a membrane. The shape and sharpness of pore size distributions were found to be useful in comparisons of ultrafiltration membranes.     

Association kinetics of wild- and mutant-type Ynd1p in relation to quality of grown crystals

The intermolecular interaction and association dynamics of the Ynd1p protein were investigated using dynamic and time-resolved static light scattering measurements. The mutual diffusion coefficients of wild- and mutant-type (a single amino acid substitution) Ynd1p monomer were measured in 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer with 5 mM MnCl2 and 7.5% (v/v) ethylene glycol. Both translational diffusion coefficients at a zero protein concentration were (40.3 +/- 0.2) x 10(-12) m2/s at 20 degrees C and a pH of 7.0, so the hydrodynamic radius of the monomers was 4.1 +/- 0.1 nm. The measured intermolecular interaction between monomers, however, showed that the mutant-type Ynd1p had a stronger attractive force. Time-resolved static light scattering measurements showed that the association of mutant-type Ynd1p yielded a larger number of aggregates than that of wild-type Ynd1p. The time dependence of aggregate gyration radius differed between the two types. Fractal dimension analysis using scattering intensity data suggested that the inner structure of the aggregates changed from loose to rigid with time. Although this phenomenon is common for wild and mutant types, the differences in the number of aggregates yielded in the initial stages and in the intermolecular interaction affected the quality of the final grown crystals. That is, single crystals of Ynd1p grew in the mutant-type protein solution and polycrystals of Ynd1p grew in the wild-type protein solution.     

Pore structure of gel chitosan membranes. I. Solute diffusion measurements

The capillary pore model of water-swollen gels was used to interpret solute diffusion through gel chitosan membranes. Diffusive permeability coefficients of 12 solutes ranging in molecular radius from 2.5 Å (methanol) to 14 Å (polyethylene glycol 4000) were measured for an untreated chitosan membrane, for four chitosan membranes crosslinked with glutaraldehyde of concentrations between 0.01 and 1% and coated with a protein and also, for comparison, for a commercial Cuprophan membrane. Through the capillary pore-model correlation of the above coefficients with the membrane water content, the following structural factors of the examined membranes were calculated: pore radius, surface porosity and tortuosity factor. Knowledge of these factors is required if the desired membranes are to be designed for a given application (e.g. dialysis).     

Membrane characterization and solute diffusion in porous composite nanocellulose membranes for hemodialysis

The membrane and solute diffusion properties of Cladophora cellulose and polypyrrole (PPy) functionalized Cladophora cellulose were analyzed to investigate the feasibility of using electroactive membranes in hemodialysis. The membranes were characterized with scanning electron microscopy, ζ-potentiometry, He-pycnometry, N₂ gas adsorption, and Hg porosimetry. The diffusion properties across the studied membranes for three model uremic toxins, i.e. creatinine, vitamin B12 and bovine serum albumin, were also analyzed. The characterization work revealed that the studied membranes present an open structure of weakly negatively charged nanofibers with an average pore size of 21 and 53 nm for pristine cellulose and PPy-Cladophora cellulose, respectively. The results showed that the diffusion of uremic toxins across the PPy-Cladophora cellulose membrane was faster than through pure cellulose membrane, which was related to the higher porosity and larger average pore size of the former. Since it was found that the average pore size of the membranes was larger than the hydrodynamic radius of the studied model solutes, it was concluded that these types of membranes are favorable to expand the Mw spectrum of uremic toxins to also include conditions associated with accumulation of large pathologic proteins during hemodialysis. The large average pore size of the composite membrane could also be exploited to ensure high-fluxes of solutes through the membrane while simultaneously extracting ions by an externally applied electric current.     

Density inhomogeneity and diffusion behavior of fluids in micropores by molecular-dynamics simulation

The density profiles and the diffusion behavior of fluid argon confined in micropores were studied by molecular-dynamics simulations. The effects of pore size (width), temperature and number density on the density profiles and the self-diffusion coefficients in micropores were simulated with pore widths from 0.6 to 4.0 nm. The density profiles are greatly affected by the pore size. Strong inhomogeneities in the channel direction and vapor-liquid phase separation in the micropores were observed when initial conditions were chosen in the coexistence region of the fluid. The self-diffusion coefficient in the channel direction in the pores was found to be much lower than in the bulk, and decreasing with decreasing pore size, decreasing temperature, and increasing density.     

Molecular dynamics simulations for water and ions in protein crystals

The spatial and temporal properties of water and ions in bionanoporous materials-protein crystals-have been investigated using molecular dynamics simulations. Three protein crystals are considered systematically with different morphologies and chemical topologies: tetragonal lysozyme, orthorhombic lysozyme, and tetragonal thermolysin. It is found that the thermal fluctuations of C(alpha) atoms in the secondary structures of protein molecules are relatively weak due to hydrogen bonding. The solvent-accessible surface area per residue is nearly identical in the three protein crystals; the hydrophobic and hydrophilic residues in each crystal possess approximately the same solvent-accessible surface area. Water distributes heterogeneously and has different local structures within the biological nanopores of the three protein crystals. The mobility of water and ions in the crystals is enhanced as the porosity increases and also by the fluctuations of protein atoms particularly in the two lysozyme crystals. Anisotropic diffusion is found preferentially along the pore axis, as experimentally observed. The anisotropy of the three crystals increases in the order: tetragonal thermolysin < tetragonal lysozyme < orthorhombic lysozyme.     

Anisotropic Brownian diffusion near a nanostructured surface

The Brownian diffusion of sub-micrometer-sized particles (diameter 0.52 microm) in the vicinity of a nanostructured surface was experimentally characterized. The surface consisted of a repeating pattern of rectangular grooves with depth 35 nm and pitch 400 nm. It was found that the one-dimensional particle diffusivity parallel to the nanogrooves was significantly higher than the diffusivity perpendicular to the grooves (1.518+/-0.274 (S.E.) microm2/s compared to 0.704+/-0.090 microm2/s). No such anisotropy in the one-dimensional Brownian diffusivity was found for particles near a flat surface.     

Modeling and simulation of the dynamic behavior of monoliths. Effects of pore structure from pore network model analysis and comparison with columns packed with porous spherical particles

A mathematical model is presented that could be used to describe the dynamic behavior, scale-up, and design of monoliths involving the adsorption of a solute of interest. The value of the pore diffusivity of the solute in the pores of the skeletons of the monolith is determined in an a priori manner by employing the pore network modeling theory of Meyers and Liapis [J. Chromatogr. A, 827 (1998) 197 and 852 (1999) 3]. The results clearly show that the pore diffusion coefficient, Dmp, of the solute depends on both the pore size distribution and the pore connectivity, nT, of the pores in the skeletons. It is shown that, for a given type of monolith, the film mass transfer coefficient, Kf, of the solute in the monolith could be determined from experiments based on Eq. (3) which was derived by Liapis [Math. Modelling Sci. Comput., 1 (1993) 397] from the fundamental physics. The mathematical model presented in this work is numerically solved in order to study the dynamic behavior of the adsorption of bovine serum albumin (BSA) in a monolith having skeletons of radius r(o) = 0.75x10(-6) m and through-pores having diameters of 1.5x10(-6)-1.8x10(-6) m [H. Minakuchi et al., J. Chromatogr. A, 762 (1997) 135]. The breakthrough curves of the BSA obtained from the monolith were steeper than those from columns packed with porous spherical particles whose radii ranged from 2.50x10(-6) m to 15.00x10(-6) m. Furthermore, and most importantly, the dynamic adsorptive capacity of the monolith was always greater than that of the packed beds for all values of the superficial fluid velocity, Vtp. The results of this work indicate that since in monoliths the size of through-pores could be controlled independently from the size of the skeletons, then if one could construct monolith structures having (a) relatively large through-pores with high through-pore connectivity that can provide high flow-rates at low pressure drops and (b) small-sized skeletons with mesopores having an appropriate pore size distribution (mesopores having diameters that are relatively large when compared with the diameter of the diffusing solute) and high pore connectivity, nT, the following positive results, which are necessary for obtaining efficient separations, could be realized: (i) the value of the pore diffusion coefficient, Dmp, of the solute would be large, (ii) the diffusion path length in the skeletons would be short, (iii) the diffusion velocity, vD, would be high, and (iv) the diffusional response time, t(drt), would be small. Monoliths with such pore structures could provide more efficient separations with respect to (a) dynamic adsorptive capacity and (b) required pressure drop for a given flow-rate, than columns packed with porous particles.     

Pore Modification in Porous Ceramic Membranes With Sol-Gel Process and Determination of Gas Permeability and Selectivity

Summary: The aim of the study was to investigate the variation in total surface area, porosity, pore size, Knudsen and surface diffusion coefficients, gas permeability and selectivity before and after the application of sol-gel process to porous ceramic membrane in order to determine the effect of pore modification. In this study, three different sol-gel process were applied to the ceramic support separately; one was the silica sol-gel process which was applied to increase porosity, others were silica-sol dip coating and silica-sol processing methods which were applied to decrease pore size. As a result of this, total surface area, pore size and porosity of ceramic support and membranes were determined by using BET instrument. In addition to this, Knudsen and surface diffusion coefficients were also calculated. After then, ceramic support and membranes were exposed to gas permeation experiments by using the CO₂ gas with different flow rates. Gas permeability and selectivity of those membranes were measured according to the data obtained. Thus, pore surface area, porosity, pore size and Knudsen diffusion coefficient of membrane treated with silica sol-gel process increased while total surface area was decreasing. Therefore, permeability of ceramic support and membrane treated with silica sol-gel process increased, and selectivity decreased with increasing the gas flow rate. Also, surface area, porosity, pore size, permeability, selectivity, Knudsen and surface diffusion coefficients of membranes treated with silica-sol dip coating and silica-sol processing methods were determined. As a result of this, porosity, pore size, Knudsen and surface diffusion coefficients decreased, total surface area increased in both methods. However, viscous flow and Knudsen flow permeability were detected as a consequence of gas permeability test and Knudsen flow was found to be a dominant transport mechanism in addition to surface diffusive flow owing to the small pore diameter in both methods. It was observed that silica-sol processing method had lower pore diameter and higher surface diffusion coefficient than silica-sol dip coating method.     

Analysis on Band Gaps of MCM-41 Type of Materials

The concept and analysis method of photonic crystals and band gaps are introduced into one-dimenslonal (1D) ordered mesoporous materials. MCM-41 type of materials are treated theoretically as photonie crystals. The formation of band gaps is exhibited and confirmed by a calculation of transfer matrix technique. PBG was found around 9-42 nm in soft X-ray region. The photonie band-gap was predicted to be dependent on incident direction, pore size and lattice constant. The mesoporous materials with different pore sizes and different lattice constants have different band-gap widths.     

Pore size distributions of cation-exchange adsorbents determined by inverse size-exclusion chromatography

The pore dimensions, pore size distributions, and phase ratios were determined for a set of cation-exchange adsorbents using inverse size-exclusion chromatography (ISEC). The adsorbents examined represent a diverse set of materials from Pharmacia, TosoHaas, BioSepra, and EM Industries, which are widely used for protein purification. The ISEC was carried out using dextran standards with relative molecular masses of 180-6,105,000. This technique provided a comparative characterization of the accessible internal pore surface area, as a function of solute size, for the adsorbents tested. Adsorbent preparation strategies in which polymers are generated in situ or grafted onto base materials were found to have significant effects on pore dimensions and phase ratios.     

Anisotropic diffusion of elongated and aligned polymer chains in a nematic solvent

The translational diffusion constant, D, of a polymer solute in a single-domain, nematic liquid crystal solvent (5CB) is measured in directions parallel and perpendicular to the nematic director using a fluorescence two-beam, cross-correlation technique. The solute under investigation is the stiff, conjugated polymer, MEH-PPV. The ratio D parallel/D perpendicular) of diffusion constants (parallel and perpendicular to the director) is observed to be 1.9 +/- 0.3. This is surprisingly small considering that MEH-PPV is known to be both elongated and highly aligned along the liquid crystal director of 5CB. We therefore argue that the structural order parameter of the solvent governs the anisotropy of the diffusion of the solute.     

Protein nanotubes comprised of an alternate layer-by-layer assembly using a polycation as an electrostatic glue

We present the synthesis and structure of various protein nanotubes comprised of an alternate layer-by-layer (LbL) assembly using a polycation as an electrostatic glue. The nanotubes were fabricated by sequential LbL depositions of positively charged polycations and negatively charged proteins into a porous polycarbonate (PC) membrane, followed by release of the cylindrical core by quick dissolution of the template with CH(2)Cl(2). This procedure provides a variety of protein nanotubes without interlayer cross-linking. The three-cycle depositions of poly-L-arginine (PLA) and human serum albumin (HSA, M(w)=66.5 kDa) into the porous PC template (pore diameter, D(p)=400 nm) yielded well-defined (PLA/HSA)(3) nanotubes with an outer diameter of 419+/-29 nm and a wall thickness of 46+/-8 nm, revealed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations. The outer diameter of the tubules can be controlled by the pore size of the template (200-800 nm), whereas the wall thickness is always constant, independent of the D(p) value. The (PEI/HSA)(3) (PEI: polyethylenimine) nanotubes showed a slightly thin wall of 39+/-5 nm. CD spectra of the multilayered (PEI/HSA)(n) film on a flat quartz plate suggested that the secondary structure of HSA between the polycations was almost the same as that in aqueous solution. The three-cycle LbL depositions of PLA and ferritin (M(w)=460 kDa) or myoglobin (Mb, M(w)=1.7 kDa) into the porous PC membrane also gave cylindrical hollow structures. The wall thickness of the (PLA/ferritin)(3) and (PLA/Mb)(3) nanotubes were 55+/-5 nm and 31+/-4 nm; it depends on the globular size of the protein (ferritin>HSA>Mb). The individual ferritin molecule was clearly seen in the tubular walls by SEM and TEM measurements.     

High Performance Size Exclusion Chromatography with Microparticulate Porous Silica Spheres. Influence of Flow Rate,Salute Mass Transfer and Polydispersity of Samples

Abstract

The contributions to peak broadening in Size Exclusion Chromatography with microparticles of porous silica spheres having narrow size distributions have been determined by measuring the plate height dependence on flow rate for toluene and for polystyrene standards covering a wide range of molecular weights. From these contributions, the diffusion coefficients of the macromolecules in the pore matrix and the polydispersities of the samples could be evaluated. It is shown that for permeating polymers the band broadening is determined by the eddy diffusion in the mobile phase, by the slow mass transfer of the solute in the stationary phase and by the polydispersity of the standards. In properly packed columns the eddy diffusion term is of minor importance compared to the other effects, whereas the solute mass transfer, which is a velocity dependent process, can be minimized only at extremely low flow rates.