An optimized reverse-phase high performance liquid chromatographic method for evaluating percutaneous absorption of glucosamine hydrochloride

A relatively simple, selective, precise and accurate high performance liquid chromatography (HPLC) method based on a reaction of phenylisothiocyanate (PITC) with glucosamine (GL) in alkaline media was developed and validated to determine glucosamine hydrochloride permeating through human skin in vitro. It is usually problematic to develop an accurate assay for chemicals traversing skin because the excellent barrier properties of the tissue ensure that only low amounts of the material pass through the membrane and skin components may leach out of the tissue to interfere with the analysis. In addition, in the case of glucosamine hydrochloride, chemical instability adds further complexity to assay development. The assay, utilising the PITC-GL reaction was refined by optimizing the reaction temperature, reaction time and PITC concentration. The reaction produces a phenylthiocarbarnyl-glucosamine (PTC-GL) adduct which was separated on a reverse-phase (RP) column packed with 5 mu m ODS (C-18) Hypersil particles using a diode array detector (DAD) at 245 nm. The mobile phase was methanol-water-glacial acetic acid (10:89.96:0.04 v/v/v, pH 3.5) delivered to the column at 1 ml min(-1) and the column temperature was maintained at 30 degrees C Using a saturated aqueous solution of glucosamine hydrochloride, in vitro permeation studies were performed at 32 +/- 1 degrees C over 48 h using human epidermal membranes prepared by a heat separation method and mounted in Franz-type diffusion cells with a diffusional area 2.15 +/- 0.1 cm(2). The optimum derivatisation reaction conditions for reaction temperature, reaction time and PITC concentration were found to be 80 degrees C, 30 min and 1 % v/v, respectively. PTC-Gal and GL adducts eluted at 8.9 and 9.7 min, respectively. The detector response was found to be linear in the concentration range 0-1000 mu g ml(-1). The assay was robust with intra- and inter-day precisions (described as a percentage of relative standard deviation, %R.S.D.) < 12. Intra- and inter-day accuracy (as a percentage of the relative error, %RE) was <=-5.60 and <=-8.00, respectively. Using this assay, it was found that GL-HCI permeates through human skin with a flux 1.497 +/- 0.42 mu g cm(-2) h(-1), a permeability coefficient of 5.66 +/- 1.6 x 10(-6) cm h(-1) and with a lag time of 10.9 +/- 4.6 h. (c) 2005 Elsevier B.V. All rights reserved.

The role of lipid composition on the interaction between a tryptophan-rich protein and model bacterial membranes

The interaction between tryptophan-rich puroindoline proteins and model bacterial membranes at the air-liquid interface has been investigated by FTIR spectroscopy, surface pressure measurements and Brewster angle microscopy. The role of different lipid constituents on the interactions between lipid membrane and protein was studied using wild type (Pin-b) and mutant (Trp44 to Arg44 mutant, Pin-bs) puroindoline proteins. The results show differences in the lipid selectivity of the two proteins in terms of preferential binding to specific lipid head groups in mixed lipid systems. Pin-b wild type was able to penetrate mixed layers of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) head groups more deeply compared to the mutant Pin-bs. Increasing saturation of the lipid tails increased penetration and adsorption of Pin-b wild type, but again the response of the mutant form differed. The results provide insight as to the role of membrane architecture, lipid composition and fluidity, on antimicrobial activity of proteins. Data show distinct differences in the lipid binding behavior of Pin-b as a result of a single residue mutation, highlighting the importance of hydrophobic and charged amino acids in antimicrobial protein and peptide activity.

Natural Membranes for Application in Biomedical Devices

Polymers from natural sources are particularly useful as biomaterials for medical devices applications. In this study, the results of characterization of a gelatin network electrolyte doped with europium triflate (Eu(CF3SO3)(3)) are described. The unusual electronic properties of the trivalent lanthanide ions make them well suited as luminescent reporter groups, with many applications in biotechnology. Samples of solvent-free electrolytes were prepared with a range of guest salt concentration. Materials based on Eu(CF3SO3)(3) were obtained as mechanically robust, flexible, transparent, and completely amorphous films. Samples were characterized by thermal analysis (thermo-gravimetry analysis (TGA) and differential scanning calorimetry (DSC), electrochemical stability, scanning electronmicroscopy (SEM), and photoluminescence spectroscopy.

Enhancement of Electrical Conductivity in Plasticized Chitosan Based Membranes

Plasticized natural macromolecules-based polymer electrolyte samples were prepared and characterized. The plasticization of chitosonium acetate with glycerol increased the ionic conductivity value from 3.0 x 10(-7) S/cm to 1.1 x 10(-5) S/cm. The conductivity temperature relationship of the samples exhibits either VTF or Arrhenius type depending on the glycerol concentration in the sample. The dielectric studies evidencing the relaxation process in the plasticized sample at low frequency region are due to the electric polarization effect. Moreover, the samples were transparent in the Vis region, showed thermal stability up to 160 degrees C and good surface uniformity.

Sodium Alginate-Based Ionic Conducting Membranes

The present study investigates gel polymer electrolytes (GPEs) based on sodium alginate plasticized with glycerol containing either CH3COOH or LiClO4. The membranes showed ionic conductivity results of 3.1 x 10(-4) S/cm for the samples with LiClO4 and 8.7x10(-5) S/cm for the samples with CH3COOH at room temperature. The samples also showed thermal stability up to 160 degrees C, transparency of up to 90%, surface uniformity and adhesion to glass and steel. Moreover, Dynamic Mechanical Analysis revealed two relaxations for both samples and the Ea values were between 18 and 36 kJ/mol. All the results obtained indicate that alginate-based GPEs can be used as electrolytes in electrochemical devices.

Ion-Conducting Membranes Based on Gelatin and Containing LiI/I-2 for Electrochromic Devices

Ionic conducting membranes of gelatin plasticized with glycerol and containing LiI/I-2 have been obtained and characterized by X-ray diffraction measurements, UV-Vis-NIR spectroscopy, thermal analysis and impedance spectroscopy. The transparent (80-90% in the visible range) membranes showed ionic conductivity value of 5 x 10(-5) S/cm at room temperature, which increased to 3 x 10(-3) S/cm at 80 degrees C. All the ionic conductivity measurements as a function of temperature showed VTF dependence and activation energy of 8 kJ/mol. These samples also showed low glass transition temperature of -76 degrees C. Moreover the samples were predominantly amorphous. The membranes applied to small electrochromic devices showed 20% of color change from colored to bleached states during more than 70 cronoamperometric cycles.

Critical review of coupled flux formulations for clay membranes based on nonequilibrium thermodynamics

Extensive research conducted over the past several decades has indicated that semipermeable membrane behavior (i.e., the ability of a porous medium to restrict the passage of solutes) may have a significant influence on solute migration through a wide variety of clay-rich soils, including both natural clay formations (aquitards, aquicludes) and engineered clay barriers (e.g., landfill liners and vertical cutoff walls). Restricted solute migration through clay membranes generally has been described using coupled flux formulations based on nonequilibrium (irreversible) thermodynamics. However, these formulations have differed depending on the assumptions inherent in the theoretical development, resulting in some confusion regarding the applicability of the formulations. Accordingly, a critical review of coupled flux formulations for liquid, current, and solutes through a semipermeable clay membrane under isothermal conditions is undertaken with the goals of explicitly resolving differences among the formulations and illustrating the significance of the differences from theoretical and practical perspectives. Formulations based on single-solute systems (i.e., uncharged solute), single-salt systems, and general systems containing multiple cations or anions are presented. Also, expressions relating the phenomenological coefficients in the coupled flux equations to relevant soil properties (e.g., hydraulic conductivity and effective diffusion coefficient) are summarized for each system. A major difference in the formulations is shown to exist depending on whether counter diffusion or salt diffusion is assumed. This difference between counter and salt diffusion is shown to affect the interpretation of values for the effective diffusion coefficient in a clay membrane based on previously published experimental data. Solute transport theories based on both counter and salt diffusion then are used to re-evaluate previously published column test data for the same clay membrane. The results indicate that, despite the theoretical inconsistency between the counter-diffusion assumption and the salt-diffusion conditions of the experiments, the predictive ability of solute transport theory based on the assumption of counter diffusion is not significantly different from that based on the assumption of salt diffusion, provided that the input parameters used in each theory are derived under the same assumption inherent in the theory. Nonetheless, salt-diffusion theory is fundamentally correct and, therefore, is more appropriate for problems involving salt diffusion in clay membranes. Finally, the fact that solute diffusion cannot occur in an ideal or perfect membrane is not explicitly captured in any of the theoretical expressions for total solute flux in clay membranes, but rather is generally accounted for via inclusion of an effective porosity, n(e), or a restrictive tortuosity factor, tau(r), in the formulation of Fick's first law for diffusion. Both n(e) and tau(r) have been correlated as a linear function of membrane efficiency. This linear correlation is supported theoretically by pore-scale modeling of solid-liquid interactions, but experimental support is limited. Additional data are needed to bolster the validity of the linear correlation for clay membranes. Copyright 2012 Elsevier B.V. All rights reserved.

Critical Review of Coupled Flux Formulations for Clay Membranes Based on Nonequilibrium Thermodynamics

Extensive research conducted over the past several decades has indicated that semipermeable membrane behavior (i.e., the ability of a porous medium to restrict the passage of solutes) may have a significant influence on solute migration through a wide variety of clay-rich soils, including both natural clay formations (aquitards, aquicludes) and engineered clay barriers (e.g., landfill liners and vertical cutoff walls). Restricted solute migration through clay membranes generally has been described using coupled flux formulations based on nonequilibrium (irreversible) thermodynamics. However, these formulations have differed depending on the assumptions inherent in the theoretical development, resulting in some confusion regarding the applicability of the formulations. Accordingly, a critical review of coupled flux formulations for liquid, current, and solutes through a semipermeable clay membrane under isothermal conditions is undertaken with the goals of explicitly resolving differences among the formulations and illustrating the significance of the differences from theoretical and practical perspectives. Formulations based on single-solute systems (i.e., uncharged solute), single-salt systems, and general systems containing multiple cations or anions are presented. Also, expressions relating the phenomenological coefficients in the coupled flux equations to relevant soil properties (e.g., hydraulic conductivity and effective diffusion coefficient) are summarized for each system. A major difference in the formulations is shown to exist depending on whether counter diffusion or salt diffusion is assumed. This difference between counter and salt diffusion is shown to affect the interpretation of values for the effective diffusion coefficient in a clay membrane based on previously published experimental data. Solute transport theories based on both counter and salt diffusion then are used to re-evaluate previously published column test data for the same clay membrane. The results indicate that, despite the theoretical inconsistency between the counter-diffusion assumption and the salt-diffusion conditions of the experiments, the predictive ability of solute transport theory based on the assumption of counter diffusion is not significantly different from that based on the assumption of salt diffusion, provided that the input parameters used in each theory are derived under the same assumption inherent in the theory. Nonetheless, salt-diffusion theory is fundamentally correct and, therefore, is more appropriate for problems involving salt diffusion in clay membranes. Finally, the fact that solute diffusion cannot occur in an ideal or perfect membrane is not explicitly captured in any of the theoretical expressions for total solute flux in clay membranes, but rather is generally accounted for via inclusion of an effective porosity, ne, or a restrictive tortuosity factor, tr, in the formulation of Fick's first law for diffusion. Both ne and tr have been correlated as a linear function of membrane efficiency. This linear correlation is supported theoretically by pore-scale modeling of solid-liquid interactions, but experimental support is limited. Additional data are needed to bolster the validity of the linear correlation for clay membranes.

Flagellar membranes are rich in raft-forming phospholipids.

The observation that the membranes of flagella are enriched in sterols and sphingolipids has led to the hypothesis that flagella might be enriched in raft-forming lipids. However, a detailed lipidomic analysis of flagellar membranes is not available. Novel protocols to detach and isolate intact flagella from Trypanosoma brucei procyclic forms in combination with reverse-phase liquid chromatography high-resolution tandem mass spectrometry allowed us to determine the phospholipid composition of flagellar membranes relative to whole cells. Our analyses revealed that phosphatidylethanolamine, phosphatidylserine, ceramide and the sphingolipids inositol phosphorylceramide and sphingomyelin are enriched in flagella relative to whole cells. In contrast, phosphatidylcholine and phosphatidylinositol are strongly depleted in flagella. Within individual glycerophospholipid classes, we observed a preference for ether-type over diacyl-type molecular species in membranes of flagella. Our study provides direct evidence for a preferential presence of raft-forming phospholipids in flagellar membranes of T. brucei.

High-frequency, spin-label EPR of nonaxial lipid ordering and motion in cholesterol-containing membranes

The EPR spectra of spin-labeled lipid chains in fully hydrated bilayer membranes of dimyristoyl phosphatidylcholine containing 40 mol % of cholesterol have been studied in the liquid-ordered phase at a microwave radiation frequency of 94 GHz. At such high field strengths, the spectra should be optimally sensitive to lateral chain ordering that is expected in the formation of in-plane domains. The high-field EPR spectra from random dispersions of the cholesterol-containing membranes display very little axial averaging of the nitroxide g-tensor anisotropy for lipids spin labeled toward the carboxyl end of the sn-2 chain (down to the 8-C atom). For these positions of labeling, anisotropic 14N-hyperfine splittings are resolved in the gzz and gyy regions of the nonaxial EPR spectra. For positions of labeling further down the lipid chain, toward the terminal methyl group, the axial averaging of the spectral features systematically increases and is complete at the 14-C atom position. Concomitantly, the time-averaged 〈Azz〉 element of the 14N-hyperfine tensor decreases, indicating that the axial rotation at the terminal methyl end of the chains arises from correlated torsional motions about the bonds of the chain backbone, the dynamics of which also give rise to a differential line broadening of the 14N-hyperfine manifolds in the gzz region of the spectrum. These results provide an indication of the way in which lateral ordering of lipid chains in membranes is induced by cholesterol.

Synechocystis HSP17 is an amphitropic protein that stabilizes heat-stressed membranes and binds denatured proteins for subsequent chaperone-mediated refolding

The small heat shock proteins (sHSPs) are ubiquitous stress proteins proposed to act as molecular chaperones to prevent irreversible protein denaturation. We characterized the chaperone activity of Synechocystis HSP17 and found that it has not only protein-protective activity, but also a previously unrecognized ability to stabilize lipid membranes. Like other sHSPs, recombinant Synechocystis HSP17 formed stable complexes with denatured malate dehydrogenase and served as a reservoir for the unfolded substrate, transferring it to the DnaK/DnaJ/GrpE and GroEL/ES chaperone network for subsequent refolding. Large unilamellar vesicles made of synthetic and cyanobacterial lipids were found to modulate this refolding process. Investigation of HSP17-lipid interactions revealed a preference for the liquid crystalline phase and resulted in an elevated physical order in model lipid membranes. Direct evidence for the participation of HSP17 in the control of thylakoid membrane physical state in vivo was gained by examining an hsp17− deletion mutant compared with the isogenic wild-type hsp17+ revertant Synechocystis cells. We suggest that, together with GroEL, HSP17 behaves as an amphitropic protein and plays a dual role. Depending on its membrane or cytosolic location, it may function as a “membrane stabilizing factor” as well as a member of a multichaperone protein-folding network. Membrane association of sHSPs could antagonize the heat-induced hyperfluidization of specific membrane domains and thereby serve to preserve structural and functional integrity of biomembranes.

Characterization of Carbon Molecular Sieve Membranes Supported on Ceramic Tubes

Carbon molecular sieve membranes have been analyzed in supported and unsupported configurations in this experimental study. The membranes were used to adsorb CO2, N2 and CH4, and their adsorption data were analyzed to establish differences in rate and capacity of adsorption between the two types of samples (supported and unsupported). Experimental results show an important effect of the support, which can be considered as an additional parameter to tailor pore size on these carbon membranes. Immersion calorimetry values were measured by immersing the membranes into liquids of different molecular dimensions (dichloromethane, benzene, n-hexane, 2,2-dimethylbutane). Similarities were found between adsorption and calorimetric analysis. The pore volume of the samples analyzed ranged from 0.016 to 0.263 cm3/g. The effect of the pyrolysis temperature, either 550 or 700 °C, under N2 atmosphere was also analyzed. Quantification of the pore-size distribution of the support was done by liquid-liquid displacement porosimetry. The composite membrane was used for CO2/CH4 separation before and after pore plugging was done. The ideal selectivity factors value (4.47) was over the Knudsen theoretical factor (0.60) for membrane pyrolyzed at 600 °C, which indicates the potential application of these membranes for the separation of low-molecular weight gases.

Analysis of Ultrasonic Techniques for the Characterization of Microfiltration Polymeric Membranes

The use of polymeric membranes is extremely important in several industries such as nuclear, biotechnology, chemical and pharmaceutical. In the nuclear area, for instance, systems based on membrane separation technologies are currently being used in the treatment of radioactive liquid effluent, and new technologies using membranes are being developed at a great rate. The knowledge of the physical characteristics of these membranes, such as, pore size and the pore size distribution, is very important to the membranes separation processes. Only after these characteristics are known is it possible to determine the type and to choose a particular membrane for a specific application. In this work, two ultrasonic non destructive techniques were used to determine the porosity of membranes: pulse echo and transmission. A 25 MHz immersion transducer was used. Ultrasonic signals were acquired, for both techniques, after the ultrasonic waves passed through a microfiltration polymeric membrane of pore size of 0.45 μm and thickness of 180 μm. After the emitted ultrasonic signal crossed the membrane, the received signal brought several information on the influence of the membrane porosity in the standard signal of the ultrasonic wave. The ultrasonic signals were acquired in the time domain and changed to the frequency domain by application of the Fourier Fast Transform (FFT), thus generating the material frequency spectrum. For the pulse echo technique, the ultrasonic spectrum frequency changed after the ultrasonic wave crossed the membrane. With the transmission technique there was only a displacement of the ultrasonic signal at the time domain.

Stability of a Nanofiltration Membrane After Contact with a Low-Level Liquid Radioactive Waste

This study investigated the treatment of a liquid radioactive waste containing uranium (235U + 238U) using nanofiltration membranes. The membranes were immersed in the waste for 24–5000 h, and their transport properties were evaluated before and after the immersion. Surface of the membranes changed after immersion in the waste. The SW5000 h specimen lost its coating layer of polyvinyl alcohol, and its rejection of sulfate ions and uranium decreased by about 35% and 30%, respectively. After immersion in the waste, the polyamide selective layer of the membranes became less thermally stable than that before immersion.

Solid-liquid interactions in microscale structures and devices

Liquid-solid interactions become important as dimensions approach mciro/nano-scale. This dissertation focuses on liquid-solid interactions in two distinct applications: capillary driven self-assembly of thin foils into 3D structures, and droplet wetting of hydrophobic micropatterned surfaces. The phenomenon of self-assembly of complex structures is common in biological systems. Examples include self-assembly of proteins into macromolecular structures and self-assembly of lipid bilayer membranes. The principles governing this phenomenon have been applied to induce self-assembly of millimeter scale Si thin films into spherical and other 3D structures, which are then integrated into light-trapping photovoltaic (PV) devices. Motivated by this application, we present a generalized analytical study of the self-folding of thin plates into deterministic 3D shapes, through fluid-solid interactions, to be used as PV devices. This study consists of developing a model using beam theory, which incorporates the two competing components — a capillary force that promotes folding and the bending rigidity of the foil that resists folding into a 3D structure. Through an equivalence argument of thin foils of different geometry, an effective folding parameter, which uniquely characterizes the driving force for folding, has been identified. A criterion for spontaneous folding of an arbitrarily shaped 2D foil, based on the effective folding parameter, is thus established. Measurements from experiments using different materials and predictions from the model match well, validating the assumptions used in the analysis. As an alternative to the mechanics model approach, the minimization of the total free energy is employed to investigate the interactions between a fluid droplet and a flexible thin film. A 2D energy functional is proposed, comprising the surface energy of the fluid, bending energy of the thin film and gravitational energy of the fluid. Through simulations with Surface Evolver, the shapes of the droplet and the thin film at equilibrium are obtained. A critical thin film length necessary for complete enclosure of the fluid droplet, and hence successful self-assembly into a PV device, is determined and compared with the experimental results and mechanics model predictions. The results from the modeling and energy approaches and the experiments are all consistent. Superhydrophobic surfaces, which have unique properties including self-cleaning and water repelling are desired in many applications. One excellent example in nature is the lotus leaf. To fabricate these surfaces, well designed micro/nano- surface structures are often employed. In this research, we fabricate superhydrophobic micropatterned Polydimethylsiloxane (PDMS) surfaces composed of micropillars of various sizes and arrangements by means of soft lithography. Both anisotropic surfaces, consisting of parallel grooves and cylindrical pillars in rectangular lattices, and isotropic surfaces, consisting of cylindrical pillars in square and hexagonal lattices, are considered. A novel technique is proposed to image the contact line (CL) of the droplet on the hydrophobic surface. This technique provides a new approach to distinguish between partial and complete wetting. The contact area between droplet and microtextured surface is then measured for a droplet in the Cassie state, which is a state of partial wetting. The results show that although the droplet is in the Cassie state, the contact area does not necessarily follow Cassie model predictions. Moreover, the CL is not circular, and is affected by the micropatterns, in both isotropic and anisotropic cases. Thus, it is suggested that along with the contact angle — the typical parameter reported in literature quantifying wetting, the size and shape of the contact area should also be presented. This technique is employed to investigate the evolution of the CL on a hydrophobic micropatterned surface in the cases of: a single droplet impacting the micropatterned surface, two droplets coalescing on micropillars, and a receding droplet resting on the micropatterned surface. Another parameter which quantifies hydrophobicity is the contact angle hysteresis (CAH), which indicates the resistance of the surface to the sliding of a droplet with a given volume. The conventional methods of using advancing and receding angles or tilting stage to measure the resistance of the micropatterned surface are indirect, without mentioning the inaccuracy due to the discrete and stepwise motion of the CL on micropillars. A micronewton force sensor is utilized to directly measure the resisting force by dragging a droplet on a microtextured surface. Together with the proposed imaging technique, the evolution of the CL during sliding is also explored. It is found that, at the onset of sliding, the CL behaves as a linear elastic solid with a constant stiffness. Afterwards, the force first increases and then decreases and reaches a steady state, accompanied with periodic oscillations due to regular pinning and depinning of the CL. Both the maximum and steady state forces are primarily dependent on area fractions of the micropatterned surfaces in our experiment. The resisting force is found to be proportional to the number of pillars which pin the CL at the trailing edge, validating the assumption that the resistance mainly arises from the CL pinning at the trailing edge. In each pinning-and-depinning cycle during the steady state, the CL also shows linear elastic behavior but with a lower stiffness. The force variation and energy dissipation involved can also be determined. This novel method of measuring the resistance of the micropatterned surface elucidates the dependence on CL pinning and provides more insight into the mechanisms of CAH.