The anomaly of oxygen diffusion in aqueous xanthan solutions

A membrane-covered polarographic oxygen electrode was used to measure oxygen diffusion coefficients in aqueous polyelectrolyte solutions of xanthan gum, sodium alginate, and sodium carboxymethylcellulose (CMC). In sodium alginate solutions, dilute xanthan solutions, and solutions containing more than 0.3 wt % CMC, oxygen diffusion coefficients decrease with increasing polymer concentrations. Interestingly, in dilute CMC solutions and concentrate xanthan solutions containing more than 0.5 wt % xanthan gum, oxygen diffusion coefficients increase with increasing polymer concentrations, and values exceeding that in pure water are generally observed.     

Characterisation of small molecules diffusion in hydrogel-membrane liquid-core capsules

The overall diffusion coefficients for several low molecular weight solutes, such as glucose, fructose, sucrose, lactose, and vitamin B(12) have been determined in Ca-alginate membrane liquid-core capsules using the unsteady-state method following the release of solutes from the capsules to a well-stirred solution of limited volume. The diffusion coefficients obtained for saccharides were 5-20% lower than the corresponding diffusivity in water while for vitamin B(12) about 50% that of water. The diffusion coefficients of the investigated capsules were not influenced by the change in alginate concentration in the capsule membrane from 0.5 to 1.0%. Lower diffusivities and higher deviations from the diffusivity in water were obtained for higher molecular weight solutes.     

Diffusion coefficients of glucose and ethanol in cell-free and cell-occupied calcium alginate membranes

The diffusivities of glucose and ethanol in cell-free and cell-occupied membranes of calcium alginate were measured in a diffusion cell. The lag time analysis was used. Diffusivities decreased with increasing alginate concentration and were comparable with those in water for a 2% alginate membrane. Glucose and ethanol concentrations had no effect on the respective diffusion coefficients. The ratio of ethanol diffusivity to glucose diffusivity in 2 and 4% alginate agreed closely with the inverse ratio of the hydrodynamic raii for the two molecules in water, indicating that the hydrodynamic theory of diffusion in liquids may be applicable to diffusion in dilute alginate gels. Also, the presence of 20% dead yeast cells had no effect on the diffusivities. The data reported can be used to study reaction and diffusion in immobilized cell reactors and cell physiology under immobilized conditions.     

Diffusion of (de)acylated antibiotic A40926 in alginate and carrageenan beads with or without cells and/or soybean meal

Effective diffusion coefficients (D(e)) of antibiotic A40926 and its deacylated derivative were determined in Ca-alginate (2% wt/wt) and kappa-carrageenan (2.6% wt/wt) gel beads with or without immobilized Actinoplanes teichomyceticus cells and/or soybean meal (SBM). The method used was based on transient concentration changes in a well-stirred antibiotic solution in which gel beads, initially free of solute, were suspended. Unsteady-state diffusion in a sphere was applied and D(e) determined from the best fit of experimental data. A40926 showed markedly different diffusion characteristics than its deacylated derivative. Diffusivity of deacyl-A40926 in alginate or carrageenan gel beads was six to seven times that of A40926. Large differences in partition coefficients (Kp) were also found. In case of beads without additions, A40926, in contrast to deacyl-A40926, strongly partitioned to the liquid phase. Introduction of SBM and/or mycelium in the gel beads decreased the effective diffusivity of deacyl-A40926, but increased its partitioning to the solid phase. Our findings indicate that a relatively moderate structural change of a lipoglycopeptide molecule could lead to a major change in its diffusion/partition characteristics.     

Gelation conditions and transport properties of hollow calcium alginate capsules

The diameter, membrane thickness, and compression intensity of hollow Ca-alginate capsules were measured at different gelation conditions, such as the reactant concentration, dropping velocity, and gelation time. The optimum operation conditions for preparing capsules were determined at 100 g/L CaCl(2), 10 g/L sodium alginate (Na-alginate), a dropping velocity of 150 droplets/min, and a gelation time of 10 min. Diffusion of some saccharide and amino acid from bulk solution into capsules was investigated, and the diffusion coefficients were calculated by the developed mathematical model. All the tested substances can diffuse easily into the capsules. The combined diffusion coefficients of the capsule D(m) are 92-99% as large as their diffusion coefficients in pure water, while the diffusion coefficients in the capsule membrane D(1) are 60-95% as large as those. By employing polyethylene glycol (PEG) and bovine serum albumin (fraction V) (BSA(V)), the molecular weight cut-off of the capsule was determined. For linear macromolecules, hollow Ca-alginate capsules have a molecular weight cut-off of 4000. No diffusion of BSA(V) into the capsules was observed.     

Optical anisotropy of calcium-induced alginate gels

Alginate gels formed by diffusion of calcium ions into solutions of sodium alginate were found to exhibit optical anisotropy depending on preparation conditions. When observed under crossed nicols, the anisotropic alginate gels showed a birefringence pattern which is characteristic of radial orientation of polymer chains. Calcium alginate gels were prepared from different concentrations of sodium alginate and calcium ion, and the conditions for formation of the anisotropic gels were determined. The gel-formation process was measured by monitoring the development of the birefringent layer and was compared with the model in which the diffusion of calcium ions dominates gel formation.     

Quantifying Pb and Cd complexation by alginates and the role of metal binding on macromolecular aggregation

The Pb and Cd binding capacity of alginates were quantified by the determination of their complex stability constants and the concentration of complexing sites using H+, Pb2+, or Cd2+ selective electrodes in both static and dynamic titrations. Centrifugation filter devices (30 kDa filter cutoff), followed by inductively coupled plasma mass spectrometry (ICP-MS) measurements of lead or cadmium in the filtrates, were used to validate the results. The influence of ionic strength, pH, and the metal-to-alginate ratio was determined for a wide range of metal concentrations. Because of their polyelectrolytic properties, alginates may adopt different conformations depending on the physicochemistry of the medium, including the presence of metals. Therefore, molecular diffusion coefficients of the alginate were determined by fluorescence correlation spectroscopy under the same conditions of pH, ionic strength, and metal-to-alginate ratios that were used for the metal binding studies. The complexation and conformational properties of the alginate were related within the framework of the nonideal competitive adsorption isotherm (NICA) combined with a Donnan approach to account for both intrinsic and electrostatic contributions.     

Alginate as immobilization material: III. Diffusional properties

The rate of diffusion of serum albumin (MW 6.9 x 10(4) D) out of beads of calcium alginate gels depends upon the concentration and uronic acid composition of the alginate (ManA/GulA ratio), the conditions under which the beads are produced, the pH, and the temperature. The diffusion coefficient decreases with increasing alginate concentration, and (ManA/GulA) ratio and with decreasing pH. Diffusion out of the beads, in which the alginate is uniformly distributed (homogeneous gel), is faster than out of the beads in which the alginate is concentrated at the surface (inhomogeneous gel). The temperature dependence of the diffusion coefficient follows the Arrhenius law, with an activation energy of approximately 23 kJ x mol(-1).     

Effective diffusion coefficient of sucrose in calcium alginate gel.

The effective diffusion coefficient of sucrose in 5% calcium alginate gel containing 41.6 g.d.c. l-1. Saccharomyces cerevisiae was investigated. Both free and immobilized S. cerevisiae in 0.175 cm and 0.3 cm diameter particles were used and the reactions were achieved in a medium containing 100 g l-1 sucrose and 0.05 M CaCl2. With the assumption that the microorganisms did not grow or die in this medium, the results were analyzed according to Michaelis-Menten kinetics and the values of the parameters were determined as: Vm = 0.256 g ml-1 gel h-1, Km0 = 0.097 g ml-1, Km1 = 0.125 g ml-1, and Km2 = 0.165 g ml-1. Using these values, effectiveness factors were calculated as eta 1 = 0.89 and eta 2 = 0.76, and effective diffusion coefficients for sucrose in calcium alginate gel were determined as De1 = 4.1 X 10(-6) cm2 s-1 and De2 = 4.0 X 10(-6) cm2 s-1, for the particle size involved.     

Transport characterization of hydrogel matrices for cell encapsulation

Current membrane-based bioartificial organs consist of three basic components: (1) a synthetic membrane, (2) cells that secrete the product of interest, and (3) an encapsulated matrix material. Alginate and agarose have been widely used to encapsulate cells for artificial organ applications. It is important to understand the degree of transport resistance imparted by these matrices in cell encapsulation to determine if adequate nutrient and product fluxes can be obtained. For artificial organs in xenogeneic applications, it may also be important to determine the extent of immunoprotection offered by the matrix material. In this study, diffusion coefficients were measured for relevant solutes [ranging in size from oxygen to immunoglobulin G (IgG)] into and out of agarose and alginate gels. Alginate gels were produced by an extrusion/ionic crosslinking process using calcium while agarose gels were thermally gelled. The effect of varying crosslinking condition, polymer concentration, and direction of diffusion on transport was investigated. In general, 2-4% agarose gels offered little transport resistance for solutes up to 150 kD, while 1.5-3% alginate gels offered significant transport resistance for solutes in the molecular weight range 44-155 kD-lowering their diffusion rates from 10- to 100-fold as compared to their diffusion in water. Doubling the alginate concentration had a more significant effect on hindering diffusion of larger molecular weight species than did doubling the agarose concentration. Average pore diameters of approximately 170 and 147 A for 1.5 and 3% alginate gels, respectively, and 480 and 360 A for 2 and 4% agarose gels, respectively, were estimated using a semiempirical correlation based on diffusional transport of different-size solutes. The method developed for measuring diffusion in these gels is highly reproducible and useful for gels crosslinked in the cylindrical geometry, relevant for studying transport through matrices used in cell immobilization in the hollow fiber configuration. (c) 1996 John Wiley & Sons, Inc.     

Diffusion characteristics of calcium alginate gels.

The diffusivity of a protein solute (bovine serum albumin) within calcium alginate gels made from sodium alginate of different guluronic acid content was determined. It was found that protein diffusion within alginate gels, prepared to be isotropic in structure, was greatest for gels prepared from sodium alginate of low guluronic acid content as opposed to those prepared from sodium alginate of high guluronic acid content. This finding was explained in terms of the difference in flexibility of the polymer backbone of the two alginates. The greater the polymer backbone flexibility, the greater the solute diffusivity within the gel.     

Evaluation of some gelling agents for immobilization of aerobic microbial cells in alginate and carrageenan gel beads

Summary Different gelling agents were used to immobilized viable cells in either alginate or -carrageenan gel beads. Based on cell leakage from the gel beads, oxygen and glucose diffusion coefficients and toxicity of the gelling agents, SrCl₂ was found to be the best for immobilization of aerobic microbial cells in, not only alginate but also carrageenan gel beads.     

Studies on the characteristics of alginate gels in relation to their use in separation and immobilized applications

Studies on diffusion of NAD and hemoglobin from calcium and barium gels are reported where alginate grade, concentration, and gel dimensions were varied. These show that NAD diffusion characteristics are unaffected by alginate and ion concentrations; however, hemoglobin diffusion is affected by alginate concentration. Both hemoglobin and NAD diffusion patterns were shown to be affected by alginate gel dimensions. Studies are reported that show that polymannuronic alginate gels posses good porosity characteristics while polyguluronic alginates from gels with lower porosity, specifically with respect to high-molecular-weight compounds. These findings are discussed with the view to the use of alginate gels for immobilization, solids separation, and diffusion chromatography techniques.     

Phase separation in calcium alginate gels

Alginates are polysaccharides consisting of beta-D-mannuronate and alpha-L-guluronate units. In the presence of bivalent cations like calcium the guluronate blocks form physically cross-linked gels. The gelation properties of alginates play an important role in the stability of extracellular polymer substances and in the food industry. When stock solutions of Ca2+ ions and alginate are mixed, the gelation starts before the Ca2+ ions are evenly distributed, which leads to non-uniform gels. In this contribution, Ca alginate gels were prepared by in situ gelation using glucono-delta-lactone and CaCO3. In this way, uniform gels could be prepared directly in the measuring cell. Below a critical concentration, highly viscous solutions were obtained, which were below the critical point of gel formation. In these solutions at low rotational speeds a Schlieren peak arose, which became smaller and steeper with increasing time until a new meniscus could be detected. This behaviour is in contrast to the peak broadening due to diffusion after a synthetic boundary was formed. Evaluation of the data leads to negative diffusion coefficients. It has been shown by others that the mutual diffusion coefficient must be negative in the spinodal region. This phenomena is known as uphill diffusion and leads to phase separation of a binary system. The formation of the gel phase in this case is therefore discussed as uphill diffusion.     

The effective diffusion coefficient and the distribution constant for small molecules in calcium-alginate gel beads

The effective diffusion coefficient, D(e), and the distribution constant, K(i), for selected mono- and disaccharides and organic acids were determined in homogeneous calcium-alginate gel with and without entrapped bacteria. Results were obtained from transient concentration changes in well-stirred solutions of limited volume, in which the gel beads were suspended. The effective diffusioncoefficients and the distribution constants were estimated by fitting mathematical model predictions to the experimental data using a nonlinear model fitting program (MODFIT). Both single solute diffusion and multiple solute diffusion were performed. A small positive effect was obtained onthe values of D(e) for the system of multiple solute diffusion; however, the values of K(i) were not significantly influenced. For the nine solutes tested, D(e) for 2% Ca-alginate gel beads was found to be approximately 85% of the diffusivity measured in water. The effects on D(e) and K(i), for lactose and lactic acid were determined for variations of alginate concentration, pH, temperature, and biomass content in the beads. D(e) decreased linearly for both lactose and lactic acid with increasing cell concentration in the Ca-alginate gel. K(i), was constant for both lactose and lactic acid with increasing cell concentration. D(e) was significantly lower at pH 4.5 than at pH 5.5 and 6.5 for both lactose and lactic acid. Furthermore, D(e) seemed to decrease with increased alginate concentration in the range of 1% to 4%. The diffusion rate increased with increasing temperature, and the activation energy for the diffusion process for both lactose and lactic acid was constant in the temperature range tested. (c) 1995 John Wiley & Sons Inc.     

Electronic speckle pattern interferometry: a tool for determining diffusion and partition coefficients for proteins in gels

The aim of this study was to demonstrate electronic speckle pattern interferometry (ESPI) as a powerful tool in determining diffusion coefficients and partition coefficients for proteins in gels. ESPI employs a CCD camera instead of a holographic plate as in conventional holographic interferometry. This gives the advantage of being able to choose the reference state freely. If a hologram at the reference state is taken and compared to a hologram during the diffusion process, an interferometric picture can be generated that describes the refraction index gradients and thus the concentration gradients in the gel as well as in the liquid. MATLAB is then used to fit Fick's law to the experimental data to obtain the diffusion coefficients in gel and liquid. The partition coefficient is obtained from the same experiment from the flux condition at the interface between gel and liquid. This makes the comparison between the different diffusants more reliable than when the measurements are performed in separate experiments. The diffusion and partitioning coefficients of lysozyme, BSA, and IgG in 4% agarose gel at pH 5.6 and in 0.1 M NaCl have been determined. In the gel the diffusion coefficients were 11.2 +/- 1.6, 4.8 +/- 0.6, and 3.0 +/- 0.3 m(2)/s for lysozyme, BSA, and IgG, respectively. The partition coefficients were determined to be 0.65 +/- 0.04, 0.44 +/- 0.06, and 0.51 +/- 0.04 for lysozyme, BSA, and IgG, respectively. The current study shows that ESPI is easy to use and gives diffusion coefficients and partition coefficients for proteins with sufficient accuracy from the same experiment.     

Influence of growth behaviour and physiology of alginate-entrapped microorganisms on the oxygen consumption

Summary The ability of alginate-entrapped microorganisms to supply oxygen was determined with regard to physiology and growth behavior of the cells. Oxygen diffusion through an alginate film containing different concentrations of Pseudomonas putida or Saccharomyces cerevisiae was measured. Oxygen diffusion decreased when the cell loading increased. Dependent on the physiological behavior of these organisms the course of the oxygen concentration under the gel film is quite different. In further experiments an Effectiveness-Factor of oxygen uptake of alginate beads with Saccharomyces cerevisiae or Aspergillus niger was determined in relation to the growth behavior of the organisms. The effectiveness factor is always higher when the biomass is concentrated in the outer region of the gel beads as if the microorganisms are distributed homogeneously in the alginate. Considering these results it is not possible to make a general statement on the ability of microorganisms in alginate to supply oxygen. The physiology and the growth behavior of the immobilized organisms have to be considered in any case.     

Measuring the translational diffusion coefficients of small DNA molecules by capillary electrophoresis

The apparent translational diffusion coefficients of four 20 base pair (bp) DNA oligonucleotides with different sequences have been measured by capillary electrophoresis, using the stopped migration method. The diffusion coefficients of the four oligomers were equal within experimental error, and averaged (120 +/- 10) x 10(-8) cm(2) s(-1) in 40 mM Tris-acetate-EDTA buffer at 25 degrees C. Since this value is nearly identical to the translational diffusion coefficient determined for a different 20-bp oligomer using other methods, the stopped migration method can accurately measure the diffusion coefficients of small DNA oligomers. The apparent diffusion coefficient of a 118-bp DNA restriction fragment was also measured by the stopped migration method. However, the observed value was approximately 25% larger than expected from other measurements, possibly because the diffusion coefficients of larger DNA molecules are somewhat dependent on the ionic strength of the solution.     

Simultaneous Characterization of Lateral Lipid and Prothrombin Diffusion Coefficients by Z-Scan Fluorescence Correlation Spectroscopy

A new (to our knowledge) robust approach for the determination of lateral diffusion coefficients of weakly bound proteins is applied for the phosphatidylserine specific membrane interaction of bovine prothrombin. It is shown that z-scan fluorescence correlation spectroscopy in combination with pulsed interleaved dual excitation allows simultaneous monitoring of the lateral diffusion of labeled protein and phospholipids. Moreover, from the dependencies of the particle numbers on the axial sample positions at different protein concentrations phosphatidylserine-dependent equilibrium dissociation constants are derived confirming literature values. Increasing the amount of membrane-bound prothrombin retards the lateral protein and lipid diffusion, indicating coupling of both processes. The lateral diffusion coefficients of labeled lipids are considerably larger than the simultaneously determined lateral diffusion coefficients of prothrombin, which contradicts findings reported for the isolated N-terminus of prothrombin.     

Lipid and water diffusion in bicontinuous cubic phases measured by NMR.

Lipid and water diffusion coefficients in bicontinuous cubic liquid crystalline phases have been determined with the NMR pulsed magnetic field gradient technique. In the monoolein-water system, a discontinuity in the variation of the water diffusion coefficient with water content is observed, which coincides with the two-phase region between the two cubic phases in this system. The degree of water association to the lipid has been determined, considering the obstruction factor for diffusion in the cubic phases. The lipid diffusion coefficient increases with increased unsaturation of the lipid, and decreases when larger amphiphile molecules like cholesterol, gramicidin-A, and lyso-oleoyl-phosphatidylcholine are solubilized in the cubic phase. In a cubic liquid crystal of monoolein (MO), dioleoylphosphatidylcholine (DOPC), and water, the individual lipid diffusion coefficients have been determined simultaneously in the same sample. The diffusion coefficients of MO and DOPC differ by a factor of two, and both decrease with increasing DOPC content. The results are discussed in relation to probe techniques for measurements of lipid diffusion.