Non-porous Supports Research Articles

High Performance Liquid Chromatography of Small and Large Molecules with Nonporous Silica-Based Stationary Phases

Nonporous monodispersed, microspherical silica particles, having mean particle diameters of 0.8 and 1.1 μm, were surfacel-modified with octadecyl or triphenyl functions as well as with lectin proteins for high performance liquid chromatography of small and large molecules. “Monomeric” or “polymeric” octadecyl-silica (C18-silica) bonded phases were formed, depending on the silane compounds used. Monomeric C18-silica stationary phases yielded high resolution in the separation of 2-pyridylamino derivatives of xyloglucan oligosaccharides and proteins. The effect of particle size of the support on resolution was also examined. For comparative study, the chromatographic behavior of large molecular weight proteins on a triphenyl-silica stationary phase was also conducted. Polymeric C18-silica stationary phases yielded higher phase ratios and better support surface coverage which proved useful in the separation of small hydrophilic species such as derivatized chitooligosaccharides of lower degree of polymerization (d.p.). The slopes of the linear dependence of logarithmic retention factor on volume percent of organic modifier were determined for the monomelic and polymeric C18-silica stationary phases with low molecular weight aromatic compounds. Lectin affinity stationary phases with lens culinaris agglutinin and wheat germ agglutinin covalently bound to nonporous silica support were examined for the applications of nonporous stationary phases in lectin affinity chromatography of glycoproteins. In all cases, rapid separations in the time scales of seconds and minutes could be obtained because of the absence of mass transfer resistance in the nonporous stationary phase matrices.

A Computational Method for Determination of The Mass-Transfer Coefficient in Packed-Bed Immobilized Enzyme Reactors

A computational method was developed that determined the mass-transfer coefficient kL or the volumetric mass-transfer coefficient kLa in packed-bed immobilized enzyme (IME) reactors. To study the performance of this method, two experimental systems were considered where an enzyme was immobilized on a non-porous support surface (surface-IME system) or within a porous support (pore-IME system). The values of kL and kLa determined in these packed-bed IME reactor systems were successfully expressed in terms of the substrate concentration at the reactor inlet and the liquid flow rate. Furthermore, the correlations obtained for kL and kLa were used to calculate the unconverted fractions of substrate at the reactor outlet. Comparison showed that the calculated results were in satisfactory agreement with the experimental values.

High-temperature reversed-phase high-performance liquid chromatographic analysis of a synthetic copolymer on a non-porous support

High-temperature HPLC using a non-porous microparticulate reversed-phase column was evaluated for characterizing an exploratory magnetic resonance imaging polymeric contrast agent. High-efficiency separations were achieved allowing resolution of over 50 individual oligomers. Separations were optimized with respect to temperature and addition of neutral salt. Results were compared to those obtained with a traditional porous HPLC support as well as a macroporous perfusion-type column. Separation efficiencies were highest with the non-porous support followed in order by the macroporous and traditional porous packing. The utility of this technique for characterizing degradation of the polymer under stress conditions was demonstrated.

Study of conformational effects of recombinant interferon gamma adsorbed on a non-porous reversed-phase silica support.

Reversed-phase chromatography is a powerful method for separating recombinant interferon gamma and one of its analogues differing only by a single amino acid residue. Structural differences of the proteins explain this separation ability as demonstrated from adsorption studies on a non-porous reversed-phase support. To reveal the structural differences occurring in the adsorbed state, two different and independent methods were employed. The variation of the retention with the slope of the linear gradient gave information about the molecular contact area of the protein with the support. For different experimental conditions, these data were correlated with the adsorbent capacities measured on an n-octadecyl-modified non-porous silica support. These supports are useful for these types of experiments because the protein is adsorbed exclusively at the external surface of the beads. Moreover, a small amount of protein is necessary to saturate the column, owing to its low capacity.

Ion transport processes and middle ear physiopathology. An experimental approach using cell culture.

The role of the middle ear (ME) epithelium is primarily to keep the cavities air-filled and fluid-free. This condition requires elimination of mucus and cellular debris from the tympanic cavity by the ciliary activity. This clearance activity may be related to active ion transport processes in the epithelium. This paper presents a review of the experimental data suggesting the existence of ion transports in ME, and demonstrating the contribution of the culture of ME epithelial cells to the investigation of ion transport properties. Primary cultures of ME epithelial cells from Mongolian gerbils (Meriones unguiculatus) were undertaken. Polarized monolayers exhibited domes on non-porous supports. On porous supports, the absorptive function was shown to be related to a Na+ absorption from apical to basal sides of the epithelium, by means of apical amiloride-sensitive Na+ channels and basolateral Na+, K(+)-ATPase. In turn, this sodium absorption would generate an osmotic gradient and drive a water flow. An amiloride-induced chloride transport is also suggested. A hormonal modulation of these ion transport properties might play a role in the development of the mucociliary impairment that initiates chronic otitis media. The establishment of a middle ea cell line using wild-type SV 40 infection is in progress. This might provide further information on the physiology of the ME.

Ion transport by primary cultures of Mongolian gerbil middle ear epithelium.

The transport properties of Mongolian gerbil middle ear epithelial cells grown in primary culture were studied. These cells formed polarized monolayers that exhibited domes on nonporous supports. On porous supports, monolayers developed an apical-negative transepithelial electric potential difference (VT = -37.2 +/- 2.7 mV) and a transepithelial resistance (RT = 519 +/- 56 omega.cm2). The short-circuit current equivalent (Ieq) was 62.4 +/- 6.2 microA/cm2 (mean +/- SE, n = 15). Na+ and Cl- accumulated in the basal bath and generated a basolateral hyperosmolarity that drove a net water flow. Amiloride (10 microM), when added to the apical but not to the basal bath, induced a 23.4 +/- 1.5 mV and 44.1 +/- 1.3 microA/cm2 decrease of VT and Ieq, respectively, while RT increased by 403 +/- 69 omega.cm2 (P less than 0.001, n = 15). Exposure of the monolayers to a low-Cl- solution (30 mM) enhanced the transepithelial potential, possibly by means of a Cl- secretion through apical Cl- channels. Isoproterenol (10(-4) M basolateral) increased intracellular adenosine 3',5'-cyclic monophosphate (cAMP) content (concentration of half-maximal response = 2.5 x 10(-7) M) and decreased VT, RT, and Ieq. The isoproterenol-induced fall of VT occurred even in the presence of low-Cl-solutions. This suggested an increase of the paracellular pathway conductance, although there was no significant modification of the mannitol permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Performance characteristics of a low sludge bioreactor for wastewater treatment

An immobilized microbial cell system was developed and tested for the treatment of industrial wastewater. A consortium of selected aerobic microorganisms was immobilized onto several different support matrices in a packed bed reactor configuration and operated in a continuous process mode. Comparison of the support matrices showed only small differences in treatment efficiency, but significant differences in sludge production and process stability. Porous polymer supports were highly resistant to feedstream upsets and produced 80% lower sludge solids as compared with non-porous supports. These results were seen at both the benchtop and pilot plant scale for treatment of complex industrial waste streams. This technology was applied, in preliminary experiments, to the treatment of a model waste stream simulating wastewater from a Controlled Ecological Life-Support System (CELSS).

Immobilization of binding proteins on nonporous supports

Four different nonporous particulate materials, nylon, polystyrene, soda-lime silicate glass, and fused silica glass, have been evaluated for their appropriateness as immobilization supports for immunoglobulins. A method of protein quantitation that is usually applied to solutions, the bicinchoninic acid (BCA) assay, was used successfully to directly measure ng amounts of protein immobilized on the supports. Two proteins, a monoclonal antibody to theophylline and the biotin binding protein avidin, were studied. Radioactive theophylline and radioactive biotin were used to measure the activity of the immobilized protein. Ligand binding capacity per mm2 of support was measured as a function of amount of protein immobilized. By measuring both the amount of protein immobilized and its ligand binding capacity, we have determined that antitheophylline antibody adsorbed on polystyrene balls loses almost 90% of its binding activity after 65 h, although little protein is lost from the balls over this time. Avidin retains nearly full activity for biotin on polystyrene. The binding activity of biotinyl-antibody conjugate immobilized on avidin-adsorbed polystyrene is stable, even when stored for over 22 wk. Antibody covalently immobilized on soda-lime silicate glass beads retains its binding activity over long-term storage, although only 0.1 mol of 3H-theophylline bind per mol of immobilized antibody. Using fused silica glass particles as the solid support, the same antibody binds approx 0.6 mol of ligand per mol of immobilized antibody protein. The structural "softness" of the immunoglobulin requires that interaction with the surface be prevented in order to maintain activity.

Immobilization of β-Glucuronidase on Pellicular Nylon

β-Glucuronidase (EC 3.2.1.31), From Patella vulgala, was immobilized on a pellicular, polyethyleneimine-derivatized nylon net. Several types of nylon nets were used in order to ensure the best relationship between activity of the immobilized enzyme and net characteristics. No differences were observed between the two most used reagents for nylon activation by alkylation under standard working conditions. The influence of attachment by several protein aminoacidic side chains was determined, and enzyme immobilization involving lysyl and tyrosyl protein residues showed better activity. Coupling conditions for these derivatives have been optimized by studying the influence of pH, temperature and reaction time on derivative activity. The saturation profiles obtained for these derivatives showed a high protein content, 30mg/g, for a non-porous support. Characterization of these derivatives against pH, ionic strength and temperature was also studied, and no differences were found between soluble and immobilized ...

Studies on the deactivation of immobilized urease

The results of experiments in a fixed-bed reactor and a CSTR containing urease immobilized on a nonporous support and conducted in the absence of diffusional limitations are reported. Kinetic parameters were established by separate batch experiments. The key observation was that the product ammonia attacked the free form of the enzyme and thereby illustrates the importance of mechanism in determining deactivation kinetics.

Immobilization of Pepsin onto Wide-Porous and Nonporous ω-Aminoalkylderivatized Inorganic Supports

Heterogeneous catalysts were prepared by covalent binding of pepsin to wide-porous and nonporous ω-aminoalkylderivatied inorganic supports by means of water-soluble carbodiimide. The amount of attached enzyme and the proteolytic activity of catalysts were determined. Nonporous catalyst, based on finely dispersed pyrogeneous silicon dioxide (Cabosil), exhibited the maximum pepsin content (22 nmol/g) and the highest proteolytic activity (5.6 units/g). Wide-porous glass-based catalyst showed the maximum retained pepsin activity (92%). The immobilized pepsin content as well as proteolytic activity of catalysts was found to increase on increasing the chain length of the alkyl spacer from C2 to C6, whereas thermal stability of catalysts (37d`C) was independent of the alkyl spacer length. All the catalysts tested were stable on storage at 8d`C, pH4.5 for 1 month.

Functional expression of the amiloride-sensitive sodium channel in Xenopus oocytes.

Expression of the amiloride-sensitive sodium channel was examined in Xenopus oocytes that were microinjected with A6 cell mRNA. Amiloride-inhibitable 22Na flux could be measured in intact oocytes 2-3 days after injection with 25 ng of poly(A)+ RNA isolated from aldosterone-treated A6 cells. The rate of 22Na uptake was approximately 15-fold greater in oocytes microinjected with 25 ng of poly(A)+ RNA than in water-injected control oocytes. An increase in 22Na uptake by mRNA-injected oocytes occurred whether the mRNA was isolated from A6 cells grown on a porous or nonporous support. In the presence of 4 mM NaCl, amiloride caused dose-dependent inhibition of 22Na uptake in mRNA-injected oocytes, which was half-maximal at 6 x 10(-8) M. Both 1 microM amiloride and 0.1 microM benzamil inhibited 22Na uptake in mRNA-injected oocytes by greater than 95%, whereas less than 50% inhibition occurred with 1 microM 5-(N-ethyl-N-isopropyl)amiloride. When A6 cell mRNA was size fractionated by sucrose density-gradient centrifugation, amiloride-sensitive 22Na uptake was expressed predominantly by oocytes injected with mRNA from two contiguous fractions.

Non-Porous Silica Supports for High Performance Liquid Chromatography

Abstract The physical and chromatographic properties of two types of non-porous HPLC support materials were examined. Loading capacities of non-porous supports were about 10 fold less than of wide-pore gels, while their physical strength was much greater, suggesting that non-porous supports could be useful for polymer separations. Such materials appeared to be less suitable for low-molecular-weight solutes, except for fundamental retention studies, where the absence of pore structure offers a less ambiguous support matrix.

Catalyst coating for nonporous supports in reactors for purification of gas discharges

In order to improve the mechanical strength of the catalyst coating, a technique has been proposed in which the catalyst is prepared on nonporous metal supports. This technique consists of application of a catalytically active suspension consisting of the catalyst, a binder, and an adhesive solution to the surface of a metal support, such as the plates of a reactor, after which the porous film formed on the metal support is dried and calcined. Recommended as the binder is the cement talyum, which is technical calcium aluminate; recommended as the adhesive is a solution of a polymethylphenylsiloxane resin in toluene, similar in composition to KO-08 varnish. The polymethylphenylsiloxane resin macromolecules making up the adhesive, after vaporization of the toluene, upon drying and calcining of the coating form an elastic, strong network that is heat-resistant to 450/sup 0/C. This network retains the catalyst and binder particles on the surface of the metal support. The elasticity of the polymer network accommodates the deformations created in the catalyst film thermal expansion or contraction of the metal coating, and also the stresses created in installing the reactor.

Preparation of membranes based on heteropolysiloxanes

Heteropolysiloxanes are a new type of material and can provide some advantages when used as membranes. The backbone consists of a siliceous network modified by the incorporation of special organofunctional groups. First results of membrane preparation from heteropolysiloxanes are presented. Variation of the composition by a choice of suitable starting materials together with proper adjustment of the reaction conditions allows tailoring of the properties of the material. This wide variability in the system can also be used advantageously for membrane preparation. For this purpose, different membrane preparation techniques can be applied. The best method for the coating of supports is reactive synthesis. This technique makes possible the preparation of membranes from monomers at the surface of a porous or nonporous support. General membrane preparation possibilities and basic properties of membranes obtained by these techniques, as well as possible applications, are described.

On the mechanism of immobilized glucose oxidase deactivation by hydrogen peroxide

The result of experiments in a fixed-bed reactor containing glucose oxidase immobilized on a nonporous support and conducted in the absence of diffusional limitations are reported. Kinetic parameters were established by separate batch experiments. The key observation was that, in every case, poisoning by product hydrogen peroxide resulted in a minimum in enzyme activity in the interior of the bed, well away from the ends. The deactivation data were interpreted by fitting the rate constant for poisoning, the only free parameter, to a previously reported theory. The theory postulates several deactivation mechanisms each of which leads to self-consistent kinetics, but the only mechanism which fitted the data assumes that peroxide attack the enzyme when it is the from complexed with glucose. Theory and experiment agreed to within the accuracy (+/- 2%) of the activity measurements.

Factors determining flow rate in chromatographic columns

It is shown that the flow in chromatography is nearly always laminar in nature. Starting from the Darcy equation, expressions are given for the flow rate in both gas and liquid chromatography columns. The concepts of specific permeability, chromatographic permeability and column resistance factor are discussed for packed as well as open tubular columns. The experimental determination of all these factoers is demonstrated. The influence of the shape and pore volume of porous and non-porous supports on the column resistance factor and the chromatographic permeability is discussed.

Immunochemical characteristics and preparative application of agarose-based immunosorbents

The physical and chemical properties of immunosorbents prepared from Sepharose 4B have been studied, with the objective of improving their utility in preparative applications. The most significant factors influencing efficient use of these immobilized reagents are loss of immunochemical reactivity with increasing protein substitution and the restrictions imposed by the porous nature of the support. Similar effects are observable with controlled-pore glass matrices, but they are of less significance when solid, nonporous supports are used. Considerable improvements in operational efficiencies have been obtained by limiting the amount of protein immobilization and restricting reaction to the more accessible surface regions of the porous supports. Improved operating protocols based on these principles have been adapted to automated chromatographic systems, resulting in a fourfold increase in productive capacity. With the addition of the continuous dialysis and concentration system described here, routine daily production of gram quantities of specific antibodies from antisera becomes possible. Similarly, with a knowledge of the basic characteristics of these immunosorbents, it has been possible to increase greatly the efficiency of small-scale separations and purifications of immunological reagents from limited sources. The general approach toward the practical exploitation of immunosorbents for preparative applications is discussed in the context of these findings.

X-ray small-angle scattering measurements on metallic catalysts

The granulometry of a metallic phase, dispersed on the surface of porous or non-porous supports was determined.

Comments on diffusive and electrostatic effects with immobilized enzymes

Shuler, Aris & Tsuchiya (1972) have recently considered the combined effects of diffusive resistance and electrostatic field on the rate of reaction catalyzed by an enzyme immobilized on a non-porous surface. They employed a potential distribution for the electrical double layer which is asymptotically valid when surface potential is small.The complete Gouy-Chapman solution, which is valid for higher surface potential, is employed here. Numerical values of the effectiveness factor calculated with this potential distribution agree very closely with the results of Shuler et al. for most cases. It is shown that the effectiveness factor can (i) attain magnitudes much greater than unity in physically realizable systems, (ii) approach the solution for “infinite” surface potential at reasonable values of surface charge density, and (iii) pass through a maximum as bulk substrate concentration is varied. This behavior leads to the existence of an optimum surface concentration for enzyme immobilized on a highly charged non-porous support such that the most effective catalytic action on a charged substrate is ensured. Finally, it is established that significant electrical and/or diffusive effects result in non-linear Lineweaver-Burk plots of reciprocal observed reaction velocity against reciprocal bulk substrate concentration. These non-linear plots cannot be interpreted in the same way as linear plots obtained when enzyme is unbound.