Stationary phase with specific surface properties for the separation of estradiol diastereoisomers

The aim of this study was to develop a procedure that enabled the separation of estradiol diastereoisomers. For this purpose a series of stationary phases with different surface properties has been utilized. Two of them contain various interaction sites, such as: cholesterol, n-acylamide, amine and silanols localised in the organic layer bonded to the surface of silica gel (SG-CHOL and SG-CHOL/AP). The other one contains mainly alkylamide ligands and also residual aminopropyl and silanol groups (SG-AP), as well as the last one consisting of hydrocarbonaceous material (SG-C(18)). In order to select the best type of stationary phase for this analysis, after chromatographic separation of 17-alpha-estradiol and 17-beta-estradiol, selectivity and resolution of the analytes were compared. The best separation of hormones was obtained for SG-CHOL packing, as a consequence of the structure and the properties of this stationary phase. To better understand the retention mechanism and the properties of the stationary phases, used in the separation of steroid compounds, the functional group contributions (tau) were compared with Hansch substituent constants (pi).     

A label-free immunosensor by controlled fabrication of monoclonal antibodies and gold nanoparticles inside the mesopores

A label-free immunosensor for the detection of α-fetoprotein (AFP) is proposed based on controlled fabrication of monoclonal antibodies of AFP (anti-AFP) and gold nanoparticles (GNPs) inside the pores of mesoporous silica (MPS). The silanol groups on the internal pore walls were grafted by aminopropyltriethoxyl silane, whereas the silanol groups on the external surface of MPS were blocked by trimethylchlorosilane (TMCS). Thus, anti-AFP and GNPs could be confined inside the mesopores of TMCS-MPS by the covalent linking with the amino groups. The prepared anti-AFP/GNPs/TMCS-MPS particles were used to modify glassy carbon electrode (GCE) to construct a label-free immunosensor. After incubating the sample AFP with the anti-AFP/GNPs/TMCS-MPS/GCE, the immunoconjugates were formed on the surface of GCE and the spatial block increased. Thus, the peak current decreased with increasing concentrations of AFP. GNPs inside the mesopores could promote the electron transportation through the pore channel. Under the optimal experimental conditions, the fabricated immunosensor could detect AFP in a linear range from 1.0 to 90 ng ml(-1) with a detection limit of 0.2 ng ml(-1) (3σ). It provided a novel alternative method for the label-free determination of other antigens.     

DETECTION OF SILICA IN PLANTS

Silica in plants can be stained by silver Chromate, methyl red, and a colorless crystal violet lactone which are adsorbed by the silanol groups resulting in red-brown, red, and blue colors, respectively. Specialized silica cells in grasses can also be detected through polarization colors due to form birefringence. Silica in the bulliform and silica cells of rice leaves is amorphous and is made up of 1–2-nm particles aggregating into 2.5 X 0.4-μm rods with oblique ends.     

General properties of silated hydroxyethylcellulose for potential biomedical applications.

The general properties of hydroxyethylcellulose (HEC) grafted with 3-glycidoxypropyltrimethoxysilane (GPTMS) or 3-glycidoxypropylmethyldiethoxysilane (GPDMS) were studied for potential biomedical applications. The graft involved a Williamson reaction between the free hydroxyl function of HEC and the epoxy function of the two silanes. As the grafted silanes are in ionic form (sodium silanolate), this product remains in gel form at basic pH (>12.3) in aqueous solution. When pH decreases, sodium silanolate is transformed into silanol (2 or 3 silanol functions are carried by silicon, depending on the silane grafted). The silanols interreact, and the gel is transformed into a cross-linking form at room or body temperature. Studies were conducted to optimise this product for specific uses. Steam sterilization was used to compare self-hardening as a function of the silane grafted. Our previous work indicated that HEC grafted with GPTMS has good reactivity, but requires high pH for dissolution, whereas dissolution occurs at lower pH with GPMDS. The rate of silanol condensation for silated HEC was then determined as a function of pH, temperature, type of silane, and the percentage grafted. Condensation rates were ascertained by the viscosity method, and gels were neutralized by different solutions to obtain buffered forms at various pH. The time required to obtain 10(5) mPa x s, with an initial state of 2500 mPa x s, was then calculated. Condensation was catalysed in acid or basic medium at a lower rate at pH 5.5-6.5, and a temperature rise increased the condensation rate, regardless of the pH or silane studied. Silanetriol was more reactive than silanediol. However, as HEC lost considerable viscosity after sterilization, further studies will be conducted to develop new polysaccharides grafted with silane.     

Water Confined in Cylindrical Pores: A Molecular Dynamics Study

Molecular dynamics simulations of water confined in two hydrophilic cylindrical pores—PH and PL—that differ in their silanol surface concentration (7.6 and 3.0 nm⁻², respectively) have been performed at 300 K. A strong interaction of interfacial water molecules with the pore was systematically found and gives rise to a layering effect, a significant distortion of both the hydrogen bond network (HBN) and the tetrahedral structure of these water molecules, and a corresponding subdiffusive mean square displacement along the main axis of the pores. By contrast, water molecules in the inner part of both pores were found to behave similarly to bulk water. The HBN and the tetrahedral configuration of water were more gradually distorted in the PL pore given the larger heterogeneity and rugosity of the surface, and the number of water–pore hydrogen bonds did not scale linearly with the silanol surface concentration of the pores, in part because of the close proximity between silanols in the PH pore. With the PL pore, the dynamic slowing down of water was found consistent with the experiment, suggesting that it provides a better model for the cylindrical MCM-41 mesopores. The structural and dynamical properties of water vary little with the silica force field.     

Analysis of the soft-tissue response to components used in the manufacture of breast implants: rat animal model.

The implant-tissue response to the silicone gel mammary prosthesis requires a more thorough evaluation in light of recent concerns related to human connective-tissue diseases, contracture, infection, and neoplasia. The silicone prosthesis is not a homogeneous implant but is a milieu of various silicone chemistries. Silicone polymer precursors and prosthesis components (silicone shells, shells extracted of their low-molecular-weight components, silica-free silicone, silicone oil, fumed silica, and silicone extract) were implanted subcutaneously using a nonhemorrhagic technique into the backs of Lew/SsN rats (n = 90), two implants per rat, for periods of 7, 14, 28, 56, and 90 days for a total of 6 implants per material per time period. Histologic analysis was performed on specimens from the harvested soft tissue. The intensity of the cellular and capsular response was lowest for the silicone oil and increased as the material's molecular weight increased and material compliance decreased. Fumed silica elicited the most highly reactive cellular response. From this study it is apparent that the polymer's molecular weight influences its migration, encapsulation, and intensity of cellular response. Further, the silicone extract distillate elicited a highly intense cellular response with pronounced lymphocyte invasion. The human relevance of this work awaits further correlation with implant retrieval and in vivo performance.     

Liquid Chromatographic Resolution of Amino Acid Esters of Acyclovir Including Racemic Valacyclovir on Crown Ether‐Based Chiral Stationary Phases

Valacyclovir, a potential prodrug for the treatment of patients with herpes simplex and herpes zoster, and its analogs were resolved on two chiral stationary phases (CSPs) based on (3,3’‐diphenyl‐1,1’‐binaphthyl)‐20‐crown‐6 covalently bonded to silica gel. In order to find out an appropriate mobile phase condition, various mobile phases consisting of various organic modifiers in water containing various acidic modifiers were applied to the resolution of valacyclovir and its analogs. When 30% acetonitrile in water containing any of 0.05 M, 0.10 M, or 0.15 M perchloric acid was used as a mobile phase, valacyclovir and its analogs were resolved quite well on the two CSPs with the separation factors (α) in the range of 2.49 ~ 6.35 and resolutions (R_S) in the range of 2.95 ~ 12.21. Between the two CSPs, the CSP containing residual silanol protecting n‐octyl groups on the silica surface was found to be better than the CSP containing residual silanol groups. Chirality 27:268–273, 2015. © 2015 Wiley Periodicals, Inc.     

Investigation of the binding mechanism of glucoamylase to alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) has been coupled to several porous silica matrices by a new covalent process using alkylamine derivatives of titanium(IV)-activated supports. In order to investigate the interaction of the titanium element with the silanol groups of the inorganic matrices, activation was performed at different times, using titanium(IV) chloride, either pure or as a 15% w/v solution, in 15% w/v hydrochloric acid at 25, 45 and 80°C, followed by washing with sodium acetate buffer (0.02m, pH 4.5) or chloroform. Using pure TiCl₄, the highest activities of all preparations were obtained at 80°C and with acetate buffer washing, resulting from a higher content of titanium coating of the carrier. When activation was performed in aqueous TiCl₄ solution, followed by a drying step, the highest activity was obtained with preparations washed with chloroform, with or without amination. When reacting pure TiCl₄ with controlled pore glass (CPG) and with porous silica (Spherosil), colour formation was observed after reaction of glutaraldehyde with the aminated support. This did not happen when Celite was used as the support. As a criterion for comparison of the different immobilized enzyme preparations, the concept of an ‘instability factor’, which measures the percentage of immobilized enzyme activity due to release of enzyme into solution, is introduced. Instability factors of immobilized enzyme preparations on Celite were always higher than those obtained with the other matrices, confirming that there was no covalent coupling of the enzyme to Celite. However, when the activation was performed with aqueous TiCl₄ solution with drying, Schiff's base formation was observed in all preparations and very stable immobilized enzyme preparations were obtained. The results of the activation of controlled pore glass and porous silica with pure titanium(IV) chloride suggest the existence of a true reaction between the titanium element and the silanol groups of these carriers by formation of a bridge, Si-O-Ti, while with the titanium(IV) chloride solution in hydrochloric acid, a coating of hydrous titanium(IV) oxide is obtained.     

Bionanocompositional chitosan-silica sorbent for liquid chromatography

Preparation of hybrid nanocompositional chitosan/silica sorbent was carried out. It was shown that formation of gel in sol-gel process of hydrolytic polycondensation of tetraethoxysilane (TEOS) with including of chitosan consists of two stages. Suppression of crystallization of chitosan in obtained two-phase system and changes in IR spectra are evidenced of interactions between molecules of chitosan and silanol groups of silica network. The resulting hybrid chitosan/silica sorbent was tested by high-performance liquid chromatography (HPLC).     

Purification of a peptide tagged protein via an affinity chromatographic process with underivatized silica

Silica is widely used for chromatography resins due to its high mechanical strength, column efficiency, easy manufacturing (i.e. controlled size and porosity), and low‐cost. Despite these positive attributes to silica, it is currently used as a backbone for chromatographic resins in biotechnological downstream processing. The aim of this study is to show how the octapeptide (RH)4 can be used as peptide tag for high‐purity protein purification on bare silica. The tag possesses a high affinity to deprotonated silanol groups because the tag''s arginine groups interact with the surface via an ion pairing mechanism. A chromatographic workflow to purify GFP fused with (RH)4 could be implemented. Purities were determined by SDS‐PAGE and RP‐HPLC. The equilibrium binding capacity of the fusion protein GFP‐(RH)4 on silica is 450 mg/g and the dynamic binding capacity around 3 mg/mL. One‐step purification from clarified lysate achieved a purity of 93% and a recovery of 94%. Overloading the column enhances the purity to >95%. Static experiments with different buffers showed variability of the method making the system independent from buffer choice. Our designed peptide tag allows bare silica to be utilized in preparative chromatography for downstream bioprocessing; thus, providing a cost saving factor regarding expensive surface functionalization. Underivatized silica in combination with our (RH)4 peptide tag allows the purification of proteins, in all scales, without relying on complex resins.     

Liquid chromatographic direct resolution of tocainide and its analogs on a (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6-based chiral stationary phase containing residual silanol protecting n-octyl groups

Optically active (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6-based chiral stationary phase (CSP) containing residual silanol protecting n-octyl groups on silica surface was applied to the liquid chromatographic direct resolution of tocainide and its analogs. The chiral recognition ability of the CSP was excellent, the separation (alpha) and the resolution factors (R(S)) for 15 analytes including tocainide being in the range of 3.02-22.92 and 3.94-20.41, respectively. In addition, the chiral recognition ability of the CSP was much greater than that of (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6-based CSP containing residual silanol groups on the silica surface. The chromatographic behaviors for the resolution of tocainide and its analogs were found to be dependent on the content and the type of organic and acidic modifiers and the ammonium acetate concentration in aqueous mobile phase.     

Insulin adsorption on coated silica based supports grafted with N-acetylglucosamine by liquid affinity chromatography

Silica beads are coated with dextran carrying a calculated amount of positively charged diethylassminoethyl groups (DEAE) in order to neutralize negative charged silanol groups at the silica surface and in this way to minimize non specific interactions between silica surface and proteins in solution. Dextran-coated silica supports are potentially excellent stationary phases for high-performance liquid chromatography of proteins. These supports combine the advantages of polysaccharide phases with the excellent mechanical characteristics of silica. These supports (silica-dextran-DEAE = SID) are easily functionalized by grafting N-acetylglucosamine (GlcNAc) using conventional coupling methods. The performances of the support bearing GlcNAc are studied by high-performance liquid affinity chromatography (HPLAC) of insulin, the hypoglycemic peptide hormone of the human organism. The study shows that these supports exhibit a reversible and specific affinity towards insulin and allow separations with high purification yields. Moreover, the influence of different physico-chemical parameters (pH, NaCl and insulin concentration) on insulin retention on the support was analysed. This allowed us to optimize the conditions of adsorption and to better understand the interaction mechanisms between insulin and GlcNAc as biospecific ligand.     

Affinity of mouse immunoglobulin G subclasses for sialic acid derivatives immobilized on dextran-coated supports

High-performance liquid affinity chromatography is a powerful method for the purification of biological compounds owing to its specificity, rapidity and high resolution. In our laboratory, we develop chromatographic supports based on porous silica beads. However, in order to minimize non-specific interactions between the inorganic surface and proteins in aqueous solution, the silica beads are coated with modified dextran. As previously reported, many affinity ligands can be covalently grafted onto dextran-coated silica. In this study, N-acetylneuramic acid, which belongs to the sialic acid family and is present in immunoglobulin G (IgG) epitopes, is used as an active ligand. The interactions of this affinity support and IgG subclasses are analyzed. This immobilized ligand enables purification of IgG3 antibodies.     

A method for the quantitation of tryptophan in Escherichia coli fermentation broth by isocratic high-performance liquid chromatography with ultraviolet detection.

A rapid, isocratic method for the determination of tryptophan in Escherichia coli fermentation broths by reversed-phase HPLC is described. Tryptophan can be measured in fermentations containing either chemically defined media or media with hydrolyzed protein supplements. The procedure was rugged and rereproducible (RSD = 1.7%). The sample response was found to be linear up to 10 mcg of tryptophan/ml. Two different columns--Vydac C18 30 mm and "deactivated" SupelcoSil LC-18-DB--were compared and evaluated for use in the analysis. The deactivated columns had the residual silanols on the silica gel chemically inactivated to reduce the interaction with basic groups or analytes. The deactivated column was found to provide better peak shape (peak assymetry factor less than 1.1) and superior efficiency (plate count greater than 40000/m) and durability (greater than 3000 injections per column) than the non-deactivated column. The procedure described was found to be more selective than a fluorometric procedure.     

Quartz fibers as templates for biopolymers.

The polymerization of silica in water solution to form quartz fibers proceeds by a dehydration process, analogous to condensation polymerization in organic high-polymers, in which monomeric Si(OH)4 groups unite through Si--O--Si bonds with the elimination of H2O. The resulting fibers are structurally polar along the direction of elongation, are enantiomorphous, and generally show stereospecific twisting around the direction of elongation. In these regards the fibers are analogues of biopolymers such as RNA and DNA. Quartz also possesses specific adsorptive relations to a wide range of organic substances including monomer amino acids, short-chain polypeptides, and proteins. These involve hydrogen-bonding between (OH) or silanol groups on the surface of the quartz with active side-groups on the organic molecules, and in part are epitaxial through dimensional coincidences in the interface. Geochemical evidence indicates that quartz was deposited in the early Precambrian ocean either by direct crystallization from seawater or by recrystallization of amorphous silica. What is of interest is the possible role of quartz fibers as a template and co-polymer in the passage of biomonomers in the pre-biotic ocean to the long-chain biopolymers such as nucleic acids and proteins that are involved in life processes.     

Insights into the chemical composition of <Emphasis Type="Italic">Equisetum hyemale</Emphasis> by high resolution Raman imaging

Equisetaceae has been of research interest for decades, as it is one of the oldest living plant families, and also due to its high accumulation of silica up to 25% dry wt. Aspects of silica deposition, its association with other biomolecules, as well as the chemical composition of the outer strengthening tissue still remain unclear. These questions were addressed by using high resolution (<1 μm) Confocal Raman microscopy. Two-dimensional spectral maps were acquired on cross sections of Equisetum hyemale and Raman images calculated by integrating over the intensity of characteristic spectral regions. This enabled direct visualization of differences in chemical composition and extraction of average spectra from defined regions for detailed analyses, including principal component analysis (PCA) and basis analysis (partial least square fit based on model spectra). Accumulation of silica was imaged in the knobs and in a thin layer below the cuticula. In the spectrum extracted from the knob region as main contributions, a broad band below 500 cm⁻¹ attributed to amorphous silica, and a band at 976 cm⁻¹ assigned to silanol groups, were found. From this, we concluded that these protrusions were almost pure amorphous, hydrated silica. No silanol group vibration was detected in the silicified epidermal layer below and association with pectin and hemicelluloses indicated. Pectin and hemicelluloses (glucomannan) were found in high levels in the epidermal layer and in a clearly distinguished outer part of the hypodermal sterome fibers. The inner part of the two-layered cells revealed as almost pure cellulose, oriented parallel along the fiber.     

Concentrations of Vacuolar Inorganic Ions in Individual Cells of Intact Wheat Leaf Epidermis

A method is described for quantitative determination of themajor inorganic constituents of individual cells of higher plants.The approach utilizes a modified pressure probe to extract samplesof undiluted vacuolar sap from single cells. Subsamples of constantvolume are taken from these sap samples and are freeze-driedon to thin films along with similarly-sized droplets of standards.The films are placed in a scanning electron microscope and elementalcontent of the freeze-dried material is determined by X-raymicroanalysis. The method has been used to compare levels ofa range of ions in two distinct types of epidermal cell froma young wheat leaf and, in association with nanolitre osmometry,was used to assess the relative importance of the inorganicions in the generation of turgor pressure in these cells. Itwas found that the concentrations of the major inorganic ionswithin the vacuole was constant both within and between twoanatomically distinct groups of epidermal cells on a leaf. Key words: Pressure probe, vacuolar ion levels, single cell sampling, wheat     

Enzymic determination of metabolites in the subcellular compartments of spinach protoplasts

A method for determining the subcellular metabolite levels in spinach protoplasts is described. The protoplasts are disrupted by centrifugation through a nylon net, releasing intact chloroplasts which pass through a layer of silicone oil into perchloric acid while the remaining cytoplasmic components remain over the oil and are simultaneously quenched as acid is centrifuged into them. Cross-contamination is measured and corrected for using ribulose 1,5-bisphosphate as a chloroplastic marker and phosphoenolpyruvate carboxylase as a cytoplasmic marker. A method for separation of intact protoplasts from the medium by silicone oil centrifugation is described, which allows a correction to be made for the effect of free chloroplasts and broken protoplasts. Methods for inhibiting chloroplast photosynthesis, without inhibiting protoplasts, are presented. It is demonstrated that ribulose 1,5-bisphosphate, ATP, ADP, AMP, inorganic phosphate, hexose phosphate, triose phosphate, fructose 1,6-bisphosphate, and 3-phosphoglycerate can be reliably recovered in the subcellular fractions isolated from protoplasts, and measured by enzymic substrate analysis.     

Variability in the properties of silicone gel breast implants.

Several generations of silicone gel breast implants have been produced by implant manufacturers. The primary material usually viewed as the base material in the manufacture of implants is polydimethylsiloxane. Polymeric reactions are notorious for their variability and nonuniformity. The elastomer used in different types of implants can have vastly different properties. Furthermore, the material properties associated with a particular type of implant can vary considerably from one lot to the next. Considering the various designs, styles, and manufacturing techniques associated with silicone gel implants, knowledge of the original properties of the implants before implantation is important in determining the effects of aging in vivo. This study was conducted to investigate differences in key mechanical and chemical properties of silicone gel breast implant materials. The two types of implants chosen for analysis were Silastic I and Silastic II control implants. Material property data were determined for both types of controls and significant differences were found in their values. Lot-to-lot variability was also investigated and found to be significant.     

Features of the mechanical treatment of rice husk for the performance of the solid-phase reaction of silicon dioxide with polyphenols

The distinguishing features of rice husk are high strength properties, chemical stability, high ash content, and low nutritional value, which are determined by the composition and structure of this type of raw material. The goal of the study was to determine optimal methods of mechanical treatment for the performance of a solid-phase reaction of silicon dioxide from rice husk with polyphenol compounds. Different regimes of the treatment of plant raw material have been compared. It has been shown that, for the solid-phase reaction of silicon dioxide from plant raw material with polyphenol compounds to occur, both the destruction of the supramolecular complex of silicon dioxide with the lignocellulose matrix, which is accomplished by fine grinding, and the plastic deformation of the silicon dioxide phase with the formation of reaction centers are necessary. These operations can be brought about using grinders with the attrition and shear modes of operation, including roller mills. It is preferable that silicon dioxide is in the composition of plant raw material since the reaction between silicon dioxide and polyphenols with the formation of surface complexes requires the presence of silanol groups. If silicon dioxide is derived from rice husk by conventional methods, most hydroxyl groups are eliminated, which significantly decreases the reactivity.