Adsorption and desorption kinetics of bovine serum albumin in ion exchange and hydrophobic interaction chromatography on silica matrices

Large scale chromatographic separation of proteins can be carried out more rapidly on rigid adsorbents than on soft gel media. The kinetics of adsorption of bovine serum albumin (BSA) have been studied on rigid adsorbents based on a wide-pore, hydrophilically-coated silica gel matrix in a packed bed (chromatographic column). Process parameters have been varied comprehensively. The effects of surface chemistry (weak anion exchanger and hydrophobic interaction), particle size and liquid flow velocity have been studied on both the adsorption and desorption processes. The relative influences of the adsorption kinetics and equilibrium isotherm on the shape of the breakthrough curve are found to vary with the process parameters in an interpretable and therefore, predictable manner. Pore diffusion resistance is dominant over the external liquid film resistance in controlling the adsorption kinetics, with Biot numbers in the range 170-2600. A two-step model based on these two resistances simulates the breakthrough curves with only limited quantitative accuracy, but gives good predictions of the effect of changes in process parameters.     

Human palatal saliva: adsorption behaviour and the role of low-molecular weight proteins

In situ ellipsometry was employed to study adsorption from human palatal saliva (HPalS) in terms of dependence on surface wettability and saliva concentration (相似文献   

Human palatal saliva: Adsorption behaviour and the role of low-molecular weight proteins

In situ ellipsometry was employed to study adsorption from human palatal saliva (HPalS) in terms of dependence on surface wettability and saliva concentration ( ? 1%). Adsorbed amounts, kinetics, and elutability with buffer and sodium dodecyl sulphate (SDS) were determined. The low-molecular weight protein content of bulk HPalS was also investigated using two-dimensional gel electrophoresis, and this revealed the presence of a large group of proteins < 100 kDa in size. Adsorption to pure (hydrophilic) and methylated (hydrophobized) silica surfaces revealed that the total adsorbed amounts were greater on hydrophobized silica. Below concentrations of 0.5 and 0.25% saliva, adsorption was concentration dependent on hydrophobized and hydrophilic surfaces, respectively. The initial adsorption ( ? 30 min) was faster on hydrophobized surfaces. Addition of SDS removed more material than buffer rinsing on both surfaces. Analysis of the adsorption kinetics indicated that the presence of low-molecular weight proteins plays a role in adsorption from HPalS.     

Evaluation of liquid chromatography column retentivity using macromolecular probes. IV. Poly(ethylene glycol) bonded phase

Interaction properties of the novel HPLC silica gel-poly(ethylene glycol) (PEG) bonded phase were evaluated applying polymeric test substances, viz. polystyrenes, poly(methyl methacrylate)s, poly(ethylene oxide)s and poly(2-vinyl pyridine)s, and eluents of different polarities. Silanols on the silica gel surface are well shielded by the PEG phase, and silanophilic adsorption of macromolecules is suppressed in comparison with most silica C(18) bonded phases. The adsorption of solutes on the -OH groups of the PEG phase seems to be low as well. The partition of macromolecules in favor of the PEG phase is inferior to that observed in case of the silica C(18) phases. The volume of the PEG bonded phase is small and it is supposed that the PEG chains assume flat conformation on the silica gel surface.     

The effects of surface and macromolecular interactions on the kinetics of inactivation of trypsin and α-chymotrypsin

The autolysis of trypsin and α-chymotrypsin is accelerated in the presence of colloidal silica and glass surfaces. It is proposed that adsorption of the enzymes (favoured by electrostatic factors) results in a conformational change that renders the adsorbed enzyme more susceptible to proteolytic attack. Although the adsorbed enzymes are more susceptible to proteolysis, their activity towards low-molecular-weight substrates is not affected, indicating a relatively minor conformational change on adsorption. The rates of autolysis in solution (i.e. in `inert' vessels) are second-order for both trypsin and α -chymotrypsin, with rate constants of 13.0mol<sup>−1</sup>·dm<sup>3</sup>·s<sup>−1</sup> for trypsin (in 50mm-NaCl at pH8.0 at 25°C) and 10.2mol<sup>−1</sup>·dm<sup>3</sup>·s<sup>−1</sup> for α-chymotrypsin (in 0.1m-glycine at pH9.2 at 30°C). In glass vessels or in the presence of small areas of silica surface (as colloidal silica particles), the autolysis of both trypsin and α-chymotrypsin can show first-order kinetics. Under these conditions, saturation of the surface occurs and the fast surface proteolytic reaction controls the overall kinetic order. However, when greater areas of silica surface are present, saturation of the surface does not occur, and, since for a considerable portion of the adsorption isotherm the amount adsorbed is approximately proportional to the concentration in solution, second-order kinetics are again observed. A number of negatively charged macromolecules have been shown similarly to increase the rate of autolysis of trypsin: thus this effect, observed initially with glass and silica surfaces, is of more general occurrence when these enzymes adsorb on or interact with negatively charged surfaces and macromolecules. These observations explain the confusion in the literature with regard to the kinetics of autolysis of α-chymotrypsin, where first-order, second-order and intermediate kinetics have been reported. A further effect of glass surfaces and negatively charged macromolecules is to shift the pH–activity curve of trypsin to higher pH values, as a consequence of the effective decrease in pH in the `microenvironment' of the enzyme associated with the negatively charged surface or macromolecule.     

Study of catalytic site structure and diffusion of radicals in porous heterogenous systems with ESR,ENDOR and ESE

The generation of paramagnetic products by adsorption of quinones on activated catalysts has been used for the diagnostic of Lewis acid sites. It has been shown that the application of ENDOR, ESE, and 2 mm-band ESR are extremely effective methods for studying the nature of observed radical species and their environment. Two applications of the ESE method for studying the diffusion of spin probes in porous media are considered. The measurement of effective diffusion coefficients of radical probes in specimens of various heterogeneous systems is described. It has been found that effective diffusion coefficients depend strongly on the mean value of silica gel pore sizes and the mobility of the probe inside the pore.     

Adsorption kinetics and equilibria of bovine serum albumin on rigid ion-exchange and hydrophobic interaction chromatography matrices in a stirred cell

Rigid adsorbents have advantages over soft gel media for downstream processing of proteins. The adsorption of bovine serum albumin (BSA) has been investigated on a rigid adsorbent based on a wide-pore, hydrophilically coated, silica-gel matrix. The effects of surface chemistry (weak anion exchanger and hydrophobic interaction chromatography) and particle size have been studied on the physical properties of the adsorbent and on the adsorption equilibria and adsorption kinetics. The rates of adsorption of BSA have been measured in a stirred cell and are found to be satisfactorily described by a two-step theoretical model, in which the mass transfer involves a pore diffusion resistance and an extra-particle film resistance. On the anion exchanger, the effective pore diffusivity decreases substantially with increasing protein concentration, approximately halving as the initial concentration rises from 0.7 to 2g/l. In the hydrophobic interaction chromatography medium, the pore diffusivity is less sensitive to protein concentration and is also reduced by a factor of about 4 by aggregation of the protein. Effective pore diffusivities with the "wide-pore" silica adsorbents in anion-exchange form are 36-94 times lower than the diffusivity in free solution and are comparable with the lower of the wide range of values published for soft gels.     

Immobilized-type chiral packing materials for HPLC based on polysaccharide derivatives

The polysaccharide-based chiral packing materials (CPMs) for high-performance liquid chromatography (HPLC) have been recognized as the most powerful ones for the analyzing and preparative separating of the chiral compounds. These CPMs have been conventionally prepared by coating polysaccharide derivatives on a silica gel support. This means that the solvents, which swell or dissolve the derivatives on the silica gel and reduce the performance of the chiral columns, do not allow to be applied as components of the eluents. Therefore, the polysaccharide-based CPMs can be used with a rather limited number of eluents. In order to enhance the versatility of the eluent selection for more practical and economical chromatographic enantioseparations, the polysaccharide derivatives must be immobilized onto the silica gel. This review summarizes our latest studies on the development of the immobilized-type CPMs via the radical copolymerization and the polycondensation of the polysaccharide derivatives bearing small amounts of vinyl groups and alkoxysilyl groups, respectively.     

The effects of surface adsorption on the thermal stability of proteins

The effect of surface adsorption on the structure and stability of proteins is a matter of increasing interest in biotechnology. Therefore, we have examined the effect of adsorption to silica on the thermal stability of 7 proteins employing differential scanning calorimetry (DSC) and front surface fluorescence (FSF) spectroscopy. In general, it was found that surface adsorption decreased the thermal stability of the bound protein. Using lysozyme for further studies, DSC, FSF, and FTIR spectroscopies, as well as enzymatic activity measurements, were used to explore the effect of decreasing surface apolarity on stability. It was observed that increasing surface apolarity produced decreasing stability and increasing structural alteration of the adsorbed protein.     

Adsorption from saliva to silica and hydroxyapatite surfaces and elution of salivary films by SDS and delmopinol

The adsorption of proteins from human whole saliva (HWS) onto silica and hydroxyapatite surfaces (HA) was followed by quartz crystal microbalance with dissipation (QCM-D) and ellipsometry. The influence of different surface properties and adsorption media (water and PBS) on the adsorption from saliva was studied. The viscoelastic properties of the salivary films formed on the solid surfaces were estimated by the use of the Voigt-based viscoelastic film model. Furthermore, the efficiency of SDS and delmopinol to elute the adsorbed salivary film from the surfaces was investigated at different surfactant concentrations. A biphasic kinetic regime for the adsorption from saliva on the silica and HA surfaces was observed, indicating the formation of a rigidly coupled first layer corresponding to an initial adsorption of small proteins and a more loosely bound second layer. The results further showed a higher adsorption from HWS onto the HA surfaces compared to the silica surfaces in both adsorption media (PBS and water). The adsorption in PBS led to higher adsorbed amounts on both surfaces as compared to water. SDS was found to be more efficient in removing the salivary film from both surfaces than delmopinol. The salivary film was found to be less tightly bound onto the silica surfaces since more of the salivary film could be removed with both SDS and delmopinol compared to that from the HA surface. When adsorption took place from PBS the salivary layer formed at both surfaces seemed to have a similar structure, with a high energy dissipation implying that a softer salivary layer is built up in PBS as opposed to that in water. Furthermore, the salivary layers adsorbed from water solutions onto the HA were found to be softer than those on silica.     

Preparative protein purification on underivatized silica

This article describes the use of underivatized silica gel as a preparative stationary phase for process purification of proteins. Although silica has been frequently used as a stationary phase backbone matrix, direct adsorption of proteins on underivatized silica has not been widely exploited for industrial applications. In this study an effort was made to fundamentally understand the interaction mechanisms between a protein and silica surface by using several proteins with a wide range of isoelectric points (pIs) and surface hydrophobicity. Interactions in silica were found to be largely dominated by a combination of ionic and hydrophobic forces. Accordingly, a predictive model was derived for describing linear retention of proteins on silica. Finally, a case study is described investigating the role of silica in an industrial purification process. It was found that the integration of the two modes of interaction confers silica with a unique selectivity that can be very effectively utilized in downstream bioprocessing.     

Bonding of the iron Lewis acid-THF adduct CpFe(CO)2 · THF to silica gel and its implication for cyclopropanation reactions

The adsorption of the iron Lewis acid-THF adduct CpFe(CO)₂ · THF (1) onto the silica gel has been observed to dramatically alter the cis:trans ratio for cyclopropanation reactions versus the homogeneous catalyzed reactions. To better understand this dramatic change in selectivity, we investigated the nature of bonding of 1 on silica with a number of analytical techniques. X-ray photoelectron spectroscopy showed the presence of a new peak at 687.7 eV for the silica-supported catalyst, which indicated possible fluorination by the anion. Further experiments using solid state NMR showed that a new boron species was also generated by the adsorption onto the silica gel. Mössbauer spectroscopy showed that adsorption of the iron Lewis acid-THF adduct onto the silica gel did not change the oxidation state to the iron; however, diffuse reflectance infrared spectroscopy showed the loss of surface hydroxyl groups and a shift in one of the C-O absorptions to higher wave numbers. The combined data suggest fluorination of the silica surface by the anion. This theory was tested by adsorption of the iron Lewis acid-THF adduct onto a polytrimethyl hydrosilylsilicate resin and sodium perchlorate treated silica. Analysis showed that both fluorination and physical adsorption of the catalysts occur, although fluorination was found to predominate for binding.     

Photochemical synthesis of simple organic free radicals on simulated planetary surfaces—An ESR study

Electron spin resonance spectroscopy provided evidence for formation of hydroxyl radicals during ultraviolet photolysis (254 nm) at −170°C of H₂O adsorbed on silica gel or of silica gel alone. The carboxyl radical was observed when CO or CO₂ or a mixture of CO and CO₂ adsorbed on silica gel at −170°C was irradiated. The ESR signals of these radicals slowly disappeared when the irradiated samples were warmed to room temperature. However, re-irradiation of CO or CO₂, or the mixture CO and CO₂ on silica gel at room temperature then produced a new species, the carbon dioxide anion radical, which slowly decayed and which was identical with that produced by direct photolysis of formic acid adsorbed on silica gel. The primary photochemical process may involve formation of hydrogen and hydroxyl radicals by means of (1) photodissociation of H₂O physically adsorbed on the silica gel, or (2) absorption of the excitation energy by the silica gel surface with subsequent cleavage of the silanol bonds, or (3) dissociation of H₂O molecules through photosensitization by the surfaces or a combination of (1) to (3). Subsequent reactions of these radicals with adsorbed CO or CO₂ or both yield carboxyl radicals, CO₂H, the precursors of formic acid. Our results confirm the formation of formic acid under simulated. Martian conditions and provide a mechanistic basis for gauging the potential importance of gas-solid photochemistry for chemical evolution on other extraterrestrial bodies, on the primitive earth and on dust grains in the interstellar medium.     

Salivary protein adsorption onto hydroxyapatite and sds‐mediated elution studied by in situ ellipsometry

Whole unstimulated saliva from two donors was investigated both with respect to adsorption characteristics and SDS‐induced elutability. Salivary protein adsorption onto hydroxyapatite (HA) discs was studied by means of in situ ellipsometry in the concentration range 0.1–20% saliva. The adsorbed amounts on HA were found to be similar to those on silica, but the rates of adsorption were lower. Protein adsorption was virtually unaffected by the presence of Na⁺, whereas Ca²⁺ induced nucleation of calcium phosphate at the surface, the deposition rate being influenced by the pellicle age but not by the presence of saliva in bulk solution. The SDS elutability of adsorbed pellicles was determined on HA as well as on silica surfaces. Desorption from both surfaces was found to occur in the same SDS concentration range, although a residual layer was observed on HA. The slight net positive charge and lower charge density of HA as compared to the strongly negatively charged silica, may, at least partly, account for this observation by causing a reduction in the repulsive force between protein‐surfactant complexes and the surface. Inter‐individual differences, observed in the adsorption as well as elution experiments, are thought to relate to the compositional differences observed by SDS‐PAGE.     

Preparation of molecularly imprinted polymers for artemisinin based on the surfaces of silica gel

Artemisinin is an effective antimalarial drug isolated from the herbal medicine Artemisia annua L. Molecular imprinting is a technique of preparing molecularly imprinted polymers (MIPs) which can specifically recognize the imprinted template molecules. In this work, silica gel were used as supporting matrix, and vinyltriethoxysilane (VTES) was grafted onto its surface. The preparation of MIPs for artemisinin was performed on the surfaces of the modified silica gel using artemisinin as the template, acrylamide (AM) and methacrylic acid (MAA) as the functional monomers, ethylene glycol dimethacrylate (EGDMA) as the cross-linker and 2,2'-azo-bis-isobutyronitrile (AIBN) as the initiator. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and pore size analysis were used to characterize the prepared MIPs. The adsorption kinetic curve, adsorption isotherm and selective adsorption were measured by static method. The adsorption reached equilibrium at about 10 h, while fast adsorption took place during the first 2-3 h. The maximum adsorption capacity has been found to be 37.13 mg/g according to calculation with Langmuir-Freundlich isotherm. The electivity coefficients of MIPs for artemisinin with respect to artemether and arteether were 2.88 and 3.38, respectively. The results showed that the MIPs possessed good specific adsorption capacity and selectivity for artemisinin.     

Ionic Partition between Surface and Bulk Water in a Silica Gel: A Biological Model

Distribution of the biologically important ions between two aqueous phases of different structure has been used as a model for ionic distribution in living tissue. When other sources of specificity had been eliminated or corrected for, surface-oriented water in a silica gel was found to have increased solvent power for water-structure-breaking ions and decreased solvent power for water-structure-making ions; and the relative solubility of an ion in the phase of enhanced structure increased regularly with the water-structure-breaking powers of the ion. The ionic selectivity was decreased in the presence of urea. The selectivity of the gel water for potassium relative to sodium increased to a maximum when the gel surface was partially ionized so that distribution of cations was not linked to distribution of anions, and then decreased as the surface changed from a hydrogen bonding to an ionic surface. It is pointed out that the distribution of ions across most living cell membranes is qualitatively the same as that found in this silica gel, and it is suggested that the membrane separates two aqueous phases of different structure, and that the enhanced structure of cell water contributes to the observed ionic distributions.     

Preparation and characterization of silica gel/chitosan composite for the removal of Cu(II) and Pb(II)

Silica gel/chitosan composite (SiCS) was prepared via., sol-gel method by mixing silica gel and chitosan and cross-linked with bifunctional cross-linker glutaraldhyde. The SiCS composite was characterized using FT-IR, SEM-EDAX, XRD and BET methods. The sorption of copper and lead ions onto SiCS has been investigated. The SiCS composite was found to have excellent metal sorption capacity than the silica gel (Si) and chitosan (CS). The sorption experiments were carried out in batch mode to optimize various parameters viz., contact time, pH, initial metal ion concentration, co-ions and temperature that influence the sorption. Langmuir, Freundlich and Dubinin-Radushkevich adsorption isotherm models were applied to describe isotherm constants. Equilibrium data agreed well with the Freundlich isotherm model. Thermodynamic studies revealed that the nature of sorption is spontaneous and endothermic. The SiCS removes metals by means of adsorption and complexation. Sorption capacity of SiCS is compared with other sorbents which suggest that this composite was useful for removing copper and lead from aqueous solution.     

Kinetic studies of protein-surface interactions: A two-stage model of surface-induced protein transitions in adsorbed biofilms

The irreversible adsorption of proteins on artificial surfaces plays an important role in a wide variety of practical problems. The simple analytical models based on definite concepts regarding the mechanisms of interfacial evolution can be used efficiently for characterization of protein-surface interactions by analyzing the intrinsic kinetics of the process. In this article, analytical expressions are derived for the adsorption kinetics that take into account the presence of more than one adsorbed state for proteins in biofilms. It is shown that the experimentally observed dependence of the adsorbed mass on the concentration of protein in solution can be reproduced with this model, and the approach provides a rapid method for obtaining quantitative parameters for the adsorption process. It is shown by analytical approximation of the kinetic curves for fibrinogen adsorption onto an unmodified gold surface studied by a surface plasmon resonance biosensor that this model is in good quantitative agreement with experiments. It is found that the rate of adsorption, controlled mainly by the mass flow from the solution, determines the contribution both to self-assembling and spreading, resulting in variations of adsorbed fibrinogen interfacial structures.     

Adsorption of glycinin and β-conglycinin on silica and cellulose: surface interactions as a function of denaturation, pH, and electrolytes

Soybean proteins have found uses in different nonfood applications due to their interesting properties. We report on the kinetics and extent of adsorption on silica and cellulose surfaces of glycinin and β-conglycinin, the main proteins present in soy. Quartz crystal microgravimetry (QCM) experiments indicate that soy protein adsorption is strongly affected by changes in the physicochemical environment. The affinity of glycinin and the mass adsorbed on silica and cellulose increases (by ca. 13 and 89%, respectively) with solution ionic strength (as it increases from 0 to 100 mM NaCl) due to screening of electrostatic interactions. In contrast, β-conglycinin adsorbs on the same substrates to a lower extent and the addition of electrolyte reduces adsorption (by 25 and 57%, respectively). The addition of 10 mM 2-mercaptoethanol, a denaturing agent, reduces the adsorption of both proteins with a significant effect for glycinin. This observation is explained by the cleavage of disulfide bonds which allows unfolding of the molecules and promotes dissociation into subunits that favors more compact adsorbed layer structures. In addition, adsorption of glycinin onto cellulose decreases with lowering the pH from neutral to pH 3 due to dissociation of the macromolecules, resulting in flatter adsorbed layers. The respective adsorption isotherms fit a Langmuir model and QCM shifts in energy dissipation and frequency reveal multiple-step kinetic processes indicative of changes in adlayer structure.     

Surface denaturation and amyloid fibril formation of insulin at model lipid-water interfaces

We consider the effects that different lipid surfaces have upon the denaturation and subsequent formation of amyloid fibrils of bovine insulin. The adsorption and unfolding kinetics of insulin being adsorbed onto the different lipid surfaces under denaturing conditions are studied using FTIR ATR spectroscopy and are compared to the bulk solution behavior of the protein. Atomic force microscopy studies are also performed to compare the fibrils growing on the different surfaces. This study shows that both the adsorption and unfolding kinetics of insulin can be described by a sum of exponential processes and that different surfaces behave differently, with respect both to one another and to the bulk protein solution. The proteins adsorbed onto the surfaces are observed to have faster unfolding kinetics than those in the bulk, and the fibril-like structures formed at the surfaces are shown to be different in a number of ways from those found in bulk solution. The beta-sheet content and growth kinetics of the adsorbed proteins also differ from those of the bulk system. An attempt is made to describe the observed behavior in terms of simple physical arguments involving adsorption, unfolding, and aggregation of the proteins.