Breakthrough performance of linear-DNA on ion-exchange membrane columns

Breakthrough performance of linear-DNA adsorption on ion-exchange membrane columns was theoretically and experimentally investigated using batch and fixed-bed systems. System dispersion curves showed the absence of flow non-idealities in the experimental arrangement. Breakthrough curves were not significantly affected by flow-rate or inlet solution concentration. In the theoretical analysis a model was integrated by the serial coupling of the membrane transport model and the system dispersion model. A transport model that considers finite kinetic rate and column dispersed flow was used in the study. A simplex optimization routine coupled to the solution of the partial differential model equations was employed to estimate the maximum adsorption capacity constant, the equilibrium desorption constant and the forward interaction rate-constant, which are the parameters of the membrane transport model. Through this approach a good prediction of the adsorption phenomena is obtained for inlet concentrations and flow rates greater than 0.2 mg/ml and 0.16 ml/min.     

Breakthrough performance of stacks of dye-cellulosic fabric in affinity chromatography of lysozyme

The breakthrough performance of stacks of dye-cellulosic fabric in affinity chromatography of lysozyme was investigated in batch and flow experiments. Breakthrough curves were significantly affected by flow rate and were not dependent on the feed solution concentration. System dispersion curves could not explain the flow-rate dependence. Breakthrough curves were analyzed by coupling the kinetic model for pore mass transfer as the only controlling resistance and a system dispersion model. From the analysis, pore film mass transfer resistance was found to be the leading rate-limiting factor when the residence time in the column is greater than 5 min. The model was used to predict the operating and design parameters needed to obtain sharp breakthrough curves. Selectivity studies using lysozyme and bovine serum albumin mixtures showed a high system selectivity for lysozyme.     

Zonal rate model for stacked membrane chromatography part II: characterizing ion-exchange membrane chromatography under protein retention conditions

The Zonal Rate Model (ZRM) has previously been shown to accurately account for contributions to elution band broadening, including external flow nonidealities and radial concentration gradients, in ion-exchange membrane (IEXM) chromatography systems operated under nonbinding conditions. Here, we extend the ZRM to analyze and model the behavior of retained proteins by introducing terms for intra-column mass transfer resistances and intrinsic binding kinetics. Breakthrough curve (BTC) data from a scaled-down anion-exchange membrane chromatography module using ovalbumin as a model protein were collected at flow rates ranging from 1.5 to 20 mL min(-1). Through its careful accounting of transport nonidealities within and external to the membrane stack, the ZRM is shown to provide a useful framework for characterizing putative protein binding mechanisms and models, for predicting BTCs and complex elution behavior, including the common observation that the dynamic binding capacity can increase with linear velocity in IEXM systems, and for simulating and scaling separations using IEXM chromatography. Global fitting of model parameters is used to evaluate the performance of the Langmuir, bi-Langmuir, steric mass action (SMA), and spreading-type protein binding models in either correlating or fundamentally describing BTC data. When combined with the ZRM, the bi-Langmuir, and SMA models match the chromatography data, but require physically unrealistic regressed model parameters to do so. In contrast, for this system a spreading-type model is shown to accurately predict column performance while also providing a realistic fundamental explanation for observed trends, including an observed increase in dynamic binding capacity with flow rate.     

Biosorption of copper(II) ions onto powdered waste sludge in a completely mixed fed-batch reactor: estimation of design parameters

Biosorption of Cu(II) ions onto pre-treated powdered waste sludge (PWS) was investigated using a fed-batch operated completely mixed reactor. Fed-batch adsorption experiments were performed by varying the feed flow rate ( 0.075-0.325 l h(-1)), feed copper (II) ion concentrations (50-300 mg l(-1)) and the amount of adsorbent (1-6 g PWS) using fed-batch operation. Breakthrough curves describing the variations of effluent copper ion concentrations with time were determined for different operating conditions. Percent copper ion removals from the aqueous phase decreased, but the biosorbed (solid phase) copper ion concentrations increased with increasing the feed flow rate and Cu(II) concentration. A modified Bohart-Adams equation was used to determine the biosorption capacity of PWS and the rate constant for Cu(II) ion biosorption. Adsorption rate constant in fed-batch operation was an order of magnitude larger than those obtained in adsorption columns because of elimination of mass transfer limitations encountered in the column operations while the biosorption capacity of PWS was comparable with powdered activated (PAC) in column operations. Therefore, a completely mixed reactor operated in fed-batch mode was proven to be more advantageous as compared to adsorption columns due to better contact between the phases yielding faster adsorption rates.     

Scanning gel chromatography: determination of transport parameters from dynamic profiles measured at low solute concentration

Direct optical scanning of solute boundaries in large zone gel Chromatography experiments provides an accurate means of determining boundary profile shapes and rates of motion. A method has been developed for correcting such boundaries to a constant time frame, eliminating the distortion which arises from finite column scanning rate Centroids or the corrected profiles can be used to determine the partition cross section for the solute of interest The partition cross section and flow rate determine translational motion within the column. The axial dispersion coefficient, L, which characterizes rate of boundary spreading may also be calculated from the profiles. In order to explore these procedures a study of four noninteracting solutes was conducted. Partition cross sections determined from rates of motion of boundary centroids were found to be in good agreement with those determined by the equilibrium saturation method on the same column.In order to explore the lowest concentration limits of the technique and to illustrate the boundary characteristics for a selfassociating solute, a study of carboxyhemoglobin was conducted over a wide concentration range. From measurements at 220 nm the lowest concentration where useful data could be obtained was 2 micrograms per ml (0.12 πM heme) These results establish validity of the procedures used in analyzing the rates of boundary transport and in studying solute transport over a wide range of conditions.     

Miniaturised expanded-bed column with low dispersion suitable for fast flow-ELISA analyses

Flow-ELISA measurements of the monoclonal antibody concentration in cultivation broth containing murine hybriboma cells were carried out using a small expanded-bed column (0.5×2.5 cm) charged with protein A. A new specialised pellicular agarose/stainless steel matrix designed for high flow rates with fast mass transport properties was used. Special care was taken to get an efficient flow distribution. The axial dispersion coefficient was very low (2×10^–6 m² s^–1 for latex particles at a linear velocity of 10 cm min^–1). Breakthrough curves for polyclonal IgG on the protein A-derivatised support (at 2–11 cm min^–1) further emphasised its advantageous properties. No significant change in dynamic capacity was found over the entire speed range.     

Hydrodynamics of vertical subsurface flow constructed wetlands: Tracer tests with rhodamine WT and numerical modelling

Typical unsteady unsaturated conditions can profoundly affect the hydrodynamics of vertical subsurface flow constructed wetlands. In this study we analyzed the hydrodynamics of a 33 m² vertical flow pilot plant, treating municipal secondary effluents. Three different saturation conditions were analyzed under several constant flux regimes: complete saturation, partial saturation with the free water table 20 cm over the bottom of the bed, and complete drainage. Tracer tests were performed in steady state conditions by dosing rhodamine WT as square input signals. Breakthrough curves were analyzed by means of both a classical residence time distribution analysis and an originally developed numerical plug-flow model with longitudinal dispersion adapted to the unsaturated conditions. We found that the degree of global mixing in the vertical flow constructed wetland increased as the water content increased; this effect was controlled by the hydraulic residence time of the system. Conversely, the degree of local mixing was inversely affected by water content; the dispersivity was 4.5, 10, and 14 cm for fully saturated, partially saturated and draining conditions, respectively. We explain the dependency of dispersivity on water content in physical terms; however, further studies are needed to mathematically include this relationship in numerical models that describe the behaviour of vertical flow constructed wetlands.     

Intestinal absorption by carrier-mediated transports: two-dimensional laminar flow model

The two-dimensional laminar flow model was adapted to the intestinal absorption of drug and biological substances by carrier-mediated transports in the single perfusion experiments and we investigated the effects of the unstirred water layer on the Michaelis constant and the maximum transport velocity. According to the calculated values, the half saturation concentration at the inlet was larger than the true Michaelis constant at the intestinal wall. The apparent values of the Michaelis constant and the maximum transport velocity obtained by the Lineweaver-Burk plots were larger than the true ones, and the relations were not linear. These deviations increased as the ratio of the maximum transport velocity to the Michaelis constant increased and as the perfusion rate decreased. In the concurrent presence of a passive transport, underestimation of the carrier-mediated transport component of the absorption rate (at steady state) was predicted. It is considered to cause the underestimation of the maximum transport velocity. When water was absorbed (or secreted), the absorption rate increased (or decreased) and did not saturate. This two-dimensional laminar flow model would enable us to analyze the experimental data to determine the true values of the Michaelis constant and the maximum transport velocity.     

Dispersion effects in preparative polymethacrylate monoliths operated in radial-flow columns

Monolithic media have found widespread use as excellent tools for fast analytical separations of small molecules, proteins, pDNA and viruses. Polymethacrylate monoliths with large channels are attractive for capturing large molecules, like immunoglobulins, DNA, and viruses. For preparative purposes, these monoliths are operated in radial flow mode. Band spreading in monoliths is extremely low and mostly dominated by the contribution of extra column effects. The model used here had a single axial dispersion coefficient which lumps together extra column effects and the intrinsic band spreading of the monolithic material to characterize the adsorption of proteins and pDNA on polymethacrylate ion-exchange monoliths. Due to the fact that the performance of the monolith was unaffected by the velocity within the applied range, and due to highly favourable adsorption isotherms, a constant pattern model could be applied to predict preparative runs on radial flow units assuming axial flow for modelling.     

Dispersion effects in preparative polymethacrylate monoliths operated in radial-flow columns

Monolithic media have found widespread use as excellent tools for fast analytical separations of small molecules, proteins, pDNA and viruses. Polymethacrylate monoliths with large channels are attractive for capturing large molecules, like immunoglobulins, DNA, and viruses. For preparative purposes, these monoliths are operated in radial flow mode. Band spreading in monoliths is extremely low and mostly dominated by the contribution of extra column effects. The model used here had a single axial dispersion coefficient which lumps together extra column effects and the intrinsic band spreading of the monolithic material to characterize the adsorption of proteins and pDNA on polymethacrylate ion-exchange monoliths. Due to the fact that the performance of the monolith was unaffected by the velocity within the applied range, and due to highly favourable adsorption isotherms, a constant pattern model could be applied to predict preparative runs on radial flow units assuming axial flow for modelling.     

Theoretical investigation of axial and local particle size distribution on expanded bed adsorption process

The general rate model was developed and solved to describe protein adsorption in an expanded bed. The model takes into account axial variation of bed porosity, particle size distribution (PSD), external and intraparticle mass transfer, and dispersion in liquid and solid phase. The analysis of the influence of the model parameters on dynamic capacity (DC) was investigated. The simulation results showed that major impact on dynamic capacity is exerted by intraparticle mass transfer (particle diameter and pore diffusivity). The external mass transfer resistance and dispersion parameters have secondary effect on DC. The replacement of axial PSD by the mean particle diameter results in error in calculation of DC, which increases remarkably with the increase of mean particle diameter. The PSD can promote a very slow approaching of plateau concentration by breakthrough curves. It was shown also that axial bed porosity variation could be replaced by average porosity with negligible error for DC calculations.     

Highly pure 1,3‐propanediol: Separation and purification from crude glycerol‐based fermentation

A new separation and purification process was developed for recovering 1,3‐propanediol (1,3‐PD) from crude glycerol‐based fermentation broth with high purity. The downstream process integrated chitosan flocculation, activated carbon decolorization, fixed bed cation exchange resin adsorption, and vacuum distillation. Breakthrough curves were measured considering the effect of sample concentration, flow rate, temperature, and resin stack height. Yoon–Nelson model was proposed to fit the fixed bed adsorption. The characteristic column parameters were calculated. Optimal condition for adsorption was 1,3‐PD, 30.0 g/L; flow rate, 1.00 mL/min; stacking height, 30.0 cm; and temperature, 298 K. Ethanol‐water (75%, 1 mL/min) was used as eluent to separate 1,3‐PD and glycerol with 95.3% 1,3‐PD elution rate. After vacuum distillation, the overall purity and yield of 1,3‐PD were 99.2% and 80.8% in the purification process, respectively. This is a simple and efficient downstream strategy for 1,3‐PD purification.     

Biosorption Processes for Natural and Waste Water Treatment – Part II: Experimental Studies and Theoretical Model of a Biosorption Fixed Bed

A model has been developed for fixed‐bed biosorption performance, i.e. combined action of adsorption of organic water contaminants and their biological destruction in a column. The model contains an adsorption isotherm of the Freundlich type, adsorption kinetics by an overall film mass transfer (Glueckauf equation), maximum bacterial growth,and biological aerobic destruction (Monod model) of the organics by exoenzymes. Bacteria can not penetrate into the pores of the adsorbent. The model was tested using the system aqueous solution of aniline/Pseudomonas putida/Polysorb 40/100. Breakthrough curves in shorter columns have been measured and a velocity‐dependent steady‐state exit concentration was achieved. These curves could be simulated with sufficient accuracy on the basis of isotherm data, mass transfer coefficients and values of biological growth and destruction activity estimated from independent measurements.     

Protein A immunoaffinity hollow fiber membranes for immunoglobulin G purification: experimental characterization

Immunoaffinity adsorption is increasingly used for protein purification and medical applications. Synthetic membranes have advantages as support matrices in comparison to conventional bead supports because they are not compressible and they eliminate internal diffusion limitations. The goal of this study was to explore in detail the performance of microporous hollow fibers composed of modified polysulfone to which protein A was immobilized for adsorption of human IgG. The internal matrix was characterized by scanning electron microscopy. The binding equilibrium constant was measured using both static and dynamic methods. Break-through curves up to ligand saturation were measured and used to study the effects of IgG concentration, presence of contaminant albumin, flow direction, flow mode, and especially filtrate flow rate and maximum IgG binding capacity. The highest binding capacities studied were comparable with that attainable with bead matrices. All of the breakthrough curves could be represented on a single figure when plotted versus the dimensionless relative throughput (the mass of IgG loaded on the membrane divided by the mass that would be bound when the entire fiber is in equilibrium with the feed concentration), and the effect of operating variables on the position and shape of the individual breakthrough curves could be understood in terms of a dimensional performance parameter (the product of membrane volume and maximum binding capacity divided by the filtrate flow rate). The best breakthrough curves were obtained with the highest values of the performance parameter. Based on the results, membranes as solid supports for immunoadsorption can be a useful alternative to the use of traditional columns for protein separations. (c) 1995 John Wiley & Sons, Inc.     

Study of multicompound sorption of Lead and Pyrene on a reconstituted soil: batch and fixed bed column tests

Many polluted sites are simultaneously contaminated with polycyclic aromatic hydrocarbons and heavy metals. In the present study, batch and continuous column experiments were performed utilizing self-composition soil to describe the sorption behavior of two contaminants: lead (Pb²⁺) and pyrene (PYR). Operational conditions such as contact time, bed depth, and flow rate were optimized. The effect of soil organic matter content on the process of adsorption of both contaminants was investigated. The presence of PYR in solution at neutral pH (6.0–7.5) decreased Pb²⁺ sorption. Similar behavior was observed for PYR in the presence of Pb²⁺ in solution. At room temperature, batch experimental data conducted as a function of contact time were analyzed using the Langmuir and Freundlich isotherms. Results revealed that Pb²⁺ sorption isotherms were fitted better by the Langmuir model and PYR sorption isotherms were fitted better by the Freundlich model. Column adsorption experiments were carried out at room temperature and under operating parameters (bed depth, flow rate, and initial contaminant concentration). Breakthrough curves were well fitted to the two-site first-order kinetic model with a sum of square errors less than 0.14. The Pb²⁺ adsorption kinetic data were processed also for the Thomas model with a good accuracy.     

Modeling, simulation, and experimental validation for continuous Cr(VI) removal from aqueous solutions using sawdust as an adsorbent

Continuous adsorption experiments were performed in a fixed-bed adsorption column to evaluate the performance of low-cost adsorbent (sawdust) developed for the removal of Cr(VI) from aqueous solutions. The effects of influencing parameters such as flow rate, mass of adsorbent, initial Cr(VI) concentration were studied and the corresponding breakthrough curves were obtained. The fixed-bed adsorption process parameters such as breakthrough time, total percentage removal of Cr(VI), adsorption exhaustion rate and fraction of unused bed-length were obtained. A mathematical model for fixed-bed adsorption column was proposed by incorporating the effect of velocity variation along the bed-length in the existing model. Pore and solid diffusion models were used to describe the intra-particle mechanism for Cr(VI) adsorption. The proposed mathematical model was validated with the literature data and the experimental data obtained in the present study and the model was found to be good for explaining the behavior of breakthrough curves.     

Development of operating conditions for protein purification using expanded bed techniques: the effect of the degree of bed expansion on adsorption performance

A strategy for the optimization of an expanded bed adsorption process has been developed by studying a model system involving the adsorption of lysozyme onto the adsorbent STREAMLINE SP. The hydrodynamic and adsorption properties of this ion exchange adsorbent in a variety of viscosities of feedstocks have been compared by analyzing bed expansion characteristics, liquid phase dispersion characteristics, equilibrium adsorption isotherms, and mass transfer characteristics. Additionally, the influences of the degree of bed expansion on adsorption performance have been investigated by frontal analysis. In these experiments, viscous feedstocks were simulated by the inclusion of glycerol in the adsorption buffers. Breakthrough curves for lysozyme were characterized and compared in terms of overall purification processing time and productivity. On the basis of these results, the relative productivities of different operating modes with the same process liquid were found to be almost the same. However, the processing time for each purification cycle decreased with increasing velocity of process liquid. It is demonstrated that an adsorption process carried out at a constant degree of bed expansion (twice its settled bed height, corresponding to bed voidage of 0.7) is more efficient, when characterized by the apparent dynamic binding capacity, than operation at a constant liquid velocity of 300 cm/h. These results have significant implications on the design and operation of the expanded bed adsorption procedures. The advantages and problems encountered in the use of expanded bed techniques for the direct extraction of proteins from unclarified feedstocks are also discussed. (c) 1996 John Wiley & Sons, Inc.     

Purification of immunoglobulins G by protein A/G affinity membrane chromatography

An affinity membrane grafted with protein A/G or protein A was characterized for human and mouse immunoglobulins G purification. Breakthrough curves up to ligand saturation were measured and used to study the effects of flow velocities, feed solution concentrations and protein A/G versus protein A membranes. Increased flow-rate did not decrease the amount of IgG bound to the membranes. Increased feed solution concentration allowed more IgG to bind prior to breakthrough. Kinetic parameters for immunoglobulins G sorption to immobilized protein A were measured in batch experiments. The static binding capacity was determined to be 6.6 mg ml⁻¹ membrane volume. Finally, this affinity membrane was used to purify IgG from cell culture supernatant. The electrophoresis of the purified IgG fractions did not show any contaminant.     

"Bridge" structures between nucleic acid molecules fixed in the structure of a liquid crystal

The binding of daunomycin and copper ions to poly(I).poly(C) molecules fixed in a particle of a liquid-crystalline dispersion was studied. A thermodynamic model of adsorption was developed, which makes it possible to describe the formation of complexes of a particular kind, "bridges" that connect adjacent nucleic acid molecules fixed in a liquid crystal. The bridges represent chelate complexes, which incorporate the molecules of the antibiotic daunomycin and copper ions. Equations describing the dependence of the concentration of these bridges in solution on the concentration of their constituents were derived. The family of dependences of experimental amplitudes of bands in CD spectra typical of "bridge" structures on the concentration of copper ions represents a set of S-shaped curves, and, as the concentration of daunomycin in solution increases, the level of saturation of these curves increases. The analysis of experimental data with the use of this model suggests that the structures of this type compete with daunomycin molecules for the binding sites on poly(I).poly(C). By using this model, the energies of formation of bridge structures were calculated.