Dispersion-enhanced chromatography refolding of denatured protein

Protein folding following the expression of insoluble gene products in bacterial cell factories such as Escherichia coli is a significant technical challenge. Preparative refolding is often conducted chromatographically using automated equipment, allowing simple exchange of buffer conditions and integrated removal of contaminants. While an understanding of the role of mixing on dilution protein refolding has recently emerged, the role of dispersion in chromatographic refolding has not been reported. Here we report that protein refolding yield during chromatography is influenced strongly by column dispersion. Specifically, modelling studies predicted that a significant yield increase could be obtained by introducing a highly dispersive gap at the top of the refolding column (i.e. between the inlet distributor and the top of the packed chromatographic bed). Experimentally, the introduction of a 15 mm gap increased yield from 47% to 64% at . This counter-intuitive strategy of dispersion-enhanced chromatography refolding (DCR) is potentially generic for other proteins, and suggests the need for gel-filtration refolding columns and resins that achieve adequate buffer exchange yet maximise, rather than minimise, peak broadening.     

A comprehensive mathematical model for the Fischer-Tropsch synthesis in well-mixed slurry reactors

A new product distribution quasi-steady-state model was proposed for the Fischer-Tropsch (FT) synthesis in slurry reactors, being applicable to their transient simulation. It may consider two chain propagation mechanisms or sites and the possibility of 1-olefin readsorption with secondary reactions, recovering the Anderson-Schulz-Flory (ASF) model, the two superimposed ASF model and the 1-olefin readsorption model as particular cases. The hydrocarbon compounds were lumped according to the number of carbon atoms in their molecules for the paraffin and 1-olefin families. The phases were assumed to be well mixed in the reactor and transient mass balances were performed for each component, allowing simulation of operation in constant liquid level and no liquid withdrawal conditions. A rigorous calculation of the vapor-liquid equilibrium (VLE) through cubic equations of state was used to describe the phase behavior. Rate expressions for the FT and the water gas shift reactions are taken from the literature and expressed in terms of fugacities. Simulation results showed that the inclusion of both the olefin readsorption and the two chain propagation mechanisms may explain the anomalies present in experimental hydrocarbon product distribution. Moreover, the effect of phase-equilibrium modeling on product distribution simulation was shown to be slightly important in the conditions analyzed. Compositional lumping schemes for the hydrocarbons were investigated to speed up the simulations. Results showed that lumping can speed up computations up to 250 times with negligible loss of accuracy.     

Cure kinetics and shrinkage model for epoxy-amine systems

Manufacture of most of epoxy resins implies that cure needs to be carried out under pressure. Due to the significance of knowing the influence of the pressure factor in cure kinetics, cure shrinkage of a stoichiometric epoxy-amine system was measured using a pressure-volume-temperature (PVT) analyzer. Recording the specific volume change in the range of temperature from 100 to 180 °C and a pressure of 200 bar we could model the cure kinetics. The Runge-Kutta method was applied to obtain the kinetic constants of the cure reaction. In addition, using the differential scanning calorimeter (DSC) for measurements of 1 bar and the PVT analyzer for pressures of 200, 400, and 600 bar, we also model the kinetic constants as a function of pressure. The results obtained show that the effect of the temperature on the kinetic constants is higher than the effect of pressure. Therefore, both PVT and DSC are complementary techniques to describe the full range of cure kinetic process of epoxy mixtures.     

Flow effects on the kinetics of a second-order reaction

Flow effects on the kinetics of an isothermal, equimolar, second-order reaction taking place in a channel were investigated using a Lagrangian numerical method. The reactants were released instantaneously from the two opposite walls of the channel into fully developed turbulent or laminar flow. The overall conversion, the residence time and reactor length required to achieve 80% conversion, and the effective reaction rate coefficient were calculated. A correlation of the efficiency ratio, defined as the effective rate coefficient divided by the reaction rate constant, with the flow parameters was found.     

Macroscopic model for multispecies ion-exchange kinetics

A model for multispecies ion-exchange kinetics based on the Nernst-Planck equation is suggested. It is analyzed in comparison with the “locally-determinate” model described by Hwang and Helfferich [1]. The model makes possible simple computation. The conditions for the occurrence of unusual kinetic curves with a maximum are clarified. The proposed model is developed for different types of kinetic problems and verified by the experimental investigation of a kinetics in ternary systems.     

Simplified design of steady-state recycling chromatography under ideal and nonideal conditions

In a recent contribution a design method for steady-state recycling chromatography in the so-called mixed-recycle mode (MR-SSR) has been proposed by Sainio and Kaspereit (2009). The method holds under ideal, dispersion-free conditions for chromatographic systems with competitive Langmuir adsorption isotherms. Here this approach is extended to practically relevant nonideal conditions with significant dispersive effects and convex-upward adsorption isotherms in general. The developed shortcut design method allows designing MR-SSR processes based on a simple procedure applied to a single conventional chromatogram. The method is validated experimentally for the separation of two cycloketones on a polymeric stationary phase. The performance of the MR-SSR process is compared to batch chromatography by means of a simulation study.     

Analysis of the kinetics of agglomerate erosion in simple shear flows

A model for the erosion kinetics of particle agglomerates in simple shear flows has been developed. The erosion rate is taken to be proportional to the difference between hydrodynamic and cohesive forces and to the rotation velocity of the dispersing agglomerate. For dispersion under identical hydrodynamic conditions, the model predicts faster erosion for larger agglomerates. Moreover, at equivalent hydrodynamic stress, erosion is enhanced at higher shear rates. Dispersion experiments using silica agglomerates of various densities and liquid low molecular weight polymers (e.g., poly(dimethyl siloxane)) of different viscosities were conducted in an oscillatory shear flow device. The experimental results validate the erosion model; the general shape of the erosion kinetic curve, and the effect of viscosity, shear rate and agglomerate size on dispersion kinetics are well predicted. The model can also predict erosion for agglomerates with fractal structure.     

Enzyme kinetics of kinetic resolution of racemic ibuprofen ester using enzymatic membrane reactor

An ultrafiltration hollow fiber enzymatic membrane reactor was employed to study the kinetics of lipase-catalyzed kinetic resolution of racemic ibuprofen ester. Lipase from Candida rugosa was employed in the hydrolysis reaction both in free form in a batch system and in immobilized form in an enzymatic membrane reactor (EMR). The half life (t_1/2) of immobilized lipase on spongy layer was 105 h at reaction temperature of and 62 h at . This value was 94 h for lipase immobilized on the inner lumen and 45 h for free lipase in batch system at . Excessive substrate was found to inhibit the reaction as an uncompetitive inhibitor. The by-product 2-ethoxyethanol was found to be non-competitive inhibitor to the reaction when it was present in an excess. Michaelis constant (K_m) and maximum reaction rate (V_max) for immobilized lipase were and , respectively; and that for free lipase were and h^-1, respectively.     

Interaction of kinetics and internal diffusion in complex catalytic three-phase reactions: Activity and selectivity in citral hydrogenation

Kinetics and mass transfer effects were studied for the complex catalytic liquid-phase hydrogenation in a semi-batch reactor, where finely dispersed and larger catalyst particles were used. Citral was used as a model molecule. The intrinsic kinetics was determined with crushed catalyst particles at and at pressures ranging from 10 to 40 bar. A kinetic model was proposed and successfully fitted to the experimental data. In order to elucidate the influence of internal diffusion on the selectivity and activity in complex reactions, citral hydrogenation was performed with larger catalyst pellets, in a pressurized autoclave equipped with a catalyst basket. As expected, the activity decreased with increasing catalyst particle size. The product distribution was shifted from the primary hydrogenation product (citronellal) to the fully hydrogenated end product (3,7-dimethyloctanol) as the catalyst particle size was increased. The concentrations of the secondary hydrogenation products were minor throughout the experiment. A complete reaction-diffusion model was developed for large pellets and complex reactions systems.     

合成甲醇液相产物的气相色谱分析

介绍了合成甲醇液相产物的气相色谱分析方法。选择高分子固定相,以避免水的干扰;运用保留值和保留指数规律定性;分别在ＦＩＤ和ＴＣＤ上分析含氧化合物杂质与水的含量,定量快速、准确。     

A Kinetic Model of Degradation of Phenol in Water by Direct Contact of Gas Nonpulsed Corona Discharge

Reaction byproducts from degradation of aqueous phenol by contact of gas d.c. corona discharge with treated water were analyzed by liquid chromatography. According to the retention time, three byproducts, pyrocatechol, hydroquinone, 1,4‐benzoquinone, were identified. Also, acetic acid was detected as a final relatively stable byproduct by gas chromatography. A simple reaction model in which relevant elementary reactions are assumed to be first order was proposed to correlate the practical behavior of degradation processes.     

Diffusion-reaction modelling of DNA hybridization kinetics on biochips

An analytical expression for the rate of DNA hybridization on the surface of DNA biochips is established for the case of a finite 1-D diffusion space. The expression allows to account for the diffusion-limited supply of unreacted probe DNA and was obtained by solving the continuous 1-D reaction-diffusion mass balance using a Finite Fourier Transform technique. The extrapolation of the presently considered 1-D case to the full 3-D case is outlined as well. By bringing the obtained result into concurrence with the results of a stochastic random walk study, the kinetic constant k of the continuous reaction-diffusion model could be expressed as a function of the basic physico-chemical parameters (persistence length and jump frequency of the Brownian motion, collision frequency, binding probability) of the individual molecules. With the availability of an analytical expression describing the full time course of the 1-D hybridization process, and by using the Damköhler number Da=kh/D_mol, the different reaction- and diffusion-limited regimes occurring during the course of a hybridization process can now be described in general, dimensionless terms, allowing to establish some very simple rules for the design of DNA biochips and flow-through biosensors.     

层析介质使用寿命缩小模型的建立和确认

在现代生物制药工艺路线中,层析是最常用的一种技术手段。层析技术所需要的层析介质是药品监管的重点项目之一,其中层析介质的使用寿命(使用次数)必须经研究确认,并经药品监管部门审核和批准。在缩小模型上进行前瞻性研究是被广泛接受的层析介质使用寿命的研究方法。本文就层析介质使用寿命缩小模型的建立和确认作一简要综述。     

Simulation of protein adsorption in a batchwise affinity chromatography with a modified rate model

A rate model was adapted to simulate the dynamics of protein adsorption. This model takes axial dispersion and film mass transfer into account where there is a nonlinear adsorption isotherm for protein. The model equations were solved with the application of orthogonal collocation method on finite elements. The model is validated with experimental adsorption of urokinase in a batchwise column chromatographic process. Adsorption kinetics and isotherm were measured in a batchwise operation. With the assumption of back mixing at the column inlet, the effect of the different flow pattern on the concentration change inside the column can be simulated with the rate model.     

A cellular automata model for liquid distribution in trickle bed reactors

A cellular automata model for liquid distribution studies in trickle bed reactors is presented. It is a potential tool for describing non-uniform distribution of gas and liquid in a trickle bed. This non-uniformity may arise from a wide range of potential sources, such as improper distribution of the feed, random or radial porosity variation, wall effect, partial wetting of the catalyst, and gas-liquid surface tension related effects. Axial and radial dispersion of the liquid flow are inherently included in the model, since the fundamental model probability parameters are directly related to the dispersion coefficients. The present model is extremely fast due to simple single-event modeling, and it is well suited for parallelization. Three examples of the model performance are shown. In the first a liquid jet spreading from a point source is followed, and in the second, effect of radial porosity profile to wall flow is examined. The third example illustrates the potential of the model to predict pulsing flow regime.     

Transient operation of a catalytic liquid-liquid plug flow reactor for kinetics measurements

A centrifugal partition chromatograph (CPC) was used as a liquid-liquid catalytic reactor for the isomerisation of hexen-3-ol into ethylpropylketone with a water soluble rhodium catalyst. Global mass transfer coefficients were measured and shown to depend on both the nature of the solute and the flow rate. Liquid-liquid partition isotherms were also determined with the CPC using elution chromatography. Finally, a reactor model was derived to account for the experimental results obtained both under stationary and transient (pulse) conditions. A parameter sensitivity evaluation is also presented.     

A variable reaction order model for prediction of curing kinetics of thermosetting polymers

Based on the study of the curing reaction of an fluorinated aliphatic cyanate ester resin using an isothermal Differential Scanning Calorimetric (DSC) method, a new kinetics model with variable reaction order was proposed to describe the curing of thermosetting resins in both the chemical controlled and diffusion controlled regions. α-T_g relationship was used to calculate the diffusion controlled reaction rate constant during cure. Then the chemical and diffusion rate constants were combined in the Simon-Gillham equation to model the overall rate constant for the entire reaction. By using the overall rate constant combined with the variable reaction order, a simple model can be built up for prediction of curing kinetics of thermosetting polymer in both the chemical and the diffusion controlled regions. The new model is simple, easy to use and has very good agreement with the experimental results.     

Concept of nano-reactor for the control of the selectivity of the free radical grafting of maleic anhydride onto polypropylene in the melt

The aim of this work was to apply the concept of nano-reactors to the reactive extrusion process of the free radical grafting of maleic anhydride onto polypropylene (PP), an important process in the polymer industry. Pre-confinement of dicumyl peroxide in the lamellar structures of organically modified montmorillonite allowed slowing down the release of primary radicals and consequently to significantly improve the selectivity of the grafting of maleic anhydride onto PP and decrease the selectivity of PP chain scission. To the authors’ knowledge, this is the first time that a non-chemical method is found to solving the inherent problem facing this complicated process: increasing the grafting degree is always concomitant to increasing PP chain scission.     

吸附床在封闭气流循环中的除湿传质动力学研究

用硅胶和4A分子筛吸附剂进行气流封闭循环实验,对吸附床的除湿传质动力学进行了研究。由湿度随时间的微分规律拟合了吸附床的除湿传质系数与吸附剂表面平衡湿度。除湿传质系数主要受操作条件的影响而随吸附剂种类变化不大,表明吸附床的除湿过程主要受外扩散控制。在层流状态下,吸附床的除湿传质系数随雷诺数近似线性增大。     

Simulation of DME synthesis from coal syngas by kinetics model

DME (Dimethyl Ether) has emerged as a clean alternative fuel for diesel. There are largely two methods for DME synthesis. A direct method of DME synthesis has been recently developed that has a more compact process than the indirect method. However, the direct method of DME synthesis has not yet been optimized at the face of its performance: yield and production rate of DME. In this study it is developed a simulation model through a kinetics model of the ASPEN plus simulator, performed to detect operating characteristics of DME direct synthesis. An overall DME synthesis process is referenced by experimental data of 3 ton/day (TPD) coal gasification pilot plant located at IAE in Korea. Supplying condition of DME synthesis model is equivalently set to 80 N/m³ of syngas which is derived from a coal gasification plant. In the simulation it is assumed that the overall DME synthesis process proceeds with steadystate, vapor-solid reaction with DME catalyst. The physical properties of reactants are governed by Soave-Redlich-Kwong (SRK) EOS in this model. A reaction model of DME synthesis is considered that is applied with the LHHW (Langmuir-Hinshelwood Hougen Watson) equation as an adsorption-desorption model on the surface of the DME catalyst. After adjusting the kinetics of the DME synthesis reaction among reactants with experimental data, the kinetics of the governing reactions inner DME reactor are modified and coupled with the entire DME synthesis reaction. For validating simulation results of the DME synthesis model, the obtained simulation results are compared with experimental results: conversion ratio, DME yield and DME production rate. Then, a sensitivity analysis is performed by effects of operating variables such as pressure, temperature of the reactor, void fraction of catalyst and H₂/CO ratio of supplied syngas with modified model. According to simulation results, optimum operating conditions of DME reactor are obtained in the range of 265–275 °C and 60 kg/cm². And DME production rate has a maximum value in the range of 1–1.5 of H₂/CO ratio in the syngas composition.