低场核磁法测定凝胶过滤介质的孔径分布

凝胶过滤介质在蛋白质、多糖等生物大分子的分离纯化过程中具有非常重要的作用,其中介质的孔径分布是决定分离纯化效果的关键因素。由于绝大部分凝胶过滤介质是软凝胶,因此很难用常规的方法如压汞法、低温氮吸附法等测定其孔径分布。本文探索了利用低场核磁共振测定凝胶过滤介质的孔径分布的方法。首先通过抽滤、自制琼脂糖凝胶块等实验确定了峰的归属,明确了介质孔内水、介质间隙水和自由水在核磁共振横向弛豫时间(T₂)图谱上的分布范围;随后与逆体积排阻层析法(ISEC)测定的结果相对比,得出层析介质孔径和介质孔内水弛豫时间的关系;最后通过高斯正态拟合得到了介质的孔径分布。实验结果证实了低场核磁共振法测定凝胶过滤介质孔径分布的可行性。该法操作简单、测定迅速,并可以为其他层析介质孔径分布的测定提供参考。     

逆体积排阻色谱法在评价色谱介质孔径结构方面的应用

介绍了影响层析介质分离纯化效果的结构参数。总结了测定色谱介质孔径结构常用的方法,如压汞法、氮气吸附法、小角X射线散射法、电镜观察法(SEM和TEM)和逆体积排阻色谱法(ISEC)。介绍了ISEC的基本原理及其涉及到的探针类型。对ISEC相关理论,尤其是溶质半径、色谱理论和体积排阻色谱法(SEC)随机理论进行了重点阐述。比较了ISEC的优缺点。介绍了ISEC在评价介质孔径结构中的应用,对ISEC的应用前景进行了展望。     

ISEC法在评价色谱介质孔径结构方面的应用

层析是生物大分子分离纯化的一种重要手段,层析介质作为层析过程的关键的一部分受到了广泛的关注。层析介质的孔径结构对其分离纯化的效果有着显著的影响,因此探究层析介质的孔径结构至关重要。介绍了测定色谱介质孔径结构常用的方法,如：压汞法、氮气吸附法、小角X射线散射法、电镜扫描法（SEM和TEM）和逆体积排阻色谱法(ISEC)。重点阐述了ISEC法的基本原理和常用的探针类型,总结了ISEC法应用中的优点及不足,介绍了ISEC法的发展历史及其在国内外的研究现状。     

体积排阻色谱法测定介孔材料的孔结构

分别合成了球形的苯基桥键和乙烷桥键两种新型杂化介孔材料.选用已知平均相对分子质量且具有窄相对分子质量分布的葡聚糖为探针溶质,采用动态光散射法测定了粒径,与经验公式计算结果接近.通过体积排阻法对两种材料的孔隙率和孔径分布进行测定并与氮气吸附法结果进行比较,实现介孔材料孔结构快速测定.结果表明,体积排阻色谱法测定苯基桥键和...     

超大孔聚苯乙烯微球固定蛋白质

采用琼脂糖对孔径为300~500 nm的超大孔聚苯乙烯(MPS)微球进行亲水化修饰,并在修饰后的表面上偶联病毒蛋白颗粒(RV),用于蛋白质的分离纯化实验. 结果表明,经过修饰的MPS层析介质具有优异的流体力学性能,层析过程的最高流速可达1120 cm/h,对病毒蛋白颗粒的固定量为0.638 mg/g,是商品化介质Sepharose 4FF的2倍. 采用RV-MPS介质对病毒蛋白颗粒抗体进行了分离纯化,收率为29.22%,高于Sepharose 4FF的18.92%.     

鸡白痢沙门氏菌兔抗血清多抗IgG的纯化与稳定

建立了离子交换层析介质一步纯化鸡白痢沙门氏菌兔抗血清获得多抗免疫球蛋白G (IgG)的方法，利用IgG与杂蛋白等电点的差异，指导阴阳离子交换层析介质筛选和操作条件优化；针对多抗IgG易失活的难题，采用差示扫描荧光法为纯化后的IgG筛选稳定剂。结果表明，通过毛细管等电聚焦测得兔抗血清多抗IgG的等电点为6.04?7.08，阳离子交换介质CM Sepharose Fast Flow层析纯化的IgG最高电泳纯度为63.5%，高效液相尺寸排阻色谱测得IgG回收率为15.5%；阴离子交换层析纯化效果更佳，pH 5.5条件下Q Sepharose XL(Q-XL)介质层析获得的IgG最高纯度达99.3%，回收率达67.5%；200 g/L山梨醇对IgG具有最佳稳定作用，IgG的2个热变性温度分别提高了5.52和8.84℃，70℃时山梨醇的稳定作用更明显；以200 g/L山梨醇为稳定剂、用阴离子交换层析介质Q Sepharose XL一步纯化所得IgG具有高纯度、高收率及高稳定性，且制备工艺简单。     

变孔径直管液体分布器的研究与设计

本文研究了均匀开孔与非均匀开孔直管液体分布器的流量分配。结果表明,改变孔径可有效地提高流量分配的均匀性。根据修正动量方程和实验数据计算并关联了动量摩擦修正系数。对小孔阻力系数进行了测定和关联。提出了变孔径直管液体分布器的设计方法,并给出设计示例。     

应用凝胶过滤筛选纯化甲型肝炎病毒介质

目的　筛选适合甲肝病毒疫苗大规模纯化凝胶过滤层析的介质。方法　采用柱层析法 ,分别以Sepharose 4FF ,Sepharose 6FF及SephacrylS 5 0 0HR三种不同的凝胶过滤介质对经过阴离子交换层析纯化的甲肝病毒样品进行纯化。结果　三种凝胶介质分离甲肝病毒的图谱不相同 ,其中以Sepharose 4FF纯化甲肝病毒 ,抗原回收率达 6 9% ,纯化倍数为 4 4倍。结论　Sepharose 4FF凝胶过滤层析可以用于甲肝灭活疫苗的大规模纯化。     

变孔径直管液体分布器的研究与设计

本文研究了均匀开孔与非均匀开孔直管液体分布器的流量分配。结果表明，改变孔径可有效地提高流量分配的均匀性。根据修正动量方程和实验数据计算并关联了动量摩擦修正系数。对小孔阻力系数进行了测定和关联。提出了变孔径直管液体分布器的设计方法，并给出设计示例。     

镁橄榄石隔热材料孔体积分形维数特征

采用压汞法研究在熔盐介质中制备的镁橄榄石多孔隔热材料中孔的分形结构,测定了多孔隔热材料中孔的分形维数,探讨了孔的分形维数与孔隙率、孔分布及材料强度之间的关系。结果表明:熔盐介质中制备的镁橄榄石多孔隔热材料的孔结构具有分形特征,孔径4.6μm的样品中孔的分形维数不具有准确性,孔径4.6μm的样品中孔的分形维数具有规律性。烧成温度不同的样品中孔径4.6μm的分形维数波动在2.563 0~2.766 1之间,且分形维数越大,显气孔率越小,耐压强度越小。不同熔盐含量样品孔径4.6μm的孔体积分形维数在2.534 1~2.972 2之间,且分形维数越小,显气孔率越大。当分形维数大于2.535 7时,分形维数越小,耐压强度越大。     

Pore size distribution from challenge coreflood testing by colloidal flow

The transport of colloidal and suspension particles and the resultant particle retention occur in a wide range of porous media. The micro scale pore throat size distribution is an important characteristic of porous media, allowing for evaluation of important transport properties. An effective method based on micro scale modelling for the determination of overall pore throat size distribution (PSD) by injection of colloidal particle suspensions into engineered porous media with monitored inlet and breakthrough particle concentrations is developed. The treatment of inlet and outlet colloidal particle concentrations obtained in coreflooding results in a good agreement between the modelling and experimental data. Yet, some deviation was observed between the obtained PSD and that calculated by the Monte Carlo simulation based on the Descartes’ theorem.     

Synthesis,gas adsorption and reliable pore size estimation of zeolitic imidazolate framework-7 using CO2 and water adsorption

Reliable estimation of the pore size distribution(PSD) in porous materials such as metal–organic frameworks(MOFs) and zeolitic imidazolate frameworks(ZIFs) is crucial for accurately assessing adsorption capacity and corresponding selectivity. In this study, the so-called zeolitic imidazolate framework-7(ZIF-7) is successfully synthesized via relatively fast and convenient microwave technique. The morphology and structure of the obtained MOF were characterized by XRD, SEM and N_2 and CO_2adsorption/desorption isotherms at 77 K and0 °C respectively. Then, to determine the PSD of the fabricated MOF, carbon dioxide isotherms are experimentally measured at various temperatures up to atmospheric pressure. Afterward, the experimental CO_2 isotherms data are utilized in two recently proposed in-house algorithms of SHN1 and SHN2 to extract the true PSD of manufactured ZIF-7. The obtained results revealed that median pore diameter of the fabricated ZIF-7 is estimated around 0.404 nm and 0.370 nm by using CO_2 isotherms at 273 K and 298 K respectively. These values are in good agreement with the real pore diameter of 0.42 nm. Moreover, experimental data of water adsorption isotherms over four different MOFs, borrowed from literature, are employed to illustrate further effectiveness of the above algorithms on successful determination of the corresponding pore size distributions. All predicted PSDs are proved to be in good agreement with those obtained from independent methods such as topology and morphology studies.     

Hydrodynamic dispersion in perfusion chromatography—a network model analysis

A network model was used to simulate the hydrodynamic dispersion, which occurs in perfusion chromatography for proteins under non-retained conditions. In order to elucidate the details of the velocity dependency of the hydrodynamic dispersion coefficient in a perfusive bed, the effects of pore-size distribution (PSD), pore inter-connectivity (PI), and solute molecular size were incorporated into the simulations. It was found that, for purification of proteins on a perfusive column, among these parameters, PI affected the dispersion results most significantly over the whole range of Peclet numbers investigated. POROS media were postulated to possibly have a very high PI, of around 4.77-5.37. PSD also played a very important role in determining the dispersion results; POROS R1 and POROS R2 behaved quite differently, although both of them possess bi-modal PSDs. At higher PI (connected-pore fraction higher than 0.8), the D_L/UL curve for POROS R1 showed double plateaus, whereas that for POROS R2 gave only one plateau. Under the experimental conditions investigated, only the macropore size distribution contributed to the dispersion results in POROS R1, whereas the composite bi-modal PSD contributed to the results in POROS R2. Based on the simulation results, size exclusion was also found to affect the dispersion results, giving rise to low dispersion coefficient in the molecular diffusion controlled region and high dispersion coefficient in the convection region. However, in the case of a perfusive column, size exclusion effects were rather insignificant. It was also found that hindered diffusion resulted in sharp increase in the dispersion data at very high Peclet number (Pe>10⁴), which was more significant for POROS R2. The simulation results corroborated the experimental data very well. These indicate that the use of a network model to obtain the hydrodynamic dispersion coefficient is a more realistic representation of the column efficiency in perfusion chromatography, one which accounts directly for microscopic information, such as pore structure and solute molecular size.     

基于逾渗理论的多孔过滤介质孔径分布估计方法

结合逾渗理论和网格模型,建立了过滤系数与过滤介质孔径分布的联系方程。利用此方程结合过滤实验数据获得过滤介质孔径分布的统计参数,同时与其他方法获得孔径分布参数以及其他文献中的数据进行比较,并利用计算机模拟过滤过程验证了获得的孔径分布参数,结果表明基于逾渗理论的过滤模型用于孔径分布参数的估计是比较准确和有效的。     

DEM Study on the effect of particle-size distribution on jamming in a 3D conical hopper

Despite the advancement on the understanding on the unwelcome phenomenon of hopper jamming, the influence of the particle-size distribution (PSD) width of the ubiquitous continuous PSDs remains unknown. Accordingly, this study investigated using discrete element method a range of PSD widths (10%≤σ/μ≤50% ) of lognormal PSDs with a constant mean particle diameter of 10 mm in a three-dimensional hopper. Results indicate that, although the monodisperse particles of 10 mm diameters do not jam, the wider PSDs (σ/μ≥ 30%) jam. The flatter arches and higher mass-averaged force that are linked to the denser packing of wider PSDs underlie the propensity to jam. To reduce the jamming probability, the friction coefficient and initial fill height can be reduced, but not changes in particle density. The results here highlight that the mean particle diameter alone is an insufficient parameter to predict jamming and the impact of PSD width is non-negligible. © 2018 American Institute of Chemical Engineers AIChE J, 65: 512–519, 2019     

Kinetic Correlation for the Hindered Diffusion of Proteins in a Porous Packing Column*

Abstract Hindered diffusion of proteins in a porous packing plays an important role in proteinchromatographic purification.The HETP method was adopted to analyze the influence of axialdispersion, film mass transfer and hindered diffosion in the porous packing employing a size-exclusionchromatography(SEC)process.The retention behavior with eight proteins of different relativemolecular mass was experimentally detected with a commercial SEC column.A correlation basedon the relative molecular mass of the proteins and the packing porosity was developed and used topredict the effective diffusion coefficient of a protein in the Porous packing.The predicted valuesof effective diffusion coefficient were very consistent with the experimental results with the averageerror of 8.6%.     

Synthesis and properties of uniform beads based on macroporous copolymer glycidyl methacrylate–ethylene dimethacrylate: A way to improve separation media for HPLC

Monodisperse beads based on hydrolyzed macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) for use as size-exclusion HPLC packings were synthesized by the method of “activated” swelling of polystyrene seeds followed by a suspension polymerization of both methacrylates. Effects of the type and fraction of the swelling agent, inert porogenic solvent, and cross-linking monomer on the uniformity of the particles, extent of the specific surface area, pore volume, pore size, and pore-size distribution and chromatographic properties (size-exclusion limit and column effciency) have been investigated. Trends leading to the synthesis of macroporous particles with predesigned properties for use in size-exclusion high-performance liquid chromatography in both aqueous and organic mobile phase were studied. © 1992 John Wiley & Sons, Inc.     

Determination of non-spherical particle size distribution from chord length measurements. Part 2: Experimental validation

In this paper, the theory on the translation of a measured chord length distribution (CLD) into its particle size distribution (PSD), which was developed in the first part of this study [Li and Wilkinson, 2005. Determination of non-spherical particle size distribution from chord length measurements. Part 1: theoretical analysis. Chemical Engineering Science 60, 3251-3265], has been validated using experimental results. CLDs were measured using the Lasentec focused beam reflectance measurement (FBRM) with three different materials, spherical ceramic beads and non-spherical plasma aluminium and zinc dust particles. Meanwhile, the particle shape and PSD of each material were also investigated by image analysis (IA). Comparison of the retrieved PSDs with the measured PSDs by IA shows that the PSD can be retrieved from a measured CLD successfully using the proposed iterative nonnegative least squares (NNLS) method based on the PSD-CLD model.