Preparation and characterization of a thermoresponsive gigaporous medium for high-speed protein chromatography

A high-speed thermoresponsive medium was developed by grafting poly(N-isopropylacrylamide-co-butyl methacrylate) (P(NIPAM-co-BMA)) brushes onto gigaporous polystyrene (PS) microspheres via surface-initiated atom transfer radical polymerization (ATRP) technique, which has strong mechanical strength, good chemical stability and high mass transfer rate for biomacromolecules. The gigaporous structure, surface chemical composition, static protein adsorption, and thermoresponsive chromatographic properties of prepared medium (PS–P(NIPAM-co-BMA)) were characterized in detail. Results showed that the PS microspheres were successfully grafted with P(NIPAM-co-BMA) brushes and that the gigaporous structure was robustly maintained. After grafting, the nonspecific adsorption of proteins on PS microspheres was greatly reduced. A column packed with PS–P(NIPAM-co-BMA) exhibited low backpressure and significant thermo-responsibility. By simply changing the column temperature, it was able to separate three model proteins at the mobile phase velocity up to 2167 cm h⁻¹. In conclusion, the thermoresponsive polymer brushes grafted gigaporous PS microspheres prepared by ATRP are very promising in ‘green’ high-speed preparative protein chromatography.     

Functional gigaporous polystyrene microspheres facilitating separation of poly(ethylene glycol)–protein conjugate

A novel sulfopropyl gigaporous polystyrene (SP-GP) microsphere enhancing the separation of poly(ethylene glycol)–protein (PEGylated protein) was first presented. The SP-GP microspheres were successfully prepared by introducing sulfopropyl groups into agarose-coated gigaporous polystyrene microspheres and used as chromatography media. Compared with a commercial medium, SP-GP microspheres exhibited improved column efficiency and reduced backpressure with increasing flow velocity, which could ensure its use in high-speed chromatography. Furthermore, a higher protein recovery and purity of the PEGylated protein could be obtained, even when SP-GP was applied at a flow velocity of 1224 cm h⁻¹. Additionally, the dynamic binding capacity (DBC) of SP-GP was significantly improved, which was higher than 10 mg mL⁻¹ medium even at a flow velocity of 306 cm h⁻¹. Further investigation using a laser scanning confocal microscope (LSCM) demonstrated that the static adsorption equilibrium of the PEGylated protein on SP-GP could be completed in 5 min, whereas a much longer period (ca. 60 min) was required for the commercial medium, indicating that the mass transfer of SP-GP was much faster with the gigaporous structure. All of these results strongly support that our developed SP-GP could serve as a promising cation exchange chromatography resin for high-speed separation, especially for biomolecules of high molecular weight.     

An effective way to hydrophilize gigaporous polystyrene microspheres as rapid chromatographic separation media for proteins

To overcome the disadvantages of protein denaturation and nonspecific adsorption on poly(styrene-divinylbenzene) (PS) medium as a chromatographic support, gigaporous PS microspheres prepared in our previous study were coated with hydrophobically modified agarose (phenoxyl agarose, Agap). Both the modification of agarose and the gigaporous structure of PS microspheres provided an advantage that facilitated the coating of Agap onto PS microspheres. The amount of Agap adsorbed onto the PS surface was examined as a function of the polymer concentration, and various samples of microspheres, differing in surface Agap density, were prepared. The adsorbed layer was then stabilized by chemical cross-linking and its stability was evaluated in the presence of sodium dodecyl sulfate. Results showed that PS microspheres were successfully coated with Agap, while the gigaporous structure could be well maintained. After coating, the nonspecific adsorption of proteins on PS microspheres was greatly reduced. Flow hydrodynamics experiments showed that the Agap-co-PS column had low backpressure, good permeability, and mechanical stability. Such a procedure could provide a hydrophilic low-pressure liquid chromatographic support for different types of chromatography, since the Agap layer may be easily derivatized by classical methods, and because of their good permeability, the coated microspheres have great potential applications in high-speed protein chromatography.     

Chemical modification and characterization of gigaporous polystyrene microspheres as rapid separation of proteins base supports

To overcome the disadvantages of protein denaturation and nonspecific adsorption on poly(styrene‐divinylbenzene) (PS) matrix as chromatographic supports, gigaporous PS microspheres, which we prepared in a previous study, were chemically modified with poly(vinyl alcohol) (PVA) through two‐step reaction. The microspheres were chloroacetylated through Friedel‐Crafts acetylation with chloroacetyl chloride and modified with hydrophilic PVA through Williamson reaction afterward. The modified microspheres were characterized by Fourier transform infrared (FTIR) spectra, X‐ray photoelectron spectroscopy (XPS), scanning electron microscope (SEM), mercury porosimetry measurements (MPM), BET nitrogen adsorption measurements, laser scanning confocal microscope (LSCM), and protein adsorption experiments. Results showed that PS microspheres were successfully coated with PVA, while the gigaporous structure could be maintained. Consequently, the hydrophilicity and biocompatibility of modified microspheres was greatly improved and nonspecific adsorption of proteins was significantly decreased. The coatings contained only stable chemical bonds (e.g., C? C, C? O? C) and easily derived hydroxyl moieties. The large pores of gigaporous PS microspheres also facilitated the modification by PVA. After further derivation, the coated gigaporous base supports could apply in various modes of chromatography and have great potentials in high‐speed protein chromatography. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 5794–5804, 2008     

超大孔离子交换制备色谱分离纯化高活性凝血因子VIII

建立了一条从人血浆中分离高活性凝血因子VIII(FVIII)的纯化工艺。基于FVIII和介质孔径的尺度比及其对蛋白质活性影响的分析,设计了以超大孔离子交换制备色谱为核心步骤的新型分离纯化工艺。分别进行超大孔离子交换色谱与传统离子交换色谱的条件优化,并对优化工艺所得产品进行了活性检测(底物显色法)和纯度检测(高效凝胶过滤和凝胶电泳)。结果表明,超大孔介质结构不但可以有效地保护蛋白质大分子结构,而且能够大幅度地提高制备色谱的传质速率,从而得到具有高凝血活性的FVIII产品。FVIII在超大孔制备色谱过程中的回收率(85%)比传统离子交换制备色谱高4～5倍,产品比活高达154 IU/mg。此外,还研究了超大孔介质的再生程序,采用5个柱体积的1 mol/L NaOH低流速清洗色谱柱,保证了色谱工艺的稳定性。本纯化工艺步骤简单,重现性好,易于放大生产。     

基于超大孔聚合物微球的混合色谱模式层析介质的制备

以甲基丙烯酸缩水甘油酯与乙二醇二甲基丙烯酸酯共聚的超大孔聚合物微球为基质,采用聚乙烯亚胺和丁基缩水甘油醚先后衍生微球的表面,制备成兼具阴离子交换与疏水相互作用的混合色谱模式层析介质。考察离子交换基团、疏水配基密度对蛋白载量、回收率的影响,结果表明,在离子交换容量0.2~0.5 mol/mL范围内,随着介质离子交换容量的增大,蛋白载量及回收率均呈增加趋势,蛋白载量最高值达40 mg/mL,回收率大于90%。当疏水配基的密度大于0.03 mol/mL时,介质开始表现疏水相互作用。此超大孔混合模式色谱介质在大于2000 cm/h 的流速下依然能保持低于2 MPa 的柱背压,同时在高流速下(2880 cm/h)纯化人血清中的抗体应用中表现良好的分辨率。此介质在高通量分离纯化应用方面具有巨大潜力。     

Cooperation of solid granule and solvent as porogenic agents. Novel porogenic mode of biporous media for protein chromatography

A novel porogenic mode, cooperation of solid granule and solvent, has been introduced to prepare a biporous medium for protein chromatography. The matrix, a ternary copolymer of glycidyl methacrylate, triallylisocyanurate and divinylbenzene, was produced by a simple in situ polymerization with granules of sodium sulfate and cyclohexanol and dodecanol as porogenic agents. Functionalized with diethylamine, the resin (denoted as Resin C) was used as an anion exchanger. The pore structure, specific surface area and chromatographic properties of Resin C were determined and compared with those of the resin with only the solvents as porogen (Resin A) and the resin with only the salt granules as porogen (Resin B). The results indicated that Resin C contained regions of micropores with a maximum at approximately 55 nm and regions of macropores with a distinct maximum near 340 nm, which swelled to about 1 microm in aqueous solution. Compared with Resins A and B, the biporous medium Resin C simultaneously possessed a high specific surface area of 37.2 m2/g and a low back-pressure at mobile phase flow velocity up to 720 cm/h. The result of dynamic porosity showed that mobile phase was able to convectively flow through the macropores in Resin C. The dynamic adsorption capacity of Resin C for bovine serum albumin was as high as 57.0 mg/ml column volume (95.0 mg/g wet resin), basically identical to its static capacity, while that of Resin A was only 1.95 mg/ml column volume (3.12 mg/g wet resin), about 3% that of its static capacity. In addition, the column efficiency of Resin C was comparable to that of Resin B, but much higher than that of Resin A, indicating that the mass transfer behavior of proteins in the column was greatly improved by convective flow through the macropores.     

Hypercross-linked polystyrene and its potentials for liquid chromatography: a mini-review

A recently introduced method [Biotechnol. Prog. 13 (1997) 429] for determining intraparticle mass transfer parameters in high speed liquid chromatography is considered in the present study for the case where the eluite adsorbs onto the stationary phase. The validity of the method was verified theoretically using simulated elution profiles and then applied to experimental data obtained using columns packed with either a macroporous or a gel-filled gigaporous stationary phase. For this purpose, experimental measurements were made using alpha-lactalbumin and bovine serum albumin as eluites at several retention factors. Apparent intraparticle diffusivities measured for the gel-filled gigaporous stationary phase were seen to increase with the retention factor, which indicates that for this material surface diffusion is a significant mechanism of mass transfer under retained conditions. Data obtained on the macroporous stationary phase revealed that the intraparticle diffusivity was independent of the retention factor, which suggests that pore diffusion remains the principal mass transfer mechanism even under conditions where proteins are adsorbed on the column packing.     

Efficiency and Resolution in Countercurrrent Chromatography

Abstract

Efficiency and resolution have been studied with a CCC apparatus classified by Y. Ito¹ as a HDES type J, and known as the HSCCC or the Multilayer Coil Separator Extractor. It has been shown that the efficiency decreases when increasing the linear velocity of the mobile phase in the column, which can be done by increasing the flow rate, or by decreasing the volume of the mobile phase in the column. The two ways of changing the linear velocity are not equivalent, since, at constant linear velocity, the efficiency increases when the volume of the mobile phase is well balanced with that of stationary phase. For each flow rate there is a minimum value for the volume of the mobile phase, and the relationship is linear. Working under these conditions, changing both the flow rate and the volume of the mobile phase leads to an efficiency which goes through a minimum at medium flow rates.

The resolution does not show this minimum: it is best at low flow rates and low volumes of mobile phase, and its variations are more important at low flow rates. This means that, if the resolution is good at moderate flow rates, it will remain good at higher flow rates. Combining three equations, it has been possible to predict the resolution when working with the minimum volume of mobile phase for a given flow rate.     

Fast reversed‐phase liquid chromatographic separation of proteins by flow‐through poly(styrene‐co‐divinylbenzene) microspheres

With the explosive growth of the bioscience and biopharmaceuticals, the demand for high efficient analysis and separation of proteins is urgent. High‐performance liquid chromatography is an appropriate technology for this purpose, and the stationary phase is the kernel to the separation efficiency. In this study, flow‐through poly(styrene‐co‐divinylbenzene) microspheres characteristic of the binary pores, i.e. flow‐through pores and mesopores, were synthesized; this special porous structure would benefit the convective mass transfer while guarantee the high specific surface area. Owing to the hydrophobic nature, poly(styrene‐co‐divinylbenzene) microspheres were suitable as the reversed‐phase stationary phase for separation of proteins. For the high permeability of the poly(styrene‐co‐divinylbenzene) microspheres packed column, fast separation of the studied six proteins in ～2 min was achieved. The recoveries of studied proteins were acceptable in the range of 79.0–99.4%. The proposed column had good pH stability of 1–13 and repeatability. Moreover, the column was applied for egg white fast separation, further demonstrating its applicability for complex bio‐sample separation. The flow‐through poly(styrene‐co‐divinylbenzene) microspheres were promising for fast separation of large molecules.     

10°锥角台锥型液相色谱柱尺寸等比例放大和柱内谱带流型的可视化研究

摘要利用改进的可视化装置, 研究了10°锥角的台锥型液相色谱柱内的谱带流型与柱参数变化的关系. 将有机玻璃柱管加工成内圆台外方型的一体结构, 选择折射率一致的色谱固定相硅胶和流动相环己烷, 使整个色谱柱成为高度清晰的透明体, 能直接观察柱中彩色样品谱带的动态三维流型. 研究结果表明, 在实验条件范围内, 流动相流速对谱带流型无影响, 填料的形状和性质对塞子状流型有一定程度的影响. 比较了柱长为5cm和等比例放大后柱长为10cm锥型柱内的流型, 发现放大后的锥型柱内仍然保持塞子状流型, 总柱效等比例增加. 表明继续按比例放大成为工业规模色谱柱后仍能保持塞子状流型.     

Poly(glycidyl methacrylate-divinylbenzene-triallylisocyanurate) continuous-bed protein chromatography

A novel continuous bed with high dynamic adsorption capacity for protein has been developed. It is a macroporous poly(glycidyl methacrylate-divinylbenzene-triallylisocyanurate) rod prepared by in situ copolymerization in a chromatographic tube. The bed matrix contained epoxy groups, so diethylaminohydroxypropyl groups were coupled to the matrix, leading to an anion-exchange continuous bed. The component, specific surface area, and the pore structure of the bed matrix were characterized by Fourier transform infrared spectroscopy, BET method and scanning and transmission electron microscopies, respectively. The flow properties, column efficiency and the dynamic adsorption behavior of the bed were studied. The results show that the continuous bed, a ternary copolymer of glycidyl methacrylate (GMA), divinylbenzene (DVB) and triallylisocyanurate (TAIC) with a specific surface area of 56.4 m2/g, consisted of a three-dimensional structure made up of continuous clusters of microspheres (300 nm) and interconnected irregular pores. The rate of mass transfer is enhanced by the convection of the mobile phase through the pores. The dynamic adsorption isotherm of the anion-exchange column for bovine serum albumin was expressed by the Langmuir equation with a dynamic capacity as high as 76.0 mg/g. Moreover, the separation of proteins, i.e. lysozyme, hemoglobin and bovine serum albumin, is little affected by mobile-phase velocity up to 902.5 cm/h; it was completed within 5 min at 902.5 cm/h.     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.     

高效液相色谱手性流动相添加法拆分阿卓乳酸对映体

采用C₁₈反相色谱柱,以磺丁基醚-β-环糊精（SBE-β-CD）作为手性流动相添加剂,建立了阿卓乳酸对映体的高效液相色谱拆分方法。考察了环糊精衍生物类型、手性添加剂浓度、流动相pH、流速和柱温对手性分离的影响,同时探讨了高效液相色谱采用磺丁基醚-β-环糊精分离阿卓乳酸对映体的分离机制及包结常数,确定了色谱条件为YMC-Pack ODS-A C₁₈色谱柱（250 mm×4.6 mm,5 μm）,流动相为含25 mmol/LSBE-β-CD的0.1 mol/L磷酸盐缓冲液（pH 2.68）-乙腈（90：10,v/v）,流速为0.6 mL/min,柱温为30 ℃,紫外检测波长为220 nm。对映体的保留时间分别为26.65和28.28 min,分离度为1.68。本方法分离度好,简便易行,且比使用手性固定相分离更加经济。     

纤维素键合手性固定相拆分6种萘满酮衍生物对映体

使用Chiralpak IC（纤维素-三（3,5-二氯苯基氨基甲酸酯）共价键合硅胶）手性柱,建立了采用手性固定相高效液相色谱拆分6种 α -芳基萘满酮类衍生物对映体的方法。考察了流动相中有机改性剂的种类和比例、柱温和流速对对映体分离的影响。结果显示6种化合物在异丙醇为改性剂的条件下均可获得较高的对映体分离度。热力学研究表明6种化合物对映体的手性拆分过程均受焓驱动影响,且低温有利于对映体分离。最终推荐分离化合物Ⅰ对映体的流动相是正己烷-异丙醇（90：10,v/v）;分离化合物Ⅱ、Ⅲ、Ⅳ对映体的流动相是正己烷-异丙醇（99：1,v/v）;分离化合物Ⅴ对映体的流动相是正己烷-异丙醇（85：15,v/v）;分离化合物Ⅵ对映体的流动相是正己烷-异丙醇（80：20,v/v）。柱温为25℃,流速为1.0 mL/min。6种化合物对映体均可在Chiralpak IC手性固定相上得到完全分离,证明该色谱柱对6种化合物具有较高的对映体选择性。     

Numerical modeling of elution peak profiles in supercritical fluid chromatography. Part I—Elution of an unretained tracer

When chromatography is carried out with high-density carbon dioxide as the main component of the mobile phase (a method generally known as “supercritical fluid chromatography” or SFC), the required pressure gradient along the column is moderate. However, this mobile phase is highly compressible and, under certain experimental conditions, its density may decrease significantly along the column. Such an expansion absorbs heat, cooling the column, which absorbs heat from the outside. The resulting heat transfer causes the formation of axial and radial gradients of temperature that may become large under certain conditions. Due to these gradients, the mobile phase velocity and most physico-chemical parameters of the system (viscosity, diffusion coefficients, etc.) are no longer constant throughout the column, resulting in a loss of column efficiency, even at low flow rates. At high flow rates and in serious cases, systematic variations of the retention factors and the separation factors with increasing flow rates and important deformations of the elution profiles of all sample components may occur. The model previously used to account satisfactorily for the effects of the viscous friction heating of the mobile phase in HPLC is adapted here to account for the expansion cooling of the mobile phase in SFC and is applied to the modeling of the elution peak profiles of an unretained compound in SFC. The numerical solution of the combined heat and mass balance equations provides temperature and pressure profiles inside the column, and values of the retention time and efficiency for elution of this unretained compound that are in excellent agreement with independent experimental data.     

Modeling of thermal processes in very high pressure liquid chromatography for column immersed in a water bath: Application of the selected models

Currently, chromatographic analyses are carried out by operating columns packed with sub-2 μm particles under very high pressure gradients, up to 1200 bar for 5 cm long columns. This provides the high flow rates that are necessary for the achievement of high column efficiencies and short analysis times. However, operating columns at high flow rates under such high pressure gradients generate a large amount of heat due to the viscous friction of the mobile phase stream that percolates through a low permeability bed. The evacuation of this heat causes the formation of significant or even large axial and radial gradients of all the physico-chemical parameters characterizing the packing material and the mobile phase, eventually resulting in a loss of column efficiency. We previously developed and successfully applied a model combining the heat and the mass balances of a chromatographic column operated under very high pressure gradients (VHPLC). The use of this model requires accurate estimates of the dispersion coefficients at each applied mobile phase velocity. This work reports on a modification of the mass balance model such that only one measurement is now necessary to accurately predict elution peak profiles in a wide range of mobile phase velocities. The conditions under which the simple equilibrium-dispersive (ED) and transport-dispersive (TD) models are applicable in VHPLC are also discussed. This work proves that the new combination of the heat transfer and the ED model discussed in this work enables the calculation of accurate profiles for peaks eluted under extreme conditions, like when the column is thermostated in a water bath.     

分子印迹整体柱快速分离烟酰胺及烟酸

以药物烟酰胺为模板分子,甲基丙烯酸为功能单体,乙二醇二甲基丙烯酸酯为交联剂,甲苯和正十二醇的混合溶液为致孔剂,采用原位聚合法制备了具有特定识别性能和分离能力的分子印迹聚合物,并将其作为高效液相色谱固定相,实现了模板分子与烟酸在2 min内的快速分离。在规格为50 mm×4.6 mm i.d.色谱柱上,以纯水为流动相（流速为7.0 mL/min）、操作温度为室温的色谱条件下,模板分子与烟酸的分离度达1.8。讨论了流动相中有机溶剂含量、醋酸及碱含量和流速对分离的影响。结果表明,原位聚合法制备的整体分子印迹聚合物在以纯水作流动相时对模板分子与其类似物有快速分离能力,这对于体内药物的分离富集研究具有很好的应用前景。     

应用质量源于设计的理念优化大鼠血浆中大黄蒽醌的分析方法

应用质量源于设计理念建立一种高效液相色谱-荧光检测法(HPLC-FLD)用于测定大鼠血浆中5种大黄蒽醌。用Plackett-Burman设计考察流动相中甲醇含量、pH值、流速、柱温和进样体积对色谱峰的分离度、理论塔板数、最末洗脱峰的保留时间和拖尾因子的影响,结果显示流动相中甲醇含量、流速和柱温对色谱系统的影响显著(p<0.05)。继而采用Box-Behnken设计结合响应面法考察上述三因素对分离度、保留时间和理论塔板数的影响。用Derringer渴求函数评价了响应值的综合作用。得出最优色谱条件为:以甲醇-0.1%(v/v)磷酸水溶液(81.4:18.6, v/v)为流动相等度洗脱,流速1.1 mL/min,柱温31℃,荧光检测激发波长为440 nm,发射波长为540 nm。建立的模型显示良好的预测性。结果表明:质量源于设计的理念可有效地应用于优化高效液相色谱分析方法。     

原子转移自由基聚合法制备超大孔聚合物微球

以甲基丙烯酸缩水甘油酯为单体(GMA)、乙二醇二甲基丙烯酸酯(EDMA)为交联剂,采用原子转移自由基聚合法(ATRP)制备了PGMA-EDMA大孔聚合物微球,采用傅里叶变换红外光谱、扫描电子显微镜及压汞法对PGMA-EDMA微球进行了表征.研究结果表明,原子转移自由基聚合法制备的PGMA-EDMA微球的孔径尺寸及比表面积均大于普通自由基聚合法(CFRP)制备的PGMA-EDMA;ATRP法制备的PGMAEDMA微球的颗粒尺寸(100~400 nm)明显小于CFRP法制备的PGMA-EDMA微球的颗粒尺寸(1000 nm).PGMA-EDMA(ATRP)的微球粒径尺寸分布优于PGMA-EDMA(CFRP).因此PGMA-EDMA(APRP)微球在快速蛋白分离纯化方面有潜在的应用前景.