Effects of urea induced protein conformational changes on ion exchange chromatographic behavior

Urea is widely employed to facilitate protein separations in ion exchange chromatography at various scales. In this work, five model proteins were used to examine the chromatographic effects of protein conformational changes induced by urea in ion exchange chromatography. Linear gradient experiments were carried out at various urea concentrations and the protein secondary and tertiary structures were evaluated by far UV CD and fluorescence measurements, respectively. The results indicated that chromatographic retention times were well correlated with structural changes and that they were more sensitive to tertiary structural change. Steric Mass Action (SMA) isotherm parameters were also examined and the results indicated that urea induced protein conformational changes could affect both the characteristic charge and equilibrium constants in these systems. Dynamic light scattering analysis of changes in protein size due to urea-induced unfolding indicated that the size of the protein was not correlated with SMA parameter changes. These results indicate that while urea-induced structural changes can have a marked effect on protein chromatographic behavior in IEX, this behavior can be quite complicated and protein specific. These differences in protein behavior may provide insight into how these partially unfolded proteins are interacting with the resin material.     

Preparation and characterization of a novel dual‐retention mechanism mixed‐mode stationary phase with PEG 400 and succinic anhydride as ligand for protein separation in WCX and HIC modes

A novel dual‐retention mechanism mixed‐mode stationary phase based on silica gel functionalized with PEG 400 and succinic anhydride as the ligand was prepared and characterized by infrared spectra and elemental analysis. Because of the ligand containing PEG 400 and carboxyl function groups, it displayed hydrophobic interaction chromatography (HIC) characteristic in a high‐salt‐concentration mobile phase, and weak cation exchange chromatography (WCX) characteristic in a low‐salt‐concentration mobile phase. As a result, it can be employed to separate proteins with both WCX and HIC modes. The resolution and selectivity of the stationary phase was evaluated under both HIC and WCX modes with protein standards, and its performance was comparable to that of conventional ion‐exchange chromatography and HIC columns. The results indicated that the novel dual‐retention mechanism column, in many cases, could replace two individual WCX and HIC columns as a ‘2D column’. In addition, the mixed retention mechanism of proteins on this ‘2D column’ was investigated with stoichiometric displacement theory for retention of solute in liquid chromatography in detail in order to understand why the dual‐retention mechanism column has high resolution and selectivity for protein separation under WCX and HIC modes, respectively. Based on this ‘2D column’, a new 2DLC technology with a single column was developed. It is very important in proteome research and recombinant protein drug production to save column expense and simplify the processes in biotechnology. Copyright © 2013 John Wiley & Sons, Ltd.     

Preparation of a weak anion exchange/hydrophobic interaction dual‐function mixed‐mode chromatography stationary phase for protein separation using click chemistry

In this study, 3‐diethylamino‐1‐propyne was covalently bonded to the azide‐silica by a click reaction to obtain a novel dual‐function mixed‐mode chromatography stationary phase for protein separation with a ligand containing tertiary amine and two ethyl groups capable of electrostatic and hydrophobic interaction functionalities, which can display hydrophobic interaction chromatography character in a high‐salt‐concentration mobile phase and weak anion exchange character in a low‐salt‐concentration mobile phase employed for protein separation. As a result, it can be employed to separate proteins with weak anion exchange and hydrophobic interaction modes, respectively. The resolution and selectivity of the stationary phase were evaluated in both hydrophobic interaction and ion exchange modes with standard proteins, respectively, which can be comparable to that of conventional weak anion exchange and hydrophobic interaction chromatography columns. Therefore, the synthesized weak anion exchange/hydrophobic interaction dual‐function mixed‐mode chromatography column can be used to replace two corresponding conventional weak anion exchange and hydrophobic interaction chromatography columns to separate proteins. Based on this mixed‐mode chromatography stationary phase, a new off‐line two‐dimensional liquid chromatography technology using only a single dual‐function mixed‐mode chromatography column was developed. Nine kinds of tested proteins can be separated completely using the developed method within 2.0 h.     

Polystyrene immobilized ionenes as novel stationary phase for ion chromatography

Several aliphatic ionenes (2-6-, 6-6-, 10-6-ionene) have been prepared as ion exchangers for the development of novel high-performance stationary phases for anion chromatography (IC). A macroporous polystyrene/divinylbenzene (PS/DVB) resin with adjusted cation exchange capacity was used as support. Therefore the immobilization of ionenes to polystyrene carriers with remaining positive surface charge became possible for the first time. Strong ion-exchange interactions, resulting in high retention times, between the stationary phase and inorganic as well as organic anionic analytes have been observed. The influence of different ionenes on the retention behaviour during the ion chromatographic separation was investigated. Additionally, partly aromatic and polar ionene backbones were prepared and their retention behaviour as anion exchanger was investigated. The highest number of theoretical plates obtained was about 90.000 per meter. The signal asymmetries were generally lower than obtained for surface functionalized anion exchangers.     

Strong complex formation of carbohydrates with metal ions at alkaline pH studied by ligand exchange chromatography

Summary At alkaline pH, carbohydrates are highly retained on a sulphonated polystyrene resin loaded with rare earth or uranyl metal ions. The complex formation is governed by the ionization of the carbohydrate moiety. pH and the type of metal ion were found to have a decisive influence on the complex formation. Binding of the metal ion to the cation exchanger and to different carbohydrates is described. The competitive complexation between solute and hydroxide to the metal ions is described as a ligand exchange reaction and a retention model is proposed. The ligand exchange column was used as precolumn in a coupled column separation system and the high selectivity is demonstrated by the separation of paracetamol glucuronide from a urine sample after filtration and direct injection.     

Retention model of protein for mixedmode interaction mechanism in ion exchange and hydrophobic interaction chromatography

A unified retention equation of proteins was proved to be valid for a mixed-mode interaction mechanism in ion exchange chromatography (IEC) and hydrophobia interaction chro-matography (HIC). The reason to form a "U" shape retention curve of proteins hi both HIC and IEC was explained and the concentration range of the strongest elution ability for the mobile phase was determined with this equation. The parameters in this equation could be used to characterize the difference for either HIC or IEC adsorbents and the changes in the molecular conformation of proteins. With the parameters in this equation, the contributions of salt and water in the mobile phase to the protein retention in HIC and IEC were discussed, respectively. In addition, the comparison between the unified equation and Melander' s three-parameter equation for mixed-mode interaction chromatography was also investigated and better results were obtained in former equation.     

Understanding and mitigating conductivity transitions in weak cation exchange chromatography

Large conductivity fluctuations were observed during a high pH wash step in a weak cation exchange chromatography process. These conductivity transitions resulted in a conductivity drop during pH increase and a conductivity rise during pH decrease. In some cases, the absolute conductivity change was greater than 6 mS/cm which was sufficient to affect target protein retention on the column. Further investigation revealed that wash buffer concentration, resin ligand density, and resin ligand pK have a profound effect on the magnitude of the conductivity transitions and the shape of corresponding pH traces. A potentiometric electrode selective for sodium ions was used to measure effluent counterion concentrations from two preparative resins during high pH washes, and the number of exchangeable counterions was compared to predictions made using ion exchange equilibrium theory. Results from this analysis show that conductivity transitions can be effectively mitigated without compromising process performance by optimizing the trade-off between wash buffer concentration and wash phase duration.     

手性配位体交换流动相添加剂法拆分对映体

综述了手性配合基交换色谱法通常采用三种手性相系统中的流动相添加剂方法。主要内容有：（Ａ）手性配合基交换机制,给出了描述对映体对在色谱系统中的保留时间和分离选择性的公式,包括手性选择剂在固定相和流动相中的各种不同情况。公式表明整个色谱往系统的对映体选择性不同于溶液中所存在的选择剂与被选择物作用的情况;（Ｂ）影响配合交换的参数,讨论了金属离子、金属离子／配位体比率、金属离子络合物浓度、固定相、流动相ｐＨ、洗脱顺序、有机调节剂、离子对试剂、流动相离子强度、温度、立体选择性和手性交互识别;（Ｃ）应用。     

离子交换色谱中混合保留机理的研究

鉴于离子交换色谱中被测离子和有机聚合物树脂之间存在的吸附作用, 选择阴离子交换色谱柱IonPacAS9-HC为研究对象, 针对离子交换色谱中的吸附保留行为, 从色谱混合保留机理的角度出发, 考虑各色谱柱固定相含量和界面吸附面积的不同, 建立柱间保留的相互关系方程. 在五根不同批号IonPacAS9-HC (250 mm×2 mm I.D.)柱上, 以含50%乙腈(V/V)的9 mmol/L Na₂CO₃为流动相, 0.25 mL/min流速, 对12个不同结构类型的无机和有机阴离子(氟离子、氯离子、硫酸根、磷酸根、二羟基丁酸、丙酮酸、乙酸、丙烯酸、苯甲酸、丙二酸、酒石酸和邻苯二甲酸)的保留行为进行研究, 并采用推导所得的混合保留机理模型对溶质这一保留特征进行表征, 结果较为理想, 其中一价离子的相关系数为0.9～0.999, 二价离子的相关系数为0.999～1, 为离子交换色谱中吸附保留行为的研究提供了新方法.     

分离蛋白质的高效离子交换色谱填料的研究(Ⅲ)──膦酸基阳离子交换色谱填料的性能

分离蛋白质的高效离子交换色谱填料的研究（Ⅲ）──膦酸基阳离子交换色谱填料的性能常建华（西北大学化学系,西安,７１００６９）关键词填料,高效阳离子交换色谱,性能,蛋白质以磷羟基为离子交换基团的色谱填料是一类中强阳离子交换色谱填料,以前报道的此类填料是使...     

Retention of some glucosinolates in anion exchange chromatography. Part I: Qualitative study on a silica-trialkylammonium exchanger

Summary The retention and separation of glucosinolates, as organic anions, were studied on a silica-based strong anion exchanger under isocratic elution conditions. All glucosinolates carry the same functional ionic group (-OSO ₃ ^– ), however they do not have the same retention in anion exchange chromatography. The plots of capacity factors of organic anions versus the reciprocal of eluent ion concentration show good linearity. From the slope and y-intercept data the major retention mechanisms are interpreted as ion exchange and reversed-phase interactions. The effects of nature and concentration of the eluent ion and the influence of organic modifier addition to the aqueous buffered mobile phase are also investigated. Direct and indirect UV detection were used.Our results open the way for the development of new systems for intact glucosinolate analysis which are easier to use than the present ion-pairing chromatographic method.     

单分散树脂基质的弱阳离子交换色谱固定相的制备及其在生物大分子分离中的应用研究

采用分散聚合法制备种子和“一步种子溶胀聚合法”制备了粒径为6~15 μm的单分散多孔氯甲基苯乙烯-二乙烯苯微球。该微球经化学改性后得到一种亲水性良好的新型高效弱阳离子交换色谱固定相。详细考察了该固定相的表面亲水性、对标准蛋白的分离性能和盐的种类对蛋白质保留行为的影响。考察结果表明该固定相是一种性能优异的弱阳离子交换色谱固定相。将其应用于鸡蛋清中溶菌酶的快速分离纯化,纯化后的溶菌酶纯度高于96%,比活高达71184 U/mg。     

弱阳离子交换色谱中疏水作用对蛋白保留的影响

选取了四种常用的弱阳离子交换(WCX)商品柱以研究标准蛋白在其上的色谱保留行为。发现在疏水色谱(HIC)模式下,蛋白在这四种WCX商品柱上也有不同程度的保留特征,且洗脱曲线呈现出保留值随盐浓度变化的"U"型。从分子力学角度定性解释了因疏水相互作用力的存在影响了蛋白在WCX色谱柱上洗脱顺序的改变。运用计量置换理论(SDT)中的两组线性方程进一步证实了WCX和HIC中蛋白与固定相间相互作用力的性质,在HIC中为非选择性作用力,而在离子交换色谱(IEC)中为选择性作用力。这四种色谱柱中的两种事实上可在WCX和HIC两种模式下,对标准蛋白进行分离且有较好的分离效果,有可能作为二维色谱柱来使用。     

Evaluation and comparison of n‐alkyl chain and polar ligand bonded stationary phases for protein separation in reversed‐phase liquid chromatography

Protein retention is very sensitive to the change of solvent composition in reversed‐phase liquid chromatography for so called “on–off” mechanism, leading to difficulty in mobile phase optimization. In this study, a novel 3‐chloropropyl trichlorosilane ligand bonded column was prepared for protein separation. The differences in retention characteristics between the 3‐chloropropyl trichlorosilane ligand bonded column and n‐alkyl chain modified (C₂, C₄, C₈) stationary phases were elucidated by the retention equation . Retention parameters (a and c) of nine standard proteins with different molecular weights were calculated by using homemade software. Results showed that retention times of nine proteins were similar on four columns, but the 3‐chloropropyl trichlorosilane ligand bonded column obtained the lowest retention parameter values of larger proteins. It meant that their retention behavior affected by acetonitrile concentration would be different due to lower |c| values. More specifically, protein elution windows were broader, and retentions were less sensitive to the change of acetonitrile concentration on the 3‐chloropropyl trichlorosilane ligand bonded column than that on other columns. Meanwhile, the 3‐chloropropyl trichlorosilane ligand bonded column displayed distinctive selectivity for some proteins. Our results indicated that stationary phase with polar ligand provided potential solutions to the “on–off” problem and optimization in protein separation.     

Selective displacement chromatography in multimodal cation exchange systems

A library of displacer analogues with varying degrees of electrostatic, hydrophobic and hydrogen bonding moieties was evaluated for their ability to enhance the selectivity of multimodal (MM) chromatography under high loading conditions. The library was screened for displacement of model proteins using a robotic liquid handling system and selective batch separations were achieved for proteins that were inseparable with linear gradient chromatography. Trends in protein displacement were identified and displacers with higher hydrophobicity and net charge exhibited improved protein displacements. Proteins that interacted with the resins primarily via electrostatic interactions were more readily displaced than those that possessed a significant hydrophobic contribution to their binding. In addition, multimodal displacers were found to be more selective than single mode electrostatic displacers. Column chromatography studies were also carried out and baseline separations were achieved for model protein pairs using selective displacement. Finally, operation of these columns in the desorption mode resulted in baseline separation of model proteins which were not separable by selective displacement chromatography. This study indicates that the inherent selectivity of MM resins can be augmented by the selectivity of the displacer under non-linear competitive binding conditions, creating new opportunities for protein separations not possible using traditional gradient operations.     

Determination of volatile compounds in Brazilian distilled cachaça by using comprehensive two-dimensional gas chromatography and effects of production pathways

Comprehensive two-dimensional gas chromatography (GC × GC) was applied to the study of cachaça production. Effects of bidistillation, and the use of charcoal filtration in the production of artisan cachaça, as well as the effects of multi-distillation on volatile products in commercial cachaça were investigated. Volatile compounds were collected and concentrated onto a polyacrylate solid-phase microextraction fibre, and analyzed using GC × GC on a non-polar (BPX5)–polar (BP20) column set. More than 100 compounds, comprising various homologous series were tentatively identified using MS library matching and comparison with retention indices. Phthalate organic contamination following the use of ion exchange resin for removal of copper ion was evident. Charcoal successfully removes this contamination product. Prediction of compounds within particular homologous series aids component identification.     

无孔单分散亲水性强阳离子交换固定相的制备及其在蛋白质快速分离中的应用研究

采用分散聚合法制备小颗粒种子及“一步种子溶胀聚合”法成功地制备了粒径为3.0 μm的无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯树脂,其表面经水解、环氧化、再水解后与氯磺酸反应,制备了一种新型的强阳离子交换色谱填料（SCX）。详细考察了该填料对标准蛋白质的分离性能及流动相中盐的种类、有机溶剂、流速等对蛋白质保留的影响。实验结果表明,在流速为4 mL/min时,采用线性梯度洗脱,1.0 min内可快速分离4种标准蛋白质,蛋白质的保留符合阳离子交换色谱规律。将SCX应用于快速纯化鸡蛋清中的溶菌酶和猪心中的细胞色素-C,取得了较好的效果。