Retention of Large Biological Molecules by Size-Exclusion Chromatography

Size-exclusion chromatography has been developed for the separation of large biological molecules including proteins, polymers, peptides, nucleic acids, and polysaccharide according to their molecular size. This study determined the retention factors for dextran (5, 25, 50, 270, 670, and 1100?kDa) and polysaccharides, such as fucoidan, alginic acid, and laminarin, in the size-exclusion chromatography stationary phase. In addition, the molecular weights and retention factor of three polysaccharides were calculated from the dextran standard curve. The largest retention factor was 4.26 using the size-exclusion chromatography columns (5?kDa dextran). The molecular weights of fucoidan, alginic acid, and laminarin were determined to be 250, 200, and 5 to 64?kDa, respectively.     

Size-Exclusion Chromatography of Asphaltenes: An Experimental Comparison of Commonly Used Approaches

Asphaltenes, the heaviest and most polar fraction of crude oil, are responsible for the clogging of oil pipelines and of underground reservoir rocks. Asphaltenes are defined as a solubility class (toluene- or benzene-soluble, n-alkane insoluble), and a consensus is still lacking on the molar mass (M) averages, distribution, and range of these materials. Size-exclusion chromatography (SEC) is among the most widely employed methods for characterizing the M averages and distributions of both natural and synthetic polymers and, as such, has seen widespread application in the study of asphaltenes. SEC analysis of asphaltenes presents a number of challenges, however, some inherent to all low-M materials and some unique to these particular analytes. Here, we have experimentally compared several of the most commonly used approaches to SEC of asphaltenes, in hope of both demonstrating the influence of experimental conditions upon the results obtained as well as to determine optimal conditions of analysis. In the end, while the former goal was met, the latter one remains an open challenge in the field.     

Size-Exclusion Chromatography of Asphaltenes: An Experimental Comparison of Commonly Used Approaches

Asphaltenes, the heaviest and most polar fraction of crude oil, are responsible for the clogging of oil pipelines and of underground reservoir rocks. Asphaltenes are defined as a solubility class (toluene- or benzene-soluble, n-alkane insoluble), and a consensus is still lacking on the molar mass (M) averages, distribution, and range of these materials. Size-exclusion chromatography (SEC) is among the most widely employed methods for characterizing the M averages and distributions of both natural and synthetic polymers and, as such, has seen widespread application in the study of asphaltenes. SEC analysis of asphaltenes presents a number of challenges, however, some inherent to all low-M materials and some unique to these particular analytes. Here, we have experimentally compared several of the most commonly used approaches to SEC of asphaltenes, in hope of both demonstrating the influence of experimental conditions upon the results obtained as well as to determine optimal conditions of analysis. In the end, while the former goal was met, the latter one remains an open challenge in the field.

     

Special Report: Standard Practice of Liquid Size-Exclusion Chromatography - SEC (GEL Permeation Chromatography - GPC

Abstract

A reversed-phase ion-pair liquid chromatographic method was applied to the analysis of bambuterol hydrochloride chemical reference substance (CRS) and tablets. The purity evaluation of the reference substance was performed with amperometric, UV, diode array and mass spectrometric detection. The total amount of impurities found was less then 0.1%.

For the determination of bambuterol hydrochloride in tablets the method reproducibility was 0.9% and the recovery was in the range 99.8–100.7%. Detection limits for conceivable degradation products were about 1 ng. The suitability of different column packing materials was investigated.     

用尺寸排阻色谱法研究蛋白质的热稳定性

用尺寸排阻色谱ＺｏｒｂａｘＧＦ－２５０凝胶过滤柱研究了核糖核酸酶Ａ,马心肌红蛋白和溶菌酶的热稳定性。测定了蛋白质在尺寸排阻系统中的分配系数Ｋｄ并用荧光检测器分析了加热引起的蛋白质内源性荧光的改变,从而计算出了蛋白质的热变性转化点温度,测定了蛋白质天然态、热变性和盐酸胍诱导变性状态下的斯托期斯半径,实验结果进一步证帝了热变性后蛋白质不是呈真正的随机线状,而是保留了部分紧密的结构区域。用本方法研究了Ｐ     

Temperature Gradient Interaction Chromatography: A Perspective

Chromatographia - The application of temperature gradient interaction chromatography (TGIC) as an advanced technique for the characterisation of polymers is discussed, in comparison to other liquid...     

Effect of Mobile-Phase Additives on Separation of Gold Nanoparticles by Size-Exclusion Chromatography

Two spherical gold nanoparticles (diameters 5.3 and 38 nm, respectively) were used to appraise the separation performance of various mobile-phase additives in size-exclusion chromatography (SEC). The three additives were sodium citrate (SC), sodium chloride (NaCl), and sodium dodecylsulfate (SDS). The problem of adsorption on gold nanoparticles occurs when SC and NaCl are used as mobile-phase additives. In contrast, the adsorption problem is overcome by using SDS as mobile-phase additive; furthermore, SDS prevents gold nanoparticles from coagulating during separation. This paper demonstrates the feasibility of using SDS as mobile-phase additive in SEC for size separation of gold nanoparticles and also demonstrates how SEC may be used to investigate interactions between a mobile-phase additive and gold nanoparticles.     

利用体积排阻快速原位表征整体固定相的孔结构

利用体积排阻色谱原理,在自制的径向色谱柱上,测定了具有窄分子量分布且已知平均分子量418～190 000的聚苯乙烯标准品的保留数据,这些数据可用于计算柱子的外部(55．1％)、内部(4．9％)和总孔隙率以及整体材料孔尺寸分布的信息。此种技术用于整体填料孔结构的表征,具有无破坏性、原位、快速的特点。整体固定相的外部和总孔隙率高于颗粒填充柱,表明整体柱的高通透性;流速与压力关系的分析,证明了整体固定相在高流速下具有低背压的特征。     

Solution Properties of Polyelectrolytes. VI. Secondary Effects in Aqueous Size-Exclusion Chromatography

Abstract

An independent analysis of different operational variables in aqueous size exclusion chromatography of poly electrolytes has been carried out using a silica-based support. The effect of polyion concentration, pH and ionic strength on sodium polystyrenesulfonate calibration plots has been investigated. Finally, a novel semi-empirical model has been developed from thermodynamic considerations which relates the support effective pore volume to the polyelectrolyte molecular weight and qualitatively describes secondary effects.     

Problems in Determining Compositional Heterogeneity of Copolymers by Size-Exclusion Chromatography and UV-RI Detection System

Abstract

The compositional heterogeneity of styrene-methylmethacrylate copolymer has been evaluated by a UV-RI dual-detector method and by a RI detector-pyrolysis-gas chromatographic method and problems associated with the determination of the chemical heterogeneity by a UV-RI method are discussed. The quotient of signals H(UV)/H(RI) of both the detectors (UV and RI) for polystyrene increased at the extreme parts of the molecular weight distribution owing to nonlinearity of UV response. The correction of the quotient of signals of both detectors for the copolymer was proposed. The UV and RI response factors for the copolymer were supposed to differ from those for homopolymers. Reproducibility for the detector responses influences the values of chemical heterogeneity much more than expected. To overcome these problems, the detection method to obtain each composition independently using different detectors will be preferable.     

High-Performance Aqueous Size-Exclusion Chromatography Using Diol-Bonded Porous Glass Packing Materials. Retention Behavior of Some Proteins

Abstract

Retention volume of proteins increased or decreased with increasing phosphate buffer or neutral electrolyte concentrations in the mobile phase. This variation suppressed or accelerated by changing pH values in the mobile phase. The behavior of proteins can be interpreted by knowing isoelectric points (pI) of proteins and pKa value of the residual silanol groups on the surface of diol-bonded porous glasses. Positively charged surface of proteins below pH 8.0 (cytochrome c, lysozyme) retarded the elution by the ion-adsorption effects and negatively charged proteins around pH 7.0 (egg albumin, bovin serum albumin) eluted earlier than expected by the ion-exclusion effects. These effects suppressed by increasing phosphate buffer and neutral electrolyte concentrations in the mobile phase. Size-exclusion separation was attained in the mobile phase over 0.1 M phosphates and 0.1 M NaCl concentrations at pH 7.0. Mcllvaine buffer and Gomori buffer showed opposite action to proteins for retention comparing with Soerensen phosphate buffer. Potassium thiocyanate showed the different action for retention of proteins comparing with other neutral electrolytes and acted like sodium dodecyl sulphonate.     

The Contribution of Catalysis for the Realization of GSC in the Twenty-First Century

The drastic climate change on global scale is becoming very serious. To curb the anthropogenic CO₂ emissions will be one of the most important issues for the human society in the twenty-first century. The chemical industry can contribute to this issue from the viewpoint of green sustainable chemistry. We have been establishing various innovative catalyst technologies and can still continue further efforts. Considering the existence of huge infrastructure of the conventional processes based on fossil resources, it would be very difficult to turn into new technologies based on sustainable resources quickly and straightforwardly. Thus it may be good to apply more practical strategies along the realistic time line. Here we propose a strategy comprised of establishing more energy-effective chemical processes based on fossil resources in short term, utilizing biomasses as chemical feedstocks in large scale in middle term, and pursuit of the artificial photosynthesis as a final goal in long term. In this article, several topics in each category are introduced.     

The Art and Science of Total Synthesis at the Dawn of the Twenty-First Century

A new millenium has begun-grounds enough to question the present state of the total synthesis of natural products. In this review we answer this question by tracing the evolution of this fine art and science from its birth to the present time. This retrospective on total synthesis should serve to demonstrate how far we have come, yet show that the science of total synthesis is still in its infancy.     

杜氏盐藻多糖的高效体积排阻色谱的保留特性及其分析方法的研究

通过对从杜氏盐藻中提取出的不同多糖级分在高效体积排阻色谱柱(Waters Ultrahydragel Linear,7.8 mm i.d.×300 mm,2根串联)上的保留特性的考察及其分离分析条件的优化,建立了高效体积排阻色谱分析盐藻多糖平均相对分子质量及其分布的方法。结果表明:流动相中盐的种类及其浓度、pH值对3种酸性多糖级分(特别是硫酸化多糖级分PD4a)的保留行为有显著影响;在柱温为45 ℃,流速为0.9 mL/min条件下,使用0.1 mol/L的NaAc水溶液作流动相基本上能消除非特异性吸附作用及分子间缔合等因素的干扰,使各多糖级分基本以非缔合状态按立体排除机制保留和分离。在优化的色谱条件下,测得的盐藻多糖5个级分的重均相对分子质量(Mw)分别为1548000,33000,67000,424000,10000;测得的硫酸化多糖级分PD4a的Mw和峰面积的相对标准偏差分别为1.7%和 0.88%(n=5)。     

Using Size-Exclusion Chromatography to Monitor the Stabilization of Au Nanoparticles in the Presence of Salt and Organic Solvent

This paper reports the use of size-exclusion chromatography (SEC) to assess the size stabilization of Au nanoparticles (NPs) in the presence of salt and organic solvent. In the absence of an adequate stabilizer for the Au NP solution, the presence of salt (NaCl) or an organic solvent (MeOH) resulted in the near disappearance of the signal of the Au NPs in the elution spectra after SEC separation, as a result of the Au NPs forming larger agglomerates under such conditions. In contrast, when the Au NPs were capped with an adequate stabilizer [i.e., 3A-amino-3A-deoxy-(2AS,3AS)-β-cyclodextrin (H₂N-β-CD)], the elution time of the signal for the Au NPs and their elution spectra after SEC separation were barely affected by the presence of salt or organic solvent. Thus, H₂N-β-CD is a good stabilizer against the coagulation of Au NPs in the presence of salt or organic solvent. In addition, this study confirms that SEC—with its short analysis times, low operating costs, automated operation, and in situ analysis—is highly applicable for the rapid analysis of Au NPs.

     

Hydrodynamic Radius Characterization of a Vinyl-Type Polynorbornene by Size-Exclusion Chromatography with a Static and Dynamic Laser Light Scattering Detector

Both absolute molecular weight and molecular sizes (radius of gyration and hydrodynamic radius) of a vinyl-type polynorbornene eluting from size-exclusion chromatography columns were determined by combined with a static and dynamic laser light scattering detector. The hydrodynamic radius of polymer fraction eluting from size-exclusion chromatography columns was obtained from dynamic laser light scattering measurements at only a single angle of 90° by introducing a correction factor. According to the scaling relationship between molecular sizes and molecular weight and the ratio between radius of gyration and hydrodynamic radius, the vinyl-type polynorbornene took a random coil conformation in 1,2,4-trichlorobenzene at 150 °C.     

Validation of an Analytical Method for the Simultaneous Determination of Hyaluronic Acid Concentration and Molecular Weight by Size-Exclusion Chromatography

The hyaluronic acid (HA) global market growth can be attributed to its use in medical, cosmetic, and pharmaceutical applications; thus, it is important to have validated, analytical methods to ensure confidence and security of its use (and to save time and resources). In this work, a size-exclusion chromatography method (HPLC-SEC) was validated to determine the concentration and molecular distribution of HA simultaneously. Analytical curves were developed for concentration and molecular weight in the ranges of 100–1000 mg/L and 0.011–2.200 MDa, respectively. The HPLC-SEC method showed repeatability and reproducibility greater than 98% and limits of detection and quantification of 12 and 42 mg/L, respectively, and was successfully applied to the analysis of HA from a bacterial culture, as well as cosmetic, and pharmaceutical products.