Glycosaminoglycans and proteins: different behaviours in high-performance size-exclusion chromatography

The influence of the conformation of globular proteins and glycosaminoglycans in high-performance size-exclusion chromatography (HPSEC) was studied. Glycosaminoglycans (heparin, chondroitin sulphate and dermatan sulphate) with different primary structures, sulphate-to-carboxyl ratios and physico-chemical properties were extracted and purified. Their physico-chemical properties and purity were evaluated by several analytical techniques. Glycosaminoglycans with different relative molecular masses (M_r) were prepared by a chemical depolymerization process. These heteropolysaccharides were evaluated by HPSEC and compared with globular proteins of known relative molecular mass. The two third-degree polynomial regression curves for proteins and glycosaminoglycans have different coefficients and the columns present different exclusion limits. In particular, under the experimental conditions, the M_r exclusion limits for high M_r are 44 000 for glycosaminoglycans and 240 000 for globular proteins. In contrast, the behaviours of these two classes of macromolecules are similar for lower M_r. In fact, the two third-degree polynomial curves show the same regression below about M_r = 1000. The behaviour in HPSEC is discussed in relation to the different steric conformations for proteins and glycosaminoglycans with different relative molecular masses.     

Influence of ionic strength and organic modifier concentrations on characterization of aquatic fulvic and humic acids by high-performance size-exclusion chromatography

Aquatic fulvic acid (FA) and humic acid (HA) were characterized by an aqueous high-performance size-exclusion chromatography (HPSEC) using a hydrophilic polymeric stationary phase and an aqueous eluent at neutral pH and low-ionic strength (5 mM Na2HPO4; final ionic strength, 13 mM). Employed HPSEC showed low sensitivity of FA to variations in ionic strength (13 and 100 mM) and contents of organic modifier (0.1 or 40% methanol) in aqueous eluent. Under these analytical conditions, peak maxima of relative UV signals versus molecular mass (Mr) defined as M'p and peak maxima of relative mass concentrations versus Mr defined as Mp of FA were shown to be located at 548-690 and 500, respectively. Organic modifier concentrations of 40% methanol in aqueous eluent enabled not only analysis of FA, but also analysis of some aquatic HA by HPSEC. Analysis showed M'p and Mp values of aquatic HAs around 1000 and 600, respectively. Measured molecular mass data of FA were found to be consistent with the recently published data describing low molecular masses of FA. Results recommend the use of the described HPSEC as a simple, rapid, reproducible, low-cost method giving consistent molecular sizes/masses of FA and some aquatic HAs.     

Electrospray ionization mass spectrometry of terrestrial humic substances and their size fractions

Electrospray ionization mass spectrometry (ESI-MS) was used to evaluate the average molecular mass of terrestrial humic substances, such as humic (HA) and fulvic (FA) acids from a soil, and humic acid from a lignite (NDL). Their ESI mass spectra, by direct infusion, gave average molecular masses comparable to those previously obtained for aquatic humic materials. The soil HA and FA were further separated in size-fractions by preparative high performance size exclusion chromatography (HPSEC) and analyzed with ESI-MS by both direct infusion and a further on-line analytical HPSEC. Unexpectedly, their average molecular mass was only slightly less than for the bulk sample and, despite different nominal molecular size, did not substantially vary among size-fractions. The values increased significantly (up to around 1200 Da) after on-line analytical HPSEC for the HA bulk sample, at both pH 8 and 4, and for the HA size-fractions when pH was reduced from 8 to 4. It was noticed that HA size-fractions at pH 8 were separated by on-line HPSEC in further peaks showing average masses which progressively increased with elution volume. Furthermore, when the HA and NDL bulk samples were sequentially ultracentrifuged at increasing rotational speed, their supernatants showed mass values which were larger than bulk samples and increased with rotational speed. These variations in mass values indicate that the electrospray ionization is dependent on the composition of the humic molecular mixtures and increases when their heterogeneity is progressively reduced. It is suggested that the dominance of hydrophobic compounds in humic supramolecular associations may inhibit the electrospray ionization of hydrophilic components. Our results show that ESI-MS is reasonably applicable to humic substances only after an extensive reduction of their chemical complexity.     

Molecular size fractionation of soil humic acids using preparative high performance size-exclusion chromatography

High performance size-exclusion chromatography (HPSEC) is useful for the molecular size separation of soil humic acids (HAs), but there is no method available for various HAs with different chemical properties. In this paper the authors propose a new preparative HPSEC method for various soil HAs. Three soil HAs with different chemical properties were fractionated by a Shodex OHpak SB-2004 HQ column with 10mM sodium phosphate buffer (pH 7.0)/acetonitrile (3:1, v/v) as an eluent. The HAs eluted within a reasonable column range time (12-25 min) without peak tailing. Preparative HPSEC chromatograms of these HAs indicated that non-size-exclusion effects were suppressed. The separated fractions were analyzed by HPSEC to determine their apparent molecular weights. These decreased sequentially from fraction 1 to fraction 10, suggesting that the HAs had been separated by their molecular size. The size-separated fractions of the soil HA were mixed to compare them with unfractionated HA. The analytical HPSEC chromatogram of the mixed HA was almost identical to that of the unfractionated HA. It appears that the HAs do not adsorb specifically to the column during preparative HPSEC. Our preparative HPSEC method allows for rapid and reproducible separation of various soil HAs by molecular size.     

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)。     

Molecular mass distribution of sodium alginate by high-performance size-exclusion chromatography

A sensitive high-performance size-exclusion chromatography (HPSEC) method with simple UV detection was developed for the molecular mass analysis of sodium alginate. It was used to evaluate alginates of varying molecular mass and the results were compared with the viscosity measurements. This HPSEC method was sensitive to serve as the stability indicating method for alginate after storage under different conditions. The information of relative molecular mass distribution of alginate was provided with reference to pullulan molecular mass standards. The comparison of the HPSEC chromatograms of alginate, pullulan and dextran revealed the effect of chemical composition of a polysaccharide and its effect on apparent molecular mass distribution.     

Advances in humeomics: Enhanced structural identification of humic molecules after size fractionation of a soil humic acid

We size fractionated a soil humic acid (HA) by preparative high performance size exclusion chromatography (HPSEC) and evaluated the analytical capacity of humeomics to isolate and identify humic molecular components in the separated size-fractions. HA and its three size-fractions were chemically fractionated to extract non-covalently bound organosoluble compounds (ORG1), weakly ester-bound organosoluble (ORG2) and hydrosoluble constituents (AQU2), strongly ester-bound organosoluble components (ORG3), and final unextractable residues (RES4). According to their solubility, the extracts were characterized by either GC–MS or on-line thermochemolysis/GC–MS techniques. The humeomic sequence showed that the analytical yields of identified compounds in either ORG or AQU extracts of size-fractions were invariably larger than for the unfractionated HA. This was attributed to a weaker conformational stability of humic suprastructures obtained by HPSEC fractionation, thereby enabling an improved separation and identification of single humic molecules. In line with the supramolecular understanding of humic substances, we found that hydrophobic compounds were mainly distributed in the largest size-fraction, while hydrophilic components were eluted in the smallest size-fraction. Furthermore, compounds with linear chains or stackable aromatic rings associated in regular structures were more abundant in the former fraction, whereas irregularly shaped compounds, that hindered association in larger size, were mostly found in the latter fraction. Thus the structural characteristics of single humic molecules determined their mutual association in humic suprastructures, as well as their conformational strength and shape. The lack of de novo synthesized macropolymers in the unfractionated soil humic matter was confirmed by the absence of RES4 fractions in the separated size-fractions. Our results indicate that humeomics capacity to reveal the complex molecular composition of humic suprastructures was significantly improved by subjecting humic matter to a preliminary HPSEC fractionation.     

Behavior of polysaccharide assemblies in field-flow fractionation and size-exclusion chromatography

Asymmetric flow field-flow fractionation (AsFlFFF) and high-performance size-exclusion chromatography (HPSEC) are techniques for separating and characterizing macromolecules; until now the latter is more utilized for analyzing polysaccharides. The demand for characterizing complex, high-molar-mass polysaccharides has raised interest in the use of AsFlFFF in analyzing polymeric carbohydrates in addition to HPSEC. In this paper, we compare the behavior of arabinoxylan aggregates present in aqueous solution in AsFlFFF and HPSEC and their effect on the obtained molecular characteristics (molar mass averages and size). Although the amount of aggregates in aqueous arabinoxylan solutions may be low, their role needs to be understood to avoid erroneous interpretations of AsFlFFF and HPSEC data. When these two separation systems were compared, AsFlFFF seemed to possess more separation power for the differentiation of aggregates from individual chains than HPSEC. To our knowledge, this is the first report on the characterization of xylans with AsFlFFF.     

胰蛋白酶水解全酪蛋白反应过程中的分析

 将高效凝胶排阻 (HPSEC)技术与水解度 (DH)概念相结合 ,对酪蛋白 胰蛋白酶水解体系的酶解反应过程进行分析 ,得到定量表征复杂酶解反应进程和不同DH值时多样性酶解产物相对分子质量分布的二维图线 ;依据蛋白质结构信息 ,结合HPSEC实验谱图 ,对胰蛋白酶作用于酪蛋白时的酶解断裂位点进行剖析 ,初步推断反应历程 ,并得到理论酶解肽段的相对分子质量分布图及酶解物中活性多肽酪蛋白磷酸肽 (CPPs)肽谱。     

不同分子质量木质素磺酸钠对煤粉的分散作用研究

应用超滤分级方法,将木质素磺酸钠分成不同分子量范围的级分。傅立叶红外光谱（FTIR）分析结果表明,分子量为5 000～10 000的木质素磺酸钠分子中亲水基团如羟基、酚羟基、磺酸根质量分数最大,随着分子量的增加,亲水基团质量分数降低。实验结果发现分子量为10 000～50 000的木质素磺酸钠级分在煤粒表面的吸附等温线近似为L型,吸附量较大,并且使煤粒的表面动电电位（ζ电位）达到-52 mV,在各个级分中对盘江煤具有最好的分散降黏作用。进一步得出结论,分散剂的分散降黏作用与其在煤粒表面的吸附量和ζ电位的高低密切相关。     

Monodisperse lignin fractions as standards in size-exclusion analysis: Comparison with polystyrene standards

The difficulty of preparing monodisperse lignin fractions on a large scale is a limiting factor in many applications. The present paper addresses this problem by examining the properties and size-exclusion behavior of lignin isolated by the acetosolv pulping process from post-extraction crushed sugarcane bagasse. The isolated lignin was subjected to a solvent pretreatment, followed by preparative gel permeation chromatography fractionation. The fractions were analyzed by high-performance size-exclusion chromatography (HPSEC) and these samples showed a great decrease in polydispersity, compared to the original acetosolv lignin. Several fractions of very low polydispersity, close to unity, were employed as calibration curve standards in HPSEC analysis. This original analytical approach allowed calibration with these lignin fractions to be compared with the polystyrene standards that are universally employed for lignin molecular mass determination. This led to a noteworthy result, namely that the lignin fractions and polystyrene standards showed very similar behavior over a large range of molecular masses in a typical HPSEC analysis of acetosolv lignin.     

Size and shape of soil humic acids estimated by viscosity and molecular weight

Ultrafiltration fractions of three soil humic acids were characterized by viscometry and high performance size-exclusion chromatography (HPSEC) in order to estimate shapes and hydrodynamic sizes. Intrinsic viscosities under given solute/solvent/temperature conditions were obtained by extrapolating the concentration dependence of reduced viscosities to zero concentration. Molecular mass (weight average molecular weight (M (w)) and number average molecular weight (M (n))) and hydrodynamic radius (R(H)) were determined by HPSEC using pullulan as calibrant. Values of M (w) and M (n) ranged from 15 to 118 x 10(3) and from 9 to 50 x 10(3) (g mol(-1)), respectively. Polydispersity, as indicated by M (w)/M (n), increased with increasing filter size from 1.5 to 2.4. The hydrodynamic radii (R(H)) ranged between 2.2 and 6.4 nm. For each humic acid, M (w) and [eta] were related. Mark-Houwink coefficients calculated on the basis of the M (w)-[eta] relationships suggested restricted flexible chains for two of the humic acids and a branched structure for the third humic acid. Those structures probably behave as hydrated sphere colloids in a good solvent. Hydrodynamic radii of fractions calculated from [eta] using Einstein's equation, which is applicable to hydrated sphere colloids, ranged from 2.2 to 7.1 nm. These dimensions are fit to the size of nanospaces on and between clay minerals and micropores in soil particle aggregates. On the other hand, the good agreement of R(H) values obtained by applying Einstein's equation with those directly determined by HPSEC suggests that pullulan is a suitable calibrant for estimation of molecular mass and size of humic acids by HPSEC.     

Capillary modified with covalently attached coating for enhanced CE separation of biopolymers

δ‐Gluconolactone was covalently coupled with aminopropyl‐derivatized capillary, creating hydrophilic brushes on the inner wall of the capillary. The hydrophilic coating provided suppression of EOF and minimized protein adsorption, resulting in the separation of basic proteins and DNA with efficiencies up to 450 000 plates/m. The intra‐ and inter‐day repeatabilities of the coating referring to the migration times of the four tested proteins were satisfactory with RSD of no more than 1.1 and 1.8% (n=5), respectively. Two hundred consecutive runs were performed with negligible change in migration times and efficiency.     

Acidic polysaccharide mimics via ring-opening metathesis polymerization

An efficient and general synthetic strategy for the preparation of high-molecular-weight hydrophilic polymers bearing both carboxylic acid and hydroxyl pendant groups is described. Specifically, poly(5,6-dihydroxyoxanorbornane carboxylic acid) with molecular weight ranging from ～100?000 to 5?000?000 g/mol was prepared by ring-opening metathesis polymerization of methyl 5-oxanorbornene-2-carboxylate in the presence of Grubbs catalyst II and subsequently modified to tune the hydrophobic/hydrophilic properties by the introduction of either hydroxyl or carboxylic acid functionalities. These polymers mimic the natural acidic polysaccharide alginate and form hydrogels with polylysine. These polymers belong to a class of carbohydrate-like polymers, which are of interest for investigating the relationships between chemical structure and rheological properties as well as for providing new synthetic polysaccharide substitutes for applications in the biotechnology and pharmaceutical industries.     

Optimization of conditions for high-performance size-exclusion chromatography of different soil humic acids.

A method of high-performance size-exclusion chromatography (HPSEC) for a wide variety of soil humic acids (HAs) was developed. Two types of soil HAs (Cambisol and Andosol HAs), which have substantially different chemical properties, showed different effects of salt and organic solvent concentrations in the eluent on chromatograms. A Shodex OHpak SB-805 HQ column with 10 mM sodium phosphate buffer (pH 7.0) containing 25% of acetonitrile (v/v) was found to be applicable for different HAs, and showed high reproducibility and recovery (87.0 - 94.5%). The Cambisol HA was fractionated into five fractions using an ultrafiltration with different molecular-weight cut-offs. The order of the molecular weights of the five fractions calculated from the HPSEC analysis corresponded to that defined by ultrafiltration. This supported the reliability of the method.     

Multielement characterization of metal-humic substances complexation by size exclusion chromatography, asymmetrical flow field-flow fractionation, ultrafiltration and inductively coupled plasma-mass spectrometry detection: a comparative approach

The use of three different separation techniques, ultrafiltration (UF), high performance size exclusion chromatography (HPSEC) and asymmetrical flow field-flow fractionation (AsFlFFF), for the characterization of a compost leachate is described. The possible interaction of about 30 elements with different size fractions of humic substances (HS) has been investigated coupling these separation techniques with UV-vis absorption spectrophotometry and inductively coupled plasma-mass spectrometry (ICP-MS) as detection techniques. The organic matter is constituted by a polydisperse mixture of humic substances ranging from low molecular weights (around 1kDa) to significantly larger entities. Elements can be classified into three main groups with regard to their interaction with HS. The first group is constituted by primarily the monovalent alkaline metal ions and anionic species like B, W, Mo, As existing as oxyanions all being not significantly associated to HS. The second group consists of elements that are at least partly associated to a smaller HS size fraction (such as Ni, Cu, Cr and Co). A third group of mainly tri- and tetravalent metal ions like Al, Fe, the lanthanides, Sn and Th are rather associated to larger-sized HS fractions. The three separation techniques provide a fairly consistent size classification for most of the metal ions, even though slight disagreements were observed. The number-average molecular weight (Mn), the weight-average molecular weight (Mw) and the polydispersity (rho) parameters have been calculated both from AsFlFFF and HPSEC experiments and compared for HS and some metal-HS species. Differences in values can be partly explained by an overloading effect observed in the AsFlFFF experiments induced by electrostatic repulsion effects in the low ionic strength, high pH carrier solution. Size information obtained from ultrafiltration is not as resolved as for the other methods. Molecular weight cut-offs (MWCO) of the individual filter membranes refer to globular proteins and molecular weight information may therefore, deviate from that given by the other methods after calibration with polystyrene sulfonate (PSS) standards.     

HPLC approach for revealing age-related changes of aquatic dissolved organic matter in sediment core

Analytical method based on HPLC has been used to characterize aquatic dissolved organic matter (DOM) from sediment core of Lake Võrtsjärv, South- Estonia. High-performance size exclusion chromatography (HPSEC) as separation method was coupled with diode-array detection (DAD) and separated molecular fractions of DOM were subject to qualitative and semi-quantitative analysis. Qualitative analysis based on UV- spectra revealed the presence of proteins and humic constituents in separated high molecular weight fraction and aromatic constituents in low molecular weight fraction. Statistical data treatment methods enabled clustering sediment layers into 4 periods according to sediment depth and age. The upper 0-30 cm sediment DOM had statistically relevant differences in comparison to other periods as revealed by lower total peak and humic substances (HS) fraction areas, and molecular weights. Samples from 80-120 cm depth differed from others by decreased low molecular weight (LMW) fraction content. The observed down-core trends suggest polymerization of LMW organic constituents and increasing humification. The statistical analyses revealed that some chromatographic and spectrometric parameters can be used to differentiate between sediment layers and to evaluate environmental changes.     

A pH Effect in the HPSEC Separation of Polyacrylamide-Based Copolymers

Abstract

Four polyacrylamide-based samples, having the same nominal molecular weight and molecular weight distribution, have been characterized by HPSEC using two column sets and a mobile phase of phosphate buffer. Depending upon the pH, the separations demonstrate that the systematic differences between samples are influenced by the level of anionic sites along the polymer chain. It is found that the HPSEC elution profiles of these samples are reversed when the pH is changed from 2.2 to 7.0. The anionicity of the samples ranges from less than 0.5 mole % carboxyl content (the homopolymer of acrylamide) to 33.0 mole %.

Capillary viscometry data support these findings in the two solvent media, indicating that no adsorption effects prevail in the chromatography. Static and on-line low angle laser light scattering (LALLS) data on the homopolymer of acrylamide also suggest that no adsorption or degradation is occurring in the chromatographic process.     

Two-step chromatographic purification of recombinant human thyrotrophin and its immunological, biological, physico-chemical and mass spectral characterization

A purification strategy for rapidly obtaining recombinant human thyrotropin (rhTSH) was designed based on size exclusion and reversed-phase high-performance liquid chromatographic (HPLC) analysis, carried out on hTSH-secreting CHO cell conditioned medium. These analyses permitted the identification of the main contaminants to be eliminated. Considering that hTSH is highly hydrophobic and elutes only with the addition of organic solvents, hydrophobic interaction chromatography was adopted as the first purification step; this resulted in the elimination of, among others, the major contaminant. A second purification step, based on size exclusion chromatography, was then utilized, being effective in the elimination of other previously identified contaminating proteins. Useful purity, as high as 99% at the chemical reagent level, and recoveries (37%) were obtained by adopting this two step strategy, which also provided adequate material for physico-chemical, immunological and biological characterization. This included matrix-assisted laser desorption ionization time-of-flight mass spectral analysis (MALDI-TOF-MS), Western blotting analysis, in vivo biological assay, size-exclusion HPLC (HPSEC) and reversed-phase HPLC (RP-HPLC) analysis, which confirmed the integrity and bioactivity of our rhTSH in comparison with the only two reference preparations available at the milligram level of native (hTSH-NIDDK) and recombinant (Thyrogen) hTSH. Thyrogen and rhTSH-IPEN, when compared to pit-hTSH-NIDDK, presented more than twice as much biological activity and about 7% increased molecular mass by MALDI-TOF-MS analysis, an accurate heterodimer mass determination providing the Mr values of 29,611, 29,839 and 27,829, respectively. The increased molecular mass of the two recombinant preparations was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPSEC analysis. Comparing the two recombinant preparations, minor though interesting physico-chemical and biological differences were also observed.     

High-performance size-exclusion chromatography of porcine colonic mucins. Comparison of Bio-Gel TSK 40XL and Sepharose 4B columns

A high-performance size-exclusion chromatography (HPSEC) method was developed for the separation of porcine colonic mucins using a Bio-Gel TSK 40XL HPSEC column (300 mm x 75 mm). In addition, porcine gastric and bovine submaxillary mucin preparations were used to describe more fully the separation characteristics of the HPSEC column. For comparison, the same preparations were also separated using a Sepharose 4B column (100 cm x 2.6 cm). The colonic and gastric mucins eluted in the void volume (V0) of both columns. Bovine submaxillary mucin was in the elution volume (Ve) of both columns. Analytical HPSEC of fractions (V0 and Ve) of the various preparations obtained by Sepharose 4B chromatography exhibited retention times identical to those for fractions obtained by HPSEC. After separation by both methods, purified mucins were obtained by CsCl2 density gradient ultracentrifugation; analytical HPSEC profiles, protein contents, and monosaccharide compositions of both gastric and colonic mucins from either column were similar. The HPSEC method, however, is ideally suited to separate microgram to milligram quantities of colonic mucin preparations quickly: 2 to 4 h, compared with 24 to 30 h for the Sepharose 4B method.