Determination of Polysaccharides in Undaria pinnatifida by Ionic Liquid-Modified Silica Gel Size Exclusion Chromatography

Ionic liquid-modified silica particles with a large pore size were synthesized and used as the stationary phase for size exclusion chromatography coupled with a refractive index detector for the determination of polysaccharides, such as fucoidan, alginic acid, and laminarin from Undaria pinnatifida (seaweed). The molecular weight of polysaccharide was determined by a dextran standard curve (5–1100?kDa). The ionic liquid-modified silica column exhibited excellent size exclusion properties in separating laminarin from other components. The 1-butyl-3-methyl-imidazolium bis-(trifluoromethylsulfonyl)-amino silica column has superior resolution in laminarin separation than the other columns because the amino-group in ionic liquid provide π–π interactions due to aromaticity of the ring structure, which enhances the effect of the hydroxyl group in the target compound separation. The concentrations of polysaccharides were calculated using a standard linear equation to be 0.332–0.484?mg/g of fucoidan, 0.207–0.301?mg/g of alginic acid, and 0.154–0.297?mg/g of laminarin.     

Application of Ionic Liquid Modified Silica for Solid-Phase Extraction of Polysaccharides from Laminaria japonica

Silica-confined ionic liquids were synthesized for solid-phase extraction of polysaccharides, which was determined by high-performance liquid chromatography coupled with RI detection. The sorbent with amino-imidazolium groups was found to be the optimal material by a comparison of the adsorption capacity of fucoidan and laminarin onto different synthetic sorbents. The proper elution solvents for both laminarin and fucoidan were decided from practical tests. The sorbent performed stably and selectively, demonstrating potential applications in the separation of hydrophilic biomacromolecules, such as polysaccharides.     

Magnetic molecularly imprinted polymers for recognition and enrichment of polysaccharides from seaweed

New magnetic molecularly imprinted polymers with two templates were fabricated for the recognition of polysaccharides (fucoidan and alginic acid) from seaweed by magnetic solid‐phase extraction, and the materials were modified by seven types of deep eutectic solvents. It was found that the deep eutectic solvents magnetic molecularly imprinted polymers showed stronger recognition and higher recoveries for fucoidan and alginic acid than magnetic molecularly imprinted polymers, and the deep eutectic solvents‐4‐magnetic molecularly imprinted polymers had the best effects. The practical recovery of the two polysaccharides (fucoidan and alginic acid) purified with deep eutectic solvents‐4‐magnetic molecular imprinted polymers in seaweed under the optimal conditions were 89.87, and 92.0%, respectively, and the actual amounts extracted were 20.6 and 18.7 μg/g, respectively. To sum up, the developed method proved to be a novel and promising method for the recognition of complex polysaccharide samples from seaweed.     

Solid-Phase Colorimetric Method for the Quantification of Fucoidan

We described the simple, selective, and rapid method for determination of fucoidans using methylene blue staining of sulfated polysaccharides, immobilized into filter paper and consequent optic density (at A ₆₆₃?nm) measurement of the eluted dye from filter paper. This solid-phase method allows selective determination of 1?C20???g fucoidan in presence of potentially interfering compounds (alginic acid, DNA, salts, proteins, and detergents). Further, we demonstrated the alternative way of using image processing software for fucoidan quantification without extraction of methylene blue dye from stained spots of fucoidan?Cdye complex.     

Characterization of dextrans by size-exclusion chromatography on unmodified silica gel columns, with light-scattering detection, and capillary electrophoresis with laser-induced fluorescence detection

Unmodified silica gel size-exclusion columns were used in an on-line combination with light-scattering detection for a size characterization of dextrans. The influence of electrostatic interactions on analyte retention was briefly investigated. Size-exclusion chromatography was also used for evaluation of the fluorescence labeling procedure for dextrans with 8-aminonaphthalene-1,3,6-trisulfonic acid. The derivatives obtained through this procedure were used for electrophoretic measurements with laser-induced fluorescence detection. A comparison between the size-exclusion data and capillary electrophoresis indicates that the effectiveness of fluorescent labeling decreases with molecular mass of the dextran analytes.     

Size-exclusion capillary electrochromatographic separation of polysaccharides using polymeric stationary phases

We report the successful size-based separations of large, neutral polysaccharides using capillary electrochromatography (CEC). As the polysaccharides possessed little chromophore for photometric detection, two separate approaches were taken. In the first approach, indirect detection was combined with size-exclusion chromatography using a sulfonated polystyrene/divinylbenzene stationary phase. The separations were performed using a 300 A pore size stationary phase under aqueous conditions. Non-size based interactions were minimal using this material, resulting in an effective calibration range of molecular masses 180 to 112 000 g.mol(-1) for pullulans. In the second approach, the polysaccharides were derivatized with phenylisocyanate and were subsequently separated on columns made using a combination of high capacity ion-exchanger and a neutral polystyrene/divinylbenzene material of various pore sizes. The sulfonated ion-exchange phase provided the electroosmotic flow, while the mixed pore size material provided the extended calibration range. The linear range for this primarily nonaqueous system using tetrahydrofuran was determined to be from molecular masses 738 to 404 000 g.mol(-1) of the original, untagged pullulan. This approach overcame the limited solubility issue associated with analysis of some polysaccharides. Analysis of pullulan and amylose samples by CEC correlated well with results obtained by conventional high-performance liquid chromatography (HPLC). The size-exclusion electrochromatographic separations provide an alternative mode for determining the relative molecular weights of polysaccharides with reduced sample and solvent consumption, as well as analysis times.     

Size exclusion (radio) chromatography of aqueous humic acid solutions with cesium and strontium

The high-performance size-exclusion chromatography (HPSEC) and radiochromatography (HPSERC) was used for the identification of radiocesium and radiostrontium interaction with humic acid. It was found that the behavior of humic acid on size-exclusion chromatography is sensitive to the salt concentration and pH of the mobile phase. At lower ionic strength and in acidic region of pH, the Aldrich humic acid exhibited three main fraction within the ranges >760 kDa, 25–100 kDa and <5 kDa. Radiocesium was found in the low-molecular fractions (<1 kDa) of humic acids but radiostrontium interacts preferably with the fractions of humic acid of molecular weight within the range 2–5 kDa.     

Molecular weight determination of hyaluronic acid by gel filtration chromatography coupled to matrix-assisted laser desorption ionization mass spectrometry.

An analytical approach has been described for the molecular weight characterization of enzymatically degraded hyaluronic acid (HA). The approach involved the combined use of aqueous gel filtration chromatography (GFC) with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Microfractions were collected across an eluting peak from the chromatography system, followed by mass spectrometric analysis of these narrow fractions. The molecular mass determined by MALDI-MS and the signal obtained from the chromatography established a calibration curve for other hyaluronic acid samples analyzed by this GFC system. Results of one HA sample were obtained from both the calibration curve and direct fraction-by-fraction MALDI-MS analysis, and comparison of these results showed reasonable agreement. In contrast, molecular weights resulted from external calibration using dextran and pullullan standards showed drastically different numbers. Therefore, the GFC-MALDI-MS approach is a reliable method for the molecular weight characterization of polydisperse polysaccharides for which suitable calibration standards are unavailable for conventional GFC analysis.     

Composition,structural characteristics,and antitumor properties of polysaccharides from the brown algae <Emphasis Type="Italic">Dictyopteris polypodioides</Emphasis> and <Emphasis Type="Italic">Sargassum</Emphasis> sp.

The polysaccharide compositions of the brown algae Dictyopteris polypodioides and Sargassum sp. from the Mediterranean Sea were determined. The principal polysaccharide of the studied algae (about 12% of the dry alga weight) was alginic acid. The content of water-soluble polysaccharides was low. The amount of fucoidan was less than 1% of the dry alga weight; of neutral polysaccharides, less than 0.25%. The monosaccharide compositions of fucoidans and neutral polysaccharides were investigated. Experiments on soft agar-agar models showed that fucoidans from D. polypodioides and Sargassum sp. exhibited antitumor activity against RPMI-7951 human melanoma cells.     

Chemical depolymerization of dextran sulfate sodium - the application of size-exclusion or ion-pair liquid chromatography

We reported previously a method for the separation and quantification of sulfated polysaccharides, dextran sulfate sodium (DSS), using fluorometric labeling and size-exclusion chromatography (SEC). In the present study, we evaluated chemical depolymerization of DSS. In fluorometric analyses using SEC, pyridylamino-DSS (PA-DSS) was depolymerized under acidic conditions, but not under alkaline conditions. In addition, we used ion-pair liquid chromatography (IPLC) and UV analyses for the separation and quantification of sulfate. Sulfate was considerably depleted from DSS under alkaline conditions, but little depleted under acidic conditions. Spectrophotometric studies using toluidine blue revealed that molecular mass is an important factor and a minimum molecular mass of 2500 is needed to induce metachromasia. Fluorometric analyses using SEC and UV analyses using IPLC allow for the detection of DSS, reactants and sulfate.     

Size exclusion chromatography/mass spectrometry applied to the analysis of polysaccharides.

A novel method for analysing polysaccharide materials is described which employs size-exclusion chromatography (SEC) followed by detection by on-line electrospray ionisation mass spectrometry (ESI-MS) and off-line matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). It is demonstrated through SEC/ESI ion trap mass spectrometry that the formation of multiply charged oligomer ions, which bind up to five sodium cations, allows the rapid analysis of polysaccharide ions with molecular weights in excess of 9 kDa. MALDI spectra generated from fractionation of the effluent collected from the same SEC separation are shown to be in good agreement with the ESI spectra with respect to molecular weight distributions and types of ions generated. ESI and MALDI mass spectra of samples obtained from sequential graded ethanol precipitation and SEC fractionation of acid and enzymatically digested arabinoxylan polysaccharides show important structural differences between polysaccharide fragments. In addition, a comparison is made between the mass spectra of native and permethylated SEC-separated fragments of acid and enzymatically treated arabinogalactan. Linkage information of the permethylated arabinogalactan oligomers can be rapidly established through the use of on-line SEC/ESI-MS( n) experiments.     

糖的基质辅助激光解吸/电离质谱(MALDI-MS)研究

通过糖类化合物3种常用基质MALDI-MS分析效果的比较以及寡糖和多糖正、负离子MALDI-MS谱的对比,找到了适合糖分析的基质2,5-DHB,探讨了糖类化合物激光解吸/电离条件下形成离子的过程,指出了Na⁺、K⁺离子在寡糖分子量测定中的重要作用,借助柱层析分离手段,成功地测出了分子量大于10000的葡聚糖的分子量。     

Seasonal variations of the composition,structural features,and antitumor properties of polysaccharides from <Emphasis Type="Italic">Padina pavonica</Emphasis> (Lebanon) as a function of composition

The variation of the polysaccharide composition of the brown alga Padina pavonica from the Mediterranean Sea was studied as a function of the collection season (April–July). It was shown that the principal polysaccharide (8.0-13.3% of dry alga weight) was alginic acid. Its content did not undergo significant changes from April to July. The content of water-soluble polysaccharides (laminarans and fucoidans) was low (<0.3% of dry alga weight). Water-soluble polysaccharides of the studied alga were practically pure fucoidan. The amount of laminarans was insignificant (<0.01% of dry alga weight). The content of fucoidans increased in alga samples from May through July. It was shown that P. pavonica is a promising source of alginic acids, which have a high capacity for gelation, and fucoidans, which exhibit antitumor action against RPMI-7951 human melanoma cells.     

Electroanalysis of cationic species at membrane-carbon electrodes modified by polysaccharides. Bioaccumulation at microorganism-modified electrodes

Membrane-carbon electrodes modified with polysaccharides suspensions entrapped between a dialysis membrane and the carbon surface were used for electroanalysis of various cationic species. Cationic complexes of ruthenium and cobalt, metallic cations (Cu²⁺, Fe³⁺, UO₂²⁺) as well as methylviologen were considered. By investigating various parameters (concentration of the suspension, pH) binding of the cations by the polysaccharides was demonstrated. Comparison of cations uptake by different kinds of polysaccharides such as alginic acid, polygalacturonic acid, pectin, dextran and agar was performed. This study has been extended to natural biomaterials, alga and lichen, which are known to contain polysaccharides. The interest of the membrane–electrode strategy is described.     

Comparison of the separation of proteins of wide-ranging molecular weight via trilobal polypropylene capillary-channeled polymer fiber,commercial superficiously porous,and commercial size exclusion columns

Reversed phase and size-exclusion chromatography methods are commonly used for protein separations, although they are based on distinctly different principles. Reversed phase methods yield hydrophobicity-based (loosely-termed) separation of proteins on porous supports, but tend to be limited to proteins with modest molecular weights based on mass transfer limitations. Alternatively, size-exclusion provides complementary benefits in the separation of higher mass proteins based on entropic, not enthalpic, processes, but tend to yield limited peak capacities. In this study, microbore columns packed with a novel trilobal polypropylene capillary-channeled polymer fiber were used in a reversed phase modality for the separation of polypeptides and proteins of molecular weights ranging from 1.4 to 660 kDa. Chromatographic parameters including gradient times, flow rates, and trifluoroacetic acid concentrations in the mobile phase were optimized to maximize resolution and throughput. Following optimization, the performance of the trilobal fiber column was compared to two commercial-sourced columns, a superficially porous C4-derivatized silica and size exclusion, both of which are sold specifically for protein separations and operated according to the manufacturer-specified conditions. In comparison to the commercial columns, the fiber-based column yielded better separation performance across the entirety of the suite, at much lower cost and shorter separation times.     

Magnetic graphene oxide modified by imidazole‐based ionic liquids for the magnetic‐based solid‐phase extraction of polysaccharides from brown alga

Magnetic graphene oxide was modified by four imidazole‐based ionic liquids to synthesize materials for the extraction of polysaccharides by magnetic solid‐phase extraction. Fucoidan and laminarin were chosen as the representative polysaccharides owing to their excellent pharmaceutical value and availability. Fourier transform infrared spectroscopy, field‐emission scanning electron microscopy, and thermogravimetric analysis were applied to characterize the synthesized materials. Single‐factor experiments showed that the extraction efficiency of polysaccharides was affected by the amount of ionic liquids for modification, solid–liquid ratio of brown alga and ethanol, the stirring time of brown alga and ionic liquid‐modified magnetic graphene oxide materials, and amount of 1‐(3‐aminopropyl)imidazole chloride modified magnetic graphene oxide materials added to the brown alga sample solution. The results indicated that 1‐(3‐aminopropyl)imidazole chloride modified magnetic graphene oxide possessed better extraction ability than graphene oxide, magnetic graphene oxide, and other three ionic‐liquid‐modified magnetic graphene oxide materials. The highest extraction recoveries of fucoidan and laminarin extracted by 1‐(3‐aminopropyl)imidazole chloride modified magnetic graphene oxide were 93.3 and 87.2%, respectively. In addition, solid materials could be separated and reused easily owing to their magnetic properties.     

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)。     

Effects of dextran polydispersity on red blood cell aggregation

B 512 F dextran fractions of different polydispersities were prepared by fractionnal precipitation and preparative elution chromatography. By combination of low angle laser light scattering (LALLS) and gel permeation chromatography (GPC), absolute average molecular weights (MWs) and molecular weight distribution functions (MWDs) were determined. Static and thermodynamical properties in terms of polymer dimensions and second virial coefficient of dilute solutions of dextran in water have been investigated. The results indicate that dextran macromolecules in water are rather compact and impenetrable coils. Measuring the disaggregation shear stress of dextran-induced red blood cell aggregates by laser light reflectometry, the macromolecular bridging energy was shown to depend upon dextran sample polydispersity. This reflects the weak and reversible character of red blood cell aggregation by dextran chains in physiological saline solution.     

Polysaccharides of algae 65. Unusual polysaccharide composition of the pacific brown alga Punctaria plantaginea

Quantitative determination revealed the presence of storage glucan (6.0%), fucoidan (19.2%) and alginate (12.7%) in the biomass of the brown alga Punctaria plantaginea collected from the Sea of Japan. The polysaccharides were isolated from the alga by fractional extraction followed by additional purification procedures. Unlike the well-known laminarans the storage polysaccharide from P. plantaginea was shown to be a linear (1→6)-β-d-glucopyranan, which is new for brown alga. The content of guluronic acid (G) residues in the alginate molecules exceeded the content of mannuronic acid residues (M), M/G = 0.5. Poly-G and poly-MG blocks were isolated from the products of partial hydrolysis of alginic acid; however, a heterogeneous mixture of polysaccharide fragments was obtained instead of the expected poly-M fraction. Preliminary data suggests that fucoidan from this alga is a new for brown algae type of sulfated polysaccharide (xylofucan) with a main backbone built of α-l-fucopyranose residues. This chain contains multiple sulfate groups and single non-sulfated β-d-xylopyranose residues as substituents.     

Quality control of clinical dextran by rapid gel-permeation chromatography

Summary Clinical dextran can be fractionated by rapid gel-permeation chromatography on Spheron^R with the aid of a high-performance liquid chromatograph. The linear calibration function is obtained by dextran fractions whose molecular weights Mw and Mn are determined by absolute methods (light scattering, membrane osmometry) and end group analysis. The transformation of elution diagrams into a molecular weight distribution is described. Characteristic data for dextran 40 and 70 were obtained from the integral distribution curves. The molecular weight distribution of dextran fractions was found to fit a Schulz-Zimm distribution which is typical for long chain polymers. For quality control purposes clinical dextran can be characterized by its molecular weight distribution with high accuracy within one hour.