Analysis of Dispersion Mechanism in Gel Chromatography

The mechanism of dispersion of solute in gel chromatography using various Sephadex gels was quantitatively studied. In order to simplify the mathematical treatment, non-ad- sorptive low-molecular weight substances such as NaCl and glucose were chosen as samples. A pulse response experiment was carried out in a column. The longitudinal dispersion coefficient and the diffusion coefficient in gel phase were determined separately by applying the moment method to the elution curve. Then, their contribution to the column efficiency characterized by HETP was studied. Particularly, the effect of gel phase diffusion was examined in detail. The gel phase diffusion coefficient was apparently much smaller than the molecular diffusion coefficient. Consequently, it was revealed that gel phase diffusion played a much more important role in gel chromatography than what was expected by other investigators.     

Apparatus and method for preparative gel electrophoresis

A new apparatus for preparative gel electrophoresis with continuous elution which includes a miniaturized electrode and elution chamber system is described. The design provides high resolution, high yield, applicability for small and large amounts of peptide material, and easy operation. Furthermore, the apparatus enables a very accurate gel column or gel gradient to be formed. A method for preparative gel electrophoresis in sodium dodecyl sulfate which allows the purification of peptides and proteins without concurrently modifying tryptophane residues or blocking N-terminal α-amino groups is also described.     

A Stereoselective Synthesis of (±)-endo-Brevicomin,a Pheromone Inhibitor Produced by Dendroctonus Bark Beetles

Prediction of elution curves in gel chromatography was attempted on the basis of a mass balance model which gave consideration to gel phase diffusion and longitudinal dispersion in a column. The basic differential equations for the model were solved by means of Laplace transformation, and then the solution in Laplace domain was inverted into time domain numerically. The calculated elution curves were in good agreement with the experimental ones of NaCl and myoglobin with various Sephadex gel columns. This indicated the validity of the calculation method and the model employed in this study.

Furthermore, the elution curves were calculated tentatively for various combinations of the parameters appearing in the mass balance model. Then, the magnitude of peak asymmetry, the shift of peak position and the maximum peak height of the elution curves were correlated with various parameters and operational variables. These correlations might permit prediction of suitable operational conditions for gel chromatography, especially for molecular weight determination.     

Separation and preconcentration of copper and cadmium ions from multielemental solutions using Nostoc muscorum-based biosorbents

Nostoc-based biosorbents (AlgaSORBs) useful as chromatographic column-packing materials were prepared by immobilizing cyanobacteria onto solid support in three different fashions: (i) cyanobacterial biofilm (Nostoc-dimethylformamide slurry) over polymer-modified silica gel, (ii) cyanobacterial biofilm over bare silica gel, and (iii) cyanobacteria as such onto polymer-modified silica gel. The materials were characterized for their stabilities and metal sorption/elution conditions under static and dynamic equilibrations. Preconcentrated metals from a test sample were detected following `standard addition' method using a differential pulse anodic stripping voltammetric technique. All sorbents showed 100% affinity for Cd²⁺ ion in a multielemental sample at pH 6.9 and a flow rate of 0.5 ml/min with a preconcentration factor varying between 28- and 75-fold. The first type of AlgaSORB was also found to be selective for Cu²⁺ ion in multielemental analysis at pH 5.2 and a flow rate of 1.0 ml/min with a preconcentration factor of 75. The low capacity and favourable kinetics of these sorbents for Cu²⁺, Cd²⁺, Zn²⁺ and Pb²⁺ ions reflect the suitability of AlgaSORB columns for satisfactory performance in single column ion-chromatography. The polymer spacer between cyanobacterial biofilm and silica gel plays a vital role in holding the immobilized biofilm resulting in better endurance and recyclability for the first type of biosorbent. This revised version was published online in November 2006 with corrections to the Cover Date.     

Separation of Alkyl β-d-Glucosides and n-Alcohols by Using a Porous Trimethylolpropane Trimethacrylate Homopolymer Gel

Alkyl β-D-glucosides and n-alcohols were separated by high-performance liquid chromatography on a porou gel of a trimethylolpropane trimethacrylate homopolymer, using a mixture of water with methanol or acetonitrile as the eluent. The effect of the methanol and acetonitrile concentrations on the separability was examined, and the optimum concentration of each to separate alkyl glucosides and alcohols with alkyl chain lengths of six to twelve was determined. A model for analyzing the elution characteristics of the solutes for various levels of methanol or acetonitrile is proposed, based upon the chemical potential of the solutes and eluent at the interface between the gel and eluent phases. Conventional column chromatographic separation of an alkyl glucoside and an alcohol was also performed by using gel with a larger particle diameter.     

Method for the characterization of size-exclusion chromatography media for preparative purification of DNA restriction fragments

A protocol has been developed to characterize the potential of size exclusion chromatography media for the separation of DNA fragments at a preparative scale. A standard DNA mixture composed of -DNA cut with the restriction enzymes, AluI and HaeIII, was chromatographed on a column packed with the gel of interest. Fractions were collected and examined by PAGE. A digitized image of the gel was analyzed with the aid of a computer software program to determine the DNA fractionation range and selectivity curve for each chromatography gel. Four gels were characterized using this protocol. The effect of the elution buffer ionic strength on the fractionation of DNA was also investigated.The US Government right to retain a non-exclusive royalty-freelicense in and to any copyright is acknowledged.     

Extracellular Lytic Enzymes of Myxococcus virescens II. Purification of Three Bacteriolytic Enzymes and Determination of their Molecular Weights and Isoelectric Points

The bacteriolytic enzymes produced by Myxococcus virescens and previously concentrated and separated from most of the non-bacteriolytic proteins have been further separated and purified. The bacteriolytic enzyme solution was concentrated by lyo-philization. When applied to a Sephadex G-100 column, three peaks of bacteriolytic activity were eluted. Polyacrylamide gel electrophoresis showed that all the three enzyme fractions were contaminated with at least four non-bacteriolytic proteins. In the first enzyme fraction the bacteriolytic enzymes could be freed from the contaminating proteolytic activity by adsorption on a hydroxylapatite column. The bacteriolytic enzymes could then be adsorbed on a CM-cellulose column. The remaining contaminating proteins passed the column un-adsorbed while the bacteriolytic enzymes could be eluted with a gradient of 0.02–0.10 M ammonium hydrogen carbonate solution. The second enzyme fraction was adsorbed on a CM-cellulose column and then eluted with 0.03–0.15 M NH4 HCO₃. After rechromatography on a new column under the same conditions, all of the contaminating proteins had disappeared. For purification of the third enzyme fraction chro-matography on one single CM-cellulose column was sufficient. The elution of the adsorbed enzymes was performed with a gradient of 0.15–0.30 M NH₄HCO₃. The recovery of activity for each of the ion-exchange chromatography separations was at least 90%. The purity of the enzymes was tested by polyacrylamid gel electrophoresis. Each of the purified enzymes gave only one coloured band which coincided with the enzyme activity assayed in sliced gels. The molecular weights of the enzymes were determined by electrophoresis on acryl-amide gels containing sodiumdodecylsulphate. The molecular weights determined in this way (about 40,000, 30,000 and 20,000, respectively) were about 10,000 daltons higher than those obtained by gel chromatography on Sephadex G-100. This discrepancy seems to depend on interactions between the enzymes and the dextran molecules probably caused by the strongly basic nature of the enzymes or by formation of enzyme-substrate complexes.     

Comparative studies of seed proteins of the genusArtocarpus with respect to lectins

Proteins from two species of the genusArtocarpus (A. integrifolia L. andA. incisa L.) were compared by ammonium sulphate fractionation, molecular sieve chromatography and SDS-polyacrylamide gel electrophoresis, with special attention to the lectins. The protein content and hemagglutinating activity were markedly different in the two seeds. The protein pattern obtained by both molecular sieve chromatography and SDS-polyacrylamide gel electrophoresis were quite different. The only similarities found were the elution volume of the lectins in the Sephadex G-100 column and the lectin bands (11 500 and 15 000 daltons) in SDS-polyacrylamide gel electrophoresis.     

两步串联层析法纯化鼠抗人CD80单克隆抗体4E5

采用阴离子交换与凝胶过滤两步串联层析法,纯化了小鼠腹水来源的CD80阻断型单克隆抗体4E5。腹水样品经离心、过滤预处理后,在Tris-HCl缓冲溶液(pH8.0, 50mmol/L)条件下上阴离子交换柱对目的单抗进行捕集,采用0-0.5 mol/L NaCl浓度分步洗脱;含目的单抗的洗脱馏分再上凝胶过滤柱纯化,用PB缓冲溶液（pH7.2, 20mmol/L）洗脱,获得目的单抗4E5,其生物学活性高、纯度大于95%,抗体总回收率达61%。     

Acyl-coenzyme A: cholesterol acyltransferase assay: silica gel column separation of reaction products

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) assays are usually performed by incubation of the enzyme with a labeled substrate followed by thin-layer chromatography separation and subsequent quantification of cholesteryl esters (CE) formed. Herein, a method is described for rapid separation of CE from other lipids, by elution from a silica gel column with a solvent mixture of petroleum ether/diethyl ether (98:2, v/v). Silica gel column chromatography is reliable and more rapid and safer than TLC. The best results were obtained when the reaction was stopped by Dole extraction followed by CE separation on a silica gel column. Assays for ACAT from rat intestinal microsomes showed that the specific activity values obtained using this method were reproducible and in good agreement with those obtained by conventional TLC method.     

Purification of Plasmodium lophurae Exoerythrocytic Merozoites by an Ion Exchange Column*

SYNOPSIS Exoerythrocytic merozoites of Plasmodium lophurae grown in embryonic turkey brain cells were successfully separated from host cell material by elution from a DEAE-cellulose column at ionic strength 0.22. Purity of parasite samples was assessed by sodium dodecyl sulphate acrylamide gel electrophoresis and electron microscopy. Increasing the ionic strength gave greater recoveries of merozoites, but host cell contamination increased.     

Subunit analysis of bovine cytochrome c oxidase by reverse phase high performance liquid chromatography

Bovine cytochrome c oxidase subunits were separated by reverse phase high performance liquid chromatography using a C4 column eluted with water and an acetonitrile gradient, both containing 0.1% trifluoroacetic acid. Subunits I and III precipitated in this solvent and could not be analyzed; the remaining eleven subunits were dissociated, denatured, soluble and could be resolved by elution from the column. The protein subunit eluting in each chromatographic peak was identified by a combination of polyacrylamide gel electrophoresis in sodium dodecyl sulfate, NH2-terminal amino acid sequencing, and amino acid analysis. Each subunit produced a single elution peak with the exception of subunit VIc (nomenclature of Kadenbach et al., 1983, Anal. Biochem. 129, 517-521), which eluted from the column as two well-resolved peaks. Sequence analysis showed that the two subunit VIc elution peaks resulted from partial chemical blockage of the alpha-amino serine residue of subunit VIc. The C4 reverse phase HPLC was used to document specific subunit removal from bovine cytochrome c oxidase either by tryptic digestion or by dodecyl maltoside extraction. The described HPLC method for separating cytochrome c oxidase subunits should be applicable for the analysis of other multisubunit proteins, especially other multisubunit membrane protein complexes.     

Preparative Electrophoresis with On-column Optical Fiber Monitoring and Direct Elution into a Minimized Volume

A “column-format” preparative electrophoresis device which obviates the need for gel extraction or secondary electro-elution steps is described. Separated biomolecules are continuously detected and eluted directly into a minimal volume of free solution for subsequent use. An optical fiber allows the species of interest to be detected just prior to elution from the gel column, and a small collection volume is created by addition of an ion-exchange membrane near the end of the column.     

Reversed-phase HPLC enantioseparation of pantoprazole using a teicoplanin aglycone stationary phase—Determination of the enantiomer elution order using HPLC-CD analyses

A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column—S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers.     

超声波及脱蛋白处理对猴头菌子实体多糖提取效果的影响

本研究以猴头菌子实体为供试材料,在采用水提醇沉法进行多糖提取的过程中,系统研究了超声波和脱蛋白处理两个关键工艺环节对多糖提取效果的影响。研究结果表明：采用超声波辅助处理可提高猴头菌多糖提取率,降低多糖的纯度,使部分由蒸馏水洗脱获得的HEFP-α组分与全部0.1mol/L NaCl洗脱获得的HEFP-β组分经Sephacryl-S400洗脱后的曲线的小峰面积增加,也使主峰处的吸光值出现偏移。3种脱蛋白方法中,复合法对蛋白的除去效率最高,但多糖的保留率最低;亚铁氰化钾-乙酸锌法操作简便且蛋白除去率与多糖保留率均较高,但会使HEFP-β-2组分的含量和回收率降低;Sevage法蛋白脱除效率最低,多糖组分的保留率相对较高,且柱洗脱后发现经Sephacryl-S400凝胶柱分离后获得的主峰整体峰值较高,该方法较为适合多糖活性组分未知时的蛋白脱除。这一研究结果将会为该类大型真菌子实体的大分子生物活性物质的提取以及相关产品的开发提供重要的参考依据。     

Pulse response in an immobilized-enzyme column: Elution profiles in reversible and consecutive reactions

A pulse of substrate solution was applied to an immobilized-enzyme column, in which the substrate was then converted by reversible or consecutive reactions. Immobilized glucose isomerase was used for the reversible reaction, and immobilized invertase and glucose oxidase for the consecutive reactions. The elution profiles of substrate and product were determined experimentally. These profiles were in good agreement with the ones predicted theoretically. The effect of some parameters on the elution profiles for reversible and consecutive reactions is discussed.     

Isolation and characterization of bovine factor VII.

Factor VII (proconvertin) has been purified approximately 5 x 10(5)-fold from bovine plasma with an overall yield of 30%. The isolation procedure involves barium sulfate adsorption and elution, DEAE-Sephadex batchwise adsorption and elution, benzamidine-agarose column chromatography, heparin-agarose column chromatography, and preparative polyacrylamide gel disc electrophoresis. The final product was homogeneous when examined by gel electrophoresis in the presence of sodium dodecyl sulfate. A minimal molecular weight of 45,500 was determined by sedimentation equilibrium. The molecular weight estimated by sodium dodecyl sulfate gel electrophoresis was 54,000. Factor VII is composed of a single polypeptide chain possessing an amino-terminal sequence of Ala-Asn-Gly-Phe-Leu-. The amino acid and carbohydrate compositions of factor VII are also reported.     

Isolation of Lectins by Affinity Chromatography with Porcine Plasma Proteins Immobilized on Agarose

To develop a convenient method to isolate lectins, we prepared an affinity gel by coupling plasma proteins with agarose beads under conditions where the pH did not exceed 7.5. The validity of the use of this affinity gel in combination with elution using a hapten saccharide was confirmed by isolation of concanavalin A from Jack bean meal. Successful application of the method was demonstrated by isolation of two novel vegetable lectins from udo (Aralia cordate) and wasabi (Wasabia japonica). The method would be useful to isolate new lectins from various sources including plant and animal tissues.     

Quantitative separation of 1:8 cineole and β-phellandrene in eucalyptus leaf extracts

A method is described for separating 1:8-cineole and β-phellandrene in extracts of eucalyptus leaves on a silica gel column by elution with selective solvents and quantitative analysis of the separates by gas-liquid chromatography.     

Isolation of a Novel Auxin,Methyl 4-Chloroindoleacetate from Immature Seeds of Pisum sativum

Human casein was separated by gel filtration on a column of Sephadex G–200 with 0.1 m Tris buffer (pH 8.5) containing 1.0 m NaCl. The effluent which increased in turbidity at 25°C was centrifuged at 25,000 × g for 30 min and the precipitate was obtained as Fraction 6. After centrifugation, the effluent was separated into 5 elution fractions.

Disc gel electrophoretic patterns of each fraction showed occurrence of secondary bands other than major bands especially in Fractions 3, 4 and 5. The casein solutions unheated and heated at 100°C for 5 and 10 min were kept at 5°C for 5 days. No marked changes of electrophoretic pattern were observed among these casein solutions. However, when a casein solution heated at 100°C for 5 min was chroma to graphed under the same condition, secondary bands also appeared.