Katsuo Komiya

Separation range and separation efficiency in high-speed gel filtration on TSK-GEL SW columns

Abstract Separation ranges for gel filtration on TSK-GEL SW columns, G2000SW, G3000SW and G4000SW, were measured for globular protein, dextran and polyethylene glycol. It was possible to separate globular proteins of molecular weights from 5000 to 7,000,000, dextrans of molecular weights from 1000 to 500,000 and polyethylene glycols of molecular weights from 500 to 250,000 by using these three columns of different pore sizes. The column having the highest separation efficiency for globular proteins was also determined. The highest separation efficiency for molecular weights of less than 30,000, 30,000–500,000 and greater than 500,000 was exhibited by G2000SW, G3000SW and G4000SW, respectively.

Evaluation of new supports for high-pressure aqueous gel permeation chromatography: TSK-GEL SW type columns

Abstract New chemically modified silica gel based supports, TSK-GEL SW type columns, for aqueous gel permeation chromatography have been evaluated in detail. Adsorption or denaturation of proteins and enzymes on these supports is negligible, the recoveries of the proteins and enzyme activities being both almost quantitative. The applications of these supports for the study of proteins, enzymes, saccharides and water-soluble sythetic polymer have been investigated.

High-speed gel filtration of proteins in sodium dodecyl sulphate aqueous solution on TSK-GEL SW type

High-speed gel filtration of proteins was performed in sodium dodecyl sulphate aqueous solutions on TSK-GEL SW type columns. The separation ranges of G2000SW, G3000SW and G4000SW were, respectively, 15,000–25,000, 10,000–100,000 and 15,000–300,000. G3000SW showed the highest separation efficiency in the molecular weight range below ca. 60,000, while G4000SW showed the highest separation efficiency above molecular weight of ca. 60,000. The elution behaviour of proteins was greatly affected by sodium phosphate concentration in the eluent and the optimum sodium phosphate concentration was 0.05–0.2 M.

Study of experimental conditions in high-performance ion-exchange chromatography of proteins

Abstract Commercial ovalbumin was measured by high-performance ion-exchange chromatography on TSK-GEL IEX-545 DEAE SIL and linear gradient elution with NaCl to investigate the effects of gradient, flow-rate, column length and sample loading on resolution and retention. As the gradient became less steep, both resolution and separation time increased. At constant gradient time, the resolution increased and the separation time decreased with increasing flow-rate, but at constant gradient volume, both resolution and separation time increased with decreasing flow-rate. Columns of 75 mm and 150 mm in length provided almost identical resolution. The maximum sample loading was approximately 0.5 mg for columns of 6 mm I.D. and almost independent of column length and injection volume.

Simultaneous analysis of monosaccharides and oligosaccharides by high-performance liquid chromatography with postcolumn fluorescence derivatization.

To develop a fluorimetric HPLC technique for the simultaneous microanalysis of reducing mono- and oligosaccharides, the technique of linear gradient elution was introduced into the postcolumn fluorimetric detemination system of reducing saccharides with benzamidine. Fluorescence measurement was performed at 288 nm for excitation and 470 nm for emission and an optimization study for this postcolumn fluorescence derivatization carried out. Under optimum conditions, the detection limits of D-glucose and maltohexaose were 1.78 and 2.59 pmol, respectively. The present method was successfully applied to saccharide analysis and should prove useful for automated simultaneous microanalysis of reducing mono- and oligosaccharides in foods.

Purification of enzymes by high-speed gel filtration on TSK-GEL SW columns

Abstract The purification of enzymes was investigated by high-speed gel filtration on TSK-GEL G3000SWG columns packed with porous silica gel deactivated by chemically bonded hydrophilic compounds. Crude β-galactosidase from bacterial cells and commercial urease were purified ca. 15-fold in a single gel filtration. These enzymes were eluted within an hour from the column and the recoveries of enzymatic activity were almost 100% although the operation was carried out at room temperature (22°). Samples up to 100 mg could be applied to the column without loss of separation efficiency.

Characterization of a new reversed-phase chromatographic column on a 2-μm porous microspherical silica gel

Abstract The physical and chromatographic properties of a reversed-phase column based on a 2-μm porous silica gel, TSKgel Super-ODS, were investigated. The Super-ODS column reveals higher column efficiencies (over 200 000 theoretical plates/m) at a higher flow-rate than that on a conventional column. High selectivity for a planar compound on Super-ODS implies that it possesses a polymeric ODS layer. Employing pure silica and a full end-capping procedure resulted in sharp peaks for basic and chelating compounds. To avoid the loss of column efficiency, the void volumes in an operating system such as connecting tubes, cell volume and sample volume, should be minimized as much as possible. The time constant of the detector also affected the column efficiency. The separation of peptides was achieved within 4 min.

Direct fractionation of proteins in particle-containing feedstocks by a filter paper pieces-based DEAE-cellulose column chromatography: Rapid, robust and low-cost capturing procedure for protein

Filter paper pieces-based (FPB) DEAE-celluloses was prepared for direct fractionation of proteins in particle-containing feedstocks. FPB DEAE-cellulose has a protein binding capacity equivalent to that of commercially available DEAE-cellulose. Crude extracts from porcine intestine and kiwi fruit pulp, which were unmanageable by commercially available chromatographic media due to rapid clothing, could be directly fractionated with FPB DEAE-cellulose column. In addition, effluents from an FPB DEAE-cellulose column were extensively clarified. The present approach can be used as a rapid, robust and low-cost capturing step for protein from particle-containing feedstocks.