Yasuyo Okada

High-performance anion-exchange chromatography of homogeneous D-gluco-oligosaccharides and -polysaccharides (polymerization degree >50) with pulsed amperometric detection

Abstract High-performance anion-exchange chromatography under alkaline conditions with pulsed amperometric detection was applied to the analyses of (1 → 2)-, (1 → 3)-, (1 → 4)- and (1 → 6)-linked homogeneous α- or β- d -gluco-oligosaccharides and -polysaccharides up to a degree of polymerization (DP) of ⩾ 50. Each series of homogeneous d -gluco-oligomers and -polymers showed a linear relationship between log k′ and DP in isocratic elution using 150 mM sodium hydroxide solution containing 100 mM sodium acetate as the eluent. An effective separation of individual members of an homologous series of linear glucans was achieved using gradient elution, accomplished by maintaining the sodium hydroxide concentration at 150 mM and increasing the sodium acetate concentration during the analysis. The detector response per HCOH group in d -gluco-oligomers (DP 2–7) was almost the same.

High-performance liquid chromatography of mono- and oligo-saccharides on a graphitized carbon column

Abstract Chromatographic behavior of several mono- and di-saccharides cyclomaltaoses on Hypercarb, a graphitized carbon column, was investigated. Monosaccharides were weakly retained on this column and were eluted with water within 1.8–3.6 min at 1 mL/min of flow rate and 30°. Nevertheless, each peak of d -xylose, d -glucose, d -galactose, and l -fucose was split into anomer peaks. Disaccharides were adequately eluted with 15:85 methanol-water or 4:96 acetonitrile-water, and each showed two peaks for the anomers. These peaks each coalesced into a single peak upon the addition of m m sodium hydroxide of the eluent. Simultaneous separation of nine glucodisaccharides was achieved on this column by gradient elution with m m sodium hydroxide solution containing 1.5–5.0% acetonitrile. Detection was by a pulsed amperometric detector. Cyclomaltohexaose (αCD), cyclomaltoheptaose (βCD), cyclomaltooctaose (γCD), and their glucosyl derivatives (G-αCD, G-βCD and G-γCD) were analyzed by using 13–15% aq. acetonitrile as the eluent. Interestingly their retention times were increased with increases in temperature, and the sharpness of their peaks was also shown to be enhanced. Furthermore, positional isomers of glucosyl-inositol and of dimaltosyl-βCD, neither of which can be separated on conventional bonded phases, were well resolved on this column.

Analyses of homogeneous d-gluco-oligosaccharides and -polysaccharides (degree of polymerization up to about 35) by high-performance liquid chromatography and thin-layer chromatography

Abstract Conditions for the separation of (1→2)-, (1→3)-, (1→4)- and (1→6)-linked homogeneous d -gluco-oligosaccharides and -polysaccharides were investigated by high-performance liquid chromatography on a 3-μm chemically modified amine column (ERC-NH-1171) and by thin-layer chromatography (TLC) on three kinds of silica gel plates. Saccharide samples were prepared by partial hydrolyses or partial acetolyses of cyclosophoraose [cyclic (1→2)-β- d -glucan], curdlan [(1→3)-β- d -glucan], amylose [(1→4)-α- d -glucan], cellulose [(1→4)-β- d -glucan], dextran [(1→6)-α- d -glucan], and luteose [(1→6)-β- d -glucan]. Each series of saccharides, other than β- d -(1→3)- and β- d -(1→4)-linked ones, whose soluble higher oligomers could not be obtained, was well resolved from glucose to the polymer having a degree of polymerization (DP) of about 35 on the amine column by using simple isocratic elution with acetonitrile—water. TLC analyses were performed on silica gel 70 and silica gel 60 TLC plates with a concentration zone using n -butanol—ethanol—water as the developing solvent, and on Si 50000 HPTLC plates using n -butanol—pyridine—water. These methods made it possible to separate simultaneously each series of homogeneous saccharides of DP up to 20–30.

Isolation and characterization of novel heterogeneous branched cyclomalto-oligosaccharides (cyclodextrins) produced by transgalactosylation with α-galactosidase from coffee bean

Transgalactosylated derivatives of cyclomalto-hexaose (alpha CD), -heptaose (beta CD), and -octaose (gamma CD) were synthesized by alpha-galactosidase from coffee bean using melibiose as a donor and alpha CD, beta CD or gamma CD as an acceptor. Mono- and di-O-alpha-D- galactosylated CDs were isolated and purified by HPLC. Their structures were elucidated by fast-atom bombardment mass spectrometry (FABMS) and 13C NMR spectroscopy. For structural determination of positional isomers of 6(1),6n-di-O-alpha-D-galactosyl-CDs, digestion products with cyclodextrin glucanotransferase were analyzed by HPLC and FABMS.

Determination of cyclic glucans by anion-exchange chromatography with pulsed amperometric detection

Abstract Anion-exchange chromatography with pulsed amperometric detection was applied to the determination of cyclodextrins ( CD s), branched CD s and cyclosophoraoses. These cyclic glucans with degree of polymerization 6–25 were well resolved in each series by using simple isocratic elution with 150 m M sodium hydroxide solution containing 140–200 m M sodium acetate. The separation mode was not only simple anion exchange, but also involved some hydrophobic interactions and, moreover, inclusion interactions also seemed to take part in the retention. The detector response per glucose unit of these cyclic glucans was almost the same, regardless of the molecular weight and linkage form. The limit of determination of the cyclic glucans was 5–10 pmol and the detection limit was 2.5–5 pmol with a signal-to-noise ratio of 3.

Separation of cyclic (1→2)-β-D-glucans (cyclosophoraoses) produced by agrobacterium and rhizobium, and determination of their degree of polymerization by high-performance liquid chromatography

Conditions for the separation of the eight components contained in cyclic (1→2)-β-D-glucans (cyclosophoraoses) from Agrobacterium and Rhizobium were investigated by high-performance liquid chromatography on a chemically modified amine column (μBondapak CH) with mixtures of acetonitrile and water as eluent, and on a reversed-phase column (Dextro-Pak cartridge) with methanol—water (4:96) as eluent. Although retention on the amine column was related to molecular mass, that on the reversed-phase column differed from it, and was probably related to the solubility in water. The degree of polymerization of each cyclosophoraose was determined by high-performance liquid chromatography of its partial hydrolysate. Sophoro-oligomers having degrees of polymerization up to 24 were well resolved on a different amine column (Finepak SIL NH2, 10 μm) within 27 min by using a simple isocratic elution of acetonitrile-water (55:45). On hydrolysis under the appropriate conditions the longest straight chain sophoro-oligomer was present in sufficient amount to be able to recognize it in the partial hydrolysate; the distinguishing last peaks in the chromatograms of the eight cyclosophoraose hydrolysates could be detected clearly as the 17th–24th peaks.

Structural studies on cyclic (1→2)-β-d-glucans (cyclosophoraoses) produced by Agrobacterium and Rhizobium☆

Abstract Eight cyclic (1→2)-β- d -glucans (cyclosophoraoses) of different molecular weights were isolated from culture filtrates of Agrobacterium and Rhizobium . By 6.2-MPa, liquid chromatography of their partial hydrolyzates, it was concluded that they were cyclosophoroheptadecaose, cyclosophoro-octadecaose, cyclosophorononadecaose, cyclosophoroeicosaose, cyclosophoroheneicosaose, cyclosophorodocosaose, cyclosophorotricosaose, and cyclosophorotetracosaose. Cyclic (1→2)-β- d -glucans from 19 strains of Agrobacterium and Rhizobium tested were divided into four classes on the basis of differences in the distribution patterns of the eight cyclosophoraoses.

Further studies on the separation of cyclic (1→2)-β-d-glucans (cyclosophoraoses) produced by rhizobium meliloti ifo 13336, and determination of their degrees of polymerization by high-performance liquid chromatography

Abstract Eight pure cyclic (1→2)-β- d -glucans (cyclosophoraoses) varying in size from 17 to 24 residues were previously isolated from culture filtrates of Agrobacterium and Rhizobium. Thereafter further studies on the separation of cyclosophoraoses by high-performance liquid chromatography (HPLC) with small-particle columns showed an occurrence of many cyclosophoraoses having degrees of polymerization (DPs) of more than 24. One sample prepared from Rhizobium meliloti IFO 13336 contained large amounts of higher cyclosophoraoses of up to at least DP 40, which were separated clearly on a 3-μm chemically modified amine column (ERC-NH-1171). Some pure cyclosophoraoses with higher DPs were isolated by liquid chromatography using reversed-phase columns, and their DPs were determined by HPLC of their partial hydrolysates.

Retention behaviour of cyclodextrins and branched cyclodextrins on reversed-phase columns in high-performance liquid chromatography

Abstract The theoretical plate numbers and the asymmetry factors of eight columns packed with C18-bonded phases were measured to obtain a reliable indication of column performance and for column-to-column comparisons. Among those columns, including silicone-coated silica gel- and porous polymer gel-based new C18 columns, silica-based, endcapped and monomeric phase columns showed the best performance. Cyclodextrins (CDs) and branched CDs were separated satisfactorily on six C18 columns with 3–7% aqueous methanol. Furthermore, multi-branched CDs could be separated from their isomers having the same molecular size. The elution order of CDs and branched CDs on C18 columns with aqueous methanol may or may not coincide with their solubilities in the mobile phases.

Preparation of cyclosophoraose-A and its complex-forming ability

Cyclosophoraoses (CySs) are unbranched cyclic (1→2)-β-D-glu-cans produced by many strains of Agrobacterium and Rhizobium. Pure CyS-A, the group member having the smallest molecular size (degree of polymerization 17), was efficiently prepared by liquid chromatography using charcoal and ODS columns from the culture fluid of the mutant strain RA-12 from R. phaseoli AHU 1133. The complex-forming ability of CyS-A was estimated from its enhancement of the solubilities of slightly soluble guest molecules in water using methods [I], [II], and [III]. In [I], an aqueous solution of CyS-A was shaken with the guest molecule, while, in [II], it was shaken with an acetone solution of the guest compound. In method [III], freeze-dried CyS-A powder was stirred with an acetone solution of the guest compound. The CyS-A cavity is thought to be able to accommodate three-dimensionally extended guest molecules, e.g., indomethacin. Method [II] was the best for obtaining CyS-A inclusion complexes, while method [III] would be recommended if the guest molecule is labile in the presence of water. Crystalline CyS-A inclusion complexes have not been obtained, but CyS-A complexes are expected to greatly enhance the solubilities of slightly soluble or insoluble guest molecules in water, because CyS-A is much more soluble than β-cyclodextrin. Method [II] or [III] may afford a useful means of obtaining oily drug, e.g., vitamin E and K1, in an amorphous state.