Keiichi Kitahara

High-performance liquid chromatographic separation of carbohydrates on a stationary phase prepared from polystyrene-based resin and novel amines

New anion-exchange stationary phases On (n = 1, 2 and 3) with a dimethylamino terminal functional group, where n is the number of oxyethylene units [-(CH2CH2O)n-], were prepared by the reaction of chloromethylated porous styrene-divinylbenzene copolymer beads and amines [(CH3)2N-(CH2CH2O)nCH2CH2-N(CH3)2]. HPLC separations of monosaccharides (sorbitol, fucose, glucosamine, mannose, glucose, galactose, fructose, allose and altrose) and disaccharides (trehalose, lactose, cellobiose and maltose) were performed successfully on these stationary phases. The ether group of the stationary phases On was found to affect the separation of carbohydrates.

HPLC Separation of All Aldopentoses and Aldohexoses on an Anion-Exchange Stationary Phase Prepared from Polystyrene-Based Copolymer and Diamine: The Effect of NaOH Eluent Concentration

To investigate the separations of all aldopentoses (ribose, arabinose, xylose and lyxose) and aldohexoses (glucose, galactose, allose, altrose, mannose, gulose, idose and talose) on the D6 stationary phase prepared by the reaction of chloromethylated styrene-divinylbenzene copolymer and N,N,N’,N’-tetramethyl-1,6-diaminohexane, we examined the effect of varying the concentration of the NaOH eluent on the elution orders. Separations of these aldoses were achieved using a 20 mM NaOH eluent. The elution behaviors of the aldoses were probably due to not only the individual pKa values, but also the chemical structures of the cyclic aldoses.

High-performance liquid chromatographic separation of carbohydrates on stationary phases prepared from polystyrene-based resin and tertiary amines : Effect of chemical structure of anion-exchange sorbents

Abstract Two types of new anion-exchange stationary phases were readily prepared by the reaction of porous particles of chloromethylated styrene–divinylbenzene copolymer with various amines. The stationary phases Mn, where n is the length of alkyl chain, were obtained by the reaction with N,N-dimethylalkylamines. The reaction with N,N,N′,N′-tetramethyldiaminoalkanes also afforded the stationary phases Dn with a terminal functional group, where n is the length of methylene bridge. The HPLC separations of monosaccharides, disaccharides and oligosaccharides were successfully performed on these stationary phases using electrochemical detection with a Ni–Ti alloy working electrode in alkaline eluents. The effects of chemical structures of stationary phases on the separation of carbohydrates are described.

Complexation behavior of mono- and disaccharides by the vinylbenzeneboronic acid-divinylbenzene copolymer resins packed in a high-performance liquid chromatographic column.

Using an HPLC column packed with monodispersed vinylbenzeneboronic acid-divinylbenzene (V-D) copolymer resins, the elution behaviors of the mono- and disaccharides were studied under different pH mobile phases. The monodispersed V-D copolymer resins were prepared by the copolymerization of 4-vinylbenzeneboronic acid and divinylbenzene in the presence of template silica gel particles (particle size: 5 microm; pore size: 10 nm), followed by dissolution of the template silica gel using a NaOH solution. Similarly, styrene-divinylbenzene (S-D) copolymer resins as the control resins were also synthesized. The transmission electron micrographs of these polymer resins revealed a good monodispersity. The complexation behavior of the saccharides was evaluated by comparison of the peak area eluted through the V-D column for that through the S-D column. Four aldopentoses (D-ribose, D-arabinose, D-xylose, and D-lyxose) and four aldohexoses (D-glucose, D-mannose, D-galactose, and D-talose) were retained completely at pH 11.9. Especially, ribose and talose were totally retained even under acidic and neutral conditions. For the disaccharides, unlike sucrose and maltose, palatinose was completely retained in basic mobile phases.

Preparation of monodispersed vinylpyridine–divinylbenzene porous copolymer resins and their application to high-performance liquid chromatographic separation of aromatic amines

For the separation of aromatic amines, two types of monodispersed porous polymer resins were prepared by the copolymerization of 2-vinylpyridine and 4-vinylpyridine with divinylbenzene in the presence of template silica gel particles (particle size 5 microm), followed by dissolution of the template silica gel in an alkaline solution. The transmission electron micrographs and the scanning electron micrograph revealed that these templated polymer resins have a spherical morphology with a good monodispersity and porous structure. Using these monodispersed polymer resins, the high-performance liquid chromatographic separation of aromatic amines in the mobile phases of pHs 2.0, 2.9, 4.1, 7.2 and 11.7 were carried out. The 2-vinylpyridine-divinylbenzene copolymer resins showed slightly stronger retentions for aromatic amines than the 4-vinylpyridine-divinylbenzene copolymer resins. Under acidic conditions (around pH 2.0), aniline and the toluidines showed no retention on these copolymer resins due to the repulsion between the cationic forms of these amines and pyridinium cations in the stationary phase, whereas less basic aromatic amines or non-basic acetanilide showed slight retentions. Above pH 4.1, the separation of aromatic amines with these polymer resins showed a typical reversed-phase mode separation. Therefore, the separation patterns of aromatic amines are effectively tunable by changing the pH value of the mobile phases. A good separation of eight aromatic amines was achieved at pH 2.9 using the 2-vinylpyridine-divinylbenzene copolymer resins.

Measurement of insulin content at different sites of an insulin preparation vial

Insulin sampled at various levels within several insulin preparation vials underwent high-performance liquid chromatography and fluorescence spectrophotometry to determine insulin content. Insulin content did not vary with Actrapid Human Insulin®, Monotard Human Insulin®, and Novolin N100®, regardless of the site of sampling. However, the insulin content of Novolin U40® tended to decrease as levels in the vial decreased. Novolin U40 was also administered subcutaneously to rabbits to determine its effect on blood glucose. Blood glucose levels and the frequency of convulsions due to hypoglycemia varied depending on the site in the insulin preparation vial. Our findings suggest that variation in insulin content at different sites of the vial may account for unstable control of blood glucose levels in diabetic patients receiving Novolin U40.

Chiral Recognition Properties of Chiral 18-Crown-6 Ethers with Aromatic Substituents

The chiral recognition properties of chiral 18-crown-6 ethers with phenyl, 1-naphthoxymethyl, 1-naphthylmethyl, or 2,3,5,6-tetramethylphenylmethyl substituents for α-phenylglycine methyl ester and 1-phenylethylamine perchlorate were investigated by a standard extraction procedure. The chiral recognition factor of 1-naphthoxymethyl substituted crown ether is 2.0 for the former salt but near 1.0 for the latter, whereas that of the other crown ethers is not so dependent on the structure of salts, which indicates the importance of the mutual relation of the structure of host and guest molecules.