Nobuharu Takai

Temperature-responsive chromatography using poly(N-isopropylacrylamide)-modified silica

A new concept in chromatography is proposed that utilizes a temperature-responsive surface with a constant aqueous mobile phase. The surface of the silica stationary phase in high-performance liquid chromatography (HPLC) has been modified with temperature-responsive polymers to exhibit temperature-controlled hydrophilic/hydrophobic changes. Poly(N-isopropylacrylamide) (PIPAAm) was grafted onto (aminopropyl)silica using an activated ester-amine coupling method. These grafted silica surfaces show hydrophilic properties at lower temperatures which, as temperature increases, transform to hydrophobic surface properties. The elution profile of five mixed steroids on an HPLC column packed with this material depends largely on the temperature of the aqueous mobile phase. Retention times increase with increasing temperature without any change in the eluent. Changes in the retention times of hydrophobic steroids were larger than those for hydrophilic steroids. The temperature-responsive interaction between PIPAAm-modified silica and these steroids is proposed to result from changes in the surface properties of the HPLC stationary phase by the transition of hydrophilic/hydrophobic surface-grafted IPAAm polymers. We demonstrate a novel and useful new chromatography system in which surface properties and the resulting function of the HPLC stationary phase are controlled by external temperature changes. This method should be effective in biological and biomedical separations of peptides and proteins using only aqueous mobile phases.

Analysis of peptides and proteins by temperature-responsive chromatographic system using N-isopropylacrylamide polymer-modified columns

A new method of HPLC using packing materials modified with a temperature responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), was developed. Homogeneous PIPAAm polymer and its copolymer with butyl methacrylate (BMA) were synthesized and grafted to aminopropyl silica by activated ester-amine coupling and they were used as packing materials. The surface properties and functions of the stationary phases are controlled by external temperature. Isocratic elution by aqueous mobile phase alone is the basis for separation of peptides and protein. The separation of the mixture of three peptides, insulin chain A and B and beta-endorphin fragment 1-27 was achieved by changing the column temperature with 0.9% NaCl aqueous solution as the sole eluent. Retention of peptides and proteins was controlled both by column temperature and by NaCl concentration in the aqueous mobile phases in this chromatographic system.

Acidic Catecholamine Metabolites and 5-Hydroxyindoleacetic Acid in Urine: The Influence of Diet

Concentrations of vanillylmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), vanillic acid (VA) and 5-hydroxyindoleacetic acid (5-HIAA) in urine from healthy subjects were determined by a high-performance liquid chromatography system with a mixed-mode (C18/ anion-exchange) column and an 8-channel electrochemical detector, in order to study the influence of diet, diurnal variation and age. The urinary excretion of 5-HIAA increased significantly after eating banana, pineapple, tomato, kiwi fruit and walnut. An increase in the urinary excretion of DOPAC and HVA after eating banana and that of VA after taking vanilla was also noted. The urinary excretion of VMA was not significantly influenced by any of the foods examined. The urinary excretion of 5-HIAA in the first-morning urine increased beyond the upper limit of the reference value when banana was taken at 2000 h the previous day. The excretion of all metabolites in the second-morning urine in the fasting state was within respective reference ranges. Diurnal variation of the excretion of VMA, DOPAC, HVA and 5-HIAA in urine was relatively small, but that of VA was large. The concentrations (mmol/mol creatinine) of VMA, DOPAC, HVA, 5-HIAA and VA in the first-morning urine from healthy subjects increased from 7 days after birth to 1 year and then decreased to adult levels at 13 years of age.

Characterization of adsorption gels prepared from plant biomaterials

The adsorption of Pb(II), Cu(II), Cd(II), and Zn(II) by new biomaterial gels synthesized in cross-linked reactions from agricultural wastes such as lemon, yuzu (Citrus junos), and coffee was investigated. Experimental data of adsorption equilibrium from the heavy metal solutions correlate well with Freundlich isotherm equations. The adsorption mechanism involved acid ion-exchange reactions between the heavy metal and the carboxylic acid of pectic acid in the lemon and yuzu gels, improving the amounts of Pb(II), Cu(II), Cd(II), and Zn(II) adsorbed onto the lemon and yuzu gels by approximately 5- and 6-fold, respectively. The maximum adsorption capacities of the yuzu and lemon gels for Pb(II) and Cu(II) were high. Among the biomaterial gels investigated, the lemon gel possessed the highest complexation ability with these heavy metals. In addition, the adsorption of nitrate ion (NO3−) was especially strong by the Pb(II)-adsorbed lemon gel, as shown by IR absorption spectrophotometry. These results indicate that lemon and yuzu gels can function, as new “green chemistry” adsorbents, which can be generated from recycled wastes, involve zero emissions, and provide effective recovery of precious resources.

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.

Analysis of ultraviolet-absorbing compounds in human urine by high performance liquid chromatography

Abstract Ultraviolet (UV)-absorbing compounds in human urine were analyzed by means of high-performance liquid chromatography using the column of macroreticular anion exchange resin maintained at 60°C. The sample was eluted with a linear gradient of water to 0.25 M ammonium perchlorate-acetonitrile (85:15) at 0–50 min and with 0.25 M ammonium perchlorate-acetonitrile (85:15) at 50–70 min and monitored with absorption at 254 nm. Reproducible chromatograms were obtained and 68 well resoluted peaks were numbered. The storage of a urine sample at −-20°C for 7 weeks did not significantly affect the chromatogram. The correlation coefficients of every pair of the numbered peaks were calculated to examine the daily variations and the individual difference of the UV-absorbing urine components.