Yasuo Ishida

New detection and separation method for amino acids by high-performance liquid chromatography

A simple system for simultaneous fluorescence detection of primary and secondary amines has been developed. The optimum conditions for fluorescing secondary amines in the two-stage reaction technique using sodium hypochlorite and o-phthalaldehyde have been studied, and then the switching flow method (sodium hypochlorite is added only to secondary amines) and the non-switching flow method (sodium hypochlorite is added to all the amino acids) have been compared. It was concluded that the non-switching flow method ensures high efficiency and reliability. This newly-developed system was applied to the detection of amino acids and gave satisfactory results.

Postcolumn fluorometric detection system for liquid chromatographic analysis of amino and imino acids using o-phthalaldehyde/N-acetyl-L-cysteine reagent

A high-performance liquid chromatographic system for the determination of amino acid and imino acid was developed using a two-step reaction with sodium hypochlorite and o-phthalaldehyde/N-acetyl-L-cysteine (OPTA/AcCys) reagent. This reagent improved the sensitivity in the analysis of proline presumably because the fluorophore is more stable to hypochlorite which has been used for the oxidative cleavage of the imino linkage. The use of OPTA/AcCys facilitated the detection of imino acids at the same concentration level as that of amino acids. The detection limit for all the amino and imino acids was a few picomoles. This detection system, together with cation-exchange chromatographic separation, was applied to the determination of amino and imino acids in biological samples.

Separatioon and detection of small amounts of catecholamines by high-performance liquid chromatography

Abstract The effects of mobiel phase pH, flow-rate, column temperature, etc., on the separation of catecholamines by high-performance liquid chromatography (HPLC) on Zipax SCX and Zorbax ODS columns were investigated. Fluorimetric detection methods based on post-column reaction using trihydroxyindole and o-phthalaldehyde methods were employed and the results are compared. Optimal reaction conditions ensuring a high sensitivity of the fluorimetric detection were established. Based on the results, a new HPLC system utilizing the trihydroxyindole method has been produced on a trial basis. The favourable detection limits (ca. 20–30 pg) achieved permit highly accurate and selective assays for noradrenalien and/or adrenaline in tissues and urine.

High-performance liquid chromatographic separation of biological macromolecules on new silica-based ion exchangers.

The fundamental characteristics of a new series of ion-exchange columns, specifically designed for high-speed analysis of biological macromolecules, have been examined. Each of these columns is packed with a chemically modified silica gel, that has a controlled pore-size distribution and a uniform particle diameter. Surface silanol groups of these materials are chemically modified with hydrophilic polymers to produce a neutral silica surface, and ionic functional groups are chemically bonded to this neutral support, producing a stable ion exchanger. The separation of biological macromolecules on these columns has been investigated, and the selection of a mobile phase is discussed.