K. Hibi

A study of micro-high-performance liquid chromatography: I. Development of technique for miniaturization of high-performance liquid chromatography

Abstract Several technical problems such as the following have been solved in order to perform micro-high-performance liquid chromatography (MHPLC): (1) a method for packing a narrow column with stationary phase and selection of suitable column material; (2) preparation of micro-flow cells suitable for a micro-column; (3) improvement of the detector system; (4) methods for the pressurized passage of mobile phase at low flow-rates and for injection of a micro-amount of sample solution; and (5) gradient elution methods suitable for micro-columns. The theory of the spreading of sample components in narrow tubes for column connections is considered and examples of experiments using MHPLC are presented.

Studies of open-tubular micro-capillary liquid chromatography: II. Chemically bonded octadecylsilane stationary phase

Abstract A chemically bonded octadecylsilane stationary phase was successfully prepared on the inner glass surface of a micro-capillary tube of I.D. 60 μm. Using these capillary columns of length 3–20 m, several aromatic compounds were separated completely with methanol—water or acetonitrile—water as the mobile phase at a linear velocity of 0.3–1.3 cm/sec.

Studies of micro high-performance liquid chromatography : III. Development of a “micro-pre-column method” for pre-treatment of samples

Abstract A sample pre-treatment method is described in which components are adsorbed on a micro pre-column, and an investigation of the method (with phthalic acid esters in water as sample components) is reported. The esters could be concentrated in the pre-column simply and rapidly, and were separated in a micro separation column. Concentrations of diethyl phthalate in the ppb-range were rapidly determined by pre-treatment of 10 ml or less of the aqueous solution. The method was suitable for use in micro-performance liquid chromatography.

Studies of micro high-performance liquid chromatography : IV. Application of the micro pre-column method to the analysis of corticosteroids in serum

The micro pre-column method, which is a useful method for the pre-treatment of samples, was applied to the analysis of corticosteroids in serum. Corticosteroids were concentrated in a micro pre-column packed with porous styrene-divinylbenzene copolymer particles and separated in a micro separation column packed with porous silica particles. Concentrations of corticosterone, cortisone and cortisol in the 20-130 ppb range were determined individually by pre-treatment of only 0.2 ml of serum.

Studies of open-tubular microcapillary liquid chromatography : III. β,β′-oxydipropionitrile and ethylene glycol stationary phases

Abstract Capillary columns coated with β,β′-oxydipropionitrile and ethylene glycols have been applied successfully in open-tubular microcapillary column liquid chromatography. These stationary phases were spread out well on a soda-lime glass surface treated with alkaline solution. The capillary columns obtained have efficiencies of about 1500 theoretical plates per metre. Xylenol isomers, aromatic amines and phthalates were separated in a normal-phase system.

Studies of micro high-performance liquid chromatography : II. Application to gel permeation chromatography of techniques developed for micro high-performance liquid chromatography

Abstract Varius micro high-performance liquid chromatographic techniques have been successfully applied to gel permeation chromatography by reducing detector cell volume, injection volume and void volume in ports and joints. Phthalic esters, various oligomers ad polymers were separated on appropriate micro columns, as well as on ordinary wide-bore columns. Use of these micro columns means that smaller amounts of expensive packing materials and carrier solvents are required, and that the chromatographic operations and instruments can be simplified.

Studies on micro high-performance liquid cromatography : V. Design of a microscale liquid chromatograph and its application to cation-exchange separation of alkali metals

Abstract A microscle liquid chromatograph has been designed and optimized by estimating band broadenings. The high-performance cation-exchange resin, Hitachi No. 2610, was packed into the column, and applied to the separation of alkali metals by radioactivity detection. Four alkali metals were separated within 26 min. using only 208 μl of 0.7 N hydrochloric acid as carrier solution.

Sensitive assay system for bile acids and steroids having hydroxyl groups utilizing high-performance liquid chromatography with peroxyoxalate chemiluminescence detection.

3 alpha- or 3 beta-hydroxysteroids, such as bile acids (free and glycine and taurine conjugates), 3 beta-hydoxy-5-cholenic acid, pregnanediol, 5-pregnene-3 beta, 20 beta-diol and 5-pregnene-3-beta,20 alpha-diol, were converted to 3-oxosteroids by enzymatic reaction using immobilized hydroxysteroid dehydrogenase, derivatized with dansylhydrazine to the corresponding dansyl hydrazones and purified by gel permeation chromatography. The dansyl hydrazones were chromatographed on a C18 column with a tetrahydrofuran-containing eluent and detected at the level of a few femtomoles by a peroxyoxalate chemiluminescence post-column reaction using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate as a chemilumigenic reagent. The dansyl hydrazones of chenodeoxycholic acid and deoxycholic acid (free and glycine and taurine conjugates) in particular, which coeluted under the chromatographic conditions above, were separated using an eluent including acetonitrile and 2,6-di-O-methyl-beta-cyclodextrin and detected in the same way.

Direct injection of blood samples into a high-performance liquid chromatographic adenine analyser to measure adenine, adenosine and the adenine nucleotides with fluorescence detection

Adenine (Ade), adenosine (Ado) and its nucleotides such as AMP, cAMP, ADP and ATP in blood or plasma were determined by a high-performance liquid chromatographic (HPLC) adenine analyser with fluorescence detection. In order to inject samples directly into the HPLC system without pretreatment except dilution, the analyser consisted of two systems each, having three columns (pre-, mini- and analytical). A precolumn with an inlet filter of pore size 40 microns was common to both systems and packed with Butyl-Toyopearl 650-M to remove hydrophobic compounds and blood cell membranes. In the system for analysis of the nucleotides, a mini-column of Hitachi anion-exchange gel 3013-N was used for adsorbing AMP, cAMP, ADP and ATP. The adsorbed nucleotides were separated by the Hitachi gel 3013-N analytical column. In the other system for analysis of Ado and Ade, they were adsorbed on a Develosil ODS-5 mini-column and separated by an Asahipak GS-320H size-exclusion analytical column. The adenine compounds in each eluate were derivatized on-line in a 15-m reaction coil at 115 degrees C with bromoacetaldehyde as the fluorescent reagent in each mobile phase for the analytical column, and detected by spectrofluorimetry. ATP, ADP and AMP were accurately determined by the direct injection of hamster, rat and human whole blood. Authentic Ade and Ado were well separated and Ado in human plasma was determined, but it was difficult to determine it in rat plasma owing to interference from an unknown compound.

Alumina and support-coated open-tubular columns in open-tubular micro-capillary liquid chromatography☆

Abstract New methods have been developed for preparing columns in open-tubular micro-capillary liquid chromatography. Layers of aluminium oxide powder were formed on the inner walls of capillary tubing, and then the tubing was drawn into a micro-capillary column. Alternatively, alumina or silica (particle diameter 10–40 nm) was dispersed on the walls of a micro-capillary and used as solid adsorbent or support material for further treatment with octadecylsilane, respectively.