Tatsuro Nakagama

Molecularly imprinted polymer as chiral selector for enantioseparation of amino acids by capillary gel electrophoresis

SummaryA capillary electrophoresis method for the enantioseparation of D,L-aromatic amino acids using molecularly imprinted polymer as chiral selector has been developed. Methacrylic acid was used as functional monomer and ethylene glycol dimethacrylate (EDMA) as crosslinking monomer. The molecularly imprinted polymer was packed in capillaries by incorporating with acrylamide gel. The composition of the polymerization mixtures affects the separation factor to some extent. The molar ratio of cross-linker to functional monomer to print molecule was 20∶4∶1, which gives the best separation for aromatic amino acid enantiomers by using L-phenylalanine anilide (L-pheAN) as print molecule. Under the same conditions, however, separation of D,L-phenylalanine using L-phenylalanine as print molecule is poorer than that using L-pheAN. Polymer particles (≤5 μm) were supported by 6% acrylamide and 5% bisacrylamide gel in the capillary. The effects of organic solvents, electrophoretic buffer and pH on separation were also investigated.

Capillary electrochromatographic separation of amino acid enantiomers using on-column prepared molecularly imprinted polymer

The use of molecularly imprinted polymer polymerized in capillary for the separation of amino acid enantiomers by electrochromatography is described. The substrate-selective polymers were prepared by using L-phenylalanine anilide as print molecule and methacrylic acid and/or 2-vinylpyridine as the functional monomers, which is believed to interact both ionically and through hydrogen bonding with the print molecule. Several aspects of the polymer preparation were investigated, including the treatment of the inside surface of the capillary, the composition of the polymers and the running conditions of the capillary electrochromatography. Such separation was highly specific and depended on the presence of both the print molecule and the functional monomer in the polymerization mixture. This preliminary report demonstrates a novel and simple method for the development of the capillary electrochromatographic separation of amino acid enantiomers using molecularly imprinted polymer.

Enantioseparation of D, L-Phenylalanine By Molecularly Imprinted Polymer Particles Filled Capillary Electrochromatography

Abstract The chromatographic properties of molecularly imprinted polymer filled capillary electrochromatography (CEC) have been studied. The network polymers were prepared using L-phenylalanine anilide (L-pheAN) or L-phenylalanine as print molecules. Methacrylic acid was used as functional monomer, such that the acid function of monomer interacts ionically with the amine function and via hydrogen bonding with the carboxyl group of the print molecules. Ethylene glycol dimethacrylate was used as cross-linker and the reaction initiator was α, α′-azobis(isobutyronitrile). The obtained polymers were ground and sieved to particles<10 μm for filling into capillary for CEC and <30μm for packing into high performance liquid chromatographic columns. The separations of D, L-phenylalanine by using the molecularly imprinted polymers against L-pheAN and L-phenylalanine were compared. It was interesting to find that the resolution of D, L-phenylalanine using L-pheAN printed polymer was higher than that using L-phenylala...

Chemiluminescence sensor with uranine immobilized on an anion-exchange resin for monitoring free chlorine in tap water

Abstract A chemiluminescence sensor using a xanthene dye immobilized on an ion-exchange resin as an indicator phase is described for the continuous monitoring of free chlorine (HOCl) in tap water. Of the dyes and ion-exchange resins tested, the immobilization giving the best dynamic response characteristics was achieved with a combination of uranine and a weakly basic anion-exchange resin (Amberlite IRA-93). The sensor, a Pyrex tube (20 mm × 3 mm i.d.) packed with the resin and placed close to a photomultiplier tube, provided a constant signal within ca. 1 min of a concentration change of free chlorine, a linear calibration graph between 1 × 10 −3 and 2 × 10 −6 M (the detection limit at a signal-to-noise ratio of 3) and an r.s.d. of 1.6% for 1 × 10 −5 M free chlorine. The sensor was unique in being insensitive to chloramines and permitted free chlorine to be determined in tap water with no interference from chloramines and other compounds.

Combined chiral crown ether and β-cyclodextrin for the separation ofo-, m-, andp-fluoro-D,L-phenylalanine by capillary gel electrophoresis

SummaryA combination of (β-CD) with a chiral crown ether, 18-crown-6-tetracarboxylic acid (18C6TA), has been used for the chiral capillary gel electrophoretic (CGE) separation of the six enantiomers and positional isomers in a mixture ofo-, m-, andp-fluoro-D,L-phenylalanine; the isomers were successfully resolved in 0.3% agarose −2×10−2 mol L−1 18C6TA −2×10−2 mol L−1 β-CD. The method has also been successfully extended to the separation of the enantiomers of 13 other amino acid or primary amine substrates. The role of the chiral crown ether was studied. The effects of the concentrations of 18C6TA, β-CD and agarose, and of pH and applied field strength on the migration time and separation factors of the analytes are reported.

Influence of the method of preparation of chiral stationary phases on enantiomer separations in high-performance liquid chromatography

Abstract Various chiral stationary phases were immobilized on supports using suitable alkyl spacers. Aminopropylsilica, which is aminopropylsilylated silica gel, is a typical spacer-carrying support for immobilization. Silanol and aminopropyl groups left unreacted on the support surface disturb the separation of enantiomers through a non-chiral inteaction. The following three types of chiral stationary phases were prepared using different preparation methods and the effect of the total structure on the chiral separation was studied: (1) a chiral group was immobilized on aminopropylsilica; (2) a chiral group connected with a spacer was immobilized on silica gel; and (3) a chiral group connected with a spacer was immobilized on dimethylchlorosilane-treated silica gel. It was shown for (1) that when the amount of chiral groups immobilized was lower, free silanol groups and unreacted amino groups would preferentially interact with sample molecules. When the amount immobilized was increased, its interaction with samples became effective, and also aminopropyl groups left unreacted were thought to enhance the interaction. Too many chiral groups, however, resulted in lower separatility. In case (2), a similar tendency to that with (1) but lower separabilities were obtained. A more effective performance was found with (3), with lower immobilization. For the preparation of an effective chiral stationary phase, the amount of chiral groups on the support and the structural and topographical conditions of the support material should be assessed.

Screening of chemiluminescent systems for the determination of some biologically important organic compounds

Abstract Screening tests are described for the development of chemiluminescence systems (oxidizing systems) capable of detecting biological organic compounds. The light emission depends strongly on the oxidizing systems employed. Acidic permanganate system gives rise to light emission for many compounds, including catechols, catecholamines, triphenols and indoles. The following oxidizing systems led specifically to chemiluminescence for hydroquinone, adrenaline or phenylpyruvic acid: 10 −1 M thiosulphate with 10 −1 M sodium hydroxide and 10 −4 M Ag (I), 0.3 % hydrogen peroxide with 10 −3 M sodium hydroxide/50% acetonitrile and 10 −4 M Fe (II), and 0.3% hydrogen peroxide with 10 −2 M sodium hydroxide/10 −2 M didodecyldimethylammonium bromide and 10 −4 M Co(II), respectively.

A flow-based enzyme-linked immunosorbent assay on a polydimethylsiloxane microchip for the rapid determination of immunoglobulin A.

A flow-based enzyme-linked immunosorbent assay (ELISA) on a polydimethylsiloxane (PDMS) microchip has been developed for the rapid determination of immunoglobulin A (IgA). The analytical principle of this integrated method is the same as the conventional sandwich-type ELISA. A primary antibody (anti-IgA) was adsorbed on the surface of a PDMS microchannel, and then an antigen (IgA) and a secondary antibody (anti-IgA HRP labeled) were reacted successively. The resulting antigen-antibody complex, fixed on the surface of the microchannel, was detected using Amplex((R)) Red and a fluorescent imaging system. The calibration curve of the IgA standard solution was linear in the range of 0-50ng/mL at the flow rate of 10muL/min. This flow rate corresponds to the reaction time of 4.8s. Compared to the conventional assay on a 96-well microtiter plate, the present assay on the microchip dramatically shortened the reaction time necessary for the enzyme-substrate reaction from 30min to 4.8s, i.e., to 1/375. The amounts of the reagent and sample were also reduced to 1/100 compared to the 96-well microtiter plate.

Enantioseparations of dansyl amino acids by capillary electrophoresis using Cu(II) complexes with L-amino acylamides as chiral selectors in electrolytes

Enantioseparations of dansyl amino acids using Cu(II) complexes with L-amino acylamides as the chiral selectors in the electrolyte solution were investigated by capillary electrophoresis. It has been demonstrated that the Cu(II) complexes with L-amino acylamides such as L-phenylalaninamide, L-prolinamide, and L-alaninamide are suitable for use as chiral selectors for enantioseparations of dansyl amino acids. The optimal separation conditions were examined. The employed chiral ligands exhibited different enantioselectivities and enantiomer migration orders. The enantiomer migration orders are discussed by using three different chiral selectors. L-enantiomers migrate faster than D-enantiomers when Cu(II)-L-phenylalaninamide and Cu(II)-L-alaninamide are used as chiral selectors, but D-enantiomers faster than their (--counterparts in the Cu(II)-L-prolinamide system.

Rhodamine 6G chemiluminescence for the determination of free chlorine in tap water by a flow-injection method

A chemiluminescence system is described for the specific determination of free chlorine by a reversed flow-injection method. The light emitted from the reaction between Rhodamine 6G and free chlorine (HOCl) under unbuffered conditions allows free chlorine in tap water to be determined with no interference from combined chlorine (chloramines). The limit of determination (signal to noise ratio = 3) is 1 × 10–7M, the linear range is two orders of magnitude, the sampling rate is 240 samples h–1 and the relative standard deviation (n= 10) is 2.0% for 1 × 10–5M free chlorine. Common inorganic species give rise to no light emison. Among oxo acids of chlorine, only chlorite ion slightly elicits light, 1 × 10–3M ClO2– providing a signal 3% of that for 1 × 10–5M HOCl. Inorganic species and chloramines co-existing in tap water do not interfere.