BUNSEKI KAGAKU Abstracts

Localized surface plasmon resonance (LSPR) scattering spectroscopy for Au nanoparticle immobilized on a glass substrate was studied. An apparatus for LSPR scattering spectroscopy, which consists of a flowcell, dark-field illumination light source and CCD spectrophotometer, was constructed in this study. A flowcell was designed to fix an Au nanoparticle deposited glass chip, so that injected fluids contact with a Au nanoparticle deposited surface inside the flowcell. The spectroscopic response of a Au nanoparticle for dielectric environments around the particle was examined by the injection of water and several organic solvents. LSPR scattering spectra revealed a red-shift of the scattering maxima and an increment of the scattering intensity at the 500〜900 nm region by increasing the refractive index of the injected solvent. Then, the time course of the scattering intensity at 620〜670 nm region was observed by the replicate injection of water and organic solvents. The scattering intensity was increased with increasing refractive index of the injected solvent. Therefore, it was confirmed that the fabricated glass chip worked as a sensitive refractive index sensor by combining with LSPR scattering spectroscopy. The LSPR scattering responses in different surface density and size of a Au nanoparticle on a glass surface were also examined. We confirmed the optimum Au nanoparticle density with uniform particle spacing and without particle aggregation for refractive index sensing.

A surface plasmon resonance (SPR) sensor for the determination of methyl parathion (MP) was developed using an indirect competitive immunoassay. A sensor chip was prepared by immobilizing a MP-BSA conjugate on a gold film on the sensor chip, followed by blocking with BSA to prevent the non-specific adsorption of MP and an anti-MP antibody onto the unmodified surface of the sensor chip. An evaluation of the adsorption equilibrium of MP-BSA onto the sensor chip by measuring the SPR angle shift revealed that the adsorption equilibrium was expressed by a Langmuir-type adsorption isotherm equation. The adsorption constant and the adsorbed amount of MP-BSA onto the sensor chip were found to be 5.0×10⁵ M^-1 and 0.95 ng/mm², respectively. An MP solution (1〜5000 ppb) containing 60 ppm anti-MP antibody was introduced into the SPR sensor chip, and then the SPR angle shift was measured. The detection limit, defined as the angle shift of 85％ for the blank, was found to be 10 ppb. The anti-MP antibody bound to the MP-BSA conjugate on the sensor chip was able to be dissociated by introducing an HCl-glycine solution (pH 2) into the sensor chip. Duplicate measurements with a single sensor chip were possible for at least 20 times.

Affinity chromatography is a powerful method for protein separation. It is based on a specific interaction between an immobilized ligand and the target proteins to be separated. Since lithocholic acid (LCA), one of secondary bile acids, has been shown to exert its function as the ligand toward nuclear receptors and a membrane-type G protein-coupled receptor, the abilities of molecular recognition and acquisition of LCA may be applicable for ligands to capture unknown functional proteins by affinity chromatography. In this study, LCA was covalently bound to an activated agarose through a bridge introduced at the C-3 and C-24 positions. The affinity absorbents were applied to capture proteins in a rat mitochondrial fraction. Structure analysis of the captured proteins after SDS-PAGE separation was carried out by liquid chromatography/electrospray ionization-tandem mass spectrometry combination with computer-assisted programs, where carbamoyl phosphate synthase I, glutamate dehydrogenase, acyl-CoA dehydrogenase, enoyl-CoA hydratase, acetyl-CoA acyltransferase and aldehyde dehydrogenase were identified. Serum albumin and cytosolic glutathione S-transferase, which were contaminated in mitochondrial fraction, were also identified.

Thermo-sensitive chitosans grafted with N-isopropylacrylamide copolymers having maleate or hydroxamate {poly(NIPAAm-co-MaA)-g-chitosan, poly(NIPAAm-co-MHA)-g-chitosan} were synthesized, and their dispersivities and adsorbtivities of metal ions were also investigated. The synthesized thermo-sensitive chitosans dispersed at 20℃, which is below the phase-transition temperatures, and aggregated at 50℃, which is above the phase transition temperatures. Every synthesized thermo-sensitive chitosan adsorbed Cu^II, Cd^II, and Pb^II ions in the neutral pH range, quantitatively. Especially, poly(NIPAAm-co-MaA)-g-chitosan kept its quantitative adsorbtivity of metal ions through replicative adsorb/desorb processes. Twenty-times preconcentrations of 1.0×10^- mol dm^- of Cu^II, Cd^II and Pb^II were achieved with over 90％ recoveries by using every synthesized thermo-sensitive chitosan. The combination of poly(NIPAAm-co-MaA)-g-chitosan used as an adsorbent and a graphite-furnace atomic-absorption spectrometry allowed to determine trace amounts of Cd^II in tap water, drinking water, and river water. The atomic spectrophotometric determination of cadmium(II) with the thermoresponsive chitosan was also successfully applied to certified reference materials of river water.

An atomic emission detector (AED) using a helium radio-frequency plasma (RFP) for gas chromatography (GC), equipped with a coaxial micro plasma torch, was developed. The torch was made by arranging two platinum tubular electrodes (1.0 mm o.d. and 0.5 mm o.d.) coaxially. The head of an optical fiber was placed on the torch, and the end was connected to a compact CCD spectrometer. The developed AED was set on a conventional GC for the measurement. When thioanisole was used as a sulfur compound, the detection limit of sulfur was obtained to be 59 pg/s (S/N＝3) at a detection wavelength of 921.3 nm with an applied power of 3 W and a flow rate of make-up gas of 1 mL/min. In the case of triethyl phosphate as a phosphorus compound, when the detection wavelength, the applied power and the flow rate of make-up gas were set at 253.4 nm, 3 W and 17 mL/min, respectively, 62 pg/s (S/N＝3) was found to be the detection limit for phosphorus. These results indicated that the applied power and/or the flow rate of plasma gas required for the developed AED for the high-sensitive detection were less than one order of magnitude smaller than that of previous reported helium RFP-AEDs.

A sensitive and selective method for the determination of sub μg/L levels of inorganic chromium species in river water is described. The proposed method is based on the cloud point extraction (CPE) with non-ionic surfactant and graphite furnace atomic absorption spectrometry. Hydrophobic chromium chelates in a 30 mL of water sample were extracted into a surfactant-rich phase of polyoxyethylene (8)octylphenyl ether (Triton X-114) after heating over the cloud point temperature (kept in a thermostated water bath at 40℃ for 15 min). Separation of the aqueous and surfactant-rich phases was accomplished by centrifugation and cooling in an ice bath. The surfactant-rich phase was dehydrated in a drying oven, and the residual extract was dissolved in a 1.0 mL of 0.1 M nitric acid/methanol solution to decrease the viscosity of the extract. A 20 μL aliquot of the resulting solution was injected into a graphite furnace. In this study, we used ammonium pyrrolidinedithiocarbamate (APDC) as a chelating agent which reacts with both Cr(III) and Cr(VI). The CPE behavior of Cr(III) and Cr(VI) according to the variation of pH and APDC concentration in sample solution was investigated. The results indicated that the major factor of CPE is the concentration of dissociated PDC^- and the chemical species of chromium, Cr(OH₂)₅(OH)^2＋ and HCrO₄^-. Cr(VI) was selectively extracted at pH 3.0, and total Cr {Cr(III)＋Cr(VI)} was extracted at pH 5.0 without an oxidation process. The optimum concentration of APDC was 0.08％ for Cr(VI) and 0.10％ for total Cr. The linear calibration curves of Cr(VI) and total Cr for ca. 30-fold preconcentration were obtained over the range of 0 to 0.5 μg/L in the initial solution. The detection limit (3σ) was 2.5 ng/L for Cr(VI) and 3.7 ng/L for total Cr. The proposed method was successfully applied to river water and certified reference material.

A flow injection system with a novel detection reaction is presented for determining trace bromate in drinking water. Bromate is used to oxidize V(IV) to V(V), and then Nitro-PAPS {2-(5-Nitro-2-pyridylazo)-5-[N-n-propyl-N-(3-sulfopropyl)amino]phenol} reacts with V(V) to form a complex that is monitored spectrophotometricaly at 592 nm in a continuous flow system. The variables related to such reactions were studied in detail, and the optimal conditions and manifold configurations were established. A linear calibration using a 2 m sample loop injection was obtained for bromate in the range of 0〜0.05 ppm. The coefficient of variation for 0.01 ppm bromate (n＝3) was 0.6％, and the estimated limit of detection (3 σ) was 0.0004 ppm. Only 4 min was required for an analytical measurement after sample injection. The present FI system was successfully in-line coupled with anion-exchange separation using 0.05 M NaCl as an eluent for a more sensitive and selective determination of trace bromate. In this case, using a 3 m sample loop, the limit of detection (3 σ) was 0.0002 ppm bromate, the rsd for 0.01 ppm bromate was 1.4％ and the time required for an analytical measurement was 9 min. Tap water samples supplied from 3 cities and a bottled water were analysed by this FI system; satisfactory recoveries of 96〜104％ were found for spiked bromate.

The partition chromatography method of electrochemical detection is the commonly used method for quantitative analysis of alcohol. The standard detectors of high-performance liquid chromatography and ion chromatography are ultraviolet-visible detector, differential refractive index detector, and electrical conduction detector. We need to develop new detectors for the analysis of alcohol. Therefore, in this study, we used the phenomenon of alcohol's signal appearing as a negative peak using the electrical conduction detector of ion chromatography. That is, we examined the quantitative analysis of alcohol by the partition chromatography method of indirect electrical conduction detection. The calibration curve obtained is a straight line whose coefficient of correlation was 0.9967〜0.9999 in the range of 0.5 mg/L〜100％, and we could analyze alcohol in a very wide range. In addition, it was found that we could also analyze organic acid and saccharide by this method.

A highly sensitive spectrophotometric semi-automated method for ammonium ion determination was proposed using flow injection analysis coupled with an on-line gas diffusion separation/ion exchange concentration technique, which was especially effective for applying to analyses of open sea or fresh seawater. Ammonium ion at the single and/or sub-ppb level in seawater samples was introduced into a gas diffusion system at first, and then subsequently concentrated on a cation exchange resign column. The collected ammonium ion was eluted by 0.5 M hydrochloric acid. After derivatization based on indophenol blue with salicylate, the color development was monitored at 660 nm. The detection limit (3 σ) for N-NH₄^＋ was 0.5 ppb at 10 minutes concentration and 0.2 ppb at 30 minutes concentration, respectively. The relative standard deviation (n＝5) was found to be 0.68％ for 1 ppb of N-NH₄^＋ at 10 minutes concentration. The proposed method could be applicable to the analysis of ammonium ion in seawater samples.

A simple and sensitive flow injection analysis (FIA) system with immobilized choline oxidase (ChO) and peroxidase (POD) enzyme reactor columns for the determination of choline has been developed. ChO was immobilized onto chitosan as a support by cross-linking with glutaraldehyde by forming a Schiff base, and choline was determined with fluorescence detection. The FIA system with the immobilized enzyme reactor columns was tested for the parameters of the optimization procedure: carrier solution, flow rate and injected sample volume. After the optimization of the FIA parameters, the response time was about 3 (min), the linear calibration curve was obtained between 0.2 μmol dm^-3 and 10 μmol dm^-3of choline (r²＝0.999) and the detection limit was 0.1 μmol dm^-3. After researching of the effects of coexisting substances by an additional recovery test, the FIA system was applied for a supplement sample and the relative standard deviations by repeated measurements (n＝5) were found to be within 0.3％. It was found that the FIA system can be applied to any samples without effecting of the coexisting substances.

A novel spectrophotometric method was established for the determination of zirconium(IV) and fluoride ion. The method was based on complex formation among salicylfluorone (SAF), Zr(IV) and fluoride ion in the presence of a cationic surfactant. In the determination of Zr(IV), Beer's law was obeyed in the range of 0.09〜0.46 μg cm^-3 in a neutral medium. The effective molar absorptivity at 550 nm and the relative standard deviation were 1.7×10⁵ dm³ mol^- cm^- and 0.32％ (n＝6), respectively. In the determination of fluoride ion, Beer’s law was obeyed in the range of 3.8〜65 ng cm^-3 in a strong acidic medium. The effective molar absorptivity at 552 nm and the relative standard deviation were 2.1×10⁵ dm³ mol^-1 cm^-1 and 1.3％ (n＝6), respectively.

The method is based on ternary complex formation among o-carboxyphenylfluorone, copper(II), and insuline. In the determination of insuline, Beer's law is obeyed in the range of 0.5〜50 μg mL^-1. The effective molar absorptivity at 550 nm and the relative standard deviation were 8.2×10⁴ dm³ mol^-1 cm^-1 and 2.2％ (n＝6), respectively. The proposed method should be useful for a simple determination of insuline.

Tobacco smoke contains more than 200 harmful compounds and 40 carcinogenic compounds. Tobacco smoke exposure is a significant risk of growth suppression for fetuses and infants. A highly sensitive and selective method for the determination of nicotine and cotinine in human serum samples, to evaluate the exposure amount of tobacco smoke, was developed. Solid-phase extraction was employed for sample preparation, and a measurement was performed by hydrophilic interaction chromatography with mass spectrometry (HILIC/MS). The limits of detection of nicotine and cotinine were 0.2 and 0.1 ng/mL, respectively. Moreover, the limits of quantification of nicotine and cotinine were 1.0 and 0.4 ng/mL, respectively. The average recoveries of nicotine and cotinine were over 90％ (RSD＜6％, n＝6). The proposed method was applied to measure the serum and umbilical cord serum of a pregnant woman. The detection rate of cotinine in the pregnant woman serum and umbilical cord serum were 51 to 14 and 11 to 1.

It has been reported that the nitrate concentration in stream water in the mountainside on Mt. Oyama (1252 m a.s.l), which is located at the western part of Kanagawa Prefecture, is higher than that in stream water in other regions, indicating that nitrogen saturation is occurring in the forested ecosystem. However, there has been no report on nitrogen saturation across the Tanzawa mountains, except Mt. Oyama. We investigated the current status of the stream water chemistry and the effect of acid deposition on the stream water chemistry across the East Tanzawa mountains. Thirty-eight samples were collected in nine mountain streams in this region. The mean pH and alkalinity in stream water in this region were 7.315 and 650 μeq/L, respectively. There was no symptom of stream-water acidification by acid deposition in East Tanzawa, probably because of the high acid buffering capacity by the weathering of carbonate and silicate minerals. Nitrate concentrations in the stream water ranged from 27.6 to 87.8 μeq/L and NH₄^＋ was detected. High nitrate concentration in stream water suggests that nitrogen saturation is occurring in the forested ecosystem in East Tanzawa mountains. We divided our sampling sites into southeast (SE), southwest (SW), and north (N) regions by the ridge line of Mt. Tanzawa - Mt. Tonodake - Shindainichi - Mt. Sannoto - Mt. Takenodai - Mt. Oyama - Mt. Mitsumine. The stream water pH was lower and the concentrations of Na^＋ and NO₃^- were higher in south regions. There were high correlations between the concentrations of Na^＋ (r＝-0.854), Cl^- (r＝-0.815) and NO₃^- (r＝-0.922) and the distance from the coast of Odawara City, Oisomachi, and Ninomiyamachi. In addition, a high correlation between the NO₃^- and Cl^- concentrations (r＝0.741), which is an indicator of atmospheric deposition, in stream water was observed. Our preliminary investigation indicates that the runoff of nitrate, originated from atmospheric deposition, might occur in the forested ecosystem in the southern regions in East Tanzawa.

Tap water samples on Shikoku Island were analyzed and evaluated with regard to their water quality. They were generally soft water with a pH value of 6.5〜8.0 and a COD value of 0.01〜1.08 mg/L. Ammonium ion was not detected. Especially, the tap water samples from Tokushima and Kochi prefectures were clean with a low COD value of 0.02〜0.51 mg/L. The classification of water type was performed for all tap water samples with the trilinear diagram method, finding 71.3％ of tap water samples to be the typical ground water type (alkaline earth hydrogencarbonate). On the other hand, the hydrothermal/fossil water type (alkaline earth nonhydrogencarbonate) was 39.1％ in north Shikoku (Kagawa, Ehime), while 14.6％ in south Shikoku (Tokushima, Kochi). It is considered that the difference is due to the geological property by the median tectonic line between north and south Shikoku Island. The water qualities were as follows, respectively: Kagawa: hardness, 23.5〜132.9 mg/L; pH, 6.8〜7.9; COD, 0.20〜1.08 mg/L, Tokushima: hardness, 14.6〜72.0 mg/L; pH, 6.6〜8.0; COD, 0.02〜0.37 mg/L, Kochi: hardness, 16.7〜66.5 mg/L; pH, 6.5〜7.9; COD, 0.03〜0.51 mg/L, Ehime: hardness, 17.8〜114.4 mg/L; pH, 6.7〜7.9; COD, 0.01〜0.61 mg/L.

The wet deposition fluxes of acidic substances were observed on the mountainside (1300 m a.s.l.) of Mt. Fuji from July 2005 to November 2006 by a solar-powered automatic rainwater collector, which was developed with emphasis on its being small-size, lightweight, and inexpensive for use in wet deposition monitoring network in mountainous area. The collector is divided into two parts, namely rainwater collection and solar battery system. The former is composed of a capacitance-type rain sensor, an originally designed automatic switching cover, a polycarbonate filter holder (80 mm in diameter), which is equipped with a 47 mm diameter and 1.2 mm pore size membrane filter, a polyethylene bottle, and a polypropylene tube connecting the holder and the bottle. The latter is composed of a 20 watt solar panel, a solar charge controller, and a 12 V lead acid battery. A comparison of the deposition fluxes, estimated by the automatic rainwater collector with those by a filtering-type rainwater collector, indicated that they were free of the influence of dry deposition without a loss of rainfall in the automatic rainwater collector. Rainfall amounts estimated by the automatic rainwater collector corresponded to those observed by a commercially available rain gauge except for winter. Annual wet deposition fluxes of H^＋, NO₃^-, and SO₄^2- in the mountainside on Mt. Fuji were 58.5, 40.4, and 78.8 meq/m²/y, respectively.

Anesthetics have in common the capacity to partition indiscriminately into all biological membranes and act as general perturbants of the membrane structure and function. However, anesthetic mechanisms have not yet been clarified. Some measurement techniques are used to investigate the interaction between anesthetic and biological materials. Thus, in this research we examined the utility of the electrode selectively sensitive to local anesthetics as a measurement technique of the interaction of the bovine serum albumin (BSA) with local anesthetics. The electromotive forces (EMF) of the local anesthetic dibucaine and tetracaine cation-selective electrodes were measured relative to an Ag-AgCl reference electrode in both the absence and presence of BSA at pH 5.5. In the absence of BSA, both the dibucaine and tetracaine electrodes showed a linear response with a Nernstian slope over the concentration of 10^-5 mol dm^-. Most of local aneshetic molecules must exist as the protonated cation at pH 5.5, judging from the pK_a values. In the presence of BSA, the response curve was shifted toward the higher concentration of anesthetics, which means a decrease of the concentration of free anesthetic cation due to binding to BSA. The binding isotherms of anesthetics to BSA were calculated from a pair of response curves. The slope of the binding isotherm increased steeply above a certain concentration of local anesthetics, which means that the binding form of local anesthetic into BSA is multiple-step.