BUNSEKI KAGAKU Abstracts

Enzyme-amplified lanthanide luminescence (EALL) is a promising method which has been developed for enzymatically amplified signal detection. A signal is generated by enzymatically transforming a substrate into a product, which forms a fluorescent lanthanide chelate. With the aim of establishing a new EALL detection system, we focused on O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid (BAPTA), which is known to be a specific chelator for calcium. BAPTA also forms some energy-transfer fluorescent complexes with lanthanide ions at physiological pH, and functions as a good sensitizer, especially for terbium(III) ion. Thus, we examined whether esterase activity can be determined by EALL detection where BAPTA and its tetraacetoxymethyl ester (BAPTA-AM) are used as the Tb(III) chelate and the reaction substrate, respectively. Regarding the rate of the Tb(III) fluorescent intensity changing per unit time (ΔI/Δt) as an initial rate of the enzymatic reaction, the determination of esterase activity was successful in the range of 0.010〜0.10 u/mL. Within this work, the suitability of the Tb(III)/BAPTA-AM system for EALL detection has been proven for determining esterase activity.

The retention behavior of Cr(III) and Cr(VI) in an alkaline solution (0.28 M Na₂CO₃/0.5 M NaOH, pH 13.5 ; US EPA Method 3060A) on four kinds of ion-exchange resins was examined. Method 3060A is an alkaline digestion procedure for extracting Cr(VI) in soils, sludges, sediments, and similar waste materials. Two commercially available ion-exchange resins were used in column operation : Dowex 50W-X8 (H^＋ form and Na^＋ form) and Dowex 1-X8 (OH⁻ form and Cl⁻ form). Column operation was made by pipetting a 15-mL aliquot of an original alkaline solution or a 10-fold diluted alkaline solution, spiked with Cr(III) and/or Cr(VI) to give 1 mg/mL, through a mini-column containing 2.0 g of ion-exchange resin. To compare the observed data, acidic solutions containing Cr(III) and/or Cr(VI) were also loaded. Then, the 1-mL fractions of the effluent were collected in small test tubes for both pH and flame AAS measurements, and break-through curves were illustrated and analyzed. It was found that both Cr(III) and Cr(VI) were not retained on the Na^＋ form cation-exchange column using both alkaline solutions. However, a selective separation of Cr(VI) from Cr(III) could be achieved by retaining Cr(III) on the H^＋ form cation-exchange column using a 10-fold diluted alkaline solution. Therefore, the separated Cr(VI) can be determined by a direct AAS measurement of the resulting effluent. On the OH⁻ form and the Cl⁻ form anion-exchange columns, both Cr(III) and Cr(VI) were retained using a 10-fold diluted alkaline solution. However, a selective separation of Cr(III) from Cr(VI) could be achieved by retaining Cr(VI) on the OH⁻ form and the Cl⁻ form anion-exchange columns using an original alkaline solution. Therefore, the separated Cr(III) could be determined by a direct AAS measurement of the resulting effluent.

Understanding the source and chemical character of the groundwater provides an important strategy for the quality management of mineral water and food materials. In order to identify a source and the flow paths of groundwater used for mineral water, the water quality and stable isotopes of hydrogen and oxygen of well water in Gotenba city were studied. The electrical conductivity and chemical character of sampled water are similar to those of well water and spring water discharged elsewhere around Mt. Fuji. The hydrogen and oxygen isotopic ratios of water samples indicate their origin to be solely meteoric and the oxygen isotopic ratios suggest that the groundwater mainly originated from the mountain-side of Mt. Fuji at altitudes of from 1500 m to 2300 m. A subsequent simulation of groundwater showed that the distribution of the total head and the Darcy velocity are down streamlines from mountain-sides toward the study area in Gotenba city. The altitudes of discharge obtained by the simulation are above 2000 m, and these correspond well with altitudes estimated from δ¹⁸O values of the samples.

An on-line coprecipitation concentrarion method has been proposed for the determination of chromium in steels based on the reaction of chromium with 1,5-diphenylcarbonohydrazide (DPC). In an analysis of trace elements in steels, a removed method of an iron matrix with 4-methyl-2-pentanone has been used mainly, but there is a trend to avoid the use of organic solvents, which has been a burden on environment in recent years. Therefore, the iron matrix removal method had been investigated without any environmental burden using Teflon filter tube. Iron ions were collected with a filter tube as Fe(OH)₃ in the flow system. After Fe(OH)₃ was removed, a sample solution with Cr(VI), which came through a Teflon filter tube, was merged with a La(III) solution. Chromium was coprecipitated at pH 9.0 with La(OH)₃, which was collected with another filter tube. The precipitation was eluted with HCl (5 M), and the chromium(VI) in the eluate reacted with a DPC. The absorbance of the obtained Cr(III)-DPC complex was measured by spectrophotometry at 542 nm. The calibration curve with 10-minute concentration operation for chromium (sample flow rate＝1.4 mL/min) was linear over the range of 0 to 200 ppb. The limits of detection and determination for chromium in the sample solution were 1.1 and 3.4 ppb, respectively. The method with a 10-minute concentration operation improved sensitivity by 17 times compared to a method with no concentration. The proposed method was applied to the determination of standard steel samples. The results of the determinations were in good agreement with the certified values.

In order to clarify the relationship between evidence from a crime and a suspect, the component of additives in adhesive tape made by polyvinyl chloride were analyzed by gas chromatography mass spectrometer. We proposed a discrimination method that consisted of three steps procedure using the kind of additives and content ratio. At first, mass chromatogram of mass-to-charge ratio (m/z) 149 was used to confirm the peaks of diisononyl phthalates. Next, mass chromatograms of five components, except for the phthalates, were checked, respectively. Finally, eight peaks on the mass chromatogram of m/z 149 from 2 to 9 minutes were analyzed and judged to discrimine the same color adhesive tapes. Using gray adhesive tapes, colorless transparent ones and blue ones made it possible to identify by this technique. Another color of adhesive tapes was identified to some extent.

Both H^＋ and alkalinity are important key parameters for evaluating environmental water quality. In this study, unified cation-exchange chromatography with conductimetric detection on an ODS-silica column (TSKgel ODS-100Z) modified with lithium dodecylsulfate was developed to simultaneously determine monovalent cations (Na^＋, NH₄^＋ and K^＋) and H^＋/alkalinity. As a result, the simultaneous determination of H^＋ as a positive peak and monovalent cations was achieved based on the cation-exchange mechanism. On the other hand, the alkalinity (OH⁻/HCO₃⁻/CO₃²⁻) was also detected as a negative peak in a vacant peak of the acidity, where alkaline species were neutralized by hydrogen ions in the eluent, while the peaks of monovalent cations and alkalinity were separated. Using the optimized eluent conditions (40 mM LiCl/0.1 mM Li-DS/0.05 mM H₂SO₄ with pH 4.0 ; flow rate, 1.5 mL/min), the simultaneous separation of monovalent cations and H^＋/alkalinity was achieved in ca. 15 min. Analytical performances tests including a calibration curve, the detection limit, reproducibility and recovery tests were carried out for monovalent cations and acidity/alkalinity under the optimized chromatographic conditions. The present method was practically applied to several acidic and basic environmental water samples (rain, hot spring and river waters) and mineral drinking water samples. Since there was a good agreement between the analytical results obtained by the present method and the conventional potentiometry for the acidity and titration method for the alkalinity in these practical samples, the usefulness of the present method was demonstrated experimentally.

This study evaluated water quality using potentiometric solid-state electrodes in order to simply and rapidly detect chemical indices, such as COD, and BOD, which were necessary to appropriately control the inflowing water quality and activated sludge properties in a sewage-treatment plant. Pt, CuS and Ag₂S electrodes, selected as solid-state electrodes have, respectively been used to detect COD and BOD. The outputs of Pt, CuS and Ag₂S electrodes have been analyzed by multiple regression analysis (MRA) in order to find correlation with the results of the COD and the BOD. It was indicated that the correlation between the experiment value and the predicted value was high. The multiple correlation coefficients of the COD and the BOD were 0.86 and 0.88, respectively.

An on-line analysis system using a solid-phase extraction column coupled to electrochemical detection has been developed for the rapid determination of small amounts of uranium in liquid waste samples of spent nuclear-fuel reprocessing plants. A sample solution with a concentration of 3 M HNO₃ was loaded onto a column : packed with U/TEVA^® resin. The interference elements were rinsed by passing 3 M HNO₃ through the column. The adsorbed uranium was eluted with 0.1 M HNO₃. The eluate was directly introduced into a flow-electrolysis cell. The reduction current of U(VI)→U(V) was monitored and recorded. The uranium concentration was calculated from the relation between the peak current and the concentration of the standard uranium solution. The result of five repeated analyses for a standard solution containing 2.5 μg (0.1 mL at 25 μg mL⁻¹) of uranium was found to be 2.5 ± 0.025 μg (mean ± 1σ). The detection limit calculated from 3-times the standard deviation of the background current was 56 ng. The analysis time required for one sample was within 5 min. The recoveries of uranium in actual nuclear waste reprocessing solutions were 92〜112％.

The conventional photolithography-chemical wet-etching method for preparing a convex mold usually uses a metal-coated glass or silicon-wafer substrate, and thus produces a waste solution containing heavy metal such as chromium. In this work, a simple photolithography-chemical wet-etching method using a slide glass without a coating of metal was proposed to fabricate a convex mold for preparing a PDMS (polydimethylsiloxane) microchip. A photoresist was directly coated on a cleaned slide glass, and was prebaked for 90 s. After covering with a photo-mask, the coated slide glass was illuminated with UV-visible light, followed by development with a developing reagent. Then, the slide glass was etched in a 1 M HF/1 M NH₄F solution for a certain time. Finally, the etched slide glass was cleaned with acetone for removing the remaining photoresist. A PDMS microchip with a cross microchannel was prepared using the convex mold. The microchip was used for the electrophoresis of two fluorescent dyes and fluorescent labeled protein A. The experimental results showed that the simple method without using a metal-coated substrate could be used for convex molds for preparing microchips.

In forensic science, highly sensitive and nondestructive methods have been required to discriminate a small amount of evidence found at criminal scenes. This doctoral thesis demonstrates that high-energy SR-XRF (synchrotron radiation X-ray fluorescence spectrometry) utilizing 116 and 75 keV X-rays at SPring-8 is a powerful technique for trace elemental analysis of forensic small samples. Typical and important samples such as glass, titanium dioxide pigments, automotive white paints, polyester single fibers, and ceramic prints on automotive glass were used to evaluate the utility of this technique for forensic discrimination. This technique allowed us to detect trace heavy elements such as rare-earth elements as well as relatively light elements in samples using K series of emission lines. Then, trace heavy elements were useful as characteristic fingerprints, showing the identity of each sample with a size of less than 0.5 × 0.5 mm². By these results, this technique has been demonstrated to be a new system for forensic examination, and is being applied to practical analyses of various samples at present.