Vol. 4 9, No. 1 2
December, 2000
Tetsuo Makino*
*Department of Clinical Chemistry, Kanagawa Prefectural College of Nursing and Medical Technology, 1-5-1, Nakao, Asahi-ku, Yokohama 241-0815
(Received 12 June 2000, Accepted 3 August 2000)A new colorimetric assay system using microtiter plates is presented for the determination of trace metals and proteins in serum samples. The use of polystyrene microwells with about 400- ml capacity and sensitive reagents made it possible to reduce the sample size and required reagent. Rapid determination methods for iron, copper and zinc in 20~50 µl of serum using a water-soluble pyridylazo dye were developed. Drug-bound iron e.g. saccharated ferric oxide, and transferrin-bound iron in serum were simultaneously determined using a combined system of kinetic and end-point assay. A system for serum zinc with a cationic porphyrin was three-times more sensitive than that with the pyridylazo dye. A latex agglutination immunoassay was utilized for the determination of α2-macroglobulin and albumin in serum. The proposed systems are sensitive, accurate, precise, simple, and relatively inexpensive.
Keywords : downsizing of sample and reagent; microtiter plates; colorimetric assay; highly sensitive reagents; trace metals; serum proteins.
Daido Ishii*
*Microlab, 1-458, Horagai, Midori-ku, Nagoya, 458-0013
(Received 7 Augast 2000, Accepted 12 October 2000)Miniaturization, in general, has numerous merits, and has long been a well-recognized direction in science and technology, as its broad range of applications including in the field of analytical chemistry. Component miniaturization is frequently sought to improve the performance versatillity of various analyzers. Commercially available flexible fused-silica capillary tubes were found to have uniform narrow bore (0.005~0.5 mm), inert inner surface, good flexilibilty and convenient performance for the miniaturization of analytical instruments. This paper reports on the practical use of fused-silica capillary tubes concerning (1) micro and ultra-micro HPLC, (2) LC using an open tubular column with rotation mixing technology, (3) micro super-critical fluid chromatography and unified fluid chromatography, (4) ultra-micro flow injection analysis (U-MFIA), (5) micro and ultra-micro photometric, conductometric and pH titration methods. Fused-silica capillary tubes could be used for micro or ultra-micro analyzers constructing materials, such as packed and open tubular columns, flow cells, reaction tubes, connecting tubes, sampling tubes, burettes and pipettes.
Keywords : capillary; micro HPLC; ultra-micro FIA; ultra-micro titration methods; miniaturization of chemical analysis.
Shigeru Taguchi, Sun Hui-Feng, Noriko Hata and Issei Kasahara*
*Faculty of Science, Toyama University, 3190, Gofuku, Toyama 930-8555
(Received 14 July 2000, Accepted 6 September 2000)Novel techniques of membrane filtration for the enrichment and determination of trace components in water are reviewed from an aspect of downsizing in analytical chemistry. One method is based on the conversion of a component in water to a hydrophobic species by a chemical reaction, and its retention on a membrane by a hydrophobic interaction between the species and the membrane material. The species retained on the membrane are eluted or dissolved together with the membrane in a small volume of solvent and determined by an appropriate instrumental method, such as spectrophotometry, GF-AAS or ICP-AES. More than a 100-fold enrichment has been easily attainable by this method. Other enrichment/determination approaches without elution and dissolution, by means of reflective spectrophotometry, tristimulus colorimetry and using a transparent membrane filter have exhibited new potentials of this method from the view point of zero emission-chemistry, in addition to a simple and rapid procedure. Some typical applications of this technique to the preconcentration and determination of trace components in environmental water samples were described. The retention behavior of ion-pairs on a membrane filter was interpreted by ion-pair solid-phase extraction equilibrium. The effects of functional groups in the ions and solid-phase materials, the structure of the ions and the organic solvent added to the aqueous phase on the extraction behavior are described.
Keywords : downsizing in analytical chemistry; membrane filter; solid-phase extraction; preconcentration; trace analysis.
Katsutoshi Ito, Masumi Mitome, Hidetoshi Arakawa and Masako Maeda*
*School of Pharmaceutical Sciences, Showa University, 1-5-8, Hatanodai, Shinagawa, Tokyo 142-8555
(Received 30 June 2000, Accepted 8 August 2000)We have developed a simultaneous determination of galactose (Gal) and galactose-1-phosphate (G1P) by a semi-micro flow-injection analysis (FIA) system using an enzyme-immobilized column. This method was applied to preliminary neonatal mass-screening for galactosemia. A galactose dehydrogenase (Gal-DH) and alkaline phosphatase (ALP) were immobilized to TSK-gel Tresyl 5PW (Tosoh Co., Tokyo) and packed into a column (1.5 i.d. × 50 mm for Gal-DH and 1.5 i.d. × 100 mm for ALP) . The enzyme-immobilized columns were connected to high-pressure 6-way valves. In the proposed method, the detection limits (S/N=3) of Gal and G1P were 3.5 × 10-13 and 5.4 × 10-13 mol/assay, respectively. The measurable ranges for Gal and G1P were 2~20 and 5.5~23.8 mg/dl in the standard dried-blood disc (3 mm i.d.) on filter paper, respectively. The accuracy of an intra-assay at each point of the standard disc (n=5) was less than 1.0%. The mean recoveries (n=10) of Gal and G1P from a dried-blood disc were 99.3 and 84.8%, respectively. Further, we measured the Gal and G1P concentrations of neonatal blood-disc samples by the proposed method and compared the results with a conventional fluorescent enzymatic method. The regression equations determined for the results were y (FIA)=0.976x (enzymatic method)-0.929 (r=0.954, n=11) for Gal and y (FIA)=1.197x (enzymatic method)-0.539 (r=0.824, n=11) for G1P, respectively. A high degree of correlation was observed between the values obtained by the proposed FIA and those by the conventional enzymatic method. The proposed method could quantitatively measure Gal and G1P in one dried-blood disc on filter paper for 13 min.
Keywords : neonatal mass-screening; enzyme-immobilized column; galactose; galactose-1-phosphate; galactosemia.
Kazuyoshi Kurihara*1, Motoichi Ohtsu*1,*2, Hideaki Hisamoto*3 and Koji Suzuki*1,*3
*1 Kanagawa Academy of Science and Technology, 3-2-1, Sakado, Takatsu-ku, Kawasaki 213-0012
*2 Interdisciplinary Graduate School of Sceience and Engineering, Tokyo Institute of Technology, 4259, Nagatsuta Midori-ku Yokohama 226-8502
*3 Department of Applied Chemistry, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama 223-8522
The preparation and response features of a micrometer-sized sodium ion-selective fiber optode based on a liquid membrane were described. The sensing membrane is a plasticized poly (vinyl chloride)-based copolymer with a neutral ionophore and an anionic dye. In order to fabricate a micrometer-sized fiber optode, a "micropipette fabrication method" was newly proposed to fix a liquid membrane-based optode on the small tip of an optical fiber probe. At the first stage of the investigation, it was found that the ionophore including a sodium ion in its cavity leached from the membrane phase. However, we have discovered that the problem can be resolved by using a "tailed" ionophore, which is an ionophore possessing a lipophilic long alkyl chain. The "tail" of the ionophore functions as an "anchor" which prevents leaching of the ionophore from the membrane phase into the water phase. The anchor effect of the tailed ionophore was clearly demonstrated with 6 µm-sized sodium ion-selective optodes. In addition, the problem of fluorescence distortion due to photobleaching and solvent effect was resolved by a ratiometric calibration in which the sensor response is monitored by the spectral shift of the dual-emission fluorescence. The sensor response of an 8 µm-sized fiber optode having ratiometric calibration was examined and successfully explained by response theory. Our method gives a general preparation for ultrasmall ion-selective fiber optodes because other ion-selective optodes can be obtained simply by replacing the tailed ionophore.
Keywords : microoptode; ion-selective optode; fiber optode; tailed ionophore; liquid membrane.
Shigeaki Nishii, Yoshihiro Soya, Kazuhiro Matsui, Takuya Ishibashi and Yoshihisa Kawamura*
*Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., 10-24, Toyo-cho, Tsuruga-shi, Fukui 914-0047
(Received 14 July 2000, Accepted 5 September 2000)Bisphenol A(BPA), which is used as a starting material of epoxy resins and polycarbonates, is suspected as being one of the endocrine-disrupting chemicals. Generally, BPA analysis requires an expensive instrument, such as GC/MS, and time-consuming processes, such as extraction, concentration, and derivatization. We have developed a rapid, simple, and highly sensitive analytical method of BPA by enzyme-linked immnosorbent assay(ELISA) using an organic solvent-resistant anti-BPA monoclonal antibody in order to solve such problems. In order to produce anti-BPA monoclonal antibodies, Hybridoma cells were produced by the fusion of mouse myeloma cells and spleen cells from mice immunized with BPA-bovine serum albumin conjugates(BPA-BSA). Some anti-BPA monoclonal antibodies were found to have resistance to methanol. They selectively react with BPA, but do not show a cross-reaction with structurally related compounds. The detection limit of BPA by ELISA using the organic solvent resistant antibody was 0.5 ppb or less, and the detection limit in 50% methanol was 1 ppb. The measurement of BPA is possible even in other organic solvents, such as ethanol, acetone, DMSO and acetonitrile, if the organic-solvent concentration in the sample is 30% or less. This ELISA system provides an analytical method which does not affect environment for detecting the BPA concentration in river water and blood specimens. In this paper, various analysis examples using the ELISA method are introduced.
Keywords : bisphenol A; ELISA; organic solvent resistant anti-bisphenol A monoclonal antibody; endocrine-disrupting chemicals.
Hideto Iketake and Akifumi Yamada*
*Department of Chemistry, Faculty of Engineering, Nagaoka University of Technology, 1603-1, Kamitomioka-machi, Nagaoka-shi, Niigata 940-2188
(Received 14 July 2000, Accepted 4 September 2000)A residual chlorine sensor with a gold electrode was developed using voltammetry. The sensor consists of a working electrode (gold wire), a counter electrode (platinum wire) and a reference electrode {saturated calomel electrode (SCE)}. The electrochemical behavior of residual chlorine was measured by cyclic voltammetry. The reduction wave of residual chlorine was observed from -0.6 V vs. SCE at pH 7. The measurements were carried out using the potential sweep method and were monitored at +0.1 V vs. SCE. A linear calibration curve was obtained in the concentration range of 0.1 to 200 mg l-1 residual chlorine. The relative standard deviation was 2.7% for 3 mg l-1 residual chlorine. The reduction current at the measurement potential was not dependent on the pH, and there was positive interference due to the existence of 0.1 mg l-1 copper ion. The working electrode was able to be used for over 300 continuous measurements. The sensor was applied to the determination of residual chlorine in tap water. The results agreed extremely well with those obtained by the o-tolidine photometric method.
Keywords : residual chlorine; voltammetry; gold electrode; tap water; on-site measurement.
Hiromitsu Hachiya*2,*5, Manabu Negishi, Tsuneaki Maeda, Satoshi Ito*2, Seishiro Ohya*3, Kinji Harada*4, Ming Yang*5, Shizuka Nakano*6, Katsumi Uchiyama and Toshiyuki Hobo*5
*2 DKK·TOA Corporation, 2-214, Sakuragaoka, Higashiyamato-shi, Tokyo, 207-0022
*3 Kanagawa Industrial Technology Research Institute, 705-1, Shimoimaizumi, Ebina-shi, Kanagawa, 243-0435
*4 Yokogawa Research Institute Corporation, 2-9-32, Nakamachi, Musashino-shi, Tokyo, 180-8750
*5 Tokyo Metropolitan University, Graduate School of Engineering, 1-1, Minami-ohsawa, Hachioji-shi, Tokyo, 192-0397
*6 Mechanical Engineering Laboratory, 1-2, Namiki, Tsukuba-shi, Ibaraki, 305-8564
A Clark-type electrochemical(EC) chlorine gas sensor has been used for a leakage-gas detector. The EC sensor, which has a PTFE porous filter as a gas permeable membrane, shows excellent performance in terms of rapid response, sensitivity, reproducibility and long-term stability. However, the size of the conventional EC sensor is as large as several centimeters. We have fabricated a micro Clark-type EC chlorine sensor using micromachining. Pyrex glass used as a sensor body was processed using a dicing-saw. Pt/Ti thin membranes adhered to the glass by means of ECR- sputtering are used as a working electrode and a counter electrode. Porous silicon of 160 µm in thickness, which had systematic φ30 µm micro-pores with a pitch of 100 µm, by means of the DeepRIE, was used as a gas-permeable membrane. It was stored in the vapor of 1,1,1,3,3,3-hexamethyldisilazane to obtain a water-repellent surface for 24 hours, and adhered to the sensor body using a silicone adhesive. The size of sensor body was 5×5×3 mm. A MBr (M: alkaline metal) solution as an electrolyte was injected to the sensor body adhered on a TO-8 package. The linear calibration curve and correlation factor in the concentrating range from 0 to 0.5 ppmCl2 was I =-16.886C-1.195 (nA) and 0.998, respectively. The lower detection limit was 0.1 ppm. The 50% response time and the 90% recovery time were within 10 s. and several minutes, respectively. The reproducibility for 2 ppm was the deviation of the sensor signals within ±1 nA. This miniaturized EC chlorine sensor showed good performances.
Keywords : microsensor; electrochemical; chlorine; gas sensor; toxic gas.
Kei Toda, Minoru Kitazaki and Isao Sanemasa*
*Department of Environmental Science, Faculty of Science, Kumamoto University, 2-39-1, Kurokami, Kumamoto 860-8555
(Received 5 July 2000, Accepted 3 October 2000)A small gas detector was developed, in which an electrode and a gas absorber were combined in a small droplet. An Au film was coated on the outside of a fused-silica capillary, and silicone was covered over the Au film for the purpose of insulation. The capillary was cut, and then a micro-ring electrode appeared on its tip. A small droplet was formed at the tip of the capillary and gas was absorbed into the droplet. The absorbed component was measured by the micro-ring electrode that was in contact with the droplet solution. In this work, the detection characteristics of the droplet micro-ring electrode were evaluated using SO2 gas. The droplet size was from sub-micro litters to a few micro-litters, and was much smaller than the droplets used as the gas absorber before. Since the gas-liquid equilibrium was quickly reacted, the measurement could be performed within a short absorbing time. A new absorbing solution was supplied at every measurement, and the electrode could be easily renewed. This system was expected to be suitable for field measurements.
Keywords : droplet micro ring electrode (DMRE); gas sensing; sulfur dioxide.
Minoru Okumura, Mie Yoshihara, Kaoru Fujinaga and Yasushi Seike*
*Interdisciplinary Faculty of Science and Engineering, Shimane University, 1060, Nishikawatsu-cho, Matsue-shi, Shimane 690-8504
(Received 14 July 2000, Accepted 5 October 2000)A simple preconcentration method for total carbonate in environmental water samples has been developed. Carbon dioxide, which was generated from an acidified sample solution, was collected on a small column packed with an OH-type anion exchange resin by bubbling nitrogen gas through the solution. After elution with a potassium nitrate solution, the total carbonate was determined using a carbon dioxide electrode. The established method is applicable to brackish and seawater samples as well as freshwater. With its application to brackish water samples taken from brackish Lake Nakaumi, the behavior of total carbonate in the lake was clarified. In this method without any expensive instruments for analysis, the use of a small resin column enables us to make the sample size smaller and to stably store the samples for a long time (at least 75 days).
Keywords : determination of total carbonate; preconcentration; carbon dioxide trapping; anion exchange resin column; carbon dioxide electrode.
Toshiaki Okabe, Yukio Yokoyama and Hisakuni Sato*
*Laboratory of Analytical Chemistry, Faculty of Engineering, Yokohama National University, 79-5, Tokiwadai, Hodogaya-ku, Yokohama 240-8501
(Received 26 July 2000, Accepted 6 September 2000)The spectrophtometric determination of polyoxyethylene non-ionic surfactants (NS) utilizing ferric thiocyanate complex formation preceded by direct toluene-extraction was miniaturized. The consumption of the reagents used was drastically decreased to ca. 1/5. Linear relationships between the concentration and the absorbance at 510 nm for the standard heptaoxyethylenedodecylether ranged from 0.1 to 2.0 mg/l. The sensitivity of the method was ca. 24-times higher than that obtained in the cobalt(II)-thiocyanate method (Japanese Industrial Standard). Although the developed method is practically acceptable for environmental analyses, the coexistence of cationic surfactants tended to cause significantly positive errors for NS determinations, which indicated that cationic surfactants should be removed before toluene extraction. The method was applied to the determination of NS in synthetic detergents.
Keywords : polyoxyethylene non-ionic surfactant; toluene extraction; ferric thiocyanate complex; small-scale of spectrophotometric determination.
Yoshitaka Yamaguchi, Noboru Taguchi, Hiroshi Yamanouchi, Toshiaki Shibata, Kazuo Hitomi and Susumu Yamagishi*
*Ship Equipment and Marine Environment Division, Ship Research Institute, Ministry of Transport, 6-38-1 Shinkawa, Mitaka-shi, Tokyo 181-0004
(Received 17 July 2000, Accepted 16 October 2000)We need to detect spilled-oil conditions on/in the sea for cleaning the ocean and protecting the coast, if oil-spill accidents like Nadhoka and Erika occur. For one possible countermeasure we have developed an oil sensor. This sensor can be applied for a remote sensing technique (LIDAR) and fluorescence analysis. This system can distinguish oil spices in laboratory. We have tried to obtain field experience for the first time using fluorescence LIDAR (F-LIDAR). The F-LIDAR system was used to carry out a field analysis around Yuge Island on board a ship (Yuge-Maru). The fluorescence response was detected but oil was not detected during this voyage. We think that cause for this result was the microbes, based on an observation of seawater by an optical microscope. F-LIDAR can observe microbes under environmental conditions. Our system is new a useful and technique for monitoring oil and microbes in the marine environment.
Keywords : environments; remote sensing; oil; microbes; monitoring on board a ship.
Susumu Shimoyama and Yasuko Noda*
*Color Material Research Laboratory, Den Material Co., LTD., 5396, Kamiogino, Atugi-shi, Kanagawa 243-0201
(Received 8 August 2000, Accepted 5 October 2000)A portable RI X-ray fluorescence spectrometer having a weight of 1800 g was constructed by using a radiation annular source (241Am sealed up with ceramics, 1.85 MBq), a small Si-PIN photodiode X-ray detector mounted on a Peltier cooling device, an amplifier and a pocket-type multi -channel pulse-height analyzer. It could also be applied to a non-destructive analysis of the blue colorant in the original Japanese votive picture "Rashoumon-zu" offered to shrine in 1682. Consequently, it was clarified that the blue colorant (X) was "Smalt" containing Ca, Fe, Co, Ni and As as major elements.
Keywords : portable RI X-ray fluorescence spectrometer; non-destructive analysis; Japanese votive picture; Smalt.
Ichiro Sakata, Yuichi Hirama, Masahiro Takahashi and Kazuo Miyamura*
*Department of Chemistry, Faculty of Science, Science University of Tokyo, 1-3, Kagurazaka, Shinjuku-ku, Tokyo 162-8601
(Received 8 July 2000, Accepted 4 September 2000)By measuring the tunnel-gap-width dependence of the tunnel current using a scanning tunneling microscope (STM), the surface barrier heights were evaluated. We measured the local barrier heights of Au, Pt and graphite (HOPG) surfaces in air, Ar and distilled water. The result of the HOPG sample varied significantly with the tip used, and the data were not reproducible at all. This was probably due to the layered structure of HOPG. The barrier height of Au and Pt, on the other hand, had sufficient reproducibility. The barrier height of Au did not change with the environment of the measurement, while that of Pt varied significantly. The result indicates that a change in the surface electronic state affected by the environment could be detected by a barrier-height measurement, and it was thus shown that the barrier-height measurement is useful for evaluating the surface electronic state.
Keywords : scanning tunneling microscope; barrier height; HOPG, Au, Pt.
Naoyoshi Egashira, Jinshun Piao, Emi Hifumi and Taizo Uda*
*School of Biosciences, Hiroshima Prefectural University, 562, Nanatsuka, Shobara-shi, Hiroshima 727-0023
(Received 30 June 2000, Accepted 11 August 2000)A miniaturized cell based on electrochemiluminescence of tris(2,2'-bipyridine)ruthenium(II) complex was prepared to determine peptides in small volume of 0.5 cm3. A calibration for proline gave a straight line in the concentration range 1.0×10-5~1.0×10-3 M (correlation coefficient: 0.991). Stronger responses for proline and histidine than other amino acids prompted us to measure some peptides. Out of six peptides, PG having proline at the amino terminal showed the strongest response.
Keywords : tris(bipyrine)ruthenium complex; electrochemiluminescence; semi-micro cell; peptides.
Yutaka Itabashi* and Takao Tsuda**
*Laboratory of Bioresources Chemistry, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate-shi, Hokkaido 041-8611
**Department of Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555
Almost complete enantiomer separations of racemic 1-O-alkylglycerols as their bis(3,5-dinitrophenylurethane) derivatives were achieved by HPLC using a slurry-packed fused-silica capillary (40 cm×0.32 mm i.d.) with a chiral phase, N-(S)-2-(4-chlorophenyl)isovaleroyl-D-phenylglycine, ionically bonded to 5 µm silica gel. The analysis was optimized by using a ternary mobile phase, n-hexane-1,2-dichloroethane-ethanol, and UV detection at 254 nm.
Keywords : alkylglycerol; enantiomer; chiral-phase HPLC; capillary column; 3,5-dinitrophenylurethane.
Haruki Shiraishi, Satoshi Kawanaka, Toshio Matsuda, Osamu Tabata* and Shigero Ikeda**
*Faculty of Science and Engineering, Ritsumeikan University, 1-1-1, Noji-higashi, Kusatsu-shi, Shiga 525-8577
**SR Center, Ritsumeikan University, 1-1-1, Noji-higashi, Kusatsu-shi, Shiga 525-8577
In this paper, we present a new fabrication method that uses a UV-sensitive epoxy resin as an etching mask for a micro-channel electrophoresis chip. We have used it to separate fluorescein and fluorescein-5-sulfonic acid. The device was fabricated in a 50 mm square of soda glass by using micro-lithographic patterning and wet etching techniques. The proposed method does not need for plating and etching of Au/Cr masks. An etched bottom plate was covered with a top plate drilled access holes into it and permanently bonded in an oven by heating at 560~580°C. The chip had a separation channel and a sample injection channel. The separation channel was 80 µm wide and 20 µm deep. Using a micro-fabricated chip with a separation length of 10 mm and a separation voltage of 800 V, the electrophoresis of a fluorescein and fluorescein-5-sulfonic acid mixture gave two well-separated peaks. Detection was achieved using a laser-induced fluorescence microscope. The HETP of this chip was 3~4 µm for fluorescence samples, and a separation coefficient of 2.2 was realized. The proposed fabrication method is very simple and easy, and has given separation and detection results for fluorescein derivatives.
Keywords : micro-channel glass chip; electrophoresis; photosensitive epoxy resin; laser-induced fluorescence detection; fluorescein.
Fumiaki Yokota and Akiko Shimizu*
*Material Department, Industrial Research Institute, Aichi Prefectural Government, Nishi-Shinwari, Hitotsugi, Kariya-shi, Aichi 448-0003
(Received 12 July 2000, Accepted 5 October 2000)In order to accurately determine major elements (Cu, Sn, Zn and Pb) in milligram amounts of bronze reference samples, an application of ICP-AES using scandium as an internal standard element was investigated. The correlation coefficient of each major element to scandium was above 0.93 and the repeatable precision in measuring the emission intensity ratio of each major element to scandium was 0.10~0.45% (RSD, %, n=25), which corresponded to 1/3~1/5 times that obtained without an internal standard. In an analysis with a 10 mg amount of the sample, the relative variances for 4 major elements {Relative variance (%)=[(Mfound-Mcert)/Mcert] ×100, where Mfound and Mcert are the found and certified values, respectively.} were 0~-1.4%, except for Pb. On the other hand, those obtained from an analysis with a 5 mg or a 2 mg amount of sample were -0.8~-6.3%.
Keywords : determination of major elements in bronze; milligram amount of sample; bronze reference sample; ICP-AES using an internal standard method; an internal standard element.
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