BUNSEKI KAGAKU Abstracts

Vol. 53 No. 10

October, 2004

Research Papers

Influence of Cl^- ion additional on methanol electrooxidation at Nafion-modified Pt microelectrode

Kohshi Kashima¹, Minoru Umeda¹ and Akifumi Yamada¹

(Received 6 May 2004, Accepted 29 July 2004)

We have studied the influence of Cl^- ion for methanol electrooxidation in direct methanol fuel cells (DMFCs) by using a Nafion-modified Pt microelectrode(φ50 µm). The microelectrode was employed to form a uniform-thick Nafion overlayer on the Pt surface. Hg/Hg₂SO₄ and Pt wire were used as a reference and a counter electrode, respectively. Current-potential curves were measured in a 0.5 M H₂SO₄ or 0.5 M CH₃OH+0.5 M H₂SO₄ solution. KCl was added into the solution at arbitrary rates. As a result, we have observed that (i)Cl^- ion adsorbs on the Pt surface, which prohibited not only Pt oxide formation but also Methanol electrooxidation. The Nafion membrane prevented Cl^- adsorption, because of it's cation exchange property. (ii)However, a small part of Cl^- ion was able to pass throughout Nafion membrane and adsorbs on the Pt surface. (iii) It has been known that a strong correlation exists between Pt surface oxide formation and methanol electrooxidation at a Pt electrode.

Keywords : direct methanol fuel cell (DMFC); methanol oxidation; Cl^- ion; Nafion membrane; Pt microelectrode.

Adsorption and desorption of chromium(VI) on active carbon

Kousuke Ishida¹ and Masaaki Harada¹

(Received 6 May 2004, Accepted 9 August 2004)

The adsorption and desorption of chromium(VI) ion from aqueous solution on active carbons were studied. The adsorption rate of Cr(VI) ion was measured at various pH values, and was proved to be proportional to its concentration. The chemical species and their respective adsorption rate constants were determined from the pH dependence of the adsorption rate. A desorption experiment of adsorbed Cr(VI) was performed by HNO₃ or NaOH. Only Cr(III) ion was desorbed by HNO₃, while both Cr(III) and Cr(VI) ions were desorbed by NaOH. This shows that Cr(VI) adsorbed on active carbon was reduced to Cr(III) and then Cr(III) was desorbed. The results of Freundlich plot supported the adsorption/desorption mechanism presumed from the experimental results mentioned above.

Keywords : chromium; adsorption; desorption; active carbon.

Observation of a metal cation entering into a phospholipid bilayer by total internal reflection with thermal lens spectroscopy

Kaori Nakamura¹, Takuya Shimosaka^1,2, Katsumi Uchiyama¹ and Toshiyuki Hobo¹

(Received 11 May 2004, Accepted 9 August 2004)

We monitored the diffusion of Cu²⁺ into a phospholipid bilayer containing a metallochromic indicator, 1-(2-pyridylazo)-2-naphthol (PAN), by using total internal reflection with thermal lens spectroscopy (TIR-TLS), which is a non-destructive and surface-selective spectrometry. When Cu²⁺ entered into the bilayer, a formation of Cu-PAN chelate resulted in decrease of the absorbance at the wavelength of the excitation beam. The decrease was detected by TIR-TLS. We measured five kinds of bilayer samples, whose molar ratios of two phospholipids, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and 1-stearoyl-2-oreoyl-sn-glycero-3-phosphatidylserine (SOPS), were 10:0, 7:3, 5:5, 3:7, 0:10. In the case of a bilayer whose ratio of DOPC to SOPS was 7 to 3, the signal intensity showed an obvious decrease compared to a blank. On the other hand, there no significant change was observed in the case of other samples. Several attempts using a 7:3 bilayer revealed a non-reproducible incorporation of Cu²⁺ into the lipid. We also proved the effect of a counter ion by comparing sulfate ion and chloride ion.

Keywords : thermal lens spectroscopy; phospholipid bilayer; Cu cation; ion transportation.

Preparation and characterization of polymer-based stationary phase modified with Thiacalix[4]arene

Natsuki Hira¹, Nobuo Tanaka¹ and Ken Hosoya¹

(Received 7 May 2004, Accepted 10 August 2004)

We have prepared a polymer-based stationary phase modified with thiacalix[4]arene. Thiacalix[4]arene (5,11,17,23-tetra-tert-butyl-25,26,27,28-tetrahydroxy-2,8,14,20-tetrathiacalix[4]arene) is an amphiphilic molecule, that consists of four p-tert-butylphenol-related groups cyclically connected through a single sulphur atom between each pair. It has a high-electron density area formed by hydroxyl groups and sulfur atoms. We studied the applicability of this interesting compound as a selector because of its specific feature for high-performance liquid chromatography (HPLC). First of all, uniformly sized polymer particles were prepared using ethylene glycol dimethacrylate (EDMA) as a cross-linker through a multi-step swelling and polymerization method. Methacrylic acid (MAA) was introduced onto the surface of polymer particles through an invented modification method. Thiacalix[4]arene was cavalently bonded to MAA using 1,4-dibromo-butane as a spacer to reduce any steric hindrance of MAA on the surface of the polymer particles. The prepared polymer-based stationary phase was evaluated with HPLC. It provided a specific chromatographic retention behavior only for catechol among positional isomers of xylene, cresol, and benzene-diol. Catecholamine showed a specific chromatographic retention behavior as catechol.

Keywords : Thiacalixarene; catechol; catecholamine; uniformly size polymer particle; surface modified.

Effect of the weather condition and volcanic gas from Miyake Island on the ionic pollution of rain and fog water at the Akita Hachimantai mountain range

Keiji Yoshimura¹, Junko Inotsume¹, Ryoei Kikuchi¹, Toru Ozeki², Masahiro Kajikawa¹ and Nobuaki Ogawa¹

(Received 7 May 2004, Accepted 16 August 2004)

Rain and fog samples were collected from the Akita Hachimantai mountain range between June and September, 2000. By classifying the course of transport of the air mass by 72 h back trajectory at the 850 hPa level, when the air masses were transported from the Korean Peninsula or near China, the ion concentrations were shown to be very high by nss-SO₄^2-. After 8 July, 2000, when a volcano erupted at Miyake Island, a high [nss-SO₄^2-]/[NO₃^-] ratio was observed in some samples. It was concluded that the samples had been influenced by volcanic gas from Miyake Island using 72 h back trajectory at the 850 hPa level.

Keywords : acid rain and fog; volcanic gas; sulfate; sulfur dioxide.

Measurement of atmospheric peroxy radicals in the polluted-urban air using a chemical amplification

Hiromi Sawada¹, Tomoki Nomaguchi¹, Kenji Okitsu¹, Norimichi Takenaka¹ and Hiroshi Bandow¹

(Received 7 May 2004, Accepted 19 August 2004)

In September 2001, January 2002 and December 2003, we measured peroxy radicals (RO₂) in urban polluted air using the Peroxy Radical Chemical Amplification (PERCA) method. Since the observation site in 2003 was much affected by automobile exhaust, during the observatiion the average NO_x concentration exceeded 90 pptv. In this observation, the daytime RO₂ cancentration was similar to that at other observation sites, and in the reported literature. However, in 2003 measurement, the nighttime RO₂ concentration was found to be below the detection limit (3.5 pptv) most of the time. This result was different from the other two measurements. Though a difference was not seen in the RO₂ concentration in the observations of 2001 and 2002, the average O₃ concentration was very different. These results obtained in this study for urban polluted air can not be explained based on general knowledge about atmospheric chemistry, especially concerning the correlation between the RO₂ and O₃ concentrations.

Keywords : peroxy radicals; ozone; the polluted-urban air; PERCA; atmospheric chemistry.

Notes

Spectrophotometric determination of p-quinones with N,N-diethy-1,4-phenylenediamine

Yusuke Fujimoto¹, Tsuyoshi Fujimoto¹, Takako Yamaguchi¹ and Yoshikazu Fujita¹

(Received 7 May 2004, Accepted 30 July 2004)

A spectrophotometric method for the determination of p-quinones was established with N,N-diethy-1,4-phenylenediamine. In the determination of 2,3-dimethoxy-5-methyl-1,4-benzoquinone (DMBQ), Beer's law was obeyed in a concentration range of 0.1~1.5 µg/ml of DMBQ. The apparent molar absorptivity was 1.1×10⁵ l mol^-1 cm^-1 at 552 nm. The relative standard deviation for six replicate determinations for 0.18 µg/ml was 0.48%.

Keywords : p-quinones; spectrophpotometry; 2,3-dimethoxy-5-methyl-1,4-benzoquinone; N,N-diethy-1,4-phenylenediamine.

A disposable voltammetric cell for determining the titratable acidity in wood-vinegars

Noriko Kitamura¹, Akira Kotani¹ and Fumiyo Kusu¹

(Received 1 May 2004, Accepted 13 August 2004)

A disposable voltammetric cell using three pencil leads as working, reference, and counter electrodes was developed for determining titratable acidity, i.e. the acid content in wood-vinegar. The materials of the pencil leads were graphite-reinforcement carbons (GRCs). The voltammetric detection of acids was based on the electrochemical reduction of quinone, 3,5-di-tert-butyl 1,2-benzoquinone (DBBQ), in an unbuffered solution. Acetic acid in an unbuffered ethanol-water (3:2, v/v) solution of DBBQ gave rise to a new peak (termed the prepeak) on a voltammogram at more positive potentials than that corresponding to the normal reduction peak of DBBQ. The prepeak current was found to be proportional to the acetic acid concentration from 0.05 to 2.7 mM with a correlation coefficient of 0.999. The cell-to-cell reproducibility for 1 mM acetic acid was evaluated with five individual disposable cells. The RSD of the prepeak current and potential were 2.0% and 1.5%, respectively. The titratable acidity in six wood-vinegar samples was determined by voltammetry using disposable cells, and was compared with that of the titratable acidity determined by the conventional potentiometric titration method. We then observed the results by both methods, and found a correlation coefficient of 0.999. As such, the voltammetry using disposable-cell required only two thousandth of the volume of a wood-vinegar sample for the titration method. The present method was superior to the potentiometric titration method in terms of facility, environment-friendly, and economy, and thus a sensor using the present cell would be useful for routine work in the quality control of wood-vinegar.

Keywords : voltammetry; 3,5-di-tert-butyl 1,2-benzoquinone; titratable acidity; wood-vinegars.

Analytical Reports

Determination of lanthanoides and heavy metals in Sagami Bay sediment and sediment reference material MESS-3

Shin Kumakura¹, Miho Tanaka¹, Susumu Hashimoto² and Masaru Maeda¹

(Received 6 May 2004, Accepted 11 August 2004)

To obtain current data of lanthanoides and heavy metals in Sagami Bay sediment, a sediment sample was collected at a location 13 km off shore. The surface sediment (0~1 cm) was decomposed with HNO₃, HF and HClO₄. The detection of these elements was achieved by inductively coupled plasma mass spectrometry. These data will be used as a comprehensive baseline data for future environmental-impact studies, such as on the discharge of accumulated sands and sediments from the dam in Sagami River. A sediment reference material, MESS-3, was also analyzed to verify the present analytical method. The obtained value for heavy metals, such as Cr and As, were in good agreement with the certified values. The concentrations of lanthanoides in MESS-3 were also presented.

Keywords : Sagami Bay; MESS-3; lanthanoides; sediment.

Accounts

Spectroscopic detection and reaction analysis of aromatic cation radicals using an electron-transfer stopped-flow method

Munetaka Oyama¹

(Received 31 March 2004, Accepted 18 May 2004)

For the purpose of elucidating and understanding the reaction dynamics of short-lived intermediates formed in the solution phase, it is very important to carry out detection and kinetic analysis in a homogeneous solution without being interfered by concentration gradients, which are usually unavoidable in electrochemical and pulse-radiolysis methods. Our group is proposing an electron-transfer stopped-flow (ETSF) method for the spectroscopic detection and reaction analysis of short-lived ion radicals formed in aprotic solvents. In the ETSF method, e.g., short-lived cation radicals are formed via electron transfer reactions with stable cation radicals in the mixing part of a stopped-flow apparatus, so that any changes in the absorption spectra are observed in an optical cell after stopping the solution flow. In this account, the principle and actual procedures of the ETSF method are described first. Then, some successful results obtained using the ETSF method in observing the absorption spectra and analyzing the reaction mechanisms are summarized for the reactions of poly-aromatic cation radicals and aromatic amine cation radicals. The former includes anthracene and pyrene derivative cation radicals formed in acetonitrile, and the latter includes aniline, N-methylaniline, N,N-dimethylaniline and diphenylamine derivative cation radicals formed in acetonitrile. Finally, as interesting examples that the ETSF analysis cannot give straightforward results, time-resolved absorption spectra observed for the oxidation processes of N,N-dimethyl-o-toluidine and N,N-dimethyl-m-toluidine in acetonitrile are shown and discussed.

Keywords : stopped-flow method; electron transfer reaction; aromatic cation radical; reaction analysis; visible absorption spectroscopy.

Research Papers

The determination and molecular weight measurment of cationic polymers used for hair shampoo by size-exclusion chromatography

Junichi Sugiyama¹, Hiroyuki Hanihara¹, Kosuke Tanaka¹ and Kuniaki Adachi¹

(Received 27 February 2004, Accepted 11 May 2004)

Cationic polymers in hair shampoo form a complex with an anionic surfactant, and adhere to the hair when the shampoo is rinsed. This complex plays an important role in the feeling of the rinse for user, and in the conditioning provided by the shampoo. Therefore, the amount and molecular weight information of the cationic polymer is important for examining both the performance and quality control. Nevertheless, no accurate method has yet been established for analyzing the cationic polymers used for hair shampoo. We used SEC-MALLS-RI (size-exclusion chromatography with a multiangle laser light-scattering detector and refractive index detector) to examine the cationic cellulose [cellulose,2-hydroxyethyl 2-{2-hydroxy-3(trimethylammonio)propoxy}ethyl 2-hydroxy-3-(trimethylammonio)propyl ether,chloride] (CC) used for hair shampoo. In this analysis, we used a column in which the stationary phases were based on vinyl polymer and a 0.3 M NaClO₄ solution. The chromatogram was clear and precise, and we verified that the CC was separated by the size of MALLS and RI(plot of molecular weight vs. retention time). There was no adsorption between the CC and the column stationary phases, and the calibration curves showed good agreement with each other. It was determined that any grade of CC could be used as a standard. We conclude that the present technique is an accurate method of measuring the amount of CC and determining its absolute molecular weight used for hair shampoo.

Keywords : cationic cellulose derivatives; determination molecular weight; SEC; polymer; shampoo; MALLS.

Precise and rapid determination of anionic and cationic surfactants by ¹H nuclear magnetic resonance using an internal standard

Ryo Koike¹, Shoichi Jo¹, Mikiko Azuma¹ and Tatsushi Wakisaka¹

(Received 8 April 2004, Accepted 31 May 2004)

A method based on proton nuclear magnetic resonance (¹H-NMR) using an internal standard (IS) was developed for the determination of anionic and cationic surfactants. In this study, potassium hydrogen phthalate or sodium 3-trimethylsilyl-2,2,3,3-tetradeutero propionic acid was added to a sample solution as an IS for the determination, and surfactants were determined by using integrations of IS and diagnostic surfactant resonances in the ¹H-NMR spectrum. This method is suitable for the determination of surfactants because there is no surfactant whose contribution corresponds completely with each analyte in many cases. By optimizing the acquisition parameters and solvent, various surfactants were determined under the same condition. Each ¹H-NMR spectrum was obtained within 5 min and the detection limit given for sodium lauryl sulfate, a typical anionic surfactant, was approximately 60 ppm. The results obtained for commercial surfactants were in good agreement with those of the JIS titration method (Epton method) and the relative standard deviations were 0.12~0.44%.

Keywords : nuclear magnetic resonance; internal standard; determination; anionic surfactants; cationic surfactants.

Precise and rapid determination of amphoteric and nonionic surfactants by ¹H nuclear magnetic resonance using an internal standard

Ryo Koike¹, Shoichi Jo¹, Mikiko Azuma¹ and Tatsushi Wakisaka¹

(Received 8 April 2004, Accepted 31 May 2004)

A method based on proton nuclear magnetic resonance (¹H-NMR) using an internal standard (IS) was developed for the determination of amphoteric and nonionic surfactants. This method does not require a tedious pretreatment, and surfactants are determined by using integrations of IS and diagnostic surfactant resonances in the ¹H-NMR spectrum. By optimizing the combination of solvent and IS, various surfactants were determined under the same condition. Each sample was measured within 5 min, and the detection limit given for lauryl dimethylamine oxide, a typical amphoteric surfactant, was 8.0 ppm. The results obtained for commercial surfactants were in good agreement with those of the conventional methods (gravimetric method etc.) and the relative standard deviations were 0.13~0.36%. Furthermore, we confirmed that the proposed method was applicable to the determination of nonionic surfactants not containing an ethylene oxide chain, such as glycerol fatty acid ester and fatty acid alkanolamide.

Keywords : nuclear magnetic resonance (NMR); internal standard; determination; amphoteric surfactants; nonionic surfactants.

Determination of individual nonylphenol isomers in water samples by using relative response factor method

Yuichi Horii^1,2, Takao Katase³, Yun-Seok Kim³ and Nobuyoshi Yamashita¹

(Received 10 February 2004, Accepted 1 June 2004)

Because technical nonylphenol (NP) preparations are constituted by complex isomers and impurities, wide varieties of isomer composition were found in the environmental matrix. Since there is not sufficient information concerning isomer specific quantification, the currently available analytical method (e.g. JIS K 0450-20-10, 2002) of NP is applicable to determine only the total concentration, which is calculated by summing three to eight peaks in a gas-chromatogram. Hence, reliable analytical methods for each isomer are necessary for a risk assessment and source determination of NP, which is one of the most important endocrine disruptors. In this study, we developed an isomer-specific quantification method of thirteen individual NP isomers based on relative response factor (RRF) quantification by gas-chromatograph mass spectrometry in combination with steam distillation extraction of non-filtered water (SD-RRF method). The remarkable points concerning the SD-RRF method are that the extraction efficiencies of chemicals using steam distillation correspond to their boiling point, not only polarity. This method enables a reliable identification/quantification of thirteen isomers of NP by using RRF (by using a suitable ion to quantify; m/z 107, 121, 135, 149, 163, 177, 191 and 220). We validated the SD-RRF method for selected water samples in Arakawa River and Hanamigawa River around Tokyo Bay; a significant variation (3.8 to 15%) of the isomer composition was found in environment water samples.

Keywords : nonylphenol; isomer; steam distillation extraction; relative response factor; water.

Technical Papers

Determination of airborne organic contaminants in clean-room air by silicon powder adsorption-solvent extraction-GC/MS analysis

Yuka Sato¹, Kimito Sakai¹, Makiko Tamaoki² and Miyuki Takenaka¹

(Received 3 March 2004, Accepted 8 May 2004)

Some airborne organic contaminants in clean rooms for semiconductor manufacturing are unstable with respect to heating, and are easily adsorbed by silicon wafers. Accordingly, in order to measure their concentration, we developed a method that uses silicon powder adsorbent and solvent desorption-GC/MS analysis. Various methods of desorption were examined, and it was found that the most efficient method was desorption by passing n-hexane through the adsorbents. This method was compared with thermal desorption using standard solutions of di(2-ethylhexyl)phthalate (DEHP) and 2,6-di-buthylhydroxyhydroxyphenol (BHT). Using these solutions, which are unstable against heating, compounds could be detected without any alteration by this method, although altered compounds from DEHP and BHT were detected by thermal desorption. The detection limits of DEHP, dibuthylphthalate (DBP) and di(2-ethylhexyl)adipate (DEHA) by this solvent desorption method were 2 ng/m³, 2 ng/m³ and 0.6 ng/m³ respectively. By applying this method to the evaluation of clean-room air, DEHP, DBP and DEHA were detected, ranging in concentration from 16 to 80 ng/m³.

Keywords : solvent extraction; silicon powder adsorbent; clean room; airborne organic contaminant.

Notes

Determination of lead in steel by high power nitrogen microwave induced plasma atomic emission spectrometry coupled with hydride generation technique

Akihiro Matsumoto¹, Tadashi Shiozaki² and Taketoshi Nakahara²

(Received 9 April 2004, Accepted 27 May 2004)

An annular-shaped high power nitrogen microwave induced plasma atomic emission spectrometry with a hydride generation method is described for the determination of lead in steel. Under the optimized experimental conditions, the best attainable detection limit at the Pb I 368.347 nm lines was 35.2 ng ml^-1 for lead. The linear dynamic range for lead was 100 to 30000 ng ml^-1 in concentrations. The presence of several diverse elements was found to cause a more or less depressing interference with the proposed technique. Of the several oxidizing agents examined, hydrogen peroxide for lead was found to be the most preferable to oxidize lead prior to hydride generation. When lead in two certified reference materials of steel was determined, a large amount of Fe(III) in the solution caused a severe depressing interference, while the presence of Fe(II) showed little or no significant interference. Of the several interference-releasing agents examined, L-ascorbic acid was found to be the most preferable to pre-reduce Fe(III) to Fe(II). The concentrations of lead in steel were determined by the use of the standard additions method. The results obtained by this method were in good agreement with their certified values.

Keywords : high power nitrogen microwave induced plasma; atomic emission spectrometry; hydride generation method; lead; steel.

Pyrolysis-gas chromatography/mass spectrometry of poly(acrylic acid) in the presence of mixture of N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane

Yoshio Kumooka¹

(Received 24 March 2004, Accepted 1 June 2004)

A mixture of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS) (99:1) is a highly sensitive derivatization reagent for carboxylic acid by gas chromatography/mass spectrometry (GC/MS), and Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) of poly(acrylic acid) (PAA) at 500°C in the presence of the above silyl reagent was conducted. It was found that the peak height of the trimethylsilyl derivative of acrylic acid (AA-TMS) was about ninety-five times that of the peak height of acrylic acid, which is one of the pyrolysates of PAA, without any derivatization reagent. The mechanism for the formation of AA-TMS was considered to be as follows. The carboxyl group of PAA and the mixture of BSTFA and TMCS reacted rapidly at room temperature. The reaction product was then pyrolyzed and AA-TMS was liberated.

Keywords : pyrolysis-gas chromatography/mass spectrometry; poly(acrylic acid); N,O-bis(trimethylsilyl)trifluoroacetamide; trimethylchlorosilane.

Technical Letters

Solid-phase extraction for the reversed-phase high-performance liquid chromatographic determination of iodoamino acids

Masahiro Miyashita¹ and Yoshiyuki Seyama¹

(Received 10 March 2004, Accepted 25 May 2004)

We studied solid-phase extraction (SPE) procedures using three SPE columns (OASIS HLB, MCX and MAX®) for the determination of iodoamino acids (iodohistidines, iodotyrosines and iodothyronines) by high-performance liquid chromatography (HPLC). Iodoamino acids could be retained on the SPE columns by hydrophobic, cation-exchange and/or anion-exchange interactions. Firstly, the effect of the sample pH on the retention was investigated. Secondly, the elution of iodoamino acids retained on each SPE column was investigated. The recoveries of iodoamino acids were evaluated using different compositions of the eluent. Good recovery (>90%) could be obtained under the optimum SPE conditions.

Keywords : iodohistidines; iodotyrosines; iodothyronines; solid-phase extraction; high-performance liquid chromatography.

A round-robin analysis for lead, cadmium and arsenic concentrations in JSAC soil CRM with the compliance test of the Ministry of Environment, Notification 19

Organizing committee of soil analysis seminar for The Japan Society for Analytical Chemistry¹

(Received 9 March 2004, Accepted 20 July 2004)

The Japan Society for Analytical Chemistry (JSAC) carried out a round-robin analysis of Pb, Cd and As concentrations in a soil certified reference material (JSAC CRM 0401 Brown Forest Soil) by a method of compliance test of the Ministry of Environment (Notification 19) based on leaching with 1 mol/l hydrochloric acid. The results were consistent among the 12 laboratories involved for Pb and Cd (inter-laboratory variation: <8%); however, that for As was more variable (20.5%), probably due to unidentified analytical problem(s) related to the use of a hydride generation technique. Based on the mean values of round-robin analysis after statistical outlier rejection and additional analyses for As, the consensus values for the fractions of Pb, Cd and As leachable by the compliance test method were determined to be 14.0, 3.80 and 1.07 mg/kg, respectively.

Keywords : soil certified reference material; round-robin analysis; lead; cadmium; arsenic; consensus value.

Digests of Doctoral Dissertation

Analytical systems that integrate biological reactions by immobilized biomolecules

Kumiko Sakai-Kato

Department of Pharmaceutical Sciences, Musashino University, 1-1-20, Shinmachi, Nishitokyo-shi, Tokyo 202-8585

(Awarded by The University of Tokyo dated March 10, 2004)

Analytical systems that integrate biological reactions by immobilized biomolecules were developed. For the immobilization of biomolecules in a micro-sized flow-through system, a novel biomolecule-encapsulation technique was developed using a sol-gel reaction. Various biomolecules were encapsulated in a tetramethoxysilane (TMOS)-based hydrogel within a capillary in a single step under mild conditions. This monolithic column showed an adequate chromatographic performance, including mechanical strength, and penetration of pressurized flow. The encapsulation of bovine serum albumin and ovomucoid in a TMOS-based matrix allowed for the separation of drug enantiomers. A trypsin-encapsulated capillary column enabled the creation of an on-line enzyme reactor with enhanced reactivity. Furthermore, by encapsulating microsomes isolated from rat liver, an on-line drug-metabolism analytical system was developed, where the metabolic reaction of drug and the determination of the metabolites were integrated in a single capillary. This thesis also presents the in situ fabrication of trypsin-encapsulated gels onto a microchip for an on-chip bioreactor, in which integrated protein digestion, separation, and detection were performed. This encapsulation technique opens a new pathway for the application to automated and high-throughput analytical schemes for a multi-analyte device or drug-screening platform.

(Received April 19, 2004)

Keywords : sol-gel; encapsulation; alkoxysilane; capillary electrophoresis; microchip electrophoresis; on-line analytical system; biomolecules.

Development of detection methods for DNA adducts of acetaldehyde by capillary electrophoresis and liquid chromatography

Shinsuke Inagaki

Measurement Technology Group, Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1, Onogawa, Tsukuba-shi, Ibaraki 305-8569

(Awarded by Gifu Pharmaceutical University dated October 14, 2003)

Acetaldehyde reacts with exocyclic amino groups of guanine in DNA to form N²-ethylguanine (N²-Et-Gua) and cyclic 1,N²-propanoguanine adduct (CPr-Gua). Until now, the ³²P-postlabeling assay is one of the most useful methods for the detection of DNA adducts. However, it is not a safe one because it requires radioactive compounds. In this study, more safe and precise methods for the detection of DNA adducts using capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) were developed. Using CE, we succeeded to develop a separation system that enabled us to control the migration time of nucleotides by changing the pH of the running buffer solution and the concentrations of buffer additives. Also, we developed an electrochemical detection cell for CE, which carried out the selective detection of normal and damaged guanines. Using liquid chromatography-mass spectrometry (LC-MS), a highly sensitive detection of DNA adducts was achieved, and it could be used to analyze real DNA samples. We considered about damage in DNA through the LC-MS analysis of DNA of cultured human cells and calf thymus DNA that were exposed to acetaldehyde. The developed detection methods are expected to replace the ³²P-postlabeling assay, or to be complementary to the assay. These results will contribute for DNA adducts studies in connection with carcinogenesis.

(Received April 9, 2004)

Keywords : DNA; acetaldehyde; N²-ethylguanine; cyclic 1,N²-propanoguanine adduct; capillary electrophoresis; liquid chromatography-mass spectrometry.

Structural effect of di-Schiff base ligands having pendant arms on ion-pair extraction selectivity of divalent metals

Syunichi Oshima

Department of Chemistry and Biology Engineering, Fukui National College of Technology, Geshi-cho, Sabae-shi, Fukui 916-8507

(Awarded by Kanazawa University dated March 25, 2003)

The structural effect of di-Schiff base ligands having pendant arms on ion-pair extraction selectivity of divalent transition metal cations was investigated by changing the ligand structure systematically. A di-Schiff base ligand, N,N'-bis(2-pyridylmethylidene)-1,2-diiminoethane (BPIE), and its analogues were used as complexation reagents. These ligands acted mainly as imine-N bidentate ones in the extraction systems, except for several cases. The effects of substituents around imine-N donor atoms, the imine-N distance, the steric restriction and the pendant arms were evaluated by comparing to extraction behavior of the BPIE analogues. (i) The introduction of substituents onto imine-C atoms led to a change in the extraction selectivity, whereas that onto ethylene-C atoms only caused an enhancement of the extractability without any change of the selectivity. (ii) A closer imine-N distance led to a higher mutual selectivity between each metal cation, whereas a longer distance decreased the selectivity. (iii) The effects of a steric restriction by geometrical and resonance structure resulted in the differences of the shield effect in extracted species and the steric distortion on the complexation, and these contributed the extraction selectivity. (iv) The pendant arms affected only the extractabiliby in the system. Furthermore, a selective extraction of capper (II) from other metal cations was achieved.

(Received April 14, 2004)

Keywords : ion-pair extraction; divalent metal cations; di-Schiff base ligands; structural effect; extraction selectivity.

Evaluation of the mechanism for enantio-separation on polysaccharide-based chiral columns in the reversed-phase mode

Masato Kazusaki

Pharmaceutical Research & Technology Center, Dainippon Pharmaceutical Co., Ltd., 5-51, Ebie 1-chome, Fukushima-ku, Osaka-shi, Osaka 553-0001

(Awarded by Kanazawa University dated September 30, 2003)

A reversed-phase HPLC method was investigated for the separation of enantiomers, the newly synthesized potential medicinal substance and corresponding optical impurity, employing polysaccharide-based chiral stationary phases (CHIRALCEL OD-RH and CHIRALPAK AD-RH). On the CHIRALCEL OD-RH column, chiral recognition was completely enthalpy-driven with a negative entropic contribution. The cavity formation effect was the major factor governing the solute distribution between the mobile and stationary phases for acetonitrile-rich mobile phase, while the effect of solvation due to acid-base equilibrium became significant in highly aqueous mobile phase. On the CHIRALPAK AD-RH column, the enantio-selectivity was exclusively driven by enthalpy above about 20°C, whereas below about 20°Cenantio-separation was achieved by the combination of enthalpy and entropy. A conformationed change in the stationary phase of the CHIRALPAK AD-RH column was observed at about 20°C. Enthalpy-entropy compensation concerning enantio-separation on both columns indicated that enthalpic gain/loss was substantially cancelled out by the entropic loss/gain. The CHIRALPAK AD-RH column was used for the determination of enantiomers (optical impurity). A low-level quantification (0.05%) of the minor enantiomer was achieved. The analytical procedure was validated and successfully applied to the detection of the optical impurity in potential medicinal substance.

(Received April 23, 2004)

Keywords : CHIRALCEL OD-RH; CHIRALPAK AD-RH; enthalpy; entropy; enantio-separation.

Optimization of high-performance liquid chromatography with electrochemical detection using a chemometric tool based on the FUMI theory and its application to the development of a highly-sensitive method for determining free fatty acids

Akira Kotani

Department of Analytical Chemistry, School of Pharmacy, Tokyo University of Pharmacy & Life Science, 1432-1, Horinouchi, Hachioji-shi, Tokyo 192-0392

(Award by Tokyo University of Pharmacy & Life Science dated March 23, 2004)

A chemometric tool based on the Function of Mutual Information (FUMI) theory can provide a relative standard deviation (RSD) from a single measurement of noise and signal was used to optimize high-performance liquid chromatography with an electrochemical detection (HPLC-ECD) system and the conditions. That the predicted RSD by FUMI theory was parallel to the observed RSD shows that FUMI theory is useful for predicting the measurement precision in HPLC-ECD without repetitive measurements. The power spectra of chromatographic baseline were used to trace the source of instrumental noise, and to select a suitable apparatus in the HPLC-ECD system. To optimize the HPLC-ECD conditions, two parameters, information content (φ) and efficiency (θ=information content/time), which were calculated from predicted RSD based on the FUMI theory, were used. Moreover, optimization methods by the FUMI theory were applied to new HPLC-ECD that was developed with the objective of providing a sensitive, simple, and rapid means for determining free fatty acids (FFAs). This method requires small sample amounts, and the procedure is quite simple without the derivatization of fatty acids, permitting time-course measurements at the FFA level in human plasma and the distribution of FFA in skin surface lipids from 50 points on a human head. The presented method using FUMI theory saves considerable amounts of chemicals and experimental time, and was found to be useful to optimize the system and the conditions of HPLC-ECD.

(Received May 6, 2004)

Keywords : HPLC; electrochemical detection; measurement precision; optimization; FUMI theory; free fatty acid.

Novel characterization method for airborne particulate matter in workplace-chemical speciation method for heavy metals and real time size classified analysis

Mitsutoshi Takaya

Department of work environment evaluation, National Institute of Industrial Health, 6-21-1, Nagao, Tama-ku, Kawasaki-shi, Kanagawa, 214-8585

(Awarded by Tokyo Metropolitan University dated February 19, 2004)

The speciation and real-time analysis of airborne inorganic particulate matter were investigated as follows. A selective determination of the concentrations of vanadium(V) and vanadium(IV) in particulate matter was achieved by connecting HPLC to ICP-AES as a detector. The method was applied to confirm the purity used for the sample particle of an animal experiment to clarify acute toxicity in lung after exposure to V₂O₅. An analytical method of acetylacetonato-chromium by micellar electro kinetic chromatography (MEKC) was developed. Using the method, 160 µg l^-1 of chromium in aqueous solution was measurable. The speciation of particulate chromium was attempted using the method. Chromium(VI) oxide was detected selectively by adding acetylacetone to the sample without solvent and chromium in potassium dichromate particle was detected by using acetylacetone with 18-crown-8. The gas change function was added to a monodisperse aerosol generator (Differential Mobility Analyzer, DMA). Aerosol particles dispersed in the argon, “arogonsol”, was made by the gas change DMA, and was directly introduced into ICP-MS without argon dilution to avoid plasma elimination. The performance of the device (DMA-ICP-MS) was evaluated by using various test particles from a pneumatic nebulizer. DMA-ICP-MS could analyze the airborne particulate matter in the range of diameters of particles 20~140 nm in real time.

(Received April 21, 2004)

Keywords : airborne particulate matter; vanadium pentoxide; hexavalent chromium; capillary electrophoresis; real time measurement; direct introduction; ICP-MS.

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