BUNSEKI KAGAKU Abstracts

A UV excitation thermal-lens microscope (UV-TLM) with an excitation beam wavelength of 266 nm and liquid chromatograph (LC) were connected to build a new system (LC/UV-TLM) for the separation and highly sensitive label-free determination of biomolecules, such as peptides. A variation in the composition of the mobile phase with time, as in gradient elution, is very commonly utilized in LC for improved separations. Thermal-lens signals are generally highly dependent on the properties of the media surrounding the photo-absorber. This effect must be carefully considered in such situations where the composition of a solution drastically changes in time. Therefore, we tested the developed LC/UV-TLM system operated in a gradient elution mode, by observing the effects of the variation in the solution composition upon TLM signal intensities. In water/acetonitrile gradient elution, no serious effects were observed as long as the acetonitrile content was less than 40％ v/v. We then tested the developed system in the separation of a standard sample solution of mixed synthetic peptides; we found that the developed system was 10-times more sensitive than a conventional system with the spectrophotometer used as a detector.

A chemical modification of beech sawdust with diphenylphosphonate- formaldehyde resin was applied to prepare a woody biomass-synthetic polymer hybrid material able to resist a fuming sulfuric acid treatment. The amount of diphenylphosphonate -formaldehyde resin required to reinforce beech sawdust was more than about 14 wt％ for a nominal concentration of materials. Thus, a cation exchanger based on about 75 wt％ beech sawdust for a nominal concentration of materials was prepared. The ion-exchange capacity was 2.4 meq g^－1-R (dry exchanger). The rate of adsorption was rapid, and the adsorption equilibrium was attained within about 5 min. Thus, the ion exchanger permitted a high flow rate due to the coarse shape. Because for density of the ion exchanger was 1.18 g mL^－1, the ion exchanger resin did not float in the water, and was able to be applied to the column operation. By using hydrochloric acid or methanol-hydrochloric acid, the ion exchanger could be utilized for the mutual separation of L^＋-K^＋, Mg^2＋-Ca^2＋-Sr^2＋-Ba^2＋, and transition metal ions, such as Cd^2＋-Zn^2＋, Cd^2＋-Co^2＋, and Cd^2＋-Ni^2＋.

The Japan Society for Chemical Analysis has developed a wastewater certified reference material for the determination of dioxins (polychlorodibenzo-p-dioxins, polychlorodibenzofurans and dioxin-like polychlorobiphenyls). This reference material is the first wastewater certified reference material for the analysis of dioxins. There has been no wastewater reference material until now because wastewater is heterogeneous, consisting of water and fine suspended substances where almost all of the dioxins are absorbed; it has been considered that the reference material should be homogeneous, in principle. Then, after setting a principle that the total content of a bottle should be analyzed in one analysis, the project team obtained a success to develop a wastewater certified reference material by studying the sampling methods of wastewater to many bottles. An interlaboratory comparison test was performed by participants from 26 laboratories. Certified values with uncertainty were obtained by treating reported data statistically after rejecting outliers. The certified value of this reference material is 13.81 TEQ pg L^－1, which is on the same level to as the wastewater quality of dioxins issued as the Japanese governmental standard.

A simple and rapid immunochromatography (IC) system for the detection of Cd in rice has been developed. The produced anti-Cd-EDTA antibody exhibited the equilibrium dissociation constant (K_d) for Cd-EDTA at 1.3×10^－8 M, which was almost 100-fold tighter than K_d for the other tested EDTA-metal complex. Cadmium in rice was extracted by HCl, and the extract was introduced into a column to remove co-existing metals (such as zinc, manganese and magnesium) while retaining cadmium. After a pretreatment, IC could detect cadmium in the range from 0.01 to 0.1 mg L^－1. Fifty brown rice samples were tested with the column treatment and the IC assay. The estimated cadmium concentrations from the assay were evaluated by a comparison with the results of a nitrate treatment and ICP-AES analysis for the samples. Two measurements were highly correlated, with a correlation coefficient of 0.94.

Silver o-ethylphosphonate was prepared by the reaction of diethylphosphonate with silver nitrate for 8 h in acetonitrile below 10℃. This compound decomposed in alcoholic potassium hydroxide to form silver(II) oxide and ethylphosphonate in the intermediate. When no reducing reagent existed, silver(II) oxide was reduced by ethylphosphonate to deposite silver, which formed a silver film on the glass base. When a reducing reagent existed, it was oxidized by silver(II) oxide. Benzaldehyde and its derivatives with an electron-withdrawing substituent were oxidized to convert corresponding carboxylic acid in a yield of over 90％. This oxidoreduction occurred at room temperature, and finished within 10 minutes. The oxidation reaction of benzaldehyde by silver o-ethylphosphonate could be utilized for the removal of benzaldehyde contained in a liquid aromatic compound. Benzaldehyde ＜2 v/v％ contained in toluene could be removed in a single oxidation reaction, and of 20 v/v％ in the operation performed four times.

We performed a space-time imaging analysis of a single colony of marine luminous bacterium using a fluorescent microscope connected to a real-color CCD camera. The bioluminescence and bright-field imaging showed that a glowing ring occurred close to the frontline of the growing colony, and that the ring spread out regularly while retaining a concentric ring shape. The glowing ring faded out when the colony terminated its growth. Furthermore, it was found that either aeration or O₂-supply triggered a new glowing ring on the dim inside of the colony, and that the newly triggered ring phases out in the absence of aeration or O₂-supply. Such a flickering pattern was confirmed to repetitively occur on the same concentric circle, corresponding to the presence and absence of aeration or O₂-supply. From these findings, it is postulated that a growing single colony possesses a certain periodic property.

The lanthanoids in shell of shellfish were determined by ICP-MS. The intensities of lanthanoids were very highly suppressed by large amounts of calcium ions, which made their determination difficult. It was necessary to separate any lantanoids from calcium. We applied the solvent-extraction method to separate the lanthanoids from the interfering calcium. The chelating agent was easy to form the chelate for lanthanoids; then, a reagent that could extract the chelate from the acid solution was chosen. 1-phenyl-3-methyl-4-trifluoroacetyl-5-pyrazolone (HPMTFP), in which the pK_a value was 2.56, was chosen as the extraction reagent. Hard tissues of shell used in the present work were treated with inorganic acids. Lanthanoids were extracted with 0.01 mol/l-HPMTFP in diisobutyl ketone at pH 2 in order to separate it from the interfering calcium. The organic solvent, including the chelate, was decomposed in inorganic acid, and was made to be an aqueous solution. Lanthanoids in the solution were determined by ICP-MS. The lanthanoids content in the shell was found to be 0.82〜124 ng/g.

Ultrafine particles of TiO₂ were prepared by the hydrolysis of titanium tetrachloride (TiCl₄) in a highly concentrated hydrochloric acid aqueous solution at a low temperature, in which the surfacing modifier did not coexist. The absorption spectrum of the prepared TiO₂ ultrafine particle showed a blue shift, which was more remarkable than that of a bulk sample. The fluorescence of the prepared TiO₂ fine particles confirmed being within the ultraviolet wavelength range with a peak at 370 nm by changing the excitation wavelength from 220 nm to 300 nm. The bulk TiO₂ of Rutile and Anatase type showed light absorption in proportion to the transition of inter-bands at 413 nm (3.0 eV) and 388 nm (3.2 eV), respectively. Because the excitation wavelength of 230 nm corresponds to 5.4 eV, it is converted into the band energy. The size of TiO₂ identified from a laser scatter measurement was 3.5±1.0 nm, which was confirmed to be the size at which a quantum size effect appears. From the XRD measurement, secondary fine particles (aggregate) of TiO₂ after separation by a membrane filter was identified to be of the Rutile type. The size of the (110), (101) and (211) crystal planes were estimated to be 5.3, 8.0 and 5.1 nm by an evaluation using the Scherrer equation for the half-value width of the diffraction peak of each crystal plane. The shape of the aggregate observed from the SEM image was tabular, of μm size. It was concluded that TiO₂ fine particles with a hierarchy of sizes from nm to μm were possible by the low-temperature hydrolysis of TiCl₄ in a high concentration of hydrochloric acid aqueous solution. Application to an ultraviolet ray protective agent for UVC, UVB and UVA is expected in the future.

Anode catalysts for a fuel-cell system were evaluated by employing a porous microelectrode technique in a 0.5 mol dm^－3 H₂SO₄ aqueous solution. An anodic current for carbon monoxide (CO) oxidation was observed under a 1000 ppm-CO-containing hydrogen atmosphere, in which the onset potential based on CO oxidation at Pt-Ru/C shifted toward cathodic direction from that at Pt/C. In a measurement of CO-stripping voltammetry, the onset potential of CO oxidation well agreed with that of CO-containing hydrogen at Pt-Ru/C. However, in the case of Pt/C, the onset potentials were not the same. This suggests that the CO adsorptions at Pt/C and Pt-Ru/C were different in terms of energetics.

Source identification of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs/PCDFs), as well as coplanar polychlorinated biphenyls (Co-PCBs) in soil and sediment samples is an essential factor to reduce those compounds released into the environment and to reduce the risk to human health and survival. However, chemical analyses of these compounds in environmental samples are ultra-trace-level analyses that required much time and labor. In this study, effective extraction and concentration methods for PCDDs/PCDFs and Co-PCBs analyses were studied, and the concentrations and sources of those compounds in paddy soil and sediment samples from major river basins in Akita were systematically investigated. Optimization studies of extraction solvents for pressurized liquid extraction (PLE), and nitrogen gas-flow rates and concentration solvents for the final concentration were performed. The proposed methods were successfully applied to analyzing the target compounds in real samples. The target compounds were ubiquitous in paddy soil and sediment in river basins, but the concentrations in those samples were much lower than that in the environmental standards. In addition, the concentrations of Co-PCBs were lower than that of PCDDs/PCDFs. Furthermore, the results obtained from homologue and isomer compositions have demonstrated that PCDDs/PCDFs originating from impurities in herbicides and atmospheric depositions could be extremely important sources of the compounds in paddy soil and sediment in Akita.

Capillary electrochromatography (CEC) is a hybrid microseparation technique that combines electrophoretic and chromatographic separation principle. CEC has not yet found wide acceptance as a practical tool in the chromatographic community. This narrow currency of CEC is mainly attributed to two factors: 1) much fewer examples of real sample analysis by CEC than those by HPLC; 2) a limited variation of the stationay phase specially designed for CEC. In this thesis, CEC with NAIP, 3-(1,8-naphthalimido) propyl-modified silyl silica gel, demonstrated suitability for the prompt screening of toxic drugs and the expeditious analysis of microdialysate. Good repeatability of the proposed method with a durable NAIP column was proved to be sufficient for real sample analysis. Moreover, SNAIP, 3-(4-sulfo-1,8-naphthal- imido) propyl-modified silyl silica gel, was specially designed as a CEC stationary phase and, as expected, the sulfonic acid group in SNAIP worked as a strong EOF generator. In addition to hydrophobic and π-π interactions, an electrostatic interaction participated in the retention mechanism of SNAIP. This multiple retentive mechanism indicated an advantage in the separation of peptides, which are difficult to be separated solely based on a hydrophobic interaction, and to be eluted early without high buffer concentration. The remarkable tolerance of current in CEC with SNAIP accomplished the purpose that a high concentration of buffer in mobile phase could be used for the accelerated elution of multiple charged peptides. The tolerance also enabled the use of a highly aqueous mobile phase for the separation of polar nucleosides and nucleic acid bases on a SNAIP column.