BUNSEKI KAGAKU Abstracts

The growing demand for high-throughput analysis in the current competitive life sciences and industries has promoted the development of high-speed HPLC techniques and tools. As one of such tools, monolithic columns have attracted increasing attention and interest in the last decade due to the low flow-resistance and excellent mass transfer, allowing for rapid separations and reactions at high flow rates with minimal loss of column efficiency. Monolithic materials are classified into two main groups : silica- and organic polymer-based monoliths, each with their own advantages and disadvantages. Organic polymer monoliths have several distinct advantages in life-science research, including wide pH stability, less irreversible adsorption, facile preparation and modification. Thus, we have so far tried to develop organic polymer monoliths for various chemical operations, such as separation, extraction, preconcentration, and reaction. In the present paper, recent progress in the development of organic polymer monoliths is discussed. Especially, the procedure for the preparation of methacrylate-based monoliths with various functional groups is described, where the influence of different compositional and processing parameters on the monolithic structure is also addressed. Furthermore, the performance of the produced monoliths is demonstrated through the results for (1) rapid separations of alkylbenzenes at high flow rates, (2) flow-through enzymatic digestion of cytochrome c on a trypsin-immobilized monolithic column, and (3) separation of the tryptic digest on a reversed-phase monolithic column. The flexibility and versatility of organic polymer monoliths will be beneficial for further enhancing analytical performance, and will open the way for new applications and opportunities both in scientific and industrial research.

A fully automated continuous monitoring system by using conventional flow-injection spectrophotometry was developed, and applied to the determination of ammonium ion in an industrial-waste brine sample. The system enables one to monitor highly concentrated ammonium ion without any dilution procedure by a manual method. In order to avoid the problem of propelling solutions and clogging the flow-lines, a peristaltic micro pump is used instead of a double-plunger pump. A flow cell with PTFE tubing of (2 mm i.d., 3 mm o.d.) and a LED of 660 nm are set in a temperature-controlled air oven, because the temperature is changeable at sampling sites. A newly designed sampling pot was used to transport a brine sample from a waste drain. A sample solution in the sampling pot is introduced into a sample loop on a six-way injection valve after passing through a suction filter (10 μm). Then, the analysis is carried out automatically. The sampling and analytical conditions could be controlled by a microcomputer installed in a FIA system. The monitoring of ammonium ion was continuously carried out for 14 days in drains from iodine manufacturing factories.

Aliphatic petroleum resin is one of the most important tackifiers. In the present work Escorez 1202, a representative aliphatic petroleum resin, was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The ionizing agent used was silver trifluoroacetate ; the matrices examined were dithranol, pyrene, trans,trans-1,4-diphenyl-1,3-butadiene, 9-nitroanthracene, anthracene, 2,5-dihydroxybenzoic acid, retinoic acid, 3-indolacrylic acid and 2-(4-hydroxyphenylazo)benzoic acid. The optimum matrix found was anthracene and the radical cations were observed without using an ionizing agent. Two adhesive tapes were treated with 2-butanone, and the 2-butanone extracts were analyzed by MALDI-TOF-MS. It was proved that an aliphatic petroleum resin was mixed in each adhesive.

In this study we examined the differentially induced protein level by elevating the calcium concentration in the human keratinocyte cell line HaCaT by proteome analysis. HaCaT cell lysates of the cultured chelex-treated medium (0.02 mM Ca^2＋) and non chelex-treated medium (1.6 mM Ca^2＋) were studied by two-dimensional polyacrylamide gel electrophoresis with molecular weights ranging between 20 and 120 kDa and isoelectric points of pH 3〜10. The identified proteins with increased expression levels were greater than 2-fold in chelex-treated medium cultured HaCaT cells and isolated protein spots from the polyacrylamide gels of non chelex-treated medium cultured HaCaT cells. Forty protein spots, corresponding to 28 different proteins, were identified peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-fight mass spectrometry and the Mascot database searching algorithm. The identified proteins were categorized into several protein groups : intermediate filament, molecular chaperone, energy metabolism, adhesion molecule, binding protein, transcription factor etc.. The proteome analysis and bioinformatics results identify Lamin A/C, KRT5/7/8/14, ZO-1/-2, SOD-1 and peroxiredoxin-1. The proteome analysis suggested that the differentially increased in HaCaT cells cultured under a high calcium condition.

An electron probe microanalyzer was applied to ammonium sulfate, ammonium nitrate and sodium nitrate to make chemical-state analyses of Kα lines of ammonium and nitrate nitrogen. The results inferred that the shape of the Kα lines in ammonium sulfate and ammonium nitrate was symmetric, and that of the Kα lines in sodium nitrate was asymmetric. To understand this asymmetry of the Kα lines in sodium nitrate theoretically, the electronic transition states of a nitrate ion cluster, related to the Kα lines of nitrogen, were calculated by the DV-Xα molecular orbital method. The results showed that the electronic transition states (transition energies and intensities), caused by chemical bonds between nitrogen and the adjacent oxygen atom, determined the shapes of the Kα lines of nitrogen.

Recentry, the demand for indium (In) is increasing mainly due to its use for electrodes of liquid crystal displays. In is toxic and causes the sickness and vomiting in the case of oral intake. Thus, the determination of In in sea water is important since In is accumulated in marine creatures and is finally taken into human beings. Therefore, the concentration of In in the seawater around Japan and the north eastern Pacific Ocean was measured by inductively coupled plasma mass spectrometry (ICP-MS). As a pretreatment, a Chelex-100 chelating resin method was used. ¹¹⁵In was used for a measurement by MS, and a high background signal was observed when the eluent was heated with hydrochloric acid to vaporize the ammonium ion so as to remove the interference by magnesium and calcium in sea-water samples. This high background is presumably due to Ar₂Cl^＋ polyatomic ion because a background intensity was very low when N₂-MIP-MS was used to determine In. However, the high background signal greatly decreased upon heating the solution with HNO₃ (1＋1) before preparing the final solution for the measurement by MS. The results obtained by the proposed method were compared with those by solvent extraction with APDC-HMAHMDC/xylene, and the results using both methods were in a good agreement with each other. The concentrations of In around Japan were considerably different between the sampling points. However, the results in the Pacific Ocean were almost equal over a wide sea area. The concentration of In (including around Japan and the Pacific Ocean) was in the ranges of 0.08〜0.72 ng/L and the relative standard deviations of the results were 8.3％〜37.5％.

The analysis of diketodipyrrole and 2,5-diketopiperazines, detected as pyrolysis products from collagen, which is a main component of natural leathers, by pyrolysis gas chromatography-mass spectrometry (Py-GCMS), was examined for the development of a forensic identification method of small bits of the natural leathers used in this study. Natural leather samples (very small pieces) from the five kinds of animals, such as cow, pig, sheep, kangaroo, and ostrich, collagen from calf-skin, collagen powder from pig-skin, wool fibers, and silk fibers were used as samples. The results of Py-GCMS analysis showed that natural leather samples from five kinds of animals were composed of collagen, which was distinguished from the other protein fibers, such as a wool fiber and a silk fiber. The discrimination of the leather samples from five kinds of animals was difficult. However, if a leather samples with a weight of more than approximately 25 μg existed as a sample, the Py-GCMS analysis examined in this study could detect a diketodipyrrole and 2,5-diketopiperazines corresponding to dipeptides, which were composed of major amino acids, such as glycine (Gly), proline (Pro), alanine (Ala), and hydroxyproline (Hyp) from collagen. Accordingly, the analytical method by Py-GCMS shown in this study can be newly applied to the forensic identification of small samples of natural leathers.

Di(2-ethylhexyl)phthalate (DEHP), despite its reported toxicity, is widely used as a plasticizer to increase the flexibility of poly(vinyl chloride) (PVC) medical devices. However, there are reports that it is easily eluted from such devices. The present study focused on the determination and assessment of DEHP exposure levels. First, a simple method to predict DEHP release from PVC medical devices was developed based on the physicochemical properties of pharmaceuticals. It was found that the results concerning the solubility of lipophilic pigments were closely related to the DEHP release potency of pharmaceuticals. Therefore, this simple method using the solubility of lipophilic pigments to predict DEHP release may be applicable to the medical field. Next, the effects of the sterilization process and the storage conditions of PVC medical devices on the DEHP release behavior were examined. Less DEHP was released when PVC medical devices were sterilized by UV or gamma-ray irradiation. However, mono(2-ethylhexyl)phthalate (MEHP) as the breakdown product was released at high levels from gamma-ray sterilized PVC medical devices, and is speculated to be more toxic than DEHP. Although the co-exposure level of DEHP and MEHP was below tolerable daily intake (TDI) in the case of PVC medical devices used with intravenous preparations, it exceeded TDI in the case of PVC medical devices used with blood preparations. The results demonstrate that not only DEHP exposure but also MEHP exposure should be assessed.

Given the trajectory of interest and innovation in this marriage of micro- and nano-chip technology and proteomics, it is speculated that micro- and nano-chip technology for proteomics are indeed on the horizon. Thus, further investigations on functional protein analysis devices by micro- and nano-chip technology are still needed to make this a success. This study focused on improving signal-to-noise (S/N) ratio so as to efficiently manipulate proteins in micro- and nano-chips to realize high-performance analysis. In detail, this study is mainly composed of 4 topics : suppression of protein adsorption on channel walls of a poly(methyl methacrylate) (PMMA) microchip (in chapter 2); high performance protein separation with cellulose derivatives (in chapter 3); a sieving matrix for microchip electrophoresis of protein (in chapter 4); and development of a highly sensitive protein detection method on microchip electrophoresis and analysis of milk protein (in chapter 5). In addition, a general introduction was added in chapter 1 and concluding remarks and future perspectives are presented in chapter 6. Namely, this thesis is composed of 6 chapters.

This research involves the development of a new approach to elucidate the health influence on humans by exposure to nanoparticles. Detailed information was obtained on the shape, particle diameter, composition of particulates in a single cell and a method was found to correlate that information to the biological response of the cell. This is a key technique to clarify the physical and chemical properties of nanoparticles, which will have a fatal influence on the human body. First, cellular oxidative stress by exposure to diesel exhaust particles was investigated by using a time-of-flight secondary ion mass spectrometry (TOF-SIMS) and a real time reverse transcription polymerase chain reaction (RT-PCR) and a scanning transmission electron microscope (STEM). From these results, it was clear that there was interaction between the cellular functions and the particle properties. Next, nanoparticles in the atmospheric environment neighboring a traffic route were investigated by using a differential mobility analyzer (DMA) and TOF-SIMS. From these results, it was revealed that there were different types (particle diameter ; 20 nm or 100 nm) of environmental nanoparticles. Thus, it is very important for this research to improve both analytical and biological techniques.