Chemiluminescence analyses of biological constituents using metal-complex catalysts developed in our laboratory are reviewed. This study was carried out in the fields of immunoassay and high performance liquid chromatography, with special reference to the determination of micro amounts of protein. The use of a synthesized metal-complex compound instead of natural substances as a catalyst was proposed in order to make immunoassay inexpensive and reproducible, as well as to make labeling easy. The immunoassay established by the use of a fiber-optic immunosensor could be successfully applied to the determination of micro amounts of protein. The method using the activity lowering of a chemiluminescence catalyst was also established for the development of a new detection system, which was applied to both indirect and direct flow injection analyses of protein. Each chemiluminescence system was utilized for indirect and direct flow injection analyses, respectively. The direct flow injection analysis, in which a protein solution was directly injected into a flow injection system, could be used to determine the protein content more easily, rapidly, and sensitively than the indirect one. Finally, the future prospect is briefly presented regarding the present subject.

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A simple spectrophotometric method has been established for the determination of chlorhexidine (CH) with the o- hydroxyhydroquinonephthalein(QP)-manganese(II) complex. The apparent molar absorptivity and relative standard deviation for 50.6μg of CH were 5.88×10⁴ 1mol^-1 cm^-1 and 0.47% (n=8), respectively. This method was applied to the determination of CH in a wide variety of pharmaceutical preparations with satisfactory analytical results. From the thermodynamic parameters (ΔG=-5.49kcal mol^-1, ΔH=0.04kcal mol^-1 and ΔS=18.60cal mol^-1 K^-1) obtained from a van′t Hoff plot, the binding between CH and the QP-manganese(II) complex was presumed to be a hydrophobic interaction.

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Magnetic circular dichroism (MCD) spectroscopy of non-metallated, dizinc, dicopper and dicobalt derivatives of 1, 8- naphthalene and 1, 8-anthracene-linked cofacial binuclear phthalocyanines is reported. Not only the spectra of neutral species, but also the spectrum of the mixed valence Co(II)Co(I) species have been recorded. These MCD spectra are, especially in the Q band region, similar in shape to those so far reported for aggregated phthalocyanines. Very intense intramolecular interaction between two phthalocyanine cores is observed in all compounds except cobalt-reduced Co(I)Co(I) species.

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Diffuse reflectance infrared Fourier transform (DRIFT) spectrometry was applied to the determination of Cu₂O and CuO in Cu powders. The peak of Cu₂O (640cm^-1) and the shoulder of CuO (580-400cm^-1) were observed in the DRIFT spectrum of oxidized Cu powder. A linear relationship between the peak heights (640 and 520cm^-1) and the concentrations of copper oxide (Cu₂O and CuO) were obtained in concentration ranges of 0-16 and 0-6wt%, respectively. Analytical curves were constructed with three size-classified powders. From the slopes of the analytical curves, the effect of particle size of the powders on the spectrum is discussed. Two determination methods of Cu₂O and CuO in Cu powders are proposed. One is a method which uses the analytical curves. The other is a method which uses the oxygen concentration and the ratio of peak heights (640 and 520 cm^-1).

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A water-soluble iron porphyrin, a meso-tetrakis (1-methyl-4-pyridyl)porphinatoiron(III) complex (FeTMPyPX₅; X=OCH₃ or halide), was successfully applied as a catalyst of indamine dye formation, which can be used for the determination of hydrogen peroxide in an acidic media. The dye formation process includes an oxidative coupling of N, N-dimethylaniline and 3-methyl-2-benzothiazolinone hydrazone with hydrogen peroxide. The absorbance of the formed dye was measured at 590nm with a spectrophotometer. The materials were stable and the reaction was specific for hydrogen peroxide without any influence of molecular oxygen at low pH, 1-2. The method showed easy handling in the concentration range of (0.3-3.7)×10^-5mol dm^-3 with a correlation factor: 0.999₈ and RSD=1.7₄%. This method can be used for the determination of hydrogen peroxide generated through enzymatic processes.

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The effect of the substituted aromatics in RCOCH₂COCF₃ (R: benzoyl, 4-chlorobenzoyl, 4-methoxybenzoyl, 3, 4, 5- trimethoxybenzoyl, 9-anthroyl) on the emission properties of tris(β-diketonato)bis(trioctylphosphine oxide) europium- (III) complexes was investigated. The relative intensities of the sharp luminescence of the ternary europium(III) complexes in hexane at 610nm occur in this order: 3, 4, 5- trimethoxybenzoyltrifluoroacetone(3, 4, 5-tmbtfa)>4- chlorobenzoyltrifluoroacetone (4-cbtfa)>4- methoxybenzoyltrifluoroacetone (4-mtbtfa)> benzoyltrifluoroacetone (btfa)>9- anthroyltrifluoroacetone (9-atfa). The Eu(9-atfa)₃(topo)₂ complex did not give the characteristic luminescence at 610nm, but did show a broad emission in the range from 400nm to 600nm. Also, the solvent effect on the luminescence quantum yield (Φ_L) of Eu(3, 4, 5-tmbtfa)₃(topo)₂ is described. The ternary complex shows the largest quantum yield in hexane, so the effect of the concentrations of Eu(3, 4, 5-tmbtfa)₃(topo)₂, free 3, 4, 5-tmbtfa and topo on the luminescence intensity, I_L or Φ_L values of Eu(3, 4, 5-tmbtfa)₃(topo)₂ in hexane could be investigated.

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A simple, rapid chemiluminescent photographic method was developed for the assay of hydrogen peroxide or glucose using a camera luminometer. Some chemiluminogenic reagents were tested regarding their ability by using a visual method. 4, 4′-Oxalyl-bis[(trifluoromethylsulfonyl)imino]trimethylene- bis (4-methylmorpholinium) trifluoromethanesulfonate (MPTQ) was found to be a suitable oxamide for the photographic method. Pyrimido[5, 4-d]pyrimidines, dipyridamole (DP), 2, 4, 6, 8-tetramorpholinopyrimido[5, 4-d]pyrimidine (MP) and 2, 4, 6, 8-tetrathiomorpholinopyrimido [5, 4-d]pyrimidine (TMP) were effective enhancers, as were perylene and Rhodamine B, on the peroxyoxalate chemiluminescent reaction examined. Fifty picomoles of hydrogen peroxide per 200μl on a micro-titer plate well could be detected as a white spot on instant film. Glucose was detected after being enzymatically oxidized to hydrogen peroxide with glucose oxidase. The detection limit of glucose was 100pmol per well using MPTQ and MP as chemiluminogenic reagents. By the proposed method, glucose in 50μl of 100-fold diluted human serum could be detected.

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Fluorescence intensities of Acridine Orange and its 10-alkyl derivatives were reduced by the presence of commercial humic acid or dissolved humic substances obtained from river water, seawater and pond water samples. Synchronous changes in the absorption spectra and the virtually unchanged fluorescence lifetime of the 10-dodecyl derivative suggested that the fluorescence quenching could be explained on the basis of a static quenching mechanism, in which the fluorescence dyes were bound to humic substances to form non-fluorescent dye aggregates. Curve fitting procedures for the upwardly-curved Stern-Volmer plots yielded the equilibrium binding constants normalized to organic carbon content. The binding constants at pH 7.0 for fractions eluting at pH 2.4-7.5 in a pH-gradient chromatographic separation of the aquatic humic substances were larger than those for fractions eluting at pH 8.2- 11.4. Some chemical properties of the binding have also been discussed in terms of the electrostatic interaction and/or the hydrophobic interaction.

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The authors have developed an analytical method for extracting trace amounts of 15 hydrophilic compounds in water using gas chromatography/mass spectrometry coupled with an activated carbon preconcentration procedure. As internal standards, perdeuterated p-dioxane and N, N-dimethylformamide were added to 11 of a water sample; the water was passed through a granular activated carbon column. Organic substances adsorbed on the activated carbon were eluted with acetone and dichloromethane; the extract was then concentrated to 10ml with a Kuderna-Danish concentrator. Quantitative analyses using GC/MS with a selected ion monitor (GC/MS-SIM) were performed to measure the recovery efficiencies of the 15 compounds spiked into water samples at concentrations of 0.2 and 2.0μg/l. The recovery efficiencies through the internal standard method of the compounds, except alcohols and acetonitrile, gave more than 90% and their relative standard deviations were less than 7% at a concentration of 0.2μg/l. In this study, it was confirmed that the activated carbon preconcentration procedure is useful for extracting trace amounts of polar compounds which have a log P value (dichloromethane-water partition coefficient) greater than -0.5, except for compounds containing a hydroxy group or having a very small molecular weight. The method was applied to the analysis of river water and seawater for polar compounds. p-Dioxane, 1-propanol, N-nitrosodimethylamine, N-nitrosodiethylamine, N, N-dimethylformamide and N, N-dimethylacetamide were detected below the μg/l level.

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A gas chromatographic method is described for the determination of di- and tributyltin compounds in seawater. Organotin compounds in seawater led to their chlorides with hydrochloric acid. The chlorides were reacted with sodium borohydride to produce hydrides and simultaneously extracted with hexane by stirring extraction. The organic layer was concentrated by a gentle stream of nitrogen and passed through a SEP-PAK silica cartridge for cleaning. The trapped organotin compounds were eluted with hexane, concentrated by a gentle stream of nitrogen and determined by gas chromatography with a flame photometric detector. Separation of the organotin compounds was successfully carried out using a DB-1 wide bore column. The detection limits of di- and tributyltin compounds as their chlorides were 0.10ng (25ng/l for 400ml of the sample) and 0.08ng (20ng/l for the same sample). Compounds yielding dibutyltin dihydride and tributyltin hydride were found in Hakata port seawater at levels of 240 to 480ng/l and 27 to 93ng/l as their chlorides.

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A practical calculation method of organic solvent composition was proposed for obtaining the same retention with different binary solvent systems in reversed phase liquid chromatography. It was shown that there is a good correlation between log k′ of each test solute and the solubility parameter δ′ of each solvent mixture calculated from the Hildebrand solubility parameter δ of each pure solvent. We established a calculation method of corresponding mobile phase composition for obtaining the same retentions, by simply inserting the solubility parameters of water and an organic solvent of interest into the equation used for δ′ calculation.

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The theoretical foundations and applications of "mixed" adsorbents for the separation and concentration of metal ions are considered. "Mixed" adsorbents are defined as those containing two or more kinds of functional groups (with different donor sets) in a controlled proportion. Varying the proportion allows one to easily regulate the affinity of the stationary phase to metals. Experiments were carried out with the use of reversed-phase thin-layer chromatography (silica plates were impregnated with solutions of macrocyclic compounds possessing different N_xO_y donor sets in a nonpolar solvent). "Mixed" stationary phases were shown to be capable of providing the strongest retention for metals which are not retained most strongly on any of the parent individual adsorbents. Also possible is an improvement in the separation when the use of individual phases leads to overlapping.

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The addition of tartaric acid to aqueous nitrate solutions containing Fe(III) produces an increase in the solvent extraction of Fe(III) by di(2-ethyl hexyl)phosphoric acid, DEHPA, in hexane solutions. This effect has been studied and it is explained in terms of chemical equilibrium between the two liquid phases by applying the following reactions: Fe³⁺+L^2-_??_FeL⁺; eq. const. Log β=7.18±0.03 and FeL⁺(HA)_2org_??_Fe(LA)_org+H⁺; log β=13.80±0.01. Here, HA stands for DEHPA and L represents tartaric acid. Such behavior determines the formation of a FeLA(HA) metal complex species of mixed ligand composition in the organic phase.

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The synergistic extraction behavior of thiocyanate complexes of gadolinium, dysprosium and erbium has been studied for a mixed system: M³⁺/NH₄SCN/tributyl phosphate (TBP)+tricaprylmonomethylammonium chloride (Aliquat-336)+ xylene. The percentage extraction and distribution ratios were measured as a function of the pH as well as the NH₄SCN, Aliquat-336 and TBP concentrations, respectively. The percentage extraction of gadolinium, dysprosium and erbium increases with increase in pH. It was significant in the pH range 4 to 6 in a thiocyanate media. The composition of a binary synergistic extracted complex of lanthanides into the organic phase is probably M(SCN)₅^2-(L⁺)₂•2TBP_o under the conditions studied. The synergistic extraction sequence as a function of the atomic number of lanthanide elements is given.

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Several N-alkylcarbonyl-substituted N-phenylhydroxylamines (R-PHAs) were synthesized for use in lanthanoids(III) extraction. They were N-octanoyl-, N-(3, 5, 5-trimethylhexanoyl)-, N-(4-butylcyclohexanoyl)-, N-(4-butylbenzoyl)-, N- (2-propylpentanoyl)-, and N-(2-hexyldecanoyl)-N-phenylhydroxylamines. The distribution ratio of a lanthanoid(III) ion between the carbon tetrachloride and aqueous phases was examined as functions of the pH and R-PHA concentration at 298K and the ionic strength, 0.1 (NaNO₃). Sodium tartrate (1×10^-2mol dm^-3) was present in the aqueous phase. Lanthanoids(III) were extracted as a self-adducted chelate, ML₃(HL)₂, with all the R-PHAs used. The conditional extraction constants and the separation factors of lanthanoids(III) with R-PHAs were evaluated. Among the R-PHAs, N-(2-hexyldecanoyl)-N-phenylhydroxylamine was most promising for the separation of heavy lanthanoids(III). Appropriate branching at the α-position of the carbonyl group seemed to transmit the R-PHAs different chemical properties with respect to the straight chain R-PHAs.

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The liquid-liquid extraction of silver ion with two cyclic and four acyclic tetrathio ethers was examined. The extractability of an acyclic tetrathio ether was superior to that of the corresponding cyclic one. The mole ratio of extracted silver complexes was Ag:ligand:picrate=1:1:1, except in the case of 1, 4, 8, 11-tetrathiacyclotetradecane (TTCT). X-Ray crystallographic analysis of the extracted Ag-TTCT-picrate complex was carried out. It becomes clear that the dimeric complex cation, [Ag₂(ttct)₂]²⁺, was formed in the solid state. Each silver(I) ion has the coordination number 4 and binds with four sulfur atoms to form a distorted tetrahedral configuration. Thus, two silver(I) ions are sandwiched between two TTCT molecules. The structure of the complex cation is similar to that of previously reported silver-13, 14-benzo-1, 4, 8, 11-tetrathiacyclopentadec-13-ene picrate.

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The analytical utility of dithizone foam for the detection, quantitative collection and semiquantitative determination of traces of bismuth and zinc in aqueous solutions has been examined. The effect of pH on the sensitivity and selectivity of the proposed foam tests has been investigated. The detection limits of bismuth and zinc by the foam batch method are 0.02 and 0.003ppm, respectively. The effect of diverse ions on the detection of bismuth and zinc has been critically investigated. A quantitative collection of bismuth and zinc from extremely dilute aqueous acidic solutions by the proposed foam column method has been successfully carried out.

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A macromolecular ion flotation method has been developed for preconcentration of thiocyanate using carboxymethylcellulose (CMC) and hexadecyltrimethylammonium chloride (HTAC), followed by spectrophotometric determination of the thiocyanate floated as the iron(III)-thiocyanate complex. The optimum conditions for macromolecular ion flotation of thiocyanate were established by varying the amounts of CMC used as a macromolecular anion and of HTAC used as a cationic surfactant, and by controlling the flow rate of nitrogen gas bubbled through the solution containing thiocyanate and flotation reagents. When 200-ml and 400-ml sample solutions were used, thiocyanate was concentrated 23.4-fold and 46.1-fold, respectively. The proposed method gave a linear calibration graph for thiocyanate and could be applied to the determination of thiocyanate in the concentration range 9×10^-8 -1×10^-5M in the case of 200-ml sample solution. The relative standard deviation was 0.50% at the 6×10^-6M thiocyanate level.

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The precipitation behavior of lanthanoid(III) ions (La-Ho except for Pm) has been studied in methanol by the use of 2, 6-diacetylpyridine bis(benzoylhydrazone) as a precipitant. The recoveries of Gd and Tb were very low compared with other lanthanoids. Rough separation of the lighter and heavier lanthanoid groups has also been examined using the (Nd+Ho)-mixed solution. For the initial solution with 0.1-9.0 of the Nd/Ho molar ratios, decontamination factors (DF) of Nd in the precipitate were estimated as 0.4-12.5, whereas those of Ho remained in the solution were estimated as 0.5-17.5. The precipitant was recovered almost completely as a metal-free form by adding the aqueous EDTA solution to the precipitate and the filtrate.

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