A novel analytical method which includes absorbance measurements by using stopped-flow apparatuses was developed, which can be applied to determine absorbance changes as low as 0.002O.D. with good precision in a time range of less than seconds, even for background absorbances of 1.5 to 2.0O.D. This method, termed "stopped-flow time difference analysis" (abbreviated to SFTDA), utilizes the progress curve of a reaction for both quantitative and qualitative analyses, based on the absorbance change and the rate of reaction, respectively. A differential kinetic analysis of mixed components is possible, due to the different reaction rates of the components, which can also be effective to exclude interference by coexisting materials. Several examples of the applications of the SFTDA method with macro-apparatuses are described, which include a determination of ascorbic acid down to 10^-7M, a highly sensitive determination of inorganic phosphate of ppb and sup-ppb level in environmental water, and an improvement in several characteristics of the methods for protein determination. Two types of micro-stopped-flow apparatuses were built, which need only 1/10 of the sample amount necessary for conventional macro-apparatuses, and still have a comparable performance. Their utility in clinical chemistry is demonstrated.

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A ¹H-NMR database comprised of NMR parameters, i.e., the chemical shifts and the coupling constants, is described. The data are compiled from the literature after full evaluation as well as from the data obtained in our laboratory, and registered to a master file, from which an access file for users is made. Spectral patterns are generated by a calculation using the parameters in the database on user′s request. The calculation program and the procedure for extraction of the parameters are discussed. Advantages and limitations of the parameter database are also pointed out.

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A ¹³C-NMR database system is described for the spectra of pure organic compounds obtained under the complete decoupling of the ¹H-NMR. In this database the peak positions, the relative intensities, and the line widths for broad lines are included, together with the spectral assignments given in the structural formula. Since the relative intensities depend on the measuring conditions, the sample and measuring conditions are also included in the database. The spectral pattern with a lorentzian line shape is generated for user′s access. The data structure and the problems in constructing the ¹³C-NMR database are discussed, especially for the spectral assignments, the splittings due to the coupling with the magnetic nuclei other than ¹H, the relative intensities, and other important points concerning the reliability of the database.

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An interface for combining a liquid ionization mass spectrometer with a liquid chromatograph (LC) was developed. In this interface, the effluent from a micro-HPLC is deposited on a needle filament in the liquid ionization ion source periodically at a constant rate. Experimental conditions, such as needle filament temperature, cycles of periodic deposition and flow rate, and composition of mobile phase, were investigated to obtain as many solute ions as possible at each cycle. Solute ions produced on the filament were introduced into a quadrupole mass spectrometer through a pinhole and mass spectra were measured continuously. Since one chromatographic peak was divided by the deposition cycle, a group of peaks appeared; the envelope of the peak groups looked similar to the corresponding peak measured by a UV detector. As an example, a sample containing three steroid hormones was measured. A satisfactory RIC (re-constructed ion current) chromatogram and mass spectrum of each component were obtained. The results indicate the usefulness of this interface in which the filament temperature is increased rapidly enough for detecting several solutes at each cycle.

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The laser two-photon ionization technique was applied to reversed-phase high performance liquid chromatography by using a free-falling jet detector (20nl). Methanol, acetonitrile and a mixture of acetonitrile-water (90:10) were used in the mobile phase. The dependences of the detection limit on such experimental parameters as the electrode spacing and applied voltage were measured. The best detection limit was obtained at 0.5mm spacing and at 60V for acetonitrile. Although these solvents showed similar leakage currents, acetonitrile gave the best detection limit. The detection limit was 60pg for pyrene, comparable with or better than that obtained by a UV absorption detector.

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The following two chemiluminescent reaction systems were investigated for the photographic detection of ultra-micro amounts of analyte on an agarose gel plate. 1) Reaction of 1, 10-phenanthroline with hydrogen peroxide, which is catalyzed by copper(II) or osmium(VIII). 2) Reaction of luminol with hydrogen peroxide, which is catalyzed by iron(III) complex. Chemiluminescent light was detected using a high-speed instant photographic film, and amounts as small as 9×10^-10mol of 1, 10-phenanthroline or 2×10^-18mol of iron complex of tetraphenylporphinetrisulfonic acid were detected by this method.

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Polyaromatic compounds which possess different base-pair selectivities in their DNA-binding (intercalating) properties were covalently bound to polyethylene glycol (PEG) via ester bonds. These PEG-bound intercalators are highly soluble in water and induced the retardation of DNA electrophoresis in agarose gel. The pattern of mobility retardation reflected the binding characteristics of the parent intercalators to DNA. Use of PEG-bound intercalators in the gel electrophoresis is discussed as a simple and convenient method for the characterization of polyaromatic DNA intercalators of low water solubility.

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An accurate and precise method for the determination of serum glucose by isotope dilution mass spectrometry was studied for use in establishing the accuracy of glucose methods in clinical laboratories. After adding uniformly labeled [¹³C₆] glucose to the serum, glucose was separated from the serum matrix and converted into α-D-glucofranose cyclic 1, 2:3, 5-bis(n-butylboronate)-6-acetate (glucose-BBA). Measurements were performed with combined capillary gas chromatography-mass spectrometry. The ratios of the peak areas at m/z 297 and 303 for samples and the calibration mixtures were used for quantification. Analytical results of the SRM serum showed satisfactory agreement with the certified value and the relative standard deviation of the replicates was within 0.5% for all of the serum samples tested.

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A facile technique was developed for the determination of penicillinase activity based on flow injection analysis (FIA). The analytical condition of the method utilizing a substrate (benzylpenicillin) solution as the carrier was optimized. Penicillinase solution was injected into the FIA system where the substrate was undergone enzymatic hydrolysis to form penicilloate. The decrease in the substrate was monitored at 229nm. A linear correlation was observed between a conventional photometric method and the present FIA method in the range of 0.04 to 6.6 unit ml^-1. The relative standard deviations (n=18) for the determination of penicillinase at 0.07, 0.5 and 2.8 unit ml^-1 were 0.65, 0.95 and 2.9%, respectively. The detection limit was 0.03 unit ml^-1 with a sample size of 5μl. The FIA method was applied to the examination of penicillinase stability in solutions.

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An enzyme sensor for L-α-glycerophosphate was prepared by covalent fixation of glycerophosphate oxidase and peroxidase by use of bovine serum albumin and glutaraldehyde onto a glassy carbon electrode silanized with 3-aminopropyl triethoxysilane. The sensor showed good linear current responses in the range of 5 to 100μM glycerophosphate (γ=0.999, n=7); the response time was about 20s. The sensor was stable for at least 60d. Sugar phosphates did not have any appreciable effect on the response of the sensor, but reducing materials such as L-ascorbic acid and L-cysteine gave some errors that changed with concentrations.

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A durable potassium ion-selective field effect transistor (ISFET) with an Urushi matrix membrane was fabricated. The potassium ion-sensing membrane is composed of valinomycin, di-2-ethylhexylphthalate, potassium tetrakis-(p-chlorophenyl)borate and Urushi. The best composition for the potassium ion-sensing membrane was determined with the results of electrochemical characteristics of Urushi ion selective electrodes. The optimal Urushi mixture was coated onto the gate region of the device, which was fabricated from a silicon-on-sapphire (SOS) wafer. The Urushi SOS/ISFET showed a linear response in the potassium ion activity range from 10⁰mol dm^-3 to 10^-4mol dm^-3 and about 50mV per decade in potassium ion activity. The prepared Urushi SOS/ISFET has good durability, for over a month, because of the strong adhesion of the Urushi membrane to the Si₃N₄ gate.

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Amberlite XAD copolymers have been used as adsorbents for organic substances and as supports for Chelating agent-impregnated resin(CIR). The adsorption mechanism and behavior of 8-hydroxyquinoline (8HQ) and its derivatives on XAD resins have been investigated by interpreting the distribution coefficients (K_d) measured under various conditions. The logK_d values of 8HQ and its four derivatives on XAD resins were found to increase up to the certain pH values, and then to decrease with increasing pH. The pH values having maximum logK_d values are very close to their isoelectric points. The adsorption of 8HQ and its derivatives was due to molecular adsorption, because the adsorption became maximum when they existed as electrically neutral molecules. Amberlite XAD resins (XAD-2, -4 and -7) impregnated with chelating agents, such as 8HQ, 5, 7-dihalo-8-hydroxyquinoline (DXHQ) and 2-methyl-8-hydroxyquinoline (MHQ), have been applied as CIR resins to the adsorption, separation, concentration and recovery of metal ions (Cu, Cd, Ni, Zn, Pb, Hg and Fe) from the aqueous solutions. All CIR resins show relatively good resistance to leaching at the pH range of 3-10. The magnitude of adsorption capacity of CIR depends on the impregnating capacity of the CIR and the adsorption mole ratio at the optimum pH condition. The metal ions adsorbed by CIR were quantitatively recovered with 3-5M hydrochloric acid. Some examples of selective separation and concentration of metal ions were performed by using CIR resins.

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In order to increase the selectivity and the sensitivity of the extractive and Spectrophotometric determination of class b and ab metals with thiacrown ethers, the effects of several kinds of counter anions on the extraction of copper(II) were examined with 1, 4, 7, 10, 13, 16-hexathiacyclooctadecane (HTCO) and 1, 4, 8, 11-tetrathiacyclotetradecane (TTCT). Tetrabromophenolphthalein ethylester (TBPE) anion was the best one among the seven kinds of dye anions examined. More than 99% copper(II) was selectively extracted with HTCO into 1, 2-dichloroethane in the range of pH 7 to 9, whereas cobalt(II), zinc(II) and cadmium(II) were not extracted at all. The molar absorptivity of the copper(II) extract was 8.8×10⁴ (at λ_max=608nm), which was the highest value found.

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The mechanism and rate of the extraction of palladium(II) with o-xylylene bis(diethyldithiocarbamate) (o-XEDTC) or 3-mercapto-1, 5-diphenylformazan (dithizone, Hdz) into chloroform were described. The extracted palladium- (II)-o-XEDTC was represented as PdCl₂ (o-XEDTC). The rate of the extraction of palladium(II) with o-XEDTC or Hdz from weakly acidic chloride medium can be represented as -d[Pd(II)]/dt=k₁[PdCl₃(H₂O)^-][Q]_orgK^-1_DR+k₂[PdCl₄^2-]× [Q]_orgK^-1_DR, where Q represents o-XEDTC or dithizone and K_DR is the distribution constant of the extractants between chloroform and water. The replacement of chloride ions and/or water molecules with these extractants should proceed through the formation of five coordinated square pyramidal intermediates, and the subsequent reaction of the release of chloride ions or/and water molecules. That the reactivity of o-XEDTC toward PdCln(H₂O)_4-n^(2-n)+ is smaller than that of dithizone may be related to a steric effect on the formation of a five coordinated intermediate. Palladium(II) was separated with o-XEDTC from other metal ions as copper(II), zinc(II), nickel(II) and cobalt(II). The interference of iron(III) can be masked with phosphate or pyrophosphate.

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Flow injection analysis has been developed for the spectrophotometric determination of silicon in silicate rocks at 415nm. A powdered sample was decomposed with small amounts of hydrochloric and hydrofluoric acids in a Tuf-tainer vial by microwave heating. The contents in the vial were diluted with water after the addition of boric acid. The sample solution thus obtained contains silicon in concentrations of 200-380μg g^-1 as silicon dioxide. It was injected to the FIA system without any dilution. Analytical results for silicon in 22 standard rocks from United States Geological Survey, Geological Survey of Japan and Canadian Certified Reference Materials Project agreed well with the proposed values. This method permitted 30 determinations per hour.

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