Plasmin was found to acquire resistance to the inhibition by _α2-plasmin inhibitor (_α2PI) on incubation at 37°C in 50% glycerin whereas its amidase activity was preserved. This resistance is ascribable to the reduction of the affinity of plasmin to _α2PI, since the ability of plasmin to form the complex with _α2PI was proved to decrease on incubation. The reduced affinity of pre-incubated plasmin was recovered by incubation with _α2PI. The pre-incubation presumably causes a reversible change in the conformation of the_α2PI-binding site of plasmin. The use of freshly prepared plasmin solution is recommended for the assay of plasma_α2PI if such an acquired resistance of plasmin is undesirable.

View full abstract

A study was performed on human urinary glycosaminoglycans (GAGs) by one- and two-dimensional 500MHz proton nuclear magnetic resonance (¹H-NMR) spectroscopy. Urinary GAGs were separated by the precipitation methods with hexadecyl pyridinium and with 85% ethanol, and purified by the β-elimination reaction in alkaline medium containing sodium borohydride to remove proteins and/or peptides. The spectrum of each purified GAG sample from healthy human urine which included chondroitin sulfates (ChS), dermatan sulfate (DS) and heparan sulfate (HS) showed the characteristic signal(s) corresponding to each GAG chain. The assignments and integrations of the structure specific signals in the spectrum made it possible to perform qualitative and quantitative analyses on human urinary GAG mixtures. Furthermore, analytical results of urinary GAGs in children, adults and the aged indicate that the present method is applicable to investigate the effect of aging on excretion of urinary GAGs.

View full abstract

Packings of spherical carbon beads were prepared by graphitizing spherical particles of cellulose. The carbon bead obtained was a carbon microbead which was physically and chemically stable and which retained the spherical shape of the original material of cellulose particles. The diameter of the microbead was found to gradually become smaller as carbonization proceeded. The diameter of the resultant graphitized carbon microbead was <5μm. This carbon bead was used as HPLC packing. Carbon microbead packing, unlike conventional carbon packings, was found not to show excessive retention or adsorption of electron-rich or hydrophobic compounds. It was also found that the peaks obtained using the carbon microbead packing became more symmetrical along with an increase in the graphitization temperature. The capability of the obtained carbon packings in retaining hydrophobic compounds was similar to that of alkylated silica gels, rather than to that of the conventional carbon packings. In addition, the carbon microbead packing, like conventional carbon packings, could separate positional isomers by the mechanism of a charge-transfer interaction.

View full abstract

This paper focuses on the relative standard deviation (RSD) of measurements obtained by the integration of noisy data in both Monte Carlo simulation and HPLC experiments. No peak overlap is considered. As the integration domain, T, which is symmetrical around the peak center is increased up to ten times the width, σ(SD), of a Gaussian peak, the integration over the peak has a minimal RSD at T=(-1.4σ, +1.4σ) in the measurement model where the white noise is the only error source. This optimal integration domain, if described as T/σ=(-1.4, +1.4) in the scale of width, is shown to be independent of the entire peak area, width and noise level. Similar results are obtained by the Monte Carlo simulation for skewed Gaussian peaks and by differentiating the RSD of a triangle signal with respect to the integration domain. The probability theory proposed here exactly predicts the RSD observed by the simulation for the integration of the Gaussian and non-Gaussian peaks. Although a minimal RSD is not evident in the HPLC data for some aromatic compounds, an important property of the model integration is retained in a trace analysis: the precision of the integration around the optimal domain (e.g., ±1.5σ) is superior to that over a wide domain (e.g., ±4σ).

View full abstract

A pentane-methanol mixture was taken as a convenient model of practical stationary phase functioning in the reversed-phase liquid chromatography (RPLC) system consisting of octadecyl-bonded silica gel (ODS) and methanol. The capacity factor (k′) of tris(β-diketonato)chromium(III) or bis(β-diketonato)palladium(II) observed in the ODS/methanol system was compared with the ratio of the solubility of the complex in a pentane-methanol mixture to that in methanol. A pentane-methanol mixture, containing methanol in a mole fraction range 0.42-0.66, was found to be a medium comparable to the stationary phase in the RPLC, because the distribution of the complex was similar. Furthermore, the incorporation of methanol into alkyl-moiety of ODS was confirmed experimentally. The above facts suggest that the stationary phase functioning in the RPLC consisting of ODS and methanol is a mixed phase of the alkyl-moiety of ODS and methanol incorporated from the mobile phase into the moiety for the complex tested.

View full abstract

Selenium is a toxic element which can cause environmental pollution. Its uptake and incorporation by Zn²⁺-induced metallothionein (MT) biosynthesis in a cyanobacterium were studied by means of high-performance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometric (ICPMS) detection. When we cultured the cells in selenite-enriched media, as much as 60% of the total selenium in the water-soluble cell components was incorporated into MT, which exists as two major isoforms but occupies a very minor fraction of the overall proteins. Existence of two pathways has been shown for the first time for selenium incorporation into the cyanobacterial MT; one is the S-Se bonding which can be reductively broken by 2-mercaptoethanol, and the other is the formation of selenocysteine followed by its incorporation into the MT peptide chain. No essential difference was noted between selenite and selenate regarding the uptake or the incorporation of Se. The present HPLC/ICPMS system can elucidate biological processes of environmental chemical importance at the molecular level.

View full abstract

Naphthalenedialdehyde (NDA) in the presence of cyanide ion reacts with primary amines to produce highly fluorescent cyano[f]benzisoindole (CBI) derivatives. Fluorescent detection with NDA as the reagent, however, has one major limitation. Namely, analytes with more than one derivatizable amine site, such as lysine, show quantum efficiencies that are much lower in value than those of mono-derivatized CBI-amines. The reason for this lowering is that the multiderivatized CBI amines undergo fluorescent quenching due to the close proximity of the CBI groups. This quenching can be circumvented, however, when these CBI-amines are deposited onto solid matrices, such as TLC plates. This method of depositing the liquid chromatographic (LC) effluent linearly on a solid matrix and then post-detecting the analytes has been referred to as a "chemical diskette" method in analogy to information storage and retrieval with computer diskettes. The implementation of this method was done by connecting a narrow bore flex-tubing from an LC microbore column to the writing head of a computer-controlled x-y recorder so that the LC effluent served as the "ink" in place of the usual writing pen. A thin layer chromatographic (TLC) plate replaced the recorder paper. The pattern of the deposition onto the TLC plate was preprogrammed and precisely controlled by a computer. The excitation light from a Xe arc lamp and the fluorescence emission to the optical detection system were transferred by optical fibers appropriately mounted on the writing head of the recorder. Thus, the deposited LC analytes could be fluorescence detected by precisely retracing the preprogrammed pattern as stored in the memory of the computer. The objective herein is therefore to evaluate the sensitivity and application of a dual wavelength excitation method (DWEM), particularly for multiderivatized CBI amines. A detection limit of 6fmol at a signal-to-noise of 2 has been demonstrated for desmosine, as one example of a multi-functional amine compound.

View full abstract

A general method has been developed for the selective coulometric detection of peptides using reversed-phase liquid chromatography. This method is based on the formation of electrochemically oxidizable copper(II)-peptide complexes by the reaction of peptides with copper(II) ions in an alkaline mobile phase through an in-line reactor containing copper metal. Di-, tri- and tetrapeptides used as model compounds (6 species) are separated on a column containing C18- bonded vinyl alcohol copolymer gel (Asahipak ODP-50, 5μm, 150mm×6mm i.d.) and an alkaline mobile phase (pH 11.0, 25mM phosphate buffer). A detection limit of 3ng was obtained for alanylalanylalanylalanine.

View full abstract

A flow injection analysis (FIA) system using a bis(2, 4, 6-trichlorophenyl)oxalate (TCPO)-hydrogen peroxide (H₂O₂) chemiluminescence (CL) system was developed for the determination of small amounts of polycyclic aromatic hydrocarbons (PAHs). The system allowed us to determine PAH over a wide range of concentrations with the detection limits (S/N=5) of 0.05pg (perylene), 65pg (anthracene), and 75pg (pyrene). On the basis of the results, an off-line reduction-HPLC-CL detection system was developed for separation and determination of dinitropyrenes (DNPs) as the specific PAH. DNPs were reduced to diaminopyrenes (DAPs), which were then analyzed by the HPLC equipped with the CL detector. A metal-free system was developed by use of plastic-frame column and all Teflon-made line. These new devices have improved the sensitivity and the reproducibility of determination; DNPs were determined over a wide range of concentration with the detection limit (S/N=5) of 0.025pg (1, 8-, 1, 6-DNP) and 0.05pg (1, 3-DNP). The present off-line reduction-HPLC system was applied to the determination of individual DNP at trace levels in the atmosphere.

View full abstract

Cationic micelles of hexadecyltrimethylammonium chloride (CTAC) markedly enhanced the intensity of fluorescein chemiluminescence (CL) catalyzed by horseradish peroxidase (HRP). No enhancement was observed with anionic micelles of sodium dodecyl sulfate or noionic micelles of polyoxyethylene(23)dodecanol. The CL enhancement was attributed to the electrostatic interaction of anionic fluorescein with the CTAC micelle. The local concentration of fluorescein onto the micellar surface increased the efficiency of the energy transfer from singlet oxygen (which was produced in peroxidation catalyzed by HRP) to fluorescein. This enhanced CL was applied to the determination of trace hydrogen peroxide. The calibration curve was linear over the range from the detection limit of 5.0nM to 1.0μM. The detection limit was more than three-times lower than that of the aqueous fluorescein CL.

View full abstract

The proposed method is based on the separation of sulfide, sulfite and thiosulfate in mixtures by using a resin-based anion-exchange column and a 5×10^-3M (M=mol dm^-3) sodium carbonate eluent, followed by a photometric measurement of any excess iodine (as triiodide) for its postcolumn reactions with the separated sulfur species. A postcolumn solution comprising 2.5×10^-5M iodine, 0.01M iodide and 0.8M acetic acid was continuously mixed with an effluent from the separating columns and then the absorbance for the remaining iodine (as triiodide) in the stream was monitored at 330nm. The chromatograms obtained for sulfide, sulfite and thiosulfate ions showed negative peaks, respectively, with detection limits (as S/N=2) of 2.09×10^-6M (0.0669ppm) for sulfide, 1.80×10^-6M (0.144ppm) for sulfite and 3.52×10^-6M (0.394ppm) for thiosulfate. The method was successfully applied to the determination of sulfide, sulfite and thiosulfate in hot-spring water samples.

View full abstract

N-(2-Hydroxybenzyl)chitosan (=HR) of an insoluble chitosan derivative was synthesized by reducing N-(2-hydroxybenzylidene) chitosan obtained from chitosan and 2-hydroxybenzaldehyde. We examined its adsorption selectivities for various metal ions in terms of the pH dependence of the distribution ratio of metal ions at 303K. The chelating ability of N-(2-hydroxybenzyl)chitosan for various metal ions was measured by a batchwise operation from 1mol dm^-3 of ammonium nitrate solution. The separations of gallium and iron(III) from zinc, and nickel from cobalt were possible because of the high selectivity of the present derivative. SinceN-(2-hydroxybenzyl)chitosan showed the high adsorption abilities for silver(I) and Copper(II), the adsorption equilibria of both silver(I) and copper(II) were investigated in order to elucidate the adsorbed species by examining the concentration dependencies of hydrogen ion and
ammonium nitrate. Silver(I) was adsorbed according to this adsorption reaction, accompanied by an ammonia molecule: AgNH₃⁺+HR_??_RAgNH₃+H⁺ while, copper(II) was adsorbed according to the following reactions: Cu²⁺+2HR_??_R₂Cu+2H⁺ and CuNH₃²⁺+2HR_??_R₂CuNH₃+2H⁺.

View full abstract

The hydration and association of tributyl phosphate (TBP) in dodecane were investigated on the basis of the water distribution between aqueous solutions and TBP-dodecane solutions at 298K. The water distribution data were analyzed as functions of the TBP concentration in the organic phase and the water activity in the aqueous phase. A highly hydrated TBP trimer was found in the organic phase at high TBP concentrations by the data analysis. At [TBP]_{org, total}≤0.02mol dm^-3, no TBP association occurred and only TBP•H2O formed as a unique hydrate. In the range of 0.02mol dm^-3<[TBP]_{org, total}≤0.3mol dm^-3, a part of the monohydrate dimerized to form (TBP)₂(H₂O) ₂. At [TBP]_{org, total}>0.3mol dm^-3, trimerization also occurred at high water activities. The trimer was found to be hydrated with six water molecules, (TBP)₃(H₂O)₆.

View full abstract

Universitätstrasse 16, CH-8092 Zürich, Switzerland Flow-through type fiber-optic chloride ion selective optodes have been developed based on chloride adducts of lipophilic organometallic compounds, including a metal: Sn, Ge, Hg or Hf and a lipophilic anionic indicator dye of the diphenyl amine type. The pellicular-type ODS beads were coated with a lipophilic organic liquid containing the lipophilic organometallic compound as the anion-selective ionophore ligand and LAD-3 as the color changeable indicator dye. The optode system was constructed by packing these color changeable ion sensing beads in the flow-through optical cell (cell volume, 7μl) having a quartz window attached directly to the distal end of a bifurcated optical fiber. The optode based on tricyclohexyltin chloride (TCTCl) exhibited the most sensitive response to chloride ion among all the sensors using the organometallic compounds investigated. This optode could detect chloride ion in the concentration ranging from 10^-4to 1M at pH 6.5 with a 0.01M sodium phosphate buffer as the flowing solution by measuring the absorbance change in the sensing beads at 516nm. This optode showed sufficient selectivity for the determination of chloride ion in normal human serum.

View full abstract

It was found, only in cases of palladium-tin and platinum-lead Intermetallic compounds, that the atomization shifted to
a higher temperature region when analytes formed an intermetallic compound with a matrix modifier, and its activity
decreased. Due to a smaller amount of data for the activity coefficient, it is difficult to confirm the above-mentioned
phenomena. In this study, tin, indium and lead were chosen as analytes, and palladium, nickel and manganese were used as matrix modifiers. On the other hand, the atomic-vapor temperature was measured by the two-absorption-line method. This temperature is close to the temperature of the sample loaded into the graphite furnace. Unfortunately, the setting temperature, or the measured temperature of the graphite furnace using an optical pyrometer, did not express the true temperature of the sample. By measuring the temperature of the alloy, we were able to verify that atomization begins in certain phases of the Intermetallic compounds, and finished in specific phases. The phases of the intermetallic compounds were then confirmed by measuring the atomic-vapor temperature. Since there has not been much data concerning the activity coeffieient being measured until now, the pressure of the atomic vapor evaporated from the Intermetallic compound was compared with the pressure from pure metal at the beginning of atomization. We also estimated whether the activity coefficient was bigger or smaller than 1.0. In other words, the absorbance profiles of the vaporization of the intermetallic compound and pure metal were compared with each other; we could thus estimate the activity coefficient. Regarding Sn, the beginning of atomization shifted to a higher temperature region in the order Mn, Ni and Pd. This order is the same as that of the decreasing activity coefficient. In the cases of In and Pb, the beginning of atomization of Pd-In and Pd-Pb only shifted to a higher temperature. The other Intermetallic compounds did not shift, because the compounds melted in the early stage of atomization. The atomization of the Intermetallic compound, whose activity coefficient is smaller than 1.0, was observed by means of high-resolution transmission-electron microscopy. As a typical example, an Intermetallic compound of Pd and Sn was chosen. Vaporization was observed in the phase of Pd₃Sn₂ beginning with atomization. The atoms in the top surface were tightened until the temperature of the intermetalic compound increased and almost reached the melting point. After the atoms had vibrated and swayed for a while, about 10 atomic layers evaporated explosively. On the other hand, the atoms gradually evaporated from the top surface in pure Sn and Pd metals. These phenomena indicate a big difference between the intermetallic compound and the pure metal.

View full abstract

An attempt was made to characterize the charge-up behavior and electron flooding effects at an α-alumina surface observed by monochromated X-ray photoelectron spectroscopy. The observed charge-up potential changed in a complicated manner with the X-ray irradiation time. The complicated features could be explained based on the balance between the electron loss from the surface caused by photoemission and electron gains by secondary electrons from the gold metal coating on the front of the input lens and by a creeping current from the ground level. The charge-up caused a retardation of the photoelectron emission rates, line-broadening of the spectra and slight changes in the differences among the binding energies of the spectral lines. The charge-up potential (ranging from 182 to 486eV) was proportional to the X-ray irradiation flux at the surface. The relationship between the low-energy electron flux from an electron flood gun and the photoelectron energies was measured; thereby, the most suitable electron flux to compensate for the surface charge could be estimated.

View full abstract

A porous cellulose tape impregnated with a coloring solution that includes hydroxylamine sulfate, Methyl Yellow (pH indicator; pH 2.9-4.0, red-yellow), glycerin and methanol has been developed to provide a highly sensitive means of detecting formaldehyde gas. Formaldehyde reacts with hydroxylamine sulfate to produce sulfuric acid. When a sample including formaldehyde was passed through the tape, the color of the tape changed from yellow to red. The
degree of color change was proportional to the concentration of formaldehyde at a constant sampling time and flow rate. The degree of color change could be recorded by measuring the intensity of reflecting light (555nm). No interference was observed from carbon monoxide (100ppm), nitrogen monoxide (50ppm), nitrogen dioxide (105ppm), carbon dioxide (4.9%), hydrogen (100%), hydrogen sulfide (32ppm), or hydrogen fluoride (6ppm). The response of acetaldehyde and propionaldehyde were one fortieth and three-hundredths of that of formaldehyde, respectively. Reproducibility tests showed that the relative standard deviation of relative intensity (n=10) was 3.4% for 4ppm formaldehyde. The detection limit was 0.1ppm for formaldehyde at a sampling time of 5min and a flow rate of 400ml/min.

View full abstract