Recent progress in mediated bioelectrocatalysis is reviewed. In a section concerning fundamentals, special attention is paid to describing the property of a steady-state current in this system from thermodynamic and kinetic viewpoints. Practical applications of the system includes biosensors for enzyme substrates, amplified detection of mediators, bioreactors, biofuel cells, enzyme kinetic measurements, and protein redox potential measurements.
In order to examine the process of the lipid oxidation, the regioisomers of hydroperoxides produced by the photosensitized peroxidation of cholesteryl oleate, cholesteryl linoleate and cholesteryl linolenate by photosensitized peroxidation and the separation of each isomer by HPLC were investigated. The olefinic parts in fatty acid chains of the above cholesteryl esters received hydroperoxidation predominantly by photosensitized oxidation. Cholesteryl oleate was subjected to hydroperoxidation at the 9 and 10 position of the oleic acid chain. Cholesteryl linoleate and cholesteryl linolenate were also subjected to hydroperoxidation at the 9, 10, 12 and 13 positions of the linoleic acid chain and at the 9, 10, 12, 13, 15 and 16 positions of the linolenic acid chain, respectively. Hydroperoxidation on the cholesteryl ring was also observed, however, their formation was negligibly small compared with hydroperoxidation of the fatty acid chain. The elution order of isomeric peroxides in HPLC was determined using a normal-phase column (Mightysil 60) eluted with hexane/2-propanol by the detected wavelength at 210 and 233 nm.
The objective of this research was to determine the true superoxide (O2-) scavenging activity of antioxidants in a sample containing xanthine oxidase (XOD) inhibitor. A previous method involving electron spin resonance (ESR) spin-trapping has generally used 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) as a spin trap and the O2- generation system of hypoxanthine (HPX)-xanthine oxidase (XOD). However, this method has a problem in that the ESR signal intensity of the DMPO spin adduct of O2- (DMPO-OOH) is decreased not only by the reaction of a sample with O2-, but also by the inhibition of XOD. Therefore, the ESR spin-trapping method combined with the SOD addition method was developed as a new experimental technique in this study. The results indicate that the O2- scavenging activity of a sample containing allopurinol as an XOD inhibitor could be determined by using this method.
An on-chip chemical micro-determination device, integrating the FIA system, was applied to L-ascorbic acid (AA) and dehydroascorbic acid (DHAA) determination. Microchannels having a 250×100 μm cross-section were fabricated in a quarts glass chip as a component in an integrated FIA system. They were put to use for flow, mixing, reaction, and detection. The reaction system was a coupled redox-complexation reaction between AA and a 1,10-phenantroline-Fe(III) mixture. Sample solutions were introduced into micro-channels by micro-syringes, the flow rate was controlled by a micro-syringe pump, and the reagents, were mixed by molecular diffusion. A photothermal microscope was used for the ultra-sensitive detection of the non-fluorescent reaction product (ferroin). For DHAA determination, dithiothreithol was used as a reducing agent and the total AA was determined. The DHAA quantity was calculated by subtraction. This method allows for the determination of AA with a linear range of up to 40 μM. The RSD for 25 μM AA (n=5) is 1%, and the limit of detection (LOD) is 0.1 μM. The procedure was successfully applied to determining the AA and DHAA content of urine and AA in vitamin C tablets. The results agree well with those obtained by reference methods.
The partition bahavior of inorganic ions in aqueous polyethylene glycol (PEG)-salt two-phase systems of different polymer and salt concentrations has been studied. Na2SO4, Na2HPO4 and K2HPO4 were used as phase-forming salts. Phase diagrams for the three aqueous PEG-salt two-phase systems were determined, and the partition coefficients for the inorganic ions in each of the systems were investigated as a function of the difference in the concentration of PEG in the two phases, ΔwPEG. It was found from the phase diagrams that the salting-out abilities of K+ and SO42- were lower than those of Na+ and HPO42-, respectively. However the former ions were more effective for the extraction of oppositely charged analyte ions to the PEG-rich phase than the latter ions when they were compared with each other under a constant ΔwPEG. On the other hand, Na+ and HPO42- were more effective than K+ and SO42-. for the extraction of the ions which have the same charge sign as theirs. These counter-ion and co-ion effects of the phase-forming ions on the partition of ionic solutes are interpreted by a model regarding the partition mechanism of ions in the presence of excess amounts of co-existing salts.
Ion-pair extraction of silver with 3,6-dithiaoctane in the presence of a univalent anion into a small volume of chloroform at pH less than 6 was studied. Univalent anions tested were 5 inorganic and 9 colored organic anions and 2 cobalt complex anions. Of these anions, perchlorate, tetraphenylborate and picrate anions and three alkyl oranges, Bromocresol Green, Bromothymol Blue, 5-Br-PAPS and cobalt-dihydroxyazobenzene complex gave a quantitative extraction (>99%) of silver. The direct nebulization of 50 μL of the chloroform extract into a fuel-lean air-acetylene flame gave a highly sensitive signal intensity without any background correction from chloroform. The present method was applied to the determination of silver in a certified botanical sample, NIES-CRM No. 9 Sargasso. The results were very satisfactory and reproducible.
A new analytical method which combines an eletrochemical quartz crystal microbalance (EQCM) and a wall jet electrode has been developed. By using a split-ring electrode, in-situ detection of two kinds of ions emitted from the working electrode can be performed during an eletrochemical measurement. In the present measurement, Cu(I) and Cu(II) dissolved from copper electrodes were determined on two half-ring electrodes, respectively; moreover, the resonant frequency change was measured simultaneously. Using the present method, the electrode reaction can be analyzed by four parameters: the working electrode current, the dissolution rates of two kinds of ions, and the electrode mass change. The amount of charge for the species accumulated on dissolving copper electrode was obtained by subtracting the dissolving components form the working electrode current. By comparing the amount of dissolved ions with the total mass change calculated from the resonant frequency change, the mass change regarding the accumulated species could be estimated.
The complicated analytical challenge of lead determination in the presence of high levels of copper, has been investigated using anodic and cathodic stripping voltammetry, on a variety of mercury free electrode substrates. CSV was found to be ideally suited towards lead determination in a predominantly copper matrix, due to the lack of interference from copper ions on the anodic electrochemistry of lead. A CSV technique, incorporating the use of power-ultrasound and a borondoped diamond electrode is presented. The sono-CSV technique has been optimised to give a lower limit of detection of aqueous Pb2+ ions of 1.6×10-9 M for a 300 s PbO2 deposition, requires no solution degassing, is mercury-free and totally free from copper-intermetallic interference. As an extreme example, the technique was applied successfully to the determination of lead content of a copper-lead alloy, after a simple acid digestion protocol. Excellent correlation was observed between the known lead level and experimental results, with the technique being sensitive towards the detection of 3.3×10-4% (w/w) lead in copper, for the analysis of a 10 mg sample.
Mica, as a bridge of the study for combining between quartz crystal microbalance (QCM) and atomic force microscope (AFM), was successfully modified onto the piezoelectric quartz crystal (PQC). This mica-modified piezoelectric quartz crystal (mica-PQC) can be stably oscillated with a shift frequency of ±1 Hz per half an hour in air. Using this mica-PQC, the processes of DNA adsorbed onto the mica surface were studied in liquid phase. The results show that a bivalent cation, such as Mn2+, can be used as an ionic bridge to immobilize DNA on mica surface. The image of DNA on the mica surface was also obtained by AFM. Mica-PQC gives the possibility of a combination between QCM and AFM in situ.
This study examined enantiomeric separation by capillary electrochromatography with charged polyacrylamide gels incorporating chiral selectors, in which electroosmotic flow would be generated. The columns are capillaries filled with a negatively or positively charged polyacrylamide gel, a so-called monolithic stationary phase, incorporating polymeric β-cyclodextrins as chiral selectors. The capillary wall is activated first with a bifunctional reagent to make the resulting gel bind covalently to the inner surface of the fused-silica tubing. Enantiomeric separations of two cationic and a neutral compounds were achieved using negatively charged polyacrylamide gel-filled capillaries incorporating poly β-cyclodextrin and carboxymethyl β-cyclodextrin polymer and a mobile phase of 200 mmol dm-3 Tris-300 mmol dm-3 boric acid buffer (pH 9.0). Enantiomeric separations of twelve anionic compounds were also achieved using positively charged polyacrylamide gel-filled capillaries incorporating poly β-cyclodextrin and a mobile phase of 200 mmol dm-3 Tris-300 mmol dm-3 boric acid buffer (pH 8.1). High efficiencies of up to 240000 plates m-1 were obtained. The within-run reproducibilities of retention time and separation factor were good.
An HPLC method utilizing reversed-phase chromatography coupled with coulometric detection was developed for the determination of alkylphenols in mineral water. Alkylphenols in water samples were extracted with ethyl acetate. The alkylphenols were completely separated by an ODS column with acidic mobile phases and detected with a dual coulometric detector. The first electrode set at 400 mV was used for sample clean up, whereas the second one set at 650 mV was used for analytical purposes. The limits of detection (signal-to-noise ratio of 2) of eight alkylphenols tested were in the range from 0.25 pmol (BPA) to 1.0 pmol (p-NP) on chromatograms. The proposed procedures were applied to mineral water in PET bottles. All nine samples contained both bisphenol A (BPA) and p-nonylphenol (p-NP). The concentrations of BPA and p-NP calculated from calibration curves were 2 - 10 ng/l and 19 - 78 ng/l, respectively. The concentrations were essentially the same before and after heating at 50°C for 8 h.