Filter furnace laser-excited atomic fluorescence spectroscopy was used for the determination of lead in whole blood. The use of a Katskov filter has enabled the direct comparison between lead fluorescence signals obtained with blood standards and water standards. Only a simple dilution of the blood was required. This direct correlation has allowed the interpolation of the lead signal of blood samples using a lead water standard calibration curve. NIST certified blood samples were analyzed using the method with excellent results.
The thermal degradation processes of the remaining substances in the hydrolysis product of aluminum alkoxide were investigated with evolved gas analysis-mass spectrometry (EGA-MS). Different patterns of the process were obtained from the hydrolysis products with various temperatures which give different products. Concerning boehmite as a 90°C-hydrolysis product, the carbon compounds are eliminated below about 300°C, before dehydration from AlOOH to Al2O3 upon heating. Whereas in the case of a boehmite+bayerite mixture as the hydrolysis product at room temperature, dehydration from Al(OH)3 to Al2O3 takes place almost simultaneously with the elimination of carbon compounds below the temperature of dehydration from AlOOH to Al2O3.
Carbon disulfide and pyrrolidine containing p-xylene was proved to be useful and convenient for metal extraction. The procedure involved forming pyrrolidine dithiocarbamic acid during the course of extraction and solidifying the organic phase by cooling in a centrifuge. Metal ions such as Cd2+, Ni2+, Co2+, and Zn2+ could be extracted into the organic phase without using any chelate extractant.
The influence of bromination at all the ortho-positions of phenols on the features of phenolsulfonphtalein and phenolphtalein was investigated. The absorption spectra of Bromphenol Blue (H2BPS) in phosphate buffers demonstrated the complete opening of the lactone ring to form yellow HBPS- at pH 1 where pK2=3.8. The reaction of hydroxide ion with blue BPS2- was found to produce colorless BPS(OH)3- required heating where pK3=9. On the contrary, the spectra of 3′,3″,5′,5″-tetrabromophenolphtalein (H2BPP) demonstrated that the lactone ring was very stable. More than 99.5% of BPP2- species consisted of a colorless lactone possessing two isolated phenol groups where pK1′=6.0 and pK2′=6.8; the reaction of hydroxide ion with BPP2- to produce BPP(OH)3- was slow where pK3=10.3. These results indicate the increased acidity of phenols and the greater tendency of the central carbon atom to act as an electron acceptor.
The strong interaction between biotin and avidin is utilized for the development of enzyme-linked competitive binding assays not only for biotin itself, but also for other biomolecules. Alkaline phosphatase, a single substrate enzyme typically used for heterogeneous types of competitive binding assays, is employed also for homogeneous types. For the biotin assay, the heterogeneous protocol offers a much improved detection capability when compared to the homogeneous type. A simple approach of simulating dose-response curves is introduced. An effective analyte concentration attached to an enzyme label is determined by using the same binder, but with a different enzyme label. The analyte system adapted to the biotin/avidin-mediated homogeneous protocol is digoxin and a monoclonal anti-digoxin antibody. The detection capabilities of these assays, particularly the homogeneous types, are shown to be limited by the detectability of the enzyme conjugate employed.
Horseradish peroxidase (HRP) was encapsulated in liposomes prepared by an extrusion technique. The liposomes were coupled covalently to anti-rabbit IgG using N-hydroxysuccinimide ester palmitic acid as a component of liposomes. The number of encapsulated HRP molecules per liposome was about 800. A large portion of HRP was encapsulated inside the liposomes for about one week at 4°C. The catalytic activity of HRP was measured by a luminol chemiluminescence (CL) method and was found to be almost constant during storage. The CL intensity per antibody in the detection of HRP encapsulated in the antibody-coupled liposomes was 125-times greater than that of HRP conjugated directly to the antibody.
A flow-injection analysis (FIA) for superoxide dismutase (SOD) activity was developed based on the use of tetrazolium salt, WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt) and an enzyme reactor packed with Sepharose 4B on which xanthine oxidase (XO) and catalase were co-immobilized. WST-1 is highly water-soluble, and no adhesion of the reduced form to the FIA line was observed during continuous operation for 3 months. As optimized conditions, a sample (9 vol) was mixed with a reagent solution (1 vol) containing 3 mM hypoxanthine and 2 mM WST-1, and the mixed solution (20 μl) was injected into a carrier stream of 50 mM carbonate buffer (pH 10.2) at a flow rate of 0.4 ml/min. Under the conditions, the concentration of the SOD preparation giving 50% inhibition (IC50) was 2.7 μg/ml and the sampling frequency was 30 samples/h. When the SOD activity in erythrocytes of rats was determined by the present FIA method, the values were linearly related to those obtained by the conventional nitroblue tetrazolium (NBT) assay (r=0.975; n=18). The enzyme reactor was stable for at least 200 repetitive injections.
The potential response of a flow-through ion sensor using a plasticized poly(vinyl chloride) membrane doped with amphiphilic anions to two synthetic polyelectrolyte cations was investigated. The potential responses to the polyelectrolyte cations were characterized in the terms of extractability of amphiphilic anion, concentration of amphiphilic anion salt in the membrane, solvent property of plasticizer, and leakage of amphiphilic anion salt. The concentration of amphiphilic anion salt in the membrane was a main factor in the potential response to polyelectrolyte cation. The examination of FTIR spectra in the plasticized poly(vinyl chloride) membrane containing amphiphilic ion salt showed that the leakage of amphiphilic ion salt from the membrane was prevented with the addition of polyelectrolyte ion. It was deduced that the potential response of polyelectrolyte cation was due to a decrease in the concentration of sodium ion, the counter ion to amphiphilic ion, as the primary ion in the plasticized poly(vinyl chloride) membrane.
The photoelectrochemical response of bacteriorhodopsin (bR) was studied in buffered electrolyte solutions. The photocurrent intensity was inversely proportional to the buffer concentration in a wide range, and the response time became shorter with increasing buffer concentration. When plotted as a function of the electrolyte pH, the ratio of the peak photocurrent in the presence of a buffer to that in the absence of the buffer exhibited a minimum at a pH nearly equal to the pK′a of the buffer. These results are explained by invoking the buffering capacity, and are in line with the view that the transient bR photoresponse originates in the pH change near the electrode due to proton release/uptake by bR molecules.
A highly sensitive fluorometric high-performance liquid chromatographic method was developed for the simultaneous determination of phenytoin and its major metabolites [5-(3-hydroxyphenyl)-5-phenylhydantoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin]. After extracting these compounds and 5-(4-methylphenyl)-5-phenylhydantoin (MPPH) as an internal standard from serum (50 μl) with ethyl acetate, they were further converted into the corresponding fluorescent derivatives by a reaction with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydrogen carbonate and dibenzo-18-crown-6 in acetonitrile. The derivatives were separated by reversed-phase chromatography on a YMC-Pack ODS-A column with a mixture of acetonitrile?50 mM phosphate buffer (pH 7.0) (4:6, v/v) as a mobile phase, and were then detected spectrofluorometrically at 448 nm with excitation at 365 nm. The detection limits for phenytoin, 5-(3-hydroxyphenyl)-5-phenylhydantoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin added to serum were 0.6, 3.0 and 0.8 ng (2.4, 11 and 3.1 pmol) ml-1 serum at a signal-to-noise ratio of three. The method was applied to determine the unbound- and total-phenytoin and the metabolites levels in the serum obtained from two healthy volunteers, after oral administration of the drug.
A method for endcapping of octadecylsilyl (ODS)-silica gels was developed using supercritical carbon dioxide as a silylation medium. The effects of temperature and pressure of carbon dioxide on the deactivation of the gels were investigated under the silylation conditions at 100 - 220°C and 11.8 - 24.5 MPa. High-performance liquid chromatographic evaluation using caffeine and phenol as test compounds indicated that the most inert gels were obtained from the endcapping under 180°C and 24.5 MPa. Endcapped ODS-silica gels, which were commercially available, could be further deactivated under these conditions. The inertness of the gels was also evaluated by supercritical fluid chromatography (SFC) using pyridine and phenol as test compounds. The effectiveness of the proposed method was clearly observed in the SFC evaluation.
The emission produced by thiourea in oxidation by permanganate in acidic solution in the presence of Ru(bipy)32+ is used to determine 1.8×10-8 to 1.8×10-6 mol/l thiourea. The limit of detection is 1.0×10-8 mol/l and the relative standard deviation is 1.1% for a 1×10-5 mol/l thiourea solution (n=10). The method was applied satisfactorily to the determination of thiourea.
The adsorption behavior of nifuroxazide on a hanging mercury drop electrode (HMDE) was critically examined using cyclic voltammetry and differential pulse techniques. The effect of different parameters on the accumulation behavior of the adsorbed species was tested. Thus, a sensitive stripping voltammetry procedure for the determination of nifuroxazide with an adsorptive accumulation on the surface of HMDE has been developed. Under optimal conditions a detection limit of 5×10-9 Mand a linear calibration graph in the range 2×10-8 - 1×10-7 M were obtained. The precision, expressed by the relative standard deviation, was 2.8% (n=10) at 1×10-7 M. The method was successfully applied for the analysis of nifuroxazide in serum.