DNA-DNA hybridization and DNA-protein or enzyme interactions are essential steps in biomolecular recognitions. These interactions have been conventionally studied by gel mobility shift assay. This technique gives us only qualitative information, we must label enzymes radioactive or fluorescent molecules, and it takes a relatively long time to analyze the results. Here we introduce a new tool of a highly sensitive 27 MHz quartz-crystal microbalance (QCM), in which the resonance frequency decreases linearly with the increase of mass on the electrode area at the nanogram level. Thus, when a host molecule is immobilized on a QCM, the binding behavior and its kinetics can be monitored from frequency changes due to the guest binding in aqueous solution. The results are also compared with those from a surface plasmon resonance sensor.
Peroxidase-like activities of glass-beads modified with metal-porphines (M-PG) were examined through a dye-formation reaction, which was catalyzed by peroxidase, of phenol and 4-aminoantipyrine in the presence of hydrogen peroxide. When M-PG was used as a catalyst in place of peroxidase, glass-beads modified with Mn3+- and Fe3+-tetrakis(4-carboxyphenyl)porphine derivatives (Mn- and Fe-TCPPG) showed relatively strong peroxidase-like activity among the M-PG examined. The percentage of the resulting quinoid dye by Mn- and Fe-TCPPG reached to about 93% and 88%, respectively, of that formed by peroxidase. Peroxidase-like activity of Mn-TCPPG was applied to the determination of hydrogen peroxide in place of peroxidase. Furthermore, the determination of serum compounds (glucose and uric acid), which form hydrogen peroxide by using their oxidases, was examined.
A new, simple and sensitive spectrophotometric method for the determination of some phenothiazine drugs has been developed. The proposed method is based on the reaction of promethazine hydrochloride (PH), chlorpromazine hydrochloride (CPH), triflupromazine hydrochloride (TPH), fluphenazine hydrochloride (FPH), trifluoperazine hydrochloride (TFPH), perazine dimaleate (PDM) and prochlorperazine maleate (PCPM) with iron(III) and a subsequent reaction with ferricyanide in an acetic acid medium to yield Prussian blue colored product with a maximum absorption at 700-720 nm. The common excipients used as additives in pharmaceuticals do not interfere with the proposed method. Analytical data for determination of the pure compound are presented together with the applications of the proposed method to the analysis of some pharmaceutical formulations. The results compare favorably with those of the official method.
Bile acid carboxy-linked glucuronides and adenylates have active ester groups, and thus may easily react with amino groups on endogenous tissue proteins resulting in physiological disorders. To identify such tissue-bound bile acids in vivo, we generated a monoclonal antibody which recognizes deoxycholic acid (DCA) residues anchored on endogenous proteins. The spleen cells from a BALB/c mouse, which was immunized with a conjugate of DCA and bovine serum albumin (BSA), were fused with P3/NS1/1-Ag4-1 myeloma to generate antibody-secreting hybridoma clones. The resulting monoclonal antibody (Ab#88; isotype γ1, λ) was specific to N-ε-deoxycholyl lysine as well as the nonamidated DCA, and enabled sensitive detection of DCA residues (270 femtograms) anchored on BSA molecules, introduced by the reaction of the DCA adenylate. The immunoaffinity column immobilizing the Ab#88 allowed selective extraction of the DCA-introduced substance P. The antibody will be useful for monitoring the formation and localization of tissue-bound DCA, which may be concern with the activity of DCA as a colon cancer promoter.
Chitin powder was dissolved in a mixed solution of N,N-dimethylacetoamide and 1-methyl-2-pyrrolidone with lithium chloride. The chitin solution was adhered to the film. Glucose oxidase having a negative charge was immobilized to the protonated chitin film in 0.1 mol dm-3 acetate buffer (pH 5.0), based on electrostatic interaction. The interaction is useful and convenient for an immobilization of the enzyme. In this study, glucose sensor was developed at a platinum electrode with a chitin/glucose oxidase film as an application of chitin in analytical chemistry. An oxidation peak of H2O2 from the enzyme reaction was observed at +0.5 V (vs. Ag/AgCl) in 0.1 mol dm-3 acetate buffer (pH 6.2). The calibration curve of glucose was linear in the range from 5×10-7 to 3×10-5 mol dm-3. The sensor was applied to determination of glucose in sports drinks.
Hydrogen ion-selective solid-contact electrodes based on alkyldibenzylamine (alkyl=butyl, hexyl, octyl, decyl, dodecyl) have been prepared. Solid-contact electrodes have been fabricated from polymer cocktail solutions based on alkyldibenzylamine. We have shown that the response ranges and Nernstian slopes are influenced by the alkyl chain length. Especially, solid-contact electrodes with decyldibenzylamine have shown the best selectivity, reproducibility of e.m.f. and long-term stability. Solid-contact electrodes containing decyldibenzylamine were shown to be linearly selective to hydrogen ion in the range of pH 2.1 - 12.4; their Nernstian slopes were 52.9 mV/pH at 20 ± 0.2°C. This electrode, coated with PVC containing decyldibenzylamine neutral carrier, showed no redox response in comparison with an uncoated Pt electrode and a poly(aniline)-coated Pt electrode. The 90% response time was less than 5 s, and their electrical resistance varied in the range of 0.1 - 2.0 MΩ.
The polarographic reduction of tin gives rise to an additional peak at more positive potentials than tin in the presence of selenite. It is found that when the selenite concentration exceeded the tin concentration, only one peak for tin was observed, enabling the determination of both these ions. A procedure for the direct determination of trace amounts of tin by the addition of selenite is described. It was possible to determine trace amounts of tin and selenium using this peak. Tin and selenium in a synthetic sample at 5 × 10-6 M Sn(II) and 2.5 × 10-6 M Se(IV) concentrations could be determined with -6.0% and -8.0% relative errors, respectively. This procedure was applied to canned corn, and 5.95 ± 0.54 µg/g tin could be determined with a 9.1% relative standard deviation.
A sequential injection analysis system has been developed for on-line monitoring of nitrite and nitrate in wastewaters, based on the Griess-Llosvay reaction and spectrophotometrical measuring of the absorbance at 543 nm. Nitrate is previously reduced to nitrite in a copperized cadmium column and analyzed as nitrite. The proposed system is fully automatized and is able to monitor nitrite and nitrate, simultaneously, in samples at a frequency of about 24 samples per hour with a relative standard deviation (RSD) better than 2.0% for nitrite and 1.3% for nitrate. The calibration graph was linear between 0.05 - 25 mg dm-3 for nitrite and 0.05 - 15 mg dm-3 for nitrate.
The multielement determination of major-to-ultratrace elements in plant reference materials (SRM Pine Needles, SRM Tomato Leaves, and NIES Tea Leaves) was carried out by ICP-AES (inductively coupled plasma atomic emission spectrometry) and ICP-MS (inductively coupled plasma mass spectrometry). The plant sample was decomposed with nitric acid and hydrofluoric acid in a Teflon beaker on a hot plate. The digest was dissolved in 1 M HNO3 solution, and then subjected to elemental analyses. As a consequence, the analytical data for about 40 elements including rare earth elements (REEs) were obtained over a wide concentration range, for example, from 49600 µg g-1 of Ca to 6.2 ng g-1 of Lu in Tomato Leaves. The enrichment factors, which were estimated by normalizing the observed concentrations of analyte elements in plant reference materials to their soil abundances, were evaluated in order to discuss the relative abundances of various elements between plants and soil. It was found that most of the elements, except for REEs in Pine Needles and Tea Leaves, were significantly accumulated in the plant reference materials. In particular, the essential elements (K, Mg, Ca, Mn, Cu, Zn, B and P) for plant growth provided large enrichment factors.
A column packed with Amberlite XAD-16 resin was used for preconcentration and determination of chemical forms of heavy metals such as Cr, Ni, Cu, Cd and Pb in lake water samples. The concentrations of the metals bound to humic substances and of free metal ions were determined after their sorption-elution on the resin. The method optimized with sodium tetraborate reagent was used in determining the free metal ions. Both flame- and graphite furnace-atomic absorption spectrometry was used to determine the metals in lake water samples. Recoveries were quantitative for all the elements studied (≥95%). The detection limits (DL, µg mL-1) were 0.007 for Cu; 0.002 for Cd; 0.022 for Pb; 0.038 for Cr; 0.008 for Co; 0.015 for Mn; and 0.054 for Ni (n=20, 3s). The precision values for the same elements were in the range of 1.3 to 2.4% (n=10).
The ion-pair extraction of univalent copper with 3,6-dithiaoctane in the presence of each of 15 univalent anions into a small volume of chloroform at a pH of less than 7 was studied. Of these anions, picric acid, Bromothymol Blue, and cobalt(III)-dihydroxyazobenzene complex gave a quantitative extraction of copper (>99%) and Bromocresol Green, EO, PO, and Co-5-Br-PAPS complex a constant, but a slightly lower, extraction (85 - 95%) at their corresponding pH ranges, respectively. The direct nebulization of 50 µL of the chloroform extract into a fuel-lean air-acetylene flame gave a sensitive signal height without any background correction from chloroform. The present method was successfully applied to the determination of copper in three standard botanical, one standard zoological, and four standard geological reference materials.
Based on a back-extraction of bis(4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedionato)copper(II), a method for the simultaneous determination of the copper(II) complexing capacity of natural water and the conditional stability constant of copper(II) complex with naturally occurring ligands was developed. The feasibility of the proposed method was confirmed using nitrilotriacetic acid and ethylenediamine-N,N′-dipropionic acid instead of naturally occurring ligands. It was then applied to a pond-water sample. The results indicated that two types of copper(II) complexes having different stabilities were formed upon the addition of copper(II) to the sample. By using the copper(II) complexing capacity and the conditional stability constant, the distribution of copper(II) species in pond water was calculated. It was found that most of the copper(II) in pond water exists as a complex with a naturally occurring ligand, and that the concentration of free copper(II) ion is extremely low.
A new on-line sample preparation system using column-switching HPLC was developed for the structure elucidation of compounds in mixtures by NMR. This system consists of three HPLC sections, separating the compounds of interest (LC-1), trapping the separated compounds on a trapping column using H2O and then replacing H2O with D2O (LC-2), and eluting the compounds from the trapping column using a deuterated solvent for an NMR measurement (LC-3). The system allows easy separation of a trace amount of the compounds from a mixture, desalting and concentration of the separated compounds, and the use of a deuterated solvent, such as CD3OD, for an NMR measurement. It was successfully applied to the structure elucidation of (±)-propranolol hydrochloride and a mixture of vitamin E derivatives as models. This new tool should be of considerable value in various fields for rapid sample preparation for NMR measurements.
It was found that Arrhenius plots were composed of two straight portions for As deposited on the PG and NPG furnace walls and of one straight portion for that on active carbon formed from the pyrolysis of ascorbic acid at a low-temperature pyrolysis. To correct the absorbance of the first peak for that of the second peak, the absorbance corresponding to the second peak in the period of the first peak was evaluated by extrapolating the straight line for the second peak to each atomization time. The activation energy (Ea) for the first peak depended on the ratio of the edge carbon species in graphite as the condensed phase based on Raman spectrometry. At a high temperature pyrolysis, the first straight portion disappeared and the Ea for the second straight portion increased in the PG and NPG furnaces. The existence of two types of atomization mechanisms as the first atomization corresponding to the desorption of As binding to active sites and the second atomization corresponding to the desorption of As intercalated within the graphite structure are proposed. On the active carbon, an increase in Ea was also observed, which is in agreement with that observed at the high-temperature pyrolysis in the graphite furnaces.
Measurement of refractive index and trace element analysis by ICP-MS were applied to the forensic discrimination of bottle glass samples from different origins. Refractive index was calculated from the matching temperature at which glass fragments became invisible in the silicone oil. Sample solution for ICP-MS was prepared by dissolving approximately 10 mg of glass fragments into a mixture of 0.5 ml of HF and 0.5 ml of HClO4 and heating for 2 h using a microwave oven, followed by dilution to 25 ml after 2 ml of HNO3 was added. Thirteen elements (Co, Cu, Zn, Rb, Sr, Zr, Ag, Sn, Sb, Ba, La, Ce and Pb) were found to be useful for the discrimination of bottle glasses. These elements in NIST SRM 612 (Trace Elements in Glass) could be determined with an RSD less than 3.2%, except for Sn and Pb. The proposed method was applied to 16 bottle glass samples. Of 120 pairs among 16 bottle samples, 13 pairs were indistinguishable by RI only. Combination of trace element analysis by ICP-MS and refractive index measurement made it possible to distinguish all the pairs.
A time-resolved quasi-elastic laser-scattering method was applied to monitor the dynamic and collective behavior of molecules of an anionic surfactant, sodium dodecyl sulfate (SDS), around the critical micelle concentration (cmc) at several water/chlorinated hydrocarbon interfaces. The time courses of the capillary wave frequencies after injection of the SDS solutions into the water phase were investigated. We found that when the injected concentration is above the cmc, the relative number density of SDS molecules decreases with an increase in the bulk aqueous concentration at all the examined interfaces. We also found that the relative number density of SDS molecules adsorbed onto the interface increases with a decrease in the permittivity of the organic phase. Our results suggested that the adsorption of SDS molecules was affected by the permittivity of the organic phase.