The fluorescence spectral characteristics and the interaction of several water-soluble metal complexes of Schiff-base with DNA are described. Among the complexes tested, Mn-Schiff-base bound to DNA showed a marked decrease in the fluorescence intensity with a blue shift of the excitation and emission peaks. Some hypochromism in the UV absorption spectra was also observed. KI quenching and competitive binding to DNA between Mn-Schiff-base and ethidium bromide (EB) were studied in connection with other experimental observations to show that the interactive model between Mn-Schiff-base and DNA is an intercalative one. The pH and salt effect on the fluorescence properties was also investigated. The linear relationship between F/F0 and the concentration of calf thymus DNA covers 3.0 × 10-6 - 2 × 10-4 mol L-1, which can be utilized for determining traces of calf thymus DNA with a detection limit of 8.0 × 10-7 mol L-1 in base pairs.
It has been proposed that acyl adenylate is first formed during activation of the carboxy group into the acyl CoA thioester, an intermediate in the formation of amino acid conjugates. Acyl CoA synthetases may be responsible for this acyl adenylate formation. Recently, we hypothesized the preferential formation of cholic acid adenylate, a major bile acid, preceding production of the corresponding CoA thioester in incubations with rat liver microsomal fractions. To verify this biosynthetic mechanism, monitoring of the incubation mixture of acyl adenylate together with both substrate and acyl CoA thioester is needed. We have developed a detection method for the simultaneous detection of these cholic acid derivatives utilizing liquid chromatography/electrospray ionization mass spectrometry. The CoA thioester of cholic acid forms a chelation complex with the divalent cations remaining on the silica gel packed into the analytical column. Both the addition of a chelating agent, such as EDTA, to the mobile phase and an adjustment of the mobile phase pH to a weak alkaline effectively removed such chelate formation, producing a sharp CoA thioester peak. For a simultaneous mass spectrometric analysis of cholic acid, the corresponding adenylate and CoA thioester, the combined use of a 300 Å particle diameter ODS column and 20 mM ammonium acetate buffer (pH 9.0)/2-propanol/acetonitrile as the mobile phase have been proved to be preferable. To avoid any degradation of the chemically unstable adenylate produced in the incubation, we employed a direct injection of the sample onto a preconcentration column. The obtained results indicated a high sensitivity of this method.
A novel cetylpyridinium chloride-selective membrane sensor consisting of cetylpyridinium-ferric thiocyanate ion pairs dispersed in a PVC matrix placticized with dioctylphthalate is described. The electrode shows a stable, near-Nernstian response for 1 × 10-3 - 1 × 10-6 mol l-1 cetylpyridinium chloride (CPC) at 25°C over the pH range 1 - 6 with a cationic slope of 57.5 ± 0.4. The lower detection limit is 8 × 10-7 mol l-1 and the response time is 30 - 60 s. Selectivity coefficients for CPC relative to a number of interfering substances were investigated. There is negligible interference from many cations, anions and pharmaceutical excipients; however, cetyltrimethylammonim bromide (CTMAB) interfered significantly. The determination of 0.5 - 350 µg/ml of CPC in aqueous solutions shows an average recovery of 98.5% and a mean relative standard deviation of 1.6% at 56.0 µg/ml. The direct determination of CPC in Ezafluor mouthwash gave results that compare favorably with those obtained by the British Pharmacopoeia method. Precipitation titrations involving CPC as titrant are monitored with a CP sensor. The CP electrode has been utilized as an end point indicator electrode for the determination of anionic surfactants in some commercial detergents.
A PVC-membrane electrode based on a recently synthesized 18-membered macrocyclic diamide is presented. The electrode reveals a Nernstian potentiometric response for Co2+ over a wide concentration range (2.0 × 10-6 - 1.0 × 10-2 M). The electrode has a response time of about 10 s and can be used for at least 2 months without any divergence. The proposed sensor revealed very good selectivities for Co2+ over a wide variety of other metal ions, and could be used over a wide pH range (3.0 - 8.0). The detection limit of the sensor is 6.0 × 10-7 M. It was successfully applied to the direct determination and potentiometric titration of cobalt ion.
A modified platinum-disk electrode coated with a non-plasticized polyacrylamide (PAA) membrane was used to study electrochemically an enzymatic reaction between tyrosinase in the PAA membrane and catechol and 3,4-dihydroxytoluene in acetonitrile (AN). Tyrosinase, a hydrophilic biofunctional material, was immobilized in the thin PAA membrane, which adhered to the platinum-disk electrode and was stable in AN. The enzymatic activity of tyrosinase in the PAA membrane to the above substrates in AN was confirmed by cyclic voltammetry and amperometry. The apparent maximum velocities (Vmaxapp) and the apparent Michaelis constants (Kmapp) were determined from the amperometric results; the apparent turnover numbers were also determined. The reduction potentials of the substrates were reported vs. the cathodic peak potential of ferrocene in AN to improve the reliability of the potential data and to make possible a comparison of the potentials in different solvents. The electrochemical system discussed in this report can be used for tracing enzymatic reactions with substrates dissolving in aprotic dipolar solvents and for investigating solvent effects on enzymatic activities.
In this study an atomic absorption spectrophotometer equipped with a selenium hollow-cathode lamp was used for analysis of BTEX (benzene, toluene, ethylbenzene and xylenes) in headspace of aqueous solutions. Initially effective factors on headspace such as volume of solution, stirring time, stirring speed, velocity of carrier gas, temperature, number of strippings, addition of salts and salt concentration were investigated and optimum conditions were selected. By addition of salt in different concentrations, different absorbances were obtained for headspace, therefore, binary mixtures of BTEX were analyzed with simultaneous equations. Obtained results agreed with actual amounts and repeatability was very good (RSD% <3). Correlation coefficients (r) for calibration curves were about 0.999. This proposed method is comparable with absorbance determination of solution with respect to correlation coefficient, linear dynamic range, limit of detection (LOD) and relative standard deviation (RSD), but this method is less susceptible to interferences and more selective.
A sensitive method involving alkaline hydrolysis and HPLC analysis has been developed to detect nanomolar levels of tryptophan dissolved in freshwater. The procedure includes 1) alkaline hydrolysis with nitrogen atmosphere in Teflon-lined Pyrex tubes with 4.2 M sodium hydroxide at 110°C for 16 h, in which ascorbic acid was used as an antioxidant; 2) a determination step, in which tryptophan was separated efficiently from other amino acids by HPLC and measured by fluorescence. The mean recovery for tryptophan standard was 91.4% with an RSD of 3.3%. Using this method, dissolved tryptophan concentrations ranged from 10.91 to 41.83 nM in water samples from Lake Biwa, Japan.
A method is described for the determination of total arsenic by hydride generation-atomic absorption spectrophotometry using a mixed acid as a pretreatment. Hydride generation is done by the flow-injection method. The authors investigated in detail the temperature and time of decomposition using inorganic, organic arsenic and environmental standard samples, pretreated with nitric-perchloric-sulfuric mixed acid. By using a mixed acid as a pretreatment agent at 220°C, the decomposition time could be shortened and the blank value of arsenic from the reagents used was reduced. The mixed acid of nitric-perchloric-sulfuric was also found to be effective as a pretreatment agent for organic arsenic compounds in which a dimethylated compound, sodium cacodylate or biological samples, is known to be one of the indecomposables. The present approach was proved to be satisfactory as a pretreatment for the quantitative analysis of trace amounts of total arsenic in liquid or solid environmental samples, such as geothermal water, sediments and biological samples.
A flow injection on-line preconcentration-graphite furnace atomic absorption spectrometric method was developed for the determination of traces of cadmium in natural water samples. Cadmium in samples was adsorbed on an iminodiacetate-type chelating resin, Muromac A-1 microcolumn (3 mm i.d. and 10 mm long), and then eluted with 2 mol l-1 HNO3. The eluate was introduced into the injection tip of an autosampler. The eluate zone with the highest analyte concentration was injected into the graphite furnace by cooperation of a peristaltic pump and a syringe pump of the autosampler, which were controlled by a programmable controller. The present system was successfully applied to the determination of cadmium in natural water samples. A detection limit of 0.2 ng l-1 was obtained with 12 ml sample loading. The recoveries were 99 and 108% for tap water (4 ml loading) and underground water (12 ml loading), respectively. Analytical results obtained for a river water reference material (JAC-0031, Japan Society for Analytical Chemistry) were close to the reference value.
The high-resolution emission spectra measured using a microscope Raman spectrometer and direct-inlet electron ionization mass spectrometry (DI-EIMS) were studied for the rapid analysis of a trace amount of various europium β-diketonates in order to utilize them as fluorescent markers. Five kinds of europium β-diketonates were distinguished from the emission spectra obtained using the Raman spectrometer. For DI-EIMS, the electron ionization mass spectra of the europium β-diketonates showed the characteristic fragment ion peaks due to the isotope effect of 151Eu and 153Eu. The DI-EIMS was very useful for identifying the compounds compared with spectrofluorometry. The scan and selected ion-monitoring modes of DI-EIMS showed the detection limits of the samples at levels of hundreds and tens of nanograms (ng), respectively.
Immobilized enzyme columns have been developed for use as high-performance liquid chromatographic enzyme reactors. Enzyme reactors were prepared by immobilizing trypsin or cytochrome-c on phospholipid columns. Dynamic coating was employed to prepare the reactors by recycling a buffer solution containing trypsin or cytochrome-c through a phospholipid-coated column, on which the enzymes were immobilized by hydrophobic binding. The immobilized trypsin column displayed hydrolytic activity which catalyzed the hydrolysis of L-amino acid esters to amino acid. The immobilized cytochrome-c column exhibited oxidation activity which catalyzed N-demethylation of N,N-dimethylaniline, codeine, and dihydrocodeine in the presence of hydrogen peroxide as an oxygenating agent. The enzyme reaction proceeded rapidly in the column; both product and substrate could be separated and detected simultaneously. The immobilized enzyme columns could be readily regenerated using the original phospholipid column by repeating the dynamic coating. These immobilized enzyme columns could be utilized as enzyme reactors in the high-performance liquid chromatographic mode. Complete hydrolysis of amino acid ester was observed with the trypsin column. Demethylation of codeine and of dihydrocodeine were observed with the cytchrome-c column.
The absorption spectra of the praseodymium complex with 1-cyclopropyl-6-fluoro-1,4-dihydro-7-(4-ethyl-1-piperazinyl)-4-oxo-3-quinoline carboxylic acid hydrochloride (NNFX) has been studied by normal and derivative spectrophotometry. The complex showed maximum absorption at 350 nm at pH 6.0. The stoichiometry of the Pr-NNFX complex was calculated by the molar ratio and continuous variation methods. The ratio of Pr to NNFX was 1:3. The absorption bands of the 4f electron transitions of the praseodymium complex with NNFX are enhanced markedly, especially the wavelength at 481 nm. Using the third-derivative spectrum, the calibration graph is linear over the range 2.5 × 10-5 - 3.5 × 10-4 mol dm-3 for praseodymium. The detection limits (signal-to-noise ratio of 2) is 1.4 × 10-6 mol dm-3. The relative standard deviation is 1.2% for 7.0 × 10-5 mol dm-3 of praseodymium. A method for the direct determination of praseodymium in rare earth mixtures with good accuracy and selectivity is described.
Based on a new electrogenerated chemiluminescence (ECL) analytical idea, this paper explains a sensitive and selective flow-injection ECL method using luminol for the determination of isoniazid, based on the sensitizing effect of isoniazid for the weak ECL emission of electrochemically oxidized luminol. Under the optimum experimental conditions, the relative ECL intensity was linear with isoniazid concentration in the range of 4.0 × 10-8 mol/L to 8.0 × 10-6 mol/L and with a detecting limit of 2.8 × 10-8 mol/L.