Chromogenic Agent Research Articles

Helicobacter pylori mutagenesis by mariner in vitro transposition.

We have developed a method for generating transposon insertion mutants using mariner in vitro mutagenesis. The gene of interest was PCR-amplified and cloned. A kanamycin-marked mariner transposon was randomly inserted into the purified plasmid in an in vitro transposition reaction. After repair and propagation in Escherichia coli, purified mutagenized plasmid was introduced into Helicobacter pylori by natural transformation. Transformants were selected by plating on kanamycin. Mutants were predominantly the result of double homologous recombination, and multiple mutants (with insertions in distinct positions) were often obtained. The site of insertion was determined by PCR or sequencing. We have made mutations in known or potential virulence genes, including ureA, hopZ, and vacA, using kanamycin- and kanamycin/lacZ-marked transposons. Colonies carrying a kanamycin/lacZ transposon appeared blue on medium containing the chromogenic agent X-gal, allowing discrimination of mutant and wild-type H. pylori in mixed competition experiments.

Helicobacter pylori mutagenesis by mariner in vitro transposition

We have developed a method for generating transposon insertion mutants using mariner in vitro mutagenesis. The gene of interest was PCR-amplified and cloned. A kanamycin-marked mariner transposon was randomly inserted into the purified plasmid in an in vitro transposition reaction. After repair and propagation in Escherichia coli, purified mutagenized plasmid was introduced into Helicobacter pylori by natural transformation. Transformants were selected by plating on kanamycin. Mutants were predominantly the result of double homologous recombination, and multiple mutants (with insertions in distinct positions) were often obtained. The site of insertion was determined by PCR or sequencing. We have made mutations in known or potential virulence genes, including ureA, hopZ, and vacA, using kanamycin- and kanamycin/lacZ-marked transposons. Colonies carrying a kanamycin/lacZ transposon appeared blue on medium containing the chromogenic agent X-gal, allowing discrimination of mutant and wild-type H. pylori in mixed competition experiments.

Determination of trace amounts of cadmium with p-acetyl-benzenediazoaminoazobenzene by primary-secondary wavelength spectrophotometry

The reaction between Cd2+and a chromogenic agent, p-acetylbenzenediazoaminoazobenzene (p-ABAA) at pH 12.5 is sensitive and selective in the presence of triethanolamine and citrate. The solubilization and sensitization of the OP emulsifier (nonionic surfactant) was observed in the above reaction. Because the absorption of excess p-ABAA interferes with the absorption of the complex, primary–secondary wavelength spectrophotometry (PSWS) was used instead of the single-wavelength method in the determination of trace amounts of cadmium. Results showed that this method gave out a higher precision and better accuracy than the single-wavelength method, because two wavelengths that are located at the peak absorption and the valley absorption of the reaction solution were used simultaneously. By analyzing samples, the relative standard deviations were less than 8% and the recovery between 96.5 and 111%.

Simultaneous spectrophotometric determination of La, Ho, Mn 5-Br-PADAP complexes using multivariate calibration with partial least-squares (PLS) data evaluation.

A simple and fast analytical pocedure is proposed for the simultaneous spectrophotometric determination of lanthanum, holmium and manganese in synthetic ceramics, (La(0.8-x) Hox Sr0.2 MnO3), by using the partial least-squares (PLS) method. As chromogenic agent 5-Br-PADAP [2-(5-bromo-2-pyridylazo)-5-diethylaminophenol] was used, which form colored complexes with the three elements studied. To avoid metal hydrolysis, a mixture of ethanol and Triton X-100 at pH 9.5 was used for all experiments. A set of 17 calibration solutions measured throughout the 400-700 nm wavelength range was used in the calibration step. The concentration range for Mn(II) was 1-12 x 10(-6) mol L-1, while the range for the rare earth elements La(III) and Ho(III) was 2-8 x 10(-6) mol L-1. In order to demonstrate the applicability of the proposed method, a set of artificial samples containing the three analytes in variable proportions was prepared and analyzed. The analytical results obtained were quite acceptable with relative errors not greater than 7% in most cases.

Spectrophotometry Determination of Ciprofloxacin in Serum Using Iron(III) Ion as Chromogenic Agent

ABSTRACT An analytical procedure for the determination of ciprofloxacin in serum without previous extraction has been developed. The determination was carried out using iron(III) nitrate as chromogenic agent, with the addition of sodium dodecylsulfate, at pH = 3.0. Absorbance was measured at 430 nm. The range of linearity was between 0.5 – 20.0 μg/mL with a detection limit 0.2 μg/mL.

Separation of Palladium by Extraction Chromatographic Technique Using Cyanex 471X (Tri Isobutyl Phosphine Sulfide) on Chromosorb-102

An extraction chromatographic procedure for the recovery of fission product palladium has been developed. The method utilizes Cyanex 471X (tri isobutyl phosphine sulfide, TIPS) impregnated on an inert support, Chromosorb-102. Using this method, quantitative uptake of the metal ion is achieved from nitric acid medium. Thiourea as a suitable stripping agent is suggested. Palladium was estimated spectrophotometrically using Arsenazo III as the chromogenic agent and incorporating a modification in the procedure when the aqueous medium contained thiourea.

Determination of Uranium and Plutonium in High Active Solutions by Extractive Spectrophotometry

Plutonium and uranium was extracted from nitric acid into trioctyl phosphine oxide in xylene. The TOPO layer was analysed by spectrophotometry. Thoron was used as the chromogenic agent for plutonium. Pyridyl azoresorcinol was used as chromogenic agent for uranium. The molar absorption coefficient for uranium and plutonium was found to be 19000 and 19264 liter/mole-cm, respectively. The correlation coefficient for plutonium and uranium was found to be 0.9994. The relative standard deviation for the determination of plutonium and uranium was found to be 0.96% and 1.4%, respectively.

Synthesis and analytical applications of 4-aminopyrazolone derivatives as chromogenic agents for the spectrophotometric determination of phenols

The synthesis of simple, extensively conjugated chromophores as substitutes for 4-aminoantipyrine (4-AAP) capable of causing hyperchromic shifts when coupled with phenols is described. The 4-aminopyrazolone derivatives synthesised are subsequently applied for the spectrophotometric assay of phenolic compounds and the advantages of each of the proposed systems are highlighted. No improvement is made on the detection of para-substituted phenols as well as polychlorophenols (e.g. pentachlorophenol). An increase in the sensitivity of coupling products is obtained for certain phenolic compounds as indicated from the comparison of the individual responses. The proposed methods have analogous analytical features and in some cases show considerable improvements compared to the 4-AAP method. Beer’s law is obeyed over a wide dynamic range (up to ca. 500 μg/l) and the relative standard deviations are <3.4%. The methods are applied satisfactorily for phenol assay in real samples, however, taking into account the intrinsic differences of the methods in the responses to the various phenolic compounds.

Study on reactivation of enzyme-inhibitor complexes by oximes using acetylcholine esterase inhibited by organophosphate chemical warfare agents

Reactivation by oximes was studied by using acetylcholine esterase (ACHE) immobilized on a cotton cloth and inhibited by organophosphate chemical warfare agents. The activity of ACHE was estimated by continuous measuring the remission changes of the cloth surface with immobilized ACHE by using the Ellman’s method with acetylthiocholine iodide as substrate and 5,5′-dithiobis(2-nitrobenzoic) acid as the chromogenic agent. The stable enzymatic chimera formed by immobilization of ACHE on the cotton cloth enables a simple isolation of the enzyme-inhibitor complex from the studied medium, in this case solutions of inhibitors and reactivators. ACHE, immobilized on cotton cloth can be recommended as a simple in vitro model for ACHE bound to cell membrane in in vivo experiments. The results obtained in this study with oximes and immobilized ACHE-organophosphate chemical warfare agents enzyme-inhibitor complexes are in accord with the published facts concerning inhibition of ACHE by organophosphate chemical warfare agents and reactivation of enzyme-inhibitor complexes by oximes. The mentioned procedure enables a simple evaluation of the reactivation effect of oximes on a given enzyme-inhibitor complex. No significant differences were found between ACHE from bovine and human tissue nucleus caudatus.

Determination of trace amounts of manganese by β-cyclodextrin polymer solid phase spectrophotometry using 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol

A sensitive and selective solid phase spectrophotometric method for the determination of trace amounts of manganese has been described. The method is based on the inclusion of the chromogenic agent, 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP) in β-cyclodextrin polymer (β-CDP). The resin can adsorb manganese to form a colored complex with a maximum absorbance of 558 nm. The working range of the calibration graph was 0–8 μg of manganese. The interference from cadmium, lead, cobalt, molybdenum, chromium, iron, nickel, zinc, tin and copper that also form colored species with 5-Br-PADAP in the resin phase was investigated. The method is useful for the determination of manganese in black rice and tea.

Post-column oxidation of purpald–aldehyde adducts at nickel electrodes

Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole), a chromogenic agent for the detection of aldehydes after TLC has been studied as a possible post-column derivatizing agent for improving the detectability of aldehydes after HPLC separation. In the first step of a two step reaction, Purpald forms a colorless adduct with aldehydes. Subsequent oxidation of the colorless adduct by air or other means yields a highly colored compound in the second step. The oxidized Purpald–aldehyde adduct is deep purple in color and has an absorption maximum at 550 nm. An on-line post-column electrochemical reactor has been investigated as a possible alternative to air or chemical oxidation for the analysis of aldehydes using HPLC. The method was shown to form colored adducts in bulk solution and on-line using electrochemical oxidation at nickel electrodes for formaldehyde, acetaldehyde, and propionaldehyde. There was a marked increase in reaction time in bulk solution as the molecular mass of the aldehyde increased. Thus, when performing chromatography using on-line electrochemical oxidation the sensitivity of the method decreased significantly as the aldehyde molecular mass increased. Reactor design, electrode configurations, electrode potentials, and optimum reaction conditions are described.

Spectrophotometric Assay of Some Antipsychotropic and Anticholinergic Phenothiazine Drugs Using Ammonium Molybdate

A simple, rapid and sensitive spectrophotometric method has been described for the quantitative determination of six phenothiazine-based antipsychotropic and anticholinergic drugs in pure form as well as in formulations. The method is based on the formation of stable phenothiazonium free radical cation by the use of ammonium molybdate as chromogenic agent. The drugs in 3.0 to 5.0M H2SO4 medium when heated in a boiling water bath react with molybdate to give intense orange or purple or blue products which possess characteristic absorption maxima at 500–638 nm. Beer's law is obeyed over the concentration range 1–60 μg.ml−1 with apparent molar absorptivities ranging from 4.37x103-9.61 × 103 l. mol−1. The proposed method has been applied successfully for the determination of examined drugs both in pure form and representative pharmaceutical formulations. The accuracy and precision of the assay method are comparable with those of the official method.

Spectrophotometric Determination of Piroxicam and Tenoxicam in Pharmaceutical Preparations Using Uranyl Acetate as a Chromogenic Agent

New simple spectrophotometric methods for the determination of piroxicam, and tenoxicam in pharmaceutical preparations based on the reaction of piroxicam, and tenoxicam with uranyl acetate have been developed. The reagent forms yellow 1:2 complexes with piroxicam and tenoxicam in alcoholic medium. The complexes are stable for more than 24 hrs. The coloured species have absorption maxima at 395 and 435 nm, with molar absorptivities of 3.8 × 103 and 0.21 × 103 1mole−1 cm−1, and the mean percentage recoveries for authentic samples were 99.11±0.79, 98.78 ± 0.62 for piroxicam and tenoxicam, respectively. The site of chelation is given, and discussed.

L-Alanine fermentation by an alanine racemase-deficient mutant of the dl-alanine hyperproducing bacterium Arthrobacter oxydans HAP-1

Arthrobacter oxydans HAP-1 produces a large amount of dl-alanine from glucose via the reductive amination of pyruvate catalyzed by l-alanine dehydrogenase (ALD). This bacterium was found to be capable of growing on either d- or l-alanine as a sole carbon source with lower growth rates than on glucose. The effects of an alanine racemase (AR) inhibitor on the accumulation and/or consumption of d- or l-alanine by the bacterium suggested that d-alanine was formed by the isomerization of l-alanine and degraded through the reverse reaction. d-Alanine non-utilizing mutants were derived from strain HAP-1 by the staining method using the chromogenic agent 2,3,5-triphenyl tetrazolium chloride. The mutants were obtained were of two kinds: ALD-deficient and AR-deficient. The former lost the ability to grow on both d- and l-alanine, indicating that the ALD of strain HAP-1 functions not only in l-alanine synthesis but also in l-alanine degradation. The latter could utilize l-alanine as a carbon source but not d-alanine; furthermore, they required d-alanine for growth in minimum or nutrient medium. A representative AR-deficient mutant, DAN 75, produced 75.6 g of l-alanine per liter with high optical purity (97% e.e.) in a fed-batch cultivation (glucose was fed to 15% in total) using a jar-fermentor. The l-alanine titer was almost equal to the sum of d- and l-alanine produced by the parental strain. The production rate of DAN 75, as well as that of the parental strain HAP-1, slowed down in the late stage of the cultivation. Examination of the influence of exogenously added alanine revealed this reduced production to be due to a deceleration in the glucose consumption rate arising from alanine accumulated in the medium.

Expression of the arylsulphatase reporter gene under the control of the nit1 promoter in Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii, the expression of the nit1 gene encoding nitrate reductase is dependent on the nature of the nitrogen source and on other environmental factors. We have fused the nit1 promoter region to the arylsulphatase (ars) reporter gene lacking its own promoter and introduced this chimeric construction (nit1/ars) into a wall-less strain of C. reinhardtii. A new and sensitive method, based on the use of alpha-naphthylsulphate as a substrate and a diazonium salt as a chromogenic post-coupling agent, was developed to detect the activity of arylsulphatase (an enzyme which is almost completely secreted in the culture medium) both in vitro and in agar plates. The transformants carrying nit1/ars did not express arylsulphatase when grown in ammonium-sufficient medium but readily accumulated the enzyme in ammonium-free medium either supplemented, or not supplemented, with nitrate or nitrite. The nit1/ars construct, however, was not expressed in the nit2 mutant lacking a specific transcription regulator controlling the expression of nit1. These results, together with the observation that the transcription of nit1/ars is initiated at the same sites as the nit1 endogenous gene, confirms the hypothesis that the regulation of nit1 expression takes place mainly at the transcriptional level. The expression of the ars gene from the nit1 promoter was high enough to allow direct measurements of arylsulphatase activities in pools of transformants without prior isolation of nit1/ars clones. This original procedure has permitted the analysis of the effects of nested deletions in the nit1 promoter region on the expression of the reporter gene. The results indicate that the -282 to -198 sequence is required for transcription to occur and that the -751 to -282 region contains several elements mediating nit1 expression.

The Application of Partial Least Squares Method (PLS) for Simultaneous Spectrophotometric Determination of Calcium and Magnesium in Human Serum

Summary Application of a partial least squares algorithm (PLS-1) is proposed in this work for simultaneous spectrophotometric determination of calcium and magnesium in human serum. Two chromogenic agents, Calmagite [1-(1-hydroxy-4-methyl-2-phenylazo)-2-naphtol-4-sulfonic acid] and o-Cresolphtalein [3′,3″-bis{[bis-(carboxymethyl)amino]methyl}-5′5″-dimethylphenolphtalein] were used, which form color complexes with the two elements studied. As expected, significant interferences were observed for the individual methods (Ca-o-Cresolphtalein and Mg-Calmagite) in the presence of the second metal. However, applying a full-spectrum chemometric method PLS-1, it was possible to eliminate these interferences in the calibration step and satisfactory prediction results were obtained for calcium and magnesium in real serum samples. The proposed procedure was validated using Refererence Serum (171760 Boehringer Mannheim, Germany). The analytical results obtained for human serum were compared with the results of AAS determination.

Low-Temperature Phosphorimetric Determination of Traces of Palladium(II) with Water-Soluble Coproporphyrin III

Abstract The characteristics of coproporphyrin III (Copro III) as a chelating agent, a chromogenic agent, and a luminescence agent for metal ions were studied. Copro III forms 1 : 1 complexes with Co(II), Cu(II), Mn(II), Ni(II), Pd(II), and Zn(II). Copper, palladium, and zinc complexes exhibit phosphorescence at liquid nitrogen temperature (77 K), and the intensity of the palladium complex (λPdmax = 659 nm) is 10 and 13 times larger than that of the copper and zinc complexes (λCumax = 685 nm, λZnmax = 702 nm), respectively. Based on these facts, a low-temperature phosphorimetric determination of the 10−8 mol dm−3 level of palladium was developed. The detection limit (S/N = 3 using a 150 W xenon arc lamp as a light source) was 8 × 10−9 mol dm−3, and the relative standard deviation was 2.8% for 6 × 10−7 mol dm−3 Pd(II) (7 determinations). Interference from many metal ions was avoided by selective extraction of palladium(II) as an ion-pair of the bromo complex with H2SO4-tetrabutylammonium bromide-methyl isobutyl ketone systems, followed by back extraction with acetate buffer solution (pH 5.0).

Separation of Cadmium Salicylate by Solvent Extraction Using Tris(2-ethylhexyl) Phosphate

Abstract Tris(2-ethylhexyl) phosphate (T2EHP) is used for the extraction of cadmium salicylate. The experimental conditions are established and the probable extractable species is ascertained by log D–log C plots. Microgram amounts of cadmium are determined in the Tris(2-ethylhexyl) phosphate phase with 1-(2-pyridylazo)-2-naphthol as the chromogenic agent. The proposed method is used for the separation of cadmium(II) from zinc(II), mercury(II), lead(II), bismuth(III), antimony(III), and thallium(III) and used for the estimation of cadmium in industrial waste water, kidney stone, cigarettes, and alloy samples.

Statistical Analysis of Spectrometric Procedures for Determination of Fe(III) and V(V) with Desferrioxamine B

Abstract For statistical evaluation of VIS-spectrometric procedures for determination of Fe(III) and V(V), with desferrioxamine B as a chromogenic agent, the slightly modified Gottschalk concept is used and found to be appropriate. Favorable metrological characteristics of both analytical procedures make them likely candidates for standard quantitative protocols.

Flow-injection spectrophotometric determination of trace iron in various salts. Elimination of blank peak effect and use of 2-(5-nitro-2-pyridylazo) -5-( N-propyl- N-sulfopropylamino) phenol as chromogenic agent

A highly sensitive flow-injection method is presented for the rapid and simple determination of iron in various salt samples. 2-(5-Nitro-2-pyridylazo)-5-( N-propyl-N-sulfopropylamino)phenol (Nitro-PAPS) was used as the chromogenic reagent for the spectrophotometric detection at 582 nm, yielding the iron(II) complex having a high molar absorptivity of 8.5 × 10 4 1 mol −1 cm −1. A large volume sample injection with 5-m sample loop was employed in a flow-injection analysis (FIA) system, which was very effective for eliminating the refractive index problem that is often found and should be solved in the FIA of samples containing an excess of salts. A linear calibration was obtained for 0–100 ng ml −1 iron in the presence of excess of sodium chloride and the relative standard deviation was 1.3% for 50 ng ml −1 iron ( n = 5). The limit of detection is 1 ng ml −1 that corresponds to 0.05 μg ml −1 based on a solid sample when a 1 g sample is taken for analysis. Lower detection limits are attainable with larger samples. No significant interference was found by the ions commonly found in most salt samples. The proposed FIA system was successfully applied to “solar” salts and purified salts with satisfactory precision. The sample throughput is ca. 15 h −1.