I. F. Dolmanova

Assay of enzyme effectors

The effects of various classes of organic compounds and of metal ions on the catalytic activity of horseradish peroxidase in hydrogen peroxide-catalysed o-dianisidine oxidation and, on the activity of alkaline phosphatase in p-nitrophenyl phosphate hydrolysis have been studied. Enzymic methods have been developed for determination of sulphur compounds at 10(-5)-10(-4)M, nitrogen compounds at 2 x 10(-7)-3 x 10(-5)M mercury at 3 x 10(-7) mu/ml and lead at 6 x 10(-4) mu/ml concentration.

Enzymic method for the determination of ethanol and methanol with spectrophotometric detection of the rate of the process

An enzymic method for the determination of ethanol and methanol and for the determination of methanol in water–ethanol solutions is described. The reaction of alcohol oxidation catalysed by alcohol oxidase conjugated with the indicator reaction of p-phenylenediamine oxidation with hydrogen peroxide was chosen for this determination. The method proposed shows reasonable limits of detection (9 µ mol l–1 for ethanol and 0.9 µ mol l–1 for methanol). The method is simple, rapid and sensitive. A test method for ethanol and methanol identification is also described.

Bioluminescent assay of creatine kinase activity using immobilized firefly extract

A bioluminescent method has been developed for creatine kinase (CK) assay using immobilized firefly extract containing the bioluminescent coimmobilized system: adenylate kinase + luciferase. ADP for the reaction with CK was produced from the initial mixture of AMP and ATP. The ATP formed in the reaction with CK was quantified using firefly luciferase. The lowest detection limit for CK activity was 0.5 +/- 0.2 U/liter in the sample. A linear range of the determined CK activities was 0.5-1000 U/liter. The correlation coefficient between the bioluminescent and spectrophotometric methods was 0.981 (n = 40). The use of immobilized firefly extract for analysis has been shown to be advantageous compared to soluble enzyme.

Sorption–catalytic testing of copper on a paper-based sorbent with attached alkylamino groups

Copper(II) was preconcentrated from aqueous solution on paper filters with chemically attached hexamethylenediamino (HMDA) groups and determined directly on the filters by its catalytic action in the oxidation of hydroquinone with H2O2 in the presence of 2,2′-dipyridyl; the reaction was monitored by measuring the absorbance of wet filters. The linear calibration graph of absorbance at 5 min versus log cCu permits the semiquantitative determination of CuII over the range 1 × 10–6–0.1 mg l–1 for a sample volume of 10 ml. Tolerance ratios for foreign ions are 3–4 orders of magnitude more favorable in the hybrid sorption–catalytic procedure than those for the catalytic determination of copper in solution without preconcentration. The procedure is simple and does not require any expensive instrumentation. Samples of tap and sea-water were analyzed.

Enzymatic method for determination of organomercury in sea water

Abstract The enzymatic determination of organomercury compounds (methyl-, ethyl-, phenyl-mercury) is based on their effect on the induction period (τind) caused by the introduction of sodium diethyldithiocarbamate to the oxidation of o-dianisidine, o-phenylenediamine and 3, 3′, 5, 5′-tetramethylbenzidine by H2O2 catalysed by native horseradish peroxidase. τind is inversely proportional to organomercury compounds concentration over a range of 0.05–10 μM. The lowest detection limit (Cmin) is 0.03 μM and the standard relative deviation (RSD) is lower than 3%. The proposed method is simple, inexpensive and does not require the preliminary conversion of organomercury compounds to elemental or ionic mercury. The developed procedure is applied successfully to methylmercury determination in water of Kara Sea.

Accelerating effect of silica on the indicator reaction o-dianisidine–H2O2

Reaction of oxidation of o-dianisidine (o-D) with H(2)O(2) which is widely used in catalytic methods of analysis in solution has been conducted on silica plates for thin-layer chromatography. The rate of the reaction catalyzed by model compounds (p-toluenesulphonyl chloride, methyl benzoate, benzoic acid, and acrylamide) is noticeably higher on silica than in solution in comparable conditions. The degree of acceleration varies depending on the catalyst and is more pronounced at its lower concentrations. By use of p-toluenesulphonyl chloride determination as an example it has been shown that the accelerating effect of silica enables to decrease the detection limit down to 0.07 nmol cm(-2) (as compared with 4 nmol.cm(-2) in solution); the accuracy is not diminished. It is concluded that catalytic indicator reactions on solid supports may represent high interest for analytical chemists.

Determination of copper by its catalytic effect on the oxidation of hydroquinone by hydrogen peroxide on supports

The reaction of oxidation of hydroquinone by hydrogen peroxide catalyzed by copper(II) in the presence of 2,2’-dipyridyl is activated by hexamethylenediamino groups that are bonded to the surface of filter paper; additional activating effect is produced by 2,2′-dipyridyl. The introduction of malonic acid dinitrile into the indicator reaction improves the sensitivity of the determination of copper and the contrast of the reaction in solution because of the formation of a blue product. Differently colored compounds are formed on a paper support at different concentrations of copper, which makes it possible to visually distinguish the quantities of copper that differ by an order of magnitude in the range 1 × 10-5-0.5 µg. Quantitative detection is possible in the range 5 × 10-6-0.1 µg (cmin = 3 × 10-6 µg). The concentration of copper (0.2-1.5 µg/mL) is determined in blood serum; the consumption of the sample per one determination is 3 µL

Adsorption-catalytic test methods

In the development of a new hybrid adsorption–catalytic method it was shown that the selective adsorption isolation of components can be combined with their catalytic determination directly on the adsorbent. To provide theoretical grounds and find fields of the application of the new method, the effect of the support matrix on the rate of catalytic (enzymatic and indicator) reactions was studied. The changes in the performance characteristics of procedures for determining inorganic and organic substances directly on the adsorbent with respect to those of procedures for determining in solutions were analyzed. It was proved that the development of catalytic test procedures with the visual detection of the analytical signal is expedient.

Enzymatic Methods for the Determination of Mercury(II)

Enzymatic methods for the determination of mercury(II) using native and immobilized enzymes from different classes and sources were considered. Enzymatic procedures were compared in sensitivity, selectivity, rapidity, and experimental techniques. The main approaches to controlling the performance characteristics of the enzymatic procedures were discussed. The application of these procedures to the determination of mercury(II) in particular samples (water and soils of different origin and biological fluids) was considered.

Adsorption–Catalytic Determination of Metal Inhibitors

(reflectance spectrometry, RSD 10%) is performed immediately after the preconcentration of their colored complexes in the dynamic mode (at P = 10–20 mm Hg). We used the colored vanadium(IV) complex of Sulfonitrophenol M (pH 3.5, 650 nm), vanadium(V) complex of Sulfonitrophenol M and hydroxylamine (mixed-ligand complex, 650 nm, pH 1.5), and mercury(I, II) complexes of tyrodine in the presence of carboxyl ions (580 nm, pH 3) at the surface of a polycaproamide membrane; the diameter of the membrane was 10 mm, b = 0.1 mm, and m = 2.7 mg. The detection limits are 0.2–0.5 ng/mL (vanadium) and 4 ng/mL (mercury). The determination of vanadium(V) at pH 1.5 is not affected by the interference from 20-fold amounts of vanadium(IV) or significant amounts of Ni, Co, Zn, Cd, Cr(III), Mn, , and F – ; the determination of mercury is not affected by the interference from V, Pb, Cd, Zn, Cu, Ni, and .