Fluorometric determination of hydrogen peroxide using resorufin and peroxidase

A fluorometric method for the determination of hydrogen peroxide using resorufin as a substrate for peroxidase is described. Two procedures were developed for the determination of hydrogen peroxide. One involves the addition of hydrogen peroxide sample to a solution of peroxidase and resorufin in phosphate buffer, pH 6.4. Fluorescence measurements are performed before and after hydrogen peroxide addition. The within-run CVs for final concentrations of hydrogen peroxide of 200 and 40 nmol/liter were 1.7 and 7.6%, respectively, and the limit of quantitation was 9 nmol/liter. The second procedure, in which the initial reaction of hydrogen peroxide with resorufin is performed in citrate buffer at pH 4.5, and then the fluorescence is measured after the pH is adjusted to 9.2 with borate buffer, has a limit of quantitation of 4.4 nmol/liter with a within-run CV of 6.5% for a final hydrogen peroxide concentration of 20 nmol/liter. The method is linear at least up to 1 μmol/liter.     

FTIR Emission Spectra, Molecular Constants, and Potential Curve of Ground State GeO

Extensive new high-temperature, high-resolution FTIR emission spectroscopy measurements for the five common isotopomers of GeO are combined with previous diode laser and microwave measurements in combined isotopomer analyses. New Dunham expansion parameters and an accurate analytical potential energy function are determined for the ground X1Sigma+ state. Copyright 1999 Academic Press.     

Comparative chromogenicities and discrepant spectra obtained between a heterogeneous and homogeneous Zimmermann reaction

A spectrophotometric study is shown for two modifications of a 17-KS procedure in which nearly all aqueous reaction media were used. Although the apparent differences in matrices of reaction between the two procedures appear to be minute, a small volume of ethanol for one is substituted for the same volume of methanol in the other, the final alkalinity of the latter procedure is stronger, and the reaction temperature was increased, wide differences in spectral results are found between the two methods. The ethanolic, lower alkalinity procedure is a homogeneous, single-phase reaction while the methanolic, higher-alkalinity procedure is a heterogeneous system in which a precipitation of detergent occurs and the precipitate becomes an integral part of the color reaction. The rate of formation of the ethanolic procedure is variable for the different 17-KS but it is constant in the methanolic procedure. The chromogenicities of color formation between the two procedures differ in that the methanolic system provides uniform and predictable spectra with more equivalent molar absorptivities. Lastly, the methanolic system shows a much greater sensitivity owing, apparently, to a more complete final reaction. This inability to form full color for several compounds in the homogeneous system can be related in some way to the large variations in ability to form that color owing to matrix composition.     

Microdetermination of Xanthates

Summary A rapid, convenient and accurate method for the determination of xanthates has been developed. N-bromosuccinimide has been used to displace iodine from potassium iodide which in turn oxidizes xanthate to dixanthogen. The p_H 4 is optimum for the determination. The maximum relative standard deviation is 6% in the case of ethyl potassium xanthate when a 0.5 mg sample was taken.

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Zusammenfassung Ein genaues Verfahren zur raschen Bestimmung von Xanthat wurde entwickelt. N-Bromsuccinimid dient zur Freisetzung von Jod aus KJ; Jod oxydiert Xanthat zu Dixanthogen. Das optimale p_H für die Bestimmung ist 4. Die größte relative Standardabweichung ist 6% bei einer Einwaage von 0,5 mg Kaliumäthylxanthat.

     

Microdetermination of thiocyanates with N-Bromosuccinimide using bordeaux red as an indicator

Summary N-bromosuccinimide has been used as a direct titrant for thiocyanates. Bordeaux red has been used as an indicator. In case of ammonium thiocyanate, nitrogen is evolved. While in case of potassium thiocyanate 0.5- to 2-mg samples were analyzed and maximum relative standard deviation was 1% in case of 0.5-mg sample. In case of ammonium thiocyanate 0.3- to 2.5-mg samples were analyzed and maximum relative standard diviation was 0.9% in case of 0.7-mg sample. The method is quick, precise and accurate.

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Zusammenfassung N-Bromsuccinimid wurde als Maßlösung zur direkten Titration von Thiocyanat verwendet. Bordeauxrot diente hierbei als Indikator. Bei der Titration von Kaliumrhodanid betrug die relative Standardabweichung 2% (bei 0,5 mg Einwaage), im Falle von Ammoniumrhodanid 0,9% (bei 0,7 mg Einwaage).

     

Study and optimization of the resazurin/diaphorase system

This study describes the resazurin/diaphorase system and its use in a kinetic assay for the determination of dehydrogenase activity. This has increased the sensitivity (limit of detection is 2 × 10⁻⁵ U/ml) and produced calibration curves which have an extended linear range and better separation of points. The specific activity of diaphorase when resazurin (6.7 μM) is used as a substrate is only about 2 × 10⁻³ that when 2,6-dichlorophenol-indophenol (DCPIP)² is used as a substrate. The specific activity of diaphorase can be increased by using higher concentrations of resazurin. The concentration chosen is influenced by the size of fluorometric cuvette used. In a 3.0-ml cuvette, concentrations above 13.4 μM result in lower signals due to the quenching of the fluorescence by resazurin. In a 1.0-ml cuvette, the quenching effect is less severe and a higher concentration (34 μM) of resazurin can be used. Diaphorase activity is first order in the concentration range up to 34 μM of resazurin, and the K_m could not be calculated from the range tested. The K_m of diaphorase with respect to NADPH is dependent on the concentration of resazurin used. It is 0.78 (± 0.04) and 1.31 (± 0.04) μM at resazurin concentrations of 6.7 and 34 μM, respectively.     

Spectrophotometric study of drug interferences in an aqueous 17-ketosteroid reaction

A spectrophotometric study of two reported drug interferences in an aqueous Zimmermann reaction for 17-ketosteroids has been described. One drug, Tegretol, did not interfere spectrally with the 17-ketosteroid determination because its Zimmermann reaction spectrum was displaced far enough toward the ultraviolet so there was no significant superimposition of its spectrum with that of 17-ketosteroids. The other drug, Cephalothin, proved to be no problem analytically, because, even though it was Zimmermann-reactive, it was unextractable from acid hydrolyzed urines and therefore could not interfere in the color-forming step.     

Catalytic determination of iodine in serum at nanogram levels using the As(III)/Ce(IV) reaction

Summary A catalytic determination of iodine using the As(III)/Ce(IV) reaction has been developed. The difference in reaction rate due to catalysis by iodide was measured spectrophotometrically at 310 nm. Optimum reaction conditions were investigated in order to obtain a greater sensitivity in the determination of iodine. The method can be satisfactorily used for samples containing iodine in the 0.4 to 10.0 ng range, with an overall error less than 6 %. The method has been used for the determination of iodine in serum, and gives results which compare favorably with a present clinical chemistry procedure.

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Zusammenfassung Eine katalytische Jodbestimmungsmethode auf der Grundlage der As(III)/Ce(IV)-Reaktion wurde ausgearbeitet. Die durch Jodkatalyse bedingte Differenz des Reaktionsausmaßes wurde spektralphotometrisch bei 310 nm gemessen. Das Optimum der Reaktionsbedingungen für eine größtmögliche Empfindlichkeit der Jodbestimmung wurde ermittelt. Das Verfahren kann für Jodmengen zwischen 0,4 und 10,0 ng mit Erfolg verwendet werden; der durchschnittliche Fehler beträgt weniger als 6%. Jodbestimmungen im Serum nach dieser Methode stimmen mit klinisch-chemisch ermittelten Werten gut überein.

     

The determination of lecithin and total choline-containing phospholipids in amniotic fluid employing enzymes as reagents

Two assays which employ enzymes as reagents have been described for the determination of lecithin and TCCP in amniotic fluid. Both of these assays are relatively quick, simple, inexpensive, precise, involve no extractions, and are amenable to automation.Hemoglobin, blood cell membranes, and bilirubin, common interferences in $\text{L}\text{S}$ ratio determinations, have been studied. Their effects on these two assays appear to be negligible.It can be inferred from preliminary studies that the described assays correlate well with each other, and with the clinical status of the fetus. The values obtained for lecithin and TCCP cmncentrations are in the same range as previous reports employing different procedures.