Colored Azo Dye Research Articles

Photometric determination of nitrite

A sensitive colored reaction for nitrite determination is developed. It involves the reaction of the titled ion with p-aminobenzoic acid to form a diazonium ion, which is then coupled with α-naphthol in alkaline medium to form a pinkish red soluble azo dye absorbing maximum at 519 nm. The described method is capable of determining 0.1–1.3 ppm of nitrite with a relative standard deviation of 0.3–1.7% depending on the concentration level, molar absorptivity of 3.5 × 10 4 liter mol −1 cm −1, and Sandell sensitivity index of 0.0013 μg cm −2. Diazotization and coupling are very fast and control of temperature is unnecessary. Moreover, the colored azo dye is stable and its intensity is in direct proportion with nitrite concentration over a wide range.

Automatic methods for determining nitrate and nitrite in water and soil extracts

Automatic methods for determining nitrate and nitrite in fresh and sea water and in soil extracts with the AutoAnalyzer are described, together with details of the analytical systems. The methods are based on a modification of the manual procedure by Wood, Armstrong and Richards, which consists in reduction of nitrate with copperised cadmium and, with the nitrite thus produced, diazotisation of sulphanilamide, the product being coupled with N-1-naphthylethylenediamine to form a highly coloured azo dye, which is measured at 520 nm. The methods are capable of analysing twenty samples per hour.

DETERMINATION OF ACID PHOSPHATASE ACTIVITY IN TISSUE HOMOGENATES

A simple and accurate method is described for the quantitative determination of acid phosphatase in tissue homogenates, utilizing stable buffered sodium α-naphthyl acid phosphate tablets as substrate, and the monocoupler Fast Red Salt B as the color developer. This technique eliminates deproteinization and ethyl acetate extraction, and produces a highly colored blue azo dye for spectrophotometry. Of the rat organs tested, the spleen had the highest acid phosphatase activity, then kidney, small intestine, liver, lung, and heart, in that order.

HEPATIC PROTEIN LOCATED BY THE OXIDIZED TANNIN‐AZO METHOD

The oxidized tannin‐azo (OTA) method was used to study the location of tannophilic protein in hepatic cells. In this method tannic acid was first attached to the tissue proteins and then oxidized with periodic acid; the oxidized tannin, which was firmly bound to the cellular proteins, was subsequently converted to a highly coloured azo dye by coupling with diazotized o‐dianisidine.Preliminary deamination with nitrous acid markedly diminished the capacity of hepatic cells to stain with OTA; this indicates that ‐NH3 groups in the tissue proteins are the principal loci revealed by the OTA method.Sections of liver, after tanning and oxidation, were also treated with 6‐amino‐3‐dimethylaminophenol; this technique produced intense colouration of the hepatic cells probably owing to the formation of an oxazine dye. The colouration of oxidized tannin by 6‐amino‐3‐dimethylaminophenol confirms the view that bound tannin is oxidized by periodic acid to a 1–2‐quinone.

The Histochemical Distribution of Protein-Bound Sulfhydryl Groups<xref ref-type="fn" rid="fn1"><sup>2</sup></xref>

A new specific method for the histochemical demonstration of protein-bound sulfhydryl groups was recently described by Barrnett and Seligman. After fixation in 80 percent alcohol - 1 percent trichloroacetic acid, it was possible to demonstrate histochemically the sulfhydryl groups of aqueous and alcohol-insoluble tissue proteins in histologic sections, and to block the histochemical reaction by the use of well-known sulfhydryl reagents. The method consisted of incubating fixed deparaffinized sections of tissue in an alcohol-barbital buffer solution containing the sulfhydryl reagent, 2, 2.-dihydroxy-6,6.-dinaphthyl disulfide (D.D.D.). After the reagent reacted with the protein-bound sulfhydryl groups in the fixed tissue sections to form a colorless substance, the excess reagent as well as the reaction by-product was removed from the sections by washing and by extraction with alcoholand ether. The colorless product of the reaction between the disulfide of the reagent and the sulfhydryls of tissue, which was alcohol-ether insoluble, was converted to an intensely colored azo dye at the sites of protein-bound sulfhydryl groups by coupling with tetrazotized diorthoanisidine. A study of the distribution of protein-bound sulfhydryl groups by this method in the tissues of the albino rat forms the subject of this communication.