Indophenol blue

Phosphatase Test. The phosphatase [9001-78-9] test is a chemical method for measuring the efficiency of pasteurization. AH raw milk contains phosphatase and the thermal resistance of this enzyme is greater than that of pathogens over the range of time and temperature of heat treatments recognized for proper pasteurization. Phosphatase tests are based on the principle that alkaline phosphatase is able, under proper conditions of temperature and pH, to Hberate phenol [108-95-2] from a disodium phenyl phosphate substrate. The amount of Hberated phenol, which is proportional to the amount of enzyme present, is determined by the reaction of Hberated phenol with 2,6-dichloroquinone chloroimide and colorimetric measurement of the indophenol blue formed. Under-pasteurization as well as contamination of a properly pasteurized product with raw milk can be detected by this test. 

Massaccesi reported the development of a two-phase titration method for the analysis of miconazole and other imidazole derivatives in pure form and in pharmaceutical formulation [14], To the sample (10 mg) are added 10 mL of water, 10 mL of 1 M-sulfuric acid, 25 mL of dichloromethane and 1 mL of 0.05% indophenol blue (C.I. No. 49700) in dichloromethane solution and the solution is titrated with 10 mM sodium dodecyl sulfate until the color of the organic phase changes from blue to pale yellow. Results obtained for the drug in pure form, tablets, suppositories, cream and lotion agreed with the expected values and the coefficient of variation (n = 6) were 0.3-0.35%. Imidazole and the other constituents of the pharmaceutical preparations did not interfere. 

Haywood and Huyser s [43] study of the indophenol blue reaction for ammonia determination showed the effect of pH variation on the final colour and emphasised the necessity for the efficient buffering to obtain reproducible results. The optimum conditions found by the authors (pH 10.8 and using sodium nitroprusside) were similar to those used by Solorzano [37]. Haywood and Huyser also replaced acetone with sodium nitroprusside. 

Berg and Addullah [47] have described a spectrophotometric autoanalyser method based on phenol, sodium hypochlorite, and sodium nitroprusside for the determination of ammonia in sea and estuarine water (i.e., the indophenol blue method). 

The interference from amino acids was investigated and found to be negligible as reported by Solorzano [37] and Haywood and Huyser [43], who employed no heating for the indophenol blue colour development. Solutions containing 50 xg N/l of urea, histidine, lycine, glycine, and alanine were analysed. The NH4-N detected ranged between 0.4% (for urea) and 2.2% (for alanine) of the nitrogen added. 

Le Corre and Treguer [49] developed an automated procedure based on oxidation of the ammonium ion by hypochlorite in the presence of sodium bromide followed by spectrophotometric determination of the nitrite. The standard deviation on a set of samples containing 1 p,g NH -N per litre was 0.02. This method was compared with an automated method for the determination of ammonia as indophenol blue. The results from the two methods are in good agreement. 

The reaction manifold describing the automated determination of ammonia is shown in Fig. 6.1. Two alternative modes of sampling are shown discrete and continuous. Discrete 5 ml samples contained in ashed (450 °C) glass vials are sampled from an autosampler (Hook and Tucker model A40-11 1.5 min sam-ple/wash). For high-resolution work in the estuary, the continuous sampling mode is preferred. The indophenol blue complex was measured at 630 nm with a colorimeter and the absorbance recorded on a chart recorder. 

The Adamski [13] procedure is semiautomated. It gives an accuracy of 8.1% and a precision of 8.2% for seawater samples spiked with 3-35 p,g/l organic nitrogen. This procedure is based on the indophenol blue method and was employed using a Technicon AutoAnalyzer II system with the appropriate accessories. Various workers have described automated procedures for determination of low levels of organic nitrogen in seawater [15,16]. 

Pelts and Belcher [46] have described a semi-automated method for the simultaneous determination of nitrogen and phosphorus in soil. Up to 40 samples are digested with sulphuric acid using a copper sulphate-sodium sulphate catalyst at 370°C for 2.25h in a thermostatically controlled block. Nitrogen is then determined in the 0.2-2.25% range as ammonia by automatic colorimetric analyses of the indophenol blue complex at 630nm. 

Summary of the indophenol blue colorimetric determination of nitrogen... 

Using autoanalysis, ammonium ions are reacted with sodium phenate and sodium hypochlorite to give the indophenol blue colour by the Berthelot reaction (Berthelot, 1859). 

Fig. 4.1. Colorimetric reaction converting protein nitrogen to the indophenol blue colour.

Colorimetry. A variety of colorimetric techniques have been used to measure ions such as NH4, SO4-, and NO7 in ambient particles. For example, nitrate can be measured by reduction to nitrite using hydrazine in the presence of a copper catalyst, followed by its conversion to a colored azo dye, which can be measured by its absorbance at 524 nm (Mullin and Riley, 1955). Sulfate has been determined using an exchange reaction between sulfate and a barium-nitrosulfo-nazo(III) chelate in aqueous acetonitrile the chelate has an absorbance peak at 642 nm and hence the decrease in this peak can be followed as a measure of the amount of sulfate present that has exchanged with the chelate (Hoffer et al., 1979). Similarly, NH4 can be measured by the indophenol blue method (Weather-burn, 1967). 

Keay and Menage [24] carried out an automated determination of nitrate and ammonia in 2 M potassium chloride extracts of soils. The sample is reacted in an autoanalyser with a 0.25% suspension of magnesium oxide the ammonia liberated from ammonium ion is absorbed in 0.1 M hydrochloric acid and determined spectrophotometrically at 625 nm by the indophenol blue method. The sum of ammonium and nitrate is determined similarly but with the addition of 4.5% titanous sulfate solution before distillation, thereby reducing nitrate but not nitrite to ammonia. The nitrate content of the soil can then be obtained by difference. 

The analysis target was the detection of ammonia in aqueous solutions, e.g. in surface water [114-116], The Berthelot reaction scheme was employed to monitor ammonia, by chemical conversion to indophenol blue, using a chlorination step first followed by coupling of two phenol moieties. The absorption of the dye was measured by a photometric-type experiment. Full conversion by efficient mixing is mandatory for a good analysis. 

The absorption spectra of (14) and wood slices treated according to the above sequence are similar, thereby providing evidence that structures (11) and (12) are present in native lignin. Quinone monochlorimide (15) reacts only with p-hydroxybenzyl alcohols (il), forming indophenol (blue) as shown below (Gierer 1954, 1956). 

The first nematic guest-host prototype nematic guest-host display device contained a nematic liquid crystal (4-butoxybenzoic acid) and a pleochroic dye (methyl red or indophenol blue) sandwiched between two (Nesa) electrodes dTn 12/im) rubbed uniaxially, but with no additional orientation layer, see Figure 3.14. One polariser was fixed to the front substrate surface with its direction of maximum absorption parallel to the rubbing direction and, therefore, the nematic director. 

Although the earliest examples of the use of US as a substitute for phase transfer catalysts in organic addition reactions were reported more than two decades ago and a number of such reactions have since been improved as a result [1-7], the sole analytical application exploiting this potential is a method for the determination of paracetamol where the drug is derivatized by hydrolysis to p-aminophenol, which reacts with o-cresol in an alkaline medium to form the Indophenol Blue dye, according to the following reaction ... 

Koroleff, E, 1970. Direct determination of ammonia in natural waters as indophenol blue. International Council for the Exploration of the Sea, ICES. Service hydrographique. Information on techniques and methods for sea water analysis. Interlaboratory Report No. 3, 19-22. 

N, P Plants UV-Vis 0.05-0.5%, 0.2-5% (w/w) Sequential determinations/indophenol blue and molybdenum blue methods [20]... 

Ammonium, nitrate Soil extracts Distillation UV—vis 0-15 mg L-1 N Air-segmented flow system 116 °C heating bath indophenol blue reaction Ti(III) addition for nitrate conversion to ammonium during distillation [525]... 

Regarding the use of the flow analyser to assist practical classes with specific examples in physical chemistry and/or analytical chemistry, reaction kinetics has been addressed in didactic experiments involving the Bertholet reaction with indophenol blue formation [410] and hex-acyanoferrate(III) reduction by ascorbic acid [411], Recently, video demonstrations have been successfully exploited aimed at the evaluation of stability constants and the classification of cations [412], These experiments require combinations of a number of different solutions. The large number of flasks required for accomplishing these experiments in the traditional manner is not necessary, reagent consumption is low and the experiments can be indefinitely repeated. 

Hydrolysis with aqueous hydrochloric acid followed by spectrophotometric estimation of the alcohol produced (using ceric ammonium nitrate) has been used136 to estimate C2-C4 alkoxy groups in various compounds including siloxanes. A spectrophotometric method137 for the determination of phenoxy groups is based on the formation of the indophenol blue. 

The phenol-hypochlorite (Indophenol-blue) method was used for ammonia analysis (Solorzano, 1969). Appropriate sample sizes were fixed with phenol within 24 hours of collection (Degobbis, 1973) and stored at all times at 4 C. Analyses were made within 2 weeks of collection. The reagent dilutions given by Presley (1971) were used, but the quantity added was doubled. All reaction and color development was done in polyethylene scintillation vials. Color development was allowed for 6-8 hr to obtain a straight-line standardization curve blanks (always <0.010 absorbance with respect to distilled water, 1-cm cell) were insignificant compared to sample absorbances and precision better than 3%. Standards were diluted immediately before use. 

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