Colour reagents

Horton, H. R. Koshland, D. E. Jr. A highly reactive coloured reagent with selectivity for the tryptophan residue in proteins 2-hydroxy-5-nitrobenzyl bromide. J. Amer. Chem. Soc. 1965, 87, 1126-1132. 

Some detection procedures involve the loss of the sample for example, in the flame ionization detectors used in gas chromatography the sample is burnt, while in liquid chromatography the addition of a colour reagent will also result in the loss of the sample. For such methods to be used in a preparative... 

Test spots can be identified by comparing their migration with that of reference samples, together with additional evidence, e.g. using a specific colour reagent. The RF value (rate of flow) is a measure of the movement of a compound compared with the movement of the solvent ... 

The same procedure described for -hydroxysteroid dehydrogenase assay can be applied with a few differences [50]. Diaphorase (33 U/ml) is dissolved in 2 ml sodium carbonate-bicarbonate buffer (0.1 M, pH 9.0). Colour reagent is deprived of diaphorase NAD+ is replaced by NADH. The reaction is started by adding 0.1 ml diaphorase. 

Serum or bile samples (0.2 ml) are incubated with 100 mg arylamine glass bound to -hydroxysteroid dehydrogenase, 200 mg arylamine-glass-bound diaphorase and 0.5 ml colour reagent in a 15 ml conical flask, in the dark at 30°C for 15 min (standard assay conditions for mixture of immobilised enzyme) under continuous stirring. The A54o of the reaction mixture is read. 

Acid solutions of zirconium salts give a brown precipitate with the reagent. If the test is conducted on filter paper, the brown precipitate remains in the pores of the paper and the excess of the coloured reagent may be washed out with dilute acid. 

There are several parameters which require critical consideration. Firstly, the colour reagent should be selective for the drug molecule itself, discriminating against degradation products, impurities, and formulation excipients likely to be present. Secondly, the effect and control of any parameters likely to affect the colour reaction should be established, i.e. 

It is important to detail the detection methods. The important feature of any planar separation technique has always been the possibility to use general colour reagents (e.g.. sulphuric acid) which can detect practically any organic compound, or to use colour reagents specific for a certain compound group (e.g.. ninhydrin for amino acids). Moreover, inexpensive film-detection of radiolabelled compounds can also be done 185). 

Specific and sensitive detection of the separated. sample components remains essential. In addition to the classical use of colour reagents, two other possibilities will... 

If, however, more than one coloured complex is formed in presence of a coloured reagent, absorbing partly in the region of Amax of the eomplex, it is difficult to find a concentration range in which Beer s law is obeyed. The values of e then have only a relatively low significanee, as they depend on the contributions of individual complexes in the mixture, and hence on conditions such as the excess of reagent or the pH value. 

The reference (comparison) solution in one-colour spectrophotometric measurements is usually the solvent (if the reagent is colourless), and in two-colour methods a reagent solution is used. As well as the spectrophotometric reagent the reference should contain other reagents and have pH identical to the analyte solution. It happens that at A-mm the absorbance of the colour reagent is zero and pure solvent may be applied as the reference. 

Quantitative structure-property relationships for colour reagents and their colour reactions with ytterbium using regression analysis and computational neural networks. Anal. Chim. Acta, 321, 97-103. 

FIGURE 3.15 Peak distortion caused by reagent depletion at the centre of the sample zone. Figure refers to a single-line flow system into which a 5.00 mg L-1 chloride solution is inserted into a coloured reagent carrier stream. For experimental details, see Ref. [71]. 

R.M. Liu, D.J. Liu, A.L. Sun, Simultaneous determination of multicomponents by flow injection analysis. Determination of copper and zinc in serum by using zincon as colouring reagent, Talanta 40 (1993) 511. 

Measuring absorbances in colorimetric analysis - if any final solution has an absorbance that is too high to be read with accuracy on your spectrophotometer (i.e. A > 2), it is bad practice to dilute the solution so that it can be measured. This dilutes both the sample, molecules and the colour reagents to an equal extent. Instead, you should dilute the original sample and re-assay. 

S. Motomizu, Fundamental Study of the Determination of Iron and Cobalt Using 2-Nitroso-5-Dimethylaminophenol as Colour Reagent by a Continuous Flow Stream Propelled by Gas Pressure [in Japanese]. Bunseki Ka-gaku, 32 (1983) 191. 

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