Effect of solvent on tetrazolium reaction.

The rate of color development of a tetrazolium formazan is shown to be inversely proportional to the dielectric constant of the solvent medium and directly proportional to the hydrogen-bonding capability of solvent mixtures having the same dielectric constant. The geometric isomers of the formazans have different absorbance maxima, and the wavelength of maximum absorbance of a mixture of formazans in different solvents depends upon which isomer predominates in that solvent. The trans-syn-isomer (blue form) of blue tetrazolium has a maximum absorbance at 625 nm in dimethylformamide while the trans-anti-isomer (red form) absorbs at 517 nm in methanol. The absorbance maxima of the corresponding isomers of the formazans of triphenyltetrazolium occur at 535 and 485 nm, respectively. Water and/or methanol (to a lesser extent) are important in the stabilization of the trans-anti-isomer, since the small size of these two substances allows them to form strong intermolecular hydrogen bonds with one or both nitrogen atoms of the azo linkage, thereby preventing the formation of the intramolecular hydrogen bonding exhibited by the trans-syn-isomer. The formazan produced by the reaction of corticosteroids with tetrazolium in strongly basic media can lose a reduction unit and be reoxidized to the tetrazolium. This reaction is solvent dependent and occurs at a much faster rate in chloroform than in alcohol USP.