From the interactions between azure A and various substrates with different physical and chemical characteristics, some suggestions are made as to the nature of the metachromatic colour reaction. The following essential factors are necessary for the appearance of metachromasia.

1. The orderly alignment of dye molecules is favoured if the molecules present hydrophobic and hydrophilic parts.

2. The distribution of electro-negative surface charges of the substrate determines the alignment of dye molecules and the degree of metachromasia. A certain minimum intercharge distance (interplanar), possibly about 5 Å, is a prerequisite for metachromasia. The degree of metachromasia increases when the intermolecular distance between adjacent dye molecules aggregated to the substrate surface becomes less than 5 Å.

3. The presence of water is essential for metachromatic interaction.

The observed spectral absorption shifts are explained by the assumption that new bonds appear between adjacent dye molecules. The necessary energy requirements are of the order of about 8 cal./mol. In the case of azure A, various types of intermolecular bonds are discussed: hydrogen bonds or perhaps hydrogen bonds plus oxygen-sulphur bridges. It seems very likely that water molecules may be intercalated between adjacent dye molecules as suggested by Scheibe and Sheppard.

Since the dye and many organic substrates form polydisperse colloidal solutions and since further the dye-substrate interactions often imply the appearance of micellar aggregates and precipitates in in vitro experiments, spectrophotometric data should be interpreted with great caution. Evidence is presented indicating that the heterogeneity of the sol systems in many instances renders spectrophotometry useless. The desired colour changes are instead conveniently registered by microscopy with transmitted light.

Available data indicate that the electro-negative surface charge density is the essential characteristic of the various substrates for the conditioning of metachromatic dye-substrate interaction.

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