Synopsis
Column chromatography on lipophilic Sephadex LH-20 of dyes labelled as Pararosaniline was carried out in a 3:I mixture of dimethyl sulphoxide and an aqueous ammonia-ammonium chloride buffer mixture. In such a mixture the dye, mainly in its leuco-form, is highly soluble. On elution with this mixture, a red-coloured band separated from fluorescent- and non-fluorescent-contaminating substances. This band contained the main component which could be precipated from the appropriate eluant fractions as a colourless base by adding excess dilute ammonia.
The purified dye showed a slightly higher molar extinction coefficient at 54I nm than the original product. By fluorescence spectrophotometry, contrary to what was found for the original product, no fluorescent components could be detected. A similar method was found to be suitable for purifying Atebrine, an acridine-type dye.
The method appears to be of general applicability for preparing purified dye fractions in quantities sufficient for histochemical and histological use.
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Van Duijn, P., Riddersma, S.H. Purification of Pararosaniline and Atebrine by chromatography on lipophilic Sephadex LH-20. Histochem J 5, 169–172 (1973). https://doi.org/10.1007/BF01012559
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DOI: https://doi.org/10.1007/BF01012559