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'Congo red' is the sodium salt of benzidinediazo-bis-1-naphtylamine-4-sulfonic acid (formula: C₃₂H₂₂N₆Na₂O₆S₂; molecular weight: 696.66 g/mol). It is a secondary diazo dye. Congo red is water soluble, yielding a red colloidal solution; its solubility is better in organic solvents such as ethanol.
It has a strong, though apparently non-covalent affinity to cellulose fibres. However, the use of congo red in the cellulose industries (cotton textile, wood pulp & paper) has long been abandoned, mainly because of its toxicity.

Contents

Behaviour in solution

Dyeing activity

Diagnostic use

Behaviour in solution

Due to a color change from blue to red at pH 3.0-5.2, congo red can be used as a pH indicator. Since this color change is an approximate inverse of that of litmus, it can be used with litmus paper in a simple parlor trick: add a drop or two of congo red to both an acid solution and a base solution. Dipping red litmus paper in the red solution will turn it blue, while dipping blue litmus paper in the blue solution will turn it red.
Congo red has a propensity to aggregate in aqueous and organic solutions. The proposed mechanisms suggest hydrophobic interactions between the aromatic rings of the dye molecules, leading to a pi-pi stacking phenomenon. Although these aggregates are present under various sizes and shapes, the "ribbon-like micelles" of a few molecules seem to be the predominant form (even if the "micelle" term is not totally appropriate here). This aggregation phenomenon is more important for high congo red concentrations, at high salinity and/or low pH.

Dyeing activity

As suggested by its intense red color, congo red has important spectrophotometric properties. Indeed, its UV-visible absorption spectrum shows a characteristic, intense peak around 498 nm in aqueous solution, at low dye concentration. Congo red molar extinction coefficient is about 45000 L/mol/cm in these conditions. Aggregation of the dye tends to red-shift the absorption spectrum, whereas binding to cellulose fibres or amyloid fibrils has the opposite effect.
Congo red also shows a fluorescent activity when bound to amyloid fibrils, which tends to be used as a sensitive diagnosis tool for amyloidosis, instead of the traditional histological birefringence test.

Diagnostic use

In biochemistry and histology, congo red is used to stain microscopic preparates, especially as a cytoplasm and erythrocyte stain. Apple-green birefringence of Congo red stained preparates under polarized light is indicative for the presence of amyloid fibrils.