Congo Red is a synthetic azo dye known for its distinctive red color and historical significance in dyeing and staining. It is widely used in various industrial, biological, and diagnostic applications due to its vibrant color.

Chemical Composition and Structure

Congo Red is an azo dye, meaning it contains one or more azo groups (-N=N-) in its molecular structure. The chemical formula for Congo Red is C₃₂H₂₂N₆Na₂O₆S₂. Its structure includes two azo groups linked to aromatic rings and sulfonate groups, contributing to its water solubility and bright red hue.

Physical Properties

Congo Red typically appears as a dark red to brown powder. It is highly soluble in water, producing a red solution when dissolved. The dye is known for its ability to bind to amyloid fibrils, a property that makes it useful in various diagnostic applications. Congo Red exhibits good stability under various conditions, including resistance to light and moderate heat.

Chemical Industrial Synthesis Process

• Preparation of reagents. The main raw materials include benzidine, sulfuric acid (H₂SO₄), sodium nitrite (NaNO₂), a base such as sodium hydroxide (NaOH), and 1-naphthylamine-4-sulfonic acid.
• Diazotization. Benzidine is treated with sulfuric acid and sodium nitrite to form a diazonium intermediate. This reaction produces a diazonium salt.
• Coupling. The diazonium salt is then coupled with 1-naphthylamine-4-sulfonic acid in the presence of a base, such as sodium hydroxide, to form the azo compound intermediate.
• Formation of Congo Red dye. The azo compound intermediate is further processed and stabilized to form the Congo Red dye.
• Filtration. The resulting suspension is filtered to separate the solid precipitate from the aqueous solution.
• Washing. The precipitate is washed with deionized water to remove any soluble impurities.
• Drying. The washed precipitate is dried at controlled temperatures to remove residual moisture and obtain a dry powder.
• Grinding. The dried product is ground to obtain a fine and uniform powder.
• Classification. The dried powder is classified to ensure a uniform particle size. This step may involve sieving or the use of air classifiers.
• Stabilization. The Congo Red powder is stabilized to ensure its stability during transportation and storage, preventing aggregation and degradation.
• Quality control. The Congo Red undergoes rigorous quality testing to ensure it meets standards for purity, color intensity, and safety. These tests include chemical analysis, spectroscopy, and physical tests to determine particle size and rheological properties.

What it is used for and where

Medical

Biological Staining. Congo Red is widely used as a histological dye in biological and medical research. It is particularly valuable for staining amyloid fibrils in tissue samples, helping in the diagnosis of amyloidosis and other conditions associated with amyloid deposits.

Congo Red is a histological dye commonly used for the detection of amyloid for the visualization and the quantification ante che in vivo of cerebral amyloids (1).

Cosmetics

Restricted cosmetic ingredient as  II/986 a Relevant Item in the Annexes of the European Cosmetics Regulation 1223/2009. Substance or ingredient reported:

• Disodium 3,3'-[[1,1'-biphenyl]-4,4'-diylbis(azo)] bis(4-aminonaphthalene-1- sulphonate). Prohibited substance in cosmetic products

Industrial Applications

Textile Dyeing: Historically, Congo Red has been used as a direct dye for cotton and other cellulosic fibers. Its vibrant color and ease of application made it popular in the textile industry, although its use has declined due to the availability of more stable dyes.

Indicator and pH Testing: Congo Red acts as a pH indicator, changing color from red at pH levels above 5.0 to blue at pH levels below 3.0. This property makes it useful in various laboratory settings for monitoring pH changes.

Paper and Inks: Congo Red is used in the paper industry to dye paper products and in the production of certain types of inks. Its bright red color and water solubility make it suitable for these applications.

Safety

 The problem associated with azo dyes (monoazo or diazo) is photocatalytic degradation leading to eventual oxidation and subsequent formation of impurities such as aromatic amines some of which have carcinogenic activity (2).

Molecular Formula  C₃₂H₂₂N₆Na₂O₆S₂

Molecular Weight    696.7 g/mol

CAS     573-58-0

UNII    3U05FHG59S

EC Number    209-358-4

Synonyms:

Direct Red 28

C.I. Direct Red 28

NSC-56651

NSC-7232

References__________________________________________________________________________

(1) Yakupova EI, Bobyleva LG, Vikhlyantsev IM, Bobylev AG. Congo Red and amyloids: history and relationship. Biosci Rep. 2019 Jan 15;39(1):BSR20181415. doi: 10.1042/BSR20181415. 

Abstract. Staining with Congo Red (CR) is a qualitative method used for the identification of amyloids in vitro and in tissue sections. However, the drawbacks and artefacts obtained when using this dye can be found both in vitro and in vivo Analysis of scientific data from previous studies shows that CR staining alone is not sufficient for confirmation of the amyloid nature of protein aggregates in vitro or for diagnosis of amyloidosis in tissue sections. In the present paper, we describe the characteristics and limitations of other methods used for amyloid studies. Our historical review on the use of CR staining for amyloid studies may provide insight into the pitfalls and caveats related to this technique for researchers considering using this dye. © 2019 The Author(s).

(2) Chung KT, Stevens SE Jr, Cerniglia CE. The reduction of azo dyes by the intestinal microflora. Crit Rev Microbiol. 1992;18(3):175-90. doi: 10.3109/10408419209114557.

Abstract. Azo dyes are widely used in the textile, printing, paper manufacturing, pharmaceutical, and food industries and also in research laboratories. When these compounds either inadvertently or by design enter the body through ingestion, they are metabolized to aromatic amines by intestinal microorganisms. Reductive enzymes in the liver can also catalyze the reductive cleavage of the azo linkage to produce aromatic amines. However, evidence indicates that the intestinal microbial azoreductase may be more important than the liver enzymes in azo reduction. In this article, we examine the significance of the capacity of intestinal bacteria to reduce azo dyes and the conditions of azo reduction. Many azo dyes, such as Acid Yellow, Amaranth, Azodisalicylate, Chicago Sky Blue, Congo Red, Direct Black 38, Direct Blue 6, Direct Blue 15, Direct Brown 95, Fast Yellow, Lithol Red, Methyl Orange, Methyl Red, Methyl Yellow, Naphthalene Fast Orange 2G, Neoprontosil, New Coccine, Orange II, Phenylazo-2-naphthol, Ponceau 3R, Ponceau SX, Red 2G, Red 10B, Salicylazosulphapyridine, Sunset Yellow, Tartrazine, and Trypan Blue, are included in this article. A wide variety of anaerobic bacteria isolated from caecal or fecal contents from experimental animals and humans have the ability to cleave the azo linkage(s) to produce aromatic amines. Azoreductase(s) catalyze these reactions and have been found to be oxygen sensitive and to require flavins for optimal activity. The azoreductase activity in a variety of intestinal preparations was affected by various dietary factors such as cellulose, proteins, fibers, antibiotics, or supplementation with live cultures of lactobacilli.