Diazo Compounds

Diazo compounds are derived from a primary arylamine.
Following treatment with nitrous acid (HNO2)
the amino group is converted to an azo group

 
Diazonium compounds have the general formula
 
They are more commonly represented in the charged state by

 

Burstone describes diazo compounds as containing

"The N2 grouping combined to only one hydrocarbon radical instead of two as in azo compounds. The formation of diazo compounds depends upon the replacement of three hydrogen atoms of the salt of an aryl primary amine by nitrogen from nitrous acid to form the so-called diazonium salts."

Examples are fast red B and fast blue B.

Due to their instability, commercially available diazonium compounds may not be supplied in the form shown above, but as some other complex. Burstone gives the following possibilities:–

 

Strictly speaking, diazo compounds are not dyes, but dye precursors. Some azo dyes are manufactured by coupling a phenol or naphthol to a diazonium compound.

In histotechnology, they have two main uses. The first is for the demonstration of enterochromaffin, traditionally using fast red B although many others will do so. The second is for demonstrating the activity of some enzymes, for example alkaline and acid phosphatases. Using naphthol phosphates as the substrate, these enzymes split off the phosphate, leaving the naphthol, which then couples to the diazo compound to form an insoluble dye at the site of activity. Similar processs are used for other enzymes as well.

In all three uses the reaction is fundamentally same. The differences are that when using azo coupling to manufacture dyes the naphthol used would have appropriate groups to ensure a soluble product. To demonstrate enzyme activity an insoluble product is needed so that it can be precipitated where the enzyme is. Enterochromaffin, being a tissue component, stays where it is with the new dye attached.

+

Diazo compound

=

β-naphthol

+  HCl

Azo dye

The azo dye in the above example has a hydroxyl group ortho to an azo nitrogen. Under these circumstances, there is some molecular rearrangement, analogous to that of lysochromes, causing the dye to become insoluble.

Azo coupling is not limited to naphthols. Burstone gives the following list of compounds capable of being coupling components with diazonium compounds:–

 

Reference
R. D. Lillie.
Conn's Biological Stains
Williams & Wilkins, Baltimore, MD., U.S.A.

Burstone, M.S.
Enzyme Histochemistry and its application to the study of neoplasms
Academic Press, New York, NY, USA

 


 

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