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Xanthoproteic Test for Amino Acids

In this xanthoproteic test for amino acids post we briefly summarise about: principle, reagents requirements, procedure, result, application and limitations of xanthoproteic test.

Xanthoproteic Test for Amino Acids

Proteins are amino acid polymers. They are nitrogen, hydrogen, carbon, and oxygen-containing organic molecules. Several procedures, including the Xanthoproteic test, can be used to validate the presence of proteins qualitatively. Certain amino acids display characteristic colour responses as a result of the existence of characteristic side chains, which are used to identify them.

The xanthoproteic test is a biochemical method for identifying amino acids like phenylalanine, tyrosine, and tryptophan that contain phenolic or indolic groups (aromatic amino acids). The test is called xanthoproteic because the yellowish substance formed by heating protein with nitric acid is called xanthoprotein. This test is used to identify amino acids like tyrosine, tryptophan, and phenylalanine that have a benzene ring in them.


To spot the difference between tyrosine and tryptophan (aromatic amino acids) and other amino acids. The presence of aromatic group containing amino acids such as tyrosine and tryptophan is detected.


When aromatic amino acids are treated with strong nitric acid, the aromatic ring is nitrated, and yellow nitro-products are formed (nitro derivatives). When a strong basic solution is introduced, the colour of the resulting products darkens (from yellow to orange). 

Xanthoproteic test reaction yields tyrosine, tryptophan, and phenylalanine as a positive result (only after extended heating time). Because it’s difficult to nitrate phenylalanine under normal circumstances, it doesn’t respond to this test unless it’s heated for a long time.

Xanthoproteic test

Xanthoproteic Test for Amino Acids Reaction



1. Concentrated Nitric acid

2. 40% NaOH

3. Test solution


1. Test tubes

2. Test tube stand

3. Pipettes


1. Add 1 ml of concentrated nitric acid to 2-3 ml of test protein solution. Heat to boil. Cool and pour half of the solution into another tube.

2. One tube is kept as control and the other as test, so as to understand the development of even faint color. To one tube add 40% NaOH or liquor ammonia (ammonium hydroxide) in excess.


Xanthoproteic test

Xanthoproteic test

A white precipitate forms on adding nitric acid, which on heating turns yellow and then dissolves to impart yellow color to the solution. Upon adding alkali the color deepens to attain orange colour.

Addition of nitric acid causes denaturation of proteins to get white precipitate. Yellow color due to nitration of benzene ring of amino acids tryptophan and tyrosine. Addition of alkali increases the ionization of compounds hence the color deepens to get final orange color


1. Xanthoproteic test  is a biochemical test for the detection of proteins and amino acids (aromatic amino acids).

2. Xanthoproteic test allows the differentiation of aromatic amino acids from non-aromatic amino acids.


1. Even though phenylalanine is an aromatic group-containing amino acid, it doesn’t give a positive Xanthoproteic test due to the highly stable phenyl group.

2. Xanthoproteic test cannot be employed for urine testing as the final color of the test and the natural color of urine are similar.

3. The aromatic amino acid Phenylalanine will not give a positive response to Xanthoproteic test even though it contains benzene ring.

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