Xanthoproteic Test: Objective, Principle, Reagents, Procedure And Result Interpretation

What Is Xanthoproteic test?

Xanthoproteic test is a biochemical test for the detection of amino acids having benzene ring in them like phenylalanine, tyrosine and tryptophan (aromatic amino acids). In the Xanthoproteic reaction, the yellowish substance produced by heating protein with nitric acid is xanthoprotein, hence the name Xanthoproteic test.  The test is a qualitative test that provides information only on the presence or absence of amino acids.


  • To detect the presence of aromatic groups-containing amino acids like tyrosine and tryptophan.
  • To differentiate tyrosine and tryptophan from other amino acids.


Xanthoproteic test aims to determine differences in types of protein and amino acids that have aromatic rings or benzene groups such as phenylalanine, tyrosine, tryptophan, etc. In this test, a solution of concentrated nitric acid (HNO3) and a base in the form of ammonia or sodium hydroxide (NaOH) are used.  Phenylalanine, tyrosine and tryptophan will form white deposits that can turn yellow when reacting with nitric acid in the presence of heat. Addition of a basic solution such as NaOH will create a very alkaline condition so that the nitro compound that has been previously formed can be ionized and the solution changes color to dark yellow or orange. The visible color appears based on the nitration reaction on the aromatic ring of amino acids.



  • Concentrated Nitric acid
  • 40% NaOH
  • Test solution

Material Required

  • Test tubes
  • Test tube stand
  • Pipettes


  1. Take 1ml test solution in dry test tube.
  2. Similarly, take 1ml distilled water in another test tube as control.
  3. Add 1 ml of concentrated nitric acid (HNO3) in all test tubes and mix well.
  4. Heat the sample using a water bath for 8-10 minutes.
  5. Cool the solution under tap water. If the sample is a protein solution, a white precipitate might develop due to the denaturation of proteins.
  6. Now add 2 ml of 40% NaOH to all test tubes.
  7. Look for the color changes.

Xanthoproteic Test, Results Interpretation

  • Positive Result: The appearance of a dark yellow or orange-colored solution represents a positive test. This indicates the presence of aromatic groups in the proteins and amino acids.
  • Negative Result: The absence of a dark yellow or orange-colored solution represents a negative test. This indicates the absence of aromatic groups in proteins and amino acids.


  • It is used in the detection of proteins and amino acids.
  • The test is useful in the differentiation of aromatic amino acids from non-aromatic amino acids.

Limitations of Xanthoproteic Test

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.