Millon’s Test: Objective, Principle, Procedure And Result Interpretation

What Is Millon’s Test?

Millon’s test is an analytical test used for detection of presence of soluble proteins. Millon’s test is one of the qualitative methods to determine differences in types of proteins and amino acids, namely the type of phenolic amino or amino acids that have phenol groups such as tyrosine and it derivatives. Tyrosine is a non-essential amino acid that has a phenyl group and is a week acid. One of these amino acids can be found in milk casein.

This test was developed by a French chemist Auguste Millon in 1849. A purplish-red deposit is precipitated upon heating a protein solution containing Millon’s reagent (a solution of mercuric nitrate in nitric acid, with an admixture of nitrous acid). The reaction is caused by the presence of phenol radicals in the amino acid tyrosine, which is a constituent of proteins.

Given that tyrosine is an amino acid occurring in nearly all proteins, Millon’s test is used for detecting proteins. Therefore, proteins without tyrosine residues cannot be detected using this test. Thus, other tests such as biuret test and Ninhydrin test to confirm presence of proteins may be necessary.

Objective of Millon’s Test

  • To detect the presence of tyrosine-containing proteins in a given sample.
  • To detect amino acid containing phenol-group (hydroxyl group attached to benzene ring).

Principle Of Millon’s Test

Millon’s test is based on the principle of nitration of the phenol group in tyrosine, which then forms complexes with heavy metals like mercury. In this test, the reagent used is referred to as Millon’s test. The Millon’s reagent is made by dissolving metallic mercury in nitric acid and diluting it with water. The resulting mercury compounds bind hydroxyphenyl groups to produce a white precipitate in a protein solution. Tyrosine in protein solution will form a brick-red solution or reddish-brown sediments when heated. In all cases, appearance of red color is a positive test. It is important to note that, all phenols (compounds having benzene ring and OH attached to it) give positive results in Millon’s test.


Reagent And Material Required


  • Test solution/sample (1% tyrosine, 1% arginine, glycine or phenylalanine).
  • Millon’s reagent: The Millon’s reagent is made by dissolving 100g of mercury nitrate in 100ml of nitric acid and diluting it with distilled water.


  • Test tubes
  • Test tube stand
  • Pipettes
  • Boiling water bath


  1. Take 1ml test solution in a dry test tube.
  2. Also, take 1ml distilled water in another test tube as control.
  3. Add 1ml of Millon’s reagent and mix well.
  4. Boil gently for 1 minute.
  5. Cool under tap water.
  6. Now, add 5 drops of 1% sodium nitrite.
  7. Heat the solution slightly.
  8. Look for any color changes.

Results Interpretation

  • Positive Result: A positive result for Millon’s test is indicated by the formation of a brick-red or reddish-brown precipitate. This confirms the presence of tyrosine or tyrosine containing protein.
  • Negative Result: A negative result for Millon’s test is indicated by the absence of reddish-brown or brick-red precipitate in the test-tube upon heating. This confirms absence of tyrosine or tyrosine-containing protein.

Limitations Of Millon’s Test

  • Compounds like salicylic acid and phenolic compounds give a positive result to this test. Thus, other tests such as biuret test and Ninhydrin test to confirm presence of proteins may be necessary.
  • Presence of chlorine in the solution might interfere with the reaction, thus the test cannot be performed on a sample containing chlorides.
  • The formation of a white or yellow precipitate might be observed immediately after the addition of Millon’s reagent due to denaturation of proteins by mercuric ions.