Millon’s Test- Definition, Principle, Procedure, Result, Uses

Millon’s Test Definition

The only amino acid with a phenol group is tyrosine, which may be found employing Millon’s test, an analytical procedure. Tyrosine is specifically detected by Millon’s test. However, it does not identify proteins since it also recognises the phenolic group, which is present in other compounds.

In addition to the Millon’s test, additional tests such as the Biuret and Ninhydrin tests must also be conducted. Tyrosine is a common component of proteins, hence the test is helpful in identifying them. The French chemist Auguste Nicolas Eugene Millon, who discovered the test, was given credit for its invention.

Objectives of Millon’s Test

  • to identify tyrosine-containing proteins in a particular sample.
  • In order to find compounds that contain phenol.
  • Tyrosine’s distinction from other amino acids

Principle of Millon’s Test

Millon’s test is predicated on the theory that nitrification of the phenol group in tyrosine leads to the development of complexes containing heavy metals such as mercury. The material utilised for the test, Millon’s reagent, is composed of mercuric and mercurous nitrates dissolved in concentrated nitric acid. The reagent’s nitric acid nitrates the phenol group on the tyrosine molecule during the test.

Mercury ions in the solution react with tyrosine that has been nitrated to form a red precipitate or solution. The first reaction between mercuric nitrate and tyrosine-containing proteins generates a white or yellow precipitate. The residue becomes red when nitric acid is applied and the combination is heated. Both of these observations, which are considered positive, indicate that tyrosine exists in the solution.



Millon’s reagent: To generate Millon’s reagent, mercuric and mercurous nitrates are dissolved in nitric acid and distilled water.

For the preparation of Millon’s reagent, combine 400 ml of concentrated nitric acid solution with 160 grammes each of mercuric and mercurous nitrate. Then, 600 ml of distilled water are added to the reagent to bring the volume to 1000 ml. The formula can be modified to fit the requirements for the performance.

Sample (1% tyrosine)

Materials Necessary

  • A test tube
  • Test tube holder.
  • Pipettes
  • a water bath

Procedure of Millon’s Test

  1. In a test tube, two millilitres of the sample solution or the 1 percent tyrosine solution are placed in a test tube.
  2. Then, 2 mL of Millon’s reagent is added. If a red precipitate is not immediately observed, the test tubes are kept in the water bath for an additional two minutes.
  3. In the tubes, the growth of the colourful precipitate is then observed.

Result and Interpretation of Millon’s Test

  • Positive result:A red or pink-tinted precipitate forming during the Millon’s test is indicative of a favourable outcome. This indicates the presence of tyrosine or a protein having tyrosine.
  • Negative result:The lack of coloured precipitate in the test tube shows that the Millon’s test was negative. This signifies the absence of tyrosine or a tyrosine-containing protein.

Uses of Millon’s Test

  • Tyrosine-containing proteins can be found in a sample using Millon’s assay.
  • In addition, the test helps to distinguish tyrosine from different amino acids.
  • The test is useful for distinguishing between casein and raw meat proteins.


  • This test is positive for salicylic acid as well as phenolic compounds; thus, adding further phenolic compounds in the test tube must be prevented. For verification, tests such as the Ninhydrin and Biuret tests must be conducted.
  • The reaction cannot be performed on a sample containing chlorides because the chlorine in the solution would inhibit the reaction.
  • As a result of the denaturation of proteins by mercuric ions, a white or yellow precipitate may develop shortly after adding Millon’s reagent.


  • Tiwari, A. (2015). Practical Biochemistry.  LAP Lambert Academic Publishing.
  • Nigam S. C. and Omkar (2003). Experimental Animal Physiology and Biochemistry. New Age International Pvt. Limited. New Delhi.
  • D (2012). Biochemistry. Fourteenth Edition. Academic Publishers. Kolkata.
  • Millon’s Reagent. R097. HiMedia Laboratories.
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