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

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Millon’s Test Definition

Millon’s test can be described as an analytical test for the identification that the amino acid tyrosine which is the sole amino acid with the phenolic group. Millon’s test is a particular test for tyrosine; however, it’s not a specialized test for protein since it also detects the phenolic component found in other substances as well. So, when conducting the Millon test it is vital to perform other tests, such as the Biuret test as well as the Ninhydrin test are also performed. Because many proteins contain Tyrosine, the test is helpful in identifying the proteins involved. The test was developed by and named after French scientist Auguste Nicolas Eugene Millon.

Objectives of Millon’s Test

  • To determine the presence of proteins containing tyrosine within a particular sample.
  • To identify the presence of compounds containing phenol.
  • To distinguish tyrosine and other amino acids.
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Principle of Millon’s Test

Millon’s test is based upon the concept of nitrification of the phenol group of tyrosine and forming complexes with heavy metals such as mercury. The reagent that is used for the test is known as Millon’s reagent and comprises mercuric Nitrate and the mercurous nitrate, which is dissolving in concentrated the nitric acid. In the test the phenol group of the molecule of tyrosine is affected by the nitric acids found within the reaction. The tyrosine that has been nitrated then mixes with mercury ions present in the solution to create an red-colored solution or precipitate. In certain proteins that contain Tyrosine, the first reaction between mercuric nitrate and tyrosine results in a yellow or white colored precipitate. When Nitric acid and the heating process the resultant substance transforms into red. Both of these results can be taken as positive results and suggest an existence of tyrosine the solution.

Requirements for Millon’s Test


  • Millon’s reagent: Millon’s Reagent comprises mercuric-based nitrate and the mercurous nitrate that is dissolved in nitric acid as well as distillation water. The preparation of Millon’s reagent is: Mix 160 grams mercuric Nitrate as well as 160g of mercurous Nitrate in 400ml concentrated solution of nitric acid. The reagent can be increased to 1000ml through adding 600ml of distillated water. The formula is adjustable according to the parameters of performance.
  • Sample (1% tyrosine)

Materials Required

Procedure of Millon’s Test

  • Around 2 ml of solution sample or the solution containing 1% tyrosine is stored in the test tube.
  • To this, approximately 2 ml of Millon’s Reagent is added. These test tubes kept in the bath for around 2 minutes , if the red-colored precipitate is not noticed immediately.
  • These tubes then are inspected to see their formation. color-changing precipitate.

Result and Interpretation of Millon’s Test

Result and Interpretation of Millon’s Test
Result and Interpretation of Millon’s Test
  • Positive result: A Positive result in Millon’s test is confirmed through the development of pink or red colored precipitate. This is a sign of the protein tyrosine, or that contains tyrosine.
  • Negative result: The Negative result of  Millon’s test is evident through the lack of colored precipitates in the tube. This means that there is no Tyrosine or any protein containing tyrosine.

Uses of Millon’s Test

  • Millon’s test is utilized to determine the presence of the presence of tyrosine-containing proteins within a sample.
  • The test can also aid in the distinction of the amino acid tyrosine from those of other amino acids.
  • The test can aid for detecting casein protein as well as the protein that is found in raw meat.

Limitations of Millon’s Test

  • Compounds such as salicylic acid or phenolic compounds yield an affirmative result for this test. Therefore all other phenol compounds that could be found within the tube must be avoid. Tests such as that of Biuret test and Ninhydrin test ought to be carried out to verify.
  • In the presence of chloride in the sample, it can affect the reaction; therefore, the test should not be carried out on a test sample that has chlorides.
  • The development of a yellow or white precipitate is possible to observe shortly after the addition of Millon’s reagent, due to the protein denaturation caused by the mercuric Ions.

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