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

Millon’s test is an analytical test used for the detection of the amino acid tyrosine, which is the only amino acid containing the phenol group. Tyrosine is one of the 20 standard amino acids that are used to synthesize proteins in living organisms. Tyrosine has a hydroxyl group attached to the benzene ring, which makes it a phenolic compound. Phenolic compounds are organic compounds that have at least one hydroxyl group attached to an aromatic ring.

Millon’s test is a specific test for tyrosine, but it is not a specific test for protein as it also detects the phenolic group present in other compounds as well. Therefore, while performing Millon’s test, it is essential that other tests like the Biuret test and Ninhydrin test also be performed. The Biuret test detects the presence of peptide bonds in proteins, while the Ninhydrin test detects the presence of free amino groups in amino acids and proteins.

The test was discovered by and named after the French Chemist Auguste Nicolas Eugene Millon. Millon was a pioneer in organic chemistry and biochemistry and made significant contributions to the study of proteins, carbohydrates, and uric acid. He also developed several other analytical tests for organic compounds, such as Millon’s reaction for aldehydes and ketones, and Millon’s base for nitro compounds.

Millon’s test is based on the principle of nitrification of the phenol group in tyrosine, which then forms complexes with heavy metals like mercury. The reagent used for the test is called Millon’s reagent, and it consists of mercuric nitrate and mercurous nitrate that is dissolved in concentrated nitric acid. In the test, the phenol group on the tyrosine molecule is nitrated by the nitric acid present in the reagent. The nitrated tyrosine then combines with the mercury ions in the solution to form a red-colored precipitate or solution.

Millon’s test can be performed on pure tyrosine or on proteins that contain tyrosine residues. In some proteins containing tyrosine, the initial reaction between mercuric nitrate results in a white or yellow colored precipitate. After the addition of nitric acid and heating, however, the residue turns red in color. Both of these results are considered positive results and indicate the presence of tyrosine in the solution.

Millon’s test is useful in the detection of tyrosine-containing proteins in a given sample. It can also help in the differentiation of tyrosine from other amino acids. The test is commonly used in biochemistry and food analysis to detect casein protein and protein found in raw meat.

The main objectives of Millon’s test are:

• To detect the presence of tyrosine-containing proteins in a given sample. Tyrosine is an amino acid that has a phenol group attached to its side chain. It is found in many proteins, such as casein, albumin, and keratin. Tyrosine is also involved in the synthesis of hormones, neurotransmitters, and pigments in the body. Therefore, detecting tyrosine-containing proteins can provide information about the nature and function of the sample.
• To detect the presence of phenol-containing compounds. Phenol is an organic compound that has a hydroxyl group attached to a benzene ring. It is widely used in the chemical industry and has antiseptic and disinfectant properties. Phenol can also be found in some natural products, such as salicylic acid and tannins. Phenol-containing compounds react with Millon’s reagent to form a red-colored complex.
• To differentiate tyrosine from other amino acids. Tyrosine is the only amino acid that contains a phenol group and gives a positive result to Millon’s test. Other amino acids do not react with Millon’s reagent and do not produce any color change. Therefore, Millon’s test can be used to distinguish tyrosine from other amino acids in a mixture or solution.

Millon’s test is based on the principle of nitrification of the phenol group in tyrosine, which then forms complexes with heavy metals like mercury. The reagent used for the test is called Millon’s reagent, and it consists of mercuric nitrate and mercurous nitrate that is dissolved in concentrated nitric acid.

In the test, the phenol group on the tyrosine molecule is nitrated by the nitric acid present in the reagent. The nitrated tyrosine then combines with the mercury ions in the solution to form a red-colored precipitate or solution.

In some proteins containing tyrosine, the initial reaction between mercuric nitrate results in a white or yellow colored precipitate. After the addition of nitric acid and heating, however, the residue turns red in color. Both of these results are considered positive results and indicate the presence of tyrosine in the solution.

The following chemical equation shows the reaction between tyrosine and Millon’s reagent:

$$\text{Tyrosine + Millon`s reagent} \rightarrow \text{Red complex + Nitrogen dioxide + Water}$$

The red complex is composed of mercury and nitrated tyrosine. The nitrogen dioxide is a brown gas that is released during the reaction.

The reaction is specific for tyrosine because it is the only amino acid that contains a phenol group. Other amino acids do not react with Millon’s reagent. However, other compounds that contain phenol groups, such as salicylic acid, also give a positive result to this test. Therefore, it is important to perform other tests to confirm the presence of protein or tyrosine in a sample.

The requirements for Millon’s test are as follows:

• Reagent: Millon’s reagent is the main reagent used for the test. It consists of mercuric nitrate and mercurous nitrate dissolved in concentrated nitric acid and distilled water. The reagent can be prepared by dissolving 160 grams of mercuric nitrate and 160 grams of mercurous nitrate in 400 ml of concentrated nitric acid solution and then adding 600 ml of distilled water to make up to 1000 ml. The formula can be adjusted to suit the performance parameters.

• Sample: The sample to be tested should contain tyrosine or tyrosine-containing protein. The sample can be a solution or a solid material. If the sample is solid, it should be finely powdered and dissolved in distilled water to make a 1% solution.

• Materials: The materials required for the test are test tubes, test tube stand, pipettes, and water bath.

The procedure of Millon’s test is as follows:

• Take about 2 ml of the sample solution or the 1% tyrosine solution in a test tube.
• Add about 2 ml of Millon’s reagent to the test tube and mix well.
• Observe the color change in the test tube. If a red or pink colored precipitate is formed immediately, it indicates a positive result.
• If no color change is observed, place the test tube in a water bath for about 2 minutes and observe again for the formation of the colored precipitate.
• Record the result as positive or negative based on the presence or absence of the colored precipitate.

• A positive result in the Millon’s test is demonstrated by the formation of a red or pink colored precipitate. This indicates the presence of tyrosine or tyrosine containing protein.
• A negative result in the Millon’s test is demonstrated by the absence of colored precipitate in the test tube. This indicates the absence of tyrosine or tyrosine-containing protein.
• The color of the precipitate may vary depending on the concentration and purity of the sample and the reagent. The color may range from light pink to brick red or brownish-red.
• The intensity of the color may also depend on the amount of tyrosine present in the sample. A higher concentration of tyrosine may produce a darker color than a lower concentration.
• The test is qualitative and does not provide any quantitative information about the amount of tyrosine or protein present in the sample.

Millon’s test is used for the following purposes:

• Detection of tyrosine-containing proteins in a given sample. Tyrosine is an essential amino acid that is involved in various biological processes such as protein synthesis, signal transduction, and enzyme catalysis. Tyrosine is also a precursor of several important molecules such as thyroid hormones, melanin, and dopamine. Therefore, detecting the presence of tyrosine-containing proteins can provide information about the metabolic and physiological status of the sample. For example, Millon’s test can be used to detect casein protein in milk or cheese, which is a major source of tyrosine for humans and animals.
• Differentiation of tyrosine from other amino acids. Tyrosine is the only amino acid that contains a phenol group, which makes it unique among the 20 standard amino acids. Millon’s test can be used to distinguish tyrosine from other amino acids based on its reaction with Millon’s reagent. Other amino acids do not react with Millon’s reagent or produce different colors. For example, tryptophan produces a violet color, histidine produces a yellow color, and cysteine produces a black color when treated with Millon’s reagent.
• Detection of protein found in raw meat. Millon’s test can be used to detect the presence of protein in raw meat samples. Protein is an essential nutrient for humans and animals, and it is composed of amino acids linked by peptide bonds. Tyrosine is one of the amino acids that are commonly found in animal proteins. Therefore, Millon’s test can be used to confirm the presence of protein in raw meat by observing the formation of a red precipitate. This can be useful for quality control and food safety purposes.

• Millon’s test is not a specific test for protein as it also detects the phenolic group present in other compounds as well. Therefore, while performing Millon’s test, it is essential that other tests like the Biuret test and Ninhydrin test also be performed to confirm the presence of protein.
• Compounds like salicylic acid and phenolic compounds give a positive result to this test; thus, any other phenol compounds that might be present in the test tube should be avoided or removed before performing the test.
• The presence of chlorine in the solution might interfere with the reaction; thus, the test cannot be performed on a sample containing chlorides. Chlorides can be removed by adding silver nitrate to the solution and filtering out the silver chloride precipitate.
• The formation of a white or yellow precipitate might be observed immediately after the addition of Millon’s reagent due to the denaturation of proteins by mercuric ions. This does not indicate a negative result, but rather an incomplete reaction. The precipitate should be dissolved by adding more nitric acid and heating the solution until a red color is obtained.
• Millon’s reagent is highly toxic and corrosive due to the presence of mercury and nitric acid. Therefore, proper precautions should be taken while handling the reagent and disposing of the waste. Gloves, goggles, and lab coats should be worn while performing the test. The waste should be collected in a separate container and treated with sodium sulfide to precipitate out the mercury ions.

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