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Millon’s Reagent and its Preparation

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An Introduction to Millon's Reagent

Millon's test was discovered by the French Chemist Auguste Nicolas Eugene Millon. Millon’s test is predicated on the principle of nitrification of the phenol group in tyrosine, which then forms complexes with significant metals like mercury. A reagent may be a compound or mixture added to a system to begin or check a chemical change.

A reagent may be used to confirm the presence or absence of a selected chemical substance as the binding of reagents to the substance or different connected substances triggers bound reactions. The reagent used for the test is Millon’s reagent, consisting of metal nitrate of mercury and mercuric nitrate that is dissolved in concentrated nitric acid.

What is a Reagent?

In terms of chemistry, a reagent is an organic or inorganic substance that triggers a series of chemical reactions once added to a combination. Reagents are used to test the presence of different functional groups in a solution. This makes various types of reagents helpful for testing functions.

The term reagent means a chemical ingredient that, once added to an organic mixture, transforms that mixture into another variety of substances. A few examples of reagents used are:

1. Collin’s Reagent

This reagent may contain Chromium(IV) pyridine in dichloromethane. It's a solid red colour compound and extremely helpful in oxidisation.

2. Grignard Reagent

This cluster has the formula \[\text{RMgX}\] , wherever X is halogen and R is alkyl or aryl group.

3. Millon’s Reagent

A few drops of this chemical agent will detect the presence of soluble proteins in a test solution. Once this happens, the chemical agent turns reddish brown or precipitates are shaped to confirm the tyrosine residue's presence.

Millon’s Reagent

Millon's reagent is an analytical reagent used to notice the presence of soluble proteins. Some drops of the chemical agent are added to the test mixture and then heated gently. A reddish-brown colouration or precipitate indicates the presence of tyrosine residue in nearly all proteins.

\[\left( \text{Hg}{{\left( \text{N}{{\text{O}}_{\text{3}}} \right)}_{\text{2}}} \right)\] is the formula of Millon’s reagent. The preparation of Millon’s reagent is done by dissolving metallic mercury in acid and diluting it with water, forming mercurous nitrate \[\left( \text{Hg}{{\left( \text{N}{{\text{O}}_{\text{3}}} \right)}_{\text{2}}} \right)\]. within the test, the phenol functional group in the side chain of tyrosine gets nitrated, and the product then complexes with \[\text{Hg}\left( \text{I} \right)\] or \[\text{Hg}\left( \text{II} \right)\] ions to convey a red-coloured precipitate.

Millon’s Reagent

Millon’s Reagent

Composition of Millon’s Reagent

The Millon’s reagent composition is as follows:

  • Mercuric Nitrate

  • Mercurous Nitrate

  • Concentrated Nitric Acid

  • Distilled water

Principle of Millon’s Test

Millon’s test depends on the principle of nitrification of the phenol cluster in amino acid, which then forms complexes with significant metals like mercury. The agent used for the test is termed Millon’s agent.

In the test, the phenol functional cluster on the amino acid molecule is nitrated by the acid present within the agent. The nitrated amino acid then combines with the mercury ions within the mixture to make a red-coloured precipitate or solution.

In some proteins containing amino acids, the initial reaction between metal nitrate ends up in a white or yellow-coloured precipitate. With the addition of acid, followed by heating, the residue turns red. Each of these results is believed to have positive results and indicates the presence of amino acids within the solution.

The Objective of Millon’s Test

1. To observe the presence of tyrosine-containing proteins in a very given sample

2. To observe the presence of phenol-containing compounds

3. To entirely differentiate tyrosine from different amino acids

Materials Needed

  • Test tubes

  • Test tube stand

  • Pipettes

  • Water bath

The Procedure of Millon’s Test

1. A certain quantity of the sample mixture or the amino acid solution is taken within the tube.

2. Millon’s agent is added to the current mixture. The test tubes are then unbroken within the water bathtub for several minutes if the red-coloured precipitate isn't discovered directly.

3. The tubes are then observed for the formation of the coloured precipitate.

Millon’s Test

Millon’s Test

Millon’s test reactions are discussed as follows:

Positive Result: Millon’s test demonstrates a positive end by forming a red or pink-coloured precipitate. This suggests the presence of amino acids or amino acids containing supermolecules.

Negative Result: Millon’s test demonstrates a negative end by the absence of coloured precipitate in the tube. This suggests the absence of amino acids or tyrosine-containing supermolecules.

Uses of Millon's Reagent

1. Millon’s test is employed to detect tyrosine-containing proteins in an exceedingly given sample.

2. The test conjointly helps in the differentiation of amino acids from one another.

3. The test is helpful in the detection of casein macromolecules and also the macromolecules found in meat.

Limitations of Millon’s Test

1. Compounds like 2-hydroxybenzoic acid and phenoplast compounds provide a positive result for the present test; therefore, the other phenol compounds present within the tube should be avoided. Tests like the Biuret and Ninhydrin tests should be performed for confirmation.

2. The presence of gas within the solution may interfere with the reaction; therefore, the test can not be performed on a sample containing chlorides.

3. The formation of a white or yellow precipitate may well be discovered right away once the addition of Millon’s chemical agent because of the denaturation of proteins by mercurous ions.


Millon’s reagent and its preparation deal with the chemical composition of the reagent. A reagent is an organic or inorganic substance that triggers a series of chemical reactions once added to a combination. Reagents are used to test the presence of different functional groups in a solution. Millon's reagent is an analytical reagent used to notice the presence of soluble proteins. The limitations and uses of Millon’s test have been discussed in this article.

Last updated date: 03rd Oct 2023
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FAQs on Millon’s Reagent and its Preparation

1. What is the difference between reactant and reagent?

A chemical process is the amendment of reactants to produce a product. These reactants will be in solid half, liquid half, or gaseous half. The term agent is used to elucidate a form of reactants. Reagents area unit is other to a reaction mixture for the progression of the reaction. However, unlike reactants, reagents are not becoming another compound. Thus, the foremost distinction between chemicals and agents is that reactants are consumed in chemical reactions, whereas reagents are not consumed throughout the progression of a reaction.

2. What is a Grignard agent?

Grignard reagents are organomagnesium or organometallic compounds that have many applications in chemistry. They have significant nucleophilic activity, so they will be used to form the latest carbon-carbon bonds. Throughout this approach, it acts as a free agent similar to organolithium. Grignard chemical agents once reply with the cluster (alkyl or aryl) and ammeter group. The compound metre is termed Hauser base, and thus the compound intentionally could be a lot more nucleophilic than the Grignard chemical agent. Grignard reagents are performed inside the aprotic solvent.

3. What is amino alkanoic acid?

The amino alkanoic acid may be a type of chemical compound that is the building block of the molecule. The amino alkanoic acid contains an amino group and carboxylic acid. In the diet, amino alkanoic acid is going to be eaten in farm products, meats, fish, eggs, nuts, beans, oats, and wheat. The body uses this to make chemical messengers that are involved in conditions poignant the brain, like mental alertness. People most generally use this in molecule supplements for hereditary diseases observed as phenylketonuria (PKU).