Hopkin’s Cole Test- Definition, Principle, Procedure, Result, Uses

Proteins are one of the most important biomolecules in living organisms, as they perform various structural and functional roles. Proteins are composed of amino acids, which are the building blocks of proteins. Amino acids have different chemical properties and structures, which determine the characteristics of the proteins they form. One of the ways to study the properties and functions of proteins is to perform biochemical tests that can detect the presence or absence of specific amino acids or groups in proteins. These tests are based on the principle that different amino acids or groups react differently with certain reagents under specific conditions, resulting in a characteristic color change or precipitate formation.

One such test is the Hopkin’s Cole test, which is a specific test used for the detection of indole ring and thus, tryptophan in proteins. Tryptophan is one of the 20 standard amino acids that are found in proteins. It has a unique structure that contains an indole ring, which is a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring. The indole ring is responsible for some of the biological activities of tryptophan, such as its role as a precursor for serotonin and melatonin, which are neurotransmitters involved in mood regulation and sleep cycle.

The Hopkin’s Cole test is also known as the glyoxylic acid test, as the reagent used in the test contains glyoxylic acid. The test was discovered by Frederick Gowland Hopkins and Sydney W. Cole in 1901 as a part of their work on the discovery of tryptophan. The test is one of the color reactions used for the detection of particular amino acids or proteins on the basis of the formation of a specific color. The test detects the indole ring that is found in tryptophan amino acid, which in turn helps in the identification of proteins containing tryptophan.

In this article, we will discuss the definition, history, principle, procedure, result, uses and limitations of Hopkin’s Cole test in detail.

Hopkin’s Cole test is a specific test used for the detection of indole ring and thus, tryptophan in proteins. The test is also termed as ‘glyoxylic acid test’ as the reagent contains glyoxylic acid. The test was discovered by Frederick Gowland Hopkins and Sydney W. Cole in 1901 as a part of their work on the discovery of tryptophan.

Tryptophan is one of the essential amino acids that cannot be synthesized by humans and must be obtained from the diet. It is also a precursor of serotonin, melatonin, and niacin. Tryptophan has an indole ring in its structure, which is a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring.

Hopkin’s Cole test is based on the reaction of indole ring with glyoxylic acid in the presence of concentrated sulfuric acid. The test produces a violet-colored compound that can be observed at the interface of two liquid layers. The test is extremely sensitive and can detect as little as 1 mg of tryptophan.

Hopkin’s Cole test is one of the color reactions used for the detection of particular amino acids or proteins on the basis of the formation of a specific color. The test detects the indole ring that is found in tryptophan amino acid, which in turn helps in the identification of proteins containing tryptophan.

The test was named after its discoverers, Frederick Gowland Hopkins and Sydney W. Cole, who were British biochemists and physiologists. Hopkins was awarded the Nobel Prize in Physiology or Medicine in 1929 for his discovery of essential amino acids and their role in nutrition. Cole was his colleague and collaborator at Cambridge University. They published their findings on tryptophan and Hopkin’s Cole test in 1901 in the Journal of Physiology.

The main objectives of Hopkin’s Cole test are:

• To detect the presence of indole ring containing amino acid in proteins. The indole ring is a characteristic feature of tryptophan, which is one of the essential amino acids for humans and animals. Tryptophan is involved in various biological processes, such as protein synthesis, neurotransmitter production, and melatonin secretion. Therefore, detecting the presence of tryptophan in proteins can help to identify the source and function of the proteins.
• To detect the presence of tryptophan-containing proteins. Some proteins, such as albumin, casein, and gelatin, contain tryptophan as one of their amino acid residues. These proteins have different roles in the body, such as maintaining fluid balance, transporting substances, and providing structural support. By performing Hopkin’s Cole test on these proteins, one can confirm their identity and purity.

The test is based on the principle that the layering of concentrated sulfuric acid over a mixture of tryptophan-containing proteins with the Hopkin’s Cole reagent results in the formation of a violet ring at the interface. The Hopkin’s Cole reagent contains glyoxylic acid, which is prepared by exposing glacial acetic acid to sunlight for a few days. The glyoxylic acid acts on the indole ring of the tryptophan molecules and combines two of them to form a condensation product. The condensation product then undergoes dehydration to form a violet colored pigment. The color developed in the test is due to the indole group of the tryptophan molecule, which is converted into a colored compound by oxidation brought about by the aldehyde group of glyoxylic acid. The H2SO4 added to the reagent helps to stabilize the glyoxylic acid and prevent its decomposition and the release of carbon dioxide.

The reaction involved in the test can be summarized as follows:

Tryptophan + Glyoxylic acid → Condensation product + H2O → Violet-coloured compound + H2O

The test is specific for tryptophan as it is the only amino acid that contains an indole ring. Other amino acids do not react with glyoxylic acid and do not produce any color change in the test.

The Hopkin’s Cole test involves a chemical reaction between the indole ring of tryptophan and the aldehyde group of glyoxylic acid. The reaction can be summarized as follows:

Tryptophan + Glyoxylic acid → Condensation product + H2O → Violet-coloured compound + H2O

The mechanism of the reaction is as follows:

• The glyoxylic acid acts as an electrophile and attacks the electron-rich indole ring of tryptophan. This forms a carbon-carbon bond between the two molecules and releases a water molecule.
• The condensation product thus formed undergoes dehydration in the presence of concentrated sulfuric acid. This removes another water molecule and forms a double bond between the two carbon atoms.
• The dehydration product is then oxidized by the aldehyde group of glyoxylic acid. This forms a violet-colored compound that has a quinoidal structure.

The reaction is shown in the figure below:

\begin{figure}
\centering
\includegraphics{hopkins-cole-reaction.png}
\caption{Hopkin`s Cole reaction mechanism}
\label{fig:hopkins-cole-reaction}
\end{figure}

The violet-colored compound is responsible for the color change observed in the test. The color intensity depends on the concentration of tryptophan in the sample. The more tryptophan present, the more violet-colored compound formed, and the darker the color.

The Hopkin’s Cole test is a specific test for tryptophan because only tryptophan has an indole ring that can react with glyoxylic acid. Other amino acids do not have this structure and do not give a positive result in this test.

To perform the Hopkin’s Cole test, the following reagents and materials are required:

• Reagents
  • Hopkin’s Cole reagent: This is a solution of glyoxylic acid, which can be prepared by exposing glacial acetic acid to sunlight for a few days. Glyoxylic acid is the main reagent that reacts with the indole ring of tryptophan to form a violet-colored compound.
  • Concentrated sulfuric acid (H2SO4): This is added along the sides of the test tube to form a distinct layer over the mixture of Hopkin’s Cole reagent and the sample. Sulfuric acid helps to stabilize the glyoxylic acid and prevent its decomposition and the release of carbon dioxide. It also acts as a dehydrating agent to facilitate the formation of the violet-colored compound.
• Sample: The sample can be any protein or amino acid solution that contains tryptophan. The concentration of the sample should be around 0.1% for optimal results.
• Materials
  • Test tubes: These are used to hold the reagents and the sample for the test. The test tubes should be clean and dry before use.
  • Test tube stand: This is used to hold the test tubes in a slanting position during the test. The test tube stand should be stable and secure to prevent any spillage or accidents.
  • Pipettes: These are used to measure and transfer the reagents and the sample into the test tubes. The pipettes should be clean and calibrated before use. Different pipettes should be used for different reagents and samples to avoid cross-contamination.

The procedure of Hopkin’s Cole test is as follows:

• In a test tube, take 2 ml of light-exposed glacial acetic acid and 2 ml of the sample liquid. The sample liquid can be a 0.1% solution of amino acid or protein.
• Carefully add concentrated H2SO4 along the sides of the test tube held at a slanting position. Make sure that two distinct layers of liquid are formed without mixing. The sulfuric acid layer should be at the bottom and the acetic acid layer should be at the top. Note: Mouth of the tube must point away from the face, as we should be careful while adding the sulfuric acid.
• Observe the test tube for the formation of a colored ring at the interface of two layers. The ring should appear within a few minutes if the sample contains tryptophan.

The procedure can be summarized in the following table:

The result of the Hopkin’s Cole test depends on the formation of a purple-colored ring at the interface of two layers of liquid in the test tube. The color is due to the oxidation of the indole ring of tryptophan by glyoxylic acid in the presence of concentrated sulfuric acid. The test can be interpreted as follows:

• Positive result: A positive result is indicated by the appearance of a purple-colored ring at the junction of two layers. This means that the sample contains tryptophan or proteins with tryptophan residues. The intensity of the color may vary depending on the concentration of tryptophan in the sample. A more intense color indicates a higher amount of tryptophan.

• Negative result: A negative result is indicated by the absence of any color change in the test tube. This means that the sample does not contain tryptophan or proteins with tryptophan residues. Alternatively, it may also mean that the reagents or the procedure were not followed correctly or that some interfering substances were present in the sample.

The Hopkin’s Cole test is a qualitative test that can only detect the presence or absence of tryptophan in a sample. It cannot measure the exact amount or concentration of tryptophan. For quantitative analysis, other methods such as spectrophotometry or chromatography are required.

• The test is used for the detection of proteins and amino acids in a sample. The test can help to identify the presence or absence of tryptophan in proteins by the formation of a characteristic violet ring. The test can also be used to estimate the amount of tryptophan in a sample by comparing the intensity of the color with a standard curve.
• The test is a simple and easy-to-perform test that helps to identify tryptophan from other amino acids. The test does not require any sophisticated equipment or reagents, and can be performed in a laboratory with basic facilities. The test is also rapid and gives results within minutes.
• The test is a specific and sensitive test that can detect even small amounts of tryptophan in a sample. The test is based on the unique indole ring structure of tryptophan, which reacts with glyoxylic acid to form a colored compound. The test does not give false-positive results with other amino acids or compounds that do not have an indole ring.

• Hopkin’s Cole test is a specific test that only detects the presence of tryptophan in proteins. It cannot differentiate between different types of proteins or amino acids that do not contain tryptophan.
• Hopkin’s Cole test is sensitive to the concentration and purity of the reagents used. The glyoxylic acid must be freshly prepared and exposed to sunlight for a few days before use. The sulfuric acid must be concentrated and added carefully to avoid mixing with the sample layer.
• Hopkin’s Cole test can be affected by the presence of other compounds in the sample that can interfere with the reaction or the color formation. Compounds like nitrites, chlorates, nitrates, and excess chlorides can prevent the formation of the condensation product or mask the purple color.
• Hopkin’s Cole test is a qualitative test that only indicates the presence or absence of tryptophan in proteins. It does not provide any quantitative information about the amount or concentration of tryptophan in the sample.

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