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Hopkins Cole Test for Proteins

In this Hopkins Cole test for proteins post we briefly summarise about: principle, reagents requirements, hopkins cole test procedure, result, application and limitations of Hopkins Cole test.

Hopkins Cole Test for Proteins

Hopkins Cole test is a specialised test for detecting the indole ring in proteins, and consequently tryptophan. Because the reagent contains glyoxylic acid, the test is also known as the ‘glyoxylic acid test.’ 

Frederick Gowland Hopkins and Sydney W. Cole discovered the Hopkins Cole test in 1901 while working on the discovery of tryptophan. The Adamkiewez reaction, which is similar in theory to this test, is very similar to Hopkin’s Cole test. 

Rosenheim questioned test, claiming that the glyoxylic acid employed in it might quickly breakdown at high temperatures into formaldehyde and carbon dioxide. 

Rosenheim was the first to utilise formaldehyde to detect tryptophan. Hopkin’s Cole test is a colour reaction that can be used to detect specific amino acids or proteins based on the production of a specific colour. 

Hopkins Cole test detects the tryptophan amino acid’s indole ring, which aids in the identification of proteins that contain tryptophan.


Hopkins Cole test is based on the idea that when concentrated sulfuric acid is layered over a mixture of tryptophan-containing proteins with Hopkin’s Cole reagent, a violet ring forms at the interface. 

By acting on the indole ring of the tryptophan molecules, the glyoxylic acid given to the sample unites two tryptophan molecules. The resulting condensation product is dehydrated to produce a violet-colored pigment. 

The indole group of the tryptophan molecule, which is transformed into a colourful product by oxidation caused by the aldehyde group of glyoxylic acid, is responsible for the colour obtained in the test. 

The addition of H2SO4 to the reagent aids in the stabilisation of the glyoxylic acid, preventing its breakdown and the release of carbon dioxide.

Hopkin’s Cole Test



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



1. Hopkin’s Cole reagent: Glyoxylic acid- It can be prepared by exposing glacial acetic acid to sunlight for a few days.

2. Concentrated H2SO4

3. Sample: 0.1% amino acid solution


1. Test tubes

2. Test tube stand

3. Pipettes


1. Take 2-3 ml of test solution add 2 drops of 1/500 formaldehyde (HCHO) and 1 drop of 10% mercuric sulfate in sulfuric acid. Mix well. Add 3 ml of concentrated sulfuric acid through the sides of the test tube.


Hopkin’s Cole Test

Hopkins Cole Test for Proteins; Aldehyde test

Positive result: The creation of a purple-colored ring at the intersection of two layers indicates a positive result. This shows that tryptophan-containing proteins are present.

Negative result: The absence of a purple-colored ring in the test tube indicates a negative result. This suggests that tryptophan-containing proteins are not present.


1. Proteins and amino acids are detected in a sample with the help of Hopkins Cole test. The Hopkins Cole test is a basic and straightforward procedure for distinguishing tryptophan from other amino acids.


1. Compounds such as nitrites, chlorates, nitrates, and excess chlorides hinder the condensation product from forming.

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