Hopkins Cole Test: Principle, Reaction, Reagents And Result Interpretation

What Is Hopkins Cole Test?

Hopkins Cole Test also referred to as glyoxylic acid reaction is one of the qualitative test methods to determine differences in types of proteins and amino acids. This test is used to identify the presence of the amino acid tryptophan in proteins; tryptophan is the only amino acid that has an indole ring group.

The reaction was first reported by Frederick Gowland Hopkins and Sydney W. Cole in 1901, as part of their work on the first isolation of tryptophan itself. In this test, a protein solution is mixed with Hopkins Cole reagent, which consists of glyoxylic acid. Concentrated sulfuric acid is slowly added to form two layers. A purple ring appears between the two layers if the test is positive for tryptophan.

Tryptophan belongs to the group of essential amino acids. Tryptophan is a precursor of niacin vitamin and an introduction to the serotonin nerve. Tryptophan functions to maximize the use of vitamin B complex, improve nerve health, stabilize emotions, increase feelings of calm, prevent insomnia and increase the release of growth hormone. One of these amino acids can be found in egg whites.


  • To detect the presence of indole ring containing amino acid in proteins.
  • To detect the presence of tryptophan containing proteins.

Principle Of Hopkins Cole Test

In this test, Hopkins Cole reagent which contains glyoxylic acid (C2H2O3) reacts with sulphuric acid (H2SO4), the tryptophan in the protein solution will be condensed with the aldehyde group (Indole group) on the glyoxylic acid with the help of strong oxidizing sulphuric acid (H2SO4). Positive result is indicated by the formation of purple rings between 2 separate layers. Glyoxylic acid is prepared by reducing oxalic acid with magnesium powder or sodium amalgam. Glacial acetic, which has been exposed to the sunlight also contains glyoxylic acid and thus can be used for this test. In this test, sulphuric acid is added to the reagent to stabilize the glyoxylic acid and prevent its decomposition and release of carbon dioxide. It is also important to note that, presence of nitrates, chlorates, nitrites and excess chlorides prevent this reaction from occurring.


Reagent And Material Required


  1. Test Reagent
  2. Dissolve 10g of magnesium powder in enough distilled water. Then, slowly add 250ml of cold saturated oxalic acid solution. Keep cool. Filter magnesium oxalate formed. Acidify the filtrate with 25 ml of glacial acetic acid and dilute it to 1liter with water. Alternatively, glacial acetic acid which has been kept in sunlight for a few minutes may be used as a test solution.
  3. Test solutions: prepare a solution containing 2% individual amino acids such as tryptophan, tyrosine, phenylalanine, glutamic acid etc.
  4. Concentrated sulphuric acid (H2SO4).

Material Required

  • Test tube
  • Test tube stand
  • Pipettes

Procedure For Hopkins Cole Test

  1. Take 2ml of the test solution in a dry clean test tube.
  2. Also, take 1ml of distilled water in another test tube as control.
  3. Add 1ml of Hopkins Cole reagent and mix thoroughly.
  4. Then, add 1ml of concentrated sulphuric acid (H2SO4) along the wall of the test tube.
  5. Observe the test tube for color development.

Hopkins Cole Test Result Interpretation

  • Positive Result: A positive result is indicated by formation of a purple-color ring at the junction of two layers. This confirms the presence of tryptophan-containing proteins.
  • Negative Result: A negative result is indicated by the absence of a purple-color ring in the test tube. This confirms the absence of tryptophan containing proteins.


  • Nitrates, chlorates, nitrites and excess chlorides prevent this reaction from occurring.