Hopkins-Cole test (Adamkiewicz–Hopkins’ test)
(Detection of tryptophan)
It is also known as the glyoxylic acid reaction. This test used for detecting the presence of tryptophan in proteins.
Principle:
The indole group of tryptophan reacts with glyoxylic acid in the presence of cone H2S04 to give a purple colour. Glyoxylic acid is prepared by reducing oxalic acid with magnesium powder or sodium amalgam. Glacial acetic acid which has been exposed to the sunlight also contains glyoxylic acid and can thus be used for this test.
Reaction:
Reagents:
- Test reagent:
- To 10 g of magnesium powder add enough water to submerge it. Then slowly add 250 ml 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 1 L with water.
- Alternatively* glacial acetic acid which has been kept in sunlight for a few minutes may be used as a test solution.
- Test solutions: Prepare solution containing 2% individual amino acids such as tryptophan, tyrosine, phenylalanine, glutamic acid, etc.
- Cone H2SO4.
Procedure:
- To 1 ml of Hopkins-Cole reagent, add 1 ml of 2% amino acid solution.
- Pour 1-2 ml H2SO4 down the side of the sloping test tube.
- Observe the formation of purple color at the interface between the two layers.
Result Interpretation:
Image source: https://www.flickr.com/photos/gabriellevelasco/4867880667
Positive test: A purple ring appears between the two layers due to presence of tryptophan.
Negative test: No appearance of a purple ring between the two layers due to absence of tryptophan.
Note:
Nitrites, chlorates, nitrates and excess chlorides prevent the reaction from occurring
References:
- https://en.wikipedia.org/wiki/Hopkins%E2%80%93Cole_reaction
- http://www.biosciencenotes.com/adamkiewicz-hopkins-test/
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1258558/pdf/biochemj01203-0043.pdf
- https://www.oxfordreference.com/view/10.1093/oi/authority.20110803095944666
- https://www.onlinebiologynotes.com/adamkiewicz-reaction-hopkins-cole-test-objective-principle-reagents-procedure-and-result/
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