[1] 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.
[2][3] Nitrites, chlorates, nitrates and excess chlorides prevent the reaction from occurring.
[4] The reaction was first reported by Frederick Gowland Hopkins and Sydney W. Cole in 1901,[5] as part of their work on the first isolation of tryptophan itself.