In the early stage of Hopkins-Cole test discovery it was confirmed that tryptophan is capable to give two distinct colored products when reacted with glyoxylic acid : With the minimum amount of the aldehyde and temperature kept below 15 o C, the first colored product is carmine. The mechanism of its formation is assumed to follow the scheme postulated in Figure . Here, the aldehyde unites the carbon atoms of the benzene rings of two tryptophan molecules to form the leuco compound, this upon oxidation forms the quinonoid configuration in the molecule responsible of the color (pigment compound (1)); the same product that appears in eq. ( ). Such suggested pathway requires the presence of a strong condensing agent like concentrated H 2 SO 4 or pure acetic acid, and a mild oxidant like Fe +3 aq . )) that appears then violet to blue; depending on water content; blue only in its solid form. Thus, the firstly formed pigment compound (1) condenses further with aldehydes of two more glyoxylic acids. This condensation, nevertheless, occurs between the nitrogen of side-chain and the carbon atom of the indole nucleus in same tryptophan molecule. It leads consequently to closure of the tryptophan side-chain and formation of a pyridine nucleus, . Such colored product might be formed according to the pathway speculated in Figure .