Ninhydrin Reaction: Ninhydrin has a polar carbonyl carbon with is electron deficient.

It is attacked by the nucleophilic nitrogren on an amino acid, temporarily combining the ninhydrin and amino acid molecule. The structure is rearranged until the origionally attacked carbon is protonated and leaves in the form of water. This creates a schiff base when the nitrogen is double bonded to the origionally attacked carbon. This molecule rearranges again so that the nitrogen is double bonded to the adjacent carbon of the amino acid. This last rearrangement produces carbon dioxide gas. It should react to this reagent. However it must be HEATED before it will react. This is due to the fact that when Ninhydrin is heated it stabilizes and the reacts with the -NH2 groups on the amino acid. Further rearrangement of the product produces ruheman's purple.

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Ninhydrin reacts with amino acids in proteins at high temperature giving a purple colored complex.

Biuret Test: Biuret test is used for detecting the presence of peptide bonds. It relies on the reduction of copper(II) ions to copper(I), the latter form a complex with the nitrogens of the peptide bonds in an alkalin esolution. A violet color indicates the presence of proteins. Two peptide bonds at least are required for the formation of this complex. The name of the test comes from the compound biuret, which gives a typically positive reaction. Biuret is obtained by heating urea to about 180 C. In a kind of chelation reaction a blue color is formed with Cu 2+.

In this experiment, alkaline copper sulphate reacts with compounds containing two or more peptide bonds to give the violet-colored complex. The depth of the color obtained is a measure of the number of peptide bonds present in the protein. The reaction is not absolutely specific for peptide bonds, since the compounds containing two carbonyl groups linked through hydrogen or carbon atom will give a positive result. Xanthroproteic: Xanthoproteic comes from the Greek word xanthos which means yellow. Boiling concentrated nitric acid reacts with tyrosine, tryptophan and phenylalanine (phenylalanine give weakly positive reaction as it contain an inactive benzene ring) to yield yellow products. The intensity of yellow color deepens upon the addition of alkaline solution that form orange colored salt in the basic medium.

Millon-Nasse: Compounds containing the hydroxybenzene radical react with millons reagent to form red complexes. The only phenolic amino acids are tyrosine and its derivatives and only these amino acids give a positive reaction. The original Millons reagent was a solution of mercuric nitrate in 50% v/v nitric acid, but modifications are now used that are less liable to interference from organic salts. Hopkins-Cole: The hopkins-cole test is used to determine the presence of the amino acid tryptophan. Tryptophan has an indole nucleus which is responsible for the violet ring found at the junction between the two layers. The indole group of tryptophan reacts with glyoxylic acid in the presence of conc. Sulphuric acid to give a purple color. Glacial acetic acid that has been exposed to the light contains glyoxilic acid. Also protein can be detected by this test as concentrated H2SO4 at the solution interface hydrolyses protein to form free tryptophan, which reacts with glyoxylic acid to form violet-purple complex.

Bromine Water: Pauly: The basic principle involved in pauly's test is diazotization. The sulphanilic acid gets diazotised in the presence of sodium nitrite and sodium carbonate with the sample. Diazotized sulphanilic acid reacts with the phenol group of tyrosine, and imidazole ring of histidine to form highly-colored azo compounds. This test reacts with histidine to give a red color and with tyrosine to give an orange color. Tryptophan gives yellow color. The diazonium compounds are only formed in the cold, so all solutions are cooled in ice before diazotization. Lead Acetate: Principle: Proteins containing sulfur (in cysteine and cystine) give a black deposit of lead sulfide (PbS) when heated with lead acetate in alkaline medium. Sakaguchi: The Sakaguchi test is a specific qualitative test for the detection of a specific type of protein with the amino acid containing the guanidinium group. In basic conditions, alpha naphthol and sodium hypobromite/chlorite react with the aforementioned compound to form red-orange complexes. In other words, The only amino acid containing the guanidine group is arginine, and this reacts with naphthol and an oxidizing agent bromide water to give a red color.