Qualitative Analysis of Amino Acids - Amrita University

Amino acids are fundamental to life, serving as protein building blocks and metabolic intermediates. They contain an amino group, a carboxylic acid group, and a variable side chain. This video outlines ten qualitative chemical tests: Ninhydrin, Xanthoproteic acid, Pauly's Diazo, Millon's, Histidine, Hopkins Cole, Sakaguchi, Lead Sulphide, Folin's McCarthy Sullivan, and Isatin tests, to identify amino acids in solution based on color changes or precipitation reactions.

The experiment requires various reagents including Ninhydrin, 40% sodium hydroxide, concentrated nitric acid, 1N hydrochloric acid, sulfanilic acid, sodium nitrite, sodium carbonate, Millon’s reagent, sodium nitrate, bromine, ammonium carbonate, acetic acid-glyoxylate reagent, concentrated sulfuric acid, alpha-naphthol, hypobromite solution, urea, lead acetate, glycine, 10% sodium nitroprusside, and isatin. Laboratory equipment such as test tubes, holders, water baths, spatulas, droppers, marble chips, Bunsen burners, filter paper, and iceboxes are also needed.

To perform the Ninhydrin test, add a few drops of an unknown solution and Ninhydrin reagent to a test tube. Heat it in a boiling water bath for 5 minutes. A blue color indicates the presence of alpha-amino acids, while a yellow color suggests imino acids.

For the Xanthoproteic acid test, add 1 ml of the unknown solution and a few drops of concentrated nitric acid in a fume hood. Add marble chips to prevent bumping during boiling. Heat the mixture, then cool it. Add a few drops of 40% NaOH; an orange color indicates aromatic amino acids.

In the Pauly’s Diazo test, combine an unknown sample, prechilled sulfanilic acid, and prechilled sodium nitrite in an ice bath. Then, add sodium carbonate solution dropwise. The appearance of a red color signifies the presence of histidine and tyrosine.

To conduct the Hopkins Cole test, mix 1 ml of the unknown solution with 1 ml of acetic acid-glyoxylate reagent. Carefully add a few drops of concentrated sulfuric acid down the side of the test tube. A purple-violet ring indicates the presence of tryptophan.

For the Lead Sulphide test, mix 1 ml of unknown amino acid solution with a few drops of NaOH solution and marble chips. Heat for 5-10 minutes, then cool. Add a few drops of 10% lead acetate solution; a black precipitate signifies cysteine.

The Isatin test involves applying a drop of unknown amino acid solution onto a filter paper strip, drying it, and then applying a drop of isatin reagent. Drying again will reveal a blue spot if imino acids are present.

For Folin’s McCarthy Sullivan’s test, combine 1 ml of amino acid solution with 40% NaOH, glycine, and sodium nitroprusside. Heat at 40°C for 15 minutes. Add 0.5 ml of 6N HCl; a red color indicates methionine.

In the Sakaguchi test, add prechilled unknown amino acid, 40% NaOH, alpha-naphthol, 5% urea, and hypobromite solution to a test tube. A red color change in the solution indicates the presence of arginine.

For the Histidine test, mix 2 ml of unknown amino acid with 5% Bromine in 33% acetic acid. Let it stand for 10 minutes, then add 2 ml of ammonium carbonate solution. Heat in a boiling water bath for 5 minutes; a blue color confirms histidine.

To perform Millon’s test, add 1 ml of amino acid solution to Millon’s reagent. Heat for 3-5 minutes, cool, and then add a few drops of concentrated nitric acid. A red color indicates the presence of tyrosine.