In this exercise, students identify two unknown amino acids by comparing their behavior with that of seven known amino acids both by paper chromatography and in chemical tests. I selected the latter for simplicity and economy in demonstrating chemical reactions characteristic of functional groups specific to the various amino acids that are used. They include tests for primary amino groups, amides, substituted benzene rings, and thiol groups. Known amino acids are 1 mg/mL solutions of alanine, cysteine, glutamine, leucine, lysine, proline, and tryptophan; unknowns contain 1 mg/mL each of two of these (pairs which are readily distinguished by the chromatography and/or the chemical tests).

For primary amines, 0.5 mL of ninhydrin solution (4 mg/mL in water) is mixed with 2 drops of each amino acid solution. The tubes are heated in a boiling water bath for 5 minutes and colors are recorded.

For amides, 0.5 mL of 20% NaOH is mixed with 5 drops of each amino acid solution. A "chimney" is prepared by inserting a 5 3/4" Pasteur pipet into a rubber stopper which is then inserted into the test tube. A strip of wet red litmus paper is pushed into the end of the pipet so that it samples vapors emitted from the tube. After heating in a boiling water bath for 5 minutes (and being careful to keep the litmus paper wet), evolution of ammonia from side-chain amides turns the litmus blue.

For substituted benzene rings, 5 drops of each amino acid solution are mixed with 4 drops of concentrated nitric acid and heated in a boiling water bath for 3 minutes. Addition of 0.5 mL of 20% NaOH results in a deep yellow or orange color in a positive test.

For thiol groups, 2 drops of amino acid solution are mixed with 0.5 mL of 1 mg/mL DTNB [5,5'-dithiobis(2-nitrobenzoic acid)] and allowed to stand for a couple minutes. A positive test is a pronounced deepening of the reagent's pale yellow color. This reagent is a little tricky to prepare; DTNB requires neutralization with NaOH to get it into solution. Too much NaOH causes a sudden deepening of the yellow color so that a positive reaction is masked; too little NaOH and the reagent isn't alkaline enough to react quickly with thiol groups. To prepare it, I add a pinch of monobasic potassium phosphate, then adjust the pH to 6.5-7.

Paper chromatography is done using Whatman #1 chromatography paper. Students are given a 5 x 8 inch piece and asked to draw a pencil line 2 cm from and parallel to a long side. They make marks at 3, 5, 7, 9, 11, 13, 15, and 17 cm from one end for applying samples. Samples applied should be small drops, giving a wet spot 1/4 to 3/8 inch in diameter. The amount drawn into a 25-microliter capillary, perhaps two to three microliters, is just right. The ends of the paper are stapled together without touching and the cylinder is placed into a wide-mouth quart jar containing 15 mL of solvent mixture (1-butanol, acetic acid, and water in 3:1:1 ratio). It takes well over an hour for the solvent to rise to near the top of the paper, when students remove them from the jars, mark the solvent front, and place them in a hood to dry. Later, I spray the papers with 2 mg/mL ninhydrin in 95% ethanol and place them in a 120-degree oven for an hour or two to develop the colors.

Results are pretty much what you would expect with a couple exceptions. I found that cysteine and lysine didn't give a strong positive reaction with ninhydrin in the test tubes; the result for proline is yellow as expected. If you use tyrosine for a known, it's necessary to make it alkaline to stay in solution; the alkaline solution gives a false positive in the DTNB test. Alanine, lysine, and leucine don't give any identifying results in the test tube reactions, but are well separated by the chromatography. Cysteine gives two spots on the chromatogram, presumably due to the presence of its oxidized form, cystine; I'm not sure whether the cysteine I used was impure or whether cysteine oxidizes so readily in water that it's difficult to avoid unless made very fresh.

At the end of the exercise, students are asked to calculate Rf values for all the knowns and their unknowns. They are asked to draw the molecular structures of the known amino acids; to discuss the relationship they observed between Rf and side chain polarity; to consider the special reaction of cysteine's thiol group and to suggest why there were two spots for cysteine; and to indicate what results they expected for each test tube reaction and whether results were as they expected. Finally, they are asked to identify their unknown based on all their results.

Revised 6/12/07