References: Boyer, 227-233; E. D. Moffat and R. Lytle, Anal. Chem., 31(1959) 926-28; D. T. N. Pillay and M. Rafia, J. Chromatogr. 47 (1970) 121; E. S. Gankina, J. Planar Chromatogr., ! (1958) 364-66. Much of this experiment was developed by Robin Anderson in her senior research in the spring of 2001.

Although this procedure should theoretically be an easy one, the fact that there are so many methods in use indicates that there is no entirely satisfactory one. Multiple reasons for this difficulty can be cited. The properties of many amino acids are so similar that their separation becomes difficult. The amino acids have no chromophore and must therefore be derivatized or detected by some color producing reagent.

All the commonly used methods have drawbacks. Ion exchange chromatography is very slow and demands expensive equipment that can’t be used for much else. High performance liquid chromatography (HPLC) is slightly faster, but requires a good instrument with a gradient system. Again, no one solvent system seems totally satisfactory and separations are still quite time consuming. Two dimensional thin layer chromatography has been used with many systems, but the main drawback has been the difficulty of running standards. Boyer advocates a method that uses a two sided polyamide plate where the unknown is run on one side and the standards on the other. Unfortunately, these plates are no longer available in North America.

The attachment of chromophoric group to one end of a peptide or protein and subsequent hydrolysis and chromatography is conceptually simple. Nevertheless the separation of these derivatives becomes quite difficult because the addition of a large chromophoric group obscures the small differences between amino acids.

Introduction: This experiment is really two procedures which will be performed in tandem. The first is to determine the amino acids present in the unknown dipeptide by hydrolysis followed by thin-layer chromatography in two directions. The second procedure will be an end-group analysis to determine which of the amino acids is in the first position, that is, at the free amino acid end of the dipeptide. This is accomplished by reacting the dipeptide with dansyl (4-dimethyl-aminonaphthalene-5-sulfonyl) chloride which adds the dansyl group to the first amino acid in the dipeptide. This is followed by hydrolysis and one-dimensional chromatography which separates dansyl derivatives into several groups. A second one-dimensional separation identifies the members of the group. The advantage of using the dansyl derivatives is that they fluoresce under ultraviolet light. These reactions are summarized on the next page.

Some problems inherent to this method: Some amino acids present extra difficulties which demand special procedures for their detection. Asparagine, glutamine and tryptophane do not survive acid hydrolysis. Sulfur-containing amino acids (cysteine and methionine) must be protected from oxidation. Leucine and isoleucine are so similar that special methods are usually employed for their separation.

To simplify our problem we will limit the amino acids in the unknown dipeptides to alanine (A), arginine (R), aspartic acid (D), glutamic acid (E), glycine (G), histidine (H), leucine (L), isoleucine (I), lysine (K), phenylalanine (F), proline (P), serine (S), threonine (T), tyrosine (Y), and valine (V).

1. Hydrolysis of the dipeptide: The unknown is divided into two equal parts, one to be used in this reaction, the other to be used in part 2 below.

One dipeptide sample in placed in a 1.5 ml plastic micro centrifuge tube and dissolved in 0.5 ml of 6 N hydrochloric acid. This is capped and allowed to sit until the next week.

2. Dansylation of the dipeptide: The second portion of the unknown dipeptide is placed in a 1.5 ml plastic micro centrifuge tube and dissolved in 0.5 ml of 0.2 M sodium bicarbonate solution. Dansyl chloride solution (0.2 ml) is added, the tube is closed and incubated at 40° for one hour.

3. Hydrolysis of the dansyl derivative: At the end of the reaction time the tube is opened and the acetone evaporated to leave a gelatinous product. To this is added 0.5 ml of 6 N hydrochloric acid. If this does not dissolve the solid, 0.5 ml of acetone can be added to bring everything into solution. The tube is capped and allowed to sit until the next week.

4. Work up of the amino acid mixture: The hydrolysis mixture (from part 1 above) is placed in a warm water bath and a stream of nitrogen is used to evaporate the liquid. When only solid remains it is dissolved in 0.5 ml of methanol and again evaporated. The sample is then dissolved in 0.1 ml of 0.3 M hydrochloric acid and chromatographed on a 10 x 10 cm silica gel HPTLC plate. The spotting is placed 1 cm from the two edges in the lower left corner.

5. Chromatography of the hydrolysis products: The solvent for the first direction is methanol, pyridine, water (4:1:1). The plate is placed in a vapor saturated tank with the spot at the lower left. The plate is left to develop until the solvent reaches the top of the plate (about). The plate is then removed and dried in a vacuum oven for 30 minutes.

The plate is then chromatographed in the second solvent (phenol, 0.06 borate buffer-pH 9.3) (4:1) for two hours. The plate is then removed, air dried and sprayed with the Moffat-Lytle mixture which contains ninhydrin, acetic acid, 2,4,6-collidine, and copper nitrate in absolute ethanol.

Amino acid	R_f x 100, 1^st direction	R_f x 100, 2^nd direction	color
Ala	46	26	brown-purple
Arg	11	11	purple
Asp	54	7	blue-purple
Glu	59	15	purple
Gly	38	22	brown-purple
His	18	16	brown-purple
Ileu	62	65	purple
Leu	62	65	purple
Lys	11	4	purple
Phe	63	61	pink-purple
Pro	48	56	yellow
Ser	37	16	purple
Thr	46	29	purple
Tyr	63	48	pink-purple
Val	59	43	purple

This system does not separate leucine and isoleucine. This can be easily done by one-dimensional chromatography on ordinary silica gel 10 x 3 cm plates using solvent D as listed below. Samples of unknown, leucine, and isoleucine should be spotted.

6. Confirming your choice: Because in any system the differences between positions of amino acids are often quite small, it is a good idea to check out your decision in another system. This is easily accomplished by one-dimensional TLC. On a 10 cm silica gel plate place a spotting of the unknown and the known amino acid that you believe to be identical. Run the plate in the solvent system indicated below. These systems have been chosen because the amino acid in question has a unique R_f value in the system. Identical R_f values will confirm your decision. A difference in R_f values shows that the decision was incorrect.

7. Work up of the dansylation product: The centrifuge tube is placed in a warm water bath and a stream of nitrogen is used to evaporate the liquid. When only solid remains it is dissolved in 0.5 ml of methanol and again the liquid is evaporated. Finally 0.2 ml of methanol is added and the tube is vortexed to dissolve the solid. The liquid, even though some solid is present, is used in the chromatography below.

8. Thin-layer chromatography of the dansyl derivatives: The separation of dansyl-amino acids is done on high performance silica gel plates with spotting of the dansylation product and the standard mixture. Two runs are made in acetone-isopropyl alcohol-25% aqueous ammonia (9:7:1). The plate must be thoroughly dried between runs. The plate is viewed under uv light and the positions of known and unknown compounds noted. Di-DNS-Lys, DNS-Ile, DNS-Thr, and DNS-Ser can be identified immediately.

Figure 1: TLC of Dansyl-amino acids and the standard set of dansyl amino acids twice developed in acetone-isopropyl alchohol-25% ammonium hydroxide (9:3:1)
of dansyl amino acids twice developed in acetone-isopropyl alcohol-25% ammonium hydroxide (9:7:1).

Those amino acids that are found in groups 3, 5, 7, and 8 are chromatographed in chloroform-benzyl alcohol-ethyl acetate-glacial acetic acid (6:4:5:0.2) using an appropriate standard. Those in groups 1 and 2 are chromatographed in chloroform-ethyl acetate-glacial acetic acid (38:4:2.8) as eluent.

Figure 2: Groups 3,8,7, and 5 chromatographed in chloroform-benzyla alcohol-ethyl acetate-glacial acetic acid (6:4:5:0.2)

Figure 3: Groups 1,2, and 4 chromatographed in chrloroform-ethyl acetate-glacial acetic acid (38:4:2.8)

Solution I (25 parts): 100 mg ninhydrin in 50 ml absolute alcohol, 10 ml glacial acetic acid, 2 ml 2,4,6-colidine.

Solution II (1.5 parts): 1 g cupric nitrate trihydrate in 100 ml of absolute alcohol.