Chromatographic Separation of Amino Acids

235750 A mobile phase for use in the chromatography was prepared by mixing 60cm3 of ethanenitrile (acetonitrile) with 40cm3 of 0.1M ammonium ethanoate. Drops of concentrated ethanoic acid were used to adjust the pH value to 7.2 after which 30cm3 of this mixture was placed in a tall form beaker and cover it with a watch-glass. Small amounts of trypsin, leucine, valine, proline, glutamic acid, glycine, and lysine amino acid solutions were prepared in water and each amino acid labeled. A faint line was drawn using a pencil on the lantern-type chromatography paper making sure that the line was above the level of the solvent in the beaker. Using a clean capillary tube each time, one small spot of each amino acid solution was put on the chromatography paper and each spot identifies and marked. An unknown sample was also spot on the paper and marked. The spots were dried using a warm air blower. The chromatographic paper was then bent around the cylinder and fixed in this shape using a paperclip. The chromatographic paper was then placed inside the beaker so that it stood on the edge without touching the sides and the chromatogram run until the solvent front moved a three-quarter way. The results above indicate that the unknown sample X is glycine. This is due to the similar Rf values of 0.64. Sample Y was identified as proline. This conclusion was arrived at by looking at the color of the spot and also the Rf values. The difference in the Rf values 0.75 and 0.76 could have been due to error in measurement.