After a protein has been hydrolysed, the mixture of amino acids can be separated and identified by chromatography, usually paper chromatography or thin-layer chromatography (TLC).A pencil baseline is drawn near the bottom of the paper or plate (ink would dissolve and run). Small spots of the hydrolysed mixture, and of known amino acids for comparison, are placed on the line. The paper is stood in a covered tank of solvent with the solvent level below the baseline. As the solvent rises, each amino acid moves a different distance, depending on its relative affinity for the stationary and mobile phases. When the solvent is near the top, the paper is removed and the solvent front is marked.Amino acids are colourless, so the chromatogram is sprayed with a locating agent, ninhydrin, and warmed. The amino acids appear as purple spots.
Rf = distance moved by spot ÷ distance moved by solvent front Rf is always between 0 and 1. A given amino acid has a fixed Rf only under the same conditions (solvent, temperature, type of paper), so unknowns are compared with known amino acids run on the same chromatogram.Worked example: a spot moves 3.6 cm while the solvent front moves 8.0 cm. Rf = 3.6 ÷ 8.0 = 0.45. Comparing this with the Rf of known amino acids in the same solvent identifies it.If two amino acids have similar Rf values, the paper can be turned 90° and run again in a second solvent (two-way chromatography) to separate them.