Ion Exchange and Thin Layer Chromatographic Separation and Identification of Amino Acids in a Mixture: An Experiment for General Chemistry and Biotechnology Laboratories

A multiday laboratory exercise is described that is suitable for first-year undergraduate chemistry, biochemistry, or biotechnology students. Students gain experience in performing chromatographic separations of bio-molecules, in both a column and thin layer chromatography (TLC) format. Students chromatographically separate amino acids (AA) in an unknown mixture using a small column of Dowex-50 resin. The AA elution profile is determined by graphical analysis of a rapid ninhydrin microplate spectrophotometric assay that converts the colorless AA into colored ninhydrin derivatives. Column fractions corresponding to elution "peaks" are further analyzed by TLC on silica gel plates alongside AA standards. Visualization of AA migration on TLC is accomplished with a ninhydrin dipping protocol. Students use their chromatography data, structural information about the resin and the various AA, and their knowledge of intermolecular attractions and acid base chemistry to determine the identity of the AA in their unknown mixture.