Experiment 31 Thin-Layer Chromatography Separation of Amino Acids

EXPERIMENT 31 THIN-LAYER CHROMATOGRAPHY SEPARATION OF AMINO ACIDS 

The amino acids are separated on a TLC sheet, e.g., silica gel, using a choice of two developing solvents. The locating reagent is ninhydrin. 

Fisher Scientific TLC Kit A or equivalent TLC sheets, sprayer (for application of reagent), developing apparatus (e.g., Fisher Scientific TLC Kit A www.fisheredu. com or equivalent). 

Obtain an unknown mixture from your instructor. The mixtures to be separated will contain approximately 1 mg of each amino acid per milliliter 0.5 M alcoholic hydrochloric acid solution (see discussion below). Spot approximately 1 /u,L of the sample solution on the Chromagram sheet about 2 cm from the lower edge. (Activation of the sheet is not necessary for this separation.) The unknown should contain acids whose Rf values are sufficiently different with the solvent system used so that they will be readily separated. Your instructor will advise you which standard amino acid solutions to run with your unknown so that you can distinguish between two amino acids that might have an value close to one in your mixture. Allow 15 to 20 min drying to ensure complete evaporation of the hydrochloric acid. 

Develop the Chromagram sheet in the solvent of choice for a distance of 10 cm or for approximately 90 min. (See the list of Rf values iu the table below and follow your instructor s directions for the solvent of choice for your unknown mixture.) Dry the developed Chromagram sheet and spray with the ninhydrin solution. Heat gently for several minutes until separated zones appear clearly visible. 

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