Biochemsistry

MAPÚA INSTITUTE OF TECHNOLOGY

School of Chemical Engineering and Chemistry

Experiment 2

SEPARATION OF AMINO ACIDS BY THIN LAYER CHROMATOGRAPHY

Introduction

The discovery of chromatography revolutionized the separation and detection of amino acids and dipeptides. The separation is based on the liquid-liquid partition of the compounds between two immiscible phases. Initially the separations were primarily conducted on filter paper and were called paper chromatography.

In paper chromatography the hydrated cellulose fibers of the paper act as the stationary phase. A polar solvent ascends in the vertically held paper by capillary action and is the mobile phase. In thin layer chromatography (TLC) a thin uniform layer of silica gel acts as the stationary phase. TLC is replacing paper chromatography because the plates are easier to use than the paper, they give a sharper separation and the amino acids or dipeptides can easily be collected from the plate. Many microscopic distributions of the compounds occur between the mobile and the stationary phases. In time equilibrium is established between the two phases and the more soluble compounds move farther along the plate; different compounds move different distances from the origin. The plate is dried, sprayed with a ninhydrin solution and heated in order to locate the amino acids. The ninhydrin reacts with the amino acids to form colored products.

The ratio of the distance moved by the amino acids to the distance moved by the solvent front from the original spot on the paper is defined as the Rf value and is characteristic of the compound. Rf values depend on several factors: type of silica gel plus binder used, water content of the thin layer, concentration of solute, temperature, manner of development and distance of the starting point from the solvent. Known compounds are usually run on the same plate as the unknowns to assist in identification of the unknowns rather than relying solely on published Rf values....