Analysis of amino acids: The thin layer chromatography technique is an analytical chromatography to separate and analyze free amino acids from proteins. In this method, the silica or alumina as a stationery phase is coated on to a glass or aluminium foil as thin layer and then a sample is allowed to run in the presence of a mobile phase (solvent). In comparison to other chromatography techniques, the mobile phase runs from bottom to top by diffusion (in most of the chromatography techniques, mobile phase runs from top to bottom by gravity or pump). As sample runs along with the mobile phase, it get distributed into the solvent phase and stationery phase. The interaction of sample with the stationery phase retard the movement of the molecule where as mobile phase implies an effective force onto the sample. Supose the force caused by mobile phase is Fm and the retardation force by stationery phase is Fs, then effective force on the molecule will be (Fm-Fs) through which it will move (Figure 32.3). The molecule immobilizes on the silica gel (where, Fm=Fs) and the position will be controlled by multiple factors.
1. Nature or functional group present on the molecule or analyte.
2. Nature or composition of the mobile phase
3. Thickness of the stationery phase.
4. Functional group present on stationery phase.
If the distance travelled by a molecule on TLC plate is Dm where as the distance travelled by the solvent is Ds, then the retardation factor (Rf) of molecule is given by:
Rf value is characterstic to the molecule as long as the solvent system and TLC plate remains unchanged. It can be used to identify the substance in a crude mixture.