Thin Layer Chromatography

Introduction
In this report, the Rf values of four amino acid standards and an unknown sample were calculated; the unknown’s molecular weight and pKa’s were also determined. These results reported are used to identify the unknown compound. In this experiment, chromatography and titration were performed to identify the unknown amino acid. Within experimental error, the results were consistent with the reference literature cited in this report.
Experimental
Thin Layer Chromatography
The amino acid standards used in this experiment were Alanine, Glycine, Serine, and Histidine. These standards and the unknown were separated by performing a method of chromatography. Thin layer chromatography (TLC) was performed by using a mobile phase and a stationary phase. The mobile phase was a nonpolar solvent composed of ethanol and ethyl acetate. The solid stationary phase was coated with silica gel, which is very polar. The amino acids were spotted onto two TLC plates; the plates were then placed in a chamber which contained the mobile phase. The plates were sprayed with 0.1% Ninhydrin and placed in an oven. The distance of the traveled spots and the solvent front were measured. With these values, the Rf value for each spot was calculated.
Amino Acid Titration
Table 1 demonstrates the color of each spot observed, and the distances measured. The Rf values were calculated by dividing the distance of the spot from the distance of the solvent front (Equation 1). Each amino acid has different Rf values because some are polar and some are nonpolar. The ones that are polar did not traveled far because the solvent is nonpolar, and polar compounds do not interact well with nonpolar compounds. In this experiment, the polar amino acids are serine and histidine; therefore, these two have the lowest Rf values. Alanine and glycine are nonpolar, hence the high Rf