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TitleTlc Separation of Amino Acids
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aspartame is sufficiently stable and fit for human consumption. Only a warning must be put on the
labels of foods containing aspartame. This warning is for patients suffering from phenylketonurea
who cannot tolerate phenylalanine.

To run a thin-layer chromatography experiment, silica gel is used in a thin layer on a plastic or glass
plate. The sample (aspartame or amino acids) is applied as a spot to a strip of a thin-layer plate. The
plate is dipped into a mixture of solvents. The solvent moves up the thin gel by capillary action and
carries the sample with it. Each amino acid may have a different migration rate depending on the
solubility of the side chain in the solvent. Amino acids with similar side chains are expected to move
with similar, though not identical, rates; those that have quite different side chains are expected to
migrate with different velocities. Depending on the solvent system used, almost all amino acids and
dipeptides can be separated from each other by thin-layer chromatography (TLC).

The rate of migration of an amino acid or a dipeptide is not actually measured, but rather, how far a
particular amino acid travels in the thin silica gel layer relative to the migration of the solvent. This
ratio is called the Rf value. In order to calculate the Rf values, one must be able to visualize the
position of the amino acid or dipeptide. This is done by spraying the thin-layer silica gel plate with a
ninhydrin solution that reacts with the amino group of the amino acid. A purple color is produced
when the plate is heated. (The proline not have a primary amine gives a yellow color with ninhydrin.)
For example, if the purple spot of an amino acid appears on the TLC plate 4.5 cm away from the
origin and the solvent front migrates 9.0 cm (see figure below), the Rf value for the amino acid is

The aspartame that will be analyzed is actually a commercial sweetener, which contains silicon
dioxide, glucose, cellulose, and calcium phosphate, in addition to the aspartame. None of these other
ingredients of the sweetener will give a purple or any other colored spot with ninhydrin.
Occasionally, some sweeteners may contain a small amount of leucine which can be detected by the
ninhydrin test.

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