Biochemistry

BMS1030 – Laboratory Practical: Studies with Amino Acids

Thin Layer Chromatography of Amino Acids

Amino Acid | Distance (mm) travelled by amino acid | Solvent front (mm) | Rf value |

Glycine | 20 | 64 | 0.31 |

Threonine | 28 | 64 | 0.44 |

Tryptophan | 40 | 64 | 0.63 |

Unknown | 25 | 64 | 0.39 |

1. Why does touching the silica gel with your hands potentially contaminate your plate? [5]

Skin is made of amino acids and would contaminate the silica on the plate. This would show after the ninhydrin spray has dried, disturbing the results of the other amino acids.

2. Why must you use a pencil to write on the TLC plate (starting line, solvent line, identification of the spots)? Why can’t you use a pen? [5]

Lead, which is made of graphite will not rise with the solvent, keeping the accuracy of the experiment as the markings are left in place. A pen would cause damage to the plate, and spread ink across it, as the ink is soluble.

3. Figure 1

Figure 1

Threonine

Threonine

Include a picture of your TLC plate below [10]

Glycine

Glycine

TLC plate

TLC plate

Solvent front 1.6 cm

Solvent front 1.6 cm

Sample spots after sprayed ninhydrin solution

Sample spots after sprayed ninhydrin solution

Unknown spot sample (No.2)

Unknown spot sample (No.2)

Starting line 1.5 cm from bottom of the plate

Starting line 1.5 cm from bottom of the plate

Tryptophan

Tryptophan

4. Why can an Rf value never be greater than 1? [5]

Because the sample spots are moving within the solvent and can never move past it. The Rf value will always be less or equal to 1.

5. Which amino acid had the highest Rf value? Explain these findings, based on your knowledge of the chemical properties of this amino acid? [10]

Tryptophan has the highest Rf value. The benzene ring structures on tryptophan’s side chains cannot form hydrogen bonds with the silica plate, hence it is hydrophobic. Instead it...