Seperating amino acids using thin layer chromatography

Question 1

What is Thin Layer Chromatography?

Answer 1

• TLC is a technique used to separate the individual components of a mixture, allowing you to identify unknown molecules.
• There are two phases: stationary phase and the mobile phase which involves an organic solvent.

Question 2

Stationary and mobile phase

Answer 2

• In stationary phase a thin layer of silica gel is applied to a rigid surface e.g glass.
• Amino acids are then added to one end of the gel.
• This end is submerged in organic solvent (a mixture of butan-1-ol, glacial ethanoic acid and water for amino acids).
• The organic solvent moves through the silica gel - this is the mobile phase.

Question 3

The rate of movement

Answer 3

• The rate at which different amino acids in the solvent move through the silica gel depends on the interactions (hydrogen bonds) they have with the stationary phase, and their solubility in the mobile phase.
• This causes different A.A to move to move different distances in the same time period, resulting in them separating out from each other.

Question 4

Method

Answer 4

1. Wear gloves; draw a pencil line on the chromatography plate near bottom. Put equally spaced points for the different A.A solutions. Handle plate only by the edges.
2. Spot the unknown amino acid onto the first pencil mark using a capillary tube. Repeat this with known amino acids.
3. Place the plate into a jar containing the solvent; the solvent shouldn’t be that deep (under the pencil line). Close the jar.
4. Leave the plate in solvent until it reached near the top. Remove plate and do a pencil line along the solvent front. Dry the plate.
5. Spray the plate with ninhydrin spray in a fume cupboard. Amino acids react with ninhydrin and a purple/brown colour is produced. Mark the spots in pencil.

Question 5

Rf value

Answer 5

Distance travelled by solute / Distance travelled by solvent