Paper chromatography of amino acids

What is chromatography used for

To separate substances based upon their differential solubility in a solvent (mobile phase) which can be a liquid or a gas.

What happens to substances that have a higher affinity for the mobile phase than the stationary phase

They are carried further by the solvent through the stationary phase.

Why is chromatography used on mixtures of amino acids

To separate and identify amino acids in the mixture

How is a mixture of amino acids usually obtained

From the hydrolysis of a protein either by protease enzymes or acid hydrolysis.

What kind of mobile phase is used in the paper chromatography of amino acids

Organic solvent

What is the stationary phase in the paper chromatography of amino acids

Paper

Why can amino acids be separated using paper chromatography

Because they each have a different R group which alters the solubility of the amino acid in a particular solvent (R groups may be polar, non-polar, or charged).

Why should the start line be draw in pencil and not in pen

Because pen ink will dissolve in the solvent and interfere with the results.

How should the spots be applied to the start line and why is this important

Using a capillary tube to apply multiple drops to 1 spot, letting them dry before each application, so that each spot is concentrated ensuring that the separated amino acids are visible.

Why must the paper not touch the sides of the container

To ensure that the solvent doesn’t run faster along the sides resulting in an uneven solvent front.

Why must the solvent not go above the start line

So that the spots don’t dissolve out of the paper and into the solvent.

How long should the chromatogram run

Until the solvent front reaches 2cm below the edge of the paper

Why should the chromatogram be left to run as long as possible

So that there is a greater separation between spots resulting in more distinct spots and more accurate measurements.

What locating agent should the paper be sprayed with and what colour does it stan the amino acids.

Ninhydrin stains the amino acids purple except proline which is stained yellow.

Where should the chromatogram be kept and sprayed, and why

In a fume cupboard as the solvent and ninhydrin are irritant.

Why should the chromatogram be handled with gloves

So oils and amino acids on the skin don’t get transferred onto the paper and interfere with the results

Why should the solvent front be drawn immediately after the chromatogram is done

Because the solvent will evaporated quickly

What can be done to aid the identification of the amino acids in the mixture

The mixture of amino acids and known standards can be run alongside each other on the same chromatogram and Rf values can be calculated and compared with known values.

What is a known standard (in terms of the chromatography of amino acids)

Solution containing only 1 type of amino acid

How are Rf values calculated

Distance the the substance (amino acid) has travelled / the distance the solvent has travelled (solvent front)