Separation of Amino acids by Thin Layer Chromatography
Theory
Chromatography is the general name applied to a series of methods that
employ a system with two phases (states) of matter: a mobile phase and
a stationary phase. The stationary phase is a solid, and the mobile phase
is either a liquid or a gas. The separation process occurs because the
components of the mixture have different affinities for the two phases
and thus move through the system at different rates. A component with
high affinity for the mobile phase moves relatively quickly through the
chromatographic system, whereas one with a high affinity for the solid
phase moves more slowly.
In this practical we will use a Thin Layer Chromatography (TLC) method
to separate amino acids from a mixture. The stationary phase is a silica
gel (hydrophilic) that is bonded on an aluminium foil. The mobile phase is
a more hydrophobic solution of butanol.
To detect the amino acids we will use a ninhydrin solution. Ninhydrin
reacts and give a violet colour with proteins, polypeptides and amino
acids.
Task
To analyse three mixtures of amino acids.
Material
100 cm3 beaker with lid. TLC foil. Capillary tubes.
Chemicals Mobile phase: A mixture of 80% butanol, 10% acetic acid and 10%
water- already prepared.
0.01% solutions of the amino acids: lysine, alanine and leucine and three
different mixtures of the three amino acids.
Ninhydrin solutions.
Procedure Pour about 1 cm the mobile phase into the beaker and put the lid on so
that the atmosphere in the beaker will be saturated with the mobile
phase. Work in a fume cupboard.
Don’t touch the TLC gel with your finger.
Cut out a piece of the TLC plate, about 3x6 cm.
Draw a base line about 1.5 cm up with a pencil
and mark 6 dots on the line.
Take up alanine solution in a capillary tube and dot it
on the TLC plate on is spot. The spot shouldn’t be
bigger than 3-4 mm. Repeat with the other solutions.
Dry the plate.
Put down the TLC plate in the beaker and put the lid on. The base line
must be over the surface of the mobile phase.
When the mobile phase is about 5 mm from the top you take it out from
the beaker. Mark the front lien of the mobile phase. Dry the plate in
drying cup board (100oC in 5 min).
Go to Helena who will spray ninhydrin solution on the plate. Dry with a
hair dryer. The amino acids will be seen as violet spots.
Calculate the Rf-value for the amino acids.
(Rf = (bas line to amino acid) / (base line to the front of the mobile
phase))
Identify the amino acids.
How do the structures of the amino acids correspond to the Rf-value?
Assessment DCP CE