KS5 Biology
Lesson Plan 7 – Thin Layer Chromatography
Science at Work in Healthcare
Post – 16 Science Education Pack
Resource Sheet 7.1 – Thin Layer Chromatography
Thin layer chromatography (TLC) is a chromatography technique used to separate out
chemical compounds.
It uses a TLC plate which consists of a thin layer of adsorbent material backed with a flat,
chemically inert, carrier sheet. This adsorbent material is most commonly silica gel, but it
could also be aluminium oxide or cellulose. The solution to be separated is then dissolved in
a solvent and is drawn up the plate by capillary action. The solution under test separates as
it travels up the TLC plate based on the polarity of the components of the compound in
question.
Note: Adsorption is the name given to one substance forming some sort of bonds to the
surface of another one.
Running TLC
In a typical set up for monitoring reactions in organic chemistry a pencil line is drawn about
0.8 cm from the bottom edge of the plate and a capillary tube is used to spot chemicals A
and B onto the TLC plate along with a spot of the reaction mixture. Labels are then added
and the plate can be heated gently with a hairdryer.
The analysis is qualitative, and it will show if the starting material has disappeared, the
product has appeared, and how many products are generated.
A tank is set up to run the plate. A simple tank can be made using a 250 cm3 beaker with a
Petri dish or watch glass on top
as a lid. A square of filter
paper is placed at the back of
the beaker and a small amount
of eluting solvent placed in the
bottom. This may be about 0.5
cm deep (it should be below
the level of the pencil line on
the TLC plate). The set up is
shown in diagram 1.
Diagram 1. Typical TLC set up
KS5 Biology
Lesson Plan 7 – Thin Layer Chromatography
Science at Work in Healthcare
Post – 16 Science Education Pack
The plate is placed into the tank very carefully using a pair of tweezers and is left to run.
This will take several minutes. The solvent can be clearly seen moving up the plate.
When the solvent front gets to within about 0.5 cm of the top of the plate, it should be
carefully removed using tweezers. Once removed from the tank the plate can be air dried or
dried with a hairdryer. Once the plate is completely dry, the spots need to be visualised.
There are a number of methods that can be used for visualising TLC plates:

Dipping the plate or spraying it with a colour reagent will cause the spots to become
visible.

Iodine vapours can be used as a non-specific colour reagent.

A small amount of fluorescent compound such as manganese activated zinc silicate
can be added to the adsorbent. The plate can be examined under UV light,
whereupon the spots of the analyte appear as dark, non-fluorescing areas.
A visualised plate might look like the one in diagram 2 below:
Diagram 2. Calculating retention
factor (Rf) using a visualised plate
The above diagram shows how to calculate a retention factor (Rf) value for a component.
The Rf value is characteristic of the component when using the same eluting solvent and the
same type of TLC plate.
KS5 Biology
Lesson Plan 7 – Thin Layer Chromatography
Science at Work in Healthcare
Post – 16 Science Education Pack
The details
Once the TLC plate has been prepared and dipped into a suitable solvent, such as ethanol
or water, it is placed in a sealed container. The solvent moves up the plate by capillary
action and meets the sample mixture, which is dissolved and is carried up the plate by the
solvent. Altogether this is referred to as the mobile phase. Different compounds in the
sample mixture travel at different rates owing to differences in solubility in the solvent, and
owing to differences in their attraction to the adsorbent material. The adsorbent material is
referred to as the stationary phase.
The silica gel of the stationary phase is a form of silicon dioxide (silica). The silicon atoms
are joined via oxygen atoms in a giant covalent structure. However, at the surface of the
silica gel, the silicon atoms are attached to -OH groups as in diagram 3 below.
Diagram 3. Giant covalent structure of silica gel stationary phase
The surface of the silica gel is very polar and, because of the -OH groups, it can form
hydrogen bonds with suitable compounds around it as well as van der Waals dispersion
forces and dipole-dipole attractions.
KS5 Biology
Lesson Plan 7 – Thin Layer Chromatography
Science at Work in Healthcare
Post – 16 Science Education Pack
How fast the compounds under test get carried up the plate depends on two things:

How soluble the compound is in the solvent. This will depend on how much attraction
there is between the molecules of the compound and those of the solvent.

How much the compound sticks to the stationary phase. This will depend on how
much attraction there is between the molecules of the compound and the silica gel.
Compounds forming hydrogen bonds will stick to the surface of the silica gel more firmly
than compounds which only participate in the weaker van der Waals reactions. They will
therefore be more strongly adsorbed and the more strongly a compound is adsorbed, the
less distance it can travel up the plate.
If both of the components of the mixture under test hydrogen bond, it is highly unlikely that
both will hydrogen bond to exactly the same extent, and be soluble in the solvent to exactly
the same extent. This means that it will still be possible to differentiate between the two
using TLC.
If compounds don't separate out very well, changing the solvent or just the pH of the solvent
already being used may help.