How to Run a Thin-Layer Chromatography

Thin-Layer Chromatography (TLC), is a chemical method that can be used to analyze mixtures of chemicals. A TLC places chemicals on a special "plate" that is coated with a material that is adsorbent. Adsorbent, unlike absorbent, means that molecules will stick to its surface, rather than being absorbed. The TLC will have chemicals adhere, or stick, to its surface. Then, when the TLC is being run, the chemicals on the surface will travel up the TLC plate at differing speeds. This then makes analyzing the chemicals easier because only chemicals that are exactly the same will travel at the same speed.

The process of TLC works via the property of polarity, or the difference in electron density across a molecule. The basic workings of TLC is that molecules that have more polarity, or have a greater difference in electron density across the molecule, will move slower than and molecules with less polarity.

Things you will need to run a TLC:

1. A Thin-Layer Chromatography plate

2. A clear and clean bottle with a cap

3. A solvent that will act as an eluent

4. The chemicals and mixtures you will be testing

5. A capillary micropipet for each chemical you are testing

6. A PENCIL (a pen will not work)

7. Tweezers

8. A source of UV light

First, draw a line across the TLC plate about 1 cm from the bottom.

This will be the line where all the materials being tested will be placed.

Make a dash along the line for every material that is being tested and create a cospot.

A cospot is there to examine how all of the various chemicals compare with each other in a single line.

Loading the TLC is a careful and difficult practice because the TLC plate is brittle and fragile.

First, apply the capillary micropipet to the sample being tested (Fig 2.1). The micropipet should suck in a small amount of the chemical. A good piece of advice for this part is to have small vials of the chemicals you are testing and to devote them exclusively to TLC.

Then take your micropipet and lightly touch the TLC plate at the labeled point for that chemical (Fig 2.2). The liquid inside the TLC will move to the plate, forming a spot. Make sure not to put too much material on the plate. A good size for the spot is around 1-2 mm.

Then apply the chemical to the cospot in the same way as the previous step.

Repeat this process with a clean micropipet for every chemical you are testing.

Eluent: A mixture of two liquid chemicals, one that is polar, and one that is non-polar that causes the spots on a TLC plate to travel up the TLC plate.

An example of two chemicals would be ethyl acetate (Fig 3.1) and hexane (Fig 3.2). Ethyl acetate is very polar since the molecule is not symmetrical and has oxygen only on one side.. Hexane is a very symmetrical molecule and so electric charge is distributed evenly.

The ratio for the eluent is determined through trial and error.

The more of the polar chemical there is in the eluent, the more the spots will travel.

A good way to know if the eluent is at the right ratio is if a test run is done on the TLC and the spots on the TLC plates are far apart to be distinguishable and that they are still far from the top of the plate.

Finding a good ratio will take multiple tries.

First get your bottle and put eluent in the bottle until the very bottom is covered in a thin layer of liquid.

Then, using tweezers, carefully place the TLC plate in the bottle and cap the bottle. Make sure that the TLC plate does not touch the sides of the bottle and that the eluent is below the spots on the plate.

The eluent will travel up the plate slowly.

When the eluent is close to the top, take out the plate using tweezers again.

Let the eluent evaporate off and then place the TLC plate under a UV light.

If the chemical being tested on the TLC plate does not show up under a UV light, a stain may be used to show the the chemicals.