Introduction to thin layer chromatography (TLC)
Chromatography is an analytical technique used to separate mixture of chemical substances into its individual compounds. Different types of chromatography are used in lab. e.g. column chromatography, thin-layer chromatography, gas chromatography etc. Among these, thin layer chromatography (TLC) is the most widely used method in chemical or biochemical laboratory.
Principles of Thin layer chromatography
Thin layer Chromatography consists of two phases: one mobile phase and one contiguous stationery phase. The stationery phase is a thin layer of silica or alumina coated on glass, plastic or metal. And the mobile gas is a suitable solvent. The compound mixture moves along with the mobile phase through the stationery phase and separates depending on the different degree of adhesion (to the silica) of each component in the sample or the compound mixture.
The stationery phase
Thin layer chromatography plate or TLC plate is commercially available. The chemical name of silica gel is silicon dioxide (silica) where silicon is attached with oxygen by covalent bond in a giant structure. TLC plate is prepared by mixing the silica gel with calcium sulfate and water. Here calcium sulfate is used as a binder. This mixture is spread over a nonreactive sheet like glass, aluminium foil or plastic. A thin layer of silica on a sheet is then dried and activated in an oven. Alumina (aluminium oxide) can also be used instead of silica.
Silica on the surface of TLC contains Si-O-H bond instead of Si-O-Si bond. The surface of TLC becomes very polar because of the presence of this -OH group. So the compounds can form hydrogen bond or can interact by van der waals dispersion forces and dipole dipole forces.
Thin layer chromatography works in few steps:
Step 1: A horizontal line is drawn near one end (about 1.5 cm from the bottom edge) of TLC plate. In figure below 6 is the horizontal line.
Step 2: The sample needs to be separated is placed as a small drop or line on to the TLC plate using capillary tube. Labelling the drop by a pencil with an alphabet or number help to identify the compound later. In figure above 3 and 4 are the drops labelled. The drops are then soaked on the plate and dried.
Step 3: The TLC plate is then placed into a sealed container with a swallow layer of suitable solvent. The solvent level must be lower than the pencil line or drop on it. The container need to be covered to stop the solvent to evaporate.
Step 4: The solvent rises up the chromatography taking each component of the sample with it. The components travel with the solvent depends on three things:
- The polarity of the sample molecule. The non polar components travel faster than the polar component.
- The attraction between the sample molecule and the solvent or solvent mixture.
- The attraction between the sample and the silica.
Suppose any sample compound mixture contains a molecule that forms hydrogen bond with silica and one only interacts with van der waals forces. In this the hydrogen bonded molecule will have tendency to stick on the surface of the TLC plate more than the other one.
Step 5: When the solvent rises near the end of the TLC plate then the plate should be taken out from sealed container and air dried. The plate with separated bands of components are then observed under UV-light.
- Thin layer chromatography is an chromatography technique used to separate mixture of chemical substances into its individual compounds.
- Chromatography consists of two phases: one mobile phase and one contiguous stationery phase.
- TLC plate (stationery phase) is prepared by mixing the silica gel with calcium sulfate and water.
- A suitable solvent (mobile phase) is moved along with compound mixture through the TLC plate according to the polarity and the degree of adhesion of each component on the stationery phase.