Chromatography is a technique that is used to separate substances of a mixture and to determine the identity of it. Even though this is the case, chromatography is not exclusively used for qualitative data, i.e. determining ‘what’ the substance is, but it can also be useful for finding R_f and R_t values.

All forms of chromatography use:

• A stationary phase – A solid or solid coated with viscous liquid that undergoes adsorption with substances carried along the mobile phase. The type of stationary phase used corresponds with the type of chromatography technique used. E.g. If a thin glass plate were to be used as the stationary phase, the technique would be called Thin Layer Chromatography.
• Mobile Phase – substance that moves over the stationary phase while carrying the sample.

Adsorption is the attraction of a substance to the surface of another.
Desorption is the breaking of bonds between a substance a surface that it is being adsorbed to.

Different compounds within a sample have different chemical and physical properties to one another and hence some may adsorb stronger to the stationary phase in comparison to other compounds. The rate of movement of components across the stationary phase depends on:

• How strongly it adsorbs to the stationary phase
• How readily it dissolves in the mobile phase.

Thin Layer Chromatography

TLC is mainly used for qualitative analysis, to determine the presence of a compound. Its stationary phase is usually a thin layer of powder such as alumina spread across a glass plate. The mobile phase is a solvent used to carry the sample across the glass plate. The origin is known as the region where the sample is placed on the glass plate. The origin should not be submerged in the solvent to ensure that the sample does not dissolve into the solvent in the beaker that it’s contained in. A watch glass is usually placed on top of the beaker to prevent the solvent from evaporating.

Depending on how readily a component of the sample adsorbs onto the stationary phase or desorbs from it, will determine the distance it travels across it. The more strongly adsorbed components will not travel as for as those that are less strongly adsorbed.

The Rf value is the distance travelled by a given component relative to the solvent front (distance moved by the solvent).

High Performance Liquid Chromatography (HPLC)

High performance liquid chromatography (HPLC) involves pumping a solvent (mobile phase) that contains the sample through a column under high pressures. The stationary phase is filled throughout the column and is generally small sized solids; 10 – 20 times smaller than normal column chromatography. The benefit of the smaller sized solid is that it increases the surface area of the surface area which allows for more frequent adsorption and desorption, giving a better separation of compounds compared to other methods of chromatography.

Smaller particles also creates considerable resistance for the mobile phase to flow through the column. This is overcome by pumping the solvent through high pressures.

Components of the sample are detected by passing the eluate (solvent that has passed through the chromatograph) through a beam of UV light. The retention time (R_t) is recorded and is the time taken for a component to pass through the column. Shorter retention times indicate that the component of the sample was less strongly adsorbed to the stationary phase in comparison to the other components of the sample.

Uses

HPLC is capable of detecting components of a sample as little as 10^-9g HPLC is used to detect:

• Drugs in blood
• Hydrocarbons in oil samples.

Gas chromatography

Gas chromatography gets its name from the fact that the mobile phase used during this procedure is an unreactive gas such as nitrogen or helium. It is essential for the gas used to be unreactive to prevent any mixing of the mobile phase with the sample.

The column of the chromatograph in gas chromatography is within an oven which vaporises the sample to allow it to be carried by the mobile phase through the column.

• Gas-liquid chromatography involves a liquid stationary phase interacting with a gaseous mobile phase. The column used in gas-liquid chromatography is filled with porous solids that has been coated with a liquid hydrocarbon or ester with a boiling point higher than the temperature at which the oven is set to prevent it from boiling off.
• Gas-solid chromatography involves a solid stationary phase interacting with a gaseous mobile phase. The column which is used is filled with adsorbent solids such as alumina.

Uses

Gas chromatography is one of the most sensitive forms of chromatography; it is capable of detecting as little as 10^-12g of a compound. GC is used to detect:

• Drugs in urine samples

See Also