Chromatography

I. Chromatography

• Chromatography is a physical separation method used to analyse and separate the components in a mixture. It is based on the principle of different components having different affinities towards the stationary and mobile phases.
• Types of Chromatography include Gas-Liquid Chromatography (GLC or GC), High Performance Liquid Chromatography (HPLC), and Thin Layer Chromatography (TLC).
• The mobile phase is usually a liquid or gas that travels through the stationary phase. The stationary phase is a solid or a liquid supported on a solid.
• In Gas Chromatography, the stationary phase is a high boiling point liquid and the mobile phase is an inert gas. The stationary phase is often coated onto the inside walls of a tube or column, and the mobile phase (carrier gas) is passed through this column.
• Components of the mixture travel through the stationary phase at different rates depending on their affinity towards it (intermolecular forces), leading to separation.
• The retention time is the time taken for a component to pass through the system from the column inlet to the detector. Different substances have different retention times.
• A chromatogram is a graphical display of detector response, in terms of retention times. The area under each peak in a chromatogram is proportional to the amount of that component in the original mixture.
• In High Performance Liquid Chromatography, the stationary phase is a high porous solid particle and the mobile phase is a liquid. It is excellent for separating compounds that are thermally unstable or non-volatile.
• Chromatography is a crucial analytical technique in pharmaceuticals, forensics, environmental testing, and food industry.
• Thin Layer Chromatography (TLC) is often used for identifying substances and determining their purity. A spot of the mixture is placed at the bottom of a plate coated with a thin layer of stationary phase. The plate is then dipped in a suitable solvent (mobile phase). As the solvent ascends the plate by capillary action, it carries the sample components with it. The components will separate, creating unique patterns on the plate depending on their affinity to the stationary phase and solvent.
• The Rf value (Retention factor) is the ratio of the distance travelled by the substance to the distance travelled by the solvent. It is characteristic of a particular substance under specific conditions and can be used for identification purposes.