Chromatographic Methods
Chromatography is a physical method for separating the components of a mixture by their different distribution between two phases: a stationary (immobile) phase and a mobile phase (a fluid that percolates through the first).
Chromatographic techniques are classified mainly according to how the stationary phase is arranged.
Main classification
Planar chromatography
The stationary phase is placed on a flat surface or a paper support:
- Paper chromatography: uses special paper as the stationary bed and a solvent as the mobile phase.
- Thin-layer chromatography (TLC): the stationary phase is a thin layer of a solid (such as silica gel) spread on a glass or plastic plate.
Column chromatography
The stationary phase is packed inside a cylindrical column. It is subdivided according to the nature of the mobile phase:
- Liquid chromatography (LC / HPLC): uses a liquid as the mobile phase to separate dissolved compounds (for example, high-performance liquid chromatography, HPLC).
- Gas chromatography (GC): uses an inert gas (such as helium or nitrogen) as the mobile phase to separate volatile or vaporizable substances.
- Supercritical fluid chromatography (SFC): uses a fluid in the supercritical state (such as carbon dioxide) as the mobile phase.
Separation mechanisms
Depending on the physicochemical interaction between the components of the sample and the phases, separation may be due to:
- Adsorption: competition for the active sites of a solid surface.
- Partition: distribution between two immiscible liquid phases.
- Ion exchange: electrostatic attraction according to the charge of the ions.
- Size exclusion: separation by size or molecular weight (the largest come out first).
- Affinity: highly specific, reversible binding (e.g. antigen-antibody or enzyme-ligand).