Column chromatography
With column chromatography (SC) ( English column chromatography ) refers to a chromatographic separation process in which, by definition, the stationary phase in a cylindrical tube - of the separation column - located. This so-called packing material can fill the entire cavity of the tube ( packed column ), or it can only be applied as a thin layer on the inner surface ( capillary column ). The latter case is restricted to analytical capillary gas chromatography.
In this definition, the mobile phase does not distinguish whether it is a liquid ( liquid chromatography ), a gas ( gas chromatography ) or a supercritical medium ( supercritical fluid chromatography ), nor is there any information about the type of interactions between the stationary phase and the chromatographed Substances differentiated; it can be both adsorption as well as to ion exchange chromatography , hydrophobic interaction chromatography , affinity chromatography or size exclusion act.
In laboratory practice, however, column chromatography is usually used to describe liquid chromatographic processes that are based on the varying degrees of adsorption of the substances in solution ( mobile phase ) on a solid support ( stationary phase ) and that are based on the preparative purification of mixtures of substances of different polarity in the microgram - to kilograms - Scale serves.
It was developed in 1978 by W. Clark Still and others.
Manual column chromatography
Stationary phase
The stationary phase is usually set up ( packed ) in a long glass tube for each use , which ends at the lower end with a frit and a tap. The use of stuffed cotton in lieu of a frit is also widespread. Mostly finely powdered silica gel or aluminum oxide is used , which is filled as evenly as possible and without air bubbles and cracks with the solvent used later as the mobile phase (see below).
Mobile phase
The polarity of the mobile phase must be adapted as precisely as possible to the specific separation problem. Mixtures of polar and non-polar solvents are used for this purpose. The most common solvent variations are for non-polar substances hexane / ethyl acetate and for polar substances to be separated from dichloromethane / methanol to aqueous solutions .
execution
The mixture of substances to be separated is applied to the upper end of the stationary phase and then carried through by the subsequently refilled solvent. Depending on their polarity, the components are repeatedly adsorbed and desorbed again and again by the stationary phase, whereby they slowly move away from each other in the flow of the solvent and are ideally separated from each other in zones at the end of the column. There the eluate can then be collected in small portions and the fractions can be combined after identification ( thin layer chromatography ).
For difficult separations it may be necessary to use a "solvent gradient", i.e. to slowly increase the polarity of the mobile phase during the separation. As a result, the non-polar fractions are initially discharged quickly, while the more polar fractions remain largely stationary. The use of an otherwise extra long and difficult to handle column can thus be avoided.
In addition to being of sufficient length, the column must have a diameter appropriate to the sample quantity in order to keep the size of the start zone as small as possible.
Automated column chromatography
Such separations can be accelerated if the mobile phase is pressed through the stationary phase with compressed air instead of the force of gravity of the supernatant solvent (so-called “flash chromatography”). The separation efficiency of the column is even increased as a result, since the zones have less time to expand by diffusion in the direction of flow.
For automated flash chromatography, device systems with gradient pumps, integrated detectors and fraction collectors are now commercially available. Pre-packed columns in the form of plastic cartridges are also available with various packing materials.
literature
- Ullmann's encyclopedia of technical chemistry. 3rd edition, Volume 2, pp. 106-112.
Web links
Individual evidence
- ↑ Entry on column chromatography . In: IUPAC Compendium of Chemical Terminology (the “Gold Book”) . doi : 10.1351 / goldbook.C01182 .
- ^ W. Clark Still, Michael Kahn, Abhijit Mitra: Rapid chromatographic technique for preparative separations with moderate resolution . In: The Journal of Organic Chemistry . tape 43 , 1978, p. 2923-2925 , doi : 10.1021 / jo00408a041 .