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 pipe - the column - is located. This known packing material may fill the entire cavity of the tube ( packed column ), or only as a thin layer on the inner surface to be coated ( capillary ).

This definition is not discriminated in the mobile phase, if there is a fluid ( liquid ), a gas (gas chromatography ), or a supercritical medium ( supercritical fluid chromatography ), nor is the type of interactions between the stationary phase and chromatographed substances differentiated; it can be both adsorption as well as by ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography or size exclusion chromatography.

In laboratory practice usually liquid chromatographic method by column chromatography, however, indicated that are based on the different degrees of adsorption of the substances in solution (mobile phase) to a solid support (stationary phase ) and a preparative purification of mixtures of different polarity in the microgram to kilogram scale used.

Manual column chromatography

Stationary phase

The stationary phase is re- recognized in the rule for every application in a long glass tube (packed), which closes at the bottom with a frit and a stopcock. Used is usually finely powdered silica gel or alumina, which is filled as evenly as possible and no air bubbles and cracks with the later as the mobile phase (see below) the solvent used.

Mobile phase

The polarity of the mobile phase must be matched as closely as possible to the specific separation problem. To mixtures of polar and nonpolar solvents. Very common is the tandem cyclohexane / ethyl acetate. Instead of the relatively expensive cyclohexane also " iso-hexane " said Hexangemische are often used.

Implementation

The substance mixture is to be separated is applied to the upper end of the stationary phase and then refilled from the solvent carried through them. The ingredients are there different degrees repeatedly adsorbed depending on the polarity of the stationary phase and desorbed again, making them slowly take off in the stream of solvent from each other and are ideally zonally separated at the end of the column. Where the eluate can be collected in small portions, and the fractions are combined after identification ( thin layer chromatography).

For difficult separations, it may be necessary to use a " solvent gradient ", ie to increase the polarity of the mobile phase during the separation slowly. Thus, the non-polar fractions are first discharged rapidly, while the more polar remain largely stationary. The use of an otherwise necessary over long and difficult to handle column can thus be avoided.

In addition to a sufficient length, the column must have a reasonable amount of sample diameter to keep the size of the starting zone as small as possible.

Automated column chromatography

Such separations can be accelerated, if the mobile phase is instead pushed by the effect of gravity of the supernatant solution with compressed air by means of the stationary ( sogn. " flash chromatography "). The separation efficiency of the column is thus even increased since the zones have less time to expand by diffusion in the flow direction.

Device systems with gradient pumps, integrated detectors and fraction collectors are now commercially available for the automated flash chromatography. Also finished prepacked columns in the form of plastic cartridges are available with various packing materials available.