Column chromatography in chemistry is a method used to purify individual chemical compounds from mixtures of compounds. It is often used for preparative applications on scales from micrograms up to kilograms.
The classical preparative chromatography column is a glass tube with a diameter from 5 to 50 mm and a height of 50 cm to 1 m with a tap at the bottom. A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles. A solution of the organic material is pipetted on top of the stationary phase. This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent. Eluent is slowly passed through the column to advance the organic material. Often a spherical eluent reservoir or an eluent-filled and stoppered separating funnel is put on top of the column.
The individual components are retained by the stationary phase differently and separate from each other while they are running at different speeds through the column with the eluent. At the end of the column they elute one at a time. During the entire chromatography process the eluent is collected in a series of fractions. The composition of the eluent flow can be monitored and each fraction is analyzed for dissolved compounds, e.g. by analytical chromatography, UV absorption, or fluorescence. Colored compounds (or fluorescent compounds with the aid of an UV lamp) can be seen through the glass wall as moving bands.
Stationary phase (adsorbent)
The stationary phase or adsorbent in column chromatography is a solid. The most common stationary phase for column chromatography is silica gel, followed by alumina. Cellulose powder has often been used in the past. Also possible are ion exchange chromatography, reversed-phase chromatography (RP), affinity chromatography or expanded bed adsorption (EBA). The stationary phases are usually finely ground powders or gels and/or are microporous for an increased surface, though in EBA a fluidized bed is used.
Mobile phase (eluent)
The mobile phase or eluent is either a pure solvent or a mixture of different solvents. It is chosen so that the retention factor value of the compound of interest is roughly around 0.75 in order to minimize the time and the amount of eluent to run the chromatography. The eluent has also been chosen so that the different compounds can be separated effectively. The eluent is optimized in small scale pretests, often using thin layer chromatography (TLC) with the same stationary phase.
A faster flow rate of the eluent minimizes the time required to run a column and thereby minimizes diffusion, resulting in a better separation, see Van Deemter's equation. A simple laboratory column runs by gravity flow. The flow rate of such a column can be increased by extending the fresh eluent filled column above the top of the stationary phase or decreased by the tap controls. Better flow rates can be achieved by using a pump or by using compressed gas (e.g. air, nitrogen, or argon) to push the solvent through the column (flash column chromatography).
Automated flash chromatography systems attempt to minimize human involvement in the purification process. Automated systems may include components normally found on HPLC systems (gradient pump, sample injection apparatus, UV detector) and a fraction collector to collect the eluent.
The software controlling an automated system will coordinate the components and help the user to find the resulting purified material within the fraction collector. The software will also store results from the process for archival or later recall purposes.
A representative example of column chromatography as part of an undergraduate laboratory exercise is the separation of three components (out of 28) in the oil of spearmint: carvone, limonene and dehydrocarveol . A microscale setup consisting of a Pasteur pipette as column with silica gel stationary phase can suffice. The starting eluent is hexane and solvent polarity is increased during the process by adding ethyl acetate.
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- Isolation of Three Components from Spearmint Oil: An Exercise in Column and Thin-Layer Chromatography Davies, Don R.; Johnson, Todd M. J. Chem. Educ. 2007 84 318. Abstract