Column chromatography

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 main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling.

The classical preparative chromatography column is a glass tube with a diameter from 5 mm to 50 mm and a height of 5 cm to 1 m with a tap and some kind of a filter (a glass frit or glass wool plug – to prevent the loss of the stationary phase) at the bottom. Two methods are generally used to prepare a column: the dry method and the wet method.

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. Fractions can be collected automatically by means of fraction collectors. The productivity of chromatography can be increased by running several columns at a time. In this case multi stream collectors are used. The composition of the eluent flow can be monitored and each fraction is analyzed for dissolved compounds, e.g. by analytical chromatography, UV absorption spectra, or fluorescence. Colored compounds (or fluorescent compounds with the aid of a UV lamp) can be seen through the glass wall as moving bands.

The stationary phase or adsorbent in column chromatography is a solid. The most common stationary phase for column chromatography is silica gel, the next most common being 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. There is an important ratio between the stationary phase weight and the dry weight of the analyte mixture that can be applied onto the column. For silica column chromatography, this ratio lies within 20:1 to 100:1, depending on how close to each other the analyte components are being eluted.

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