Exclusion Chromatography


One of the most common problems in biochemical research is to separate the many components — usually macromolecules — in cell extracts and the like. Methods for separating the components of a mixture exploit such differences as of the molecules in it.

One example: Electrophoresis which separates such macromolecules as proteins and DNA by their charge (and sometimes size as well). Link to an example of separating serum proteins by electrophoresis.

Chromatographic separation by size of molecule.

Exclusion chromatography separates molecules on the basis of size. A column is filled with semi-solid beads of a polymeric gel that will admit ions and small molecules (blue) into their interior but not large ones (shown in red). When a mixture of molecules and ions dissolved in a solvent is applied to the top of the column, the smaller molecules (and ions) are distributed through a larger volume of solvent than is available to the large molecules. Consequently, the large molecules move more rapidly through the column, and in this way the mixture can be separated (fractionated) into its components.

The porosity of the gel can be adjusted to exclude all molecules above a certain size. Sephadex and sepharose are trade names for gels that are available commercially in a broad range of porosities.

Link to discussions of other chromatographic techniques: affinity chromatography and paper chromatography.
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1 February 2011