Column chromatography is an analytical chemistry technique. It is used most often to separate protein molecules based on their tendency to bind to a glass or plastic column lined with a ligand specific to a certain protein.

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An analytical chemistry technique commonly used in organic chemistry in order to separate or purify substances. In general a column is a vertical tube (usually glass or Pyrex), that contains a stationary phase, such as silica gel, and a mobile phase (sometimes called the elutent), which is generally a solvent or mixture of solvents. While one is sending the mobile phase across the stationary phase one is said to be "running the column". Due to the differences in certain chemical properties, typically polarity, each component of the sample will elute at a different time. The solvent coming out of the column is collected in discrete quantities called fractions, and each fraction is then individually analyzed using some other method (such as Thin layer chromatography) to find out which component or components is currently eluting. Once a component has finished eluting, all the fractions containing only that component can then be combined and concentrated, providing a purified sample.

Column Theory

The general operating principle of the column is that each component in sample running through the column will have interactions both with the stationary phase and the mobile phase. The more a component interacts with the stationary phase the longer it will take it to move down the column. Take for example a column with a stationary phase of silica gel, being eluted with a mixture of Hexanes (a non-polar solvent) and Ethyl Acetate (a polar solvent): Given a sample with a polar component and a non-polar component, the non-polar component will be pulled down the column relatively quickly by the hexanes, whereas the polar component will be slowed down as it will have a tendency to bind to the Silica. The initial fractions collected will contain only the solvent solution. As time progresses the fractions will contain increasing concentrations of only the non-polar compound. Eventually the amount of non-polar substance in the column will run out, and there will be another period where fractions will contain only a solvent mixture (this is the ideal case and is called baseline separation). Finally fractions will be collected contain increasing concentrations of the polar compound, again until all has been collected.


The selection of solvents and ratio when mixing the solvents is an important part of running a column, as one wants to choose a mix that will provide adequate separation in a reasonable amount of time. In general a large volume of a non-polar solvent (such as hexanes) and a smaller volume of a polar solvent (such as Ethyl Acetate, Methylene Chloride, or Methanol) are used so that the more polar compounds will not elute too quickly from the column. If there is too much of a polar solvent the more polar components may co-elute with the less polar components and there will not be well defined separation. As running the column progresses and some of the less polar components have been collected, it is common to change this mixture in order to speed up the rest of the column.

Variants of Column Chromatography
  • Gravity Column - A standard means of running a column. Gravity is the force pulling the solvent through the column.
  • Flash Column - Increased air pressure is used to push the solvent through the column at a more rapid rate. This is an extremely common technique used to accelerate the results of a gravity column.
  • HPLC - High Performance Liquid Chromatography - A machine-automated form of chromatography that is very popular because once set up and programmed, it completely automates the entire separation and analysis process.

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