Column Chromatography: Theory


All chromatographic techniques share the same fundamental operational principles. (GC theory) Mixed samples are passed over an immovable or stationary phase that interacts to some extent with all components within the sample. This interaction depends on the relative polarities of the stationary phase and sample components.

Alumina, a polar finely divided solid, is often used as the stationary phase in column chromatographic separations. Thin layers of sand are usually positioned at the top and bottom of the alumina layer to support it and protect it from disturbances produced when pouring liquids into the top of the column. Prior to application of the actual sample, the column is filled with a non-polar solvent.

The sample is first introduced as a highly concentrated liquid solution (known as the sample plug) that is poured directly in the top of the column. As the sample moves down the column, polar sample components interact strongly with the alumina while non-polar components are carried away by the non-polar mobile phase. Consequently, the non-polar sample components are the first to be collected in the filter tube at the bottom of the apparatus.

To remove the polar sample components trapped in the alumina phase, a polar solvent is used to flush the column. As the polar solvent moves down the column, it carries with it the polar sample component that is collected in a new filter tube at the bottom of the apparatus.