Topics - Gradient Elution

Gradient Elution Gradient elution is used in LC for the same purpose that temperature programming is used in GC, that is, to accelerate the elution of strongly retained solutes. Depending on the interactive character of the stationary phase, the interactive character of the mobile phase is continually changed to have interactive properties more like those of the stationary phase during the development of a separation. As an example, if a reversed phase (e.g. silica bonded with long hydrocarbon chains) was used as the stationary phase in a liquid chromatography separation, the solutes would be held and retained by predominantly by dispersion. To allow the dispersion forces to dominate in the separation process and retain the solutes, the mobile phase would be made strongly polar, for example, a 10% solution of acetonitrile in water. Now the solvent that has the greater dispersive character in the mobile phase mixture is acetonitrile. Thus, if the proportion of acetonitile is increased during chromatographic development, the dispersive interactions in the mobile phase will become larger until they can compete with those of the stationary phase and elute the dispersively held solutes. Conversely, if the stationary phase was polar (e.g. silica gel) the mobile phase would be made dispersive (e.g. 5% ethyl acetate in n-heptane). The mobile phase would then be made progressively more polar by increasing the ethyl acetate content, so that the polar interactions between the ethyl acetate and the solutes would be able to compete with the polar interactions of the solutes with the hydroxyl groups of the silica gel and allow them to be eluted more quickly. There is a wide range of solvent mixtures that can be used in gradient elution and the two examples given are extreme to illustrate the basic factors controlling gradient elution. In practice, were the interactive difference between solutes is very subtle, very subtle mixtures of solvents may be necessary to make the gradient elution process successful.