Liquid Chromatography

General Scheme

Components within a mixture are separated in a column based on each component's affinity for the mobile phase. So, if the components are of different polarities and a mobile phase of a distinct polarity is passed through the column, one component will migrate through the column faster than the other. Because molecules of the same compound will generally move in groups, the compounds are separated into distinct bands within the column. If the components being separated are colored, their corresponding bands can be seen. Otherwise as in high performance liquid chromatography (HPLC), the presence of the bands are detected using other instrumental analysis techniques such as UV-VIS spectroscopy¹. The following figure shows the migration of two components within a mixture:

In the first step, the mixture of components sits atop the wet column. As the mobile phase passes through the column, the two components begin to separate into bands. In this example, the red component has a stronger affinity for the mobile phase while the blue component remains relatively fixed in the stationary phase. As each component is eluted from the column, each can be collected separately and analyzed by whatever method is favored. The relative polarities of these two compounds are determined based on the polarities of the stationary and mobile phases. If this experiment were done as normal phase chromatography, the red component would be less polar than the blue component. On the other hand, this result yielded from reverse phase chromatography would show that the red component is more polar than the blue component.