As a sample solution flows through a column with the mobile phase, the components of that solution migrate according to the non-covalent interactions of the compound with the column. The chemical interactions of the mobile phase and sample, with the column, determine the degree of migration and separation of components contained in the sample. For example, those samples which have stronger interactions with the mobile phase than with the stationary phase will elute from the column faster, and thus have a shorter retention time, while the reverse is also true. The mobile phase can be altered in order to manipulate the interactions of the sample and the stationary phase. In isocratic elution compounds are eluted using constant mobile phase composition.
All compounds begin migration through the column at onset.
However, each migrates at a different rate, resulting in faster or slower elution rate. In gradient elution different compounds are eluted by increasing the strength of the organic solvent. The sample is injected while a weaker mobile phase is being applied to the system. The strength of the mobile phase is later increased in increments by raising the organic solvent fraction, which subsequently results in elution of retained components. This is usually done in a stepwise or linear fashion.