Gradient Elution Chromatography

Abstract Elution chromatography can be carried out in either isocratic or gradient modes. In isocratic elution, the mobile‐phase composition is held constant during separation of the sample, e.g. 60% v acetonitrile–water. In gradient elution, the mobile‐phase composition will be varied during sample separation, e.g. changing from 0 to 100% v acetonitrile–water. Gradient elution requires special chromatographic equipment, as well as somewhat greater care on the part of the operator, but it has important advantages for many separations. Thus, in isocratic elution (Figure 1a), sample peaks tend to “bunch up” at the beginning of the chromatogram (often with decreased resolution) and to broaden at the end of the chromatogram (with reduced detection sensitivity). Gradient elution (Figure 1b), on the other hand, provides a more even spacing of peaks, similar widths throughout the chromatogram, and often a shorter run time. For these and other reasons, gradient elution is preferred for the separation of many samples.