Gradientelution

Gradient elution is a mode of high-performance liquid chromatography in which the composition of the mobile phase is varied during the separation, in contrast to isocratic elution, where the solvent composition remains constant. It is widely used in liquid chromatography, including HPLC and UHPLC, to improve peak resolution and shorten analysis times for complex mixtures.

In gradient elution, the mobile phase strength is increased during the run by mixing two or more solvents with different elution power. In reversed-phase chromatography, the aqueous or weakly polar solvent is gradually replaced by a more organic modifier, allowing analytes with a wide range of polarities to elute in a single run. The gradient affects retention by reducing retention of late-eluting compounds and sharpening peaks.

Common gradient patterns include linear gradients, in which the solvent strength changes at a constant rate, and nonlinear or step gradients, which use discrete increases. Programs specify initial and final mobile phase compositions, gradient time, flow rate, and any hold or re-equilibration steps between runs. Gradient performance is influenced by system dead volumes and mixing efficiency.

Typical solvent systems use a strong solvent B (for example acetonitrile or methanol) and a weaker solvent A (often water with modifiers). The gradient ramp might go from a low percentage of B to a high percentage (for instance from 5% to 95% B over several minutes), followed by a re-equilibration period.

Advantages of gradient elution include better separation of complex mixtures, improved peak shapes, higher sample loading capacity, and shorter analysis times compared with isocratic methods. Limitations include more complex method development, potential baseline drift, and longer system equilibration between runs due to the need to re-establish initial conditions.

Applications encompass pharmaceutical analysis, proteomics, environmental and food analysis, and metabolomics, where gradient elution is especially helpful for separating compounds with a broad range of polarities or affinities.