Developing Focused Gradients for Isolation and Purification
Chromatographic separations for isolation and purification are governed by the same physical and chemical principles as analytical separations. In prep experiments, however, scientists isolate compounds at high mass loads, often on large columns, and require better resolution to enhance purity and recovery of the collected materials. Although creating a shallower gradient is a good first approach to enhancing resolution, changing the gradient slope for the whole separation leads to broader peaks and an increase in total run time. Focused gradients, and alternative to universally shallower gradients, decrease the gradient slope for only that portion of the chromatogram that needs increased resolution, providing more resolution between closely eluting peaks without increasing the total run time. A focused gradient can be defined based on a scouting run or directly from a first prep run.