The aim of this module is to explain the problems that can be encountered when using isocratic HPLC, and demonstrate how these problems can be overcome using gradient HPLC. We will illustrate how gradient elution HPLC works, define its parameters and show how the retention and elution processes differ from isocratic HPLC. We will demonstrate why peak shapes in gradient HPLC are more efficient than those obtained from isocratic HPLC. The effects of gradient steepness will be examined, and it is shown how the various gradient parameters can be practically determined. We will interactively illustrate the use of scouting gradients in HPLC method development and optimization. Also, we will look at the pitfalls and advantages of gradient elution HPLC in a practical situation.