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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.


Topics include:

  • Isocratic HPLC analysis
  • Gradient HPLC analysis
  • Gradient elution parameters
  • Optimizing gradient parameters
  • Gradient elution principles
  • Peak shape in gradient HPLC
  • Scouting gradients
  • Optimizing initial gradient conditions
  • Gradient steepness
  • Optimizing gradient analyses
  • Practical gradient HPLC
  • Changing method parameters
  • Gradient suitability
  • Method transfer between HPLC systems
  • Gradient method transfer calculator
  • Non-linear gradients
  • Segmented gradients
  • Isocratic hold
  • Peak capacity
  • Estimating gradient parameters
  • Gradient performance checks