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The CHROMacademy reversed phase chromatography module is a comprehensive guide to this ubiquitous technique. It will guide the novice, or seasoned chemist, from the basic concepts of the technique through to more complex topics on solvent and buffer selection, the effects of temperature on chromatography, and working with ionizable analytes. All of the topics will provide a fundamental understanding of the science behind the technique, often explained with the use of interactive learning tools and animations; as well as providing practical knowledge on how to develop reversed phase HPLC methods. 

The aim of this module is to give an overview of the mechanism of reversed phase high performance liquid chromatography (RP-HPLC), and explain the basis behind the retention mechanism. Retention order in RP-HPLC is described, and the influence of mobile phase composition on retention illustrated. The module will show how the mobile phase composition and constituents might be manipulated to optimize chromatographic separations. The principles used to select appropriate stationary phases and column geometry are explored. We will also look at how how mobile phase pH affects the retention of ionizable compounds. Typical RP-HPLC applications will also be highlighted. 

At the end of this section you will be able to interactively demonstrate how to use mobile phase pH to optimize separations, as well as choose buffers for RP-HPLC. You will learn how to effectively use the principles of ion-suppression to optimize separations.


Topics include: 

  • Mechanism of reversed phase HPLC
  • Applications of reversed phase HPLC
  • Analyte retention in reversed phase HPLC
  • Retention order in reversed phase HPLC
  • Reversed phase mobile phase solvents
  • Mobile phase strength and retention
  • Changing the organic modifier
  • Eluotropic series
  • Selecting reversed phase columns
  • Reversed phase HPLC of ionizable samples
  • Controlling extent of ionization
  • The 2 pH rule
  • pH vs retention in reversed phase HPLC
  • Basic analytes and ion suppression
  • Buffers for reversed phase HPLC
  • Henderson-Hasselbalch equation
  • Buffer preparation using the Henderson-Hasselbalch equation
  • Getting started with ionizable compounds
  • Optimizing separation selectivity of ionizable analytes
  • Effect of temperature on reversed phase HPLC