Basics of HPLC

HPLC: high-performance liquid chromatography

HPLC (or high-performance or high-pressure liquid chromatography) is an analytical separation technique, based on the progressive migration of compounds in a column that withstands high pressures.

The interactions used to retain and elute compounds play out between:
  • The compound: C
  • The mobile phase: MP
  • The stationary phase: SP
Illustration of the exchanges between compound C, mobile phase PM and stationary phase PS. Double arrows symbolize the exchanges between C and PM, C and PS, PM and PS.

Different separation modes exist in HPLC. The chosen mode depends on the characteristics of the molecules to be separated.

The different types of chromatography commonly used are:

  1. Partition chromatography (most commonly used)
  2. Ion-exchange chromatography
  3. Size-exclusion chromatography
  4. Adsorption chromatography (rarely used, intended for specific applications)

The simplified diagram below helps to identify the HPLC method best suited to the separation of the compounds according to their characteristics.

Decision tree of the type of HPLC according to the molecule. If M > 2000, organosoluble, then steric exclusion. If M > 2000 and hydrosoluble, slors steric exclusion or reverse phase partitioning. If M < 2000, Hydrosoluble and ionic, then ionic. If M < 2000, water-solubleand nonionic, then normal or reverse phase partitioning. Finally, if M < 2000, organosoluble, then normal or reverse phase partitioning.
The quality of a chromatographic separation can be evaluated thanks to different chromatographic parameters that can be found in the drawer "Chromatographic parameters".
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