Forward and Reverse Actions in High-Performance Liquid Chromatog

High Performance Liquid Chromatography (HPLC) can be either Normal Phase HPLC or Reverse Phase HPLC, depending on the nature of the chromatographic column and the mobile phase used.

 

Normal Phase Chromatography

In Normal Phase HPLC, the column packing material is polar (e.g., silica gel), and the mobile phase is a non-polar or moderately polar solvent. In this setup, polar molecules have a longer retention time in the column as they have stronger interactions with the polar stationary phase.

 

Reverse Phase Chromatography

In contrast, Reverse Phase HPLC uses non-polar or weakly polar chromatographic column packing materials (like hydrophobic alkyl silanes, such as C18), and the mobile phase is polar. In Reverse Phase Chromatography, non-polar molecules have a longer retention time in the column as they have stronger interactions with the non-polar stationary phase.

 

In practical applications, Reverse Phase HPLC is more common, especially in bioanalysis and organic molecule analysis. This is because reverse phase columns have better compatibility and separation efficiency for most biomolecules and organic compounds.