Applications of Normal and Reverse Phase High Performance Liquid Chromatography
High Performance Liquid Chromatography (HPLC) can be either Normal Phase HPLC or Reverse Phase HPLC, depending on the properties of the column and the mobile phase used.
1. Normal Phase Chromatography:
In normal phase HPLC, the column packing 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 due to stronger interactions with the polar stationary phase.
2. Reverse Phase Chromatography:
In contrast, reverse phase HPLC uses non-polar or weakly polar column packing materials (such as hydrophobic alkyl silane compounds, e.g., C18), and the mobile phase is polar. In reverse phase chromatography, non-polar molecules have a longer retention time in the column because 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 offer better compatibility and separation efficiency for most biomolecules and organic compounds.
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