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Reversed-Phase Chromatography: General Introduction For Improved Method Development

Today, more than 80% of all analytical chromatographic separations are performed using reversed-phase sorbents which have shown higher versatility compared to normal-phase chromatography sorbents. Reversed-phase sorbents have found their use in a wide range of applications such as process purification, isolation of active biomolecules, analytical separation of drugs and metabolites as well as extraction of various contaminants in environmental samples.

However, reversed-phase chromatography includes a large number of different phases that differ significantly in both chemical and physical properties which will have a significant impact on their chromatographic behaviours. Thus, one chromatographer will have to consider all of the following stationary phase properties while gathering information on the sample to be analyzed or purified:

  • Hydrophobicity
  • Particle and Pore Diameter
  • Bonding Chemistry (Surface Coverage, Polymeric vs Monomeric)
  • Particle Geometry (Irregular vs Spherical)
  • Silica Purity
  • Analyte pKa
  • Molecular Weight
  • Sample Matrix (pH, salts, concentration, interferents)

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