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AAT Bioquest

What is the difference between normal-phase chromatography and reversed-phase chromatography?

Posted June 1, 2020


Answer

In a normal-phase chromatography, a polar stationary phase (such as silica) and a non-polar, non-aqueous mobile phase (e.g. chloroform and hexane) are employed. The separation is based on analyte’s ability to engage in polar interactions (such as hydrogen-bonding or dipole-dipole interactions) with the stationary surface. Non-polar molecules have weaker retention and would be eluted first.

Reversed-phase chromatography, in contrast, has a non-polar stationary phase and an aqueous, moderately polar mobile phase. One popular stationary phase is C18 modified silica, which retains molecules through hydrophobic interaction. Acetonitrile, methanol, and their mixture with water are commonly used as the mobile phase. Molecules with less polarity have longer retention time, while polar molecules elute more readily.

Additional resources

Antibody development services

Miller, J. M. (2003). Chromatography. digital Encyclopedia of Applied Physics, 1055-1102.

Janson, J. C. (Ed.). (2012). Protein purification: principles, high resolution methods, and applications (Vol. 151). John Wiley & Sons.