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Frequently Asked Questions

What is the difference between Reversed Phase (RP) HPLC and hydrophobic interaction chromatography (HIC)?

In both cases, the stationary phase is more hydrophobic than the mobile phase. The hydrophobicity of RP-stationary phases is generally higher than that of HIC media. Elution in RP is obtained by adjusting the polarity of the mobile phase with a water miscible solvent, e.g. an alcohol or acetonitrile. Elution takes place either under isocratic (constant modifier concentration) or a under gradient conditions (increase in modifier concentration).

HIC media has weaker hydrophobic character and elution is induced by decreasing the polarity in the mobile phase by virtue of a decrease in salt concentration. HIC is generally applied in protein separations, where exposure to organic solvents under prolonged time might induce irreversible unfolding, thus loss of biological activity of the proteins.

For analytical purposes, RP-HPLC is an unchallenged method when it comes to separating closely related substances, including proteins.