What is the Difference Between RP HPLC and HIC?

The main difference between Reversed Phase (RP) HPLC and Hydrophobic Interaction Chromatography (HIC) lies in the type of stationary phase and mobile phase used, as well as the conditions under which the separation occurs. Here are the key differences between RP HPLC and HIC:

1. Stationary Phase: RP HPLC uses a less hydrophobic stationary phase, while HIC uses a more hydrophobic stationary phase.
2. Mobile Phase: RP HPLC employs a more polar mobile phase, such as water, while HIC uses a less polar mobile phase.
3. Separation Mechanism: In RP HPLC, molecules are separated based on their hydrophobicity, with more hydrophobic molecules eluting first. HIC also separates molecules based on hydrophobicity, but the conditions for binding are different. High salt concentrations are used to increase surface tension and promote the binding of hydrophobic molecules to the stationary phase.
4. Selectivity: The selectivity of HIC columns can be easily manipulated by changing mobile phase variables, such as salt concentration and type. The selectivity of RP HPLC is affected by factors such as matrix ligand density.

In summary, RP HPLC and HIC are both chromatographic techniques used for separating components in a mixture, but they differ in their stationary and mobile phases, as well as the conditions under which separation occurs. RP HPLC uses a less hydrophobic stationary phase and a more polar mobile phase, while HIC uses a more hydrophobic stationary phase and a less polar mobile phase, with separation occurring under high salt concentrations.

Comparative Table: RP HPLC vs HIC

The main differences between Reversed-Phase HPLC (RP HPLC) and Hydrophobic Interaction Chromatography (HIC) are the mobile phase and stationary phase properties, as well as the elution conditions. Here is a comparison table highlighting the key differences between the two techniques:

Both RP HPLC and HIC use a hydrophobic stationary phase to separate molecules based on their hydrophobicity. However, the mobile phase and elution conditions differ significantly. In RP HPLC, a highly polar mobile phase (often water) is used, while HIC employs high salt concentrations as the mobile phase. The elution process in RP HPLC is driven by an increasing gradient of organic solvent, whereas in HIC, it is driven by a decreasing gradient of salt.