Retention mechanism
In general, HPLC is a dynamic adsorption process. Analyte molecules, while moving through the porous packing bead, tend to interact with the surface adsorption sites. Depending on the HPLC mode, the different types of the adsorption forces may be included in the retention process:
 | Hydrophobic (non-specific) interactions are the main ones in reversed-phase separations. |
| Dipole-dipole (polar) interactions are dominated in normal phase mode. |
| Ionic interactions are responsible for the retention in ion-exchange chromatography. |
All these interactions are competitive. Analyte molecules are compete with the eluent molecules for the adsorption sites. So, the stronger analyte molecules interact with the surface, and the weaker the eluent interaction, the longer analyte will be retained on the surface.
SEC (size-exclusion chromatography) is a special case. It is the separation of the mixture by the molecular size of its components. In this mode any positive surface interactions should be avoided (eluent molecules should have much stronger interaction with the surface than analyte molecules).
Basic principle of SEC separation is that the bigger the molecule, the less possibility for her to penetrate into the adsorbent pore space, so, the bigger the molecule the less it will be retained.
