What is the primary mechanism of solute separation in HPLC with a C18 column?

In High-Performance Liquid Chromatography (HPLC) utilizing a C18 column, the primary mechanism of solute separation is through partitioning. C18 columns are composed of octadecyl (C18) chains bonded to silica particles, which provide a hydrophobic environment for the separation of compounds.

During the HPLC process, the sample is introduced into a mobile phase that moves through the stationary phase (the C18 column). As the mobile phase interacts with different solutes in the sample, compounds will partition between the mobile phase and the hydrophobic stationary phase depending on their hydrophobic characteristics. Polar or less hydrophobic substances will remain longer in the mobile phase, while more hydrophobic substances will tend to associate more with the stationary phase. This differential partitioning leads to their separation as they travel through the column at different rates.

In contrast, anion and cation exchange mechanisms involve ionic interactions between charged solutes and the resin used in the column, which is not applicable to the C18 mechanism. Size exclusion primarily relies on molecular size and does not involve hydrophobic interactions, making it unsuitable for describing the separation in a C18 column application.