What is the principle behind reverse-phase HPLC?

The principle behind reverse-phase high-performance liquid chromatography (HPLC) is separation based on hydrophobicity. This technique utilizes a stationary phase that is non-polar, typically composed of C18 alkyl chains bonded to silica particles, while the mobile phase is more polar, often a mixture of water and organic solvents like acetonitrile or methanol.

In reverse-phase HPLC, compounds in the sample interact differently with the stationary phase depending on their hydrophobic characteristics. More hydrophobic molecules will have a stronger interaction with the non-polar stationary phase, leading to longer retention times, as they tend to stay in the stationary phase longer before eluting. Conversely, more polar compounds interact less with the stationary phase and elute faster. This method effectively separates compounds based on their relative hydrophobicity, making it a popular choice for purifying and analyzing a wide range of organic and biological molecules.

This is distinct from other separation techniques that focus on different properties, such as molecular size, charge, or binding interactions, which utilize different mechanisms and conditions for separation.