ABSTRACT
Of the various modes of high performance liquid chromatography (HPLC), reversed phase (RPLC) is the most commonly used, with normal phase, ion exchange, gel permeation, chromatofocusing, metal interaction, and affinity chromatography being the principal alternative modes. Normal phase is a phrase used to indicate that the stationary phase is more polar than the solvent, and reversed phase indicates that the stationary phase is less polar than the solvent. The development of aqueous-organic gradient generation, which allows the polarity to be varied over a wide range during a single run, has made the distinction between reversed and normal phase somewhat artificial, although the distinction is still useful. In general, adsorption of a solute to a reversed phase is driven by hydrophobic interactions, while adsorption to normal phase is often driven by hydrogen bonding between the solute and stationary phase. Normal phase and reversed phase HPLC predominate in the analysis of small organic molecules. Reversed phase was extended long ago to the analysis of macromolecules, such as proteins, while the extension of normal phase to macromolecules has been slower. A considerable variety of very efficient reversed phase columns is available. The present chapter will examine the theory and practice of reversed phase chromatography, beginning with an examination of the separation of small molecules.
