TABLE OF CONTENTS The IUPAC definition understandably states that reversed phase chromatography is an elution procedure in which the mobile phase is significantly more polar than the stationary phase . This is a somewhat simplistic statement that covers a wealth of possibilities mirrored by the broad range of reversed stationary phases available, to what is without doubt the most commonly applied mode of preparative HPLC. A brief survey suggests that there are over five hundred different reversed phase materials commercially available. However, the eventual scale of operation may limit the number of suitable stationary phases to less than ten.
Reversed phase media are designed to operate in aqueous buffers where the hydrophobic adsorption between analyte and stationary phase is disturbed by an increasing concentration of a miscible organic solvent. The most common application of reversed phase HPLC is in the purification of peptides and proteins where the analyte is often desorbed under gradient elution. The most common aqueous buffer is water containing low levels of trifluoroacetic acid (typically 0.1% v/v) and the eluting mobile phase or polar modifier as it is often termed is usually acetonitrile containing trifluoroacetic acid. The trifluoroacetic acid serves two purposes: firstly, it forms a strong ionpair with the analyte thus counteracting the cation exchange with any free silanol groups on silica supports; and secondly, by virtue of the ion-pair, it reduces the conformational variations in peptides and proteins consequently improving peak shape. The added advantage of trifluoroacetic acid lies in its transparency to...
