Ag + -HPLC of conjugated linoleic acids on a silica-based stationary phase. Part III: Model compounds During the development of a method for the direct analysis of the conjugated linoleic acids (CLAs) in their free fatty acid form, it was necessary to include a competing acid (acetic acid) in the hexane-based mobile phase in order to achieve resolution of the isomers in a reasonable time. Thus it was clear that the CLAs were retained by two mechanisms. In this study we examine the relative retentions of the CLAs, their methyl esters (CLAMEs, which is the form generally analyzed), and oleic and stearic acids. The primary aim was to investigate the nature of CLA retention in terms of the Ag + -p bond interactions (that might be approximated by the retention of the CLAMEs) and the 1COOH-surface silanol hydrogen bonding that should be the same as stearic acid, if the retention mechanisms are independent. Allowing for some retention of the ester group in the CLAMEs on the silica, it may have been expected that the sum of the retention of the CLAMEs and stearic acid would slightly exceed that of the CLAs. On the contrary, it was found that the retention of the CLAs far exceeded the sum for the CLAMEs and stearic acid. Some synergy is suggested, rather than a third retention mechanism.

In the previous paper on the Ag+‐HPLC of conjugated linoleic acids (CLAs) on a silica‐based stationary phase, we examined the relative retentions of the CLAs, their methyl esters (CLAMEs), and stearic acid. The retention of the CLAs far exceeds the sum of the retention of the CLAMEs and stearic acid. In this paper we isolate the interactions at the silica surface by determining the retentions of the same compounds on the NH4+ cation exchanger that is the precursor to the Ag+ column in the manufacturing process. On the NH4+ column, the CLAMEs are very weakly retained, but the sum of the retentions of the CLAMEs plus stearic acid is equal to that of the CLAs. It was determined that stearic acid is retained to the same degree on both the NH4+ and Ag+ stationary phases. By implication, the CLA carboxylic acid functionality does not interact with Ag+ and thus three‐centre complexes of the Ag+ with the CLA π bonds and carboxyl group do not occur. (Independent supporting evidence is also presented.) A model for CLA retention on the Ag+ stationary phase that explains the synergy of retention is presented. It involves a random arrangement of collapsed Ag+ alkyl sulphonate bonded phase on the silica surface. The nature of retention on the NH4+ stationary phase is also discussed. Partial separation of the CLAs on the NH4+ stationary phase shows that π bond interactions with the silica surface also contribute to the separations of the CLAs, the CLAMEs, and other unsaturated analytes. As was found on the Ag+ column in the absence acetonitrile, normal phase elution occurs and log k′ versus log (% strong solvent) plots for all analytes are linear on the NH4+ column. (Where the specific displacement mechanism of unsaturates from the Ag+ by ACN was present, the log‐linear plots were straight lines.)