AG⁺‐HPLC of conjugated linoleic acids on a silica‐based stationary phase. Part IV: A reference stationary phase and retention mechanisms

Abstract

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.)

Silver Ion Chromatography of Fatty Acids

“…Gunstone and co-workers (1967) were the first to demonstrate that the double bond position affected the retention of isomeric octadecenoic methyl esters in argentation TLC. Later, Cross and Zackari (2003) used the same concept in their efforts to explain the separation of conjugated linoleic acid (CLA) isomers in Ag-HPLC. (ii) The other hypothesis assumes that one silver ion complexates with two reaction centers in the same molecule, namely the double bond in the alkenyl chain and the free electron pair at the carbonyl oxygen in the ester moiety, thus forming a chelate-type complex (Nikolova- Damyanova et al 1996).…”