Characterisation of π–π interactions which determine retention of aromatic compounds in reversed-phase liquid chromatography

Abstract

This paper presents the influence of π–π interactions on the retention behaviour of aromate-rich and aromate-poor analytes separated on various chromatographic sorbents. The presence of π–π interactions between the analytes and a polymer based sorbent can be very useful for separating compounds with similar retention behaviour on silica based reversed-phase sorbents (where this type of interaction is absent). Various C 4 and C 18 modified silica sorbents (5 μm and 15 μm) are compared with some poly(styrene-divinyl benzene) (PS-DVB) sorbents. For this purpose five aromates and four non-aromates were selected. The retention times of the nine uncharged test compounds (hydrocortisone, hydrocortisone acetate, testosterone, testosterone propionate, nitrobenzene, anisole, toluene, valerophenone, cumene) increase with increasing hydrophobicity (log P). On silica based reversed-phase sorbents this relationship is the same for aromates and non-aromates. On PS-DVB sorbents this relationship is different: the aromatic compounds need more acetonitrile to elute from the column than the non-aromatic compounds with a similar hydrophobicity: at log P 2.7 the difference in acetonitrile concentration is approximately 18–20% for all tested polymer matrices which have no groups covalently bound to their PS-DVB moieties (Vydac™, Polymer Laboratories, Amersham Pharmacia Biotech). This difference is probably due to the presence of an π–π interaction between the sorbent and the analyte. The importance of the π–π interaction decreases when more acetonitrile is needed to elute an aromatic analyte from the column. The retention of these mono-aromates largely depends on hydrophobic interactions and π–π interactions. The higher the log P of the analyte (containing an aromatic group), the more important the hydrophobic interaction becomes. Although covalent coupling of functional groups to the PS-DVB sorbent (with a phenyl, isopropyl or ether linked via a –CH 2CHOHCH 2O– spacer) leads to a more hydrophilic sorbent, it does not lead to a decrease in π–π interaction since the surface still accessible for the compounds tested. A comparison of the retention behaviour of the aromatic test compounds with the non-aromatic test compounds on silica based reversed-phase sorbents with that on PS-DVB based sorbents shows the importance of determining π-π interactions.