Mixed-mode ion-exchangers and their comparative chromatographic characterization in reversed-phase and hydrophilic interaction chromatography elution modes

Abstract

A set of particulate silica-supported mixed-mode RP/weak anion-exchangers (RP/WAX) (obtained by bonding of N-undecenoylated 3-aminoquinuclidine, 3-aminotropane and 2-dimethylaminoethylamine as well as of N-butenoyl-(2S,4S,5R)-2-aminomethyl-5-[(2-octylthio)ethyl]-quinuclidine to thiol-modified silica) were chromatographically characterized in comparison to selected commercially available columns using two distinct isocratic elution modes, viz. an aqueous-rich RP-type elution mode (with 40% ACN and 60% buffer) as well as an organic solvent-rich hydrophilic interaction chromatography (HILIC)-type elution mode (95 and 90% ACN). The mixed-mode RP/WAX phases showed multimodal applicability, unlike a polar embedded RP material (Synergi Fusion RP), amino phases (Luna NH(2), BioBasic AX) or typical HILIC packings (ZIC-HILIC, TSKGel Amide-80). Principal component analysis (PCA) of the RP test data confirmed that the in-house developed RP/WAX columns as well as the Acclaim Mixed-Mode WAX-1 phase resemble each other in their chromatographic characteristics having slightly lower hydrophobic selectivity (alpha(CH2) of 1.5) than the tested Synergi Fusion RP (alpha(CH2) approximately 1.8). In contrast, a decrease in mixed-mode character due to lowered ion-exchange capacity and concomitantly increased RP-like behavior could be identified for other mixed-mode phases in the order of Obelisc R > Primesep B2 > Uptisphere MM3. PCA on HILIC data revealed that the RP/WAX phases behave dissimilar to TSKGel Amide-80, ZIC-HILIC and polysulfoethyl A under the chosen elution conditions. Hence, they may be regarded as complementary to these commercial stationary phases with applicability profiles for hydrophilic but also hydrophobic solutes.