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

2008; Wiley; Volume: 31; Issue: 14 Linguagem: Inglês

10.1002/jssc.200800178

1615-9314

Michael Lämmerhofer, Martin Richter, Junyan Wu, Raquel F. Pupo Nogueira, Wolfgang Bicker, Wolfgang Lindner,

Protein purification and stability

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‐(2 S ,4 S ,5 R )‐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 (α CH2 of 1.5) than the tested Synergi Fusion RP (α CH2 ∼︁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.