Characterization and multi-mode liquid chromatographic application of 4-propylaminomethyl benzoic acid bonded silica—A zwitterionic stationary phase

4-Propylaminomethyl benzoic acid bonded silica (4-PAMBA-silica) was synthesized by reacting aminopropyl modified silica with 4-carboxybenzaldehyde and reducing the resulting Schiff base with sodium cyanoborohydride in situ. The structure of this bonded phase was confirmed by 13C cross polarization magic angle spinning (13C CP MAS) NMR. Elemental analysis indicated a coupling efficiency of about 79%. Chromatographic characterization of a 4-PAMBA-silica stationary phase revealed that at a mobile phase pH of 3.0, basic compounds were unresolved and co-eluted near the void volume, while aromatic sulfonates were retained and were well-resolved. By contrast, at a mobile phase pH of 7.0, the aromatic sulfonates were unresolved and eluted at the void volume, while basic compounds were retained and were well-resolved. To further understand the chromatographic retention mechanism the retention factors for a series of cationic and anionic compounds were measured at pH 7.0 and 3.0 as a function of the charge and concentration of competing ions in the mobile phase. A plot of the logarithm of the retention factor versus the logarithm of the eluent ion concentration was linear with a negative slope that is equal to the ratio of effective charges of the solutes and the eluent ions. This indicates that an ion exchange mechanism contributes to the separation of both cations and anions at pH 7.0 and pH 3.0, respectively. The increase in retention of alkanoic acids with their number of carbons at a mobile phase pH of 7.0 and exclusion of alkanoic acids at a mobile phase pH of 3.0 suggests that an ion exclusion mode and hydrophobic interaction mode are also operational with 4-PAMBA-silica. The amino acids, l-arginine, l-phenylalanine, and l-tyrosine were retained and well-resolved with a mobile phase containing a high concentration of organic solvent. This behavior was further studied by measuring the retention factors of polar and charged compounds as a function of the organic solvent content in the aqueous mobile phase. An increase in retention with decreasing water content in the mobile phase was observed, consistent with a hydrophilic interaction liquid chromatographic (HILIC) mode of separation.