Investigation of mixed-mode monolithic stationary phases for the analysis of charged amino acids and peptides by capillary electrochromatography

The potential of N,N-dimethylacrylamide–piperazine diacrylamide-based monolithic stationary phases bearing sulfonic acid groups for electroosmotic flow generation is investigated for the separation of positively charged amino acids and peptides. The capillary columns were used under electrochromatographic but also under purely chromatographic (nano-HPLC) conditions and the separations interpreted as the result of possible chromatographic and electrophoretic contributions. The stationary phases were found to be mechanically stable up to pressures of 190 bar and chemically stable towards a wide variety of organic and hydro-organic mobile phases. In order to investigate the retention mechanism, the salt concentration and the organic solvent content of the (hydro-)organic mobile phase were varied in a systematic manner, taking three aromatic amino acids (phenylalanine, tryptophan, histidine) as model analytes. The respective contributions of electrostatic and hydrophobic and/or hydrophilic interactions were further investigated by varying the charge density and the hydrophobicity of the standard stationary phase. The former was done by varying the amount of charged monomer (vinylsulfonic acid) added during synthesis, the latter by (partially) replacing the interactive monomer (N,N-dimethylacrylamide) by other more hydrophobic monomers. A mixed mode retention mechanism based primarily on electrostatic interactions modified in addition by “hydrophilic” ones seems most suited to interpret the behavior of the amino acids, which stands in contradistinction to the previously investigated case of the behavior of neutral analytes on similar stationary phases. Finally the separation of small peptides was investigated. While the separation of Gly–Phe and Gly–Val was not possible, the separation of Phe–Gly–Phe–Gly and Gly–Phe but also of the closely related Gly–His and Gly–Gly–His could be achieved.