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

Center of Biotechnology, Swiss Federal Institute of Technology Lausanne, 1015 Lausanne, Switzerland.
Journal of Chromatography A (Impact Factor: 4.26). 08/2003; 1004(1-2):195-208. DOI: 10.1016/S0021-9673(03)00563-6
Source: PubMed

ABSTRACT 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.

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Available from: Ruth Freitag, Jul 28, 2014
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    • "Their observed critical mobile phase composition are around 20% and 5% ACN in water, respectively [20,23]. 1,4-bis(acryloyl)piperazine (PDA) is another polar crosslinker (solubility in water: 0.560 g/100 mL at 25 • C) [27] and has been successfully employed for preparing organic polymers such as monoliths resulting from N,N-dimethylacrylamide (DMAA) and methacrylamide (MAA) as the functional monomer [28] [29]. Compared to EDMA, PDA could also better match the solubility of highly hydrophilic functional monomers in water. "
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    Journal of Chromatography A 11/2014; 1373. DOI:10.1016/j.chroma.2014.11.007 · 4.26 Impact Factor
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    • "Another approach to create a macroporous acrylamide monolith was studied by Hoegger and Freitag [27]. The addition of a lyotropic salt to aqueous polymerization feed manifested itself in the phase separation during a redox-initiated polymerization [28] [29] [30]. Maruska also developed a sturdier acrylamide monolithic material [31] and demonstrated its application in chiral ligand-exchange CEC separations [32] [33] [34] [35]. "
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