Comprehensive isotachophoresis of 20 natural amino acids for preconcentration in column-coupling ITP-CE-MS

KLER, PABLO A.; HUHN, CAROLIN

Congreso; 20th INTERNATIONAL SYMPOSIUM ON ELECTRO- AND LIQUID PHASE- SEPARATION TECHNIQUES; 2013

Comprehensive isotachophoresis of 20 natural amino acids for preconcentration in column-coupling ITP-CE-MS Pablo A. Kler, Carolin Huhn* Central Institute for Engineering, Electronics and Analytics, ZEA-3: Analytics, Forschungszentrum Jülich; Wilhelm-Johnen-Str., 52428 Jülich, Germany *c.huhn@fz-juelich.de, Tel: +49-2461-61-6873, Fax: +49-2461-61-2560 The analysis of amino acid is required in various fields such as clinical diagnosis, metabolic studies of different living organisms, and food science, among others. These organic compounds may be present at trace levels and in concentration ranges spanning up to 6 orders of magnitude [1] in complex biological matrices and often only small sample volumes are available, which makes them difficult to be analyzed. In the last decade with the impressive progresses made by electromigrative separation techniques, several successful applications of CE for the analysis of amino acids were reported, especially CE-MS coupling was shown to offer unique features for identification and quantification of the 20 natural human amino acids. Ideally it would be combined with ITP offering complementary features in terms of the high sample loadability and a strong pre-concentration effect [2], and thus giving rise to impressive limits of detection, matrix removal, high separation efficiency and resolution. However, so far a comprehensive preconcentration of all 20 natural amino acids has not been achieved using ITP, as extremely low or high pH-values would be required to assure all amino acids being positively or negatively charged respectively [3]. Under these conditions of pH, accomplishing ITP is impossible due to the fact that the conductivities of leading (LE) and trailing electrolyte (TE) are ruled by the high mobility H+ or OH- ions. As a consequence, the conductivity gap between LE and TE is neglectable and the conditions for a successful ITP-stacking are not satisfied [4], instead CE conditions are obtained. We overcame this problem by reducing the mobility of H+ or OH- in the ITP system and by changing the pH and pKa regime in the electrolyte system. In order to assure proper ITP conditions we developed a novel mixed (aqueous-organic) electrolyte system working at extreme acidic conditions using oxalate as counterion for extremely acidic conditions enforcing cationic charge for all amino acids, impossible in purely aqueous ITP systems. The key here is to dilute water by the addition of diploar aprotic solvents or high dielectric constant as the lack of hydrogen bonding hinders the fast ?motion? of H+ via flipping bonds. In addition acid-base equilibria are advantageously shifted in these solvents. We here show comprehensive column-coupled ITP-CE-MS analysis of amino acids using a novel hybrid capillary-microchip approach for interfacing, that includes intermediate contactless conductivity detection and enables robust separation and identification processes [5]. [1] T. Soga, et al., Electrophoresis 25 (2004) 1964-1972. [2] P. Gebauer, et al., Electrophoresis 30 (2009) 29-35. [3] J. Prest, et al., J. Chrom. A. 1051 (2004) 221-226. [4] J. Petr, et al., J. Sep. Sci. 29 (2006) 2705 ? 2715. [5] P. Kler, et al., J. Chrom. A. http://dx.doi.org/10.1016/j.chroma.2013.04.046.