Enantiomeric separation of homocysteine and cysteine by electrokinetic chromatography using mixtures of gamma-cyclodextrin and carnitine-based ionic liquids

Abstract

Two new chiral ionic liquids based on the ester of the non-protein amino acid carnitine as cationic counterpart were synthesized, characterized and evaluated as chiral selectors for the enantiomeric separation of homocysteine and cysteine derivatized with 9-fluorenylmethoxycarbonyl chloride by electrokinetic chromatography. The use of both ionic liquids, L-carnitine methyl ester bis(trifluoromethane)sulfonimide and L-carnitine methyl ester L-(+)-lactate, as sole chiral selectors in the separation buffer, did not allow the enantiomeric separation of the studied amino acids. However, the combined use of L-carnitine methyl ester bis(trifluoromethane)sulfonimide ionic liquid with gamma-CD forming a dual system led to the enantiomeric separation of both analytes and showed the existence of a strong synergistic effect. On the contrary, the dual system gamma-CD plus L-carnitine methyl ester L-(+)-lactate did not improve the enantiomeric separations with respect to those obtained using the CD alone. The influence of different experimental variables such as buffer composition and pH, and ionic liquid concentration was investigated. Also, the nature of the anionic moiety was evaluated by comparing the results obtained with both ionic liquids when combined with gamma-CD. The use of 2 mM gamma-CD combined with 5 mM L-carnitine methyl ester bis(trifluoromethane)sulfonimide in a 50 mM phosphate buffer at pH 7.0 enabled to achieve the simultaneous enantiomeric separation of homocysteine and cysteine with high resolution values (> 6.0) in analysis times close to 12 min.