Research Article Enantiomeric separation of FMOC-amino acids by nano-LC and CEC using a new chiral stationary phase, cellulose tris(3-chloro-4-methylphenylcarbamate) A novel polysaccharide-based chiral stationary phase (CSP), cellulose tris(3-chloro- 4-methylphenylcarbamate), also known as Sepapak-2 or Lux Cellulose-2, has been evaluated for the enantiomeric separation of FMOC derivatives of amino acids. After mobile-phase optimization in nano liquid chromatography (nano-LC) the column enabled the enantiomeric separation of 19 out of 23 amino acids tested, indicating the high chiral recognition power of this new CSP. Subsequently, a comparison of the driving force employed (pressure or voltage) was carried out comparing nano-LC and CEC under the same conditions. Better peak efficiencies and resolution were observed by using CEC experiments, which enabled the chiral discrimination of 20 out of 23 amino acids tested. Finally, in order to show the potential of this new CSP, the determination of the content and the enantiomeric purity of the non-protein amino acid citrulline in food supplements was performed. For that purpose, the method was optimized, evaluated and applied to different commercial samples. Keywords: Amino acids / Citrulline / FMOC / Nano-liquid chromatography / Polysaccharide- based chiral stationary phase DOI 10.1002/elps.201000701 1 Introduction CEC and nano-LC are attractive separation methods which offer the advantages of microseparation techniques as low sample and solvent consumption, high column efficiency and good sensitivity when coupled with mass spectrometers. CEC is a promising microseparation technique, which combines the high selectivity of HPLC and the high efficiency of CE showing a great potential for enantiomeric separations. Different types of chiral stationary phases (CSPs) have been used for CEC separations as cyclodextrins, proteins, molecularly imprinted polymers, macrocyclic antibiotics, chiral acrylamides, anion and cation exchangers, Pirkle-type CSPs and polysaccharide derivatives [1, 2]. Polysaccharide-type CSPs have shown to be powerful for enantioseparation in HPLC due to their enantioselectivity towards different analytes, their compatibility with organic and aqueous mobile phases, the possibility of high loading of a chiral selector onto inert carrier, and the easy availability of their natural sources [3]. Typically, these CSPs are prepared by coating or covalent-bonding of polysaccharide phenyl esters or phenylcarbamates on the surface of silica particles [4]. Using this type of CSPs the major forces involved in chiral recognition are considered to be hydrogen- bonding, p–p, dipole–dipole, and steric interactions, and in the RP mode also hydrophobic interactions [2, 5]. The simultaneous introduction of electron-donating and elec- tron-withdrawing groups, such as methyl or chlorine, in the phenyl group of polysaccharide phenylcarbamates has shown a favourable effect on their enantioselectivity [6–8]. Cellulose tris(3-chloro-4-methylphenylcarbamate), also known as Sepapak-2 s or Lux Cellulose-2, is a novel CSP which combines both electron-donating and electron-withdrawing substituents. This CSP has been employed only in a few works for HPLC [3, 5–10] and CEC [11, 12] separation of enantiomers of chiral pharmaceutical compounds. Enantio- mers of some amino acid derivatives have been separated with this CSP in normal-phase HPLC [3] but its resolving potential for amino acid derivatives under RP HPLC and CEC conditions has not yet been studied. Amino acids are important molecules that play essential roles in nature. It is well known that amino acids are chiral Elena Domı´nguez-Vega 1 Antonio L. Crego 1 Ketevan Lomsadze 2 Bezhan Chankvetadze 2 Maria L. Marina 1 1 Department of Analytical Chemistry, Faculty of Chemistry, University of Alcala ´ , Madrid, Spain 2 Institute of Physical and Analytical Chemistry, School of Exact and Natural Sciences, Tbilisi State University, Tbilisi, GA, USA Received December 31, 2010 Revised February 16, 2011 Accepted February 16, 2011 Abbreviation: Cit, citrulline; CSP, chiral stationary phase; RLOD, relative LOD Correspondence: Professor Maria L. Marina, Department of Analytical Chemistry, Faculty of Chemistry, University of Alcala ´, Ctra. Madrid-Barcelona Km. 33.600, 28871 Alcala ´ de Henares, Madrid, Spain E-mail: mluisa.marina@uah.es Fax: 134-91-8854971 & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2011, 32, 2700–2707 2700