title: Method development and optimization on cinchona and chiral sulfonic acid-based zwitterionic stationary phases for enantiomer separations of free amino acids by high-performance liquid chromatography.
abstract: CHIRALPAK ZWIX(+) and ZWIX(-) are cinchona alkaloid-derived zwitterionic chiral stationary phases (CSPs) containing a chiral sulfonic acid motif which serves as negatively charged interaction site. They are versatile for direct enantiomer resolution of amino acids and many other ampholytic compounds by HPLC. The synergistic double ion-pairing between the zwittrionic chiral selector and the ampholyte is the basis for interaction and chiral recognition mechanisms. ZWIX(+) and ZWIX(-) type CSPs or columns behave pseudo-enantiomerically and provide the feature of reversing enantiomer elution order by column switching. In the current study, extensive experimental work was carried out with the aim of developing schemes for an efficient generic screening and proposing straightforward approaches for method optimization on these ZWIX columns. Various chromatographic parameters were investigated using a large series of diverse amino acids and analogues for the purpose. The role of methanol (MeOH) as the protic solvent in the mobile phase is confirmed to be essential. The presence of water in a low percentage is beneficial for peak shape, resolution, analysis speed, sample solubility and MS detection performance. The involvement of acetonitrile (ACN) or tetrahydrofuran (THF) can help for adjusting retention time and selectivity. Incorporation of a suitable pair of acidic-basic additives at a right ratio in the mobile phase is determinant as well for the double ion-pairing mechanism. 50 mM formic acid+25 mM diethylamine (or ammonium hydroxide) in MeOH/ACN/H₂O and in MeOH/THF/H₂O at 49:49:2 (by volume) are recommended as the starting mobile phases for method development. Some other parameters are also considered in the proposed scheme to achieve successful enantioselective or stereoselective separation of the ampholytes.
authors: Chiral Technologies Europe, Bd. Gonthier d'Andernach, 67400 Illkirch, France. Electronic address: tzhang@chiral.fr.
authors: Tong
authors: T
authors: Zhang
authors: Chiral Technologies Europe, Bd. Gonthier d'Andernach, 67400 Illkirch, France.
authors: Emilie
authors: E
authors: Holder
authors: Chiral Technologies Europe, Bd. Gonthier d'Andernach, 67400 Illkirch, France.
authors: Pilar
authors: P
authors: Franco
authors: Department of Analytical Chemistry, University of Vienna, 1090 Vienna, Austria.
authors: Wolfgang
authors: W
authors: Lindner
mesh_terms: D000596:Amino Acids / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002929:Cinchona / Q000737:chemistry; D013237:Stereoisomerism; D013451:Sulfonic Acids / Q000737:chemistry
chemical_list: D000596:Amino Acids; D013451:Sulfonic Acids
keywords: Approaches for method development; Enantiomer separation by HPLC; Free amino acids; Hydro–organic mobile phase systems; Method optimization; Zwitterionic chiral stationary phases based on cinchona alkaloids