Quinine‐Based Zwitterionic Chiral Stationary Phase as a Complementary Tool for Peptide Analysis: Mobile Phase Effects on Enantio‐ and Stereoselectivity of Underivatized Oligopeptides

Abstract

Peptide stereoisomer analysis is of importance for quality control of therapeutic peptides, the analysis of stereochemical integrity of bioactive peptides in food, and the elucidation of the stereochemistry of peptides from a natural chiral pool which often contains one or more D-amino acid residues. In this work, a series of model peptide stereoisomers (enantiomers and diastereomers) were analyzed on a zwitterionic ion-exchanger chiral stationary phase (Chiralpak ZWIX(+) 5 µm), in order to investigate the retention and separation performance for such compounds on this chiral stationary phase and elucidate its utility for this purpose. The goal of the study focused on 1) investigations of the effects of the sample matrix used to dissolve the peptide samples; 2) optimization of the mobile phase (enabling deriving information on factors of relevance for retention and separation); and 3) derivation of structure-selectivity relationships. It turned out that small di- and tripeptides can be well resolved under optimized conditions, typically with resolutions larger than 1.5. The optimized mobile phase often consisted of methanol-tetrahydrofuran-water (49:49:2; v/v/v) with 25 mM formic acid and 12.5 mM diethylamine. This work proposes some guidance on which mobile phases can be most efficiently used for peptide stereoisomer separations on Chiralpak ZWIX. Chirality 28:5-16, 2016. © 2015 Wiley Periodicals, Inc.

Exploring the enantiorecognition mechanism of Cinchona alkaloid‐based zwitterionic chiral stationary phases and the basic trans‐paroxetine enantiomers

“…In previous work, we already recognized the crucial contribution of SELF‐SA in explaining the observed EEO . Very importantly, the computational data produced by the present study strongly support the outstanding role of the conformational energy of the chiral selector as it interacts with the analyte.…”

“…ZWIX(+) [QN-(S,S)-ACHSA] and ZWIX(-) [QD-(R,R)-ACHSA] (where QN is quinine and QD is quinidine) are commercially available from Chiral Technologies Europe (under the trade name of CHIRALPAK ZWIX(+) and CHIRAL-PAK ZWIX(-), respectively), while ZWIX(-A) [QD-(S,S)-ACHSA] and ZWIX(+A) [QN-(R,R)-ACHSA] were prepared as reported elsewhere [6]. Profitably, CHIRALPAK ZWIX(+) and CHIRALPAK ZWIX(-) are well performing in the enantioseparation of chiral acids [7][8][9][10][11], bases [6,7,12], and zwitterion molecules [7,13,14].…”

State-of-the-art enantioseparations of natural and unnatural amino acids by high-performance liquid chromatography

“…Another interesting field of application of zwitterionic CSPs that may be expected to gain in importance in the near future is the stereoselective separation and analysis of small peptides [48][49][50][51].…”

Liquid chromatographic enantiomer separations applying chiral ion-exchangers based on Cinchona alkaloids

“…Similar to the AX-type columns, the zwitterionic phases are mostly employed with MeOH-and/or ACN-based eluent systems using acid and/or base additives ( Table 2). Other organic solvents like EtOH [42], IPA [42,98] and THF [53,108,116,120] have been tested to improve separation performance. On Chiralpak ZWIX(+)™, the replacement of MeOH with 2 v% EtOH or IPA resulted in slightly reduced retention factors.…”