Resolution of acylated dipeptide stereoisomers by capillary electrophoresis using sulfobutylether derivatized β‐cyclodextrin

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Abstract
The separation of enantiomerically and diastereomerically related stereoisomers of acylated Asp-Phe dipeptides was explored using capillary electrophoresis (CE). This series of dipeptides included the α-L,L parent compound and the three other potential Asp containing stereoisomers (α-D,D, α-L,D, and α-D,L), as well the four possible isoAsp containing stereoisomers (β-L,L, β-D,D, β-L,D and β-DL). The separation of these substances was explored using both neutral and charged cyclodextrins as the stereoisomer selector added to the running electrolyte. The major experimental parameters investigated included pH, the cyclodextrin type, and the cyclodextrin concentration. Due to differences in the pKa values of the carboxylic acid groups, adjustment of the separation buffer to between pH 3.0 and 4.0 provided for sufficient electrophoretic mobility differences to result in excellent separations of the diastereomerically related peptides in this pH region. The resolution of the enantiomerically related peptide stereoisomers was accomplished using low concentrations (1 mM) of the anionic cyclodextrin derivative, sulfobutylether-β-cyclodextrin (SBE-β-CD). This negatively charged cyclodextrin was found to be superior for the resolution of the enantiomerically related peptides as compared to native β-cyclodextrin or the neutral derivatives, dimethyl β-cyclodextrin and hydroxypropyl β-cyclodextrin. An alternative approach using anionic or neutral surfactants in conjunction with the SBE-β-CDs was also explored and found to be successful but problematic.