LC and LC-MS Separation of Peptides on Macrocyclic Glycopeptide Stationary Phases: Diastereomeric Series and Large Peptides

Previous work on the LC separation of peptides had shown that macrocyclic glycopeptide stationary phases to be selective for peptides of five to thirteen amino acids in length. In this work, the selectivity of the teicoplanin stationary phase is compared to that of a C18 stationary phase for seven diastereomeric enkephalin peptides. The teicoplanin stationary phase separated all seven diastereomeric enkephalin peptides in a single chromatographic run. The insertion of d-amino acids into the primary enkephalin sequence produced areas of hydrophobicity that influenced retention order on the C18 stationary phase. However, analogous trends are not observed on the teicoplanin stationary phase, which is more polar and structurally diverse. Optimization of the mobile phase and the use of a step-gradient for the enkephalin separation on the teicoplanin stationary phase is discussed. Also, the selectivity of macrocyclic glycopeptide stationary phases for peptides of 14, 28, 30, and 36 amino acids also is investigated and compared to separation on a C18 stationary phase. A method for eluting peptides with multiple basic amino acids, which tend to be strongly retained on the macrocyclic glycopeptide stationary phases, is presented.