Capillary electrochromatographic separation of peptides using a macrocyclic polyamine for molecular recognition

A macrocyclic polyamine, 1,5,9,13,17,21,25,29-octaazacyclodotriacontane ([32]ane-N-8), in the bonded phase was employed as a molecular receptor for CEC separation of oligopeptides. Parameters affecting the performance of the separations were considered. Baseline separation for the mixture of angiotensin I, angiotensin I, [Sar(1), Thr(8)]-angiotensin II, beta-casomorphin bovine, P-casomorphin human, oxytocin acetate, tocinoic acid, vasopressin, and FMRF amide could be achieved using phosphate buffer (30 mM, pH 7) as the mobile phase. Column efficiency with average theoretical plate numbers of 69 000 plates/m and RSDs of < 1% (n = 6) was achieved. [Met(5)]-enkephalin and [Leu(5)]-enkephalin, which have identical pI values and similar masses could be completely separated using acetate buffer (30 mM) with pH gradient (pH 3 inlet side and pH 4 outlet side). The results suggest that the mechanism for the peptide separation was mediated by a combination of electrophoretic migration and chromatographic retention involving anion coordination and anion exchange. After long-term use, the deviation of the EOF of the column after more than 600 injections was still within 6.0% of that for a freshly prepared column.