Capillary electrochromatography analysis of hormonal cyclic and linear peptides

The retention behavior of linear and cyclic peptides has been studied by capillary electrochromatography (CEC) with a variety of different n-alkyl silica reversed-phase sorbents and also with mixed-mode phases containing both strong cation-exchange (sulfonic acid) and n-alkyl groups bonded onto the silica surface, using eluents ranging from pH 2.0 to pH 5.0. Depending upon the amino acid sequence, electrochromatographic retention of the peptides was strongly affected by the composition of the eluent, its pH value, and the choice of sorbent packed into the capillaries. The dominant separation processes operating with these charged analytes could be modulated inter alia by the content of organic modifier, acetonitrile, in the eluent, with peptide resolution predominantly arising from electrophoretic migration processes at high acetonitrile content. As the concentration of acetonitrile was decreased, chromatographic retention processes became more pronounced. With the n-alkyl silica CEC columns used in this study, silanophilic interactions between the sorbents and the charged peptides could be suppressed by increasing the molarity of the buffer and by adjusting the pH of the eluent to lower values. On the other hand, electrostatic interactions between basic peptides and the surface of strong cation-exchanger, mixed-mode materials can be suppressed at low pH values by using higher ionic strength conditions in the eluent. Different selectivity behavior was achieved with desmopressin and the other peptides with Spherisorb C18/SCX and Hypersil mixed-mode materials when an identical eluent composition of 60% (v/v) acetonitrile with 7.6 mM triethylammonium phosphate, pH 3.0, was used. These findings confirm that the surface charge density of the sorbent fulfills an important role in the modulation of peptide selectivity in CEC. These studies also confirm that the dependency of the logarithm of the CEC retention coefficients, i.e., log Kcec, of a peptide separated with n-octadecyl silica sorbents under CEC conditions, on the volume fraction, psi, of the organic solvent modifier, acetonitrile, within the range of 0.20 < or = psi < or = 0.60, can be approximated by a linear relationship. Moreover, these studies show that the selectivity differences of peptides separated by CEC with nonpolar sorbents in packed capillary systems can be discussed in terms of semiempirical dependencies that link peptide retention behavior with their molecular descriptor properties, e.g., their hydrophobicity, surface charge anisotropy, surface area, molecular mass and intrinsic charge, and thus to their corresponding linear free energy relationships.