The resolution of several structurally related synthetic peptides, derived from the loop 3 region of the activin βA-βD subunits, has been studied using capillary electrochromatography (CEC) with Hypersil n-octadecylsilica as the sorbent. The results confirm that the CEC migration of these peptides can be varied in a charge-state-specific manner as the properties of the background electrolyte, such as pH, salt concentration and content of organic modifier, or temperature are systematically changed. Acidic peptides followed similar trends in retention behaviour, which was distinctly different to that shown by more basic peptides. The CEC separation of these peptides with the Hypersil n-octadecyl-silica involved distinguishable contributions from both electrophoretic mobility and chromatographic retention. Temperature effects were reflected as variations in both the electro-osmotic flow and the electrophoretic mobility of the peptides. When the separation forces acting on the peptides were synergistic with the electroosmotic flow, as, for example, with the positively charged peptides at a particular pH and buffer electrolyte composition, their retention coefficient, κcec, decreased with increasing capillary temperature, whereas when the separation forces worked in opposite directions, as for example with negatively charged peptides, their κcec values increased slightly with increasing temperature. Moreover, when the content of organic modifier, acetonitrile, was sufficiently high, e.g. >40% (v/v) and nonpolar interactions with the Hypersil n-octadecyl-silica sorbent were suppressed, mixtures of both the basic and acidic synthetic peptides could be baseline resolved under isocratic conditions by exploiting the mutual processes of electrophoretic mobility and electrostatic interaction. A linear relationship between the ln κcec values and the volume fractions, ψ, of the organic modifier over a limited range of ψ-values, was established for the negatively charged peptides under these isocratic conditions. These findings thus provide useful guidelines in a more general context for the resolution and analysis of structurally related synthetic peptides using CEC methods.
- Activin loop peptides
- Capillary electrochromatography
- Fmoc-based synthesis
- Retention behaviour
- Solvent and buffer effects
- Synthetic β-sheet polypeptides