The CEC behaviour of several synthetic peptides related to the activin β_A–β_D subunits

Abstract

The resolution of several structurally related synthetic peptides, derived from the loop 3 region of the activin betaA-betaD 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 electro-osmotic flow, as, for example, with the positively charged peptides at a particular pH and buffer electrolyte composition, their retention coefficient, kappacec, decreased with increasing capillary temperature, whereas when the separation forces worked in opposite directions, as for example with negatively charged peptides, their kappacec 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 kappacec values and the volume fractions, psi, of the organic modifier over a limited range of psi-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.

Recent developments in capillary electrophoresis and capillary electrochromatography of peptides

“…The CEC separations of several structurally related synthetic peptides derived from activin subunits using CEC with Hypersil n-octadecyl silica as the sorbent [158] has shown that the CEC elution/migration of peptides can be varied in a charged-state-specific manner as the properties of BGE, such as pH, salt concentration and content of organic modifier, or temperature, are systematically changed. Depending on the separation conditions, the contributions from both electrophoretic migration and chromatographic retention could be distinguished in the resulting CEC separation.…”

Electrochromatographic retention of peptides on strong cation‐exchange stationary phases

“…Systematic studies of retention and selectivity in peptide CEC were reviewed by Walhagen et al [36,37]. It was concluded that peptide CEC separations depend on an interplay between electrostatic interactions, hydrophobic interactions, and electrophoretic mobility.…”

Capillary electrochromatography of proteins and peptides

“…When the separation forces acting on the peptides were synergistic with the EOF (i. e., positively charged peptides), their retention coefficient decreased with an increase in capillary temperature, whereas when the separation forces worked in opposite directions (i. e., negatively charged peptides), their retention coefficient increased slightly with increase in temperature. When the content of the organic modifier, ACN, was high (i. e., more than 40% v/v) and nonpolar interactions with the C18 sorbent were suppressed, mixtures of both the basic and acidic synthetic peptides could be baseline resolved under isocratic conditions by utilizing the mutual processes of electrophoretic mobility and electrostatic interaction [29].…”