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The resolution of several structurally related synthetic peptides, derived from the loop 3 region of the activin β[sub A]-β[sub D] 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, κ[sub cec], decreased with increasing capillary temperature, whereas when the separation forces worked in opposite directions, as for example with negatively charged peptides, their κ[sub cec] 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 In κ[sub cec] values and the volume fractions, ψ, of the organic modifier over a limited... [ABSTRACT FROM AUTHOR]

Abstract: In this study, we describe the application of a new analytical procedure based on capillary electrochromatographic (CEC) techniques for the characterization of different basic and acidic peptides using isocratic eluent conditions containing acetonitrile and ammonium acetate buffers of different molarities between pH 3.8 and 5.2. In particular, 10 immunogenic peptide analogs with isoelectric points ranging from 3.7 to 10.1 were investigated; nine of these peptides, 1–9, were truncated analogs of the parent peptide, 10, which is a peptidomimetic related to a HIV-1 gp120 epitope. Several of these peptides have the propensity to form α-helical secondary structures in solution. Electrochromatographic separations of these peptides were achieved with packed fused silica capillaries (25 cm packed length, 100 µm i.d.) containing 3 µm n-octadecylsilica particles. The influence of temperature on the CEC elution behavior of these peptides, as well as the impact of changes in the eluent composition, e.g. pH, buffer concentration and acetonitrile content, were examined. The results confirm that improvements in the resolution and analysis of synthetic peptides by CEC procedures result from the increase in electro-osmotic flow (EOF) as the temperature is increased. These findings emphasize the dominant influence of the temperature-dependent viscosity parameter, η, on the EOF and thus on peptide resolution in CEC. Moreover, these investigations have shown that eluent properties can be specifically chosen to favor either electrophoretic mobility or chromatographic retention, with the overall CEC selectivity peptides of different sequence or composition reflecting the summated contributions from both separation mechanisms. Over the pH range 4.0–5.0, and using eluents with ionic strengths ranging from 6.2 to 15 mm ammonium acetate but containing a fixed volume fraction, ψ, of acetonitrile above ψ=0.40, the CEC retention behavior of peptides 1–10 correlated with a linear relationship linking the retention coefficient, κ[sub cec], and the differential frictional size-to-mass ratio parameter, ξ[sub fric], of these peptides. However, using eluents with a low acetonitrile content and low pH values, linear correlations were also observed between the incremental retention coefficient, Δκ[sub cec], and the product term [- 0.66(Δ∑χ[sub n]) log(M[sub i]/M[sub j])], which links the difference in intrinsic hydrophobicities and molecular masses of two peptides, P[sub i] and P[sub j]. This study thus demonstrates the power of CEC procedures in the analysis of synthetic bioactive peptides and provides a general experimental framework to evaluate, using CEC procedures, the influence of the key molecular attributes of peptides on their structure-retention dependencies. Finally, these studies provide additional, practical insights into the use of CEC procedures for the analysis, resolution and biophysical characterization of closely related peptide analogs derived from solid-state peptide synthesis under conditions of different eluent composition or temperature. [ABSTRACT FROM AUTHOR]