Influence of temperature on the behaviour of small linear peptides in capillary electrochromatography

The influence of temperature, T, on the retention times, peak widths, peak symmetry coefficients and theoretical plate numbers of two small linear peptides, [Met5]enkephalin and [Leu5]enkephalin, has been studied with capillary electrochromatography (CEC) capillary columns of 100 μm I.D. and 250 mm packed length with a total length of 335 mm, containing 3 μm Hypersil n-octadecyl bonded silica. With increasing column temperature from 15 to 60°C, the electroosmotic flow (EOF) and the column efficiencies increased, whereas the retention coefficients (κcec) of both peptides decreased. A linear relationship was found between the EOF value and the square root of the temperature over this temperature range, with a linear regression correlation of 0.998. Non linear Van ’t Hoff plots (ln κcec versus 1/T) were observed for these peptides between 15 and 60°C, suggesting that a phase-transition occurred with the n-octadecyl chains bonded on the silica surface, affecting the CEC retention behaviour of these peptides. In CEC systems, the κcec values of peptides incorporate contributions from both electrophoretic migration and chromatographic retention. Positive and negative κcec values can, in principle, thus arise with these charged analytes. However, the κcec values of the enkephalin peptides under all temperature conditions studied were positive with an eluent composed of water–50 mM NH4OAc/AcOH, pH 5.2–acetonitrile (5:2:3, v/v) and therefore the chromatographic component dominates the retention process with these small peptides under these conditions.