Multivariate evaluation of the separation performance in micellar electrokinetic capillary chromatography of peptides: Experimental screening

Critical parameters for the separation of enkephalin related peptides by micellar electrokinetic chromatography were identified by using statistical experimental design. Nine experimental variables: micelle concentration, pH of the background electrolyte, addition of organic modifier to the background electrolyte, injection length, temperature of the capillary, applied voltage, ionic strength of the buffer, the composition of the injected solution and stacking were investigated in a two-level fractional factorial design. The pH was found to be of fundamental importance but a great variation in separation performance was observed, subsequent modeling revealed two subgroups with respect to pH. For further screening of significant factors, the pH was kept constant and by using D-optimal design it was possible to study six interaction terms together with eight main effects in only 20 experiments. The effect of the interaction terms were often found to be as high as the main effects, thus exemplifying the complexity of the system. The effect of sodium dodecyl sulfate (SDS) concentration on efficiency and resolution was highly dependent on the amount of organic modifier, temperature and ionic strength. The migration factor calculated from obtained migration times was found to be a non-linear Function of the factors involved. A complete separation within reasonable analysis time was obtained at the centre point.