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

Abstract

SummaryCritical 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 twolevel 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.

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

“…A) From the CCD according to Table I B) From the CCD without axial points tion to the micelles, c.f. [12]. The peptides studied all have their isoelectric point (pI) at 6.0 except for the peptide containing arginine which has pI > 9.0 [14].…”

“…The level of the factors were based on a previous experimental screening [12], the experimental domain was defined according to Table I. The SDS concentration was varied from 30 to 60 mM, the acetonitrile percentage between 5-15 % v/v, the ionic strength of the buffer between 0.02 to 0.04, the injected plug length was varied from 1.9 (3 s 40 mbar) to 4.7 min (7 s 40 mbar), the temperature between 25 and 40 ~ and the composition of the injected solution was without or with micelles.…”

“…For a given migration time window there is an optimal value for the migration factor which maximizes the resolution. Previous studies, using D-optimal design to study the resolution, showed that it was necessary to choose an experimental domain where all migration factors were either positioned on the same side of this optimal value or to use more sophisticated models [12]. In this work the separation performance in MEKC for enkephalin related peptides, was optimized utilizing central composite design, where all migration factors were on the lower side of the optimal value for the migration factor.…”

Precision study on capillary electrophoresis methods for metacycline

“…Experimental design offers an efficient alternative for identifying the conditions yielding the best resolution [18][19][20]. Many factors can be screened simultaneously to determine which one has a significant effect on the separation.…”

Experimental design methodologies to optimize the CE separation of epinephrine enantiomers

“…However, this step-by-step approach, although widely used, involves a large number of independent runs and, since multiple interacting parameters determine the observed separation, a multivariate optimization was considered necessary [12,[15][16][17][18][19][20]. In particular experimental design offers an efficient route for identifying the zone in factor space for the best resolution in CE [21][22][23][24][25][26][27]. It allows a large number of factors to be screened simultaneously to determine which of them has a significant effect on the separation.…”