CEval: All-in-one software for data processing and statistical evaluations in affinity capillary electrophoresis.

We introduce CEval software (downloadable for free at echmet.natur.cuni.cz) that was developed for quicker and easier electrophoregram evaluation and further data processing in (affinity) capillary electrophoresis. This software allows for automatic peak detection and evaluation of common peak parameters, such as its migration time, area, width etc. Additionally, the software includes a nonlinear regression engine that performs peak fitting with the Haarhoff-van der Linde (HVL) function, including automated initial guess of the HVL function parameters. HVL is a fundamental peak-shape function in electrophoresis, based on which the correct effective mobility of the analyte represented by the peak is evaluated. Effective mobilities of an analyte at various concentrations of a selector can be further stored and plotted in an affinity CE mode. Consequently, the mobility of the free analyte, µA, mobility of the analyte-selector complex, µAS, and the apparent complexation constant, K('), are first guessed automatically from the linearized data plots and subsequently estimated by the means of nonlinear regression. An option that allows two complexation dependencies to be fitted at once is especially convenient for enantioseparations. Statistical processing of these data is also included, which allowed us to: i) express the 95% confidence intervals for the µA, µAS and K(') least-squares estimates, ii) do hypothesis testing on the estimated parameters for the first time. We demonstrate the benefits of the CEval software by inspecting complexation of tryptophan methyl ester with two cyclodextrins, neutral heptakis(2,6-di-O-methyl)-ß-CD and charged heptakis(6-O-sulfo)-ß-CD.

Determination of relative enantiomer migration order using a racemic sample.

We developed a method that enables us to distinguish between the same or the opposite enantiomer migration order (EMO) of two enantiomers of a chiral compound with two different selectors. The method is applicable to racemic samples and thus a standard of the pure enantiomeric form(s) is not required. First, complexation constants and mobilities of complexes of the two enantiomers with the first and second selector are determined. However, for a racemic sample it is not possible to deduce whether the first migrating enantiomer with one selector is the same one as the first migrating enantiomer with the second selector. A specific mixture of the two selectors is designed to resolve this. In case the two enantiomers exhibit the same, respectively the opposite EMO in the two selectors, the mixture does, respectively does not separate the racemic sample. Thus two peaks are detected in the first case, while a single coalescent peak is recorded in the opposite case. We demonstrate the method on a racemic sample of amphetamine. Its relative EMO is determined with three cyclodextrins, heptakis(2,3,6-tri-O-methyl)-ß-cyclodextrin, (2-hydroxypropyl)-ß-cyclodextrin and heptakis(2,3-di-O-acetyl-6-O-sulfo)-ß-cyclodextrin.