Pre-equilibrium capillary zone electrophoresis or frontal analysis: Advantages of plateau peak conditions in affinity capillary electrophoresis

Abstract

The feasibility of using the affinity CE methodologies pre-equilibrium CZE and CE frontal analysis was tested on interaction systems exhibiting rapid on-and-off kinetics. Experimentally, the methodologies differ only with respect to the volume of sample introduced into the capillary. Pre-equilibrium CZE has been considered amendable to interactions with slow on-and-off kinetics only; however, it has recently been applied in studies of interactions with fast on-and-off kinetics. The effect of varying the sample volume introduced hydrodynamically into the capillary on the apparent degree of complexation was studied. For two different binding systems, the fraction of free analyte was found to be overestimated using pre-equilibrium CZE as compared to volumes providing plateau peak conditions as used with frontal analysis. Results indicate that frontal analysis conditions lead to more robust binding assays and thus more reliable data. The validity of data obtained by pre-equilibrium CZE may be low, thus the use of an experimental setup providing plateau peaks is highly recommended. It is suggested that the effect of altering the sample volume on the degree of binding should be investigated as part of method development and validation.

Bioanalytical interaction studies executed by preincubation affinity capillary electrophoresis

“…In the majority of these studies [63][64][65][66] peak heights were used for quantitation because peak areas were almost constant. However, in a study comparing pre-eq CZE with CE-FA (Section 2.4) the degree of binding was found to vary with sample volume for two different binding systems when zone electrophoresis conditions prevailed [68]. Thus, the use of pre-eq CZE for assaying interactions with fast off-kinetics must be discouraged.…”

“…The only experimental difference between pre-eq CZE and CE-FA is the volume of pre-equilibrated sample introduced into the capillary. Hence, CE-FA is performed by introduction of a relatively large volume of sample into the capillary, typically between 20 and 200 nL [13,68] depending on the dimensions of the capillary, i.e. 5-20% of the total capillary volume.…”

“…In cases, where complete separation is achieved between plateau peaks the area is proportional to the total ligand concentration [73]. An attractive feature of CE-FA is that it is insensitive to changes or fluctuations in migration times, EOF, and applied voltage [68]. Inside the plateau peak a nondispersed zone of ligand is maintained which ads to the robustness of the method as compared to CZE.…”

Binding of Low-Molecular-Weight Cationic Ligands to Chondroitin Sulfate as Studied by Capillary Electrophoresis Frontal Analysis

“…Due to the different charge states the analyte is easily separated from the chondroitin sulfate. Frontal analysis conditions [53], where plateau peaks are observed, were achieved using injection times above 8 s and 5 s for standards and samples, respectively. In addition to propranolol, a UV signal for chondroitin sulfate and EOF was observed in the sample electropherograms (Fig.…”

“…Ishwar et al used mobility shift affinity CE to investigate the affinity of two dodecapeptides for chondroitin sulfate and heparin [53]. To the best of our knowledge this study represents the first application of CE-FA for investigation of chondroitin sulfate complexation.…”