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Pre‐equilibrium capillary zone electrophoresis or frontal analysis: Advantages of plateau peak conditions in affinity capillary electrophoresis
Author(s) -
Østergaard Jesper,
Hansen Steen H.,
Jensen Henrik,
Thomsen Anne E.
Publication year - 2005
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200500287
Subject(s) - capillary electrophoresis , analyte , chemistry , kinetics , plateau (mathematics) , capillary action , chromatography , volume (thermodynamics) , analytical chemistry (journal) , thermodynamics , mathematics , mathematical analysis , physics , quantum mechanics
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.