Abstract
The choice between bare and coated capillaries is a key decision in the development and use of any methods based on capillary electrophoresis. In this work several permanently and dynamically coated capillaries were successfully implemented in a previously developed micellar electrokinetic chromatography (MEKC) assay of the plant membrane enzyme chlorophyllase. The results obtained demonstrate the rationale behind the use of capillary coating, which is crucial for successful optimization of both the off-line mode and the on-line/electrophoretically mediated microanalysis assay mode. The application of an amine permanently coated capillary (eCAP) is a simple way to significantly increase the repeatability of migration times and peak areas, and to ensure a strong electroosmotic flow that considerably decreases the overall analysis time. A dynamic coating (CEofix) allows one to apply an on-line incubation to control the reaction progress inside the capillary, and to increase the signal-to-noise ratio and peak efficiency. The dynamic coating is possible with use of both the normally applied uncoated silica capillary and the precoated amine capillary, which ensures more repeatable migration times. The strong points of the uncoated silica capillary are its attractive price and wide range of pH that can be applied. The characteristics presented may simplify the choice of capillary modification, especially in the case of hydrophobic analytes, MEKC-based separations, and other enzymatic assays.
Highlights
Capillary electrophoresis (CE) has become a routine instrumental technique applied in biochemical analysis
The separation enabling reaction monitoring in the conventional off-line reaction mode was achieved with the bare silica capillary, the amine capillary, and the same capillaries subjected to dynamic coating by a polyanionic layer
The differences between the migration times obtained for chlorophyll a (Chla) and Chlidea, shown in Fig. 3, suggest a similar mechanism of separation in all capillaries
Summary
Capillary electrophoresis (CE) has become a routine instrumental technique applied in biochemical analysis. There are many reports on CE-based assays of watersoluble enzymes [14,15,16,17], which are easier to use than hydrophobic membrane enzymes because of the need to maintain a hydrophobic environment for both the reaction mixture and the separation medium. In the latter case the use of capillary coating is especially advisable to minimize potential undesirable effects; for example, the formation of protein aggregates that may adsorb onto the capillary inner surface, leading to the deterioration of the repeatability of both migration times and peak areas, and to the loss of catalytic activity. The development of CE enzymatic assays of membrane proteins in combination with a rational choice of capillary modification is an important direction of research
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