Capillary coating as an important factor in optimization of the off-line and on-line MEKC assays of the highly hydrophobic enzyme chlorophyllase

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.

Keywords: Capillary coating; Capillary electrophoresis; Chlorophyll; Chlorophyllase; Electrophoretically mediated microanalysis; Micellar electrokinetic chromatography.

Fig. 1

The reaction of chlorophyll hydrolysis catalyzed by chlorophyllase

Fig. 2

The electrophoretically mediated microanalysis approach, including the four sequential steps (see the text for details)

Fig. 3

Electropherograms presenting separations between chlorophyll a (substrate, S) and chlorophyllide a (product, P) in various capillaries at 430 nm, and the peak deconvolution performed for the on-line reaction. DC dynamically coated, phosp. phosphate

Fig. 4

The average reaction progress analyzed off-line in various capillaries (see the text for details). DC dynamically coated, phosp. phosphate