Combination of Transverse and Longitudinal Diffusion as a Generic Approach for Reactants Mixing inside the Capillary
| Authors | |
|---|---|
| Year of publication | 2011 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | Applications of on-line enzymatic reactions in capillary electrophoresis (CE) are of particular interest in modern bioanalytical chemistry since resulting automatization of the assays minimize both sample consumption and possibility of experimental error. However the development of an effective way of reactants mixing inside the capillary forming a nano scale reactor is a challenging task, which prevents wider practical implementation of this interesting concept. Several approaches based on differential electrophoretic mobilities and transverse diffusion have been already presented [1,2]. Unfortunately they are not able to provide homogenous reaction mixture which composition could be simply changed during given experimental task. To solve this limitation, we introduce the generic methodology of in-capillary reaction mixture mixing based on combination of longitudinal and transverse diffusion. Conceptually, the solutions of reaction mixture components are injected by relatively low pressure as a series of consecutive narrow plugs having parabolic profiles due to laminar flow inside the capillary. Resulting character of plugs with rather longitudinal interfaces then enable creation of homogenous reaction mixture by both longitudinal and transverse diffusions within minute period. The goal of this study was to introduce a method based on the above described principle for monitoring of on-line reaction of cytochrome P450 2C9 with diclofenac as a probe substrate. A sandwich consisting of four plugs of diclofenac and NADPH solution injected alternately with three enzyme solution plugs was selected as an appropriate layout for the formation of homogenous reaction mixture. Separation of reaction product 4’-hydroxydiclofenac was achieved within 13 minutes analysis with background electrolyte containing 55 mM SDS in 18 mM phosphate-borate buffer pH 8.6. Presented approach thus constitutes a promising tool for on-line inhibition studies of CYP2C9 requiring routine changes in reaction mixture composition. |
| Related projects: |