Simulation and experimental study of enzyme and reactant mixing in capillary electrophoresis based on-line methods

Abstract

The establishment of an efficient reaction mixture represents a crucial part of capillary electrophoresis based on-line enzymatic assays. For ketamine N-demethylation to norketamine mediated by the cytochrome P450 3A4 enzyme, mixing of enzyme and reactants in the incubation buffer at physiological pH was studied by computer simulation. A dynamic electrophoretic simulator that encompasses Taylor-Aris diffusivity which accounts for dispersion due to the parabolic flow profile associated with pressure driven flow was utilized. The simulator in the diffusion mode was used to predict transverse diffusional reactant mixing occurring during hydrodynamic plug injection of configurations featuring four and seven plugs. The same simulator in the electrophoretic mode was applied to study electrophoretic reactant mixing caused by voltage application in absence of buffer flow. Resulting conclusions were experimentally verified with enantioselective analysis of norketamine in a background electrolyte at low pH. Furthermore, simulations visualize buffer changes that occur upon power application between incubation buffer and background electrolyte and have an influence on the reaction mixture.

Computer simulation of the enantioselective separation of weak bases in an online capillary electrophoresis based microanalysis configuration comprising sulfated cyclodextrin as selector

“…Separation of ketamine and norketamine enantiomers was achieved anionically using highly sulfated γ‐cyclodextrin (HS‐γ‐CD) as chiral selector. In the previous work, the 1D dynamic electrophoresis simulator GENTRANS , extended to include Taylor–Aris diffusivity to account for transverse diffusion associated with pressure‐driven plug injection , was employed to characterize mixing occurring during plug insertion and short application of voltage . In the present communication, a first simulation approach of the electrophoretic processes that take place after incubation, including the enantiomeric separation of the analytes ketamine and norketamine, is reported.…”

“…The steps in a typical online assay are as follows: (1) consecutive hydrodynamic injection and mixing of enzyme and reactant solution plugs, (2) incubation of the reaction mixture, (3) voltage application and separation, and (4) detection of the reaction products. Two different CE‐based online assays were developed in our laboratory for monitoring the enantioselective N ‐demethylation of ketamine to norketamine, which is mediated by the cytochrome P450 3A4 (CYP3A4) enzyme. Separation of ketamine and norketamine enantiomers was achieved anionically using highly sulfated γ‐cyclodextrin (HS‐γ‐CD) as chiral selector.…”

“…In the studied online assays, enzymatic reactions take place at physiological pH, and enantiomeric separation of the analytes takes place at low pH. Thus, electrophoresis proceeds in a discontinuous buffer system . The discontinuities of the buffer system are accompanied by changes in pH, conductivity, and ionic strength, in which the latter has a large impact on chiral separations by influencing the effective mobilities of all components, and potentially also the values of binding constants .…”

“…The configuration studied was the online system with four initial plugs . As initial concentration profiles, GENTRANS data obtained after mixing (see Fig.…”

Electromagnetophoretic Micro-convection around a Droplet in a Capillary

“…4 To develop electromagnetophoretic applications more extensively to various microscale systems, it will be crucially important to investigate in detail the phenomena of the electromagnetophoretic convection of the medium. Any study of the convection of a liquid in micro-systems will have a more important meaning as a novel technique of micro-mixing, since it has been widely studied as a bottle-neck technique in μTAS, 11 lab-on-a-chip, 12,13 capillary systems, [14][15][16] and many microchannel methods. [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] Some reviews have also been reported concerning effective mixing in microchannels 34,35 and in small-scale production.…”