Modeling of electroosmotic and electrophoretic mobilization in capillary and microchip isoelectric focusing.
Our dynamic capillary electrophoresis model which uses material specific input data for estimation of electroosmosis was applied to investigate fundamental aspects of isoelectric focusing (IEF) in capillaries or microchannels made from bare fused-silica (FS), FS coated with a sulfonated polymer, polymethylmethacrylate (PMMA) and poly(dimethylsiloxane) (PDMS). Input data were generated via determination of the electroosmotic flow (EOF) using buffers with varying pH and ionic strength. Two models are distinguished, one that neglects changes of ionic strength and one that includes the dependence between electroosmotic mobility and ionic strength. For each configuration, the models provide insight into the magnitude and dynamics of electroosmosis. The contribution of each electrophoretic zone to the net EOF is thereby visualized and the amount of EOF required for the detection of the zone structures at a particular location along the capillary, including at its end for MS detection, is predicted. For bare FS, PDMS and PMMA, simulations reveal that EOF is decreasing with time and that the entire IEF process is characterized by the asymptotic formation of a stationary steady-state zone configuration in which electrophoretic transport and electroosmotic zone displacement are opposite and of equal magnitude. The location of immobilization of the boundary between anolyte and most acidic carrier ampholyte is dependent on EOF, i.e. capillary material and anolyte. Overall time intervals for reaching this state in microchannels produced by PDMS and PMMA are predicted to be similar and about twice as long compared to uncoated FS. Additional mobilization for the detection of the entire pH gradient at the capillary end is required. Using concomitant electrophoretic mobilization with an acid as coanion in the catholyte is shown to provide sufficient additional cathodic transport for that purpose. FS capillaries dynamically double coated with polybrene and poly(vinylsulfonate) are predicted to provide sufficient electroosmotic pumping for detection of the entire IEF gradient at the cathodic column end.
Thormann
W
W
Department of Clinical Pharmacology, University of Bern, Bern, Switzerland. wolfgang.thormann@ikp.unibe.ch
Caslavska
J
J
Mosher
R A
RA
D000665:Ampholyte Mixtures; D002021:Buffers; D003198:Computer Simulation; D004129:Dimethylpolysiloxanes; D053841:Electroosmosis / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D046208:Microchip Analytical Procedures; D009994:Osmolar Concentration; D019904:Polymethyl Methacrylate
D000665:Ampholyte Mixtures; D002021:Buffers; D004129:Dimethylpolysiloxanes; D019904:Polymethyl Methacrylate