Capillary Coatings: Choices for Capillary Electrophoresis of DNA

One of the most distinguishing features of capillary electrophoresis (CE) is the electroosmotic flow (EOF). This electrokinetic phenomenon originates from the presence on the capillary surface of weakly acid hydroxyl groups attached to the silicon atoms. Different authors have reported an overall pKa value of 5.3 ( 1 ) or 6.3 ( 2 ) for surface silanols. In practice, when the capillary surface is bathed in any buffer solution with a pK higher than 2, immobilized negative charges are formed on the wall. The presence of this layer of immobilized charges causes adsorption of positive counter ions from the buffer solution. As a result, an electric double layer is generated at the silica-buffer solution interface consisting of an immobile layer of cationic species, tightly held to the ionized silanols, and a diffuse layer of hydrated ions loosely bound to the surface. Figure 1 shows a schematic representation of the electrical double layer structure at the surface-solution interface. Two planes can be envisioned in the double layer: the so-called (inner) Helmholtz plane (IHP) located in the center of the compact layer, and the outer Helmholtz plane (OHP) that delimits the compact portion of the double layer. In the diffuse part of the double layer, the ion concentration diminishes until it reaches that of the bulk solution. When an electric field is tangentially applied to the surface, the diffuse ions of the double layer migrate towards the cathode with their surrounding water molecules generating a flow of liquid in the capillary. This overall solution transport phenomenon is called EOF and has dramatic influences on the time the analytes reside inside the capillary ( 3 ). As a consequence of the forces exerted by the electric field on the ions of the diffuse layer, a plane of shear is formed between the diffuse and the mobile layer regions and the potential generated at this shear plane is termed zeta potential (ζ). In a typical buffer for DNA electrophoresis, the double layer is about 3 nm thick. A velocity gradient develops owing to the differences in the magnitude of frictional and electric forces within the double layer. The flow velocity that is zero across the wall increases in the region of the double layer reaching a maximum value at a very small distance from the charged surface. The bulk of the solution migrates with this maximum velocity. This means that the flow stream that originates at the wall of the capillary has an almost flat velocity distribution across the capillary diameter, deviating only within a few nanometers from the capillary surface. Unlike the parabolic hydrodynamic flow, EOF is almost plug-like and does not necessarily cause a deterioration of the separation.

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