Effect of ionic strength, pH and polymer concentration on the separation of DNA fragments in the presence of electroosmotic flow

Abstract

DNA separations in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions have been demonstrated. During the separations, PEO entered capillaries filled with Tris–borate (TB) free buffers by EOF and acted as sieving matrices. We have found that ionic strength and pH of polymer and free solutions affect the bulk EOF and resolution differently from that in capillary zone electrophoresis. The EOF coefficient increases with increasing ionic strength of the free TB buffers as a result of decreases in the adsorption of PEO molecules. In contrast, the bulk EOF decreases with increasing the ionic strength of polymer solutions using capillaries filled with high concentrations of free TB buffers. Although resolution values are high due to larger differential migration times between any two DNA fragments in a small bulk EOF using 10 m M TB buffers, use of a capillary filled with at least 100 m M TB free buffers is suggested for high-speed separations. On the side of PEO solutions, 1.5% PEO solutions prepared in 100 to 200 m M TB buffers are more proper in terms of resolution and speed. The separation of DNA markers V and VI was accomplished less than 29 min in 1.5% PEO solutions prepared in 100 m M TB buffers, pH 7.0 at 500 V/cm using a capillary filled with 10 m M free TB buffers, pH 7.0.