Examination of band dispersion during size-selective capillary electrophoresis separations of DNA fragments.

ABSTRACT

Versatile capillary electrophoresis instrumentation that permits the rapid and precise translation of a laser fluorometric detection zone along the capillary wall has been used to examine the factors which cause band broadening during size-selective separations of DNA fragments. Separations are performed using capillaries containing entangled polymer solutions. The scanning capabilities of this instrumentation facilitates the determination of diffusion coefficients under static conditions without the need to discontinue and reapply an electric field. The ability to rapidly translate the detection zone along the column allows the monitoring of the separation at various points along the capillary which enables the examination of the sources of band dispersion under kinetic conditions. Results from experiments utilizing various concentrations of both high and low molecular mass methyl cellulose polymers as sieving media are presented. It is shown that axial diffusion, even when adjusted for kinetic conditions using the Einstein relationship, does not account for the total observed band variance. Possible explanations for this behavior are presented.