Modulation in capillary electrophoresis and analysis of DNA.

Abstract

Analyte velocity modulation, a general modulation technique for capillary electrophoresis, results when a sinusoidal AC voltage is superimposed onto the driving DC voltage. The principal motivation for the work has been exploration of voltage modulation as a technique for improving signal/noise ratios of existing detectors. The technique is especially effective with excess noise limited detectors such as the refractive index detector. One advantage of an electrical modulation technique, over an optical one, is its applicability to all detection schemes. The mathematical theory of analyte velocity modulation is presented in this work. The main idea is that the superimposed AC field forces the electroosmotic flow profile to oscillate between laminar and plug flow at the modulation frequency. The changing profile induces a radial movement of sample species to and from the capillary surface. The induced sample concentration gradients can be monitored by carefully probing the capillary surface. The resulting signal is a derivative of the normal shaped peak. Derivative shaped peaks can be observed with cations, but not with anions. Anions are unable to approach the double layer region and therefore are unaffected by the modulation process. The results indicate that the double layer thickness in free solution capillary electrophoresis might be larger than the nominally calculated 3 nm. Analyte velocity modulation can be used as a variation of pulsed field electrophoresis in gel-filled capillaries. This technique provides superior resolution of DNA fragments and shortens their migration times. We demonstrate subpicogram detection limits for nucleic acids, with very high efficiencies. Theoretical plate numbers are in the range of 10$\sp5$ to 10$\sp6$. We also demonstrate that since wave modulation gives resolution similar to square wave modulation for frequencies between 10 and 100 Hz. Although a square wave contains high frequency harmonics, we show that in general its coupling to DNA motions is not always inferior to that of a sine wave.