Contact conductivity detection of polymerase chain reaction products analyzed by reverse-phase ion pair...

We describe the development of an integrated microelectrophoretic system consisting of a contact conductivity detector mounted on-chip for monitoring the separation of double-stranded (ds) DNA fragments produced via the polymerase chain reaction (PCR) using microcapillary electrochromatography as the separation mode. The separation was carried out in a polymer-based microfluidic device, hot-embossed into poly(methylmethacrylate) (PMMA), whose walls were functionalized to produce a C(18)-terminated surface to act as the stationary phase (open channel format). The carrier electrolyte contained the ion-pairing agent, triethylammonium acetate (TEAA) to allow the separation to be carried out using reverse-phase ion-pair capillary electrochromatography (RP-IPCEC). The microelectrophoretic separations were investigated utilizing various solvent strengths (acetonitrile/water) with 25 mM TEAA to observe the effects on the separation efficiency as well as the chromatographic development time and detector performance. The field strength significantly affected the quality of the separation, with no separation observed at 333 V/cm for a low mass dsDNA sizing ladder, but baseline separation achieved using a field strength of 67 V/cm. It was observed that the solvent strength affected the retention behavior of the polyanionic molecules as well as the electroosmotic mobility. Higher acetonitrile compositions in the run buffer resulted in reduced plate numbers, which produced lower chromatographic resolution. The use of conductivity detection allowed mass detection sensitivities in the range of 10(-21) mol with a separation efficiency of 10(4) plates and the performance of the detector independent of the acetonitrile content used in the carrier electrolyte.