Ultra high voltage capillary electrophoresis

Conventional capillary electrophoresis is performed in fused silica capillaries with potentials of 30 kV or less. Analysis at these potentials can generate separation efficiencies of about half a million theoretical plates for a small molecule. Increasing the potential should increase the efficiency of the separation. However, it would also cause dielectric breakdown of the fused silica capillary unless preventative measures were taken to shield the capillary wall from the radial field generated.; An ultra high voltage capillary electrophoresis instrument was built to perform separation with potentials of up to 150 kV. This required the assembly of a high-voltage power supply, electrical shielding for the capillary, and an air-tight chamber for sample introduction.; Initially, peptides from protein digests, tagged with a fluorescent label, were separated at 120 kV by zone electrophoresis. These separations generated up to 6 million theoretical plates. The separations at 120 kV were compared to identical separations performed at conventional potentials. The efficiency was found to be 3.8 times higher at 120 kV than at 28 kV, and the resolution improved by a factor of 2.1.; Glycan pools derived from human Ribonuclease B and human IgG were analyzed in a mixed-mode separation, by complexation with borate and micellar electrokinetic chromatography at 100 kV and at 20 kV. Again, the ultra high voltage separation was compared to the conventional separation. The use of 100 kV was found to provide a factor of 4.4 improvement in efficiency, and a factor of 2.2 improvement in resolution, relative to the separation at 20 kV.; Hyaluronic acid, a polydisperse, linear carbohydrate, was analyzed by capillary gel electrophoresis at 95 kV and at 15 kV. The use of the higher potential was found to improve the efficiency by a factor of 10 and the resolution by a factor of 3 to 5 for the smaller hyaluronic acid molecules. The improvement in the efficiency and resolution decreased with increasing size of the hyaluronic acid molecules.