Gradient elution isotachophoresis for enrichment and separation of biomolecules

A novel format for performing capillary isotachophoresis (ITP) is described -- gradient elution ITP (GEITP). GEITP merges the recently described electrophoretic separation technique of gradient elution moving boundary electrophoresis (GEMBE) with an ITP enrichment step. GEMBE utilizes a combination of continuous sample injection with a pressure-controlled counterflow; as the counterflow is reduced, analytes are sequentially eluted onto the separation column and detected as boundary interfaces. By incorporating leading electrolytes into the counterflow and terminating electrolytes into the sample matrix, an ionic interface can be formed near the capillary inlet. The discontinuous buffer system forms highly enriched analyte zones outside of the capillary, which are then eluted onto the separation capillary as the counterflow is reduced. Separation of fluorescent analytes was achieved either through discrete electrolyte spacers added to the sample or by using ampholyte mixtures to form a continuum of spacers. As the ITP process occurs off-column, extremely short length separations can be achieved, as demonstrated by a separation in 30 microm. The effects of various parameters on the GEITP enrichment process are investigated, including initial counterflow rates, electric field, leading electrolyte concentration, and counterflow acceleration, which is an adjustable parameter allowing for highly flexible separations. Typical enhancements in limits of detection and sensitivity were greater than 10,000-fold and were achieved in less than 2 min, yielding low-picomolar detection limits using arc lamp illumination and low-cost CCD detection. An optimized system afforded greater than 100,000-fold improvement in detection of carboxyfluorescein in 8 min. Specific examples of enrichment and separation demonstrated include the following: small dye molecules, DNA, amino acid mixtures, and protein mixtures.