Preparative capillary zone electrophoresis using a dynamic coated wide-bore capillary

Preparative capillary zone electrophoresis separations of cytochrome c from bovine and horse heart are performed efficiently in a surfactant-coated capillary. The surfactant, dimethylditetradecylammonium bromide (2C(14)DAB), effectively eliminated protein adsorption from the capillary surface, such that symmetrical peaks with efficiencies of 0.7 million plates/m were observed in 50-microm id capillaries when low concentrations of protein were injected. At protein concentrations greater than 1 g/L, electromigration dispersion became the dominant source of band broadening and the peak shape distorted to triangular fronting. Matching of the mobility of the buffer co-ion to that of the cytochrome c resulted in dramatic improvements in the efficiency and peak shape. Using 100 mM bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane phosphate buffer at pH 7.0 with a 100-microm id capillary, the maximum sample loading capacity in a single run was 160 pmol (2.0 microg) of each protein.