Flow counterbalanced capillary electrophoresis using packed capillary columns: resolution of enantiomers and isotopomers

A method with the ability to increase greatly both the resolution and efficiency of a given capillary electrophoretic system is described. This method differs from traditional capillary electrophoresis (CE) in that a counterflow is induced in the direction opposite to the electrokinetic migration of the analyte. This has the effect of extending not only the time the analytes migrate in the electric field but also the effective length and the effective applied voltage of the system. Previous work in our group with flow counterbalanced capillary electrophoresis has utilized an open tube of small inner diameter to reduce peak broadening caused by hydrodynamic flow. Narrow-diameter capillaries (5-10 microm) restricted analysis to fluorescent analytes and laser-induced fluorescence detection. The method described here uses a capillary of much larger inner diameter (75 microm) that has been packed with nonporous silica particles. The packing material reduces the amount of band broadening caused by pressure-induced flow relative to that experienced in an open tube. A larger diameter capillary allows the detection of analytes by UV absorption, not only eliminating the need to tag analytes with fluorescent tags but also allowing for the detection of a much broader range of analytes. The system was evaluated by studying the separations of several enantiomers using only beta-cyclodextrin as the chiral selector. The system was also used to resolve the two naturally occurring isotopes of bromine and to resolve phenylalanine from phenylalanine-d8. Relative to traditional CE, large improvements in resolution and separation efficiency have been achieved with this method.