Trace analysis of proteins by capillary zone electrophoresis with on‐column transient isotachophoretic preconcentration

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Abstract
The qualitative and quantitative aspects of transient isotachophoretic (ITP) sample preconcentration in the capillary zone electrophoretic analysis of protein samples have been demonstrated. By the proper selection of components of the background electrolyte and/or additives to the sample solution, two basic electrolyte arrangements have been employed. In the first, a typical isotachophoretic electrolyte system consisting of a leading and terminating electrolyte was used, and after focusing and preconcentration, the terminating electrolyte was replaced by the leading electrolyte, with the separation being continued in the zone electrophoretic mode. In the second, only one background electrolyte was used, containing a co-ion with low electrophoretic mobility, and the sample was supplemented with a salt of a highly mobile co-ion. In this case transient isotachophoretic migration of the sample ions took place at the beginning of the migration and gradually changed to the zone electrophoretic mode. Sample mixtures containing basic (positively charged) or acidic (negatively charged) proteins were examined using surface-coated fused-silica capillaries. For acidic proteins, bare silica was also tested. The isotachophoretic sample stacking permitted injection and preconcentration of sample volumes two to three orders of magnitude higher than usual in capillary zone electrophoresis. For example, up to 1 μL was injected into a 75 μm ID capillary. This approach afforded quantitative analysis of protein samples in the concentration range of 10−7–10−8 M, with detection limits of approximately 10−9 M. Furthermore, with constant sample volume injected, good reproducibility of migration times was obtained. Finally, the determination of trace components in the presence of a major sample component using transient ITP preconcentration has been demonstrated.