Selective preconcentration for capillary zone electrophoresis using protein G immunoaffinity capillary chromatography.

ABSTRACT

Capillaries with 150 microns inner diameter were packed with a perfused protein G chromatographic support and used as immunoaffinity preconcentrators for capillary zone electrophoresis. Antibody was loaded onto the protein G support to form an immunoaffinity stationary phase. Injection of samples onto the column caused selective retention and preconcentration of antigen. Injection of appropriate buffers onto the column caused desorption of the antibody and antigen which were then separated by capillary zone electrophoresis. The combination was used on-line and off-line. For on-line combination, a flow-gated interface coupled the two columns and allowed injection of desorbed zones onto the electrophoresis system. Off-line coupling required collection of desorbed fractions and then injection onto the electrophoresis system. Flow rates as high as 100 microL/min were used to load sample onto the affinity column. Desorbing flow rates had to be 1 microL/min or less to prevent excessive dilution during desorption. Using the system, 1 mL insulin samples could be loaded onto the affinity column and desorbed in volumes as small as 1 microL for 1000-fold preconcentration. The use of the preconcentrator with serum samples spiked with insulin was demonstrated.