Whole-column imaging capillary electrophoresis of proteins with a short capillary

Whole-column imaging capillary electrophoresis with a short capillary is discussed. A short capillary (3-6 cm) coated with either fluorocarbon or polyacrylamide was used as a separation capillary. The whole capillary was illuminated with 280 nm light, and the transmitted light was monitored by a linear charge-coupled device (CCD). For the short capillary, hydrodynamic flow caused by a subtle height difference between the anodic and cathodic reservoirs affected the sample migration in the capillary greatly. Several sample injection methods, including use of a cross connection, sealing of the capillary ends with a gel, and use of a gel-filled capillary, have been discussed. The experimental results showed that the peak height decreased and peak width increased with the electromigration distance. Therefore, higher sensitivity was obtained in a short capillary rather than a long capillary. The whole-column imaging CE with the short capillary has been applied for the study of conjugation reactions of protein cytochrome c with sodium dodecyl sulfate (SDS) and the dye Congo Red. The method has also been used for in situ monitoring of the electrophoretic protein desorption process. Our technique is a unique tool for the study of protein binding reactions and the interaction between analyte and inner wall of the capillary.