Application of capillary isoelectric focusing with absorption imaging detection to the analysis of proteins.

A capillary isoelectric focusing instrument with an on-line optical absorption imaging detector was used to analyse protein samples. The separation column was a 4 cm x 100 microns I.D. capillary. The light source of the imaging detector was a argon ion laser. The light beam from the laser was focused into the capillary by a cylindrical lens. An 1024-pixel charge-coupled device (CCD) measured intensity of light beam passing through the capillary. The optical alignment of the detector was optimized to eliminate interference produced by the refractive index gradient created by sample zones inside the capillary. The signal-to-noise ratio of the detector was enhanced by averaging 30 scans of the CCD every 3 s. The on-line imaging detector allows simultaneous separation and detection so that the analysis time for a sample is only 2-4 min. Several protein samples were analyzed by the instrument, including human hemoglobin variants, cytochrome c, myoglobin and transferrin.