EXAMINATION OF VARIABLES AFFECTING THE PERFORMANCE OF ISOELECTRIC-FOCUSING IN UNCOATED CAPILLARIES

The effects of different variables on protein separation by isoelectric focusing in uncoated capillaries were investigated. Four model proteins were used to study the effects. The variables included applied electric field, column length, % methyl cellulose, % tetramethylethylenediamine (TEMED), and buffer concentration. Methyl cellulose showed a bimodal effect on the separation performance in terms of migration time and peak width. It was determined that the optimal % TEMED and applied field depended strongly on each other. Furthermore, longer column lengths led to better resolution at constant field and % TEMED. Using a grid, the applied electric field and % TEMED were optimized concurrently at a fixed column length.