PRECOLUMN DERIVATIZATION OF PROTEINS TO ENHANCE DETECTION SENSITIVITY FOR SODIUM DODECYL-SULFATE NON-GEL SIEVING CAPILLARY ELECTROPHORESIS

Pre-column derivatization of proteins with fluorescamine, naphthalene-2,3-dicarboxyaldehyde, and o-phthaldialdehyde was used to enhance absorption and fluorescence detection after separation by SDS non-gel sieving capillary electrophoresis. When compared with underivatized proteins, absorption sensitivity increased by as much as a factor of 22 at 280 nm, and 1.74 and 4.71 at 200 and 220 nm, respectively. Under favorable conditions, absorption detection limits with the labeled proteins at 280 Mn were approximately equivalent to the detection limits of underivatized proteins at 200 nm. Fluorescence detection provided attomole detection limits with the best results being obtained with high-molecular-mass proteins. Pre-column labeling decreased the efficiency of the separation, but did not give rise to multiple peaks from heterogeneous labeling. The migration velocity of labeled proteins was slightly different from the unlabeled molecules, but did not significantly degrade molecular mass determinations. Pre-column derivatization with fluorescence detection allowed the proteins in a fertilization membrane isolated from a single amphibian embryo to be easily characterized.