PRECOLUMN DERIVATIZATION OF PROTEINS TO ENHANCE DETECTION SENSITIVITYFOR 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 abso rption and fluorescence detection after separation by SDS non-gel siev ing capillary electrophoresis. When compared with underivatized protei ns, absorption sensitivity increased by as much as a factor of 22 at 2 80 nm, and 1.74 and 4.71 at 200 and 220 nm, respectively. Under favora ble conditions, absorption detection limits with the labeled proteins at 280 Mn were approximately equivalent to the detection limits of und erivatized proteins at 200 nm. Fluorescence detection provided attomol e detection limits with the best results being obtained with high-mole cular-mass proteins. Pre-column labeling decreased the efficiency of t he separation, but did not give rise to multiple peaks from heterogene ous labeling. The migration velocity of labeled proteins was slightly different from the unlabeled molecules, but did not significantly degr ade molecular mass determinations. Pre-column derivatization with fluo rescence detection allowed the proteins in a fertilization membrane is olated from a single amphibian embryo to be easily characterized.