ONLINE CHEMILUMINESCENCE DETECTION OF PROTEINS SEPARATED BY CAPILLARY ZONE ELECTROPHORESIS

In a capillary zone electrophoretic (CZE) experiment in phosphate buffer (pH 3.5), Eosin Y was found to migrate together with protein in a capillary tube as a supramolecular complex. This finding gave the possibilities not only of overcoming the problem of protein determination but also of measuring the Eosin Y comigrating with protein by a chemiluminescence (CL) method with high sensitivity. Labelling of protein with dyestuff was achieved simply by mixing the protein and a dyestuff. On-line CL detection of the protein separated by CZE was feasible by measuring the CL intensity of a bis(2,4,6-trichlorophenyl) oxalate (TCPO)-H2O2-dyestuff system by means of an interface between CZE and CL detection. Several xanthene dyestuffs including Eosin Y were examined and Rose Bengal was found to be more sensitive than Eosin Y used in previous work. Using the present method, 5.10(-7)-1.10(-4) mol dm(-3) of bovine serum albumin (BSA) could be determined using Rose Bengal in about 20 min with a detection limit of 2.10(-7) mol dm(-3) (signal-to-noise ratio = 3), corresponding to 4 fmol of BSA. The results for fifteen kinds of proteins including BSA are reported.