High-sensitive analysis of heme proteins separated by capillary electrophoresis with on-line chemiluminescence detection using a luminol and hydrogen peroxide system

In the past several years, capillary electrophoresis (CE) has been shown to be a powerful and efficient analytical separation technique. One of the major areas of study is the development of sensitive detection methods. On-column optical detection modes, such as UV absorption and fluorescence detection, are the most commonly used, on account of the extremely small sample zone and the small capillary dimensions in CE. Chemiluminescence (CL) has been verified to be a highly sensitive detection method in both now injection analysis (FIA) and highperformance liquid chromatography (HPLC).(1) Due to its simple optical system and low background nature, CL is expected also to be an ideal detection method for CE. Recently, the applicability of CL detection in CE has been successfully demonstrated. Several CL reagents, such as luminol(2,3), acridinium(4) and peroxyoxalate(5,6) have been utilized. The present authors have for the first time reported on a CL detection of proteins separated by CE.(5,6) The bis(2,4,6-trichlorophenyl)oxalate(TCPO)-hydrogen peroxide(H2O2) system was used together with fluorescent compounds, such as Eosin Y, rhodamine B isothiocyanate, tetramethylrhodamine isothiocyanate isomer R and fluorescamine. The proteins labeled with them were quantitatively analyzed by the CE-CL detection method with detection limits of 10(-8)-10(-7)mol dm(-3) orders.