Development of a quantitative bio/chemiluminescence spectrometer determining quantum yields: re-examination of the aqueous luminol chemiluminescence standard.

We have developed a bio/chemiluminescence spectrometer with a cooled charge-coupled-device (CCD) detector to obtain a quantitative luminescence spectrum as the absolute number of all emitted photons at each wavelength. The integrated area of the spectrum divided by the number of reacted substrate molecules gives the quantum yield. Calibration of the absolute sensitivity of the CCD-spectrometer system was performed by using lasers and a tungsten lamp with calibrated powers as primary light standards, and calibration of the light-collection efficiency of the spectrometer with several kinds of cells for liquid samples was achieved by introducing a simple reference double-plate cell. The reference cell is not convenient for final bio/chemiluminescence measurements but is useful for the calibration because it has well-defined angular dependence of light emission, allowing accurate calculation of the light-collection efficiency. Using this CCD-spectrometer system, we re-examined the quantum yield of aqueous luminol chemiluminescence with H2O2 catalyzed by horseradish peroxidase. The quantum yield was constant for a wide range of luminol concentrations, whereas it changed and had an optimum against H2O2 concentrations. The optimum quantum yield was 1.23(+/-0.20)%, which is in good agreement with previously reported values.

Carbon dioxide effects on luminol and 1,10-phenanthroline chemiluminescence.

Luminol and 1,10-phenanthroline are widely used chemiluminescent (CL) reagents for the analysis of a wide range of metals and inorganic and organic complexes. While the fundamental mechanism for luminol and 1,10-phenantholine chemiluminescence is understood, the analytical application of these reagents is largely empirical and often poorly described mechanistically. For example, CL signals observed from metal-luminol systems are strongly dependent on the pH of the sample, even though the final pH of the reaction mixture is controlled to a narrow range by a buffer. Other investigators report significant changes in CL signal due to freshness and the acidity of reagents. Our work shows that many of these effects are due to dissolved CO2 present or formed in the analytical system. The hypothesis that carbon dioxide plays a pivotal role in enhancing luminol CL is supported by direct manipulation of CO2(aq) concentrations by the addition of CO2(g) or carbonic anhydrase. In contrast, Cu(II) analysis using the CL reagent 1,10-phenanthroline is completely quenched in the presence of CO2(aq). A plausible mechanism for these observations involves the reaction between superoxide, produced in these analytical systems, and CO2(aq) to form the peroxycarbonate radical, *C04-. The formation of *CO4- has very important analytical implications since this species appears to enhance or quench the CL signal from luminol and 1,10-phenanthroline, respectively.

A study of common interferences with the forensic luminol test for blood.

A wide range of domestic and industrial substances that might be mistaken for haemoglobin in the forensic luminol test for blood were examined. The substances studied were in the categories of vegetable or fruit pulps and juices; domestic and commercial oils; cleaning agents; an insecticide; and various glues, paints and varnishes. A significant number of substances in each category gave luminescence intensities that were comparable with the intensities of undiluted haemoglobin, when sprayed with the standard forensic solution containing aqueous alkaline luminol and sodium perborate. In these cases the substance could be easily mistaken for blood when the luminol test is used, but in the remaining cases the luminescence intensity was so weak that it is unlikely that a false-positive test would be obtained. In a few cases the brightly emitting substance could be distinguished from blood by a small but detectable shift of the peak emission wavelength. The results indicated that particular care should be taken to avoid interferences when a crime scene is contaminated with parsnip, turnip or horseradish, and when surfaces coated with enamel paint are involved. To a lesser extent, some care should be taken when surfaces covered with terracotta or ceramic tiles, polyurethane varnishes or jute and sisal matting are involved.