Design, construction and application of an instrument for simultaneous measurement of molecular fluorescence, absorbance and chemiluminescence

Graduation date: 1985An instrument was constructed to measure molecular fluorescence (FL) or chemiluminescence (CL) and absorbance (ABS) simultaneously. The lower, middle, and upper portions of the test solution are probed to obtain ABS, FL, and CL data. Optical fibers are used extensively. The ABS and FL or CL spectra are acquired in 400 ms or more with diode array detectors mounted on spectrographs. The FL and CL signals can also be detected with photomultiplier tubes after wavelength selection with filters and/or a monochromator. Two microcomputers in a master- slave arrangement control data acquisition. The performance of the multiple signal spectrometer was evaluated using quinine sulfate (QS) solutions and the CL reactions of lucigenin (Lc) and luminol. Chemiluminescence and FL signals which cannot be spectrally resolved are measured sequentially by blocking the FL excitation (EX) beam. Absorbance can be measured from 300 to 800 nm. Noise and stray light limit accuracy above 1.5 A.U. The EL detection limit for QS ranges from 1.2 to 40 pg/mL, depending on the detection scheme. Software was developed to utilize the measured ABS in appropriate equations to automatically correct the luminescence signals for attenuation (inner-filter effects) caused by absorption of the EX or emission (EM) radiation by the lumophore or other species. Pre- viously developed correction equations were modified for situations in which the EX or EM beam cannot be considered to be monochromatic and for collection geometries involving optical fibers. The correction scheme was tested in the absence and presence of chromophores (gentisic acid, methyl red, K₂Cr₂O₇ ). Even when conditions are such that the FL signal is attenuated by a factor of 10, the correction scheme is accurate to 2% or better. These corrections extend the linear range of the QS calibration curve by a factor of over 100. Finally, the kinetics of the reaction of Lc with H₂O₂ in the presence of KOH were studied. Time resolved ABS, FL, and CL spectra were obtained. Although some of the spectral shifts occurring during the reaction are due to changing inner-filter effects, the corrected spectra demonstrate that methyl acridone is not the species respon- sible for CL, as some researchers claim