The effect of excitation beam absorption on measured values of fluorescence has been studied wlth a computer-centered spectrofiuorimeter capable of measuring fluorescence and absorbance slmuitaneously. This effect appears to be independent of the nature of the absorbing species and the excitation and emisslon wavelengths. A model is proposed and tested which corrects fluorescence, observed at 90°, for the attenuation of the excitation beam caused by the absorbance of the fluorophore and any chromophores present in the cell. The resuitlng absorptlon-corrected fluorescence is linear with the concentration of the fiuorophore in solutions wlth total absorbances as high as 2.0.

A computer centered spectrophotometer-spectrofluorimeter combination instrument has been fabricated that will allow simultaneous absorption and fluorescence measurements.The dedicated computer applies corrections to the data collected for many of the instrumental and photophysical variables of fluorescence measurements.

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