The absolute fluorescence quantum yields, {, of a number of highly fluorescent organic dyes and aromatic compounds have been measured in various solvents, utilizing a calorimetric technique in which the heating rates under monochromatic irradiation are compared for the fluorescent solution and for an inert reference absorber. The technique yields the absolute fluorescence yield for air-saturated solutions with an accuracy of ±0.02. The compound/solvent systems studied provide reference fluorescence values of 0.50 or greater for solvents of refractive index varying between 1.33 and 1.50 and at excitation wavelengths ranging from 366 to 633 nm.

The mechanism of the enhancement of the fluorescence of ethidium bromide on binding to double helical RNA and DNA has been investigated. From an examination of the effect of different solvents on the fluorescence lifetime, quenching of fluorescence by proton acceptors, and the substantial lengthening of lifetime observed upon deuteration of the amino protons, regardless of the medium, we conclude that proton transfer from the excited singlet state is the process primarily responsible for the approximately equal to 3.5-fold increase in the lifetime of free ethidium bromide in going from H2O to D2O; the fact that addition of small amounts of water to nonaqueous solvents decreases the fluorescence whereas addition of small amounts of D2O enhances the fluorescence; and the enhancement of the ethidium bromide triplet state yield on binding to DNA. Other proposed mechanisms are shown to be inconsistent with our findings.

Publication costs assisted by the Petroleum Research Fund and the National Science Foundation We measure ( for the oxazine dye cresyl violet to be 0.54s in methanol, at 22 °C, for excitation from 540 to 640 nm. With a mean emission wavelength near 638 nm, this should be a useful luminescence quantum yield standard for the red. We judge our absolute accuracy to be well within 10% and probably better than 5%. The yield is rather insensitive to conditions even including choice of solvent. It is constant from extreme dilution up to above 10~4 M; but absorption-emission overlap makes it a convenient standard only for relative measurements which use "dilute" procedures. We employed two independent and completely different calorimetric methods to achieve an absolute measurement which is independent of all previous luminescence yield data: photomicrocalorimetry, an optimized conventional technique, and thermal blooming, a new laser approach. We report also determinations relative to presently accepted standards.

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