The Determination of the Fluorescence Lifetimes of Dissolved Substances by a Phase Shift Method

Abstract

The lifetimes of fluorescent substances are determined by exciting the fluorescence with light modulated at a high frequency and measuring the difference in phase between the exciting light and the resulting fluorescence. The exciting light is modulated by an ultrasonic standing wave in a liquid which causes the latter to act as an intermittent diffraction grating. Photomultiplier tubes are used to pick up the exciting and fluorescent light. Tuned circuits are used to isolate a single harmonic of the signal from the phototubes and the phase shift determined by comparison with a calibrated phase shifting circuit. The phase angle φ is related to the lifetime by tanφ=ωτ. In which ω is 2π times the frequency of modulation and τ is the lifetime of the excited state. The data show that lifetimes can be determined with a precision of 1 to 2×10⁻¹⁰ second. The lifetimes of acridone in neutral solution (saturated to 10 percent saturated) and quinine sulfate (4×10⁻⁶ to 4×10⁻⁵M) in 0.01 M nitric acid are independent of the concentration. The values found are acridone 1.59±0.01×10⁻⁸ second, and quinine sulfate 2.28×0.02×10⁻⁸ second. The lifetime of fluorescein in 0.005M potassium hydroxide increases with concentration over the range 10⁻⁶ to 5×10⁻⁴M. This fact can be explained by assuming that some of the fluorescent light is absorbed and re‐emitted. The lifetime of fluorescein extrapolated to zero concentration is 4.5±0.1×10⁻⁹ second.