Quantum yield measurement against standard

This method of luminescence quantum yield measurement against a standard emitter is simple and easy to implement with computer-linked fluorimeters, in particular for the integration of the spectra. Its accuracy should however not be over-rated. It is, in the best cases, of the order of 5% (and often far worse). It remains at this time the most widely used technique in photophysics. 

To study the excited state one may use transient absorption or time-resolved fluorescence techniques. In both cases, DNA poses many problems. Its steady-state spectra are situated in the near ultraviolet spectral region which is not easily accessible by standard spectroscopic methods. Moreover, DNA and its constituents are characterised by extremely low fluorescence quantum yields (<10 4) which renders fluorescence studies particularly difficult. Based on steady-state measurements, it was estimated that the excited state lifetimes of the monomeric constituents are very short, about a picosecond [1]. Indeed, such an ultrafast deactivation of their excited states may reduce their reactivity something which has been referred to as a "natural protection against photodamage. To what extent the situation is the same for the polymeric DNA molecule is not clear, but longer excited state lifetimes on the nanosecond time scale, possibly of excimer like origin, have been reported [2-4],... 

The dynamics of populations of the electronic states in a 4,4 -bis(dimethylamino) stilbene molecule (two-photon absorption) was studied against the frequency, intensity, and shape of the laser pulse [52]. Complete breakdown of the standard rotating wave for a two-photon absorption process was observed. An analytical solution for the interaction of a pulse with a three-level system beyond the rotating wave approximation was obtained in close agreement with the strict numerical solution of the amplitude equations. Calculations showed the strong role of the anisotropy of photoexcitation in the coherent control of populations that can affect the anisotropy of photobleaching. The two-photon absorption cross section of an ethanol solution of a trans-stilbene and its derivatives exposed to radiation of the second harmonic of a Nd YAG laser (532 nm) of nanosecond duration has been detected [53]. In experiments, the method based on the measurement of the photochemical decomposition of examined molecules was used. The quantum yield of the photoreaction (y266) of dyes under one-photon excitation (fourth harmonic Nd YAG laser 266 nm) was detected by absorption method. 

The absolute fluorescence yields for QHj derive with one exception from excitation at 2536 A. Two of these are based on calibration against the phosphorescence yield of biacetyl,which has been a gas phase standard for many years. The first reports a yield of about 0.23 0.05 at 11 torr, but this was subsequently remeasured by Poole as 0.17 in further evolution of that technique in the same laboratory. A later measurement independent of the biacetyl standard is reported by Noyes, Mulac, and Harter. They observe (in their Method B) that f = 0.18 0.04 at 11.5 torr. Further support for this value comes from its use in calibration of singlet relaxation quantum yields in fluorobenzene and toluene in which the sum of observed radiative and nonradiative yields is unity within experimental error. A higher benzene fluorescence yield would push that sum paradoxically above unity. 

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