Environ. Sci. Technol. 1996, 30, 1407-1408
Comment on “Solubility Enhancement and Fluorescence Quenching of Pyrene by Humic Substances: The Effect of Dissolved Oxygen on Quenching Processes” SIR: In a recent research communication, Danielsen et al. (1) suggest that the dynamic quenching of pyrene by oxygen in aqueous solution is somehow enhanced by the presence of humic (HA) or fulvic acids (FA). However, there is no need to invoke novel mechanisms to explain the reported data; within expected experimental error, the results presented are entirely consistent with static quenching of humic-bound pyrene in conjunction with dynamic quenching of aqueous-phase pyrene by oxygen. The standard view of the interactions of fluorescent compounds with humic materials is that two distinct populations of fluorophore exist in solution: one consists of molecules bound to the humic matrix, and the other consists of those remaining free in solution. Fluorescence of the first group is completely quenched (static quenching) (2-6). The second group has no interaction with the humic material, but of course remains vulnerable to other quenchers that may be present in the solution (dynamic quenching). Danielsen et al. (1) apparently postulate that their FA solutions contain either a third fluorophore population uniquely vulnerable to quenching or an unprecedented increase in the concentration of dynamic quenchers, specifically oxygen, in these samples. Considerable evidence has accumulated demonstrating that static interactions between humic or fulvic acids and hydrophobic organic solutes are primarily responsible for the quenching of solute fluorescence (2-6). Four types of experiments have strongly supported this conclusion: (1) substantial quenching of polyaromatic hydrocarbon (PAH) fluorescence is observed with very low quencher (HA or FA) concentrations; (2) as the FA:PAH ratio is increased, the PAH fluorescence intensity asymptotically approaches zero (2, 3, 6); (3) little or no increase in quenching is detected with increasing temperature (4); and (4) fluorescence lifetimes of aqueous-phase (unbound) fluorophores are constant in the presence and absence of HA (4). Danielsen et al. (1) considered four types of fluorescence decay curves of pyrene in aqueous solution: argon-sparged solutions with and without FA and air-saturated solutions with and without FA. In all cases, they determined the lifetimes by the single-photon counting technique, but with data analysis restricted to times longer than 115 ns after the excitation pulse. In this way, they limited their view solely to unbound pyrene molecules, i.e., those vulnerable to dynamic quenching. The fluorescence lifetime of this population was determined to be 190 ns, consistent with other reported values for aqueous pyrene. In argon-sparged solutions, the presence of FA had no effect on the fluorescence lifetime of pyrene at long times, as expected. Also predictably, the lifetime of this aqueous-phase pyrene was unaffected by the quantity of FA in solution (ref 1, Figure 2). The maximum concentration reported, about 45 mg of C/L, corresponding to approximately 30 µM FA, could at most be responsible for the quenching of ≈5% of
0013-936X/96/0930-1407$12.00/0
1996 American Chemical Society
the pyrene fluorescence, assuming diffusion-controlled kinetics. It is well known that pyrene in aerated solutions is dynamically quenched by oxygen with a diffusion-controlled second-order rate constant, kq, of approximately 1 × 1010 M-1 s-1. The decrease in fluorescence lifetime in the presence of oxygen is given by the Stern-Volmer equation:
τ0/τ ) 1 + kqτ0[O2] where τ0 and τ are the fluorescence lifetimes in the presence and absence of quencher, respectively. This equation can be employed to predict the expected fluorescence lifetime in air-saturated pyrene solution at 25 °C, in which the O2 concentration is 266 µM (7). Taking τ0 as 190 ns, as reported, and kq ) 1 × 1010 M-1 s-1, we calculate τ for pyrene in aerated solutions to be 126 ns. Lifetimes of 122-133 ns were measured by Danielsen et al. (1) for pyrene in airequilibrated SRFA solutions, precisely as expected for simple dynamic oxygen quenching (ref 1, Figure 2). The crux of the argument in this paper centers on the very slight difference in pyrene fluorescence lifetime in the absence (144 ns) and presence (132 ns) of SRFA in aerated solutions. As the shorter lifetime corresponds to the expected value for air-saturated solutions, the observed difference may simply be the result of an undersaturation of oxygen before the addition of SRFA. No indication was given as to how air-equilibrated solutions were prepared and maintained or whether the temperature was carefully controlled throughout the experiments. Indeed, the fact that lifetimes in the absence of SRFA ranged from 139 to 148 ns (ref 1, Figure 2) indicates that among samples either there were variations in oxygen concentrations and temperature and/or inconsistencies in the data fitting procedure. The high sensitivity to environmental factors of lifetimes of very long-lived excited states (>100 ns) requires that “extreme caution” be used in determination of decay kinetics (8). Despite the fact that the authors base their conclusions on an 8-10% difference in lifetimes, they provide no estimates of the reproducibility or precision of the data collected under “air-saturated” conditions. The issue of fitting becomes important in this case because data were only fit at times longer than 115 ns. For a fluorescence lifetime of 130 ns, 59% of the photons are emitted before 115 ns. In addition, a close examination of the fluorescence decay curves shown (ref 1, Figure 3) suggests that the difference between reported lifetimes of 144 and 132 ns relied heavily on points at times greater than 300 ns after the excitation pulse. By 300 ns, 90% of the photons have already been emitted. Thus, the kinetic fits included only the last half of the decay, and major interpretations were apparently strongly influenced by the final 10% of the curves. The authors suggest that oxygen sequestered in or transported by the FA matrix is responsible for additional quenching of pyrene in aerated solutions. This implies either that sufficient excess oxygen is introduced by the FA to significantly increase the total O2 concentration in solution or that there are FA-associated pyrene molecules that are not quenched by FA but are quenched by O2 which
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is associated with the humic matrix. Both possibilities are extremely unlikely based on current information. If oxygen concentrations within the FA matrix were 10 times normal aqueous values, then FA would have to make up an incredible 1% of the total solution volume to effect the ≈10% change in oxygen concentration required to reduce the pyrene fluorescence lifetime from 144 to 132 ns. The specific volume of hydrated FA is on the order of 0.50.6 cm3/g (9); from this we calculate the volume fraction of FA in a 12 mg of C/L solution to be
