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CORRESPONDENCE Comment on “Fluorescence Quenching of Synthetic Organic Compounds by Humic Materials” SIR: The authors of this paper (1)have done a service to the environmental community by pointing out that the fluorescencequenching method for measuring the binding of organic compounds to humic materials cannot be universally applied. They are correct in emphasizing that Stern-Volmer plots must be linear in order to get reliable equilibrium constants. However, parts of the manuscript imply that the fluorescence quenching method cannot be applied at all with humic materials. The experimental basis for this is the observation that Stern-Volmer plots for difenzoquat binding to humic materials show significant upward curvature. The authors attribute this to the micellar nature of humic material. If curvature is indeed due to the humic material, then one might expect that it would occur with other organics. A critical point, dismissed by the authors in the last paragraph on page 1790, is that difenzoquat is a cation, while the fluorophores that have been previously studied by this method are neutral. The issue is not static vs dynamic quenching. For both neutral fluorophores and difenzoquat, quenching clearly involves a ground-state interaction. This is readily concluded from the magnitude of the quenching which is orders of magnitude greater than is possible from diffusion-limited excited-state in-
SIR: The interpretation of data obtained through the application of Stern-Volmer theory to humic quenchers is not straightforward. We, therefore, not only welcome the thoughtful comments of Dr. Seitz on our recent manuscript (1) but also feel that such comments are necessary for the effective application of fluorescence quenching to these complex systems. There are a number of specific issues addressed by Dr. Seitz on which we disagree. Although our emphasis in this manuscript was on difenzoquat, a cationic fluorophore, we showed that 1-naphthol quenching by humic acid was also best represented by aquadratic and not a linear SternVolmer plot (Table 111). In addition, data from Traina et al. (2) originally interpreted as producing a linear SternVolmer relationship were shown to be better fit by inclusion of a quadratic term (Table 111). Upward curvature of Stern-Volmer plots is therefore not specific to humic acid quenching of cationic fluorophores but also occurs with nonionic fluorophores.
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teractions. Instead the difference is due to the nature of the interaction between the organic fluorophore and the humic material. The attraction between neutral organics and humic materials is hydrophobic in nature, while there is an electrostatic interaction between a cation and a negatively charged humic acid. This neutralizes the charge on the humic acid, reducing its solubility in water, presumably causing a change in conformation. If the charge is completely neutralized, the humic acid will come out of solution. This is usually accomplished by adding protons but applies equally well with other cations. This process does not occur with neutral organics. I have submitted this comment because I fear that the manuscript in question might discourage other scientists from using the fluorescencequenching method for systems where it is in fact applicable. In any event, the linearity of the Stern-Volmer plot needs to be confirmed before deriving equilibrium constants from fluorescence quenching data.
Literature Cited (1) Puchalski, M. M.; Morra, M. J.; von Wandruska, R. Enuiron. Sci. Technol. 1992, 26, 1787-1792.
W. Rudolf Seltz Department of Chemistry College of Engineering and Physical Sciences University of New Hampshire Durham, New Hampshire 03824-3598
There also appears to be some confusion related to the nature of the quenching and whether a ground-state association is responsible for the observed decrease in fluorescence intensity. We argue in this manuscript as well as in a previous publication (3)that quenching cannot be described as a true diffusion-limited process, because the fluorophore and quencher are in close physical association. Quenching is not caused by a ground-state reaction but by immediate deactivation of the fluorophore on excitation. In such a system, the magnitude of quenching cannot be used to infer the type of interaction responsible for decreased fluorescence intensity. Indeed, other investigators working with micellar systems ( 4 )have shown even greater quenching than observed in our work and still consider that quenching dynamic in nature. This has been shown to occur not only with nonionic fluorophores in micellar systems but also with cationic fluorophores in combination with anionic surfactants, not unlike the situation of difenzoquat and humic acid. In summary,
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while quenching appears static, no true ground-state association occurs at the molecular level. The final aspect that deserves comment concerns the possible conformational change and precipitation of humic acid as a direct result of the addition of a cationic fluorophore. The lack of such a mechanism to explain nonlinear Stern-Volmer plots with nonionic fluorophores, as previously described, would seem to preclude this possibility. In addition, humic acids behave as uncharged colloids under electrolyte concentrations in excess of 0.05 M (5), while difenzoquat in our system was present at a concentration of only 6.9 X M. We feel that the continued use of fluorescencequenching with humic materials is justified but that binding constants derived from such analyses must be carefully evaluated.
Literature Cited (1) Puchalski, M. M.; Morra, M. J.; von Wandruszka, R. Environ. Sci. Technol. 1992, 26, 1787-1792.
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(2) Traina, S. J.; Spontak, D. A.;Logan, T. J. J . Enuiron. Qual. 1989, 18, 221-227. (3) Morra, M. J.; Corapcioglu, M. 0.;von Wandruszka, R. M.; Marshall, D. B.; Topper, K. Soil Sci. SOC.Am. J. 1990,54, 1283-1289. (4) Eftink, M. R.; Ghiron, C. A. J. Phys. Chem. 1976,80,486493. (5) Ghosh, K.; Schnitzer, M. Soil Sci. 1980, 129,266-276.
Michele M. Puchalskl and Matthew J. Morra' Division of Soil Science University of Idaho Moscow, Idaho 83843
Ray von Wandrusrka Department of Chemistry University of Idaho Moscow, Idaho 83843
