CORRESPONDENCE/REBUTTAL pubs.acs.org/est
Comment on “HILIC-NMR: Toward the Identification of Individual Molecular Components in Dissolved Organic Matter” e read with great interest the recent article by Woods et al.1 that employed a new separation technique (hydrophilic interaction chromatography or HILIC) to separate Suwanee River DOM (SRDOM) into 80 fractions. The separated fractions were then analyzed by NMR and by fluorescence excitation emission matrix spectroscopy (EEMs) combined with Parallel Factor Analysis (PARAFAC). To “aid in identifying possible fluorophores,” HILIC fractions were additionally spiked separately with model quinones (anthroquinone-2,6-disulphonate (AQDS), p-benzoquinone, lawsone, and juglone) and further subjected to EEMs and PARAFAC analysis. The authors observed that four components of the seven-component PARAFAC fit “were found to contain signals influenced by the addition of a variety of model quinones” and subsequently labeled them Q1, Q2, Q3, and Qb, implying a structural assignment of these components to quinones. Three sentences later, the authors refer to these components as quinones, further reinforcing the idea of a structural assignment based on the fluorescence data. This presentation within the main text is highly misleading, and could convince some investigators to believe that these components do result from a direct emission contribution from quinones similar to the past claims of Cory and McKnight,2 despite several recent papers providing strong evidence contradicting these claims.3,4 Only upon delving into the Supporting Information does one find that AQDS, lawsone and juglone were each found to “affect” three of the PARAFAC components, a pattern incompatible with a direct emission contribution from these quinones. Further, no information was provided as to how these PARAFAC components, or the original EEMs spectra, were “affected” by quinone addition. Indeed, no controls were performed to test whether the model quinones even exhibited significant fluorescence emission (ref 3; see also below). Only in the Supporting Information do the authors concede that the results are “complex” and allude to the possibility of other explanations.5,6 We previously demonstrated that the fluorescence quantum yields of p-benzoquinone and AQDS, as well as those of an extensive series of structurally diverse quinones, were either indistinguishable from zero or were far, far smaller than those of humic substances.3 In Figure 1, we show further that lawsone (2-hydroxy-1,4-napthoquinone) does not emit, while juglone (5-hydroxy-1,4-napthoquinone) emits only very weakly, far less than that of Suwanee River fulvic acid (SRFA) at the same excitation wavelength. Based on the data in Figure 1 and the known quantum yield of SRFA at this wavelength (∼0.007 at 425 nm; refs 3,6), we roughly estimate the quantum yield for juglone to be 0.0012, ∼6-fold lower than that of SRFA. In addition, none of the spectra of the four model quinones employed by Woods et al. (see Figure 1 and ref 3) match any of their PARAFAC components (Supporting Information in ref 1). In summary, existing studies1,2 have failed to provide indisputable evidence of a significant, direct emission contribution from quinones to the fluorescence of humic substances or chromophoric dissolved organic matter.3,4
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r 2011 American Chemical Society
Figure 1. Optical properties of SRFA (50 mg/L), lawsone (∼4.5 mg/L) and juglone (∼23 mg/L) in phosphate buffer (10 mM, pH 7.5). A Shimadzu UVPC 2401spectrophotometer was employed to obtain the absorption spectra (1 cm cell). An Aminco-Bowman AB-2 fluorescence spectrometer was employed to acquire the emission spectra (excitation and emission bandpasses both 4 nm). The emission spectra were corrected employing manufacturer provided correction factors. Arrows indicate excitation wavelengths.
Indeed, we found in preliminary experiments that addition of juglone (∼2.3 mg/L) and lawsone (∼0.23 mg/L) to SRFA (5 mg/L in 10 mM phosphate buffer, pH 7.5) produces partial quenching of SRFA fluorescence in emission spectra recorded over a range of excitation wavelengths including the ultraviolet. We suspect that this quenching is the likely origin of the “influenced” PARAFAC components in the Woods et al. study.1 Because the fluorescence lifetimes of SRFA are very short6 and low quinone concentrations were employed, this quenching must be “static” and occur through sorption of these quinones to SRFA. Finally, we note that three of the seven PARAFAC components obtained in the model of Woods et al. (Qb, Tyr, and TRP1) exhibit a lowest-lying excitation (absorption) band whose maximum
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dx.doi.org/10.1021/es201531v | Environ. Sci. Technol. 2011, 45, 5908–5909
Environmental Science & Technology
CORRESPONDENCE/REBUTTAL
is at a wavelength comparable to or longer than the emission maximum, a result that is not physically meaningful, thus raising further questions about the suitability of the model itself. In our view, this study represents another example where the indiscriminant use of EEMs and PARAFAC modeling has produced more confusion than clarification. The advantages, as well as the limitations, of the use of EEMs and PARAFAC modeling is discussed in Stedmon and Bro7 and nicely demonstrated in the recent study by Murphy et al.8 Neil V. Blough†,* and Rossana Del Vecchio§,* †
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20770, United States
§
Earth System Science Interdisciplinary Center, University of Maryland, College Park, Maryland 20770, United States
’ REFERENCES (1) Woods, G. C.; Simpson, M. J.; Koerner, P. J.; Napoli, A.; Simpson, A. J. HILIC-NMR: Toward the identification of individual molecular components in dissolved organic matter. Environ. Sci. Technol. 2011, 45, 3880–3886. (2) Cory, R.; McKnight, D. Fluorescence spectroscopy reveals ubiquitous presence of oxidized and reduced quinones in dissolved organic matter. Environ. Sci. Technol. 2005, 39, 8142–8149. (3) Ma, J; Del Vecchio, R.; Golanoski, K. S.; Boyle, E. S.; Blough, N. V. Optical properties of humic substances and CDOM: Effects of borohydride reduction. Environ. Sci. Technol. 2010, 44 (14), 5395–5402. (4) Maurer, F.; Christl, I.; Kretzschmar, R. Reduction and reoxidation of humic acid: Influence on spectroscopic properties and proton binding. Environ. Sci. Technol. 2010, 44 (15), 5787–5792. (5) Del Vecchio, R.; Blough, N. V. On the origin of the optical properties of humic substances. Environ. Sci. Technol. 2004, 38, 3885–3891. (6) Boyle, E. S.; Guerriero, N.; Thiallet, A.; Del Vecchio, R.; Blough, N. V. Optical properties of humic substances and CDOM: Relation to structure. Environ. Sci. Technol. 2009, 43, 2262–2268. (7) Stedmon, C. A.; Bro, R. Characterizing dissolved organic matter fluorescence with parallel factor analysis: A tutorial. Limnol. Oceanogr. Methods 2008, 6, 572–579. (8) Murphy, K. R.; Hambly, A.; Singh, S.; Henderson, R. K.; Baker, A.; Stuetz, R.; Khan, S. J. Organic matter fluorescence in municipal water recycling schemes: Toward a unified PARAFAC model. Environ. Sci. Technol. 2011, 45, 2909–2916.
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