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Oversimplification of Dissolved Organic Matter Fluorescence Analysis: Potential Pitfalls of Current Methods Fernando L. Rosario-Ortiz*,† and Julie A. Korak‡ †
Department of Civil, Environmental and Architectural Engineering, University of Colorado, Boulder, Colorado 80309, United States Bureau of Reclamation, U.S. Department of the Interior, Denver, Colorado 80225, United States first approaches includes peak picking, which compares fluorescence intensity in four wavelength regions commonly observed in DOM samples (see Figure 1b): humic substances (Peaks A and C) and so-called protein peaks (tryptophan (Peak T) and tyrosine (Peak B)).3 Quantitative procedures to analyze EEMs include fluorescence regional integration (FRI) and parallel factor (PARAFAC) analysis. The FRI method assigns regions of an EEM to specific DOM fractions: humic acid-like, fulvic acid-like, soluble microbial products and aromatic proteins4 (see Figure 1c). It is important to note that the FRI method defines three regions at excitation wavelengths less than 250 nm, but many commercial fluorometers do not provide correction factors for excitation wavelengths less than 240 nm where lamp output is low, making signals in this area unreliable. PARAFAC decomposes EEMs into n independent components representing groups of fluorophores with similar spectra.5 Given the complexity of DOM fluorescence, these methods are very attractive to scientists and engineers as they offer the opportunity to relate fluorescence behavior to a reduced number of chemical entities. However, caution is recommended when using these methods for the analysis of DOM composition by f luorescence. ver the past 25 years, there has been a significant increase Interpretation of fluorescence data needs to consider that in the use of fluorescence spectroscopy to characterize FDOM is a very small subset of DOM. Based on reported the physicochemical properties of dissolved organic matter fluorescence quantum yields for DOM of diverse origins, less (DOM) in natural and engineered systems. Fluorescence than 3% of the photons absorbed (i.e., CDOM) are emitted as intensity measured across a range of excitation and emission fluorescence.2 Although there are clear trends in behavior wavelengths produce thousands of data points per sample that between FDOM and DOM as a whole, the limitations of are displayed using a three-dimensional contour plot, known as drawing conclusions about an entire system based on f luorescence data alone have to be considered carefully. an excitation−emission matrix (EEM) (see Figure 1a). Therefore, when using fluorescence as a tool to characterize Fluorescence is an attractive analytical approach due to its DOM, it has to be understood that the analysis only applies to ease of use, but the complexity associated with the FDOM and may not be representative of DOM as a whole. interpretation of an EEM is often oversimplified. In many The association of EEM regions with operationally defined cases, the use of EEMs has replaced targeted analytical methods DOM fractions (i.e., humic acids (HA), fulvic acids (FA) and (e.g., fractionations) used to assess the chemical composition of proteins) is an oversimplification that cannot be supported for DOM, at the expense of certainty regarding the chemical aquatic systems. HA and FA are not distinct chemical identity of specific constituents.1 subgroups in the sense that proteins and carbohydrates are The photophysics of DOM are complex. Only a subset of the separate groups with different chemistries. FA and HA are molecules that comprise DOM contain functional groups operational definitions based on their adsorption to XAD resins capable of absorbing light in the ultraviolet−visible wavelengths and solubility at low pH. Although we know that, in general greater than 200 nm (this subset is known as chromophoric terms, FA are smaller and more polar than HA, they can be DOM (CDOM)). In turn, only a subset of CDOM fluoresces seen as a continuum of similar chemical structures, albeit with (FDOM) upon relaxation from an excited singlet state. different degrees of oxidation. Fluorescence analysis of FA and Different models could be invoked to describe the optical HA show that both fractions fluoresce in the same regions properties of DOM, including superposition of individual, (Peaks A and C/Regions III and V) (see Figure 1b and c). noninteracting fluorophores or charge transfer (CT) interactions between electron donor−acceptor complexes.2 To simplify the analysis of DOM EEMs, several qualitative Received: December 4, 2016 and quantitative approaches have been developed. One of the ‡
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© XXXX American Chemical Society
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DOI: 10.1021/acs.est.6b06133 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Environmental Science & Technology
acids (tyrosine and tryptophan) that fluoresce in this region have optically active functional groups (phenols and indoles). These functional groups may be present in environmental systems yet be unrelated to microbial activity (e.g., plantderived polyphenols, phenol-containing mixture of hydrocarbons). Environmental sample context and a consideration of other possible fluorophores should be discussed when interpreting fluorescence in this region. Additional validation of this approach could be done by combining EEM analysis with established methods for the quantification of amino acids in environmental samples. PARAFAC analysis is a powerful tool, but discretion is needed to identify suitable data sets to model and to recognize interpretation limitations. Small data sets and those with little spectral variability often yield models with few (≤3) components, which may be an artifact of the data set and may underrepresent the complexity of the system. Additionally, PARAFAC relies on an underlying assumption that components are independent, which may not reconcile with current theories regarding DOM fluorescence.2 Additional work is needed to better understand the underlying fluorescence mechanisms to strengthen model interpretations. Although there is a well-developed field of inquiry into the use of PARAFAC for DOM analysis, the possibility exists that simplification of DOM fluorescence to a limited number of components may be masking more complicated behavior and biasing the analysis of DOM chemistry. Although a vast amount of knowledge has been gained from FDOM analysis conducted in tandem with other analytical methods, care must be taken in interpreting DOM fluorescence data in isolation. Fluorescence as a characterization method should be applied within the context that only a small fraction of DOM fluoresces. It is also important to consider the statistical variability in some of the metrics that are used for fluorescence analysis.8 Interpretations that relate fluorescence signals to specific DOM fractions not only have contextual limitations, but may not be scientifically acceptable without further analytical validation.
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Figure 1. EEMs of (a) natural organic matter, (b) humic acid, and (c) fulvic acid isolates from the Suwannee River with peak picking (b) and FRI (c) approaches illustrated. FRI Regions I and II labels not shown for clarity, and excitation wavelengths less than 240 nm not analyzed due to range of instrument correction factors from manufacturer.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Fernando L. Rosario-Ortiz: 0000-0002-3311-9089
Furthermore, there are three lines of evidence that suggest that Regions III and V are both more representative of FA than HA. In fractionated aquatic DOM samples, FA are several times more abundant than HA on a carbon mass basis.6 Per unit carbon, FA exhibit higher specific fluorescence intensities in both Regions III and V. Per unit absorbance, FA have higher fluorescence quantum yields than HA in both regions.2 Interpreting fluorescence signals as representative of specifically the HA fraction cannot be substantiated for most aquatic systems based on the lower abundance and lower fluorescence efficiency of HA. Therefore, assigning EEM regions to HA or FA is an oversimplification, because HA and FA fluorescence signals overlap and are dominated by FA (in both abundance and intensity) for aquatic systems. Analysts interested in the quantification of these fractions using fluorescence should validate the approach using the classical techniques for DOM fractionation.7 In a similar sense, fluorescence in the so-called protein region may not indicate the presence of amino acids. The two amino
Notes
The views, analysis, recommendations, and conclusions in this report are those of the authors and do not represent official or unofficial policies or opinions of the United States Government and the United States takes no position with regard to any findings, conclusions, or recommendations made. The authors declare no competing financial interest.
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REFERENCES
(1) Leenheer, J. A. Systematic approaches to comprehensive analyses of natural organic matter. Annals Enviro. Sci. 2009, 3, 1−130. (2) Del Vecchio, R.; Blough, N. V. On the origin of the optical properties of humic substances. Environ. Sci. Technol. 2004, 38 (14), 3885−3891. (3) Coble, P. G. Characterization of marine and terrestrial DOM in seawater using excitation-emission matrix spectroscopy. Mar. Chem. 1996, 51 (4), 325−346. (4) Chen, W.; Westerhoff, P.; Leenheer, J. A.; Booksh, K. Fluorescence excitation - Emission matrix regional integration to
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DOI: 10.1021/acs.est.6b06133 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.est.6b06133 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
