Grantee Research Project Results
Final Report: Spectral Characterization of Humic Substances
EPA Grant Number: R822251Title: Spectral Characterization of Humic Substances
Investigators: McGown, Linda B.
Institution: Duke University
EPA Project Officer: Packard, Benjamin H
Project Period: October 1, 1995 through September 30, 1998
Project Amount: $332,309
RFA: Exploratory Research - Chemistry and Physics of Water (1995) RFA Text | Recipients Lists
Research Category: Water , Land and Waste Management , Safer Chemicals
Objective:
The purpose of this project was to obtain new, fundamental information about humic substances and their binding interactions with organic compounds and metals that will increase our ability to evaluate and predict their role in environmental processes. Specific goals included the following:1. Identification of photophysical properties of humic substances that are good for classifying environmental water samples and discriminating among them, to serve as the basis for predictive modeling.
2. Elucidation of binding interactions of humic substances with metals and
the effects of metals on the fluorescence properties of humic
substances.
Summary/Accomplishments (Outputs/Outcomes):
Fluorescence Spectral and Lifetime Characterization of Humic Substances. Humic substances are large, heterogeneous molecules formed by the decay of plant and animal biomass in aquatic and terrestrial systems. They are comprised of high-molecular-weight organic compounds, both aromatic and aliphatic, that are rich in oxygen-containing functional groups (e.g., COOH, phenolic and/or enolic OH, alcoholic OH, and quinolic C=O). They account for approximately 60-70 percent of the total organic carbon in soils and 40-60 percent of dissolved organic carbon in natural waters.Humic substances can be operationally defined according to their solubility in water: humic acids (HAs) are insoluble in aqueous solution with pH < 2 but soluble at higher pH values, fulvic acids (FAs) are soluble in water under all pH conditions, and humins are insoluble in water at any pH. In spite of having many common functional groups and structural features, humic and fulvic acids differ significantly. FAs have lower molecular weights (500 - 2000 daltons) and a smaller number of total and aromatic carbons than their HA counterparts (2000 - 5000 daltons) which in turn have longer-chain fatty-acid products and therefore a higher hydrophobicity than FAs.
Chemical and physical studies of humic substances and their interactions with
environmental contaminants in the naturally occurring humic matrix are essential
in order to elucidate their impact on the environmental fate, bioavailability
and toxicity of organic and metal contaminants. Elucidation of the important
role of humic substances in environmental contamination and remediation is
complicated by their highly heterogeneous nature. The vast diversity in
structural components of humic substances is a result of the random
polymerization of a variety of decomposed materials. Moreover, the composition
of humic substances varies according to their
source and method of
extraction. It is this structural heterogeneity that has made the structural and
conformational characterization of humic substances extremely challenging.
The sensitivity and non-destructive nature of fluorescence techniques are ideally suited to studies of the intact humic matrix. The intrinsic fluorescence of humic substances contains information relating to structure, conformation, and heterogeneity of humic substances, as well as dynamical properties related to their intramolecular and intermolecular interactions. Several studies have investigated the intrinsic fluorescence spectral properties of humic substances in order to obtain information relating to their molecular structure and conformation, as well as dynamic interactions in the humic matrix. Excitation and emission spectra have provided limited discrimination among various humic fractions, identifying only broad classes of humic and fulvic acids. Synchronous fluorescence spectroscopy has been used in attempts to resolve the broad, significantly overlapping peaks in the excitation and emission spectra. Total luminescence spectroscopy, which provides a complete representation of the fluorescence spectral features of a sample in the form of an excitation-emission matrix (EEM), has been applied to humic substances as well. In the EEM, fluorescence intensity is presented as a function of excitation wavelength on one axis and emission wavelength on the other.
In this project, we have investigated a new approach to the characterization of humic substances that combines fluorescence lifetime and total luminescence spectral information using Phase-Resolved Fluorescence Spectroscopy (PRFS). PRFS is a frequency-domain fluorescence lifetime technique that incorporates both spectral and lifetime information in a single, time-independent measurement of phase-resolved fluorescence intensity (PRFI). Through manipulation of the lifetime dimension in PRFS, the spectral contributions of various fluorescent components can be selectively suppressed or enhanced. In HA research, PRFS has been used to suppress the spectral contributions of particular (unidentified) lifetime components, thereby resolving the broad peak that dominated the steady-state emission spectrum into two distinct peaks in the phase-resolved emission spectrum.
We first applied steady-state total luminescence spectroscopy to the fluorescence characterization of humic substances obtained from the International Humic Substances Society (IHSS). Results show that total luminescence spectra, represented as excitation-emission matrices (EEMs), may be used to discriminate between soil-derived and aquatic-derived IHSS humic substances and between humic and fulvic acids derived from the same source (soil or aquatic). Ionic strength in the range of 0-1 M KCl and humic substance concentration in the range of 5-100 mg/l had little effect on the fluorescence spectral characteristics of the humic substances, while pH had significant effects as expected. Absorbance correction was shown to be essential for accurate representation and comparison of the EEMs of the humic substances at high concentrations.
Direct measurement of fluorescence lifetimes of humic substances recovered three ranges of lifetime components in the humic substances, < 1 ns, 2-5 ns, and 8-14 ns, which are consistent with previously reported lifetimes. All of the IHSS samples studied were found to exhibit similar fluorescence decay that could be modeled by three or four decay components. In general, the lifetime components are slightly longer for the FAs than for the HAs from the same source. The decays are relatively independent of ionic strength and humic substance concentration. pH shows some effect, primarily in the weighting of the contributions from the different lifetime components to the total decay.
In the next phase of the research, we introduced fluorescence lifetime into the EEM through PRFS to yield a lifetime-spectral representation of fluorescence that we refer to as a phase-resolved excitation emission matrix, or PREEM. We showed that the PREEM provides a unique visual representation of the intrinsic fluorescence properties of HAs under a variety of solution conditions. The calculation of spectral peak ratios in PREEMs as well as steady state EEMs was shown to be a convenient means for quantitating differences between the spectra with good precision. Absorbance correction was found to be essential for accurate comparison among spectral features of different samples. Increased detail is available from PREEMs at various modulation frequencies, which reveal the distribution of fluorescence lifetime contributions across the spectral surface. PREEMs, which provide a concise "survey" of how the lifetimes change across the spectrum, may aid in pinpointing spectral regions that provide the best lifetime discrimination among samples.
Metal Binding to Humic Substances. Humic substances often form complexes with metals through carboxylic and phenolic groups with a wide range of pKa's. These complexation reactions are an important determinant of the environmental fate of trace and heavy metals. There are a variety of sites of different binding strengths in humic substances and the complexation isotherms can be explained by both continuum and multiple site binding models.
The intrinsic fluorescence of humic substances provides a powerful means for studying their interactions with metals. Metal complexation can alter the fluorescence of humic substances, resulting in either quenching or enhancement depending on the metal cation. Advantages of using fluorescence to study complex samples such as humic substances include low detection limits and minimal sample perturbation and preparation. However, a potential disadvantage is that the fluorescence signal may not represent the entire sample. Indeed, the low fluorescence quantum yield of humic substances has led to an estimate that only one percent of the molecules is fluorescent.
Studies have shown that the optical properties of humic substances depend upon molecular weight and that the fluorescence in different spectral regions is associated with different types of ligand. Therefore, results of fluorescence studies will depend upon the choice of excitation and emission wavelengths. In one study, gel permeation chromatography showed that the highest molecular weight fraction of an aquatic humic substance was not fluorescent at the wavelengths chosen for the study, and that this fraction was at least partially composed of aggregates of smaller molecules. Low molecular weight fractions are more fluorescent than high molecular weight fractions, raising the possibility of self-quenching in the latter. Dynamic quenching mechanisms have been proposed for humic substance-bound pyrene and may be similar to self-quenching mechanisms in large humic aggregates.
In this project, we investigated the relationship between fluorescence spectra and aggregation processes of HAs. Specifically, steady-state fluorescence anisotropy was used to study whether cation induced aggregates of HAs are fluorescent; if they are, then the anisotropy should detect a change in particle size upon aggregation. Previous investigations by other researchers used steady-state and time-resolved anisotropy in attempts to monitor conformational changes in solutions of FAs as a function of concentration and pH. The results were contradictory.
We chose to use Al, which is a well known and powerful coagulant of humic substances and is known to enhance the fluorescence of HAs, indicating that ligands associated with the metal are fluorescent. To increase the rate of aggregation we employed a heating step in the reaction between the HAs and Al. Production of stable aggregates (over 48 h) was verified by light scattering. Thus, our solutions contained both aggregates and metal-associated fluorophores. The anisotropy data was analyzed using a model for non-spherical particles to determine whether these aggregates contribute to the fluorescence signal. Our results show that in solutions of HAs, Al induced aggregates are fluorescent.
Aluminum induced aggregates of terrestrial and aquatic HA standards from the IHSS were shown to be fluorescent by means of a multi-wavelength fluorescence anisotropy experiment in which the data was treated with a model for non-spherical particles. While aggregates of aquatic HAs appeared in the fluorescence signal at both short and long excitation wavelengths, aggregates of terrestrial HAs were detected only at the long wavelength. Furthermore, the results indicate that emission obtained at longer excitation wavelengths was representative of smaller particles. At pH 4, the aquatic HAs appeared to exist in an extended conformation whereas the terrestrial HAs showed less extension.
The size and shape of the fluorescent particles display a complex dependence on Al concentration. Both enhancement and quenching of fluorescence were observed in the Total Luminescence Spectra upon Al addition. However, quenching was shown to be the result of decreased HA concentration due to precipitation by Al rather than photophysical processes.
We conclude from this work that Al induced aggregates of HA do fluoresce. Decreased intensity in the fluorescence spectra upon Al addition may be due to HA self-quenching in larger aggregates but is at least partially due to loss of material to precipitate. The lack of detection of fluorescent aggregates at 325/420 nm in the terrestrial HA samples could be the result of coiling processes or quenching in aggregates, but there is no way to distinguish between the two from these experiments. Finally, the differences and trends in particle size at 325/420 nm and 457/500 nm indicate that different ligand types are observed in different spectral regions and highlights the need to investigate multiple wavelengths when using fluorescence to study humic substances.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
Other project views: | All 16 publications | 4 publications in selected types | All 4 journal articles |
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Gjessing ET, Alberts JJ, Bruchet A, Egeberg PK, Lydersen E, McGown LB, Mobed JJ, Munster U, Pempkowiak J, Perdue M, Ratnawerra H, Rybacki D, Takacs M, Abbt-Braun G. Multi-method characterisation of natural organic matter isolated from water: characterisation of reverse osmosis-isolates from water of two semi-identical dystrophic lakes basins in Norway. Water Research 1998;32(10):3108-3124. |
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Hemmingsen SL, McGown LB. Phase-resolved fluorescence spectral and lifetime characterization of commercial humic substances. Applied Spectroscopy 1997;51(7):921-929. |
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Mobed JJ, Hemmingsen SL, Autry JL, McGown LB. Fluorescence characterization of IHSS humic substances: total luminescence spectra with absorbance correction. Environmental Science & Technology 1996;30(10):3061-3065. |
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Sharpless CM, McGown LB. Effects of aluminum-induced aggregation on the fluorescence of humic substances. Environmental Science & Technology 1999;33(18):3264-3270. |
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Supplemental Keywords:
water, groundwater, humic acid, environmental chemistry, modeling, fluorescence., Scientific Discipline, Water, Hydrology, Physics, Environmental Chemistry, Chemistry, Engineering, Engineering, Chemistry, & Physics, humic substances, spectroscopic studies, metal speciation, organic compounds, spectroscopy, molecular probe techniques, groundwater contamination, surfactants, water qualityProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.