Fluorescence of humic substances

Antunes, M.C.G. and da Silva, J.C.G., Multivariate curve resolution analysis excitation-emission matrices of fluorescence of humic substances. Anal. Chim. Acta, 546, 52, 2005. 

Figure 14 shows the improvement in Raman spectra via near-infrared excitation due to the elimination of fluorescence of humic substances. The top spectrum was taken with 514.5 -nm excitation, and the Raman signal is completely obscured by fluorescence. However, the bottom spectrum that was taken with 1064-nm excitation with an FT-Raman system provided two clearly observable Raman bands. The peak in the 1300-cm range (referred to as the D band) proves to be extremely valuable because it indicates the amount of disorder of carbon networks. Because of the presence of this band and the absence of second-order Raman bands of humic substances (2000-3000-cm range), Yang and Wang [99] have concluded that the backbones of humic substances are structurally disordered carbons (i.e., graphitelike) for most cases. 

Oxidized quinones are relatively poor fluorophores however, the reduced forms, hyd-roquinones, are highly fluorescent, a property that has been utilized in developing photochemical-reaction fluorescence detectors for liquid chromatography (Poulson and Bitks, 1989). Klapper et al. (2002) used the differences in fluorescence properties between oxidized and reduced samples of humic substances isolated from a range of environments to infer the presence of quinone-like moieties. These authors proposed that quinone moieties contribute significantly to the fluorescence of humic substances and that fluorescence analyses could be used to assess the redox status of humic substances. 

Sharpless, C.M. and McGown, L.B. (1999). Effects of aluminum-induced aggregation on the fluorescence of humic substances. Environ. Sci. TechnoL, 33(18), 3264-3270. 

Lead, J. R., Wilkinson, K. J., Starchev, K., Canonica, S. and Buffle, J. (2000). Diffusion coefficients of humic substances as determined by fluorescence correlation spectroscopy role of solution conditions, Environ. Sci. Technol., 34, 1365-1369. 

Kenworthy, I. P., and Hayes, M. H. B. (1997). Investigations of some structural properties of humic substances by fluorescence quenching, In Humic Substances, Peats, and Sludges. Health and Environmental Aspects, Hayes, M. H. B., and Wilson, W. S., eds., The Royal Society of Chemistry, Cambridge, pp. 39 15. 

Fuentes, M., Gonzalez-Gaitano, G., and Garcfa-Mina, J. M. (2006). The usefulness of UV-visible and fluorescence spectroscopies to study the chemical nature of humic substances from soils and composts. Org. Geochem. 37,1949-1959. 

Figure 8.3. Fluorescence micrographs of Pinus sylvestris roots at 1mm (top) and 2 mm (bottom) behind the root tip after a treatment with LMS (low-molecular-size humic substances). Note the much smaller rate differentiation of the roots treated with LMS than the roots treated with HMS (high-molecular-size humic substances). Reprinted from Nardi, S., Pizzeghello, D., Remiero, F., and Rascio, N. (2000). Chemical and biochemical properties of humic substances isolated from forest soils and plant growth. Soil Sci. Soc. Am. J. 64,639-645, with permission from the Soil Science Society of America.

Structural and Interaction Studies of Humic Substances. Changes in structural aspects and interactions with other compounds usually affect fluorescence... 

Senesi, N., Miano, T. M., Provenzano, M. R., and Brunetti, G. (1991b). Characterization, differentiation, and classification of humic substances by fluorescence spectroscopy. Soil Sci. 152(4), 259-271. 

Norden, M. and Dabek-Zlotorzynska, E. (1997) Characterization of humic substances using capillary electrophoresis with photodiode array and laser-induced fluorescence detection. Electrophoresis, 18, 292-299. 

Burdon542 has surveyed the current hypotheses for the structure of humic substances and has concluded that the various products from chemical degradations and NMR data are all consistent with their being mixtures of plant and microbial materials and their microbial degradation products. The examination of soil carbohydrates, proteins, lipids, and aromatics supported this view the presence of colour, fluorescence, ESR signals, mellitic acid, and other features do not contradict it. Regarding the Maillard reaction, some free monosaccharides and the necessary amino species are present in soil, so it may proceed, but only to a small extent it is not a major process. However, in marine environments, the relative abundance of carbohydrates and proteins makes them more probable precursors of humic substances than lignin or polyphenols. 

Binding studies between humic substances and polluants have also been performed by following the variation in fluorescence lifetime for the polluant as a function of increased concentration of humic substances. Binding means static quenching, and so no variation in the fluorescence lifetime should be observed (Figure 10.13). 

Size exclusion chromatography (SEC) has been used to measure molecular weight (MW) distribution of humic substances (3, 6-9). Coupled with detection methods such as molecular fluorescence spectroscopy and dissolved organic carbon analysis (7), electrochemical detection (9), and atomic emission spectroscopy (5), SEC has been used extensively to study humic-metal complexes. A major disadvantage of SEC is that it does not provide adequate resolution for separating humic materials as they do not appear to be made up of distinct fractions with large differences in MW. 

The potential role of humic substances and colloids on the fate of radiotoxic pollutants should be evaluated in particular around a nuclear waste repository. The different processes involving these entities such as complexation or sorption can strongly affect the behavior of radionuclides. In particular, results on the complexation of actinides with humic substances, investigated by laser induced fluorescence and spectrophotometry will be presented as well as data on the retention of colloids in the presence or not of heavy elements on mineral surfaces measured by Rutherford Backscattering spectrometry. From these studies, the impact of colloids and humic substances on radiotoxic element behavior will be discussed in terms of confinement or enhancement of migration in the geological media. 

Aqueous organic carbon analyzers helped revolutionize humic studies during the last decade. Not only could organic carbon measurements be made quickly and accurately on any water sample, but organic carbon analysis, used as a monitoring detector like conductivity and fluorescence, opened a new dimension in experimental design and approaches. In conjunction with other methods, the amount and distribution of humic substances in water in relation to other carbon-containing compounds could be determined. 

Many workers have observed fluorescent areas or fractions during electrophoresis experiments (e.g., Waldron and Mortensen, 1961), and this behavior is usually associated with the more mobile materials. It is not clear whether the fluorescence is due to the presence of closely associated non-humic components, or is an innate characteristic of particular fractions of humic substances. It is possible that all fractions do in fact fluoresce, but that this fluorescence is masked by the intense light absorption of the gray-brown components in fractions which do not emit measurable fluorescence. 

As with UV-visible absorption spectroscopy, the fluorescence spectra of humic substances represent the summation of the signals from many different fluorescing molecules, and it is not possible, at present, to provide any detailed interpretation of these spectra. Functionality has been inferred from... 

The ill-defined nature of the UV-visible and fluorescence spectra of humic substances mitigates against their use for providing detailed information concerning the functionality of humic substances. No direct information relating to the functionality of humic substances can be obtained from these methods and only inferential information, based, for exampb, on the variation of the spectra as a function of pH, can be acquired. 

Ghosh, K. and Schnitzer, M. (1980a). Fluorescence excitation spectra of humic substances. Can. J. Soil Sci. 60, 373-379. 

---