Humic materials fluorescence

In this chapter, we present the theory and results of measurements on humic acid fractions using fluorescence techniques. The fluorescence techniques are attractive for this application because of the natural fluorescence of humic materials, the hi sensitivity of fluorescence detection, and the ability to directly observe the morphology of the molecule in aqueous solutions without the need for drying or applying harsh chemical conditions. Several interesting types of information are obtained from fluorescence measurements ... 

Gauthier, T.D., Shane, E.C., Guerin, W.F., Seitz, W.R., Grant, C.L. (1986) Fluorescence quenching method for determining equilibrium constants for polycyclic aromatic hydrocarbons binding to dissolved humic materials. Environ. Sci. Technol. 20(11), 1162-1166. 

Puchalski, M. M., Morra, M. J. von Wandruszka, R. (1992). Fluorescence quenching of synthetic organic compounds by humic materials. Environmental Science Technology, 26, 1787-92. 

Although the problem of identifying the molecular components responsible for the fluorescence of HA and FA is a complex task, some hypotheses can be suggested concerning the possible chemical nature of the fluorescent structure in several humic materials already examined. 

Grimm, D.M., Azarraga, L.V, Carreira, L.A. and Susetyo, W. (1991) Continuous multiligand distribution model used to predict the stability constant of Cu(II) metal com-plexation with humic material from fluorescence quenching data. Environ. Sci. Technol, 25,1427-1431. 

Finally, a variety of zooplankton have recently been shown to produce colored (chromophoric) DOM (CDOM), a fraction of which is fluorescent (FDOM) (Steinberg et al, 2004 Urban-Rich et al, 2004, 2006). Although the nitrogenous composition of CDOM/FDOM is little known, fluorescent humic-hke material (likely fulvic and humic acids) is produced during grazing, influencing the optical characteristics of coastal waters (Urban-Rich et al, 2006). A recent culture study shows that humic-N can be a source of N for coastal phytoplankton (See et al, 2006). 

Total protein assays have the advantage of being relatively straightforward compared to molecular-level analyses. Methods with fluorescence-based detection are also highly sensitive, and thus amenable direcdy to DON. Quantitative interpretation for environmental mixtures such as seawater, however, may be problematic for some samples. Most methods react with specific moieties (e.g., coomassie blue binds to lysine and arginine) and thus results obtained can depend on protein composition, size distribution, and even conformation (Sapan et ai, 1999), making the careful choice of calibration standards important. In addition, common components of natural samples, such as humic materials (e.g., Mayer et ai, 1986), carbohydrates (Sapan et ai, 1999), or NH3 may interfere with quantification. Overall, colorimetric methods can be very useful as quick, Hkely semi-quantitative estimates of total protein or peptide. However, potential biases inherent in the mechanism of a specific method should be considered before one is chosen, and appHcation of newer molecular assays (e.g., CBQCA) should be carefully examined in terms of natural sample matrix (Nunn et ai, 2003). 

Ventry, L. S. Metal Ion Binding by Humic Materials An Integrated High Performance Liquid Chromatography and Fluorescence Quenching Study. 1989, Northeastern University. PhD. 

Molecular fluorescence has been shown to be a valuable tool for studying the binding of certain metal ions to both isolated humic materials (1-4) and natural organic matter present in samples collected from lakes, rivers and other bodies of water (5,6). Paramagnetic metal ions tend to reduce or quench fluorescence which has been demonstrated for Cu " (1-5), Co " (4,6), Mn " (4), Ni (1), Fe " and Fe " (7) with various samples of humic materials. Diamagnetic metal ions, on the other hand, may quench, show no effect or even enhance humic material fluorescence depending on the metal, the source of the humic material and other experimental factors (1,8-13)... 

According to the initial results reported here, a fluorescence enhancement effect for humic material binding with aluminum can be observed, while under different conditions a quenching of fluorescence predominated. Another very interesting fluorescence phenomenon described here is that after complexation with AE, the maximum intensity of the humic material fluorescence shifts to longer excitation wavelengths and to shorter emission wavelengths in an excitation emission matrix (EEM). 

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. 

Larson, R. A. and Rockwell, A. L. (1980). Fluorescence spectra of water-soluble humic materials and some potential precursors. Arch. Hydrobiol. 39, 416-425. 

Visser, S. A. (1983b). Fluorescence phenomena of humic matter of aquatic origin and microbial cultures. In Aquatic and Terrestrial Humic Materials (R. F. Christman and E. T. Gjessing, eds.), Ann Arbor Science, Ann Arbor, MI, 183-202. 

Table 1. Fluorescence lifetimes of humic materials and natural waters...

The fluorescence spectra of many freshwater humic materials have been found to be surprisingly similar to one another (Larson and Rockwell, 1980 Table 1.10). The fluorescence does not resemble that of other naturally occurring polymers, such as lignin, nor does it show the mirror-image absorption/fluorescence relationship characteristic of pure compounds. This suggests that the active fluorophores contribute only a small fraction of the light absorption (Visser, 1983). The quantum efficiency... 

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