Shape, humic fractions

Any factor that affects the size or shape of a molecule, the hindered movement of a fluorophore within a molecule, or the energy transfer within the molecule will affect the measured depolarization of its fluorescence emission. Therefore, the conformation of humic fractions in solution can be studied as a function of pH, ionic strength, temperature, and other factors by depolarization measurements. The principle of the method is that excitation of fluorescent samples with polarized light stimulates... 

Spectra of fulvic acids must be interpreted carefully purification of this fraction leads to losses that can reach 50%. Comparison of spectra of different humic fractions of the same sample, such as a sediment from the Oman Sea (Fig. 6) illustrates several differences. Oxygenated functional groups are more important in fulvic and humic acids than in stable residues. Particularly important are the absorption bands at 3400 cm (OH from alcohols, acids, etc.), 1710 cm (C==0 from quinones, ketones, carboxylic acids), 1250 cm (C—O from alcohols, esters, ethers) and 1050 cm (C—O from carbohydrates). Absorption at 1050 cm is nearly absent in stable residues. Aliphatic content increases from fulvic acids to humic acids and stable residues (bands between 2870 and 2960 cm ) and the shape of the aliphatic bands (2900-2950 1450 1375) indicates that fulvic acids contain mainly CH groups. 

This chapter presents new information about the physical properties of humic acid fractions from the Okefenokee Swamp, Georgia. Specialized techniques of fluorescence depolarization spectroscopy and phase-shift fluorometry allow the nondestructive determination of molar volume and shape in aqueous solutions. The techniques also provide sufficient data to make a reliable estimate of the number of different fluorophores in the molecule their respective excitation and emission spectra, and their phase-resolved emission spectra. These measurements are possible even in instances where two fluorophores have nearly identical emission specta. The general theoretical background of each method is presented first, followed by the specific results of our measurements. Parts of the theoretical treatment of depolarization and phase-shift fluorometry given here are more fully expanded upon in (5,9-ll). Recent work and reviews of these techniques are given by Warner and McGown (72). 

Humic substances account for 40-70% of the DOC in rivers and 5-25% of the DOC in the ocean (Table I). It is important to note that recoveries of adsorbed humic substances from XAD resins are not quantitative, so the chemical characteristics of the recovered humic substances are not necessarily representative of all the humic substances retained by the resin. Tangential-flow ultrafiltration retains 45-80% of the DOC in rivers and 25-40% of the DOC in the surface ocean (Table I). Essentially all of the DOC retained during ultrafiltration is recovered for chemical characterization. In general, ultrafiltration recovers a larger fraction of the DOM from these systems. These methods also isolate DOM based on different mechanisms. Adsorption onto XAD resins at low pH chemically fractionates the DOM and isolates the more hydrophobic components, whereas ultrafiltration principally separates components of DOM on the basis of size and shape. 

Calvin, M., and Philip, R. P. (1976). Possible origin for insoluble organic (kerogen) debris in sediments from insoluble cell-wall material of algae and bacteria. Nature 232,134-136. Cameron, R. S., Thornton, B. K., Swift, R. S., and Posner, A. M. (1972). Molecular weight and shape of humic acid from sedimentation and diffusion measurements on fractionated extracts. /. Soil Sci. 23,394 108. 

The particle size distribution for the humic acid fraction is depicted in Figure 4. No material sedimented out until the most extreme conditions were applied (40,000 rpm for 24 hr), when some lightening of color at the top of the solution was observed. The sedimented particles had a Stokesian diameter of around 2 nm, which means that a particle size gap of three orders of magnitude exists between these and the next largest particles detected (5 xm). From the experimentally determined coal particle density of 1.43 g/cm, it was calculated that a solid sphere of diameter 2 nm would have a molecular mass of 4000. If the molecules were rod-shaped, even smaller molecular masses would be predicted. Literature values of the molecular mass of regenerated humic acids range between 800 and 20,000, with the values clustering around 1,000 and 10,000 (i5, 16, 17). 

The results show that, depending on fractions, several observations can be drawn. The different responses are related to the diffuse absorption response of solids and colloids. The general tendency is that the slope of the spectrum generally decreases as the particle size increases. Spectra corresponding to suspended solids are flat, confirming a mineral nature. Adsorption phenomenon can be seen mainly on fine colloids, and the shape of soluble fractions (artificially denitrified with the deconvolution method see Chapter 2) is related to the probable presence of humic-like substances (see Chapter 6). 

Answers to these questions have been developed slowly in comparison to rapid advances in our knowledge of air particulates based on studies completed in the late 1960 s. This is partly a result of superior sampling devices for size and chemical characterization of air particulates, and partly a result of the more heterogeneous nature of water particulates. The size spectrum of particulates in water extends from colloidal humic substances 1 nm in size, to large aggregates such as fecal pellets or marine snow with sizes up to 10" m. The distribution of shapes, densities, surface chemical properties, and chemical composition may vary widely with size. Some fractions of the size spectrum may be living, and all particulates are subject to diverse physical-chemical and biological processes that can alter size distributions, shape, or chemical composition. 

A variety of spectroscopic techniques have been applied to DOC isolated from seawater by cross-flow ultrafiltration or adsorption onto XAD resins. The two techniques isolate very different organic fractions from seawater. Hydrophobic fractions (such as marine humic material) are isolated on XAD resins [48], whereas the organic matter extracted by ultrafiltration is retained primarily on the basis of its molecular size and shape [49], resulting in isolates rich in nitrogen and carbohydrates (polysaccharides). Nuclear magnetic resonance (NMR) spectroscopy has proven successful in distinguishing between the specific structures of XAD-bound humics and the carbohydrates concentrated into colloidal size fractions. 

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