Solvent fractionation humic substances

Recall from Chapter 23.2.4 that humic substances are isolated from seawater by adsorption on a hydrophobic resin followed by elution using solvents of varying pH. The desorbed compounds are fractionated into two classes, humic acids fulvic acids based on their solubility behavior. A model structure for a humic acid is illustrated in Figure 23.10a in which fragments of biomolecules, such as sugars, oligosaccharides. 

Figure 1 gives a typical infrared spectrum of lake humic acid. The interpretation of infrared spectra of humic substances is discussed in depth by -MacCarthy and Rice in Chapter 21. The similarity of infrared spectra of humic acids from different lakes suggests a similarity of the aspects of chemical structure that are related to their infrared absorptions. However, infrared spectroscopy is not sensitive enough to uncover minor structural differences among humic acids. In fact, humic acids were separated by organic solvents (chloroform, methylethylketone, methanol, dimethylformamide) into various fractions (Ishiwatari, 1969b, 1973). Infrared spectra of two of these fractions, the chloroform-soluble fraction and the methylethylketone-... 

Application of solubility parameter data is hindered by the multicomponent nature of humic substances, and homogeneous fractions are needed to obtain the necessary data for the macromolecules. Nevertheless, the available information shows that the best solvents for humic acids have polar,... 

In the experiments of Hayes et al. (1975) DMSO was marginally better than DMF or sulfolane for dissolving humic substances (Table 4). In the ESR there was evidence of a higher free radical concentration in DMSO than in either DMF or sulfolane. Because DMSO would not be expected to generate free radicals, it is reasonable to infer from the ESR data that humic components, which are insoluble in the DMF- and sulfolane-water systems, were dissolved in this solvent. Elemental contents were similar for the humic and fulvic acids of the DMSO extracts, and these data infer that the major difference between the two fractions was one of molecular size. However, some fulvic acid materials were observed to precipitate during dialysis, as was noted for the DMF and sulfolane systems. 

In a further set of experiments Hayes et al. (1975) exhaustively extracted an H -exchanged sapric histosol with water, and then exhaustively with the series DMF-, sulfolane-, DMSO-, pyridine-, and EDA-water, 1 1 (v/v) mixtures in that order. The cumulative amounts of humic substances extracted for any succession of solvents were the same as the amounts extracted by the last solvent in the series without the aid of the others. This would suggest that the materials dissolved in the less efficient solvents were dissolved also in the more efficient members of the series. The analytical data for the different fractions closely resemble those in Table 5 for the substances extracted by the single solvent systems. 

Determination of the Flory-Huggins interaction parameter (x) for solvent-polymer pairs again requires careful fractionation of the humic macromolecules. In the view of this author, much can be learned about the ways in which humic substances are associated through determination of Flory-Huggins and solubility parameters of carefully fractionated humic substances, and through applications of empirical equations such as those of Flory-Huggins [Equation (6)] and Hildebrand-Scatchard [Equation (7)]. 

Organic solvents have long been used for extraction and sequential extraction, which is fractionation of a sort (Flaig et al., 1975 Schnitzer, 1978). While the direct use of organic solvents in fractionation has not been widespread, nonetheless, the technique has received some attention. For instance, the separation of hymatomelanic acid from precipitated humic acid is obtained by extraction with ethanol (Oden, 1919). Ethanol has been used to bring about fractional precipitation by addition to alkaline solutions of humic acid (Kyuma, 1964 Kumada and Kawamura, 1968). There is no reason why other water-miscible solvents such as acetone and methanol should not be used in this way. Solvents that are highly immiscible with water (e.g., hexane and benzene) do not appear to remove any substantial fraction of humic substances. These are perhaps best used to remove nonhumic substances (such as fats and waxes) prior to extraction. However, recent work by Allen and MacCarthy (personal communication) has shown that more polar water-immiscible solvents, such as methyl isobutyl ketone and diethyl ether, can be used successfully to purify and fractionate humic substances. 

Any fractionation obtained by the use of organic solvents is again likely to be rather crude and the method is not likely to find great favor as a preparative technique. Nonetheless, solubility in nonaqueous solvents can be a very useful adjunct to the chemists armory when physicochemical measurements (such as molecular weight and viscosity) need to be made. In this context, Ifiwer-molecular-weight, hydrogen-ion saturated humic substances are most likely to be useful. 

Fractionation of aquatic humic substances by adsorptive interactions has been the most successful method for fractionation as well as concentration and isolation. Humic solutes readily interact with various adsorptive surfaces without the requirement of crossing the interface surface as is necessary with solvent partitioning or absorptive interactions. 

Little has been published in this area in relation to humic substances. Humates tend to have retention times close to, or before, the solvent front in most reverse-phase columns. In one report (Rodgers et al., 1981), a fulvic material was separated into seven fractions on a silanized BioSil column. The fractions were analyzed by infrared spectroscopy. One of the fractions was patently not a fulvic acid, although it co-precipitated with fulvic acid. 

The reported spin concentrations correspond to one free radical per 1100 molecules (number average molecular weight of 951) for an untreated fulvic acid in the work of Schnitzer and Skinner (1969) and one free radical per 250 molecules of humic acid (1.4 x 10 spins/g, with a molecular weight of 20,000) in the work of Steelink (1964). Consequently, the ESR spectrum is providing data on only a small fraction of the total molecules in the humic mixture, as pointed out by Riffaldi and Schnitzer (1972). Hayes et al. (1975) found that the free radical contents of humic and fulvic acids vary with the solvent used in the extraction, suggesting that the free radical in a humic substance may be an artifact of the extractive procedure. With the severity of these limitations in mind, any generalization concerning the functionality or structural nature of humic substances based on ESR data must be conservative. 

Humins - the fraction of humic substances that is not soluble in water at any pH value. On this basis, humins include any humic-type material that is dissolved in non-aqueous solvents after the soil has been exhaustively extracted with basic aqueous solvents. Humins are often considered to consist mainly of humic materials strongly associated with the soil inorganic colloids. 

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