ABSTRACT
Extraction with an oxygen-free NaOH solution is a common method for releasing organic matter from soil [1]. The release occurs through the hydrolytic action of the alkaline extractant, which results in the disruption of linkages between humus constituents and inorganic soil components. However, the recovery of humic material is incomplete; only humic and fulvic acids can be removed (in the form of water-soluble sodium salts), whereas the alkali-insoluble humin fraction is left together with soil minerals. The isolation of unextractable humin materials from soil was attempted by
digesting the mineral component with a mixture of concentrated HF and HC1
[2,3]. In this procedure, however, humin materials were subject to alterations. Rice and MacCarthy [4] and Xie et al. [5] proposed a milder procedure wherein humin was separated from the alkali-extracted soil after resuspension in a NaOH-water solution by liquid-liquid partitioning between the aqueous phase and meth-ylisobutylketone (MIBK). By this approach, the following four organic fractions were collected: a solvent-extractable lipid fraction (bitumen), a humic acid-like fraction (bound humic acid), an unextractable lipid fraction (bound lipids), and a mineral component (the insoluble residue). It is not clear, however, how much of the total humin could be extracted in each fraction by this method.The recovery of soil organic matter by supercritical fluid extraction was first reported by Spiteller and Ashauer [6]. Supercritical fluids have the ability to penetrate throughout complex organic materials and solubilize specific components that are otherwise insoluble in the respective solvents. Depending on the polarity of the supercritical fluids, Schnitzer et al. [7] and Schnitzer and Preston [8] observed preferential extraction of either aliphatic compounds and carbohydrates or components with increased aromaticity. The most effective supercritical extractant, composed of acetone and H20 (40:60 v/v), released 50% (wt/wt) of soil organic matter; other supercritical solvents, such as n-pentane or aqueous ethanol, removed between 4 and 34% of organic material that consisted mainly of alkanes or carbohydrates [8].Chemical derivatization has been widely used for the quantification of common polar substituents, such as carboxyl, amino, hydroxyl, and sulfhydryl groups, present in the aliphatic or aromatic constituents of humus. Because of their high oxygen content (between 30 and 50%), humic substances are very polar and, thus, insoluble in aprotic organic solvents. They may be solubilized, however, through derivatization of the polar groups with lipophilic reagents. Diazomethane, for instance, was commonly used for derivatization of carboxyl-, hydroxyl-, and amino-substituted organic chemicals with the formation of N-methylated products [9]. A sample of fulvic acid methylated with diazomethane in methanol was found to be soluble in this solvent after the evaporation of excess diazomethane. Humic acids that were largely insoluble in methanol could be methylated and solubilized in dimethylformamide [10].Because of the polyfunctional character of humic substances, methylation of hydroxyl groups, especially the tertiary ones, is usually incomplete unless very strong methylating agents are used, such as methyl iodide in combination with sodium hydride [10]. In a study with aquatic humic substances, Thom [11] had to resort to a supplementary methyl iodide/sodium hydride methylation after the diazomethane treatment failed to result in quantitative methylation of phenolic constituents.After methylation with 13C-enriched diazomethane and methyl iodide-sodium hydride, humic substances showed strong bands in the 50-60 ppm region of the 13C NMR spectrum, which represented methoxylated carboxyl groups (COOCH3) and aromatic or aliphatic OCH3 moieties [12,13]. The occurrence and
intensities of these relatively discrete signals constituted clear evidence for the presence of hydroxyl functionalities and their considerable abundance in the analyzed organic matter.To summarize, derivatization of functional groups is beneficial for the following reasons: 1. It introduces unique nuclei or chromophores that enable the detection of specific functional groups.2. It reduces the polarity of humic substances and makes them soluble in organic solvents commonly used in fractionation by liquid chromatography.3. It reduces interactions caused by hydrogen bonding and other sorption mechanisms that interfere with the interpretation of NMR and IR spectra.4. It, therefore, facilitates the application of molecular spectroscopy (NMR and IR) for structural evaluation of humic materials.
