Humic material/substances precipitation

Acidification of aqueous concentrates and extracts to pH near 1 is the standard procedure to precipitate humic from fulvic acid, and this procedure also has been applied to aquatic humic substances (Thurman and Malcolm, 1981). Aquatic humic substances that interact significantly with metal ions can be precipitated from water by addition of lead(Il) nitrate (Klocking and Mucke, 1969). Co-precipitation of aquatic humic materials with aluminum, copper, iron, and magnesium hydroxides has been used to recover aquatic humic substances from various types of water (Jeffrey and Hood, 1958 Williams and Zirino, 1964 Zeichmann, 1976). Humic acids can also be precipitated from an unconcentrated water sample by adding acetic acid and isoamyl alcohol to a sample contained in a separatory funnel, and after shaking, humic acid precipitates at the alcohol-water interface (Martin and Pierce, 1971). Precipitation methods are among the crudest of fractionation methods... 

During the first part of the nineteenth century considerable attention was paid to the chemistry of humic substances. A detailed description of the analysis of humic acid with particular reference to its acidic properties was given by Sprengel. One of his innovations still in vogue today was the pretreatment of soil with dilute mineral acid prior to its extraction with alkali. Sprengel also observed that after removal of all the mineral acid, freshly precipitated humic acid partially dissolved in cold water, but after drying, the humic material was converted into a less water-soluble form for which the term humus coal was proposed. 

Organic matter extracted from earth materials usually is fractionated on the basis of solubility characteristics. The fractions commonly obtained include humic acid (soluble in alkaline solution, insoluble in acidic solution), fulvic acid (soluble in aqueous media at any pH), hymatomelamic acid (alcohol-soluble part of humic acid), and humin (insoluble in alkaline solutions). This operational fractionation is based in part on the classical definition by Aiken et al. (1985). It should be noticed, however, that this fractionation of soil organic matter does not lead to a pure compound each named fraction consists of a very complicated, heterogeneous mixture of organic substances. Hayes and Malcom (2001) emphasize that biomolecules, which are not part of humic substances, also may precipitate at a pH of 1 or 2 with the humic acids. Furthermore, the more polar compounds may precipitate with fulvic acids. 

The first attempt to isolate humic substances from soil appears to have been made by Achard (1786), who extracted peat with alkali and obtained a dark, amorphous precipitate upon acidification. This alkali-soluble, acid-insoluble material became known by a number of names, of which the term humic acid survived. 

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. 

The insolubility of humic substances in nonpolar organic solvents has limited the use of solvent extraction as a method of isolating humic substances from water. The most effective method for solvent extraction was reported by Eberle and Schweer (1974). Humic acid was efficiently extracted with trioc-tylamine/chloroform at pH 5 and was recovered by back-extracting with water at pH 10 or above. Butanol has been used to extract freeze-concentrated humic substances however, not all the material was extracted (Shapiro, 1957). Another method involves acidification of a sample with acetic acid, followed by extraction with isoamyl alcohol. Humic acid precipitates at the interface (Martin and Pierce, 1975). This method is slow 5 hours were required to extract 100 mL of sample. No data on the behavior of fulvic acid in this solvent extraction were presented. 

The classic definitions of soil humic and fulvic acids are based on solubility (Schnitzer and Khan, 1972). Thus, humic acid is the alkali-soluble material in soil, which is precipitated at pH 1. The material which remains soluble in the extract at pH 1 is fulvic acid. A more recent definition for aquatic humic substances is given by Thurman and Malcolm (1981). Here the material which adsorbs on an XAD column from an acid aqueous solution is defined as aquatic humus. That part of the adsorbed material which is soluble in acid and base is fulvic acid the portion insoluble in acid is humic acid. Another definition of an aquatic humic substance is based on adsorption by DEAE-cellulose columns (Miles etal., 1983). 

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. 

Acid hydrolysis has been used extensively to remove ash, protein, and carbohydrate from humic substances (see reviews by Schnitzer and Khan, 1972, 1978 Stevenson, 1982 Kononova, 1961). In theory, this method should remove peripheral groups and co-precipitated materials and leave the core of humic or fulvic acid. What actually happens may be quite disconcerting. Structural changes may take place, as well as the removal of contaminants. Depolymerization may occur, but acid-catalyzed condensation may also take place. These changes can be seen by the loss of hydrogen content and by a decrease in the E IEf, ratios. All of these events are best summarized in Table 5 (Stevenson, 1982). 

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