Laboratory Procedures
Extraction and Purification
EarthFax employs procedures for humic substance extraction and purification which were adapted from methods developed by the International Humic Substances Society (IHSS). The adaptations made by EarthFax were developed in collaboration with Dr. Michael H. B. Hayes, University of Limerick in Ireland and Dr. Kenneth Day, an agronomist and humic substances expert, to make the test procedure more rapid, repeatable, and affordable. If needed, EarthFax will also attempt to accommodate requests for alternative extraction and/or purification procedures.
Characterization
Functional Groups - The reactive nature of humic substances is due in large part to their high content of oxygen-containing functional groups. It is through these groups that humic materials react with metals, minerals, and organic compounds. These groups include COOH, phenolic-, and/or enolic-OH, alcoholic OH, and C=O of quinones, hydroxyquinones, and ketones (Stevenson, 1982).
Fulvic acids contain more functional groups of an acidic nature than humic acids, particularly COOH groups. The total acidities of fulvic acids (900-1400 meq/100g) are also higher than those of humic acids (400-870 meq/100g). In addition, while the oxygen in fulvic acids occurs primarily as a component in the functional groups, a high proportion of the oxygen in humic acids occurs as a structural component (i.e. unknown ether linkages) of the polymer.
The procedures employed in humic and fulvic acid functional group analyses are those detailed by Schnitzer (1982). Some analyses require determination of additional functional groups. For instance, determination of phenolic OH groups requires a coincident determination of total acidity and COOH groups. Similarly, determination of the alcoholic OH groups requires analyses of total acidity, COOH groups, and total OH groups.
The E4/E6 Ratio - The E4/E6 ratio is the ratio of absorbencies of humic materials at 465 and 665 nm. E4/E6 ratios of humic acids are generally less than 5.0 and those of fulvic acids typically range from 6.0 to 8.4 (Schnitzer, 1971). The ratio decreases with increasing molecular weight and has been used as an index of the "degree of condensation" or "humification" of aromatic constituents. This ratio is determined using a UV/vis spectrophotometer.
Infrared Spectroscopy - Infrared spectra of humic substances and their derivatives contain a variety of bands that are diagnostic of specific molecular structures. These spectra can (1) provide key information regarding the nature, reactivity, and structural arrangement of oxygen-containing functional groups; (2) establish the occurrence of protein and carbohydrate constituents; (3) demonstrate the presence or absence of inorganic impurities (e.g., metal ions, clay, etc.); and, (4) be used for quantitative analysis (Schnitzer, 1982). IR spectra are useful for (Stevenson 1982):
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The gross characterization of humic materials of diverse origins
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Evaluation of the effects of different chemical extractants; evaluation of chemical modifications such as methylation, acetylation, and saponification; and evaluation of the formation of derivatives
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Detection of changes in the chemical structure of humic materials following oxidation, pyrolysis, and similar treatments to ascertain and characterize the formation of metal-humate and clay-humate complexes
- Determination of possible interactions of organic compounds (e.g. pesticides herbicides, pollutants) with humic materials.
IR spectra are determined using an infrared spectrophotometer.
References
Schnitzer, M. 1971. Characterization of Humic Constituents by Spectroscopy. pp. 60-95 In A. D. McLaren and J. Skujins (eds), Soil Biochemistry, Vol. 2. Marcel Decker, New York.
Schnitzer, M. 1982. Organic Matter Characterization. pp. 581-594 In: Methods of Soil Analysis, Part 2. Chemical and Microbiological Properties. Agronomy Monograph No.9 (2nd Edition).
Stevenson, F. J. 1982. Humus Chemistry - Genesis, Composition, Reactions. John Wiley and Sons. New York.
