Fractionation of aquatic humic substances

A conceptual DOM pie diagram (Fig. 1) represents the chemical view of the DOM pool of a typical riverine sample. The specific chemical classes or wedges can be separated based upon several different operational fractionation schemes (Thurman and Malcolm, 1981 Aiken, 1985 Aiken et al., 1992 Benner, 1998). For typical natural waters, the dominant fraction comprising DOM is fulvic acid, which is defined as the yellow, moderate molecular weight organic acid fraction of aquatic humic substances that is soluble at all pH values (Aiken et al., 1985). In surface waters receiving... 

Aster, B., Burba, E and Broekaert, J.A.C. (1996) Analytical fractionation of aquatic humic substances and their metal species by means of multistage ultrafiltration. Fresenius J. Anal. Chem., 354, 722-728. 

Most concentration and isolation techniques, except for evaporative and freeze-concentration techniques, are also the first steps in chemicfd and physical fractionation of aquatic humic substances. This chapter will concentrate primarily on techniques used to subfractionate and chromatographi-cally separate aquatic humic substances previously isolated as crude fractions. 

Trial and error approaches seldom have been successful for fractionations of aquatic humic substances. However, fractionations can be designed using the following general considerations about the nature of aquatic humic substances. 

Size fractionations of aquatic humic substances definitely need to be performed on-site to minimize changes in aggregation during sample preservation, transport, and storage if the size fractionation data are to be related to environmental conditions. If additional aggregation or precipitation or both occur after an on-site size fractionation, the sample should not be refractionated, but needs to be treated as if the on-site fractionation is valid. 

Examples of successful fractionations of aquatic humic substances where only one fractionation mechanism was operative include utilization of the hydrophobic properties of XAD resins (Mantoura and Riley, 1975 Aiken et al., 1979), hydrogen bonding of weak-acid functionalities of humic constituents to weak-base anion-exchange resins (Kim et al., 1976), and use of ion-exchange celluloses for ion-exchange fractionation of aquatic humic substances without hydrophobic matrix adsorption (Sirotkina et al., 1974). These examples of successful fractionations demonstrate the potential for chromatography of aquatic humic substances when fractionations are designed carefully to avoid undesirable interactions. 

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. 

Adsorption chromatography of aquatic humic substances is still in its developmental stage. For progress to be made, aggregation phenomena and undesirable adsorbent interactions will have to be minimized. However, the rapid advances being made in the field of liquid chromatography undoubtedly will have applications to the fractionation of aquatic humic substances. 

An extraction method for isolating humic substances from water by using XAD-8 has been proposed by Thurman and Malcolm (9) (see box). Humic substances in natural waters represent almost the entire hydrophobic acid fraction. This method has been used to isolate 4.25 g of humic substances from 24,500 L of ground water from the Fox-hills-Laramie aquifer and to obtain 500 g of humic material from 10,400 L of the Suwannee River (Table II). The sample from the Suwannee River was collected as a reference sample of aquatic humic substances by the International Humic Substances Society. In both of the examples cited, a fc cutoff of 100 was used. 

Burba, R, Aster, B.,Nifant eva,T.,Shkinev, V., and Spivakov, B. Y. (1998). Membrane filtration studies of aquatic humic substances and their metal species A concise overview. Part 1. Analytical fractionation by means of sequential-stage ultrafiltration. Talanta 45, 977-988. 

Burba, P., Shkinev, V and Spivakov, B.Ya. (1995) On-line fractionation and characterization of aquatic humic substances by means of sequential-stage ultrafiltration. Fresenius J. Anal. Chem., 351, 74-82. 

Beckett R, Bigelow JC, Jue Z, Giddings JC (1989) Analysis of humic substances using flow field-flow fractionation. In MacCarthy P, Suffet IH (eds) Influences of aquatic humic substances on fate and treatment of pollutants. American Chemical Society, Washington, DC, pp 65-80... 

Thurman and Malcolm (1981) proposed a large-scale preparative method for isolation of aquatic humic substances, in which XAD-8 resin is used to adsorb humic substances from acidified water samples. The aquatic humic substances are then back-eluted with NaOH, and fractionated into aquatic HAs (insoluble at pH 1) and aquatic FAs (soluble at pH 1). The two fractions are further purified to remove inorganic solutes and freeze-dried separately. 

Ikeda K., Arimura R., Echigo S., Shimizu Y., Minear R. A., and Matsui S. (1999) The fractionation/concentration of aquatic humic substances by the sequential membrane system and their characterization with mass spectrometry. Water Set Technol. 42, 383-390. 

Adsorption of aquatic humic substances onto surfaces changes their molecular weight distribution pattern. However, there is apparently little knowledge of how the chemical composition and the microbial availability is altered by this process under environmental conditions. Photolysis by UV radiation also results in changes of the molecular weight distribution, but, there is some contradictory evidence concerning which molecular weight fractions are affected most. 

Fractionation techniques for aquatic humic substances have not been developed to the same extent as concentration and isolation techniques. Many organic fractionation techniques presuppose a concentrated sample, but aquatic humic substances exist naturally at dilute concentrations in the presence of greater suspended sediment and inorganic solute concentrations. Now that efficient preparative concentration and isolation techniques for aquatic humic substances have been developed, as reported by Aiken in Chapter 14 of this book, renewed emphasis can be given to group fractionations with the ultimate hope that the chromatographic separation of aquatic humic substances into individual compounds can be achieved. 

Macro- as well as microfractionation techniques need to be developed, as necessary steps in attaining the goal of organic structure elucidation. Now that several hundred grams of aquatic humic substances can be isolated from water at reasonable time and cost, it is not unrealistic to plan to process this quantity of material to obtain milligram quantities of pure substances for structural studies. Once some structures have been determined, it should be possible to miniaturize the fractionations and use mass spectroscopy for structure identification. 

It is usually desirable to fractionate aquatic humic substances by only one interaction mechanism operating at a time. Unfortunately, the complexity of aquatic humic substance structures and properties usually causes multiple... 

Electrophoretic separations of solutes are determined primarily by the mass-to-charge ratio of the solute. However, certain electrophoretic separations use gel or paper supports for the electrolyte, and adsorptive and molecular-size fractionations may be as significant as electrophoretic fractionation in these systems. Hall (1970) found that polyacrylamide gel electrophoresis of aquatic humic substances gave size fractionations corresponding to Sepha-dex gel fractionation however, individual fractions were poorly resolved in polyacrylamide gel electrophoresis. Clapp (1957) and Mortensen and Schwendinger (1963) used electrophoresis with electrolyte supporting curtains to separate colorless polysaccharides from humic substances in soil-water extracts. 

The preparative fractionation scheme will apply the chromatography developed for the analytical scheme to larger samples. From 10 to 20 mg of the pure compound isolated from aquatic humic substances needs to be the objective of the preparative fractionation. The preparative fractionation might have to begin with as much as a kilogram of initial material. After structural determination of a number of aquatic humic substance components, the analytical fractionation, combined with spectral determinations. 

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