Isolation, of humic substances

D.H. Stuermer and G.R. Harvey, The isolation of humic substances and alcohol-soluble organic matter from seawater, Deep-Sea Res. 24 (1977) 303-309. 

Hydride generation AAS is a well-established technique for the determination of selenium because of its selectivity and sensitivity. The detection limits are improved by concentrating the hydride prior to the transfer to the spectrometer. Cryogenic condensation in a U-tube at liquid nitrogen temperature is a preferred method of pre-concentration. However, selenium hydride is formed essentially only from Se. The selectivity for Se has been combined with various chemical preparation steps to determine the sum of SeIV and Se and the total selenium content of samples. Se concentrations are measured by the difference between the (Se + Se ) and Se contents. The difference between total selenium and (Se + Se ) contents represents the concentrations of Se° and Se2- species. These methods have been used for determinations of selenium species in natural waters. Soluble organic matter in some groundwater samples interferes with the hydride generation determination of selenium. Isolation of humic substances by their adsorption on resins has overcome this problem. 

Parson, J.W. (1988) Isolation of humic substances from soils and sediments. In Humic Substances and their Role in the Environment (Frimmel, F.H., and Christman, R.F. eds.), pp. 3-14, John Wiley, New York. 

Equipment. Silver filters of 0.45-pm pore size and 47-mm and 142-mm diameter were obtained from Osmonics, Inc., Minnetonka, MN. Stainless-steel pressure filtration equipment (47-mm and 142-mm plate filtration units) was obtained from Millipore (Bedford, MA). XAD-8 resin was purchased from Supelco, Inc. (Bellefonte, PA) and cation-exchange resin was purchased from Fisher Scientific (Ag-MP-5, Springfield, NJ). A Millipore XX80 peristaltic pump was used in the isolation of humic substances (Bedford, MA), and the freeze drying unit was a FTS Systems Flex Dry freeze drier, an FTS Systems shell freezer, and an FTS model VP62A vacuum pump (Stone Ridge, NY). The specific conductance meter (model 604) was obtained from Amber Science, Inc., Eugene, OR. 

Compared to the isolation of humic substances from soil or peat, the extraction of reasonable quantities of humic substances from aquatic sources poses severe difficulties due to their very low concentrations in natural waters. However, in recent years significant advances have been made in the technology of isolating humic substances from aquatic environments as discussed by Aiken in Chapter 14. 

A useful procedure for the isolation of humic substances from groundwater is that of Thurman and Malcolm (1981) with the following modifications. Hydrochloric acid should be added immediately to the water sample to prevent the precipitation of iron hydroxide, and the sample should be evacuated with a vacuum pump to remove hydrogen sulfide. If the sulfide is not removed, it can react to form both elemental sulfur and polysulfides that adsorb onto and clog the XAD resin (Leenheer and Noyes, in press Thurman, 1979). 

The isolation of humic substances from estuarine waters has usually been performed by acidification of the water sample followed by either filtration, to yield humic acids only, or adsorption onto a resin such as XAD-2, which appears to allow recovery of both the humic and fulvic acid fractions (Stuer-mer and Harvey, 1977a). Table 1 provides a listing of determinations of humic and fulvic acids in estuarine waters by a variety of investigators, along with the techniques employed. The concentrations of dissolved humic and fulvic acids found typically range from undetectable to less than 2 mg C/L, with most values in the tens of fig C/L for the humic acid fraction and hundreds of fig C/L for the fulvic fraction. Higher values are usually found at lower salinities. However, it must be stressed that few analyses are available most of these are from estuaries of the northeastern United States and a different isolation technique has been used in virtually every study cited. 

Any attempt to design an effective procedure for the isolation of humic substances from soil should take account of the properties of the solvents or extractants to be used, of the solutes or material to be extracted, and of the types of associations that can exist between these solutes and other soil colloidal constituents. This chapter outlines some aspects of the composition, properties, and associations of soil humic substances which are relevant to their extraction, and it considers some of the properties of solvents that are used or might be considered for use as extractants of humic substances, or of molecules associated with these substances. It then compares the effectiveness and outlines some of the limitations of a selected number of solvents and procedures for dissolving and extracting humic substances. 

Advantages and disadvantages of the commonly used methods to isolate and concentrate aquatic humic substances have been presented in this chapter. For most waters, the process of producing low-ash humic material involves filtration, concentration, isolation of humic substances from inorganic and other organic solutes, and lyophilization. Development of... 

While ultrafiltration has been used extensively by water chemists in the isolation of humic substances (Schnitzer, 1978), its use with soil materials has been more limited (Cameron et al., 1972b Wake and Posner, 1967). From the foregoing discussion it should be clear that, as a technique for preparative fractionation and desalting, ultrafiltration is very attractive and will rival gel permeation chromatography in this type of work. Only time and a considerable amount of effort will tell which is the superior technique. 

Chemical characterization of DOM has been studied extensively, providing a wealth of information regarding its chemical properties [17,20,25,60,64-89]. CDOM is usually characterized as aquatic humic substances, such as humic and fulvic acids, owing to the presence of multiple double bonds in aromatic, aldehyde, and ketone groups. Isolation of humic substances involves their separation by adsorption on macroporous resins (e.g., XAD-8 or XAD-4) and elution at various pH [90]. Humic acids are soluble above a pH of 2, while fulvic acids are soluble at any pH. Solid phase extraction (SPE) onto Cig resin is also employed to isolate CDOM [44,73]. Amador and coworkers have shown that... 

Lepane, V., Comparison of XAD resins for the isolation of humic substances from seawater, J. Chromatogr. A, 845, 329-335, 1999. 

A comparison of the NEXAFS spectra of isolated humic substances and pristine soil organic molecules for a pine ultisol indicate that the undisturbed soil samples indicate much greater concentrations of carboxylic acids when compared to that of aromatic C (Fig. 13, details discussed later). Similar behavior is observed for N, P, S and Cl functional groups (discussed later). These studies suggest that chemical extraction procedures used in the isolation of humic substances preferentially concentrate the aromatic C over the carboxylic groups. 

The classical method for isolation of humic substances from solid-phase source materials such as soils, peat, and leonardite is alkaline extraction with aqueous NaOH, followed by precipitation of humic acid at low pH, and a series of desalting steps involving cation exchange, dialysis, etc., to obtain fulvic acid. Through this procedure, samples extracted from solid-phase materials include both... 

Hayes, T. M., M. H. B. Hayes, J. O. Skjemstad, R. S. Swift, and R. L. Malcolm. 1996. Isolation of humic substances from soU using aqueous extractants of different pH and XAD resins, and their characterization by 13C-NMR. In Humic Substances and Organic Matter in Soil and Water Environments Characterization, Transformations, and Interactions Proceedings of the 7th International Conference of the International Humic Substances Society, University of the West Indies, St. Augustine, Trinidad and Tobago, 3-8 July 1994, ed. C. E. Clapp, M. H. B. Hayes, N. Senesi, and S. M. Griffith, 13-24. St. Paul, MN International Humic Substances Society. 

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