Isolation of IHSS Soil Fulvic and Humic Acids

exerpt from — Swift, R.S. 1996 Organic matter characterization (chap 35). pp. 1018-1020. In: D.L. Sparks et al. (eds) Methods of soil analysis. Part 3. Chemical methods. Soil Sci. Soc. Am. Book Series: 5. Soil Sci. Soc. Am. Madison, WI

© Soil Science Society of America, used with permission.

Reprints can be obtained from: Roger S. Swift, CSIRO, PMB 2, Glen Osmond 5064, Australia.

International Humic Substance Society Method

A number of methods for the extraction of humic substances from soil using sodium hydroxide solution have been published. These methods are generally successful and yield comparable results. The following is a method which has been developed by the International Humic Substance Society (IHSS) as an acceptable method for the extraction of humic substances from soils.

It has been clearly stated by IHSS that this is not meant to be a recommended or approved method, but a method that has been found to be satisfactory for most soil types and one which can be performed in most laboratories. It produces relatively high yields and can be used as a standard method for comparisons between and within laboratories. An important component of this method is the use of an adsorbent resin in the purification process. This can be replaced by dialysis if the resin is unavailable (See note about dialysis at end of text).


1. Hydrochloric acid (HCl), 1 M, 6 M

2. Sodium hydroxide, 1 M, 0.1 M

3. Potassium hydroxide (KOH), 0.1 M

4. Potassium chloride (KCl)

5. Hydrofluoric acid (HF) concentrated, 0.3 M

6. XAD-8 resin (Rohm & Haas Co., Philadelphia, PA)

7. Visking dialysis tubing (Visking Co., Chicago, IL) [MWCO (molecular weight cut-off)] 10,000 dalton


Remove roots and sieve the dried soil sample to pass a 2-mm sieve. Equilibrate the sample to a pH value between 1 to 2 with 1 M HCl at room temperature. Adjust the solution volume with 0.1 MHCl to provide a final concentration that has a ratio of 10 mL liquid/1 g dry sample. Shake the suspension for 1 h and then separate the supernatant from the residue by decantation after allowing the solution to settle or by low speed centrifugation. Save the supernatant (FA Extract 1) for the isolation of fulvic acid using XAD-8.

Neutralize the soil residue with 1 M NaOH to pH = 7 then add 0.1 M NaOH under an atmosphere of N2 to give a final extractant to soil ratio of 10:1. Extract the suspension under N2 with intermittent shaking for a minimum of 4 h. Allow the alkaline suspension to settle overnight and collect the supernatant by means of decantation or centrifugation. Acidify the supernatant with 6 M HCl with constant stirring to pH = 1 and then allow the suspension to stand for 12 to 16 h. Centrifuge to separate the humic acid (precipitate) and fulvic acid (supernatant – FA Extract 2) fractions.

Redissolve the humic acid fraction by adding a minimum volume of 0.1 M KOH under N2. Add solid KCl to attain a concentration of 0.3 M [K+] and then centrifuge at high speed to remove the suspended solids. Reprecipitate the humic acid by adding 6 M HCl with constant stirring to pH = 1 and allow the suspension to stand again for 12 to 16 h. Centrifuge and discard the supernatant. Suspend the humic acid precipitate in 0.1 M HCl/0.3 M HF solution in a plastic container and shake overnight at room temperature. Centrifuge and repeat the HCl/HF treatment, if necessary, until the ash content is below 1%. Transfer the precipitate to a Visking dialysis tube by slurrying with water and dialyze against distilled water until the dialysis water gives a negative Cl test with silver nitrate AgNO3. Freeze dry the humic acid.

Pass the supernatant designated “FA Extract 1” through a column of XAD-8 (0.15 mL of resin per gram of initial sample dry weight at a flow rate of 15 bed volumes per h). Discard the effluent, rinse the XAD-8 column containing sorbed fulvic acid with 0.65 column volumes of distilled H2O. Back elute the XAD-8 column with 1 column volume of 0.1 M NaOH, followed by 2 to 3 column volumes of distilled H2O. Immediately acidify the solution with 6 M HCl to pH = 1. Add concentrated HF to a final concentration of 0.3 M HF. The solution volume should be sufficient to maintain the fulvic acid in solution.

Pass the supernatant designated “FA Extract 2” through a column of XAD-8 (1 mL of resin per gram of initial sample dry weight). Repeat the back elution and acidification as for “FA Extract 1” above. Combine the final eluates from each of the fulvic acid extracts and pass this solution through XAD-8 resin in a glass column (column volume should be one-fifth of sample volume). Rinse with 0.65 column volumes of distilled H2O. Back elute with 1 column volume of 0.1 M NaOH followed by two column volumes of distilled H2O. Pass the eluate through H+-saturated cation exchange resin [Bio-Rad AG-MP-5 (Bio-Rad, Richmond, CA) using three times the mole of Na ions in solution]. Freeze dry the eluate to recover the H+-saturated fulvic acid.


XAD-8 is a nonionic, macroporous (pore size 25 µm), methyl methacrylate ester resin (see “Fractionation of Humic Substances Adsorption”). Because it is sometimes difficult to obtain, it may be necessary to use an alternative resin such as Polyclar, which is a cross-linked poly(vinylpyrrolidone) (PVP) (De Nobili et al., 1990; Watanabe & Kuwatsuka, 1991) or other equivalent resin.

Extensive purification procedures of the resins are required before use. These methods and methods used to store the resin are detailed by Thurman & Malcolm (1981).

If it is not possible to purify the fulvic acid using resin treatments, exhaustive dialysis against distilled H2O is an alternative but less satisfactory method of purification. (See note about dialysis at end of text.). If there is a significant concentration of polyvalent cations such as Al3+ present, these may form insoluble metal-humate complexes as the solution is neutralized. Therefore, the dialysis should be carried out against dilute HCl initially until the concentration of any polyvalent cations has been significantly reduced, before finally dialyzing against distilled H2O. Technically, a fraction obtained in this way should be referred to as a fulvic fraction, rather than fulvic acid, as it is likely to contain significant amounts of unbound soil polysaccharide.


De Nobili, M., G. Bragato, J.M. Alcaniz, A. Puigbo, and L. Comellas (1990). Characterization of electrophoretic fractions of humic substances with different electrofocusing behavior. Soil Sci. 150:763-770.
Thurman, E.M., and R.L. Malcolm (1981). Preparative isolation of aquatic humic substances. Environ. Sci. Technol. 15:463-466.
Watanabe, A., and S. Kuwatsuka (1991). Fractionation of soil fulvic acids using polyvinyl-pyrrolidone and their ionization difference spectra. Soil Sci. Plant Nutr. 37:611-617.

Note on HF treatment: More recently a filtration step has been added for removal of clays from the NaOH soil extract. This removes removes ash without HF. The NaOH extract is filtered through a 0.2 micrometer polyethersulfone membrane filter (Gelman Supor) under pressure (nitrogen gas). The extract should be filtered twice through the same membrane. The partial plugging of the filter with the first filtration helps remove fine clay and eliminates the need for the KOH-KCl and HF-HCl treatments to reduce the ash content.

Note on dialysis of fulvic acids. Research on dialysis of fulvic acid shows much of fulvic acid will pass through commercially available dialysis membranes. Dialysis is probably not a good substitute for the resin treatment.