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. An important component of this method is the use of an adsorbent resin in the purification process.

XAD-8 is a nonionic, macroporous (pore size 25 ?m), 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. As an alternative to XAD-8, the DAX-8 resin may also be used.

Purification Protocol:

Remove roots and sieve the dried soil sample to pass a 2.0-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 M HCl to provide a final concentration that has a ratio of 10 mL liquid/1 g dry sample. Shake the suspension for one hour 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 resin (Rohm & Haas Co., Philadelphia, Pa.).

Neutralize the soil residue with 1 M NaOH to pH=7.0 then add 0.1 M NaOH under an atmosphere of N 2 to give a final extractant to soil ratio of 10:1. Extract the suspension under N 2 with intermittent shaking for a minimum of four hours. 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=1.0 and then allow the suspension to stand for 12 to 16 hours. 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 N 2 . 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=1.0 and allow the suspension to stand again for 12 to 16 hours. 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 AgNO 3 . 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 hour). Discard the effluent, rinse the XAD-8 column containing sorbed fulvic acid with 0.65 column volumes of distilled H 2 O. Back elute the XAD-8 column with 1 column volume of 0.1 M NaOH, followed by 2 to 3 column volumes of distilled H 2 O. Immediately acidify the solution with 6 M HCl to pH=1.0. 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 (1.0 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 H 2 O. Back elute with 1 column volume of 0.1 M NaOH followed by two column volumes of distilled H 2 O. Pass the eluate through H+-saturated cation exchange resin (Bio-Rad AG-MP-5 (Bio-Rad, Richmond, Calif.) using three times the mole of Na ions in solution). Freeze dry the eluate to recover the H+-saturated fulvic acid.