Isolation of IHSS Aquatic Humic and Fulvic Acids

Adapted from: Aiken, G.R. (1985) “Isolation and concentration techniques for aquatic humic substances”, in G.R. Aiken, D.M. McKnight, R.L. Wershaw, and P. MacCarthy (Eds.), Humic substances in soil, sediment and water: geochemistry and isolation. Wiley-Interscience, New York.

  1. Filter water with a 0.45 µm silver or polymer membrane filter.
  2. Lower pH to 2.0 with HCl.
  3. Pass sample through column of XAD-8 resin to retain humic and fulvic acids. The preparative cleaning of the resin is described by Thurman and Malcolm (1981).
  4. Elute HA and FA from the column with 0.1 M NaOH, in the reverse direction. Acidify immediately with HCl to avoid oxidation of humic substances.
  5. Re-concentrate on a smaller XAD-8 column. Elute with NaOH and acidify. The eluted DOC should contain more that 500 mg C/L.
  6. Adjust pH to 1.0 with HCl. Centrifuge to separate the humic acid (HA) from the fulvic acid (FA) fraction. Wash HA with water until wash is negative to the AgNO3 test for chloride. Add sufficient 0.1 M NaOH to dissolve HA and then acidify by passing through a strong acid resin column.
  7. Adjust FA fraction to pH 2.0 with NaOH and re-adsorb FA fraction on XAD-8. Wash with one void volume of distilled water to remove the salt.
  8. Reverse flow and elute column with 0.1 M NaOH.
  9. Immediately pass FA eluate through cation-exchange resin and hydrogen saturate. Pass HA in 0.1 M NaOH through cation-exchange resin and hydrogen saturate. Repeat until Na+ is less than 1 mg/L.
  10. Freeze dry HA and FA samples.



Thurman, E.M., Malcolm, R.L. (1981) Preparative isolation of aquatic humic substances. Environ. Sci. Technol. 15:463-466.