f you are not a soil science researcher, your first question is likely to be “What is humic acid?” A physical chemist recently gave a science reporter the following pithy but nonrigorous definition of humic acid (HA): “Humic acid is a complex polyelectrolyte of variable composition found in nearly every scoop of dirt”(1). A soil humic substance (HS) is operationally classified by its aqueous solubilities. Three major humic fractions exist in soil: HA, fulvic acid (FA), and humin. HA is the fraction of a HS that is insoluble in water at pH ≤1, FA is water-soluble under all pH conditions, and humin is water-insoluble under any conditions (2). Typical arable soil will contain 5–6% organic matter, which is 80% HS. The other 20% is composed of polysaccharides, proteins, resins, fats, and waxes. HAs perform various roles in soil chemistry. They act as redox reagents and pH buffers, retain water, bind metal ions, sorb organic solutes, photosensitize soil reactions, stimulate plant growth, and biotransform toxic pollutants. When leached into surface waters, they also play a pivotal role in the aquatic environment. For example, they bind and transport metal ions. Classified as a natural or biopolymer, HA is not a well-defined molecular species— it is polydisperse (composed of many molecular weights [MWs]), irregular in structure, and varies in elemental composition with its natural origin.
This article will focus on the various analytical probes that relate to the structure and function of soil HAs, because there is a reasonably narrow range of elemental composition within this species (3). Characteristics of HAs from coal, lignite, marine, and aquatic sources vary considerably from soil humates. Although FAs play a vital role in soil and aqueous environments, adequately examining them would require another entire article.
Isolation and purification of soil HA Analytical advances have helped surmount some extraordinary challenges that face those studying natural biopolymers, and now there are many approaches to analyze a multifunctional, environmentally active, complex biopolymer. Fifty years ago, when HA was assumed to be a monomeric species, destructive chemical degradation was the primary method of probing its structure. From the fragments of the reaction mixture, scientists attempted to piece together the shape of the original molecule. Since then, nondestructive analytical probes have been developed. Chromatographic, spectrometric, and spectroscopic methods have been used to elucidate structure and function. As an example, electron paramagnetic resonance (EPR) studies have revealed the extent of internal quinhydrone structure and the bonding nature with metal ions, such as copper.
Cornelius Steelink University of Arizona J U N E 1 , 2 0 0 2 / A N A LY T I C A L C H E M I S T R Y
327 A
The isolation protocol depends on the acidic properties of the HA (4). Soxhlet extraction of dry soil with benzene–methanol removes trapped lipids, nucleic acids, polysaccharides, proteins, and smaller organic molecules. The soil is treated with dilute HCl and water. When HCl reduces the pH to 1, the residual soil is repeatedly extracted with dilute NaOH until the supernatants stop depositing a brown gel. The gel is HA and contains 98% water. Vacuum-drying, freeze-drying, or supercritical CO2 drying yields solids with different densities, surface areas, and morphologies (5). The supernatants at pH ≥1 contain the soluble FAs (6). Another common extraction scheme uses aqueous sodium pyrophosphate—a milder reagent than NaOH, which causes fewer chemical changes in the alkali-sensitive HS—at pH 7. However,
