Method 54 Determination of fulvic and humic acids

Microorganisms break down plant and animal residues in the soil to form a stable dark brown organic material called humus. It is composed of a mixture of large complex molecules (molar masses 20,000-100,000 g mol-1). Lignin-type precursors result in benzene ring (aromatic) compounds substituted with hydroxyl (—OH), methoxyl (—OCH3) and carboxyl (—COOH) groups such as gallic and vanillic acids. These react by oxidation and/or polymerization to form dark brown substances (Flaig, 1997) typical of water leached through peat.

The main sources of negative charge on the humus particle arise from the —COOH and —OH (phenolic) groups, with only the —COOH being significantly charged below pH 7. The sources of these charges may be mainly separated into the fulvic and humic acid fractions. These are not distinct chemical species, but rather two groups of complex organic soil substances with some common chemical characteristics, which are separated and differentiated purely by their solubility in sodium hydroxide and then hydrochloric acid under the stated conditions. Aiken et al. (1985) have defined fulvic acids as 'the fraction of humic substances that is soluble under all pH conditions', and humic acids as 'the fraction of humic substances that is not soluble in water under acid conditions, but becomes soluble at greater pH'. In general, fulvic acid has a lower molecular weight, lower N content and is possibly more aromatic than humic acid. With spectroscopic techniques, IR shows broad bands from functional groups, but little information on the composition of humic substances (HS); UV-Vis using 465/665 nm ratios shows differences depending on source, but no meaningful chemical interpretation of the spectra; NMR has yielded more information and shown the persistence of lignin- and tannin-type residues. Wet chemical and some spectroscopy procedures have been reviewed by Hayes and Swift (1978), Swift (1996) and Hayes (1997).

The ratio of fulvic to humic acid varies between soils and between horizons of the same soil. Humic fractions are involved with solubilization of the sesquioxides (especially gibbsite, Al(OH)3; goethite, FeOOH; haematite, Fe2O3; and ferrihydrite, Fe2O3.nH2O). It is therefore desirable to determine the Al and Fe associated with these fractions. The scheme of separations is shown in Fig. 5.1.


Soluble fraction in -4-supernatant

0.5 M NaOH


Insoluble residue HUMIN

6 M HCl

Adjust supernatant to pH 2

Soluble fraction in <--► Insoluble residue supernatant ^nMuge HUMIC ACID


Fig. 5.1. Scheme of separating the soil fulvic and humic acid fractions.

Procedure. Weigh 10 g air-dry sieved (^2 mm) earth into a 250-ml plastic centrifuge bottle, add 200 ml 0.5 M NaOH and shake overnight. Centrifuge at 2000 rpm for 20 min to allow sedimentation of the insoluble humin and decant all the supernatant into a clean centrifuge bottle. Adjust to pH 2.0 with 6 M HCl, then centrifuge at 2000 rpm for 20 min to cause sedimentation of the humic acid. Decant the solution of fulvic acid into a 250-ml volumetric flask. Wash the sediment of humic acid with 30 ml 0.5 M HCl, centrifuge, add supernatant to the volumetric flask, and make up to the mark with water and mix. Read the optical density at 465 nm, diluting if necessary to bring on scale. The approximate concentration of fulvic acid in mg l-1 is given by comparing with the graph of optical density vs. concentration (Professor W.A. Adams, Aberystwyth, 2001, personal communication) as shown in Fig. 5.2. (The fulvic acid is in a solution of NaCl, therefore gravimetric determination is not possible.) It is recommended, however, that the extinction (absorption) coefficient should be determined for humic substances from different origins, from the modified Beer-Lambert expression (Schnitzer and Khan, 1972):

E = OD/c/ where E is the extinction coefficient, OD is the optical

1 cm density, c is a 0.001% solution of the humic compound in the stated reagent, and / is the internal cell length of 1 cm.

Wash the humic acid residue with 200 ml 0.5 M HCl, centrifuge and discard the supernatant. Wash the humic acid out of the centrifuge bottle with 60% IMS into a pre-weighed oven-dry 100 ml glass beaker. Evaporate to dryness carefully on a hotplate, avoiding loss by spitting, cool in a desiccator

Fulvic acid mg 100 ml-1

Fig. 5.2. Relationship between optical density at 465 nm and concentration of ash-free sedge peat fulvic acid.

Fulvic acid mg 100 ml-1

Fig. 5.2. Relationship between optical density at 465 nm and concentration of ash-free sedge peat fulvic acid.

and reweigh. The difference in weights gives the weight of humic acid plus ash.

Ignite in a muffle furnace at 500°C overnight to burn off the humic acid fraction, cool in a desiccator (leave lid slightly open for the first minute to allow air to expand) then reweigh the beaker containing the residual ash. Subtract this weight from the weight of beaker and residue before ashing to obtain the weight of ash-free humic acid.

Add 20 ml 6 M HCl dropwise on to the ash and warm carefully to dissolve, and then make up with water to 50 ml in a volumetric flask. Using suitable dilutions where necessary, analyse for Ca, Fe and Al by AAS. It will be necessary to use a nitrous oxide-acetylene flame for aluminium, otherwise use the titration method as in Method 5.3.

Calculation. Ash-free fulvic acid y mg 100 ml-1 is read from the chart. This solution resulted from 10 g air-dry soil in 250 ml solution. Therefore 250 ml solution contains y x 250/100 mg fulvic acid from 10 g air-dry soil, which converts to 25y mg fulvic acid 100 g-1, or 0.025y% air-dry soil. This must be multiplied by any dilution factor before reading the optical density, also converted to percent oven-dry soil (see Method 5.2, Calculation (2)).

The weight of ash-free humic acid was derived from 10 g air-dry soil, therefore should be multiplied by 10 to convert to percent air-dry soil and further converted to percentage oven-dry soil. The fulvic and humic acid content may be expressed as a percentage of total soil organic matter, which is quantified as soil organic carbon (SOC). SOC is conveniently determined from the loss on ignition, where the correlation is:

Loss on ignition = 1.94 SOC + 16.0 (r2 = 0.96) McGrath (1997)

The ratio of fulvic to humic acid is also significant; both, together with carbohydrates (uronic acids and sugars), are linearly related to the SOC. The humic acid increases and the fulvic acid decreases as the SOC increases (McGrath, 1997).

The Al, Ca and Fe may be expressed as a percentage of the humic acid content.

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  • Mantissa Labingi
    How to detect humic acid in solution?
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