Extraction of Soil Organic Matter
- The properties of soil organic matter, and especially humic substances,
can be studied only
- in free state, that is, when freed of inorganic soil components.
Therefore, the first task of the researcher is to separate organic matter from
the inorganic matrix of sand, silt, and clay.
- A variety of techniques have been employed, depending upon the nature of
the material to
- be examined. Thus nonpolar compounds like fats, waxes, resins, etc. can be
extracted with such organic solvents as hexane, ether, alcohol-benzene
mixtures, and others. Hydrolysis prosedures have been used for isolating
individual monomers, such as amino acids and sugars.
- The ideal extraction method is one which meets the following objectives:
- the method leads to the isolation of unaltered material
- the extracted humic substances are free of inorganic contaminants, such
as clay and polyvalent cations
- extraction is complete, thereby insuring representation of fractions
from the entire molecular-weight range
- the method is universally applicable to all soils
Reagents used for extraction of organic constituents from soil
- NaOH solution of 0.1 to 0.5N concetration in water and a soil to
extractant ratio of from
- 1:2 to 1:5 (g/ml) have been widely used for recovering organic
matter.Repeated extraction is required to obtain maximum recovery. Leaching
the soil with dilute HCl, which removes Ca and other polyvalent cations,
increases the efficiency of extraction of organic matter with alkaline
reagents. As a general rule, extraction of soil with 0.1 or 0.5N NaOH leads to
the recovery of approximately two-thirds of the soil organic matter.
- Undesirable features of alkali extraction are as follows:
- Alkali solutions dissolve silica from the mineral matter and this silica
contaminates the organic fractions separated from the extract.
- Alkali solutions dissolve protoplasmic and structural components from
fresh organic tissues and these become mixed with the humified organic
- Under alkaline conditions, autoxidation of some organic constituents
occurs in contact with air both during extraction and when the extracts are
allowed to stand.
- Other chemical changes can occur in alkaline solution like condensation
between amino acids and aldehydes or quinones.
- The more alkaline the solution and the longer the extraction period the
- greater will be the chemical changes.
- The amount of organic matter extracted from soil with caustic alkali
increases with time of
- Several milder and more selective extractans have been recommended in
recent years as
- alternatives for the classical extraction with strong alkali. Included are
salts of complexing agents (Na4P2O7 and EDTA),
organic complexing agents in aqueous media (acetylacetone), and organic
solvents of various types.Whereas less alternation of organic matter may
result, these extractans are much less effective than alkali hydroxides in
removing organic matter. The main exepction being the illuvial (B) horizon of
the Spodosol. As was the case with alkali extraction, recovery of organic
matter frequently can be increased by pretreating the soil with mineral acids
to remove carbonates (HCl) or silicates (HCl-HF mixtures).
- For certain ivestigations, a mild extractant is definitely preferred; for
others, a more
- complete extraction is caustic alkali. At now, many investigators are
using a sequence of extractants in which part of the organic matter is
recovered by a mild reagent before alkali extraction.
Na4P2O7 and other neutral salts
- In many soils, Ca and other polyvalent cations (Fe, Al) are responsible
- organic matter in a flocculated and insoluble condition. Accordingly,
reagents that inactivate these cations by forming insoluble precipitates or
soluble coordination complexes lead to solubilization of the organic matter.
Such reagents as ammonium oxalate, sodium pyrophosphate and salts of weak
organic acids have been used for this purpose.
- Of the various neutral reagents, Na4P2O7
has been the most widely used. As note earlier,
- the amount of organic matter recovered (<30%) is considerably less than
with caustic alkali, but less alteration occurs.To minimize chemical
modification of the humic material, extraction should be carried out at pH
Formic acid - HCOOH
- Extensive research on the extraction of soil
- organic matter with formic acid shows that under certain circumstances up
to 55% of the organic matter in mineral soils and as much as 80% of that in
composts can be extracted with formic acid containing LiF, LiBr or HBF4
- Advantages of anhydrous formic acid for
- extraction of organic matter is that it is a polar compound that neither
exhibits oxidizing nor hydrolytic properties. Furthermore, formic acid is a
good solvent for a wide variety of compounds, including polysaccharides. Large
quantities of Ca, Fe, Al and other inorganic components are dissolved from the
soil along with the organic matter and thus far it has not been possible to
remove the inorganic material completely.
- Formic acid is most efficient with soils where
- much of the organic matter is only partially humified.
Organic chelating agents
- Organic compounds such as acetylacetone, cupferron and hydroxyquinoline,
- capable of forming chelate complexes with polyvalent metal ions, have been
used for extracting illuvial organic matter from Spodosols. The organic matter
in the B horizon of these soils occurs as complexes with Fe and Al and the
complexing of these metals by chelating agents releases the organic matter to
soluble forms.Organic chelating agents are rather ineffective for extracting
organic matter from other soil types.
Outline of extraction procedures in IHSS method
- Step 1. Equilibrate the sample to a pH value between 1-2 with 1 M
HCl at room teperature.
- Adjust solution volume with 0.1 M HCl to provide a final concentration
that has ratio of 10 mL liquid/1 g dry sample. Shake the suspencion for 1
- Step 2. Separate supernatant from the residue
- by decantation after allowing solution to settle (or by low speed
centrifugation). Save supernatant for XAD-8 isolation.
- Step 3. Neutralize the soil residue with 1 M NaOH to pH=7.0 then
add 0.1 NaOH under an
- athmosphere of N2 to give a final extractant to soil ratio of
- Step 4. Extract the suspension under N2 with
intermittent shaking for a minimum of 4 hours.
- Allow the alkaline suspension to settle overnight and collect the
supernatant by means of decantation or centrifugation.
- Step 5. Acidify the supernatant with 6 M HCl with constant stirring
to pH=1.0 and then allow the
- suspension to stand for 12-16 hours.
- Step 6. Centrifuge to separate the humic acid (precipitate) and
fulvic acid (supernatant
- - FA Extract 2) fractions.
- Step 7. Redissolve the humic acid fraction by adding a minimum
volume of 0.1 M KOH
- under N2. Add solid KCl to attain 0.3 M (K+) and
then centrifuge at high speed to remove suspended solids.
- Step 8. Reprecipitate the humic acid as in step 5. Centrifuge and
- Step 9. Suspend the humic acid precipitate in 0.1 M HCl/0.3 M HF
solution in a plastic
- container. Shake overnight at room temperature.
- Step 10. Centrifuge and repeat HCl/HF treatment(step 9), if
necessary, until the ash content is
- below 1 percent.
- Step 11. Transfer the precipitate to a Visking dialysis tube by
slurring wiyh water and dialyze
- against distilled water until the dialysis watr gives a nagative Cl-
- with the AgNO3.
- Step 12. Freeze-dry the humic acid.
- Step 13. Pass the supernatant from step 2 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 water.
- Step 14. Back elute the XAD-8 column with 1 column volume of 0.1 M
NaOH, followed by
- 2-3 column volumes of distilled water.
- Step 15. Immediately acidify with 6 M HCl to pH=1. Add concentrated
HF to a final
- concentration of 0.3 M HF. Solution volume should be sufficient to
maintain fulvic acid solubility.
- Step 16. Pass the supernatant from step 6 through a column of XAD-8
(1.0 mL of resin
- per gram of initial sample dry weight).
- Step 17. Repeat steps 14 and 15
Step 18. Combine the final eluates from steps 15 and 17 and pass this
solution through XAD-8
- resin in glass column (column volume should be 1/5 of sample volume).
Rinse with 0.65 column volumes of distilled water.
- Step 19. Back elute with 1 column volume of 0.1 M NaOH followed by
2 column volumes of
- distilled water. Pass eluate through H+- saturated cation
exchange resin (Bio-Rad AG-MP-5) using three times the mole of Na ions in
- Step 20. Freeze-dry the eluate to recover the H+-
saturated fulvic acid.