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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 (Stevenson 1982)

Type of material	Extractant	Organic matter extracted %
Humic substances	NaOH	to 80%
	Mild extractans:
	Na4P2O7 and other	to 30%
	Organic chelates:acetyloacetone, cupferron, hydroxyquinoline	to 30%
	Formic acid (HCOOH)	to 55%

Alkali extraction

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 matter.
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 extraction.

Mild extractans

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 for maintaining 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 7.0.

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 (see picture).

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, which are 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 hour.

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 10:1.

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 discard supernatant.

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- test
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 solution).

Step 20. Freeze-dry the eluate to recover the H+- saturated fulvic acid.