The Nature of Organic Materials in Intimate Associations with the Soil Clay Fraction Michael H.B. Hayes 1, Andre J. Simpson 2, Guixue Song 1 1 Chemical and Environmental Sciences University of Limerick, Ireland 2 Department of Chemistry University of Toronto at Scarborough Toronto, Canada
Classical Definitions Humus: Components of Soil Organic Matter so transformed as to bear no morphological resemblances to the materials of origin Humic Substances: Amorphous, brown, polymeric substances that are differentiated on the basis of solubility properties into: HUMIC ACIDS, Precipitated at ph 1 from solution in aqueous base; FULVIC ACIDS, soluble in aqueous media at all ph values; HUMIN, insoluble in aqueous media
COMPOUNDS BELONGING TO RECOGNISABLE CLASSES, SUCH AS POLYSACCHARIDES, PEPTIDES, ALTERED LIGNINS THAT CAN BE SYNTHESIZED BY MICROORGANISMS OR CAN ARISE FROM MODIFICATIONS OF SIMILAR COMPOUNDS IN THE ORGANIC DEBRIS ARE NOT CONSIDERED TO BE HUMIC SUBSTANCES. The concept of Humic Substances as polymeric or macromolecular materials is now challenged and there is growing acceptance of pseudo-macromolecular assemblies arising from molecular associations.
HUMIN IS GENERALLY CONSIDERED TO BE A HUMIC COMPONENT IN INTIMATE ASSOCIATION WITH THE SOIL CLAYS. IT WILL BE SHOWN THAT SUCH MATERIALS DO NOT SATISFY THE CRITERIA OUTLINED IN THE CLASSICAL DEFINITIONS OF HUMIC SUBSTANCES
Any humic material recovered from soil by whatever means, after exhaustive extractions in aqueous base, can be considered to be Humin
Hydrogen Bonding and cation bridging are major mechanisms that inhibit the solvation of humic and other organic molecules in soils
Divalent and polyvalent cations can be displaced by H + -exchanging or by chelation. Hydrogen bonds can be broken by urea and by some organic solvents
Uses of Urea Urea (8M) is used in Biological Chemistry to break Hydrogen bonds. After exhaustive extractions of H + - exchanged soil, we use urea, in 0.1M NaOH. There were little differences between the amounts of additional organic materials isolated by 8M compared with 6M urea.
Urea (Continued) Optical density of Extractions Optical density 0.8 0.6 0.4 0.2 0 Molarity of Urea 1st Extraction 6M 2nd Extraction 6M 1st Extraction 8M
Thus we use 6M urea in 0.1M NaOH in our exhaustive extraction procedures (following exhaustive extraction in 0.1 M NaOH)
In the classical definitions the material isolated in the 0.1M NaOH + 6M urea system described would be regarded as Humin
Principles Involved in Solvation When solvent molecules cluster around a solute molecule solvation takes place. Solvation is governed by properties of the solvent and the solute. The relative polarities of solvent and of solute are important in considerations of solubilities
Table 1 Relevant Properties of Organic Solvents
Table 2
Good Organic Solvents The Absorbance Values listed were compiled by multiplying the optical density observed by the dilution factor (using the solvent as diluent). Consider AVs of 18 and above as Good Solvents All these have molar volumes of 77 or less. All have viscosity values of 2 or less. All have Electrostatic Factor values of 140 or more. All have pk HB values >2 (a measure of abilities to break hydrogen bonds)
Good Organic Solvents (Continued) Thus DMF, Formamide, and DMSO are good solvents, But Solutes cannot be recovered from these solvents by freeze drying. Boiling points are too high for solvent evaporation to be effective. Resin technology can be used.
Again Any material that can be considered to be humic that is extracted in organic solvents, after exhaustive extractions in base, would, in the classical definitions, be HUMIN
We have seen that DMSO is the best of the organic solvents tested for the solvation of H + -exchanged humic acid
But, DMSO is a poor solvent for anions (though a good solvent for cations) Thus it is essential to have the soil/clay medium H + -exchanged in order to use DMSO.
DMSO plus concd H 2 SO 4 (94:6 in volume): An excellent solvent for aqueous base insoluble humin fractions DMSO is a excellent solvent to penetrate clay minerals A small amount of H 2 SO 4 protonates Ionized HS allowing Solvation in DMSO. High solvation (45-65%) takes place Exothermic reaction avoided for dry clay samples. Mild solvent for extraction of recalcitrant humin. No significant structural changes observed.
Flow chart for sequential exhaustive extraction of SOM
H + -exchanged soil Sequential exhaustive Extraction at ph 7, 10.6 and 12.6, under N 2 Alkaline extracts Dilute <20 ppm, adjust ph 2 XAD-8 Crude humin (soil residue) HAs at ph7, 10.6 and 12.6 XAD-4 FAs at ph7, 10.6, and 12.6 Insoluble humin XAD-4 acids 0.1M NaOH + 6 M Urea in N 2 Urea extracts Dry humic-rich clay DMSO + 6% H 2 SO 4 Dilute <20ppm, adjust ph 2.5 Insoluble DMSO extract XAD-8 10% HF H 2 O DMSO insoluble Humin Insoluble dialysis Soluble XAD-8 resin Urea HA XAD-4 Urea FAs DMSO Humin DMSO FAs Urea XAD-4 acids
In Summary Our approach isolates soil organic substances at different ph values giving fractionation on the basis of charge density differences. Then in the sequential extraction we use 0.1M NaOH + 6M urea. Then DMSO + 6% H2SO4 The Urea and DMSO isolates are fractionated into operationally defined humic, fulvic and XAD-4 fractions. The residual material is recovered by dissolving the clay in HF
13 C Chemical Shift (ppm) 13 C Chemical Shift (ppm) 13 C Chemical Shift (ppm)
Variable Amplitude Cross Polarization(VACP) 13 C NMR Solid-state 13 C nuclear magnetic resonances (NMR) experiments were carried out using a Varian Inova spectrometer at 13 C and 1 H frequencies of 100.5 and 400.0 MHz, respectively. Jackobsen 5 mm MAS double-resonance probe heads were employed. The Variable Amplitude Cross Polarisation with Total Sidebands Suppression technique (VACP-TOSS) was applied with a contact time of 1 ms, a spinning speed of 5 khz, acquisition times of 13 ms, recycle delays of 500 ms.
We now show VACP spectra for humic acids isolated from soil at different ph values ranging from ph 7 to 12.6, in 0.5 M NaOH and 0.1M NaOU + Urea. The sequential extraction process was used
56 174 153 129 74 30 104 Urea HA 163 urea HA 0.5M NaOH HA ph 12.6 HA ph 10.6 HA ph 7 3 0 0 2 5 0 2 0 0 1 5 0 1 0 0 5 0 0-5 0 1 3 C C h e m ic a l s h ift (p p m ) VACP-TOSS 13 C NMR spectra: HAs sequentially/exhaustively extracted at different ph and with 0.1M NaOH + 6M urea from maize-amended soil
Note how the evidence for lignin residues (O-aromatic and methoxyl resonances) increase as the ph of the extractant increased. That means that lesser humified humic substances were isolated at the higher ph values. Again, note the similarities between the base and urea extracts.
And, What about the DMSO + H 2 SO 4 system? Is it likely to degrade the organic materials and give rise to artefacts? Our evidence, based on NMR spectra for lignin, suggests that structural alterations do not occur
Comparison of VACP 13 C NMR spectra of lignin, and Of Lignin after sequential treatment with 0.1M NaOH+6M urea and with DMSO+6%H 2 SO 4 (a) lignin base/urea treated DMSO+6%H 2 SO 4 treated 4000 3500 3000 2500 2000 1500 1000 500 (b) Cellulose 250 200 150 100 50 0-50 13 CChemicalShift(ppm) FTIR spectra of lignin (a) and Cellulose (b). Cellulose (DMSO+6% H 2 SO 4 treated) 4000 3500 3000 2500 2000 1500 1000 500
128 HAs 171 IHSS Mollisol FAs 173 129 72 56 24 55 25 a * * 72 VACP DD ph 7 d VACP DD b 172 56 130 151 29,23 109 15 VACP DD ph 12.6 e 128 146 103 VACP DD 173 72 c 72 56 22 VACP 0.1M NaOH + 6M urea f 127 102 24 VACP DD DD 300 250 200 150 100 50 0-50 -100 13 C Chemical shift (ppm) 300 250 200 150 100 50 0-50 -100 13 C Chemical shift (ppm)
173 Humic acids 128 Fulvic acids 129 72 56 24 171 d a 55 25 72 ph 7 VACP DD * VACP b 172 56 130 151 29,23 109 15 DD VACP DD ph 12.6 e 128 146 103 VACP DD 173 72 c 72 56 22 VACP 0.1M NaOH + 6M urea f 127 102 24 VACP DD DD 300 250 200 150 100 50 0-50 -100 13 C Chemical shifts (ppm) 300 250 200 150 100 50 0-50 -100 13 C Chemical shift (ppm)
Based on the NMR and IR evidence shown, we are reasonably confident that the DMSO + H 2 SO 4 system does not give rise to artefacts in the systems we work with
Let us look now at DMSO Humin, i.e. materials isolated in DMSO + 6% H 2 SO 4 after the soil had been exhaustively extracted in the solvent sequence ending with 0.1M NaoH + 6M urea
33 30 DMSO Humin 173 72 56 25 DMSO Fulvic Acids full vacp CSA filter 130 153 148 102 full vacp 173 132 102 72 56 30 23 DD 30 23,19,14 DD 153,148 110 56 30 250 200 150 100 50 0-50 250 200 150 100 50 0-50 VACP-TOSS spectra of DMSO humin (a) and DMSO Fulvic acids, an uncultivated grassland surface soil from Clonroche, Ireland.
(a) DMSO humin VACP-TOSS DD Oak Park 33 VACP-TOSS spectra (thin line) and corresponding DD spectra (thick line) of DMSO humin (a), DMSO insolulbe humin (b) and an expanded CSA filtered spectrum of DMSO insoluble humin (c) (c) DMSO insoluble humin. CSA-filtered 33 30 173 153,148 129 104 73 56 15 104 73 56 25 15 250 200 150 100 50 0-50 (b) DMSO insoluble humin VACP-TOSS DD 125 100 75 50 25 0-25 -50 a Oak Park Transmittance% b 3395 2849 2918 1377 1724 1463 1541 1632 1058 1230 250 200 150 100 50 0-50 13 C Chemical shift (ppm) 4000 3500 3000 2500 2000 1500 1000 500 wavelength (cm -1 ) FT-IR spectra of DMSO humin (a), and DMSO insoluble humin (b).
VACP-TOSS DD VACP-TOSS DD a b 250 200 150 100 50 0-50250 200 150 100 50 0-50 13 C Chemical shift (ppm) 13 C C hem icalshift (ppm )
DMSO/H 2 SO 4 extracted significant amount of DMSO humin, and a small amount of water soluble DMSO fulvic- acids like fractions (<5%). DMSO humin is dominanted by highly aliphatic moieties, low aromatic carbon, and by strong contributions from polysaccharides,and polypeptides. CSA filtered spectra are well resolved, indicating strong resonances from anomeric C (carbohydrates), crystalline polymethylene aliphatic (33 ppm) and amorphous polymethylene aliphatic carbon from long chain alkanes, waxes, culticular material and/or long chain fatty acids, etc. DD spectra showed lower resonances from 55 ppm and in the O- aromatic region, suggesting low altered lignin residues, and the contribution from polypeptides and lignin or lignin derived mateiral. Compared with DMSO humin, DMSO fulvic acids are more oxidized, have more mobile polysaccharide-like structures.
Liquid State NMR of Humin Materials We now look at liquid state NMR spectra of Urea- and DMSO/H 2 SO 4 isolated Humin soil components
amide phenylalanine Aromatic in lignin Urea other protons in C DMSO 1 2 3 4 5 6 7 8* 9 11 10 12 Anomeric protons in C α protons in P methoxyl in lignin P-OCO- (CH2) in LP N-acetyl group in PG -(CH2)n Urea humic acids, IHSS Mollisol -CH3 -γ(ch2) a Aliphatic C: carbohydrate P: polypeptides LP: lipoprotein PG: peptidoglycan DMSO humin, IHSS Mollisol b deuterium exchanged N-H to N-D due to addition of 4% D 2 SO 4 aromatic, amide carbohydrate, peptides, lignin, DMSO LP* PG 9 8 7 6 5 4 3 2 ppm
ppm 5 4 2 3 1 1 2 3 4 5 6 7 Total Correlation Spectroscopy (TOCSY) spectrum of DMSO humin isolated from DMSO+ 6% H 2 SO 4. General assignments are as follows: (1) Aliphatic Couplings; (2) Couplings from aliphatic alcohols and ethers - some amino acid side chains overlap in this region; (3) Couplings between α- protons and amino acid side chains in peptides/proteins. Couplings from ester will also overlap in this region; (4) Couplings from double bonds and, (5) Couplings from amide in peptides. Note these couplings are weak as most of the amides have been exchanged by the addition of D 2 SO 4. 8 8 7 6 5 4 3 2 1 ppm
ppm DMSO humin, IHSS Mollisol 2 PG 10 DMSO 20 6 5 30 40 50 3 1 60 4 70 80 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm Heteronuclear Multiple Quantum Coherence (HMQC) spectrum of the DMSO humin fraction, Mollisol IHSS standard. Assignments are as follows: (1) Aliphatic (for detailed assignment of this region; (2) R-OCO-CH 2 R methylene unit adjacent to the carbonyl in lipids (including lipoproteins and cutins); (3) CH 2 carbohydrate; (4) CH carbohydrate; (5) methoxyl; (6) α H-C in peptides/protein.
What can we Conclude? The procedures outlined show that, using sequential exhaustive extractions at increasing ph values, humic fractions can be isolated that are compositionally different, though still mixtures. Additional humic acids and fulvic acids are isolated in 0.1M NaOH + 6M urea systems. The mechanisms of release of these materials from the humin matrix may involve conformational alterations or cleavages of hydrogen bonding constraints. The residual materials released in DMSO + 6% H 2 SO 4 are compositionally different from the extracts in aqueous systems. These isolates would be classed as Humin Materials However, these do not satisfy the Operational definitions of Humic Substances
Conclusions (Continued) Thus, these so called humin materials are likely to be the most recalcitrant components of SOM. These are protected because of intimate associations between the molecules and clay surfaces. It is evident that, for the most part these are carbohydrates, peptides, peptidoglycans, waxes, lipids, aliphatic hydrocarbons, cutins, etc., but lignin derived (humic) subatances are likely to be there only because of entrapments. In other words the components in associations with the clays are predominantly organic molecules, whose structural types are well known.