2000 Conte Et Al Adv Environ Res

7/31/2019 2000 Conte Et Al Adv Environ Res

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Reprinted from

Advances in Environmental ResearchAn international journal of research in environmental science, engineering and technology

Advances in Environmental Research, 3 (4) 2000, 508-521

PERGAMON

An im pr in t of Elsevier Science

MOLECULAR SIZE OF HUMIC SUBSTANCES. SUPRAMOLECULAR

ASSOCIATIONS VERSUS MACROMOLECULAR POLYMERS

ALESSANDRO PICCOLO* AND PELLEGRINO CONTE

Dipartimento di Scienze Chimico-Agrarie, Universit di Napoli Federico II

Via Universit 100, 80055 Portici, Italy


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MOLECULAR SIZE OF HUMIC SUBSTANCES. SUPRAMOLECULAR

ASSOCIATIONS VERSUS MACROMOLECULAR POLYMERS

ALESSANDRO PICCOLO* AND PELLEGRINO CONTE

Dipartimento di Scienze Chimico-Agrarie, Universit di Napoli Federico II

Via Universit 100, 80055 Portici, Italy

This work illustrates recent experiments conducted by low- and high-pressure size exclusion chromatography(HPSEC) on humic substances of different origin and molecular structure. Solutions of humic substances wereeither injected as such into the HPSEC system that operated with eluents which differed in composition butwere constant in ionic strength, or were previously modified with mineral and organic acids and then eluted inthe HPSEC operating with a constant solution. The findings indicate that humic substances behave as associations

of relatively small molecules rather than as macromolecular polymers. The weak association of humic moleculesinto supramolecular structures is stabilized by weak hydrophobic forces. Interactions with mineral and organicacids of different degrees of aliphaticity may reversibly disrupt the supramolecular association and separatesmaller sub-units. The recognition that soil humus possesses supramolecular rather than polymeric properties isof importance in further understanding the function and reactivity of soil organic matter in the environment.

Key words: humic substances, supramolecular associations, self-association, size-exclusion chroma-tography, waters, soils, transport, contaminants

*Corresponding author. Fax: 0039-081-7755130; Email:[email protected]

508 Advances in Environmental Research, 3 (4) 2000Published by Elsevier Science Ltd. All rights reserved.

INTRODUCTION

Humic substances (HS) are natural organic

substances that are ubiquitous in water, soil, andsediments. Because of the beneficial effects of HSon the physical properties of soil, their role in the soilenvironment is significantly greater than that attrib-uted to their contribution to sustaining plant growth.HS are also recognized for their role in controllingboth the fate of environmental pollutants and thebiogeochemistry of organic carbon in the globalecosystem (Piccolo, 1996). Despite the obvious im-portance of the substances in sustaining life, theirbasic chemical nature and reactivities are still poorlyunderstood.

Because of their chemical heterogeneity and

polydispersity, our knowledge of the secondary chem-ical structure and absolute molecular weight valuesare inadequate. The heterogeneity can be expectedbecause HS of different origins are found to havevariable compositions, and detailed information onmolecular weights and sizes can be useful when

comparing different humic materials and when study-ing their reactivities with other chemical species.Several reviewers (e.g. Wershaw and Aiken, 1985;

Stevenson, 1994) have pointed out problems associ-ated with the techniques used to determine molecularweight values (such as freezing point depression,vapor pressure osmometry, light scattering, and sed-imentation methods) and molecular sizes (such assize exclusion chromatography, ultrafiltration, small-angle X-ray scattering) of HS.

Since humic matter is a complex mixture ofnaturally occurring organic substances, there is nomodel compound of known composition and mo-lecular weight (MW) that can be used to calibratethe various physicochemical techniques. As a resultthere is considerable uncertainty with regard to the

sizes and real molecular weight values. Data in theliterature suggest that these values may vary from500 daltons for some aquatic HS to more than 106

daltons for soil humic acids (Stevenson, 1994), andthere is not agreement between the different methodsused to evaluate MW values.

Pergamon


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Advances in Environmental Research, 3 (4) 2000

SUPRAMOLECULAR STRUCTURE OF HUMIC SUBSTANCES

The objective of this contribution is to furtherour understanding of the sizes and conformationsof HS on the basis of recent experimental resultsobtained by means of size exclusion chromatography.

MODELS OF SIZES AND SHAPES OFHUMIC SUBSTANCES

The concept of the polydispersity of HS has hadtraditional acceptance (Dubach and Metha, 1963),but there has also been a widely held assumption thatthe substances are macromolecular polymers in themode of other biological macromolecules of nature,such as proteins, polysaccharides, nucleic acids, andlignin (Swift, 1989). This view has been rationalizedby the hypothesis that humus synthesis is based oneither the lignin or the polyphenolic theories (Flaig etal., 1975). These theories rely on the supposition that

progressive polymerization of humic matter takesplace via covalent bonding, and that the processesare often mediated by soil enzymes. This represen-tation has fostered in the scientific community theperception that HS are macromolecular polymers inwhich basic (though heterogenoeous) monomericunits progressively build up into high MW polymersby random condensation and oxidation processes.Stevenson (1994) has listed a series of monomericand polymeric molecular structures which have beenproposed in the literature by a number of authors.However, even though the chemistry of these mono-mers or network structures has varied, depending on

the humic materials and/or the physicochemicaltechniques which provided the experimental data(Hatcher et al., 1980; Schulten et al., 1998), the ma-cromolecular structural concept, based on covalentpolymerization processes, has not until recently beenseriously questioned through sound experimentaldata. In the hypothetical model of humus formation,the randomness of the covalent polymerization ofmonomers accounted for the observed large poly-dispersity of humic macromolecules. Furthermore,the multiple conformational foldings that a polymericchain, either linear or branched, would assume in thesoil environment provided a plausible explanation for

its resistance to microbial degradation and the con-sequent long residence time observed for soil humiccomponents (Insam, 1996).

The success during the nineteen sixties of physi-cal biochemistry in explaining spatial molecular be-haviour of bio-polymers (DNA, proteins) introducedappealing models of conformational structure whichwere readily applied to humus chemistry. Visser(1964) used the concept of rigid globular particles

to account for the apparent macromolecular struc-tures of HS. Cameron et al. (1972) used low-pres-sure size exclusion chromatography (gel permeation)and ultrafiltration to decrease the polydispersity of ahumic acid (HA) and then measured by means of

sedimentation ultracentrifugation the MW values forthe fractions isolated. They obtained fractions withrelatively low polydispersities, and with MW valuesranging from 2 x 103 to 1.5 x 106 daltons. Never-theless, by plotting the frictional ratio values for eachsize-fraction versus the mean MW, they obtained arelationship which they interpreted to be indicativeof a random coil solution conformation and with adegree of branching at the higher MW values (wherethe data points deviated from linearity). Experimentsby others were interpreted as indicative of macro-molecularity with random coil conformations whichexhibited variations from linear, to spheroidal, to fil-

amentous shapes depending on the sample concen-tration, and the pH and ionic strengths of the media(Chen and Schnitzer, 1976; Ghosh and Schnitzer,1980).

Wershaw (1986) was first to postulate an alter-native description for the macromolecular structureof HS. His suggestions were based less on directmeasurements than on observations of experimentaldata from the literature which could not be explainedby the random-coil model. He proposed (Wershaw,1993) that HS in solution form mixed aggregates ofamphiphilic molecules of plant degradation productsand lignin-carbohydrate complexes. In his view, hu-

mic aggregates are held together by weak bondingmechanisms such as H- bonding, and hydrophobicinteractions, which would account for the unsuccess-ful attempts to separate monodisperse fractions fromhumic solutions. By considering the well establishedmicelle concept (Tanford, 1991), he suggested thathumic aggregates are like micelles in which liquid-like interiors are composed of hydrophobic portionsand highly charged components are positioned at theexternal surfaces. While the micellar model was morelikely than the polymeric-coil model to explain anumber of the findings described in the literature, theactual size of the humic aggregate and of its inner

and outer components was still undefined. Barak andChen (1992) maintained the random-coil polymericmodel in their explanation of dissociation constantsof HAs, but they did take account partially of a mi-celle-like behaviour.

Laboratory observations have indicated that whenacetic acid is added to HS that have been extensivelydialyzed, further small-sized components are releasedduring subsequent dialysis (Nardi et al., 1988). It was


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A. PICCOLO AND P. CONTE

found that the low molecular size fractions thusobtained stimulated specific biological properties inplants, and were more biologically active than thehumic materials from which they were separated(Piccolo et al., 1992). By means of low-pressure size

exclusion chromatography, Piccolo et al. (1996a,b)showed that a number of organic acids, as well asacetic acid (when added to progressively lower thepH values of humic solutions), were able to alter theoriginal molecular size distributions and to shifttowards higher elution volumes the UV-detectablehumic materials. This behaviour was explained byinvoking the amphiphilic properties of organic acidswhich are able to interact with both the hydrophilicand the hydrophobic domains of humic aggregatesthereby disrupting the weak forces that stabilize thehumic conformations and allowing the chromato-graphic separation of the small-size sub-units that

form the humic aggregate.Piccolo et al. (1996a,b) have shown that the

disrupting effect on humic size of acetic acid wasreversible since the original size distribution wasattained as the pH was progressively returned toalkalinity (Figure 1). Moreover, both disruption andaggregation processes appeared generally indepen-dent of ionic strength (Figure 1), which suggestedthat the observed phenomenon was due to the sep-aration of loosely-bound small humic sub-units ratherthan to retardation effects caused by gel-solute inter-actions, or to modifications of gel pore sizes.

In another approach Kenworthy and Hayes

(1997) used the fluorescence quenching of pyreneby bromide to investigate the nature of humic asso-ciations. They found that pyrene in solution in thepresence of HS was protected from bromide quench-ing. This protection was lost, however, when aceticacid, followed by base, was added to the medium.The authors considered that hydrophobic associationsof the humic molecules protected the pyrene fromthe bromide. Treatment of the humic solution withacetic acid removed that protection. This suggestedthat the HS in solution were associations of low mo-lecular weight masses held together by hydrophobicbonding and protecting the enveloped pyrene.

These preliminary experiments (Piccolo et al.,1996a, b; Kenworthy and Hayes, 1997) were thefirst direct evidence that the macromolecular struc-ture of HS may not be entirely polymeric. The highmolecular sizes that are often observed may be con-sidered to arise from associations of smaller mole-cules held together by weak and easily disruptableforces. Much of the behaviour of HS, and theirreactivities with other chemical species that had been

explained based on the folding or coiling of polymericstructures (Engebretson et al., 1996; Ragle et al.,1997; Lebouef and Weber, 1997; Chien and Bleam,1998), could well be explained by means of a modelin which small molecules associate to form larger

aggregates.

Figure 1. Low-pressure size exclusion chromatograms of a

humic acid treated with acetic acid and eluted with a 0.02M Na4B2O7 solution at pH 9.2 (I) and with a 0.1 M Na4B2O7solution at pH 9.2 (II). Humic acid was treated before elu-tion as follows: (A) dissolved at pH 11.8 (B) titrated withacetic acid to pH 6 (C) to pH 4.5 (D) to pH 3.5 (E) to pH 2;(F) the material brought to pH 2 was further back-titratedwith KOH to pH 3.5 (G) back-titrated to pH 4.5 (H) back-titrated to pH 6 (I) back-titrated to pH 8.5; (J) the lattermaterial at pH 8.5 was further roto-evaporated to attemptthe elimination of the residual acetic acid.


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HIGH-PERFORMANCE SIZE EXCLUSION

CHROMATOGRAPHY (HPSEC)

The conformational model of humic associationsintroduced by Piccolo et al. (1996a,b) was based onresults obtained using low pressure size exclusionchromatography. That technique, when used for an-alytical purposes, has severe limitations because itusually is poorly reproducible (adsorption on thepolysaccharide gel matrix), time consuming (about16-20 h for one run), and it requires careful handling(to avoid preferential flow) and frequent changes ofthe gel bed (at least every three or four runs). Theseshortcomings have prevented the low-pressure gel

permeation chromatography technique from provid-ing a more definite contribution to humus chemistry.

Conversely, the use in modern HPSEC systemsof pre-packed columns and rigid gel phases allowschromatographic runs to be made more rapidly and,because of slower diffusion of the sample in thecolumn, higher and more reproducible resolutionsare obtained than for low pressure chromatography.There have been applications of HPSEC to determinethe molecular sizes of HS and these have helped toset the best operational conditions (eluent concen-tration, ionic strength, etc.) in order to obtain truesize exclusion chromatograms for humic materials

(Saito and Hayano, 1979; Miles and Brezonik, 1983;Becher et al., 1985; Berden and Berggren, 1990;Rausa et al, 1991; Chin and Gschwend, 1991; vonWandruszka et al., 1999).

Conte and Piccolo (1999a) have compared thecapacities of two commercial HPSEC columns tomeasure accurately and precisely the molecular sizesof HS. They determined for each column the chro-matographic parameters, such as peak asimmetry

factors (As), the number of theoretical plates (N),

the coefficient of distribution (kd), and the columnresolution (R s). Both columns had been calibratedwith polysaccharides of known MW, and so it waspossible to obtain very reproducible weight-average(Mw) and number-average (Mn) molecular weightvalues for various HAs (Table 1) even though thechromatographic resolution differed according to thegel pore size of each column (Figures 2 and 3). Infact, the molecular sizes of the HAs were distributedin the TSK column (Figure 2) over a wider volumerange than in the Biosep column (Figure 3). However,the shape of the chromatographic peak for a singleHA sample differed in the two columns. In the case

of the TSK column, the HA2 and HA3 showed ahigher intensity for the first peak than for the second(diffused) peak, whereas in the case of the Biosepcolumn the same humic material gave a diffused peakthat had a higher intensity than the first peak. Thisdifference in peak intensities of HAs between thecolumns was not justified by the higher resolution ofthe TSK column. Moreover, both columns showed asimilar molecular size distribution for HA1 and HA4without the inversion in peak intensities noted for HA2and HA3. This discrepancy in the chromatographicbehaviour of the four samples was considered toprovide evidence that the different UV response be-

tween the effluents from these columns was due todifferent stabilities of humic associations rather thanto differences in column properties. If the latter wastrue a consistent difference in elution behaviour shouldhave been observed for all humic materials.

The differences between the HA2 and HA3 chro-matograms from the two columns can be explainedby considering the response of HS and biomoleculesto electron excitation. While it is known that humic

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substances do not strictly follow the Beers law andthat their molecular absorptivities vary with molec-ular size (Stevenson, 1994), a recognized property ofbiomolecules is that they show higher molecularabsorptivity when in random coil arrangements or

when represented by a mixture of monomer com-ponents in close association (Cantor and Shimmel,1980). This is because the close interaction and thereciprocal orientation of the transition dipole momentof an absorbing chromophore and the induced dipolesof neighboring chromophores may either increase(hyperchromism) or decrease (hypochromism) themolecular absorptivity (Cantor and Shimmel, 1980;Freifelder, 1982). The observed decrease in peakabsorbance in the effluent from the TSK columnindicates that the molecular absorptivity of the firstpeak is lower than in that from the Biosep column. Aprobable reason for the decrease in the UV-reading

(hypochromism) is that a separation of molecules (orchromophores) from the high-molecular size arrange-ment may have taken place during elution from theTSK column, thereby causing an alteration of thereciprocal orientation of the dipole moments among

chromophores. This can be attributed to the higherresolution capacity of the TSK column.

These results were in agreement with the asso-ciation model proposed by Piccolo et al. (1996a,b)and indicated that humic materials are loosely-boundmolecular aggregates which may be disrupted duringHPSEC elution. Humic molecules may be separatedbecause of interactions with the stationary phasewhich can take place when a humic aggregate dif-fuses through gel pores smaller than the hydrodynamicradius of the aggregate. The molecular absorptivityappears thus decreased, as is the absorbance readingof the eluting fraction of that molecular size. Although

Figure 2. HPSEC chromatograms of four different humicacids eluted through a TSK column.

Figure 3.HPSEC chromatograms of four different humicacids eluted through a Biosep column.


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shear degradation of water-soluble polymers duringthe passage of a sample through a HPSEC columnhad not been reported (Barth, 1980), high molecular-weight polystyrene was shear degraded in non-aque-ous HPSEC (Kirkland, 1976). Shear disruption ofthe weakly-bound humic conformation and conse-

quent hypochromism may thus explain the lower UVreadings for the diffused peaks of HA2 and HA3from the TSK column. The Biosep column has alower separation capacity, and it did not give the samemolecular disaggregation and hypochromic effect forthe humic materials diffusing through the gel pores.Thus the absorbance reading of its second (diffuse)peak remained higher than the first peak.

The findings of Conte and Piccolo (1999a) showedthat the behaviour of HS in both high- and in low-pressure chromatography is consistent with the modelfor association of small heterogeneous molecules intoapparently large high-molecular size structures. Since

the apparent macromolecular structure of HS reflectsa holding together of molecules by weak forces,HPSEC chromatograms might provide a measure ofthe conformational stabilities of humic materials fromdifferent sources.

Recently a HPSEC experiment has been carriedout by Conte and Piccolo (1999b) to verify the sta-bilities of various humic solutions when the mobilephase (0.05 M NaNO3, pH 7) was modified by smalladditions of methanol (4.6 x 10-7 M to a pH of 6.97),HCl (2.0 x 10-6 M to pH 5.54) and acetic acid (4.6x 10-7 M to a pH of 5.69) in order to keep constantthe ionic strength of the eluting solution. UV-Vis and

Refractive Index (RI) detectors were used to obtainthe chromatograms of the molecular size distributionof the humic materials. The objective was to comparethe chromatographic behavior of chromophores (UV-Vis) with that of the bulk of humic mass (RI).

Modifications of the mobile phase resulted in theprogressive decreases in the molecular sizes of humicsolutions in going from the control solution to thoseto which methanol, HCl, or acetic acid were added.

The HPSEC chromatograms obtained by means ofthe UV-Vis and RI detectors for a HA isolated froma Danish agricultural soil are shown in Figures 4 and5, respectively. The decrease in molecular size wasrevealed by the shift toward increasingly larger elu-tion volumes for both the UV and RI detectors, and

by the concomitant reductions of peaks absorbanceat the UV-Vis detector. The variation in molecularsize distribution in the different mobile phases wasreflected in the weight-average molecular weightsof the humic material calculated from the chromato-grams (Table 2).

The observed changes were not attributable tovariations in ionic strength because this was keptconstant in the different mobile phases, but could beattributable to the interactions of the added chemicalswith the weakly-associated macromolecular structureof the humic matter. In the case of the methanol ad-dition, no new ions were introduced, nor were there

changes in pH. Hence, the alteration of molecularsize distribution of the humic materials can be attrib-uted only to the capacity of the CH3OH to form bothvan der Waals bonds with the hydrophobic humiccomponents and hydrogen bonds with the oxygen-containing functional groups in the HA. Thus a verysmall amount of methanol in the eluting solution coulddisrupt the weak forces which temporarily stabilizedhumic associations in aggregates. The result can beinterpreted as a dispersion of the aggregates into thesmaller component humic molecules, the diffusionof these through smaller gel pores and an overalldecrease in molecular size (Table 2). This effect was

confirmed by the RI chromatogram (Figure 5) whichshowed a shift of humic mass towards elution volumestypical of lower molecular-weight materials.

While the RI detector has begun to be used inHPSEC studies of humic substances (von Wandrusz-ka et al., 1999) as an essential means to evaluatemass rather than chromophoric distribution of HS,the apparent confirmation of the association modelas evidenced by results from the RI detector may

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not be readily accepted by proponents of the poly-meric structure and the random-coil properties of HS.In fact, it could be argued that even the RI resultsmight not rule out the possibility that conformationalchanges (rather than the dispersion of weakly-asso-

ciated small molecules) might give a dramatic coilingdown of large macromolecules. Such an effect, bydecreasing the hydrodynamic radius, could lead toelution at higher elution volumes. That type of ex-planation has previously been used to account forshifts in gel permeation to higher elution volumeswhen HS were subjected to cation additions (Swift,1989), or to changes in ionic strength (Berden andBerggren, 1991).

However, the traditional polymeric model of HScan not explain the experimental observation of thedecreased peak intensities (seen in UV chromato-grams where the various treatments were applied)

compared with those for the control solution (Figure

4). In fact, the substantial reduction in peak intensityrevealed by the UV detector should be regarded asevidence for the hypochromic effect described above.Not only were the peaks shifted to higher elutionvolumes (lower molecular sizes), but the decreases

in molecular absorptivities of the humic fractionsindicated that the chromophores were drawn apartfrom each other because of the disrupting effect ofmethanol on the loosely associated humic structures.Conversely, if the humic samples had been composedof polymeric macromolecules which, despite theconstant ionic strength, had coiled down to give thechanges observed in the elution profiles, one wouldhave expected an increase in molecular absorptivityand UV readings with respect to the control solution.

The greater changes produced by decreasing thepH of the control solution (with addition of HCl) to5.54 were due to a larger disruption (than for meth-

anol) of the humic molecular associations (Figures 4

Figure 4. HPSEC chromatograms of a humic acid from a Danish soil recorded with the UV-Vis detector. A = control mobilephase (0.05 M NaNO3, pH=7, I= 0.05); B = same as A but 4.6x10

-7 M in methanol (final pH 6.97); C = same as A but to pH 5.54with HCl; D = same as A but 4.6x10-7 M in acetic acid (final pH 5.69).


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and 5). The additional hydrogen ions in this mobilephase protonated the humic carboxylic functionalities(which were in their dissociated forms at pH 7 of thecontrol mobile phase). A number of negative chargeswere then neutralized and hydrogen bondings were

concomitantly formed between the complementaryfunctionalities in the humic molecules. In that waythe conformational stability that existed in the controlsolution was disrupted. Due to the parallel observationthat a reduction in peak absorbance (hypochromism)was seen in the UV chromatograms, and that a shiftof humic mass to high elution volumes was visible inRI chromatograms, this change could not simply bedue to a volume decrease of the random coil, as waspreviously suggested (Swift and Posner, 1971; Ghoshand Schnitzer, 1980; Berden and Berggren, 1991).Again, another explanation is that the heterogeneoushumic conformation collapsed into molecular associa-

tions of smaller dimensions, but of greater thermo-

dynamic stabilities than for the control solution. Thechemical rationale for this behaviour lies in the energygained in hydrogen bond formation (ranging from 10to 20 kJ mol-1) compared with van der Waals bonding(Schwarzenbach et al., 1993). Humic molecules,

protonated by HCl addition, abandoned the looseconformation assumed at pH 7 of the control solutionand formed relatively strong intermolecular hydrogenbonds. The concomitant large decrease in molecularsize suggests that the weak association of apparentlyhigh molecular size, as observed for the HA in thecontrol solution, must therefore have been due pre-dominantly to weak intermolecular hydrophobic forces(van der Waals bonding) holding small moleculestogether.

The further decrease in molecular size distributionwith acetic acid addition (Figure 4 and Table 2) canbe attributed to the methyl group of the acetic acid.

As for HCl addition, acidification of the solution

Figure 5. HPSEC chromatograms of a humic acid from a Danish soil recorded with the RI detector. A = control mobile phase(0.05 M NaNO3, pH=7, I= 0.05); B = same as A but 4.6x10

-7 M in methanol (final pH 6.97); C = same as A but to pH 5.54 withHCl; D = same as A but 4.6x10-7 M in acetic acid (final pH 5.69).


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favours hydrogen bonding formation. Moreover, theweak acidity of CH3 COOH (pKa 4.8) will allow asmall number of undissociated species to exist at pH5.69 and thus the formation of mixed intermolecularhydrogen bondings with humic molecules. As de-

scribed already, such energy-driven rearrangementswill outweigh the weak humic associations in thecontrol solution that were stabilized mainly by hydro-phobic forces. However, the shift to higher elutionvolumes, and the general reduction of molecular

absorbivity in the UV chromatogram (hypochrom-ism), as well as the large shift to the total columnvolume of the humic mass in the RI chromatogram,would suggest that the methyl group of acetic acidplayed an additional role in further disrupting the

weakly-bound humic associations. The apolar methylgroup of acetic acid must have altered the residualhydrophobic forces which still stabilized the humicassociations even after the hydrogen bonding re-arrangement had taken place.

Figure 6. HPSEC chromatograms of a humic acid from an Italian volcanic soil eluted in a mobile phase made of 0.05 MNaNO3, pH=7, I= 0.05. A, control humic sample dissolved in the mobile phase prior to injection; B, as for A but with HCladded to lower pH of solution to 3.5; C, as for A but with formic acid added to lower pH of solution to 3.5; D, as for A butwith acetic acid added to lower pH of solution to 3.5; E, as for A but with propionic acid added to lower pH of solution to3.5; F, as for A but with butyric acid added to lower pH of solution to 3.5.


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Table 3). Conversely, HAs containing more hydro-phobic carbons were reduced to lower molecularsizes by the action of monocarboxylic acids withhigher numbers of carbon atoms, such as propionicand butyrric acids.

The findings of Piccolo et al. (1999) representadditional evidence that HS do not behave as po-lymeric random coils. They also indicate that theHPSEC technique can be used to reproducibly de-crease the apparently large dimensions of humicassociations into fractions of smaller molecularsizes as the result of simple interactions with mono-carboxilic acids. Moreover, the extent of the sizereductions of humic associations is dependent on thealiphatic chain lengths of the acids, and on thehydrophobicities of the HS.

The efforts to improve soil physical stability inorder to fight erosion and desertification can be

supported by considerations of a model involvingsupramolecular aggregation of long-chain hydro-phobic compounds in soils. In fact, soil aggregatestability was found to be improved more by hydro-phobic than by hydrophilic waste materials (Piccoloand Mbagwu, 1999) in either temperate or tropicalclimates (Spaccini et al., 1999). Finally, the supra-molecular nature of humic molecules in soil and watermay well account for the short- and long-term bindingof pesticides and other organic contaminants tonatural organic matter, and for the mobility of thesein terrestrial and aquatic environments. Piccolo etal. (1996c; 1998) have shown that adsorption and

desorption of widely used pesticides, such as Glypho-sate and Atrazine, depends on the aliphatic characterof humic substances and on their conformationalarrangement.

CONCLUSIONS

The experimental evidence collected in previousworks, first by low pressure and then by HPSECchromatography, casts doubt on the concept that HSisolated from soils and from other sources consist ofmacromolecular polymers as they are commonlyreported (Macalady and Ranville, 1998). Our view

is that the polidispersity of HS is not due to randomvariations in high MW values during the enzyme-assisted covalent polymerization of small molecules(Bollag and Loll, 1983; Swift, 1989), but rather to theself-assembly of different small molecules into supra-molecular structures of different sizes.

Humic supramolecular associations are tempo-rarily stabilized in aqueous solutions by weak forces,such as hydrogen bonds and van der Waals forces,

depending on the pH of the solution and on the hydro-phobic character of humic components, respectively.It is conceivable that the most polar components formassociations which are sufficiently hydrated to bestable and mobile in aqueous solutions, and can

separate from the bulk of the humic supramolecularstructure. These would represent the small-size frac-tions of the humic polydisperse system. Fulvic acidsare excellent examples of such low-molecular sizefractions. Conversely, less polar components reducethe free energy of solvation by withdrawing fromwater and self-assembling into supramolecular as-sociations of larger dimensions which are stabilizedby relatively strong hydrophobic forces. This type ofhigh molecular size association well describes theHA fraction of the HS.

HPSEC experiments have suggested that thehigh-molecular size fraction eluted at the void vol-

ume of the column (the first peak) is composed mainlyof apolar components that are self-associated intohydrophobic domains, while the lower-sized fractionswhich elute in the diffused peak would then be com-posed of polar compounds in hydrophilic associations.The weak forces which stabilize both hydrophilic andhydrophobic domains, and determine the polydisper-sity of the samples, are easily overcome by inter-actions with amphiphilic molecules. The originalsupramolecular structure of HS is thus disrupted todifferent extents, which depend on the stability ofhydrophobic domains and on their affinities with thedisrupting molecules. The polydispersity is concom-

itantly reduced because a larger number of small-size associations, or even single molecules, may hencebe liberated.

The consequences to soil science of the conceptof humus supramolecular structure may be far reach-ing. It provides a physicochemical framework to thegrowing consensus on the hypothesis that organicmatter accumulates in soil because of selective pres-ervation of recalcitrant compounds of plant origin(Hatcher et al., 1981; de Leeuw and Largeau, 1993;Almendros et al, 1996; Piccolo et al., 1999b) and torecent results based on 13C measurements (Licht-fouse et al., 1998) and on NMR spectra (Almendroset al., 1998) which concomitantly indicate that re-sistant highly aliphatic microbial compounds are thehydrophobic components of humic substances. Thesupramolecular aggregation of resistant aliphaticstructures, and hence their confinement from waterand microbial activity, represents an important mech-anism of organic matter accumulation in soil andprovides an alternative physicochemical explanationfor the long residence time of soil humus (Piccolo,1996; Piccolo et al., 1999b).


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ACKNOWLEDGMENTS

This work was partially supported by the ItalianMinistry of University and Scientific and Techno-logical Research (MURST) through the project no.

9807352092. The second author is grateful to theNational Inter-University Consortium Chemistry forthe Environment for his post-doc fellowship.

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