conformational arrangement of dissolved humic substances. influence of solution composition on...

Conformational Arrangement ofDissolved Humic Substances.Influence of Solution Compositionon Association of Humic MoleculesP E L L E G R I N O C O N T E A N DA L E S S A N D R O P I C C O L O *

Dipartimento di Scienze Chimico-Agrarie, Universita diNapoli “Federico II”, Via Universita 100, 80055 Portici, Italy

Both the primary chemical structure and the conformationalstructure of humic substances are still a matter ofdebate. A traditional assumption is that humic substancesare large polymers and may present linear or coiledconformations according to solution properties. We studiedthe conformational changes of humic and fulvic acids ofdifferent chemical nature by high-pressure size-exclusionchromatography (HPSEC) after dissolution in mobilephases differing in composition but constant in ionic strength(I ) 0.05). Modification of a neutral mobile phase (0.05M NaNO3, pH 7) by addition of methanol (4.6 × 10-7 M, pH6.97), hydrochloric acid (<2 × 10-6 M, pH 5.54), andacetic acid (4.6 × 10-7 M, pH 5.69) produced, in the order,a progressive decrease in molecular size. Size diminishingwas shown by increasingly larger elution volumes at arefractive index detector and by concomitant reductionsof peaks absorbance at a UV-vis detector. The decreaseof molecular absorptivity (the phenomenon of hypochromism)proved that size reduction of dissolved humic substanceswas due more to disruption of an only apparent high-molecular-size arrangement into several smaller molecularassociations than to coiling down of a macromolecularstructure. The most significant conformational changesoccurred in acidic mobile phases where hydrogen bondingsformation was induced, suggesting that the large andeasily disruptable humic conformation was held togetherpredominantly by weak hydrophobic forces. The size ofmolecular association varied with humic samples indicatinga close relation between humic chemical compositionand stability of conformational structure. Our results showthat humic substances in solution are loosely bound self-association of relatively small molecules, and intermolecularhydrophobic interactions are the predominant bindingforces. The stability of such a conformation in solution isattributed to the entropy-driven tendency to exclude watermolecules from humic association and thus decreasetotal molecular energy. This model of dissolved humicsubstances based on the reversible self-association of smallmolecules rather than on the macromolecular randomcoil represents a new understanding that should contributeto predict the environmental behavior of contaminants inassociation with natural organic matter.

IntroductionThe characteristics and quantity of humic substances greatlyaffects the environmental fate of organic pollutants in soilsand natural waters (1-3). The molecular properties ofdissolved humic substances have been recently recognizedto influence removal of synthetic organic compounds frommunicipal and industrial wastewaters (4, 5). The molecularsize of humic substances were found responsible for thebinding and environmental transport of both nonpolar (6,7) and slightly polar (pesticides) organic contaminants (8,9).

Despite their prominent environmental role, humicsubstances molecular and conformational structures are farfrom being elucidated. A common belief is that they are coiled,long-chain molecules which may be slightly cross-linked (10).Formation of negative charges from ionization of carboxylgroups is responsible for the mutual repulsion and expansionof the coil (11). The random coil model (12) depicts the humicmacromolecules as most densely coiled at high concentra-tion, low pH, and high ionic strength, whereas they behavelike flexible linear polymers at neutral pH, low ionic strength,and low concentration. This model has explained resultsobtained by gel permeation chromatography (13, 14) or bydiffusion through ultrafiltration membranes (15). A generalobservation was that by increasing the ionic strength ofmobile phase, the molecular size distribution of humicmolecules changed considerably by retarding elution at largerpermeation volumes. This change was attributed to coilingdown of the humic conformation with consequent reductionof the hydrodynamic radius and enhanced diffusion throughsmaller gel pores. The same phenomenon was observed byvarying ionic strength of mobile phase in High PerformanceSize Exclusion Chromatography (HPSEC) experiments withorganic colloids from natural waters (16, 17). Anotherconformational model describes the aggregation of humicmacromolecules as micelle-like or membrane-like structures(18, 19). The humic polymers are then thought to presentstructural voids where apolar organic compounds may beentrapped (20) and quench their fluorescence activity (21).Recent results (22, 23) have indicated that humic aggregatesare composed of relatively small subunits mainly heldtogether by weak hydrophobic forces. The high moleculardimension of humic material was only apparent since it wasfound to be reversibly disrupted by an organic acid undera rapid change from acidic to alkaline pH. These findingssuggested that dissolved humic substances, rather than beingpolymeric coils as previously believed (10-12) for analogyto biological macromolecules (24), appear to reflect thestructure of randomly self-associating small heterogeneousmolecules.

This study had the objective to gain further informationon the association modes of humic material in solution.Changes in the conformational behavior of dissolved humicand fulvic acids of different molecular structure are describedwhen the mobile phase of a size-exclusion chromatographysystem was varied in composition but not in ionic strength.

Materials and MethodsHumic Substances. Humic acids (HAs) were obtained from(HA1) a North Dakota Leonardite (Mammoth, Chem. Co);(HA2) a Danish agricultural soil (Roskilde, DK) classified asHaplic Luvisol; and (HA3) a German agricultural soil (Munich,DE) classified as Haplic Luvisol. Fulvic acids (FAs) wereobtained from (FA1) the above Danish and (FA2) Germansoil and (FA3) an Italian soil (Caserta, IT) classified as Eutric

* Corresponding author phone: 39-081-7885236; fax: 39-081-7755130; e-mail: [email protected].

Environ. Sci. Technol. 1999, 33, 1682-1690

1682 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 33, NO. 10, 1999 10.1021/es9808604 CCC: $18.00 1999 American Chemical SocietyPublished on Web 04/13/1999

Regosol. Humic substances were extracted by standardprocedures (10). Original materials were shaken overnightin a 0.5 M NaOH and 0.1 M Na4P2O7 solution under N2

atmosphere. Humic acids (HAs) were precipitated fromalkaline extracts by lowering the pH to 1 with 6 M HCl. HAswere extensively purified by three cycles of dissolution in 0.1M NaOH and subsequent precipitation in 6 M HCl. HAs werethen treated with a 0.5% (v/v) HCl-HF solution for 36 h,dialyzed (Spectrapore 3 dialysis tubes, 3500 MW cutoff)against distilled water until chloride-free, and freeze-dried.Fulvic acids (FAs), the humic material left in solution afterprecipitation of HAs at pH 1, were purified by absorbing ona Amberlite XAD8 resin (25), eluting by a 1 M NaOH solution,and, after adjusting the pH to 5, dialyzing in Spectrapore 3tubes against distilled water until chloride-free and freeze-dried. Both HAs and FAs were then redissolved in 0.5 M NaOHand passed through a strong cation-exchange resin (Dowex50) to further eliminate divalent and trivalent metals andfreeze-dried again. All humic samples were characterizedfor their elemental content using a Fisons EA 1108 ElementalAnalyzer. The ash content was less than 5% in all humicsamples. Both HA and FA samples (50 mg) were subsequentlysuspended in distilled water (50 mL) and titrated for 2 h topH 7 with a CO2-free solution of 0.5 M NaOH by an automatictitrator (VIT 90 Videotitrator, Radiometer, Copenhagen)under N2 atmosphere and stirring. The resulting sodium-humates were then filtered through a Millipore 0.45 µm andfreeze-dried.

Solid State NMR Spectroscopy. Cross-Polarization MagicAngle Spinning Carbon-13 Nuclear Magnetic Resonance(CPMAS 13C NMR) experiments were performed on a BrukerAMX400 operating at 100.625 MHz on carbon-13. The rotorspin rate was set at 4500 Hz. A recycle time of 1 s and anacquisition time of 13 ms were used. All experiments wereconducted with Variable Contact Time (VCT) pulse sequencein order to find the Optimum Contact Time (OCT) for eachsample and to minimize errors on evaluation of peak areas(26). OCT ranged from 0.8 to 1 ms. A line broadening of 50Hz was used to transform the FIDs. The 110-140 ppm regionwas subtracted by the sideband area of the 190-230 ppminterval.

Molecular Size Determinations by HPSEC. Molecularsize distribution of humic materials was evaluated by highperformance size exclusion chromatography (HPSEC). Thisconsisted of a Perkin-Elmer LC-200 solvent pump followedby two detectors in series: a UV-vis variable wavelengthdetector (Perkin-Elmer LC-295) set at 280 nm and a refractiveindex (RI) detector (Fysons Instruments, RefractomonitorIV). Humic solutions were loaded by a rotary injector witha 100 µL sample loop. Size exclusion separation occurredthrough a G3000SW (600 mm × 7.5 mm i.d.) TSK column(Toso Haas). The stationary phase is a rigid spherical silicagel chemically bonded with hydrophilic compounds (27) withan alleged low residual hydrophobicity and minimal ionexchange capacity. The column was preceded by a 7.5 cmTSK Guard-Column (7.5 mm i.d.) packed with G3000SW andby a 0.2 µm stainless steel inlet filter. The column system wasthermostated at 25 °C. The column manufacturer only reportsa calibration for globular proteins of 5-300 KD as nominalseparation range. Experimental results have shown thatcolumn separation range varied with calibration standardsand their molecular-size (28).

Absolute measurements of molecular weights of humicsubstances by column calibration is made futile by ignoranceof their chemical structures. For any possible calibrationstandard, the hydrodynamic radius and the interaction withthe stationary phase are bound to be different from those ofhumic substances (11). A number of investigators (17, 29, 30)have used the globular protein standards despite theirrecognized overprediction of humic substances molecular

weights. Polystyrene sulfonates (PSS) are also popularstandards in exclusion studies of humic substances (29-31).These compounds were reported to have a coiled configu-ration similar to a single fulvic acid from the Suwannee Riverat pH 6.8 and I ) 0.1 M (32). However, PSS have an aromaticC content much greater than FA molecules (31) and a ratherdifferent branching and cross-linking degree (17). Further-more, the average pKa of PSS (benzenesulfonate has a pKa

of 0.7 according to the Handbook of Physics and Chemistry,1976) is rather different than the average pKa interval, about4-7, usually attributed to humic substances (10). Thus, asimilarity in charge density between PSS and humic sub-stances cannot be generally accepted. Inconsistencies in size-exclusion behavior of PSS with I and pH changes have beenalready described and only partially explained by hydro-phobic gel-solute interactions (17, 31). Nonionic, hydrophilicbiomolecules such as polysaccharides have been successfullyused to evaluate molecular size distributions of dissolvedhumic substances from different sources when eluted indilute salts such as a 0.05 M NaNO3 at pH 7 and constantionic strength (33). This neutral mobile phase was suitablefor rapid and efficient HPSEC of humic substances and foravoiding phenomena of solute-stationary phase interactions(31, 33). Moreover, the 0.05 M concentration was consideredthe best compromise between sample solubility and abilityof humic molecules to form a fully coiled conformation insolution (12).

Polysaccharides of known MW (186 KD, 100 KD, 48 KD,23.7 KD, 12.2 KD, 5.8 KD) were used as calibration standards(Polymer Sciences Laboratories, UK) whereas void volume(V0 ) 10.7 mL) and total volume (Vt ) 24.7 mL) wasdetermined with blue dextran and acetone, respectively.Calibration curves were semilog linear over the range definedby our standards and evaluated the molecular weight of ananalyte, Mi, at some eluted volume i. Elution was at the flowrate of 0.6 mL‚min-1 (33).

Four different mobile phases were used for sampledissolution and HPSEC elution: (A) a solution at pH 7.01 of0.05 M NaNO3 and 4.0 × 10-3 M NaN3 (the latter to suppressmicrobial activity); (B) as in A but 4.6 × 10-7 M in methanol(final pH was 6.97); (C) as in A but brought to pH 5.54 withconcentrated HCl; and (D) as in A but 4.6 × 10-7 M in aceticacid (final pH was 5.69). All mobile phases had the sameionic strength (I ) 0.0504 M) as control solution A, since thelow ionic changes introduced in solution C and D (e2.0 ×10-6 M) did not significantly vary I values. Column wascalibrated by polysaccharides standards for all mobile phases.No significant differences were found in calibration curves,thereby showing that column behavior was not affected bycomposition of solution. All mobile phases were made withMilliQ water and HPLC-grade reagents, filtered throughMillipore 0.45 µm filters and He degassed.

Sodium-humates and fulvates at pH 7 (see above) wereused to exclude the random occurrence of negative chargeson solute molecules when dissolved into the mobile phase.Uncontrolled formation of negative charges on the solute isbelieved to change ionic strength of humic solutions therebyaffecting sample exclusion (35). In this study, charge densitywas the same for each humic sample dissolved in all mobilephases except for the modifications brought about bydifferences in mobile phases themselves. Freeze-dried so-dium salts of humic samples were dissolved in the samesolution of the mobile phase under study, immediately passedthrough a 0.2 µ filter (PVDF Millipore), and injected into theHPSEC system operating with one of the four mobile phases.Matching the conditions of injectate to that of mobile phaseis reported to minimize the propensity of colloids to changetheir degree of coiling and allows consistent size exclusionto occur (17, 36). A concentration of 0.5 g L-1 was used foreach humic solution because it was found not to influence

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the chromatogram’s shape and not to show adsorptivephenomena between sample and stationary phase (16, 33).Humic solutions were prepared anew before each injection.Three replicates of each humic sample were run for eachmobile phase. Relative standard deviation of Mw values insucceeding sample injections never exceeded 5%, therebyconfirming the extreme reproducibility of HPSEC analysesof humic substances reported earlier (30, 32, 34). Suchprecision is also an evidence for absence of reversible andirreversible solute adsorption on stationary phase (34, 35).

Size exclusion chromatograms for both the UV-vis andRI detectors were evaluated by using Perkin-Elmer-Nelson-Turbochrom 4-SEC peak integration and molecular weightsoftware. Calculation of weight- (Mw) and number-averaged(Mn) molecular weights and polydispersity (Mw/Mn) weredone by the method of Yau et al. (36) using the followingequations

and

where hi is the height of the size exclusion chromatogramof each sample eluted at volume i. Based on the describedmethod (36), the system dispersion was found to be lessthan the error (<5%), and the chromatograms were evaluatedwithout additional correction factors.

Results and DiscussionMolecular Structure of Humic Substances. The structuralfeatures of HAs and FAs shown by CPMAS-13C NMR spectra(Figures 1 and 2) were not different from those reported forterrestrial humic substances by other investigators (37-39).In general, HAs showed sharper resonances for aliphaticcarbons (19-23 ppm) than FAs, which instead revealed moreintense signals for peptides and carbohydrates signals (atabout 50 and 66 ppm, respectively). In particular, Leonarditehumic acid (HA1) had molecular composition substantiallydifferent from that of the other two HAs. HA1 signals foraliphatic (23 ppm) and aromatic (125 ppm) carbons weredistinctly more intense than for soil-derived HA2 and HA3,whereas nitrogen-bound carbons (40-50 ppm) were hardlyvisible for HA1. Differences in humic chemical nature were

also shown by their elemental composition and C/H andC/N ratios (Table 1). Quantitative evaluation of NMR signals(Table 2) showed that HA1 from lignite had a higher contentof aliphatic (0-40 ppm) and aromatic (110-140) carbonsthan HA2 and HA3 from arable soils. Conversely, the latterHAs showed larger carbon content in the 40-110 ppm range,while no significant differences were found for the carboxylcarbons (160-190). A slight increased aliphatic and aromaticcarbon content in FAs was observed when passing fromnorthern to southern European soils (Table 2). Aromaticityof HA1 was twice that of HA2, four times that of HA3, andabout six times that of FAs (Table 2) thereby confirming thestructural differences existing among humic materials. Aro-maticity values were largely below those shown by otherinvestigators which reported aromaticity for river FAs rangingfrom 12.6 to 24.8% (32, 40) and for soil and lignite HAs ashigh as 50-58% (6, 7). The discrepancy between literatureand this study may be attributed to applied NMR state. Recentresults (41) indicated a significant difference betweenquantitative data obtained by liquid- or solid-state 13C NMRspectra of humic substances. In liquid-state NMR, theconformational rigidity of aliphatic carbons in poorly dis-solved humic hydrophobic domains prevents the fast spin-lattice relaxation of these carbons (40), thereby reducingspectral signals in the 0-40 ppm range. Underestimation ofaliphatic carbons leads to overestimation of aromatic carbonsas it may have been the case for the high aromaticity reportedin the literature (6, 7, 32, 40). Conversely, in solid-state CPMASNMR, carbon signals do not depend on spin-lattice relax-ation and hydrophobicity of humic microsites but rather onthe number of adjacent hydrogens. All carbons, exceptquaternary ones, are hence quantitatively evaluated inCPMAS NMR, thereby appearing more reliable than liquid-NMR for molecular investigation of humic substances.

FIGURE 1. CPMAS NMR spectra of humic acids.

Mw ) ∑i)1

N

hi(Mi)/∑i)1

N

hi

Mn ) ∑i)1

N

hi/∑i)1

N

hi/Mi

FIGURE 2. CPMAS NMR spectra of fulvic acids.

TABLE 1. Elemental Analyses of Humic Samples Used in ThisStudy

samplea %C %H %N C/H C/N

HA1 54.06 4.39 0.96 12.4 47.8HA2 50.26 5.31 4.70 9.5 10.7HA3 49.54 5.51 4.64 9.0 10.7FA1 37.80 4.72 4.72 8.0 8.0FA2 38.60 4.42 4.12 8.7 9.2FA3 21.96 4.39 3.29 5.0 6.7

a See meaning in text.

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Data from solid-state NMR can correctly show the humicstructural differences that may affect the conformationalstructure of the HAs in aqueous solution. As in the case ofproteins, which reduce the free-energy of solution byassuming the conformation that best excludes hydrophobiccomponents from water (42), humic substances in solutionshould arrange their conformation to confine most of theirhydrophobic constituents away from water. The concept ofhumic matter as a self-association of small heterogeneousmolecules (22, 23) leads to a conformational arrangement inwhich hydrophobic domains which are stabilized by weakforces such as van der Waals bondings are contiguous tohydrophilic domains which are stabilized by hydrogenbondings. The size and number of both domains aredependent on the molecular nature of the humic componentswhich ultimately determine the reactivity of a specific humicmaterial.

Changes of Molecular-Size Distributions with MobilePhases. UV chromatograms show the distribution of mo-lecular absoptivity of humic chromophore groups absorbingat the chosen UV wavelength, whereas RI chromatogramsprovide the overall mass distribution of humic matter. Formobile phase A, both UV and RI Mw values showed thatmolecular sizes of HAs were higher than for FAs and so wasthe polydispersity (Table 3). HA1 had the lowest Mw of theHAs with good agreement between UV and RI values.Differences in molecular properties are readily inferred bycomparing chromatograms of HA1 and HA2 in both UV andRI mode (Figures 3-6). UV chromatograms (Figures 3 and

5) obtained in mobile phase A (control) appeared composedof two peaks for both humic acids. However, HA1 showeda small first peak (high-molecular-size material eluting atthe void volume) and a large second peak (low-molecular-size material diffusing through smaller column pores),whereas HA2 showed an intense and sharp first peak and acomparably large second peak, thereby revealing a muchhigher variability in molecular size than HA1. Such molecular-size difference were confirmed by comparing RI chromato-grams (Figures 4 and 6). Similar Mw values and polydisper-sities were found for all FAs in mobile phase A (Table 3). Thiswas shown by both UV (Figures 7 and 9) and RI (Figures 8and 10) chromatograms. The significantly lower molecularsize of FAs, as compared to HAs, was indicated both by higherelution volumes of their diffused peak in UV chromatogramsand by the considerable material eluting at total volumes inRI chromatograms of both FA2 and FA3. No material elutingat total volumes was in fact observed in the RI chromatogramsof the HAs (Figures 4 and 6).

Mobile phase B (solution A made 4.6 × 10-7 M in methanolbut with very similar pH) modified chromatograms of bothHAs and FAs and their respective calculated Mw values (Table3). UV and RI chromatograms of HA1 (Figures 3 and 4) werealtered by methanol addition much more than for HA2(Figures 5 and 6). The first peak in the UV chromatogram ofHA1 completely disappeared, whereas the second (diffused)peak was more than halved in intensity and elution of itsmaximum retarded from about 18 mL (in solution A) to 21mL. The RI chromatogram of HA1 confirmed these changes

TABLE 2. Distribution of C Intensity in Different Regions (ppm) of 13C-NMR Spectra of Humic Samples

sample0-40

aliphatic40-110

C-O, C-N110-140aromatic

140-160phenolic

160-190carboxyl

hydrophobicindexa aromaticityb

HA1 44.1 18.6 13.6 8.5 15.2 1.36 22.02HA2 38.5 37.2 3.8 6.4 14.1 0.73 9.7HA3 40.8 41.3 1.1 4.3 13.0 0.72 5.4FA1 36.6 42.7 1.2 2.4 17.1 0.60 3.6FA2 34.6 42.0 0.9 2.8 14.9 0.59 3.6FA3 40.2 36.6 2.4 2.4 18.3 0.74 4.9

a [(0-40) + (110-140)]/[(40-110) + (140-190)]. b (110-140)/(0-190).

TABLE 3. Weight-Average Molecular Weight (Mw), and Polydispersity (P) of Humic Samples in Different Mobile Phases asDetermined by UV and RI Detectorsa

Ab Bc Cd De

sample Mw Pf Mw Pf % Mw Pf % Mw Pf %

HA1UV 17000 2.0 7900 1.5 53.5 9000 1.8 47.0 3500 1.1 79.4RI 16650 3.5 7790 1.7 53.2 7990 1.5 52.0 2200 1.0 86.7

HA2UV 35000 3.6 34000 3.9 2.8 6500 1.9 81.4 5300 1.4 84.8RI 51130 5.2 49670 5.3 2.9 3100 1.1 93.4 10720 1.3 79.0

HA3UV 39000 3.6 38000 3.9 2.5 6900 1.5 82.3 4600 1.2 88.2RI 56980 5.3 55270 5.3 3.0 25240 2.3 55.7 2190 1.0 96.1

FA1UV 10000 1.6 9000 1.6 10.0 7000 1.5 30.0 5100 1.3 49.0RI 5470 2.0 5100 1.9 6.7 4200 1.5 23.2 1500 1.3 72.5

FA2UV 12000 1.8 11000 1.8 8.3 8600 1.7 28.3 6700 1.4 44.2RI 6550 2.1 6230 1.9 4.8 5170 1.6 21.0 1490 1.1 77.2

FA3UV 11000 1.7 9600 1.7 12.7 7000 1.5 36.4 5100 1.3 53.6RI 6000 1.9 4910 2.1 18.2 4280 1.5 28.7 3480 1.7 42.0

a Mw changes (%) as compared to control mobile phase (A) are reported. b A: NaNO3 (pH 7). c B: CH3OH (pH 6.97). d C: HCl (pH 5.54). e D: AcOH(pH 5.69). f Pc polydispersion (Mw/Mn).


and even showed the appearance of low-molecular sizefractions eluting at the total volume. UV and RI chromato-grams of HA2 showed less modifications than for HA1, buta new RI peak at the total volume was again observed (Figure

6). Methanol slightly shifted the main diffused peak to higherelution volumes for both FA2 and FA3, but a significantreduction in peak intensity was noticeable more in the UV

FIGURE 3. Size exclusion chromatograms of HA1 recorded with theUV-vis detector. A ) control mobile phase (0.05 M NaNO3, pH )7, I ) 0.05); B ) same as A but 4.6 × 10-7 M in methanol (final pH6.97); C ) same as A but to pH 5.54 with HCl; and D ) same as Abut 4.6 × 10-7 M in acetic acid (final pH 5.69).

FIGURE 4. Size exclusion chromatograms of HA1 recorded with theRI detector. A ) control mobile phase (0.05 M NaNO3, pH ) 7, I )0.05); B ) same as A but 4.6 × 10-7 M in methanol (final pH 6.97);C ) same as A but to pH 5.54 with HCl; and D ) same as A but4.6 × 10-7 M in acetic acid (final pH 5.69).

FIGURE 5. Size exclusion chromatograms of HA2 recorded with theUV-vis detector. A ) control mobile phase (0.05 M NaNO3, pH )7, I ) 0.05); B ) same as A but 4.6 × 10-7 M in methanol (final pH6.97); C ) same as A but to pH 5.54 with HCl; and D ) same as Abut 4.6 × 10-7 M in acetic acid (final pH 5.69).

FIGURE 6. Size exclusion chromatograms of HA2 recorded with theRI detector. A ) control mobile phase (0.05 M NaNO3, pH ) 7, I )0.05); B ) same as A but 4.6 × 10-7 M in methanol (final pH 6.97);C ) same as A but to pH 5.54 with HCl; and D ) same as A but4.6 × 10-7 M in acetic acid (final pH 5.69).


chromatogram of FA3 than for FA2 (Figures 7 and 9). RIchromatograms (Figures 8 and 10) also showed a differentmass distribution for FAs.

Mobile phase C (solution A brought to pH 5.54 with HCl)was generally very effective in reducing Mw values of all humicsamples for both UV and RI detectors (Table 3). The UVchromatogram of HA1 (Figure 3) showed the disappearanceof the small peak at the void volume and a further slightretardation in the second peak elution. However, the latterpeak showed a higher intensity than for solution B, therebyjustifying a higher Mw value (Table 3). The UV chromatogramof HA2 was greatly modified in mobile phase C since the very

intense and sharp absorbance at the void volume completelydisappeared, while the second peak was dramatically reducedin intensity and substantially retarded at higher elutionvolumes (Figure 5). The RI chromatogram of HA1 (Figure 4)showed that in mobile phase C less humic material wasdetected at higher elution volumes and at the total volumethan in B. Conversely, the RI chromatogram of HA2 (Figure6) showed that most of this humic material was eluted at thetotal volume when in mobile phase C, thereby indicating asubstantial size reduction. Mobile phase C induced a shiftof the main peak to higher elution volume in the UV

FIGURE 7. Size exclusion chromatograms of FA2 recorded with theUV-vis detector. A ) control mobile phase (0.05 M NaNO3, pH )7, I ) 0.05); B ) same as A but 4.6 × 10-7 M in methanol (final pH6.97); C ) same as A but to pH 5.54 with HCl; and D ) same as Abut 4.6 × 10-7 M in acetic acid (final pH 5.69).

FIGURE 8. Size exclusion chromatograms of FA2 recorded with theRI detector. A ) control mobile phase (0.05 M NaNO3, pH ) 7, I )0.05); B ) same as A but 4.6 × 10-7 M in methanol (final pH 6.97);C ) same as A but to pH 5.54 with HCl; and D ) same as A but4.6 × 10-7 M in acetic acid (final pH 5.69).

FIGURE 9. Size exclusion chromatograms of FA3 recorded with theUV-vis detector. A ) control mobile phase (0.05 M NaNO3, pH )7, I ) 0.05); B ) same as A but 4.6 × 10-7 M in methanol (final pH6.97); C ) same as A but to pH 5.54 with HCl; and D ) same as Abut 4.6 × 10-7 M in acetic acid (final pH 5.69).

FIGURE 10. Size exclusion chromatograms of FA3 recorded withthe RI detector. A ) control mobile phase (0.05 M NaNO3, pH ) 7,I ) 0.05); B ) same as A but 4.6 × 10-7 M in methanol (final pH6.97); C ) same as A but to pH 5.54 with HCl; and D ) same as Abut 4.6 × 10-7 M in acetic acid (final pH 5.69).


chromatogram of both FA2 and FA3 (Figures 7 and 9).However, peak intensity was slightly increased in FA2 butreduced substantially in FA3. RI chromatograms confirmedthe general molecular size reduction of both FA2 and FA3(Figures 9 and 10) in mobile phase C.

Mobile phase D (solution A made 4.6 × 10-7 M in aceticacid with a of pH 5.69) had the largest effect on Mw valuesof all humic samples (Table 3). UV chromatograms of bothHA1 and HA2 (Figures 3 and 5) showed a further shift tohigher elution volumes and a even more dramatic reductionof peak intensity. RI chromatograms confirmed these changesby showing that the bulk of material was shifted to totalvolume with hardly any humic matter still diffusing throughthe bed volume (Figures 4 and 6). Similarly, the large peakof both FAs was shifted to higher elution volumes andsubstantially reduced in intensity (Figures 7 and 9). RIchromatograms confirmed the UV findings in that most ofthe FAs were eluted at the total volume (Figures 8 and 10).

These results show that if the composition of the elutingsolution was only slightly changed, though the ionic strengthremained constant, the molecular-size distribution of thehumic substances was largely affected. When the controlmobile phase was only 4.6 × 10-7 M in methanol (solutionB), no new ions nor pH changes were introduced. Thealteration of humic molecular-size distribution is explainedwith the capacity of CH3OH to form both van der Waals bondswith the hydrophobic humic components and hydrogenbondings with the oxygen-containing functions present inhumic colloids (43). Such weak interactions seemed sufficientto modify the appearances of UV chromatograms wherebypeaks were both shifted to higher elution volumes or reducedin absorbance (280 nm). A very small amount of methanolin the eluting solution was thus able to disrupt the weakforces which temporarily stabilized humic associations intoapparently large aggregates. The result appeared to be thedispersion into smaller humic molecules, their diffusionthrough smaller gel pores, and an overall reduction inmolecular size. This effect was confirmed by RI chromato-grams which showed a shift of humic mass toward elutionvolumes typical of lower molecular-weight material. However,results from the RI detector would not rule out the possibilitythat humic conformational changes, rather than proceedingthrough dispersion of weakly associated small molecules,could be through a coiling down of large macromoleculeswhich, by assuming a reduced hydrodynamic radius (radiusof gyration, Rg), would elute at higher elution volumes. Thispossibility should be excluded by the general and sometimeslarge (HA1) reduction of peaks intensity observed in UVchromatograms. While it is well-known that molecularabsorptivity of humic substances vary with molecular size(10, 44), a recognized property of biomolecules is to showhigher molecular absorptivity when they are in random coilarrangements (45) or when they are reduced to a mixture ofmonomer components in close association (46). This effectis called chromism and is related to the interaction betweenone particular electronic excited state of a given chromophoreand different electronic states of neighboring chromophores(47, 48). A close interaction between the transition dipolemoment of an absorbing chromophore with induced dipolesof neighboring chromophores increases molecular absorp-tivity (hyperchromism), whereas this is decreased (hypo-chromism) when chromophores are separated (47, 48).Therefore, if humic samples were composed of macromol-ecules which, despite the same ionic strength, had been coileddown by changes in elution composition, the molecularabsorptivity should have been increased and the readings atthe UV detector should have been higher than in controlsolution. Conversely, a reduction in peaks absorbance is anevidence that the total molecular absorptivity of the elutingmaterial is lower (hypochromism) than in control mobile

phase. This must be attributed to a separation of molecules(or chromophores) rather than to their compaction into acoil. A logical conclusion is that methanol addition to themobile phase disrupted humic molecular associations andsmaller-size aggregates were formed.

The greater changes produced by decreasing the pH ofcontrol solution to 5.54 (mobile phase C) was due to adisruption of humic molecular associations larger than thatoccurred in methanol. The additional hydrogens in thismobile phase protonated humic carboxylic functions whichwere in their dissociated forms at the pH 7 of control mobilephase. A number of negative charges were then neutralized,and hydrogen bondings were concomitantly formed amongthe complementary functions of humic molecules, therebyaltering the conformational stability existing in controlsolution. Due to the parallel observation that a reduction inpeak absorbance was shown in UV chromatograms (hypo-chromism) and that a shift of humic mass to high elutionvolumes was visible in RI chromatograms, this change couldnot be simply due to a volume reduction of the humic randomcoil as previously accounted for (12, 31, 35). Again, the logicalexplanation is that humic conformation collapsed into amolecular association of smaller dimension but of greaterthermodynamic stability than in control solution. Thechemical rationale of this behavior lies in the energy gainedin hydrogen bond formation that ranges from 10 to 20 kJmol-1 in comparison to a van der Waals bond (49). Protonatedhumic molecules in mobile phase C abandon the looseconformation assumed at pH 7 of control solution whileforming relatively strong intermolecular hydrogen bond-ings. The concomitant large molecular-size reduction sug-gests that the weak conformation of apparently high mo-lecular size shown by HAs in control solution must havebeen predominantly due to weak intermolecular hydrophobicforces (van der Waals bonding) holding together smallmolecules.

The much smaller variation in molecular-size distributionshown by FAs suggests a profound different molecularcomposition from HAs (Table 3). The FAs absorbing chro-mophores must be too few and spread out for their addi-tive absorptivity to be affected by conformational changesand give the large hypocromic effect shown by HAs. Thesmall changes in UV chromatograms of FAs (Figures 7 and9) can be related to the low content of highly absorbingelectronic systems such as aromatic or olephinic structures(Table 2). The more sensitive UV changes observed for FA3must be ascribed to its higher aromatic content. Neverthe-less, RI chromatograms were self-evident in showing aprogressive shift to higher elution volumes with changes inmobile phase, thereby indicating some decrease in molecularsize also for FAs. The use of poorly UV-absorbing fulvic-likeDOM and the lack of RI detectors prevented earlier studiesto appreciate size reduction in mobile phases of varying pHs(31).

The further effect of mobile phase D at a pH similar tosolution C is to be accounted to the methyl group of aceticacid. Contrary to HCl, the weak acidity of CH3COOH allowsboth its undissociated and dissociated forms at pH 5.69 andthus the formation of mixed intermolecular hydrogen bond-ings (50) with humic molecules. As for mobile phase C, suchenergy-driven rearrangement outweighed the weak humicassociations in solution A, which were stabilized by hydro-phobic forces. However, the shift to higher elution volumesand the general reduction of molecular absorptivity in UVchromatograms (hypochromism) as well as the large shift tototal volume of the mass of all HAs in RI chromatogramsindicate that the methyl group of acetic acid played anadditional role in further enhancing the disruption of humicassociations. The apolar methyl group of acetic acid musthave altered the residual hydrophobic forces which still


stabilized humic conformations even after the hydrogenbonding rearrangement. Previous results have already shownthat the number of carbons in an interacting organic acidaffects the molecular size of humic substances (22, 23).

Our results indicate that conformational changes indifferent mobile phases were dependent on specific mo-lecular composition of humic samples. The higher reductionof HA1 molecular size in the methanol-added solution ascompared to HA2 and HA3 may be explained by the largecontent of aromatic and carboxyl carbons of HA1 (Table 2).This implies that methanol not only largely interacted withHA1 by forming hydrogen bondings but also a conformationalchange was facilitated by the high aromaticity which mayhave destabilized hydrophobic associations. The fewercarboxyl groups of HA2 and HA3 and their lower aromaticitiesallowed an extensive molecular-size change only in solutionC where a lower pH forced the formation of hydrogenbondings and a consequent conformational disrupture. Thestrongest hydrophobic forces were overcome in all HAs inmobile phase D by both the acidity and partial apolarity ofacetic acid. In the case of FAs, the higher aromaticity of FA3(Table 3) may explain why the reduction of its UV molecularabsorptivity in the methanol-added solution was greater thanFA2. This may suggest that even in the highly acidic fulvicmaterials part of the molecular association must be attributedto hydrophobic forces as in HAs. In FAs, however, the weakhydrophobic interactions are less important than in HAs asit is indicated by the relatively minor changes in molecularsize observed in mobile phase C and D.

This study showed that the molecular size distribution ofhumic substances by HPSEC must be interpreted by usinga combination of two factors: the elution volume and themolecular absorptivity of the chromatographic peaks. Thisresulted by comparing UV and RI size-exclusion chromato-grams of various humic and fulvic acids in mobile phases ofdifferent composition but of constant ionic strength. Previousworks have failed to indicate the combination of the abovefactors because either humic acids of too much similar naturewere only UV detected in less sensitive low-pressure size-exclusion systems (31) or poorly UV absorbing fulvic acidsand/or aqueous DOM were studied in HPSEC systemswithout the support of a RI detector (16, 17, 30-32, 40).

Our experiments indicated that by adding to a mobilephase an un-ionized alcohol such methanol, a mineral acid,and an organic acid without changing ionic strength themolecular size of humic substances varied significantly. Theirsize reduction, proved by a RI detector, was accompanied bya significant decrease in molecular absorptivity by a UVdetector and could only be attributed to a disruption of thehumic conformational arrangement into smaller associations.Our controlled experimental procedure excludes that suchsize reduction be explained with a compaction of a coilconformation. Furthermore, specific humic molecular struc-tures determined the conformational stability and hence theextent of size reduction.

These results confirm previous findings (22, 23) whichsuggested that humic substances in solution are looselybound self-association of relatively small molecules andintermolecular hydrophobic interactions are the predomi-nant binding forces. The stabilization of such a conformationshould be attributed to the entropy-driven tendency toexclude water molecules from humic association and thusdecrease total molecular energy (42, 49, 51). Such a modelof dissolved humic substances based on the reversible self-association of small molecules rather than on the macro-molecular random coil represents a new understanding thatshould contribute to predict the environmental behavior ofcontaminants in association with natural organic matter.

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Received for review August 20, 1998. Revised manuscriptreceived December 8, 1998. Accepted February 2, 1999.

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