conformational changes of dissolved humic and fulvic superstructures with progressive iron...

Journal of Geochemical Exploration xxx (2013) xxx–xxx

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Journal of Geochemical Exploration

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Conformational changes of dissolved humic and fulvic superstructures withprogressive iron complexation

Assunta Nuzzo a, Antonio Sánchez b, Barbara Fontaine a, Alessandro Piccolo a,⁎a Centro Interdipartimentale di Ricerca sulla Risonanza Magnetica Nucleare per l'Ambiente, l'Agroalimentare ed i Nuovi Materiali (CERMANU), Via Università, 100, 80055 Portici (NA), Italyb Departamento de Agroquímica, Bioquímica, Universidad de Alicante, Ap. Correos 99, 03080 Alicante, Spain

⁎ Corresponding author at: DISSPAPA, Università di N100, 80055 Portici, Italy. Tel.: +39 081 2539160x169x4

E-mail address: [email protected] (A. Picc

0375-6742/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.gexplo.2013.01.010

Please cite this article as: Nuzzo, A., et al., Conation, Journal of Geochemical Exploration (2

a b s t r a c t
a r t i c l e i n f o
Article history:Received 21 June 2012Accepted 23 January 2013Available online xxxx

Keywords:Metal-complexationDissolved humic substancesSupramolecular associationsHeavy metalsMolecular SizesSize-exclusion chromatography

Conformational changes of a humic acid (HA) and a fulvic acid (FA) induced by iron complexationwere followedby high-performance size exclusion chromatography (HPSEC) with both UV–vis and refractive index (RI) detec-tors. Molecular size distribution was reduced for HA and increased for FA with progressive iron complexation.Since interactions of Fe with humic components are electrostatic, it is likely that the triple-charged Fe ionsformed stronger complexes with the more acidic hydrophilic and hydrated FA than with the less acidic andmore hydrophobic HA. The large content of ionized carboxyl groups in FA, thus favored the formation of intra-or intermolecular bridges between the negatively charged fulvic acid molecules, and led to more compact andlarger size network than for HA. Conversely, iron complexation with HA disrupted the humic conformationalarrangements stabilized by onlyweak hydrophobic bonds into smaller-size aggregates of greater conformationalstability due to formation of strong metal complexes. These results confirmed that humic molecules in solutionwere organized in supramolecular associations of relatively small molecules loosely bound together by disper-sive interactions and hydrogen bonds, and they specifically responded to chemical changes brought about bymetal additions. The present study revealed themolecular changes occurring in superstructures of natural organ-ic matter when in metal complexes and contributed to understand and predict the environmental behavior inwaters and soil of metal complexes with natural organic matter.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Humic substances (HS) are ubiquitous natural compounds arisingfrom the chemical and biological degradations of plant and animalresidues (Piccolo, 1996). Based on their solubility in aqueous solutionsas a function of pH, HS can be divided in three fractions. Humic acid(HA) is the fraction that is soluble under alkaline and sub-neutral pHconditions, fulvic acid (FA) is the fraction soluble in water regardlessof pH, and humin is the fraction insoluble at any pH value.

HS have long been recognized to have an impact on migrationand bioavailability of heavy metals in the environment throughcomplexation (Parker et al., 1995; Tipping, 2002) which is usuallyattributed to the large content of oxygen-containing functional groups(carboxylic, phenolic, carbonyl) and, to a lesser extent, nitrogen- orsulfur-containing functional groups (Evangelou and Marsì, 2001). Theformation and stability of these complexes have a major effect on themobility of metals ions as environmental contaminants through soilsand aquifers (Buffle, 1988; Sposito, 1986), but the innate heterogeneityof HS limits a detailed understanding of the involved processes (Korshin

apoli Federico II, Via Università48; fax: +39 081 2539186.olo).

rights reserved.

formational changes of disso013), http://dx.doi.org/10.101

et al., 1998). Therefore, the metal interactions with HS have been thesubject of several studies (Hering and Morel, 1988; Pinheiro et al.,2000). Most abundant divalent and trivalent metals in soils are Ca,Mg, Al and Fe, which influence humic matter structures to a muchgreater extent than monovalent cations and other trace elements suchas Zn (Stevenson, 1977). Various analytical techniques have been ap-plied to study the changes of humic structures when in complexationwith metal ions (Senesi and Loffredo, 2008). Advanced X-ray spectros-copy (EXAFS) was found to be sensitive to changes in Cu geometrywhen in transition from aqueous to humic complex state (Korshinet al., 1998). Measurements of time-resolved fluorescence anisotropyrevealed alteration of humic conformations in the presence of metalions (Kolokassidou et al., 2007). A combination of spectroscopic, chro-matographic, and capillary electrophoresis–inductively coupled plasmamass spectrometry (CE–ICP-MS) techniques confirmed that humiccomplexing sites varied with humic samples and their size (Dua et al.,2005).

The possible effect of multivalent ions such as Ca, Mg, Fe and Al onthe conformation of humic substances has received considerable atten-tion, since itwould also contribute to the understanding of the structur-al complexity of humic molecules. A general consensus regards HS assupramolecular associations of heterogeneous and relatively small(b1000 Da) molecules, which are held together in only apparently

lved humic and fulvic superstructures with progressive iron complex-6/j.gexplo.2013.01.010

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2 A. Nuzzo et al. / Journal of Geochemical Exploration xxx (2013) xxx–xxx

large molecular sizes by weak forces, such as hydrogen and hydro-phobic bonds (Piccolo, 2001, 2002), and whose conformations can bedisrupted by the action of weak organic acids (Piccolo et al., 1999). Anumber of studies based on low- and high-pressure size exclusionchromatography, (Piccolo and Spiteller, 2003; Piccolo et al., 1996,2002), NMR spectroscopy (Peuravuori, 2005; Simpson et al., 2001;Smejkalova and Piccolo, 2008) low- and high-resolution mass spec-trometry with electrospray ionization (ESI/MS) (Kujawinski et al.,2002; Piccolo et al., 2010) and other methods (Baalousha et al., 2006;Jung et al., 2005; Kucerík et al., 2009; Liu et al., 2011), have confirmedthe supramolecular nature of HS. Complexation of metals by humicsubstances has then been further studied in light of the supramolecularassembly of humic molecules (Aquino et al., 2011; Kalinichev et al.,2011; Nebbioso and Piccolo, 2009).

This work had the objective to investigate the effects of iron com-plexation on the conformational structure of both a humic acid and afulvic acid by means of HPSEC in order to gain further information onthe conformational associations of dissolved humic material when inmetal complexes.

2. Material and methods

2.1. Humic matter

A humic acid (HA) was isolated from a volcanic soil (TypicXerofluvent) near Rome (Italy) and a commercial fulvic acid (FA) wasisolated from leonardite (Welgro Humus™). Humic substances wereextracted by standard procedures (Piccolo, 1988). Original materialswere shaken overnight in a 0.5 M NaOH and 0.1 M Na4P2O7 solutionunder N2 atmosphere. The HA was precipitated from the alkalineextract by lowering the pH to 1 with 6 M HCl, purified by three cyclesof dissolution in 0.1 M NaOH and precipitation in 6 M HCl, treatedwith a 0.5% (v/v) HCl-HF solution for 36 h, dialyzed against deionisedwater until chloride-free (Spectrapore 3 dialysis tubes, 3500 Mwcut-off), and freeze-dried. The FA, the humic material left in solutionafter precipitation of HA at pH 1, was purified by absorbing on anAmberlite XAD8 resin, eluting by a 0.1 M NaOH solution, and, afteradjusting the pH to 5, dialyzing in Spectrapore 3 tubes against deionisedwater until chloride-free and freeze-dried. Both HA and FA were thenredissolved in 0.5 M NaOH and passed through a strong cation ex-change resin (Dowex 50) to further eliminate divalent and trivalentmetals and freeze-dried again. An aliquot of both HA and FA sampleswere subsequently suspended in deionised water obtaining a finalconcentration of 0.25 g/L and titrated (VIT 90 Videotitrator, Radiometer,Copenhagen, Denmark) to pH 7 with a CO2-free solution of 0.5 MNaOH. After having reached the constant pH 7, the resulting sodium-humates were left under titration for 2 h, filtered through a Millipore0.45 μm and freeze-dried. The total acidity of both HA and FA sampleswas determined by treating 0.6 g/L of freeze-dried humic solutionswith a CO2-free solution of 0.1 MNaOH for 2 h to pH 9.2 by an automat-ic titrator (VIT 90 Videotitrator, Radiometer, Copenhagen, Denmark)under N2 atmosphere and stirring (Piccolo et al., 2001). Titration topH 9.2 required 0.9924 meq/L and 0.9339 meq/L of the NaOH solutionfor HA and FA, thereby representing total acidity for HA and FA,respectively.

2.2. Infrared spectroscopy

Diffuse reflectance infrared spectroscopy (DRIFT) was carried outon a Perkin-Elmer Spectrum-One FT-IR spectrometer. Freeze-driedHA and FA samples were prepared by mixing 5 mg of sample with200 mg of KBr in an agate mortar. Spectra were acquired with 16scans, automatic subtraction of water, smoothing, and baselinecorrection.

Please cite this article as: Nuzzo, A., et al., Conformational changes of dissoation, Journal of Geochemical Exploration (2013), http://dx.doi.org/10.101

2.3. Complex formation

A0.2 g/L solution of the freeze-dried humic sampleswas then addedwith increasing amount of a 0.01 N FeCl3·6H20 solution in order toneutralize, with the Fe3+ cation, progressively larger fractions (40, 50,60%) of HA and (80, 90, 100%) of FA total acidities, respectively. Anamount of cation greater than 60% of total acidity could not be addedhere to HA, because of incipient formation of a precipitate. Metal solu-tionswere added to humic solutions dropwise, and pHwas continuous-ly brought back to 7 by automatic titration, to ensure interaction of Fecations with humic matter. All solutions, with a final volume of 25 mLwere freeze-dried.

2.4. HPSEC

Freeze-dried iron complexes of humic and fulvic samples weredissolved in the same eluting solution to reach a concentration of0.5 g/L, passed through a 0.2 μm filter (PVDF Millipore), and injectedinto the HPSEC system. The latter consisted of a Perkin-Elmer LC-200solvent pump and two detectors in series: a UV–vis variable wave-length detector (Perkin-Elmer LC-295) set at 280 nm and a refractiveindex (RI) detector (Fisons Instruments, Refractomonitor IV). Arheodyne rotary injector, equipped with a 100-μL sample loop, wasused to load the calibration standards and humic solutions. A Polysep-GFC-P-3000 (600×7.5 mm i.d.) column (Phenomenex), preceded by aPolysep-GFC-P-3000 (75×7.5 mm i.d.) pre-column and by a 0.2 μLstainless steel inletfilter, was used. The column systemwas thermostat-ed at 25 °C. The elution flow rate was set to 0.6 mL/min for an elutingsolution made of 0.1 M NaH2PO4, buffered at pH 7, filtered throughMillipore 0.45 μm, and degassed with He. Polysaccharides of knownmolecular weight (0.7 kDa, 5.8 kDa, 11.8 kDa, 22.8 kDa, 47.3 kDa,212.0 kDa) were used as the column calibration standards (PolymerSciences Laboratories, UK).

Size-exclusion chromatograms for both the UV and RI detectorswere automatically recorded and evaluated by using a Perkin-Elmer-Nelson-Turbochrom 4-SEC peak integration andmolecular weight soft-ware. Calculation of the apparent weight-average molecular weightvalues (Mw) was done by the method of Yau et al. (1979) using thefollowing equation:

Mw ¼XN

i¼1

hi Mið Þ.XN

i¼1

hi

where hi is the height of the size exclusion chromatogram of each sam-ple eluted at volume i. The relative standard deviation of calculatedvalues among triplicates of each chromatogram varied to a maximumof 7%, thereby confirming the reproducibility of HPSEC analyses ofhumic substances.

3. Results and discussion

DRIFT (diffuse reflectance infrared Fourier transform) spectra of theHA and FA used in this study are reported in Fig. 1. Both infrared spectrashow the stretching (3550–3450 cm−1) vibrations of the O\H groups,the aromatic C_C stretching bandnear 1600 cm−1 and the 1044 cm−1

absorption for the C\O stretching vibration due to the presence of car-bohydrates. The infrared spectrum of FA shows stronger absorptionbands possibly attributable (Piccolo, 1988) to carboxylic acids (theC_O stretching band at near 1700 cm−1 and the C\O stretching vibra-tions or O\H deformation vibrations at 1397 cm−1 and 1215 cm−1).In general, both the more intense and numerous bands in the DRIFTspectrum of FA may be ascribed to a larger content of oxygen-containing functional groups (carboxylic, hydroxyl, phenolic) than forHA.



4000 3200 2400 1800 1400 1000 600

Wavenumber (cm-1)

T (%)

35151618

1034

3513

1583 1397

1215

10441710

b

a

Fig. 1. DRIFT spectra of HA (a) and FA (b) samples.

Elution time (min)

Abs

orba

nce

(mV

)

a

b

dc

abcd

70

60

50

40

30

20

10

18 20 22 24 26 28 30 32 34 36 380

Fig. 3. UV-detected HPSEC chromatograms of control FA (a) and FA complexed for 80%(b), 90% (c) and 100% (d) with Fe.

3A. Nuzzo et al. / Journal of Geochemical Exploration xxx (2013) xxx–xxx

Progressive iron addition to humic samples, modified substantiallythe elution profiles of HA and FA, bringing about considerable variationsin peak absorbance and elution times (Figs. 2 and 3). The UV-detectedHPSEC chromatograms of HA subjected to progressive iron complexa-tion show that the diffuse peak of control solution at about 26.96 minof elution time (Fig. 2a) was increasingly shifted to larger elutiontimes and considerably reduced in intensity with greater Fe additions(Fig. 2b, c and d). The decrease in absorbance with increasing per-centage of iron complexation is attributed to the phenomenon ofhypochromism, whereby the absorbance of a mixture of chomophoresdecreases in intensity when chromophore dipole moments no longerinteract with each other. It has been observed for humic moleculesthat the close interaction of the transition dipole moment of an absorb-ing chromophore and the induced dipoles of neighboring chromophoresdepends on their reciprocal orientation and may either increase(hyperchromism) or decrease (hypochromism) the molar absorptivity,depending on their proximity (Piccolo, 2001, 2002). Thus, the hypochro-mic effect observed here in the HPSEC chromatograms of HA and theconcomitant reduction in molecular size of humic associations (largerelution volumes), can be explained with a separation of neighboringchromophores induced by formation of iron-humic complexes. The sep-aration is due to the fact that smaller humic aggregates containing iron

Elution time (min)

Abs

orba

nce

(mV

)

a

b

d

c

abcd

50

40

30

20

20 22 24 26 28 30 32 34 36 38

10

0

Fig. 2. UV-detected HPSEC chromatograms of control HA (a) and HA complexed for40% (b), 50% (c) and 60% (d) with Fe.


complexes are more conformationally stable than the large associationsof control, which are instead loosely held together by weak dispersivebonds (Piccolo et al., 1999).

Contrary toHA, progressive iron complexation of FA shifted the peakeluted at about 26.75 min in control chromatogram to lower elutiontimes, while it did not change substantially the intensity of absorbance(Fig. 3a, b, c and d). The fact that FA was less subjected to the hypo-/hyper-chromic effect than HA has been already described earlier(Piccolo, 2001, 2002) and it was attributed to the larger hydrophilicityand, thus, hydration of fulvic molecules, which prevented a sufficientintermolecular proximity to influence absorbance.

The observedmodifications of HA and FA elution profiles induced byFe addition were reflected in the apparent weight-average molecularweight (Mw) values calculated from the UV-HPSEC chromatograms(Table 1). In fact, the HA solutions complexed for 40%, and 50% oftheir total acidity revealed a decrease in the Mw values in respect tocontrol, whereas the Mw value of the 60% complexation showed atrend of re-aggregation into greater molecular size. In line with thechromatographic profiles, the Mw values of FA increasingly addedwith iron resulted larger than control, thereby indicating a molecularsize increase favored by intermolecular complexation.

The RI-detectedHPSEC chromatograms of HA and FA samples beforeand after Fe complexation confirmed the UV findings (Figs. 4 and 5). Infact, the HPSEC chromatograms recorded by RI detector showed aprogressive shift of the diffused peak of HA to larger elution times anda significant decrease in intensity with increasing metal content.Conversely, the main diffused absorption of FA showed a significantincrease in intensity and shift to lower elution times with progressionof iron complexation. Therefore, since RI provides a real overall massdistribution of humic matter, the chromatograms revealed by this de-tector suggested that themolecular size of HA and FAwas, respectively,reduced and enhancedwith progressive Fe complexation. These resultsconfirm those obtained by theUV detector that showed the distribution

Table 1Apparent weight-average molecular weight (Mw) and elution time (min) of HA and FAsamples at increasing iron complexation (%), as calculated from UV-detected HPSECchromatograms.

HA FA

% complexation Mw Elution time % complexation Mw Elution time

0 12,882 26.96 0 6222 26.7540 8944 27.26 80 8461 26.5350 8292 27.10 90 8476 26.5260 11,523 27.21 100 8552 26.51



Elution time (min)

Abs

orba

nce

(mV

)

a

b

d

c

abcd

50

45

40

35

30

30 32 34 36 38 40 4442

25

20

15

10

5

Fig. 4. RI-detected HPSEC chromatograms of control HA (a) and HA complexed for 40%(b), 50% (c) and 60% (d) with Fe.

4 A. Nuzzo et al. / Journal of Geochemical Exploration xxx (2013) xxx–xxx

of molecular absorptivity of humic chromophores in HA and FA absorb-ing at the selected UV wavelength.

The alteration of the original humic conformations brought aboutby metal complexation is explained with the enhanced thermody-namic stability acquired by associations of humic components whenin complexes with Fe. Humic substances contain multiple functionalgroups that can interact with metal ions through complex formationor chelation, and carboxyl groups are the most effective metalcomplexing agents of HS (Stevenson, 1977). Considering that, withintotal acidity, FA have generally a larger content of carboxyl functionalgroups and, hence, a greater charge density than HA, our results maybe explained by the formation of a compact FA network of intra- andinter-molecular complexes with iron cations. At progressive Fe addi-tion, the highly hydrated and poorly associated fulvic molecules werethus mutually associated into larger molecular sizes by formation ofiron complexes. Conversely, the less acidic and poorly hydrated HAmolecules aremutually associated at neutral pHmainly byweak hydro-phobic interactions. Formation of iron complexes with their acidicfunctional groups forced humic molecules to abandon their previousweakly-held conformations and re-aggregate in smaller andmore ther-modynamically stable associations (Piccolo, 2001).

The HPSEC behavior of HA and FA indicated that the changes insize distribution of dissolved humic substances by iron complexationwere dependent on the molecular composition of humic materials.Addition of Fe to humic substances was capable of modifying the sta-bility of humic conformations in solution and alters their molecular

Elution time (min)

Abs

orba

nce

(mV

)

a

b, c, d

abcd

30

25

20

15

10

5

32 34 36 38 42403028

Fig. 5. RI-detected HPSEC chromatograms of control FA (a) and FA complexed for 80%(b), 90% (c) and 100% (d) with Fe.


size distribution. The extent of changes in humic molecular size wasa function of humic molecular composition and, hence, of the thermo-dynamic stability of the new supramolecular associations induced bycomplex formation with Fe. These findings are in line with the supra-molecular model of humic substances that depicts humus as a self-assembling association of relatively small heterogeneous moleculesheld together by weak forces (Piccolo, 2001, 2002), which specificallyresponded to chemical changes brought about by metal additions.Moreover, these results are useful to further enlarge the understandingof molecular dynamics occurring in humic superstructures when inmetal complexes and may contribute to predict the environmentalbehavior of metal contaminants in association with natural organicmatter.

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