Journal of Molecular Structure 982 (2010) 169–175

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Journal of Molecular Structure

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Structural characterization of humic-like substances with conventionaland surface-enhanced spectroscopic techniques

Paolo Carletti a,⇑, Maria Lorena Roldán b, Ornella Francioso c, Serenella Nardi a, Santiago Sanchez-Cortes b

a Dipartimento di Biotecnologie Agrarie, Università di Padova, Agripolis, Viale dell’ Università 16, 35020 Legnaro, Padova, Italyb Instituto de Estructura de la Materia, CSIC, Serrano 121, 28006 Madrid, Spainc Dipartimento di Scienze e Tecnologie Agroambientali, Università degli Studi di Bologna, V.le Fanin 40, 40126 Bologna, Italy

a r t i c l e i n f o a b s t r a c t

Article history:Received 7 May 2010Received in revised form 17 August 2010Accepted 17 August 2010Available online 22 August 2010

Keywords:Humic-like substanceFluorescence spectroscopySurface-enhanced Raman scatteringSurface-enhanced fluorescence

0022-2860/$ - see front matter � 2010 Elsevier B.V. Adoi:10.1016/j.molstruc.2010.08.028

⇑ Corresponding author. Tel.: +39 049 8272934; faxE-mail address: paolo.carletti@unipd.it (P. Carletti

Emission–excitation, synchronous fluorescence spectroscopy and surface-enhanced Raman scattering(SERS) combined with surface-enhanced fluorescence (SEF) were applied to aqueous solutions of ahumic-like substance (HLS) extracted from earthworm faeces. All measurements were acquired in a widerange of pH (4–12) and analysed by the linear regression analysis. Diffuse Reflectance Infrared FourierTransform (DRIFT) spectra were also acquired to assist in the structural characterization of this HLS.The emission and excitation spectra allowed the identification of two main fluorophores in the analysedsample. Moreover, a close correlation between fluorescence intensities of each fluorophore with pH var-iation was observed. SERS and SEF, in agreement with the fluorescence spectroscopy, showed that theHLS at low pH values exists in an aggregated and coiled molecular structure while it is dispersed anduncoiled at alkaline conditions. The obtained spectra also evidenced that different conditions modifythe functional groups exposed to the surrounding aqueous environment.

� 2010 Elsevier B.V. All rights reserved.

1. Introduction

Humic substances (HS) represent the more refractory fraction ofsoil organic carbon. The main characteristic of these macromole-cules is their extreme stability to biochemical or chemical degrada-tion over time, whether in soil [1] or in coals [2] or composts [3].The chemical and structural properties of HS are still poorly under-stood. In particular it is debated if the HS in aqueous solution arefound in aggregated or dispersed phases.

The conceptions of molecular aggregation [4] and of supramo-lecular structure [5] suggest that HSs consist of relatively smallmolecules of amphiphilic nature, which can form molecular aggre-gates or supramolecular associations in solution and on mineralsurfaces [6].

The state of aggregation seem to depend not only on molecularstructure, but also on the solvation conditions under which the HSexist, such as ionic strength, pH and the presence of metallic ions[7,8], thus the behaviour of HS at different pH values is of a greatimportance.

Most of the results on the structure and conformation of HS arebased on the application of spectroscopic techniques. The sensitiv-ity and non-destructive nature of fluorescence techniques have al-

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lowed the identification of different aromatic fluorophores withelectron-donating functional groups [9,10] as well as the study ofthe dynamic properties of HS under different environments [11].The position and broadness of fluorescence peaks are related tostructural signatures such as the molecular size or polycondensa-tion and the content of aromatics, phenolics, carboxyl and hydro-xyl functional groups. The synchronous fluorescence hasimproved the peak resolution and increased the selectivity of thestructural component of HS compared to those of emission andexcitation spectra [9,10,12]. However, the presence of multiplepH-dependent fluorophores in HS has complicated its application[13]. A statistical multivariate approach is often applied to resolveand improve the interpretation of HS spectra [14]. However, theseprocedures are complex and do not give a clear cut linear interpre-tation of investigated phenomenon. In our experience a simple lin-ear model can explain the relationship between fluorescence andinvestigated variables.

In recent years, several optical spectroscopy techniques(Raman, IR and fluorescence) have undergone a renaissance dueto the notable characteristics of metal nanostructures (MNs) wherelocalised surface plasmon (LSP) can occur. LSP leads to a remark-able local electromagnetic field enhancement owing to the highabsorption of light in the vicinity of metal nanoparticles and thusinduces a huge enhancement of spectroscopic signals, mainly inthe case of emission signals of molecules placed in the proximitiesof the surface [15]. Raman scattering is greatly enhanced as a con-

170 P. Carletti et al. / Journal of Molecular Structure 982 (2010) 169–175

sequence of LSP. For this reason, the surface-enhanced Ramanscattering (SERS) technique can be applied to the study of highlyfluorescent molecules in water such as HS, due to the fluorescencequenching occurring on the metal surface via a charge-transfermechanism [16–20].

The surface-enhanced fluorescence (SEF) technique has notbeen employed as much as SERS because of the energy transfer ef-fects taking place on the surface, which, in turn, lead to an actualquenching of the fluorescence. SEF effect is also induced by LSP res-onance near a metal surface [21]. Although the fluorescence ofadsorbates is in general quenched in the presence of Ag nanoparti-cles, the net effect seems to vary depending on the distance be-tween the fluorophore and the surface, as well as on the intrinsicquantum yield of the fluorophore [22]. In general, the fluorescenceis not quenched if the molecule is relatively far from the surface.The estimated optimal distance for a good SEF enhancement isachieved above 10–100 Å [23]. At distances further than this value,SERS and SEF signals can be simultaneously emitted and registeredfor molecular species placed in the vicinity of metal nanoparticles[23]. Since the average size of humic acid is larger than this value[24], the fluorophores included in the HS structure could be goodcandidates to give rise to intense SEF + SERS joint emission spectra,without the intrinsic quenching observed on a surface. This is pos-sible due to the fact that the HS cross sections of either Raman orfluorescence are of the same order on plasmonic metalnanoparticles.

In previous works we have employed the SERS or combinedSERS + SEF technique in the study of humic substances of differentorigins [16,18]. The aim of the present work is to apply differentspectroscopic techniques (emission–excitation, synchronous fluo-rescence spectroscopy and surface-enhanced Raman scatteringcombined with surface-enhanced fluorescence) in the character-ization of humic-like substance (HLS) extracted from earthwormfaeces in aqueous medium at different pH.

2. Materials and methods

2.1. Humic-like substances extraction and characterization

HLS were extracted from the faeces of Nicodrilus (=Allolobopho-ra (Eisen) = Aporrectodea (Oerley) caliginosus (Savigny) and Allol-obophora rosea (Savigny), collected from the Ah horizon of anuncultivated couchgrass (Agropyron repens L.) growing in soils clas-sified as Calcaric Cambisol (CMc-FAO classification). The extractionand purification procedures were described in a previous paper byMuscolo et al. [25].

The elemental analysis (C, H, N, O) was carried out using an ele-mental analyser (CHNS-O mod. EA 1110). All analyses were per-formed in triplicate.

2.2. DRIFT spectroscopy

The DRIFT spectrum was recorded with a Bruker TENSOR seriesFT-IR Spectrophotometer (Ettlingen, Germany) equipped with anapparatus for diffuse reflectance (Spectra-Tech. Inc., Stamford,CT). An amount of 4 mg of HLS was mixed with 200 mg of KBr (Al-drich, Chemical Co.). The KBr was used to obtain a background ref-erence spectrum. The spectrum was collected from 4000 to400 cm�1, averaged over 100 scans (resolution ±4 cm�1) and con-verted into Kubelka–Munk units. The second-derivative (2d) ofthe IR spectrum was used for wavenumbers determination of over-lapped bands. Moreover, the 2d spectrum was multiplied by �1 toget the bands pointing upwards for convenience [26]. Analyses ofspectral data were performed with Grams/386 spectral software(Galactic Industries, Salem, NH).

2.3. Fluorescence spectroscopy

Different HLS concentrations were tested for best fluorescencesignal to noise ratio. HLS solution for following analyses was thenprepared by dissolving 100 mg/L of lyophilised sample in distilledwater also according to literature [27]. The pH variations wereachieved adding 0.1 N HCl or 0.1 N NaOH. Fluorescence spectrawere recorded using a LS 45 luminescence spectrometer (Perkin El-mer, Norwalk, CT, USA) with FL WINLAB software. Emission spectrawere recorded over the range 380–550 nm at a fixed excitationwavelength of 360 nm [10]. Excitation spectra were obtained overa scan range of 270–480 nm, by measuring the emission radiationat a fixed wavelength of 520 nm. The effect of varying Dk on theshape of the synchronous-scan spectra was investigated in a preli-minary study. As previously observed [13], a value of Dk = 20 nmresulted in the best overall spectral resolution thus synchronous-scan excitation spectra were recorded with excitation wavelengthsfrom 290 to 550 nm and Dk = 20 nm. The excitation-emission andsynchronous-scan spectra were taken with a 5-nm slit width onboth excitation and emission monochromators. The wavelengthstep size was 1 nm, and spectrum intensity was measured as theaverage of three signal samplings.

2.4. SEF–SERS spectroscopy

The SEF–SERS spectra of the HLS were recorded using Lee-Mei-sel colloid (LMC) [28]. An amount of 1 mg of HLS was dissolvedwith 10 ml of tri-distilled water. Samples for Raman measure-ments were prepared by adding 10 ll of the HLS solution to 1 mlof the silver colloid. The activation of LMC was performed by add-ing a solution of paraquat (Sigma–Aldrich) at the final concentra-tion of 10�4 M. The samples pH was adjusted with 0.1 M HNO3

or 0.1 N NaOH. The presence of paraquat increases largely the SERSintensity as already demonstrated in previous work [29].

The SEF–SERS spectra were recorded with a Renishaw RM2000Raman spectrometer (Gloucestershire, UK). As the excitation linewe employed the 514.5 nm line provided by an Ar+ laser. The sam-ples were measured in a quartz cell with 1 cm optical path lengthplaced in a macro-sampling accessory with a focalization lens of15 mm. The laser power at the sample was 2 mW. The resolutionwas set at 2 cm�1 and the geometry of Raman measurementswas 180� [19]. The SERS spectra were baselined to withdraw thecontribution of the fluorescence by using the algorithm providedby the Origin 6.0 Program.

2.5. Data analysis

A linear regression model (Y = a + bX) was applied to describethe best fitting between emission or excitation fluorescence anddifferent pH values. The multiple regression procedure (Y = b0 +b1 X1 + b2 X2 + bk Xk) was performed to describe the impact of a sin-gle quantitative factor X on the dependent variable Y.

Statistical analysis was performed by using Statgraphics version5 plus (statistical graphics system by statistical graphicscorporation).

3. Results and discussion

3.1. HLS elemental composition

The elemental composition (mean ± SD) of the HLS was55.97 ± 0.17% carbon; 4.41 ± 0.16% nitrogen; 3.12 ± 0.10% hydro-gen and 36.5 ± 0.12% oxygen. As expected the composition of Cand N was in the range of HLS extracted from earthworm compostof Eisenia foetida [30]. The high C/N and low H/C ratios found in this

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Fig. 2. Fluorescence emission spectra of humic-like substances (HLS) at differentpH values (bottom). The linear regression analysis (Y = a + b � x) described the bestfitting between maximum-intensity of emission at 455 nm and different pH values(upper).

P. Carletti et al. / Journal of Molecular Structure 982 (2010) 169–175 171

sample suggested a high stability and presence of aromatic struc-tures [1].

3.2. DRIFT spectroscopy

The DRIFT spectrum of the HLS (Fig. 1) showed the typicalvibrations of functional groups of HS investigated in previous pa-pers by Canellas et al. [30] and Carletti et al. [31]. More detailedinformation on functional groups could be observed in the 2d spec-trum (Fig. 1A). The spectrum was dominated by strong bands at1729 and 1220 cm�1 assigned to m(C@O) and m(CAO) stretch vibra-tions of COOH groups, respectively [17,18]. The strong band at1653 cm�1 might denote the presence of m(C@O) vibration in qui-nonic and ketonic [32] or amide I groups. The peak at 1542 cm�1

was due to secondary amide deformation [32]. The presence ofthe bands at 1514 cm�1 suggest the existence of C@C stretchingvibrations of aromatic moieties. In addition, the band at1269 cm�1 might also be due to guaiacyl ring breathing [17]. Theregion between 1480 and 1300 cm�1 was assigned to CH2 andCH3 bending, CAOH deformation of COOH, and COO� symmetricstretch. The peaks between 1153 and 1033 cm�1 were assignedto CAO stretching of polysaccharide-like components [1].

3.3. Fluorescence spectroscopy

The fluorescence emission spectra were obtained by exciting at360 nm, which corresponds to the wavelength at which a maxi-mum emission is observed (Fig. 2). These emission spectra ap-peared as a very broad and featureless band showing amaximum at 455 nm confirming previously published data on sim-ilar substrates [10,13]. Best fitting between maximum-intensity ofemission at 455 nm and different pH values (Fig. 2A) was obtainedby using the linear regression model. In fact, the estimation ofregression (b ± s.e. = 4.345 ± 0.507, P < 0.001) and correlation (r =0.967) coefficients indicated a close linear relationship betweenthe two variables.

The intensity increase may be associated to the macromolecularnature of HLS. According to the supramolecular theory [5,33] at thealkaline pH the intermolecular hydrogen bondings are completelydisrupted, as consequence the HLS in solution are in disperse formor random coil. Thus, at these conditions the reabsorption of pho-tons by the own matrix is less probable leading to a relative in-crease of the emitted fluorescence. The supramolecular changesof HLS are supported by the low H/C ratio and the presence of

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Fig. 1. (A) DRIFT spectrum of humic-like substances (HLS) extracted from theearthworm faeces and (B) second-derivative (2d) of spectrum.

the bands at 1600 and 1514 cm�1 in the DRIFT spectrum (Fig. 1),which accounts for the existence of aromatic structures in thissample which were the responsible for the intense fluorescenceemission. At acidic pH a partial blue-shift and a quenching of fluo-rescence are observed. This phenomenon could to be attributed toan increase of electron-withdrawing groups in the HS structureupon the protonation, such as the carboxylic ones and the morehydrophobic environment inside the more compact HLS matrix.Fluorescence quenching may be then attributed to the ring stack-ing inside the HS structure and the aggregation of different molec-ular species at acid pH.

The increase of a shoulder at 420 nm at pH 4–5 was attributedto the formation of hydrogen bonds between non-ionized phenolicand carboxylic groups. Such hydrogen bonds could enhance therigidity of phenols and thereby contributed to the moderate fluo-rescence at acid pH [34,35]. The lack of the above shoulder at pHalkaline was probably due to the reduced number of hydrogenbonds and, consequently to the lower rigidity of HS due to the exis-tence of phenolate and carboxylate anions.

The fluorescence excitation spectra (Fig. 3) of HLS were charac-terized by a multi-peak profile, with two main maximums centredat ca. 350 nm, 390 nm and a minor shoulder at ca. 436 nm. The firstband is assigned to chromophores with high carboxylic group con-tent, while the second is associated to structures containing phe-nolic groups [36]. The presence of these chromophores was alsowell supported by strong bands of COOH groups (1729 and

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Fig. 3. Fluorescence excitation spectra of humic-like substances (HLS) werecharacterized by two main maximums centred at ca. 350, 390 and a shoulder at436 nm (bottom). The linear regression analysis (Y = a + b � x) described the bestfitting between maximum-intensity of excitation at 390 nm and different pH values(upper).

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Fig. 4. Synchronous fluorescence spectra of humic-like substances (HLS) atdifferent pH values. The spectra were characterized by a very complex spectro-scopic pattern with a prominent shoulder at 364 nm, three peaks at 394, 437,457 nm and two minor shoulder at 500 and 520 nm.

172 P. Carletti et al. / Journal of Molecular Structure 982 (2010) 169–175

1220 cm�1) and that of phenol (1260 cm�1) shown in the DRIFTspectrum (Fig. 1A). The excitation spectra showed a significant var-iation with the pH (Fig. 3A), which was better understood by per-forming the corresponding linear regression analysis. The bestfitting was observed on the excitation peak at 390 nm(b ± s.e. = 7.119 ± 0.821, P < 0.005; r = 0.968). Instead the linearmodel was not statistically significant (P 6 0.05) for the excitationpeak at 350 nm and it explained only 78% of the variance for theshoulder at 436 nm (b ± s.e. = 4.916 ± 1.142, P < 0.01; r = 0.887).

The multiple linear regression between pH and three excitationpeaks resulted in an only one statistically significant partial regres-sion coefficient (P < 0.05) relative to the excitation peak at 390 nm(b ± s.e. = 0.191 ± 0.052). This suggested that the peak at 390 nmhas the greatest weight on the observed variations in the fluores-cence excitation spectra and that the linear model is adequate inexplaining the observed data. The increase of pH induces an in-crease of the band at 390 nm and a subsequent decrease of thatat 350 nm. In addition, both bands undergo a red-shift as corre-sponds to less coiled structures. The strong decrease of the bandat 350 nm can be related to the ionization of carboxylic com-pounds, while the increase of the band at 390 nm is associated tothe appearance of phenolate groups, which fluorescence emissionis enhanced and red-shift upon ionization.

Synchronous fluorescence spectra (SFS) of HLS were obtainedby using the Dk = 20 nm between the excitation and the emissionmonochromators as this value is thought to be optimal for SFS

measurement of humic acids and it has been recommended in sci-entific literature for a long time [37]. By using the Dk = 20 nm it ispossible to reveal the presence of fluorophores which consist ofcondensed rings or simple aromatics which are in mutual intermo-lecular contact affecting their spectral fluorescence characteristics.These groups are those which are more intense in SERS and SEF byexciting at 514 nm.

SFS showed a very complex spectroscopic pattern characterizedby a prominent shoulder at 364 nm, three peaks at 394, 437,457 nm and two minor shoulder at 500 and 520 nm (Fig. 4).Although the synchronous-scan spectra of HLS appeared to bebroad, the spectra were better resolved than those of the emissionspectra. In general, the changes observed on varying the pH arealso related to the ionization of acidic groups. The synchronousspectral peak intensity and red shift of bands appearing in the450–520 nm region are related to the presence of high molecularweight and polycondensed components existing in the HLS sample[10]. Thus, an increased fluorescence intensity and red shift of thisregion might suggest a high molecular weight in the investigatedhumic materials, as well as polycondensed aromatic moieties. Onincreasing the pH a relative decrease is observed for the bandappearing at 360 nm, as a consequence of the ionization of benzoicgroups. On the contrary, the bands appearing above 430 nm arerelatively intensified, and this can be again associated to the disso-ciated of phenolic and quinone types of organic compounds[10,36].

P. Carletti et al. / Journal of Molecular Structure 982 (2010) 169–175 173

3.4. SERS and SEF spectra of HLS

Fig. 5 shows the SERS + SEF combined spectra from HLS at dif-ferent pHs. These spectra were normalized to the solvent band,i.e. water, at 3400 cm�1. As can be seen, the SERS and SEF intensityunderwent an opposite behaviour with the pH. While the fluores-cence SEF signal was progressive quenched on increasing the pH,on the contrary, the SERS intensity underwent an enhancement,as well as a profile change. This behaviour could be better seenin Fig. 5A, where the emission intensities at about 4400 cm�1

(SEF emission) and that at 1612 cm�1 (SERS emission) were com-pared at the different studied pHs. Interestingly the main changesin SEF and SERS spectra were observed in the range of pH between5.0 and 6.0. Recently, Terashima et al. [38] have demonstrated thatthe apparent pK (pKapp) of humic acids (HA) is approximately 5.5and, as a consequence, at pH = 5.5 drastic changes take place inseveral physical properties, such as surface tension, surface areaoccupied by HA molecules when adsorbed onto a surface and HAhydrophobicity. A similar pH value was reported by other authors,when studying the aggregation tendency of HA [39,40]. Addition-ally, this is related to the changes observed for the fluorescenceemission in Fig. 3, where a significant variation of the emissionband at 390 nm was observed in the 5.0–6.0 interval of pH. At thispH a massive ionization of carboxylate groups is expected (pK = 5),thus leading to important structural changes in HLS [41].

At pH lower than 5.0 the protonation of carboxylic groups wasexpected leading to the formation of hydrogen bonds. In this con-dition the HS might adopt a micellar structure lacking of a netcharge. This negatively charged humic functional groups were re-pulsed by the colloid surface (also negatively charged) increasingthe distance between the two, therefore leading to a lower SERSsignals. In contrast, the SEF signal was intensified due to the longerdistance of fluorophore groups regarding the surface and the com-pacted structure adopted by the HLS, where the fluorophoregroups were placed under a more hydrophobic environment whichfavoured the fluorescence emission. Molecular aggregation couldalso occur together with contraction of the humic matrix at lowpH, since the intermolecular charge repulsion effects were de-creased due to the poor dissociation of H+ from the carboxyl groups[20]. Viscosimetry studies carried out for several fractions of HLSrevealed conformational change of these structures when the pHis lowered [8] thus confirming this hypothesis.

At alkaline pH, humic substances appeared to have a linear oruncoiled structure [42]. The HS uncoiling or structural expansion

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occurring in the pH range between 7.5 and 12 provoked an oppo-site response on the fluorescence and Raman intensities. By onehand, it favoured the approach of the inner PAH residues of HSto the metal leading to an enhancement of the SERS signal. Onthe other hand, this expansion induced the fluorescence quenchingdue to the higher exposure of fluorophores to the aqueous polarmedium and the energy and charge-transfer induced by the closermetal surface.

The variation of SEF intensity with the pH is then opposite tothat observed for the excitation-emission fluorescence (Figs. 2and 3). This is due by one hand to the fact that the observed SEFemission bands are attributed to polycyclic aromatic moietiesexisting in HLS, since they are observed upon excitation at514.5 nm. On the other hand, it is to note the higher importanceof structural factors in the SEF intensity than the relative changesinduced by the own ionization, since the SEF intensity is highly af-fected by the fluorophore distance to the metal surface, which inturn is modified by the tridimensional structure of the HLS matrix.

The SEF spectra of HLS displayed broad bands at ca. 4100, 4650and 5650 cm�1 (Fig. 5). These bands corresponds to those groupsexcited at 514.5 nm, which consist mainly of those moleculargroups mainly seen in the SERS spectrum, i.e. polycondensed aro-matic moieties. While the two first bands moved to lower wave-numbers, the latter bands become lower when raising the pH.Thus, the last band can be attributed to an exciplex emission in-duced by the molecular associations occurring inside the HS mi-celle structure between aromatic polycyclic groups, and whichwere more probable when the HS was under a coiled conformationat low pH. This behaviour is similar to what happens in polymerscontaining polycyclic residues when increasing the temperatureor varying the pressure [42,43].

The uncorrected SERS spectra of HS at different pH’s are shownin more detail in Fig. 6. At acidic pH, the SERS spectra showed twobroad features centred at about 1600 and 1380 cm�1 which couldbe attributed to ring stretching vibrations and symmetric m(COO�)vibration, respectively [17] suggesting that the interaction HS/col-loid took place through carboxylate groups [20]. From a pH 7.5 nar-rower bands at 1310 cm�1 and 1612 cm�1 were recorded andassigned to aromatic ring stretching motions of polycyclic aromaticcompounds existing in the HS core [16], together with shoulders at1570 cm�1 (assigned to aromatic ring vibrations) and at1650 cm�1, corresponding to aromatic m(C@O) [20]. These bandsincreased as the pH becomes more alkaline, being very intense atpH 12.0.

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174 P. Carletti et al. / Journal of Molecular Structure 982 (2010) 169–175

Fig. 7 shows the corrected SERS spectrum registered at pH 12.This spectrum was obtained by subtracting the SERS at pH 12.0that obtained at 3.6. Three different regions could be observed inthe difference spectrum. In the low wavenumber region (Fig. 7A)a strong band appears at 338 cm�1 which could be assigned to askeletal vibration of polycyclic aromatic compounds, also associ-ated to the intense bands appearing at 1310 and 1612 cm�1. Thebands at 738 and 840 cm�1 were due to in-plane stretching bandor aromatic compounds. The most intense bands appeared in themedium wavenumber region (Fig. 7B) where the aromatic vibra-tions at 1310 and 1612 cm�1 appeared. Besides, other weakerbands were seen at 1166 and 1192 cm�1 attributed to in-planeCAH bending; at 1254 cm�1, attributed to CAO stretching motionsin phenols. The observation of these bands indicated that at this pHthe interaction HS with the colloid surface took place through aro-matic groups. At pH 9.0 and pH 12 the bands at 1250, 1310,1610 cm�1 showed a further enhancement thus supporting thehypothesis of the involvement of the aromatic moieties in theinteraction with the silver colloid surface. Finally, the bands ob-served in the high wavenumber region (Fig. 7C) were mainly dueto overtones and combination bands of the intense polycyclic aro-matic groups observed at lower wavenumbers, which were furtherintensified by a resonance effect. However, fundamental band dueto CAH stretching in aromatic compounds (at 3088 cm�1), or toOAH stretching (at 3440 and 3635 cm�1) were also identified.

In relation to other humic substance with different origins stud-ied by SERS [i.e. 16], the HS studied here showed more intensebands corresponding to oxygenated groups linked to aromatic cy-

cles. This was the case, for instance, of the band at 1252 cm�1 (CAOstretching in aromatic rings) and the structural bands at 840 and738 cm�1.

4. Conclusions

Conventional fluorescence spectroscopy and SERS–SEF tech-niques applied in this research evidenced modifications of thetested HLS substances which appeared coiled and aggregated atacid pH, uncoiled and dispersed in alkaline conditions. This studydemonstrated that the different fluorescence techniques (conven-tional and surface-enhanced one) can afford rich structural infor-mation on these important molecules. The conventionalfluorescence spectra undergo a variation with the pH mainly in-duced by the intrinsic ionization of the acidic groups existing inHLS, although with a small contribution from the higher or lowerexposure to the environment of the fluorophores. On the contrary,the SEF emission is mainly related to the structural modification ofthe HLS structure which drives the distance to the metal surface. Inaddition SEF can be used to selectively study the polycyclic aro-matic moieties existing in HS.

DRIFT and conventional fluorescence techniques are then help-ful techniques to investigate the overall composition of humic sub-stances, in terms of aliphatic and aromatic groups bearing differentstructure. SERS and SEF can be employed as a complement to thisstructural characterization, since the aromatic moieties are moreenhanced in these spectra. Furthermore tridimensional informa-

P. Carletti et al. / Journal of Molecular Structure 982 (2010) 169–175 175

tion of HS can be also obtained due to the extreme sensitivity of theSERS and SEF signals with the distance to the surface.

After the application of all these techniques we have concludedthat HLS have a higher content of aromatic moieties with a highamount of oxygenated groups, also indicated by elemental analy-sis, as compared to other HS studied elsewhere e.g. [16].

Acknowledgements

This work has been supported by the Spanish Ministerio de Cien-cia e Innovación (Grant FIS2007-63065) and Comunidad de Madridthrough the MICROSERES II network (Grant S2009/TIC-1476).M.L.R. also acknowledges a MAEC-AECID post-doctoral fellowshipfrom the Ministerio de Asuntos Exteriores.

P. Carletti gratefully thanks professor J.V. Garcia-Ramos and allthe staff of Istituto de Estrutura de la Materia for the help duringthe preparation of this work.

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