is nitrogen functionality responsible for contrasted responses of riverine dissolved organic matter...
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Journal of Analytical and Applied Pyrolysis 97 (2012) 62–72
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Journal of Analytical and Applied Pyrolysis
journa l h o me page: www.elsev ier .com/ locate / jaap
s nitrogen functionality responsible for contrasted responses of riverineissolved organic matter in pyrolysis?
. Templiera,∗, F. Miserqueb, N. Barréc,1, F. Mercierc,1, J.-P. Crouéd, S. Derennea
Laboratoire de Biogéochimie et Ecologie des Milieux Continentaux, UMR CNRS 7618, Université Pierre et Marie Curie, Paris, FranceLaboratoire de Réactivité des Surfaces et des Interfaces, CEA, Centre d’Etudes de Saclay, FranceLaboratoire « Analyse et Environnement », UMR 8587, CEA/CNRS/Université d’Evry, Centre d’Etudes de Saclay, FranceLaboratoire Chimie et Microbiologie de l’Eau, UMR CNRS 6008, ESIP, Université de Poitiers, France
r t i c l e i n f o
rticle history:eceived 9 December 2011ccepted 1 May 2012vailable online 10 May 2012
eywords:issolved organic matter (DOM)urie point pyrolysisC/MS1s and N1s XPSP/MAS 13C and 15N NMR
a b s t r a c t
Fractions of dissolved organic matter (DOM) from the Loire and the Gartempe rivers were obtained usingAmberlite XAD resin fractionation procedure. According to the eluting system used and to the polarityof their components, the fractions were termed hydrophobic (HPO) and transphilic (TPI) for the Loire(elution with acetonitrile/water mixture) and hydrophobic acid (HPOA) and transphilic acid (TPIA) forthe Gartempe (elution with NaOH). In addition, for the Loire, colloids (COL) were pre-isolated througha dialysis step. The composition of the three fractions from the Loire was investigated with solid statecross polarisation/magic angle spinning (CP/MAS) 13C NMR and Curie point pyrolysis at 650 ◦C withand without tetramethylammonium hydroxide (TMAH). Separation and identification of the releasedcompounds were performed using gas-chromatography/mass spectrometry (GC/MS) and focussed onnitrogen-containing pyrolysis products (N-products). Quantitative differences were observed betweenthe N-product distribution of the HPO and TPI fractions, whilst the few N-products from the COL fractionexhibited different structures corresponding to peptidoglycan contribution. Comparison with previousresults obtained for two DOM fractions (HPOA and TPIA) from the Gartempe river (France) revealed thatpyrolysis detection of nitrogen containing molecules is not only related to the nitrogen content of thefractions, even in the case of similar hydrophobicity, but also likely to the functionality of nitrogen in themacromolecule sources. To correlate the molecular level information about N-containing moieties withthe functionality of nitrogen in the macromolecular sources, the five fractions of DOM were investigatedthrough X-ray photoelectron spectroscopy (XPS) and solid state cross polarisation/magic angle spinning(CP/MAS) 15N NMR. C1s XPS and 15N NMR analyses revealed an important contribution from amidenitrogen in all the DOM fractions, with a large increase from the hydrophobic fractions to the transphilic
15
and colloids ones. Moreover, N NMR revealed an additional pyrrole nitrogen contribution in the HPOfraction of the Loire and in the TPI and TPIA fractions of both rivers. For the two rivers, the ı 15N valueswere maximal for the fraction containing the highest proportion of amide N, and decreased in parallelwith increasing pyrrole N contribution. Only the hydrophobic acid fraction of the Gartempe, which didnot contain any pyrrole N was characterised by a lack of N-containing pyrolysis products, suggesting thattheir detection could be dependent on the presence of pyrrole N in the macromolecule sources.. Introduction
Organic nitrogen accounts for the major part of nitrogen in
oils, sediments and the aquatic environment [1,2]. The dynam-cs of nitrogen is tightly linked to the composition and reactivityf organic matter (OM) as the latter plays a major role in the∗ Corresponding author. Tel.: +33 1 44 27 51 72; fax: +33 1 44 27 41 64.E-mail address: [email protected] (J. Templier).
1 Present address: UMR CNRS 8608, Institut de physique nucléaire d’Orsay (IPN),niversité de Paris 11 Paris-Sud, 91406 Orsay Cedex, France.
165-2370/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jaap.2012.05.002
© 2012 Elsevier B.V. All rights reserved.
stabilisation of nitrogen by sequestration of inorganic nitrogenand mineralisation of nitrogen-containing organics [3,4]. Dissolvedorganic matter (DOM) is involved in material transport and carbonbalance in aquatic ecosystems but, as a complex and heteroge-neous mixture of natural macromolecular organic material, it ispartly unidentified [5], especially dissolved organic nitrogen (DON).Organic nitrogen occurs mainly in complex high molecular weight(MW) structures, most of which are non-hydrolysable [6]. Thus,
despite their environmental importance, the origin and struc-ture of nitrogen-containing organic compounds remain widelyunknown, partly because of the refractory nature of the sourcemacromolecules and the limitations in analytical procedures. In
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ecent years, according to the importance of nitrogen as nutrientor biological productivity, its nature in soil organic matter (SOM)as been investigated. Recent studies using 15N NMR spectroscopyointed to a dominance of amide functional groups in soils andediments [7–11], thereby supporting the idea that N is engagedn polar building blocks [12]. However, the chemical functionali-ies of organic matter and consequently of organic nitrogen maye modified according to environmental processes, fossilisationnd vegetation fires, resulting in the formation of heteroaromatic
[13,14]. X-ray photoelectron spectroscopic (XPS) studies havelso underlined the dominance of peptide/amide nitrogen in humicubstances (HSs) [15] and a positive correlation has been foundetween the increasing proportion of heterocyclic N and the degreef humification of these substances [1,16]. At the molecular level,arious pyrolysis methods, combined with GC/MS analysis, haveeen applied to the study of refractory OM from different sources,nd some of them were focused on N-containing macromolecules3,17–22]. Recent microscale sealed vessel pyrolysis (MSSVpyr)tudies of natural organic matter have contributed to improve thefficiency for the detection of N-containing products [21,22]. How-ver, the origin of the limited detection afforded by more traditionalurie point pyrolysis techniques is not yet understood. In additiono the thermal analytical conditions, the functionality of nitrogen inhe macromolecular sources could be responsible for low pyrolysisesponses.
The objective of this study was to improve the understand-ng of pyrolysis responses for nitrogen-containing compounds. The
ajor point of interest was to understand the origin of contrastedesponses obtained from different fractions of natural OM con-aining similar amount of nitrogen, when using a commonly usedyrolysis method, namely Curie-point pyrolysis (CuPy), and possi-ly decipher the relationship between nitrogen functionality andyrolysis detection. We used on-line CuPy at 650 ◦C, with andithout methylating reagent, to investigate the released nitrogen-
ontaining products, and to correlate the data with the functionalityf nitrogen in the DOM, determined through spectrometric meth-ds (solid state CP/MAS 15N NMR and XPS). Five fractions of DOMbtained from two French rivers and following two different frac-ionation processes were investigated to address this question.
. Materials and methods
.1. Sample collection and fractionation
The water samples were collected in France, from the Gartempeiver during winter in Lathus (dissolved organic carbon: 6.5 mg l−1)nd the Loire River at the beginning of the spring in Belleville (dis-olved organic carbon: 2.85 mg l−1). Two fractions were obtainedrom the Gartempe River, using an extension of the conven-ional XAD isolation protocol [23,24] based on a two column arrayf Amberlite XAD-8 and XAD-4 resins [25,26], as described byemplier et al. [27]. The DOM fractions isolated from the resinsorrespond to the hydrophobic acid fraction or HPOA and to theransphilic acid fraction or TPIA (resin elution conducted with.1 N NaOH), as termed by Croué et al. [28], respectively. Threeractions from the Loire River were separated. The high MW30,000–50,000 Da) colloid (COL) fraction, operationally definedy the protocol, was pre-isolated using membrane dialysis with a.5 kDa cut off [29]. The lower MW DOM (1000–2000 Da) separatedrom the colloids by dialysis was then pumped through XAD-8 andAD-4 resins to isolate the hydrophobic (HPO) and transphilic (TPI)
ractions, thanks to elution with an MeCN/water (75/25%, v/v) mix-ure. HPOA and TPIA fractions correspond to acidic structures only,hereas HPO and TPI fractions include acidic, basic and neutral
tructures.
Applied Pyrolysis 97 (2012) 62–72 63
2.2. On-line Curie point pyrolysis (CuPy)
CuPy/GC/MS was carried out with a Fischer 0316 Curie pointflash pyrolyser. Samples (2–3 mg) were pyrolysed for 10 s in a ferro-magnetic tube (10 mm × 2 mm) with a Curie temperature of 650 ◦Cunder a He flow of 5 ml min−1. The pyrolysis unit was directlycoupled to the GC/MS system. The pyrolysis products were sep-arated with a Hewlett-Packard 5890 Series II gas chromatograph,equipped with a Rtx-5Sil MS column (30 m × 0.25 mm i.d., 0.5 �mfilm thickness), He was used as carrier gas. The analysis conditionsused were: injector temperature 280 ◦C, splitless mode, oven tem-perature maintained at 50 ◦C (10 min) and then programmed from50 to 300 ◦C at 4 ◦C min−1, constant pressure of 15 psi. Detectionwas via a Hewlett-Packard 5889A mass spectrometer coupled withthe gas chromatograph, with a heated interface (250 ◦C), electronenergy 70 eV and ion source 220 ◦C, scanning from m/z 35 to 600 at2 scan s−1. Compounds were assigned on the basis of their massspectra, comparison with the NIST library mass spectra and GCretention times.
To perform TMAH-CuPy/GC/MS, samples were mixed withtetramethylammonium hydroxide (TMAH 25% (w/v) in MeOH) in1/1 (w/w) ratio and freeze dried before loading the ferromagnetictube. This ratio was chosen after several tests, and corresponds toa compromise. The more TMAH is added, the more efficient themethylation is and the more DOM sample is pyrolysed, the morepyrolysis products are detected. However, only a limited amountof material can be loaded in the tube and the 1/1 (w/w) ratio allowsobtaining both a significant detection of pyrolysis products and asuitable efficiency of TMAH.
2.3. XPS analysis
DOM samples, as fine powder, were fixed on double-sidedtape. XPS spectra were recorded with a VG ESCALAB 220i XLspectrometer (ThermoFisher Scientific). The X-ray source was thenon-monochromatic ray K� of Al (1486.6 eV). The beam diameterwas around 6 mm × 7 mm and its power 20 mA × 15 V = 300 mW.For high resolution spectra the pass energy was 20 eV and thestep size 0.1 eV. The spectrometer was calibrated in energy to thesilver Fermi level (0 eV) and to the 3d5/2 electronic level of Ag(368.3 eV). The analyser chamber pressure was in the range of 10−9
to 10−10 Torr. Spectra were recomposed using Avantage Software(ThermoFisher Scientific). The baseline was drawn according toShirley method. The binding energy of the C1s peak of aliphaticgroups was fixed at 285.0 eV in order to compensate for the chargeeffect. C1s and N1s high-resolution spectra of the samples weremeasured simultaneously (30 scans). The N1s spectra of refer-ence compounds, d-l pyroglutamic acid (>99% TCI), cytosine (>99%,Sigma–Aldrich), protoporphyrin disodium salt (Interchim) andserum albumin bovine (SAB, fraction V, 96–99%, Sigma-Interchim)were obtained under the same conditions.
2.4. Solid state NMR
Solid-state 13C NMR spectra of the Loire samples were obtainedwith a Bruker AV300 spectrometer at 75.47 MHz using a sequenceof variable amplitude (VA/CP/MAS) with a 5 s pulse delay, a contacttime of 1 ms and a spinning rate of 14 kHz (rotor diameter 4 mm).As for the Gartempe samples, a Bruker DSX400 spectrometer at100.62 MHz was used with a 1 s pulse delay, a contact time of 1 msand a spinning rate of 10 kHz (rotor diameter 4 mm). The chemicalshift was referenced to tetramethylsilane (TMS). Between 5 and
10 × 103 scans were accumulated and a line broadening of 50 Hzwas applied.All the solid-state 15N NMR spectra were obtained with a BrukerAV400 spectrometer at 40.56 MHz with a 500 ms pulse delay, a
6 al and Applied Pyrolysis 97 (2012) 62–72
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Fig. 1. TIC traces of 650 ◦C Curie-point pyrolysates: (a) hydrophobic (HPO), (b)
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4 J. Templier et al. / Journal of Analytic
ontact time of 0.8 ms and a spinning rate of 4 or 5 kHz (rotoriameter 7 mm). Between 4.3 and 5.8 × 105 scans were accumu-
ated and a line broadening of 50 Hz was applied. The chemicalhift was referenced to the nitromethane scale (0 ppm) and wasdjusted with 15N labelled glycine (−347.6 ppm). Deconvolutionf spectra was performed using the “dmfit program” [30].
.5. Elemental and isotopic analyses
Total C and N and the ı 13C and ı 15N isotope ratio values wereeasured for the five DOM fractions using elemental analysis-
sotope ratio mass spectrometry (EA-IRMS; Carlo-Erba NA-1500C Elemental Analyzer on line with a Fisons Optima A66 isotope
atio mass spectrometer). The ı 13C values are reported in per mil‰) relative to Vienna Pee Dee Belemnite (VPDB). The ı 15N valuesre reported in per mil (‰) relative to air N2. Analytical precisionas ±0.2‰. Data reproducibility was checked by replicates and a
yrosine laboratory standard.
. Results and discussion
.1. On-line CuPy
The organic nitrogen content, C/N atomic ratio and ı 15N val-es for the HPO, TPI and COL fractions of the Loire River arehown in Table 1. The pyrograms obtained by CuPy-GC/MS ofhese fractions and focussed on nitrogen-containing products (N-roducts), are shown in Fig. 1. The major pyrolysis productsre labelled and the N-products are reported in Table 2. Majorifferences were observed among the three fractions. However,etween HPO and TPI the difference is mainly related to theelative abundance of the compounds. Only three N-productsere identified from the HPO fraction (Fig. 1a). These were 4,5-imethyl-3(2H)-isoxazolone (10), 3,4-dimethylpyrrole-2,5-dione11), and 3-ethyl,4-methylpyrrole-2,5-dione (12), all of which areeterocyclic compounds also containing an oxygen atom. The TPIyrolysate yielded the same products, although in higher relativebundance, as well as 3-ethenyl,4-methylpyrrole-2,5-dione (13),,6-dimethyl-2(1H)-pyridinone (14) and trimethyl-isooxazole (15)Fig. 1b). These results can be compared with those previouslybtained for the HPOA (N: 1.9%, N/C: 0.03) and TPIA (N: 2.6%,/C: 0.05) fractions from the Gartempe [27] (Fig. 2). Although
he hydrophobic fractions from the two rivers were characterisedy similar nitrogen content, virtually none of the identified N-ontaining products in the Loire could be detected in the case of theartempe. Only traces of dimethyl-2-pyrrolidinone (9) and indoleerivatives (17, 18) were identified as coeluted products. As for the
ransphilic fractions, all the N-products detected from the LoirePI fraction were also detected from the Gartempe TPIA fraction,nd notwithstanding its lower nitrogen content, the latter addi-ionally yielded other N-containing heterocyclic products includingable 1lemental composition (C, N), atomic ratio and bulk isotope analyses (13C and 15N) for th
DOM fraction (river) Elemental content
Ca Na
HPOA (Gartempe) 47 1.9
HPO (Loire) 51.2 1.8
TPIA (Gartempe) 43.2 2.6
TPI (Loire) 47.4 3.7
COL (Loire) 46.4 3.7
a In wt.%.b Atomic ratio.c In ‰; standard deviations ı 13C: 0.2‰, ı 15N: Loire 0.3‰, and Gartempe 0.5‰.
transphilic (TPI) and (c) colloids fractions of DOM from Loire River ( , N- andO-heterocycles; �, N-containing compounds; P, peptidoglycan pyrolysis products;numbers refer to Table 2).
dimethyl-2-pyrrolidinone (9), and several pyridine, pyrrole andindole derivatives.
The marked difference in the number and abundance of N-products between the HPOA and TPIA fractions of the GartempeRiver might indicate more efficient XAD resin separation of their N-containing structural precursors than for the corresponding LoireRiver fractions. However, the low yield of N-products does notreflect, and strongly underestimates, the nitrogen content of the
HPOA (1.9%) fraction, which is slightly higher than that for theLoire river HPO fraction (1.8%). In the same way, the large num-ber of nitrogen compounds detected in substantial abundance inTPIA fraction (2.6%) led us to assume an underestimation of the TPIe different fractions of DOM.
C/Nb N/Cb ı 13Cc ı 15Nc
24.7 0.03 −29.7 3.728 0.03 −28.9 216.6 0.05 −28.4 0.712.8 0.07 −27.3 3.712.5 0.07 −28 4.9
J. Templier et al. / Journal of Analytical and Applied Pyrolysis 97 (2012) 62–72 65
Table 2Summary of the N-products released upon pyrolysis from the different fractions of DOM.
Peak number Main mass fragmentsa Identified compounds
1 67, 41/39 1H-pyrrole2 79, 52 Pyridine3 80/81, 53 1H-pyrrole, 3-methyl4 80/81,53 1H-pyrrole, 2-methyl5 96, 42, 68, 54 4(3H)-Pyrimidinone6 94/95, 80, 67, 55/53 2,5-Dimethyl-(1H)-pyrrole7 67, 43, 109 1H-pyrrole, trimethyl8 94, 109, 66 1-(1H-pyrrole-2yl)-ethanone9 56, 113, 85 Dimethyl-2-pyrrolidinone10 42/43, 113, 70, 52 4,5-Dimethyl-3(2H)-isoxazolone11 125, 54 3,4-Dimethyl-(1H)-pyrrole-2,5-dione12 67, 139, 53, 124 3-Ethyl-4-methyl-(1H)-pyrrole-2,5-dione13 137, 66, 94, 109 3-Ethenyl-4-methyl-(1H)-pyrrole-2,5-dione14 123, 94 1,6-Dimethyl-2(1H)-pyridinone15 111, 54/55, 68, 96 Trimethyl-isoxazolone16 117, 90/89, 63 1H-indole17 130/131, 77, 51, 103/102 1H-indole, 3-methyl18 76, 147, 104, 50 1H-isoindole-1,3(2H)-dione19 104/105, 133, 78, 51 1,3-Dihydro-2H-indol-2-one20 147, 118, 91, 65 1-Carboxaldehyde, 2,3-dihydro-1H-indol21 118, 161, 90 Methyl-1H-isoindole-1,3(2H)-dioneP1 59, 44/43 AcetamideP2 43, 97, 69, 139 3-Acetamido-5-methyl-furan
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P3 43, 125, 167
a Base peak underlined.
3.7%) nitrogen content. The source of these N-products is not com-letely ascertained, and several origins can be considered. Thus,yrroles, pyrrolediones and indoles derivatives can derive from
roteinaceous material [31–33], or melanoidin structures [34,35]ut another biochemical source such as tetrapyrrole pigments (i.e.hlorophyll) for pyrroles [36], cannot be ruled out.ig. 2. TIC traces of 650 ◦C Curie-point pyrolysates: (a) hydrophobic (HPOA) and (b)ransphilic (TPIA) fractions of DOM from Gartempe River ( , N- and O-heterocycles;, N-containing compounds; P, peptidoglycan pyrolysis products; numbers refer toable 2).
Unknown
CuPy-GC/MS of the Loire River colloids yielded a very differ-ent N-product distribution from the HPO and TPI fractions. TheN-products included, in order of elution (Fig. 1c), pyrrole (1) and
acetamide (P1), with traces of 2,5-dimethyl pyrroles (6) and 3-acetamido-5-methyl-furan (P2), and based on the mass spectra,probably a higher homologue of the latter (P3). Acetamide inpyrolysates are typical for N-acetyl amino sugars [37] of microbialFig. 3. TIC traces of 650 ◦C TMAH/Curie-point pyrolysates: (a) colloids fraction ofDOM from Loire River and (b) peptidoglycan from Staphylococcus aureus ( , N- andO-heterocycles; numbers refer to Table 2 and letters to the main text).
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ell walls (i.e. peptidoglycans), which have been shown throughpectral characterisation to dominate colloidal NOM fractions29,38,39]. These differences are consistent with COL as a distinctraction from HPO and TPI, defined by the isolation process with aigher MW (30,000–50,000 Da) than HPO and TPI (1000–2000 Da)nd revealed by their ı 15N values. Indeed, the ı 15N values ofhe three fractions are different from HPO (2‰) to TPI (3.7‰)nd colloids (4.9‰). This difference could be related to differentitrogen-containing macromolecule sources, but pyrolysis prod-cts reflect only a difference for colloids.
To improve the detection of the more polar compounds, Curieoint pyrolysis was performed in the presence of TMAH. No addi-ional information could be obtained for the hydrophobic andransphilic fractions from both rivers (data not shown). On the con-rary, TMAH analysis of the COL fraction revealed two additional-product groups (Fig. 3a). The first comprises the methylatederivatives of the dimethyl-2-pyrrolidinone (9), 4,5-dimethyl-(2H))-isoxazolone (10) and 3,4-dimethyl-1H-pyrrole-2,5-dione11) identified in the hydrophobic and transphilic fraction with-ut addition of TMAH. This likely reflects a very low abundance ofhese compounds in the COL fraction, under the detection thresh-ld without TMAH. The structure of the second group of pyrolysisroducts has not been completely elucidated but they have been
orrelated to peptidoglycan, as they are dominant in the TMAHyrolysate of a commercial peptidoglycan from Staphylococcusureus (Fig. 3b). The mass spectra of the compounds A [m/z 42,42 (M+•), 127, 57/56] and C [m/z 98, 41, 157 (M+•), 42, 70] cor-ig. 4. XPS C1s spectra: experimental curve and Gaussian-Lorentzian components (—), fitrom Loire River; (d) hydrophobic (HPOA) and (e) transphilic (TPIA) fractions from Garte86.3–286.5; (C O) 287.4–287.6; (C(O)N) 288.5–288.7; (C(O)O) 289.2–289.5.
Applied Pyrolysis 97 (2012) 62–72
respond to 3-methyl-N,N-dimethyl-2,4-imidazolidinedione and toN-methyl-5-oxoproline methyl ester, a major pyrolysis product ofglutamic acid [33], respectively. The B and D compounds, both withm/z 128 as base peak and with m/z 170 and 184 as M+•, respec-tively, could also correspond to amino acid derived products andneed more investigation.
It appears that, pyrolysis of two hydrophobic fractions of DOM,with similar organic nitrogen content showed major differences inthe abundance of N-pyrolysis products. The two transphilic frac-tions afford similar N-products but with an abundance inverselyrelated to the nitrogen concentration. Finally, the N-product pro-file of the COL fraction (Loire) was very different to that of the TPIfraction, despite the same nitrogen content.
Therefore, the analysis of these five different fractions of riverineDOM clearly demonstrates that the detection of nitrogen contain-ing compounds is not only related to the nitrogen content of thefractions, even in the case of similar hydrophobicity. Detectiondifferences may reflect the functionality of nitrogen present inthe source macromolecules. The latter was therefore investigatedthrough XPS and solid state 15N NMR.
3.2. Spectroscopic studies
3.2.1. C1s X-ray photoelectron spectroscopy (XPS)C1s XPS and 13C CPMAS NMR spectroscopy are powerful tools
to determine the nature of the different types of C. Moreover, C1sXPS complements 13C CPMAS NMR by providing information on
(+++): (a) hydrophobic (HPO), (b) transphilic (TPI) and (c) colloids fractions of DOMmpe River. Binding energy range from the spectra, in eV: (C C) 285; (C O+C N)
J. Templier et al. / Journal of Analytical and
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ig. 5. Solid state CP/MAS 13C NMR spectra: (a) hydrophobic (HPO), (b) transphilicTPI) and (c) colloids fractions of DOM from Loire River.
he speciation of nitrogen-linked carbon. Indeed, the C1s core levelpectrum enables the distinction between amide and acid/esterarbons, which 13C NMR cannot do, and can thus be used to deter-ine amide contribution [15].The C1s spectra exhibit a rather complex shape (Fig. 4) and thus,
s commonly performed, they were deconvoluted. According to theolid-state 13C NMR spectra of the Loire fractions (Fig. 5), thosereviously reported for the Gartempe fractions [27] and literatureata [15], four types of C were initially considered to deconvolutehe spectra: aliphatic and aromatic carbon (C C, C C, C H), carbon
ound to oxygen or nitrogen (C O;C N), amide carbon [C(O)N] andarboxyl carbon [C(O)O], which comprises acid and/or ester. Theine shape and peak position were then adjusted using the auto-tting function of the data analysis software on the most complexable 3elative distribution of carbon functionalities in the different fractions of DOM determine
DOM fraction (river) Functional group distribution (BEa, RPb)
C C, C C+C H C O+C N C O
BE (eV) RP (%) BE (eV) RP (%) BE (eV) RP (%)
HPOA (Gartempe) 285 52 286.34 25.3 287.43 7.5
HPO (Loire) 285 60.6 286.36 17.1 287.38 7.5
TPIA (Gartempe) 285 47.2 286.41 26.8 287.55 7.5
TPI (Loire) 285 48.9 286.41 24.2 287.41 8.5
COL (Loire) 285 39.4 286.51 37.8 287.57 8.2
a Binding energy (an energy correction was made to account for sample charging basedb Relative proportion of each functional group corresponding to fitted curves in Fig. 5.
Applied Pyrolysis 97 (2012) 62–72 67
spectra. This four component fit was not entirely satisfactory, espe-cially for the highest energies, and an additional contribution ofcarbonyl carbon (C O) was considered.
The same five contributions were then used to deconvoluteall the spectra. The resulting high resolution C1s spectra of thefive DOM fractions are shown in Fig. 4, with the binding energies,assignments and quantitative data for the different peaks in Table 3.
For the Gartempe, both fractions (HPOA, TPIA) have rather sim-ilar profiles, the main contributions being aliphatic and aromaticcarbon (C C, C C, C H), along with carbon bound to oxygen ornitrogen (C O;C N) with a high predominance of the former. Themajor difference between the two fractions is a large increase in therelative abundance of amide carbon over aliphatic and aromaticcarbon in TPIA. In the latter fraction a slight enhancement in theC O;C N carbon contribution is also apparent, as well as a cleardecrease in carboxyl carbon compared to HPOA.
A similar trend is apparent for the Loire fractions from HPO toTPI and COL, however the enhancement in C O;C N carbon contri-bution is more pronounced, especially for colloids. When comparedto the carbon bound to oxygen or nitrogen (C O;C N), the aliphaticand aromatic carbons (C C, C C, C H) are highly dominant in thehydrophobic fraction, with a (C O;C N)/(C C, C C, C H) ratio of0.28, which rises up to 0.49 for TPI and reaches 0.96 for the colloidswhere the two contributions become equivalent. The increase inCO;CN contribution from HPO to TPI to COL is most likely a resultof an increase in the CO contribution, according to the evolution ofthe 13C NMR peak around 70 ppm (C O) (Fig. 5). These results sug-gest an important contribution of polysaccharides in the transphilicfraction, as previously observed for the Gartempe [27], and of pep-tidoglycan in the COL fraction.
The C1s spectra of the hydrophobic fractions of the two riverswere generally similar, although there was a higher relative abun-dance of CO;CN carbon for the Gartempe and of C(O)N carbon forthe Loire. An even stronger similarity is noted between the twotransphilic spectra. As observed for the Gartempe River, the rela-tive abundance of amide carbon in the TPI fraction is approximatelytwice that of the HPO fraction. Conversely the C(O)O carbon contri-bution is greater in the HPO fraction. Acid titration revealed morecarboxylic groups in the TPI fraction than the HPO [40], implyingthat the relative proportion of acid and ester functions is quite dif-ferent between the two fractions – in TPI most of the carboxylC belongs to acid groups and the contribution of ester functionsbecomes minor. However, when the sum of the amide C and car-boxyl C contributions is considered, it increases in agreement withthe rise in the 175 ppm NMR peak [C(O)O;C(O)N].
Analysis of the C1s spectra of these five fractions provides newinformation with respect to 13C NMR. First, it should be noticed that
an additional C O contribution had to be considered to fit the XPSspectra. Such a functional group could not be evidenced from NMR,likely due to its rather low abundance combined with a relativelypoorer detection of this type of quaternary carbon when using thed using XPS.
C(O)N C(O)O Amide+carboxyl Total C O
BE (eV) RP (%) BE (eV) RP (%) RP (%) RP (%)
288.52 5.9 289.23 9.3 15.2 22.7288.62 7.3 289.36 7.5 14.8 22.3288.69 13.7 289.36 4.8 18.5 26288.67 13.7 289.43 4.7 18.4 26.9288.58 10.8 289.48 3.8 14.6 22.8
on the carbon peak at 285 eV).
68 J. Templier et al. / Journal of Analytical and
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3
stNvbiapapaaaorf3ts
btTp
ig. 6. XPS N1s spectra: (a) pyroglutamic acid, (b) cytosine, (c) protoporphyrin andd) serum albumin bovine.
P sequence. Second, XPS revealed the increasing contribution ofmide carbon from the hydrophobic fraction to both transphilic andolloids fractions.
.2.2. N1s XPSCommercial compounds were first analysed as reference
tandards. The N1s spectra of pyroglutamic acid, cytosine, pro-oporphyrin and serum albumin bovine with binding energy of
functional groups are shown in Fig. 6. Reported XPS energyalues of organic nitrogen [1,16,41–45] indicate three groups ofinding energy, one centred around 400 eV for peptide bond N,
ncluding other amides, secondary and tertiary amines, imidesnd pyrroles, one for lower binding energy down to 398.6 eV foryridinic N including imines, pyridines and aromatic amines, and
third with higher binding energy of 401–402 eV attributed torotonated amines and quaternary N. In agreement with thesessignments, the pyroglutamic acid spectrum shows a symmetricalmide peak at 399.8 eV and the cytosine spectrum shows amide andromatic amine contributions at 399.8 and 398.7 eV. The spectrumf protoporphyrin is also consistent with previous studies [46] andeferences therein] and illustrates that the protonated N (pyrroleorm) is clearly distinct from the unprotonated N (imine form), at99.8 eV and 397.7 eV, respectively. The contribution at 399.4 eV inhe serum albumin bovine spectrum can be related to the lateralide chain of amino acids [16,47].
The N1s spectra of the five riverine fractions were analysed
y fitting Gaussian–Lorentzian curves to each signal to estimatehe relative proportion of nitrogen functionalities (Fig. 7, Table 4).he spectrum of the HPO fraction shows a single symmetricaleak. It is centred at 400.1 eV and can be attributed to amides,Applied Pyrolysis 97 (2012) 62–72
imides, secondary and tertiary amines or pyrrole N. The otherspectra have asymmetrical profiles. Besides a major peak centredfrom 399.9 to 400.1 eV, they require an additional contribution athigher energy (from 401.5 to 401.9 eV), corresponding to proto-nated amines and/or quaternary N, to provide a satisfactory fit. TheTPI and COL fractions of the Loire River show a very similar contri-bution of protonated amines, whilst such a contribution occurs inthe HPOA and TPIA fractions of the Gartempe, with a higher pro-portion for the TPIA fraction. No pyridinic contribution (<399 eV)was needed to obtain a good fit for any of the five fractions.
These results indicate a high predominance of amide and/orpyrrole N in all the DOM fractions from the two rivers, and agreewith nitrogen functionalities commonly reported for HSs [10,45].Pyridinic N has not been systematically observed for the organicnitrogen from soils or aquatic environments. Although similar XPSspectra with no evidence of pyridinic N were obtained for humicacids (HAs) from a number of soils and sediments [15,48–50],pyridinic N was detected as a constituent of HSs from other soils[1,3,10,16], kerogen, peat [47] and sediments [43]. In the case ofsoil HSs, such heterocyclic N C is relatively more concentrated inthe non-hydrolysable fraction and can be positively related to thedegree of humification [1,10]. For DOM, nitrogen functionalities dif-fer, depending on the source of organic matter. Fimmen et al. [51]showed that aquatic fulvic acid (FA) derived mostly from precur-sor plant material subjected to partial degradation and has a N1sspectrum dominated by peaks with binding energy <399 eV cor-responding to aromatic nitrogen. On the contrary, the spectrumof FA of microbial origin is completely devoid of such a contribu-tion and can be compared with the present spectra. Moreover, theXPS-based N composition of ultra filtered dissolved organic mat-ter (UDOM) from rivers and estuarine environments, reveals animportant pyridinic N contribution [45], the source of this aro-matic N being unclear according to the authors. The concentrationof aromatic N was tentatively correlated with the 13C NMR-basedaromatic C concentration [1] but, according to the diversity of theresults and the limited number of samples analysed, this has notbeen confirmed [14,15,27,51]. In the present study, no differencecan be evidenced between the N1s spectrum of the HPOA fraction,containing an important concentration of aromatic compounds(lignin-derived products), and the TPIA fraction, mainly composedof polysaccharides [27], or the colloids fraction, mainly derivedfrom peptidoglycan.
3.3. Solid-state 15N NMR analysis
The 15N NMR spectra of the five fractions of DOM (Fig. 8) exhibitimportant differences. The spectra of the Loire fractions show adegree of complexity increasing from the COL to HPO. The COLspectrum is dominated by a narrow signal peaking at −262 ppm,assigned to amide-N, with an additional signal at −348 ppm asso-ciated with free amino groups, indicative of a peptide-like structure[7]. The peak at −309 ppm can be attributed to a downfield shift ofamine N after protonation or to NH groups from nucleic acid [51];indeed, the XPS data support the presence of protonated aminegroups.
The spectra of the Loire HPO and TPI fractions are more complex,revealing a contribution from several nitrogen species. In bothspectra, the main peak is broad (−289 to −222 ppm), overlappingthe chemical shift regions of amide N (−239 to −283 ppm) andheterocyclic N such as indoles and pyrroles (−170 to −250 ppm;[12]) with a large shoulder centred at −208 ppm, which can beassigned to pyrimidines, pyrrolidinediones, imidazoles and/or
N-substituted pyrroles [52]. The contribution of the differentnitrogen functionalities was estimated by way of deconvolutionof the main signal based on the single amide peak from the LoireCOL fraction with additional contributions from Gaussian peaks.
J. Templier et al. / Journal of Analytical and Applied Pyrolysis 97 (2012) 62–72 69
Fig. 7. XPS N1s spectra: (a) hydrophobic (HPO), (b) transphilic (TPI) and (c) colloids fractions of DOM from Loire River; (d) hydrophobic (HPOA) and (e) transphilic (TPIA)fractions of DOM from Gartempe River. Binding energy range from the spectra, in eV: amide N 399.9–400.1; protonated amine N 401.5–401.9.
Table 4Relative distribution of nitrogen functionalities determined by XPS in the different fractions of DOM.
Fractions of DOM (river) Functional group distribution (BEa, %b)
Amide Protonated amineBE (eV) RP (%) BE (eV) RP (%)
HPOA (Gartempe) 400.08 89 401.47 11HPO (Loire) 400.13 100TPIA (Gartempe) 399.88 84 401.57 16TPI (Loire) 399.87 91 401.68 9Colloids (Loire) 399.9 90 401.92 10
basede fitte
Tdsa
TR
a Binding energy, an energy correction was made to account for sample chargingb Relative proportion of each functional group in percentage corresponding to th
he relative proportions of the different functionalities are quite
ifferent (Table 5). In addition to HPO, the TPI spectrum showsignals related to protonated amine and free amino groups, centredt −302 and −348 ppm, respectively.able 5elative distribution of nitrogen functionalities determined by solid state CP/MAS 15N NM
Fractions of DOM (river) Functional group distribution (ıa, %b)
Substituted pyrrole Pyrrole
ı (ppm) RP (%) ı (ppm) RP (%)
HPOA (Gartempe)
HPO (Loire) −208.03 24 −230.0 13−243.55 35
TPIA (Gartempe) −207.83 11 −233.13 12−246.45 9
TPI (Loire) −208.03 7 −232.00 5−245.55 25
Colloids (Loire) −232.00 6
a Chemical shift in ppm.b Relative proportion of each functional group in percentage corresponding to the fitte
on the carbon peak at 285 eV.d curves shown in Fig. 7.
In contrast, in the case of the Gartempe DOM fractions, the
spectrum of HPOA is quite different from that of TPIA. The HPOAspectrum exhibits a single amide N peak, whereas the TPIA spec-trum is rather comparable to the Loire TPI spectrum. EvidenceR for the different fractions of DOM.
Amide Protonated amine Amine
ı (ppm) RP (%) ı (ppm) RP (%) ı (ppm) RP (%)
−262.61 100−265.61 28
−260.89 68
−265.61 48 −302.00 7 −348.20 8
−262.00 74 −309.40 7 −348.20 13
d curves shown in Fig. 8.
70 J. Templier et al. / Journal of Analytical and Applied Pyrolysis 97 (2012) 62–72
amide amid e
pyrrol esubs�tute dpyrrol e
amine
proton atedamine
pyrrol e
subs�tute dpyrrol e
d)
b)
a)
e)
-20 0 -30 0 -40 0ppm
-20 0 -30 0 -40 0ppm
c)
Fig. 8. Solid state CP/MAS 15N NMR spectra: (a) hydrophobic (HPO), (b) transphilic (TPI) and (c) colloids fractions of DOM from Loire River; (d) hydrophobic (HPOA) and (e)t m thea .4; am
fTaw−pue1
3
paipfboHlvaftcTe
ransphilic (TPIA) fractions of DOM from Gartempe River. Chemical shift range frond −243.5 to −246.4; amides −260.1 to −265.6; protonated amines −302 to −309
or amine and protonated amine is not clear in all the samples.his could be a result of the small quantity of sample availablend the rather low signal/noise ratio. In all the spectra, no signalas detected in the chemical shift region of pyridinic N between25 and −90 ppm [53]. This is in agreement with XPS results,articularly since 15N NMR has been reported for heterocyclic Nnderestimation [54] and especially for pyridinic N [45,47]. How-ver, such underestimation was recently contested by comparing5N Bloch decay and CP MAS NMR spectra [55].
.4. Discussion
From these results it appears that the relative proportion ofyrrolic N to amide N shows large variations depending on the rivernd the fraction. For the Loire, we observe a dominance of pyrrolesn HPO, with 72% of the detected N functionalities, and a decrease inyrroles contribution for TPI at 37% and for colloids at 6%. The dif-erent pyrrole contribution in Loire DOM, with HPO > TPI > COL, maye related to the variations observed in the 15N isotopic signaturesf the fractions. Indeed, the increase in the bulk ı 15N value from thePO fraction (2‰) to the TPI (3.7‰) and COL (4.9‰) fractions paral-
els their decreasing pyrrole contribution, suggesting a lower ı 15Nalue for pyrrole N than for amide N. For the Gartempe, amide N islways predominant. In contrast to Loire HPO, the only detectableorm of nitrogen in the Gartempe HPOA fraction is amide, whilst
he pyrrole contribution to the Gartempe TPIA fraction (32%) isomparable to that of the Loire TPI. The ı 15N values increase fromPIA (0.7‰) to HPOA (3.7‰), whereas pyrroles are no longer appar-nt. As a result, the pyrrole contributions (Fig. 8; Table 5) allowspectra, in ppm: substituted pyrroles −207.8 to −208.3; pyrroles −230 to −233.1ine −348.2.
understanding of the reverse trend in the ı 15N values noticed fromHPO to TPI on one hand and HPOA to TPIA on the other hand. Theimportant difference noticed between the two rivers, with highlycontrasted nitrogen functionalities between HPOA and TPIA, is pos-sibly related to the fractionation procedure. The HPOA and TPIA areonly acidic fractions from the total DOM, whereas the HPO and TPIfractions contain acidic, basic and neutral structures, after colloidsseparation. Heterocyclic nitrogen has previously been detected inorganic matter from different sources. For soil organic matter, itsorigin has been positively correlated with the degree of humifica-tion [13,56,57], or with fire induced transformation of N-containingbiochemical precursors [14,18,58–60]. The presence of a significantamount of heterocyclic N was also reported for freshwater and estu-arine DOM, but its origin is not as clear [45]. The latter authorssuggest a possible input from soot/charred material produced bywildfire events, which is unlikely in the present study. For the Loireand the Gartempe samples, the source of pyrroles is more proba-bly related to diagenetic alteration of the DOM. Fimmen et al. [51]showed that the proportion of non-peptide nitrogen can accountfor up to 50% of total organic N in diagenetically altered terrestriallyderived DOM.
From the proportion of amide N determined from 15N NMR anal-ysis and the N/C ratio values established from elemental analysis,the proportion (%) of amide C can be evaluated for each fraction.For the Loire samples, the amide C approximates to <1% in HPO,
>3% in TPI and about 5% in the colloids and, for the Gartempe 3%and 3.4% in HPOA and TPIA, respectively. These values cannot bedirectly compared to the amide contribution derived from the C1sspectra. Indeed, a contribution of pyrroles C to this peak cannot be
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xcluded (in the C1s spectrum of protoporphyrine, the C N carbonontribution is centred at 287.85 eV whereas the amide carbon ofyroglutamic acid is at 288.04 eV, data not shown). This underlineshe limits of each technique, especially when the functionalities areot evaluated as distinct peaks. Consequently, the data have to beonsidered with caution.
From these NMR based determinations, the presence and rela-ive contributions of the different nitrogen functionalities can beelated to the occurrence of N-products. In contrast to the HPOAnd TPIA fractions, which exhibit quite different pyrograms, LoirePO and TPI fractions show comparable detection of N-products.owever, whilst the proportion of amide N, determined from the
5N NMR and nitrogen content, was similar for the HPOA andPIA fractions (1.9% and 1.8%, respectively), it was very differentor the HPO and TPI fractions (0.5% and 1.8%), respectively. Theignificant difference observed upon pyrolysis in the detection of-products between HPOA and TPIA and the comparable identi-cation revealed for HPO and TPI cannot therefore be related tomide functionality. On the contrary, HPOA is the only fractionhich does not contain any pyrrole N and is characterised by a
ack of N-products. Taken together these observations suggest thathe presence of N-containing pyrolysis products could be depen-ent on the presence of pyrrole N in the source macromolecules.rom these results it would be interesting to consider the amountf pyrrole N instead of total N content to anticipate the release of-products upon pyrolysis of DOM.
. Conclusions
Pyrolysis of five different fractions of riverine DOM clearlyhows important differences in the occurrence of N-productsetween fractions containing a similar amount of nitrogen. Itppears that the presence of N-products is not only related tohe nitrogen content of the fractions, even in the case of similarydrophobicity, but is likely related to the functionality of nitrogen
n the source macromolecules. In contrast to 13C NMR, C1s and N1s-ray photoelectron spectroscopy allows detection of the amideccurrence. Amide contribution to DOM was observed in all fiveractions. 15N NMR reveals the importance of this amide contri-ution, which is low in HPO, in similar amounts in TPI, TPIA andPOA and highest in COL. In addition, solid-state 15N NMR analysis
eveals the presence of different nitrogen functionalities such asmine and pyrrole. However, the relative proportion of the differ-nt functionalities varied largely among fractions. The only fractionhich did not release any N-products (Gartempe HPOA) was alsoevoid of pyrrole N. Thus, it is proposed that the presence or notf N-products in pyrolysates of DOM could be partly related to theresence or not of pyrroles in the macromolecular sources.
cknowledgements
We gratefully acknowledge J. Maquet (LCMC, UPMC Paris,rance) for NMR technical support and G. Bardoux (BioEMCo, INRArignon, France) for nitrogen isotope analysis.
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