Analytica Chimica Acta 843 (2014) 46–58

Exploration of cardanol-based phenolated and epoxidized resins bysize exclusion chromatography and MALDI mass spectrometry

Thierry Fouquet *, Laura Puchot, Pierre Verge, João A.S. Bomfim, David RuchPublic Research Centre Henri Tudor, Department of Advanced Materials and Structures, ZAE Robert Steichen, L-4940 Hautcharage, Luxembourg

H I G H L I G H T S G R A P H I C A L A B S T R A C T

� Phenolated and epoxidized cardanol-based resins were analyzed by SECand MS.

� A nomenclature was proposed forthe main products of both resins.

� Unsaturation patterns of cardanol-based species were simulated.

� Comparison of simulated anddetected unsaturation patternprovided insight into the changesinduced by the chemical treatments.

� SEC peaks convolution andMALDI artifacts were evidenced bySEC–MALDI off line coupling.

A R T I C L E I N F O

Article history:Received 29 April 2014Received in revised form 15 July 2014Accepted 19 July 2014Available online 24 July 2014

Keywords:Mass spectrometrySize exclusion chromatographyCardanolPolyphenol resinPolyepoxy resinBioresin

A B S T R A C T

Cardanol and cardanol derivatives are among the most important biobased materials currentlyinvestigated in green chemistry, as renewable and promising building blocks in lieu of traditional rawmaterials from non renewable resources, in particular owing to the olefinic linkages on the C15 alkylside-chain. Despite the increasing interest they arouse, analytical chemistry dedicated to cardanol andassociated resins has been rarely reported in the literature, found even poorer when dealing withchromatography and mass spectrometry. In this work, a thorough molecular characterization wasconducted using matrix assisted laser desorption ionization (MALDI) mass spectrometry, size exclusionchromatography (SEC), and SEC–MALDI coupling to gain insights into the composition of phenolated,epoxidized, and epoxidized phenolated cardanol. A nomenclature was proposed to properly describe thenumerous species found in these materials, while simulations of the unsaturation patterns and theircomparison with the detected patterns in MALDI-MS gave useful details about the phenolation treatmentexpected to occur on the polyunsaturated C15 side chain. Finally, the SEC–MALDI off-line couplingallowed SEC peaks to be deconvoluted by mass spectrometry and MALDI artefacts related to matrixadduction to be pointed out.

ã 2014 Elsevier B.V. All rights reserved.

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* Corresponding author. Tel.: +352 42 59 91 4598.E-mail address: thierry.fouquet@tudor.lu (T. Fouquet).

http://dx.doi.org/10.1016/j.aca.2014.07.0300003-2670/ã 2014 Elsevier B.V. All rights reserved.

1. Introduction

Cashew nutshell liquid (CNSL) is an abundant agriculturalnon-food by-product generated during the processing of cashewnutshells and forms the basic raw material for a vast number ofindustrially important chemicals and chemical intermediates, as

Scheme 1. Structures of (a) native cardanol with its polyunsaturated C15 side chain (tri-, di-, mono-unsaturated and saturated components naturally found in cardanol),(b) epoxidized cardanol (commercially named as NC513), idealized structures of (c) phenolated cardanol (or polyphenol resin, commercially available as NX5266), and(d) epoxidized phenolated cardanol (or polyepoxy resin, sold as NC514).

T. Fouquet et al. / Analytica Chimica Acta 843 (2014) 46–58 47

natural pesticides/insecticides [1], antioxidants [2], or plasticizers[3]. The main constituent of this family of bio-phenols is cardanol,an unsaturated alkylphenol (Scheme 1a). It is composed of fourdifferent meta-alkyl phenols differing in the degree of unsatura-tion in the side chain: 2% of 3-(pentadecyl)-phenol, 34% of3-(8Z-pentadecenyl)-phenol, 23% of 3-(8Z, 11Z-pentadecadienyl)-phenol, and 41% of 3-(8Z, 11Z, 14-pentadecatrienyl)-phenol [4].

The phenolic hydroxyl group and olefinic linkages on the C15alkyl side-chain make cardanol a promising starting material forthe production of chemical building blocks for the polymerindustry made for instance by esterification, nitration, hydrogena-tion, epoxidation, sulfonation, decarboxylation, and etherification[5]. Thanks to these tailorable chemical modifications, formation offibers and nanotubes, self assembled gels, liquid crystals,surfactants and even antibacterial coatings can be reached withsuccess [6], underlining the potential of this bio-synthon.

The characterization of cardanol derivatives remains achallenging task. NMR is fully appropriate for the characterizationof single cardanol moieties [7], but peaks assignments andespecially integrations become harder and harder with theincrease of the unsaturated alkyl side chains, preventing from aneat peaks integration. Generally speaking, the use of NMR for thecharacterization of bio-based compounds remains a field of activefundamental research. Notwithstanding this clear difficulty,and despite being traditionally considered as perfectly comple-mentary to NMR spectroscopy, mass spectrometry (MS) has beensurprisingly poorly presented as an efficient tool for the finecharacterization of cardanol and cardanol derivatives.

By the past, we reported the characterization by massspectrometry of cardanol-designed surfactants which was partic-ularly efficient thanks to the fingerprint pattern of cardanolthoroughly discussed later in the article [8]. A few papers deal withthe analysis of cardanol using electrospray [9] and electron impact(EI) following a gas chromatography (GC) separation [10]. Theleanness of this topic indicates that significant investigations havestill to be performed.

This paper deals with the first full MS and chromatographiccharacterization of poorly described cardanol derivatives– epoxidized cardanol, phenolated, and epoxidized phenolated

cardanol-based resins – essential raw materials for new buildingblock chemistries. In particular, a detailed attention has been givenon the unsaturation pattern analysis and its simulation to evidencethe modification induced by the phenolation treatment. To the bestof our knowledge, such simulations of patterns arising from thepoly-unsaturated side chains of cardanol moieties are not reportedin the literature.

2. Experimental

2.1. Chemicals

Cardanol (Lite 2013), epoxidized cardanol (NC513), phenolatedcardanol or polyphenol resin (NX5266) and epoxidized phenolatedcardanol or polyepoxy resin (NC514) were kindly supplied byCardolite (Zhuhai, China). HPLC grade tetrahydrofuran andmethanol were from Acros (Geel Belgium), while diethylenetri-amine (DETA), trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-prope-nylidene] malononitrile (DCTB), silver trifluoroacetate (AgTFA)and lithium trifluoroacetate (LiTFA) were purchased fromSigma–Aldrich (St Louis, MO). Dithranol was from Bruker Daltonics(Leipzig, Germany). All chemicals were used as received withoutfurther purification.

2.2. HPLC and HPLC-MALDI-MS offline coupling

The HPLC apparatus consisted of a 600E controller and pumpingunits (Waters, Versailles, France) followed by a 486 tunable UV–visdetector (Waters). Separation was performed on a uniqueC18 reverse phase HPLC column (Eclipse XDB-C18 5 mm,150 mm � 4.6 mm, Agilent) at room temperature. MeOH at a flowrate of 1 mL min�1 was used as eluent, while 20 mL of a 1 g L�1 resinsolution was injected in the HPLC system – allowing reproducibleHPLC-MALDI-MS data to be recorded in a single run. Fractions ofaround 200 mL were collected directly after the UV detector at themaximum peak detection and submitted to a solvent-free/solvent-based mixed MALDI sample preparation. 50 mL of each aliquotwere mixed with grains of DCTB and AgTFA, gently ground togetherusing mortar/pestle until the solvent fully evaporates, the resulting

Fig. 1. (a) HPLC chromatogram of native cardanol in methanol (isocratic elution) recorded from a UV–vis detector (l = 280 nm). HPLC–MALDI mass spectra of the three mainpeaks detected in the chromatogram: (1) tri-unsaturated cardanol (elution time: 4.55 min), (2) di-unsaturated cardanol (elution time: 5.18 min), and (3) mono-unsaturatedcardanol (elution time: 5.98 min) (off-line coupling, mass range: m/z 402–414, positive mode, DCTB, and AgTFA as matrix and cationizing agent). (b) Direct MALDI massspectrum of cardanol using the above-detailed conditions.

48 T. Fouquet et al. / Analytica Chimica Acta 843 (2014) 46–58

powder further deposited onto the MALDI target and pressedgently with a small spatula to produce a homogeneous thincoverage.

2.3. SEC and SEC-MALDI-MS offline coupling

Using the same LC apparatus as for the HPLC analyzes, SECseparation was performed on a unique mixed bed size exclusionchromatography column (PLgel Mixed-D 300 mm � 7.5 mm,particle size 5 mm, Agilent) with precolumn (PLgel Mixed-D50 mm � 7.5 mm) at room temperature. THF at a flow rate of1 mL min�1 was used as eluent, while 20 mL of a 15 g L�1 resinsolution was injected – allowing reproducible SEC-MALDI-MS datato be recorded in a single run. Several fractions of 500 mL werecollected directly after the UV detector and submitted to a solvent-free/solvent-based mixed MALDI sample preparation (vide supra).

2.4. Mass spectrometry

MALDI mass spectra were recorded using a Bruker Autoflex IIImass spectrometer (Bruker Daltonics, Leipzig, Germany)equipped with a frequency-tripled Nd-YAG laser (l = 355 nm)operating at a pulse rate of 50 Hz. Ions were accelerated by apositive 19 kV voltage with a pulsed ion extraction device(10–100 ns). The time-of-flight analyzer was operated in reflectronmode and ions were detected using a microchannel plate detector.External and internal calibrations were performed using mono-disperse PEGs standards (Sigma–Aldrich, 600, 1.5k and 4k, external

calibration: solvent-free deposit, with CHCA as matrix and LiTFA ascationization salt; internal calibration: mixed with the analyte,DCTB, and AgTFA). FlexControl software version 3.0 (BrukerDaltonics) was used for instrument control and data acquisition,while both FlexAnalysis software version 3.0 (Bruker Daltonics)and mMass version 5.5 [11] were used for data processing. DirectMALDI-MS analyzes were done using a solvent-free samplepreparation with DCTB and LiTFA or AgTFA as matrix and dopingsalt, respectively. Solvent-free (for direct MALDI analyzes) andhybrid solvent-free/solvent-based (for LC–MALDI coupling)sample preparations have been chosen since: (a) insoluble orpoorly soluble components could constitute a part of the cardanol-based resins and would thus be not detected in a solvent-basedapproach; (b) solvent-free MALDI is less sensitive to the matrix-to-analyte molar ratio [12,13], totally unknown when analyzing thealiquots collected from the LC run; and (c) low matrix-to-analytemolar ratios are usually reached in a solvent-free samplepreparation with no further dilution, leading to matrix peakssuppression and consequently simpler mass spectra [14].

Post source decay (PSD) experiments were conducted using theFAST method implemented in FlexControl. The precursor ion wasselected using an ion gate with a �20 Da selection. The m/z400–800 mass range was analyzed from high to low m/z values infour segments by applying a decreasing reflectron voltage (21 kVfor the first segment containing the precursor ion, further loweredto 19.43 kV, 16.80 kV, and 13.77 kV). Full PSD–MALDI mass spectrawere then recalibrated and overlaid using the FlexAnalysissoftware.

Fig. 2. (a) HPLC chromatogram of NX5266 cardanol-based polyphenol resin inmethanol (isocratic elution) recorded from a UV–vis detector (l = 280 nm). (b)HPLC–MALDI mass spectra of the peaks noted (1) (elution time: 2.03 min) and (2)(elution time: 3.01 min) detected in the chromatogram (off-line coupling, massrange: m/z 480–710, positive mode, DCTB and AgTFA as matrix and cationizingagent). In insets are given the structures of the main species detected in theHPLC–MALDI mass spectra. * Designates matrix peaks.

Fig. 3. SEC chromatograms of (a) epoxidized cardanol (gray line) and epoxidizedphenolated cardanol (black line) and (b) native cardanol (gray line) and phenolatedcardanol (black line) recorded from a UV–vis detector (l = 280 nm) using THF as themobile phase.

T. Fouquet et al. / Analytica Chimica Acta 843 (2014) 46–58 49

3. Results and discussion

3.1. First chromatographic considerations

Reverse phase HPLC has been reported as an efficient tool forthe separation of the four components constituting the pristinecardanol [15]. In Fig. 1a is depicted the HPLC chromatogram ofnative cardanol eluted with methanol in isocratic mode. Threemain peaks are readily detected (noted 1, 2, and 3) after 4.55, 5.18,and 5.98 min of elution. Their chemical nature was assessed byHPLC–MALDI off line coupling, the HPLC–MALDI mass spectra ofaliquots collected from peaks 1–3 being depicted in Fig. 1a(see Table S1a in the Supplementary data for peaks assignments).

Tri-, di-, and mono-unsaturated species were observed at m/z 405/407, m/z 407/409, and m/z 409/411 in that order of elution inaccordance with their expected decreasing polarity, with thedetection of two peaks per mass spectra due to the 107/109 silverisotopes convolution. The direct MALDI mass spectrum of nativecardanol is reported in Fig. 1b (mass range m/z 402–414),exhibiting the expected and so-called “unsaturation pattern”due to the varying degree of unsaturation of the C15 side chain.Four peaks are detected at m/z 405 (107Ag tri-unsaturated n = 3),m/z 407 (107Ag di-unsaturated n = 2, and 109Ag tri-unsaturatedn = 3), m/z 409 (107Ag mono-unsaturated n = 1 and 109Ag di-unsaturated n = 2) and m/z 411 (107Ag saturated n = 0 and 109Agmono-unsaturated n = 1), actually the addition of the three HPLC–MALDI mass spectra previously commented (see Table S1b in theSupplementary data for peaks assignments). The full MALDI massspectrum of cardanol is depicted in Supplementary data Fig. S1.

If HPLC with a simple isocratic MeOH elution readily discrim-inates the components of cardanol regarding their polyunsaturat-ed C15 side chain, a poorly resolved HPLC chromatogram wasrecorded for the phenolated cardanol resin, as depicted in Fig. 2awith highly polar compounds eluted within the first 5 min of thechromatographic run and a long tail probably formed of dimeric oroligomeric species far more apolar eluted for the following 10 min.Such isocratic elution was conducted for sake of comparison withthe pure cardanol chromatogram, highlighting a dramatic changein terms of chemical composition and polarity. HPLC–MALDI massspectra recorded from the peaks noted 1 and 2 in Fig. 2a aredepicted in Fig. 2b.1. and Fig. 2b.2., respectively. The expected maincomponent of phenolated cardanol (Scheme 1c) is readily detectedin both spectra as a silver adduct at m/z 499 (+unsaturatedcongeners), but a clear discrimination has occurred during theHPLC run regarding the C15 side chain. While the di-unsaturatedphenolated cardanol at m/z 499 (inset of Fig. 2a for its structure) isobserved as the main species in peak 1 (Fig. 2b.1 and inset for azoom on the unsaturation pattern), peak 2 (Fig. 2b.2 and inset) ispredominantly composed of the saturated phenolated cardanol atm/z 503. In addition to this separation process based on the C15side chain unsaturations, another discrimination based on the

Fig. 4. (a) MALDI mass spectrum of the epoxidized cardanol. Structures of the threemain components (the targeted monoepoxidized cardanol at m/z 463, a cardanol/epoxidized cardanol dimer at m/z 763 and a hydrated epoxidized cardanol/epoxidized cardanol dimer at m/z 835) are given as insets. (b) SEC–MALDI massspectra of the fractions #1 (13.7 min elution time) and #2 (14.6 min elution time)using DCTB and AgTFA as matrix and doping salt. See Table S2 for peak assignments.

50 T. Fouquet et al. / Analytica Chimica Acta 843 (2014) 46–58

functionalization of this side chain (i.e., the number of phenolgroups attached which influence the polarity of the so-formedspecies) is clearly effective, as evidenced by the detection on a

di-phenolated cardanol product at m/z 595 in Fig. 2b.1. (peak 1)(inset of Fig. 2a for its structure) and its absence in Fig. 2b.2. (latereluted peak 2, less polar).

With the occurrence of these different separation processesand despite being a fully appropriate technique for thefractionation of cardanol-based products according to thecomplex but promising results previously mentioned obtainedwith basic conditions, a whole methodological developmentwould be mandatory and should deserve a study of its own, out ofthe scope of this first exploration of unknown resins. Moreover,chemical modifications performed on the C15 side-chain, as it isthe case for phenolated cardanol, could lead to noticeablepolymerization pathways and/or to the occurrence of severalcardanol moieties per molecule.

Size exclusion chromatography (SEC), another chromatographicseparation process based on the hydrodynamic radius discrimina-tion (eventually related to molecular weight for a given chemicalfamily) instead of polarity issues – already proposed by Jaillet et al.[16] for their characterization of the epoxidized phenolatedcardanol was hence adopted here as a “simpler” chromatographictechnique – of lower resolution, though.

The SEC chromatograms of epoxidized cardanol (gray line) andepoxidized phenolated cardanol (black line) are depicted in Fig. 3a.Jaillet et al. [16] used native cardanol as a reference compound andits SEC chromatogram was compared to epoxidized phenolatedcardanol, but owing to its structure, epoxidized cardanol wasthought to be a better standard to be compared to.

Two fine peaks were readily detected in the SEC chromatogramof epoxidized cardanol, at 13.7 and 14.6 min of elution. In the orderof elution, hypothetic “dimeric” species and monoepoxy cardanol(Scheme 1b) are hence constituting the commercial epoxidizedcardanol. Such molecular attribution will be validated later in thearticle with the (SEC-)MALDI-MS analyzes. Looking at the SECchromatogram of the epoxidized phenolated cardanol, in additionto those two peaks above-mentioned, a broad signal from 12 to14 min was also observed, highlighting the presence of polymericmaterial as previously reported by Jaillet et al. The presence ofpolymeric chains probably dramatically decreased the HPLCresolution capabilities as discussed previously. Epoxidizedphenolated cardanol will hence be mentioned as polyepoxy inthe following text.

A quite similar result was obtained for the phenolated cardanolSEC analysis (Fig. 3b, black line), compared to the chromatogram ofa native cardanol (Fig. 3b, gray line). A unique fine peak is detectedfor cardanol (elution time �14.4 min), while a similar fine peak aswell as a “dimer-like” peak and a broad signal are all observed forthe phenolated sample, hence containing cardanol-like structure,cardanol-based dimers and polymeric components and now calledpolyphenol resin. It is worth mentioning the main fine peak in thepolyphenol resin chromatogram seems slightly shifted toward theshorter elution times as compared to cardanol, while a shoulderappears concomitantly to the cardanol elution. Several species ofdifferent chemical nature would hence be convoluted into thispeak. A SEC–MALDI coupling detailed later in the article willhighlight this chemical composition.

Since their chemical nature and structure are expected to beclose, polyepoxy and polyphenol should exhibit similar hydrody-namic radii in solution and their associated SEC chromatogramscould hence be directly compared without the need of intermedi-ate re-calibration. The molecular weight of the polyphenol resinseems quite lower and its high molecular weight populationsmaller than in polyepoxy – the epoxidation treatment potentiallyleading to additional polymerization reaction(s). To gain furtherinsights into the molecular composition and variations betweenboth resins, a thorough exploration at a molecular level by massspectrometry is thus mandatory.

Table 1Computed abundances of tri-, di-, mono-unsaturated and saturated components of monoepoxidized cardanol adducted with Ag+ (107/109 isotopes). Summing theseabundances leads to its simulated unsaturation pattern. Simulations of the isotope patterns of each component were done separately using the Bruker Daltonics isotopepattern add-on.

Tri-unsaturation Di-unsaturated Mono-unsaturated Saturated Simulated Unsaturation Pattern

Abundance Abundance Abundance Abundance Abundance

m/z Abs. Rel. m/z Abs. Rel. m/z Abs. Rel. m/z Abs. Rel. m/z Abs. Norm.

461 100 41.0 461 41.0 65.5462 26.4 10.8 462 10.8 17.3463 96.7 39.6 463 100 23.0 463 62.6 100464 24.9 10.2 464 26.4 6.1 464 16.3 26.0465 3.5 1.4 465 96.7 22.2 465 100.0 34.0 465 57.7 92.1466 0.4 0.1 466 25.0 5.7 466 26.5 9.0 466 14.9 23.8

467 3.5 0.8 467 96.7 32.9 467 100.0 2.0 467 35.7 57.0468 0.4 0.1 468 25.0 8.5 468 26.5 0.5 468 9.1 14.5

469 3.5 1.2 469 96.7 1.9 469 3.1 5.0470 0.4 0.1 470 25.0 0.5 470 0.6 1.0

471 3.5 0.1 471 0.1 0.1472 0.4 0.0 472 0.0 0.0

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3.2. Preliminary MS analysis: the unsaturation pattern

Before entering the thorough exploration of the targetedphenolated and epoxidized phenolated cardanols, a few commentshave to be made about the MS analysis of a simple system, in thatcase the epoxidized cardanol used above as standard for the SECanalysis. Below is depicted its direct MALDI mass spectrum usingsilver as cation (Fig. 4). Three main species are actually observed inthis sample. The targeted epoxidized cardanol is detected as the“complex” unsaturation pattern at m/z 461/463/465/467, arising

Fig. 5. MALDI mass spectra of (a) epoxidized cardanol and (b) an epoxidized cardanol/diconditions (positive mode, DCTB and AgTFA as matrix and doping salt). Insets show tcardanol/cardanol dimer, and (3a) a tetra-addition of epoxy onto a DETA moiety aexperimental patterns. In inset (3b) is depicted the hypothetic structure of an epoxidizedmost reactive centers [17].

from the tri-, di-, and mono-unsaturated and saturated side chainpeaks, respectively, convoluted by the 107Ag/109Ag isotopes, asstated before for pure cardanol (see Fig. 1 and Fig. S1 in theSupplementary data). The additional species found at m/z 759/761/763/765/767/769 and m/z 833/835/837/839/841/843 wereassigned to a dimer of native and epoxided cardanol (opening ofthe epoxy group by the phenol) and a dimer of hydrolyzedepoxided and epoxided cardanol. Ions are listed in Table S2 whiletheir structures are given as insets in Fig. 4a, with the SEC–MALDImass spectra of the two aliquots collected during the run of

ethylenetriamine (DETA) mixture allowed to react for a few minutes under ambienthe unsaturation patterns of (1) silverated epoxidized cardanol, (2) an epoxidizeddducted by silver. Simulated patterns are mirror-depicted under corresponding

cardanol adducted four times to a DETA core considering the primary amines as the

Fig. 6. MALDI mass spectrum of NC514 (cardanol-based polyepoxy resin) recorded in the positive mode, using DTCB and LiTFA as matrix and doping salt. (a) Mass range: m/z450–1000 and (b) mass range: m/z 1000–1800. The two main structures proposed by Jaillet et al. were found in the sample and reproduced in insets 1 and 2. Additionalstructures were noted using the aforementioned (a,b,c,d) nomenclature ((a): epoxidized cardanol moiety, (b): native cardanol, (c): epoxidized phenol, and (d): native phenol).An example is depicted in inset 3. Ions are listed in Table S7 (Supplementary data).

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epoxidized cardanol (Fig. 3b). The two dimers and the monoepoxycardanol were readily detected in fractions 1 (Fig. 3b1, peak 1 at13.7 min) and fraction 2 (Fig. 3b2, peak 2 at 14.6 min), respectively,hence validating the attributions postulated in Section 3.1.

Owing to the richness of the mass fingerprints – especially fordimers which bear two cardanol moieties – a simulation of theunsaturation pattern should and could be computed, and would beof high interest for a better understanding of the MS results, buthas surprisingly never been reported in the literature – to the bestof our knowledge. Here is briefly clarified the simulation of theunsaturation pattern of the main monoepoxy cardanol. For each ofthe four tri-, di-, mono-unsaturated and saturated components, theisotopic pattern could be simulated separately (using the BrukerDaltonics isotope Pattern add-on) considering its chemicalcomposition C24H(40–2n)O2 (with a defined n unsaturation valuefrom 3 to 0, respectively) and the silver isotopes 107Ag/109Agadduction. Peaks abundances are reported in Table 1 for thefour components (column “Absolute Abundance”). Absoluteabundances should then be convoluted considering the occurrenceof the four species in native cardanol (Supplementary data,Table S3), the resulting value being reported in Table 1column “Relative Abundance”. Eventually, summing these lastabundances leads to the simulated unsaturation pattern of acardanol-containing moiety.

A simulation of the unsaturation pattern for the epoxidizedcardanol/native cardanol dimer is detailed in the Supplementarydata. In this case, one has to calculate the probability for the dimerto contain, for instance, two tri-unsaturated side chains, or atri-unsaturated and a di-unsaturated side chains, or all the possiblecombinations of the two side chains compositions. Results arelisted in Tables S4 and S5 and the related simulated patterndepicted in Fig. S3.

These simulated patterns (gray lines, bottom) were thencompared to the observed signal (black line, top) and depictedin insets 1. and 2. in Fig. 5 and found to perfectly match in bothcases.

Making a digression to demonstrate this unsaturation patternsimulation efficiency and check at the robustness of thedeconvolution, a more complex sample composed of largerepoxidized cardanol adducts was synthesized by the facile reactionbetween the epoxy groups from the epoxidized cardanol anddiethylenetriamine (DETA) – the mixture reacting in a few secondsunder ambient conditions – and further mass-analyzed by MALDImass spectrometry. When only dimers are seen in the pristineepoxidized cardanol, up to five cardanol moieties per molecule(from pentadeca-unsaturated to fully saturated congeners) areexpected to be synthesized, exhibiting highly complex unsatura-tion patterns and consequently testing severely the simulation

Scheme 2. Examples of structures found in the NC514 cardanol-based polyepoxysample and their associated (a,b,c,d) value, where (a) represents the epoxidizedcardanol, (b) numbers the native cardanol, (c) the epoxidized phenol, and (d) thenative phenol. (a) Epoxidized cardanol bearing an additional phenol moiety, hencenoted (1,0,0,1). (b) Epoxidized cardanol containing both an epoxidized and a nativephenol on its side chain, further noted (1,0,1,1). (c) Epoxidized cardanol linked to anative cardanol, called (1,1,0,0), and (d) two epoxidized cardanol linked by a nativephenol, noted (2,0,0,1).

T. Fouquet et al. / Analytica Chimica Acta 843 (2014) 46–58 53

procedure. The resulting MALDI mass spectrum is depicted onFig. 5b and exhibits the five expected additions at m/z 565(+unsaturated congeners, one epoxidized cardanol moiety),m/z 920 (two epoxy), m/z 1275 (three epoxy), m/z 1635 (fourepoxy), and m/z 1994 (five epoxy). Ions are listed in theSupplementary data Table S6. The observed pattern associatedwith a tetra-addition of epoxidized cardanol is reproduced in inset3a. and compared with its simulated counterpart, the hypotheticstructure being depicted in inset 3b. (Fig. 5). A very satisfactorycorrelation is readily seen, ensuring both the simulation to berobust and the analysis of the unsaturation pattern to provideuseful information – each peak could be assigned to anunsaturation degree and a silver isotope couple (Supplementarydata Fig. S4 for more details about the deconvolution). This wouldbe of high interest for the exploration of a cardanol-basedpolyphenol – which would supposedly contain several cardanolmoieties and an undefined number of unsaturations along the sidechains – especially because the phenol grafting occurs precisely onthese unsaturations and their numeration would provide insightsinto the reaction schemes and product structures.

It should be noted that the occurrence of dimeric species inNC513 would actually lower its expected reactivity as they do not

contain epoxy rings anymore, and worse, potentially induceadditional epoxy ring openings to occur leading to an even lowerepoxy equivalent. Out of the scope of the present paper, theirquantitation should hence be quite useful and readily done usingeither the SEC or MS results, providing these dimers are isolatedand further used as standards in a calibration procedure for aternary mixture.

3.3. Mass spectrometry analyzes of polyepoxy and polyphenol resins

3.3.1. Polyepoxy resinMass spectrometry exploration was first conducted on the

NC514 polyepoxidized cardanol, as its main chemical structure wasalready proposed by Jaillet et al. based on 1H and 13C NMR results.The knowledge about such epoxidized resin will then be directlyused for the understanding of the NX5266 polyphenol resin as thelatter is the precursor of NC514, supposedly epoxidized usingepichlorhydrin to further produce a polyepoxy resin. The MALDImass spectrum of NC514 is depicted in Fig. 6a (mass range: m/z450–1000) and Fig. 6b (mass range: m/z 1000–1800), recorded inthe positive mode and using DCTB and lithium trifluoroacetate asmatrix and doping agent. Lithium was found to readily ionizeNC513 and NC514 components and would likely lead to “simpler”unsaturation patterns regarding the isotopic pattern of Li (7Li is themain isotope, 6Li arises with a limited 8% relative abundance).

Two structures have been proposed to account for thecomposition of NC514 [16], described as a polyepoxy cardanol (amain epoxidized cardanol bearing one, two, or three epoxidizedphenols along the C15 side chain) (Schemes 1 and 2) and apolymeric structure composed of epoxidized cardanols linkedtogether by phenols bearing opened epoxy groups. These specieswere found in the MALDI mass spectrum of NC514, their structuresbeing depicted in insets 1. and 2. in Fig. 6a above the correspondingpeaks. However, a closer look at the MALDI mass spectrum allowedseveral other structures to be highlighted, suspected to bearnon-epoxidized cardanol and phenol groups. For sake of clarity, anomenclature will be adopted in the rest of the paper to properlyname the so-detected components, precising the number of (a)epoxidized cardanols, (b) native cardanol, (c) epoxidized phenol,and (d) native phenol they contain by a (a,b,c,d) value. Examples ofthese structures with their associated nomenclature are reportedin Scheme 2, while all the main peaks detected in Fig. 6a wereassigned with their (a,b,c,d) value. It is worth mentioning thatepoxidized cardanol/phenol and cardanol/epoxidized phenol pairswould obviously exhibit the same chemical composition and m/zvalues. These isomeric issues are nevertheless not taken intoaccount in the present study for sake of simplicity.

The numerous peaks detected in Fig. 6b (i.e., in the upper massrange of the MALDI mass spectrum of NC514) were tentativelyassigned with the above-defined nomenclature, but the complexi-ty of the spectrum prevented us from assigning all the productsdetected. An example of a (2,0,2,1) structure at m/z 1113.36(+congeners) formed of two epoxidized cardanol moieties, twoepoxidized phenol moieties and one native phenol is depicted ininset 3.

Despite the inherent richness of the sample, four main structurefamilies could hence be now proposed from the analysis of NC514by mass spectrometry: (a) epoxidized cardanol bearing nativephenol and/or epoxidized phenol, (b) epoxidized cardanol bearingnative cardanol and/or epoxidized cardanol, (c) epoxidizedcardanol bearing phenol with open (chlorinated) epoxy groups,and (d) a polymeric structure whose repeating unit is a(epoxidized) cardanol/(epoxidized) phenol.

The complexity of this polyepoxy cardanol-based resin mainlyarises from the epichlorhydrin treatment – seemingly notquantitative and leading to residual native phenol and cardanol

Fig. 7. MALDI mass spectra of the phenolated cardanol recorded in the positive mode, using DCTB and AgTFA as matrix and doping salt. (a) Mass range: m/z 400–1100 and (b)mass range: m/z 1000–1800. Main peaks detected in both MALDI mass spectra were annotated using the simplified (b,d) nomenclature (b: cardanol moieties, d: phenolmoieties) detailed in Scheme 3 with generic structures. Peaks annotated with * are matrix and salt peaks. See Table S8 in the Supplementary data for peak assignments.

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moieties. The analysis of the polyphenol resin should hence beeasier as it is actually the precursor of the NC514 product, madefrom a phenolation of cardanol with no epoxy functionalization.

3.3.2. Polyphenol resinThe MALDI mass spectrum of the polyphenol resin is depicted

in Fig. 7 ((a) m/z 400–1000 and (b) m/z 1000–1800), recorded in thepositive mode, using DCTB and AgTFA as the matrix and dopingagent. As for the native cardanol MALDI mass analysis, silver cation– well-known to readily ionize aromatic and unsaturatedcompounds such as polystyrene [18,19] was mandatory to recorda satisfactory signal (lithium adduction leading to poor or even nosignal detection) while the epoxidized cardanol and the polyepoxyresin were readily analyzed using such lithium salt. This couldhighlight the location of the Li+ cation along the cardanol-basedstructure, near of the epoxy group for NC513 and NC514, and thelimited capability of the same Li+ cation to efficiently interact withthe phenol moiety for native cardanol. Similarly, Li+ would hencenot be found along the C15 side-chain (Scheme S1). As a shortdigression in the question of ionization efficiency, no signal wasrecorded in the negative mode for any cardanol-based product,either with no salt to promote deprotonation or with the adductionof anions (trifluoroacetate CF3CO2

� and bromide Br�). Generic

structures of the three main components of the polyphenol resinare depicted in Scheme 3 and named using a simplified (b,d)nomenclature.

As already suspected in the MALDI-mass spectrum of nativecardanol (Fig. S1 and Table S1b), DCTB readily produces (non-covalent) adducts with the phenolated cardanol compounds of thepolyphenol resin (Fig. 7 and Table S8). Side reactions betweenprimary/secondary amines and DCTB were previously described byLou et al. [20], with a 184 Da mass shift assigned to a chemicalreaction between the analyte and the matrix, both in liquid and gasphase. In our case, adduction of intact DCTB was indirectlysupported by the absence of any peak at a +250 Da mass shift fromthe (1,1) species at m/z 503 (+congeners) using dithranol (Fig. 8a)when a m/z 753 adduct (m/z 503 + 250 Da) is readily detected ifDCTB is used as matrix (Fig. 8b) [21]. Using internal calibrationwith two consecutive silverated PEG 14- and 15-mers at m/z741.282 and m/z 785.308 surrounding the peak of interest– following the calibration procedure proposed by Charles [22]– this hypothetical DCTB adduct was accurately mass measured atm/z 753.353, in good accordance with the theoretical value at m/z753.354 (C44H58AgN2O2, error: – 1.3 ppm). Finally, submitted to apost source decay (PSD) MALDI-MS experiment [23] (i.e., a tandemmass spectrometry analysis where metastable precursor ions are

Fig. 8. MALDI mass spectra of the phenolated cardanol resin recorded in the positivemode restricted to a m/z 400–800 mass range, using (a) dithranol and (b) DCTB asmatrix (AgTFA as doping salt in both cases). Peaks of interest are annotated using thesimplified (b,d) nomenclature (b: cardanol moieties, d: phenol moieties). (c) PSD–MALDImassspectrumofthe(1,1)DCTBadductatm/z753(+congeners)detectedin(b).

Scheme 3. The three main structures proposed to constitute the cardanol-basedpolyphenol resin. (a) One cardanol bearing one–three (undefined x) phenol groupsnoted (1,x), (b) one cardanol bearing several (x) cardanol moieties, i.e., apolycardanol with no phenol (x + 1,0), and (c) random copolymeric structure withcardanol and phenol as the co-monomers (y + 1,x).

T. Fouquet et al. / Analytica Chimica Acta 843 (2014) 46–58 55

selected using an ion gate and undergo dissociation processesduring their flight in the first drift region of the TOF analyzer, theresulting product ions being separated by the reflectron devicethanks to a step-by-step decrease of its voltage), the adduct at m/z753 readily expulsed a 250 Da neutral (Fig. 8c), further validatingthe non-covalent nature of the DCTB adduction.

The special propensity of DCTB to form adducts withpolyphenol resin but not (or in a so lower extent they were notdetected in the MALDI-mass spectra) with polyepoxy resinremains unclear at the time of writing. Specific interactionsimplicating the phenol moiety should take place, but no clear dataare at our disposal to better describe this phenomenon. Practicallyspeaking, the expected simplification of the mass analysis of apolyphenol instead of the polyepoxy resin (no epoxidation step,precursor of the latter) was actually counterbalanced by thismatrix adduction. DCTB remains, however, the best matrix for theanalysis of cardanol-based polyphenol resin regarding the numberof detected species and spectral quality as compared to other

Fig. 9. Top: unsaturation patterns detected in the MALDI mass spectrum of the polyphenol resin: (a) “native” cardanol (1,0), (b) mono-phenolated cardanol (1,1), and (c)di-cardanol (2,0) using the above-defined nomenclature. Bottom: simulated unsaturation patterns (see the Supplementary data for the procedure).

Fig.10. SEC–MALDI mass spectra of fractions #1–#3 collected in the high molecularweight area (shorter elution times) during the elution of the polyphenol resin inTHF. The SEC chromatogram is depicted as an inset with the position of the collectedaliquots.

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matrices tested (dithranol, a-cyano hydroxycinnamic acid CHCA,and 2,5-dihydrobenzoic acid DHB).

3.3.3. The unsaturation patterns issueIt has been extensively shown in Section 3.1 that a thorough

analysis of the unsaturation patterns as detected by MALDI-MScould be of high interest for a better understanding of the structureof cardanol-based products. A similar study based on anexperimental/simulated unsaturation patterns comparison hasbeen conducted here for the specific case of polyphenol resin. Sincethe phenolation of cardanol (and partial polymerization) isexpected to occur on the unsaturations of the C15 side-chain[24], the unsaturation patterns of the resulting products shouldbe dramatically modified – in a simplification way. Detected

unsaturation patterns of the compounds named (1,0), (1,1) and(2,0), i.e., “native” cardanol, mono-phenolated cardanol anddi-cardanol are depicted in Fig. 9 (top) and compared with theirsimulated patterns (bottom).

In the three cases, it is worth mentioning a remarkabledepletion of the most unsaturated components (tri- anddi-unsaturated for cardanol, di-unsaturated for phenolated carda-nol, penta- and tetra-unsaturated for di-cardanol) and anassociated over-detection of the saturated chains, compared tothe predictions. Such depletion/overabundance is in obviousaccordance with the higher reactivity of the double bond richside-chains: the more the unsaturations along the chain, the morethe propensity of these species to be phenolated or incorporatedwithin a polymeric structure with a phenol/cardanol repeatingunit. A similar trend was noted for the polyepoxy (data not shown).

3.3.4. SEC–MALDI coupling for complex mixtures: MALDI artifactsrevealing and SEC peaks deconvolution

No high molecular weight products were detected in the SECchromatograms of either polyepoxy or polyphenol resins, but abroad shoulder from “dimers” peaks – the estimated molecularweight was in both case around 1000 g/mol. An off-lineSEC-MALDI-MS coupling was tempted and confirm the absenceof structures with mass larger than 2000 Da. SEC-MALDI-MSspectra of three fractions collected during the elution of thepolyphenol resin are depicted in Fig. 10. It is worth mentioning twopoints highlighted by these mass spectra comparison. First, quasi-isobaric interferences between “real” products and matrix adductscould be avoided. A species detected at m/z 1303 in fraction #1 ofthe SEC elution (+unsaturated congeners, Fig. 10 #1) was describedas a (4,0) tetra-cardanol structure in Fig. 7b and Table S8. A similarm/z 1303 (+unsaturated congeners) pattern is seen in the followingfractions 2 and 3 (Fig. 10 #2 and #3) but together with a m/z 1053(+unsaturated congeners) species ((2,1) + DCTB). The concomitanceof their detections strongly suggests that the latter species at m/z1303 arises from a DCTB adduction to the m/z 1053 main product,hence of different nature than the (4,0) component seen in fraction#1. Performing accurate mass measurement as proposed inSection 3.3.2 undoubtedly validated this assumption, with peaksdetected at m/z 1303.810 in fraction #1 (C84H124AgO4, (m/z)th:1303.855, error: �34.5 ppm) and at m/z 1303.729 in fraction #2(C82H108AgN4O3, (m/z)th: 1303.747, error: �13.8 ppm). Second,different species of close molecular weights are detected indifferent fractions, as a deconvolution of the direct MALDI-MSspectrum of phenolated cardanol in Fig. 7b. For instance, the same(4,0) species at m/z 1301 in Fig. 10 #1 is eluted before the (4,1) at m/z 1395, the latter being of slightly higher molecular weight butprobably of smaller size in solution due to the phenolic moiety. A(limited) discrimination based on the hydrodynamic radius of poly(cardanol) and poly(cardanol-co-phenol) is hence suspected tooccur during the SEC elution. Further analyzes using ion mobilityspectrometry–mass spectrometry (IMS–MS) and LC–IMS–MScoupling (either HPLC or a rather new SEC–IMS–MS coupling)should be of nice added value for the exploration of the gas phaseconformation of these cardanol-based polyphenols, and willconstitute the topic of a forthcoming paper.

Apart from this MALDI artifact highlighting and a simplificationof the direct MALDI-mass spectrum, a SEC–MALDI coupling wasalso found of great interest for the deconvolution of the main SECpeak recorded from the polyphenol resin. As stated at Section 3.1,this peak maximum was slightly shifted toward the shorter elutiontimes as compared to the reference cardanol, with a shouldernicely correlating the cardanol peak (Fig. 3b, gray and black lines).A fine aliquot collection allowed this peak to be divided into twofractions further mass-analyzed. Their SEC-MALDI-mass spectraare depicted in Fig. 11 with the SEC chromatogram of the

Fig. 11. SEC–MALDI mass spectra of fractions #1 and #2 collected in the main lowmolecular weight area (longer elution times) during the elution of the polyphenolresin in THF. The SEC chromatogram is depicted as an inset with the position of thecollected aliquots.

T. Fouquet et al. / Analytica Chimica Acta 843 (2014) 46–58 57

polyphenol resin. Mass fingerprint of the peak maximum (noted 1in the inset) clearly exhibits the (1,1) species and its (1,1) + DCTBmatrix adduct, while the mass spectrum of the following shoulder-like part of the peak (noted 2 in the inset) only exhibits the (1,0)native cardanol peak and its DCTB adduct. Low molecular weightspecies from the polyphenol resin are hence constituted of theexpected phenolated cardanol and some traces of remainingunreacted cardanol, which was suspected from the SEC analysisbut nicely and unambiguously confirmed here with a molecularcharacterization.

4. Conclusion

Cardanol is a versatile biomolecule with a high potential ofchemical modification, reaching it at a broad applications field. Itsinherent chemical structure and particularly its varying degree ofunsaturation of the side-chain represent, however, a strongdifficulty for fine-tuning chemistries and especially for theidentification of the formed products. In this work, we proposedkey solutions, even if not exhaustive, to describe by SEC, MALDI-MSand SEC-MALDI coupling the products of cardanol phenolation,followed by epoxidation reactions. In particular, the simulation ofthe so-called unsaturation patterns (arising from the presence oftri-, di-, mono-unsaturated and saturated side chains) and their

comparison with the detected patterns – surprisingly neverreported in the literature – was found quite efficient and usefulfor a better understanding of the treatments conducted on thecardanol-based resins. HPLC- or SEC–MALDI off-line couplingtechniques also appeared as an essential asset for the developmentof the cardanol-based chemistry. The next step would be a tandemmass spectrometry (MS/MS) and ion mobility spectrometryexploration of these resins to gain insights into the structuresand isomeric richness potentially arising from the phenolation [24]and epoxidation route. A notable lack of MS/MS fragmentationpathways – a few papers deal with MS/MS of cardanol-relatedspecies in the literature [25] – will imply these dissociationschemes to be clearly understood and described for referencecardanol-based structures prior to any attempt to fully characterizethe structures of the species described from a molecular point ofview in the present article.

Acknowledgement

Cardolite Specialty Chemical is thanked for the supply of thematerials used in this study.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.aca.2014.07.030.

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