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Journal of Analytical and Applied Pyrolysis xxx (xxxx) xxxxxx

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Journal of Analytical and Applied Pyrolysis


journal homepage: www.elsevier.com/locate/jaap

Py-GC/MS and HPLC-DAD characterization of hazelnut shell and cuticle:


Insights into possible re-evaluation of waste biomass
Marco Mattonai, Domenico Licursi, Claudia Antonetti, Anna Maria Raspolli Galletti,

Erika Ribechini
Dipartimento di Chimica e Chimica Industriale, Universit di Pisa, via Moruzzi 13, 56124 Pisa, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: The chemical characterization of shell and cuticle from Tonda Gentile Romana hazelnut was carried out using
HPLC-DAD analytical pyrolysis in the presence of hexamethyldisilazane followed by gas chromatography-mass spectrometry
Py-GC/MS analysis (Py(HMDS)-GC/MS) and high-performance liquid chromatography with diode-array detection (HPLC-
Hazelnut DAD). The aim was to acquire a comprehensive picture of the chemical composition of shell and cuticle, which
H/L ratio
are produced as wastes by hazelnut confectionery industry, in view of a suitable exploitation strategy. Such
Polyphenols
waste biomass could be a promising substrate to produce value-added chemicals and biofuels. The pyrogram of
the shell fraction showed the typical pyrolysis products of a lignocellulosic matrix. The cuticle pyrogram showed
intense peaks that were attributed to fatty acids. Holocellulose-to-lignin (H/L) content ratios were determined
both by Py-GC/MS and by a traditional wet-chemistry method. While the H/L values obtained by the two
methods were similar for the shell fraction, those of the cuticle fraction were very dierent. To further in-
vestigate this behaviour, HPLC-DAD analyses were carried out on ethanol extracts of the two fractions. High
concentrations of catechin, epicatechin and procyanidins were found in the cuticle fraction. The presence of
phenolic compounds was hypothesised to determine the overestimation of the H/L ratio in the Py-GC/MS
technique. 1,3,5-trihydroxybenzene was found to be a plausible marker for the presence of polyphenolic ex-
tractives in lignocellulosic materials. Based on the results obtained from the dierent analysis methods, possible
exploitation strategies are suggested for the two waste fractions. The high lignin and carbohydrate content of the
shell fraction could be used to produce value-added chemicals, biofuels and biochars. The high extractives
content of the cuticle fraction, on the other hand, makes it a possible starting material to produce pharmaceutical
and nutraceutical products, as well as cosmetic emulsions.

1. Introduction and health benets of dierent tree nuts was reported by Singh and
Kaur [9].
Shell and cuticle represent the main waste fractions originating Analytical characterisation of hazelnut samples in the literature is
from hazelnut industrial processing. These fractions are usually burned usually focused either on the lignocellulosic or the lipid fractions
to generate energy, which is used within the same processing plant. [68,1013].
However, a more green and sustainable approach would be the con- Analytical pyrolysis with on-line gas chromatography and mass
version of these waste biomasses into bio-fuels and value-added bio- spectrometric detection (Py-GC/MS) already represents a well-estab-
chemicals such as hydroxymethylfurfural, levulinic acid, and glycerol lished technique for the investigation of biomass composition [1417].
[15]. A conversion strategy of hazelnut shell into biofuels is available In-situ derivatisation with a methylating or silylating agent also im-
in the literature [68], showing that this is a very promising exploita- proves the chromatographic performances in terms of peak shape and
tion strategy. The choice of the best biomass conversion route strongly resolution [18]. To the best of our knowledge, analytical pyrolysis with
depends on its starting composition, which also deeply inuences the in-situ silylation has not yet been used for the characterisation of whole
optimization of the process itself. Therefore, a detailed investigation of hazelnut shell and cuticle.
the chemical composition is essential to any type of exploitation. An A complete characterisation of these materials must also consider
useful summary of the chemical composition, phytochemical content the presence of minor components, such as extractive compounds.


The paper is dedicated to the memory of Sara Rapiti, friend and colleague, who collaborated to the present work.

Corresponding author.
E-mail address: erika.ribechini@unipi.it (E. Ribechini).

http://dx.doi.org/10.1016/j.jaap.2017.07.019
Received 12 January 2017; Received in revised form 19 July 2017; Accepted 21 July 2017
0165-2370/ 2017 Elsevier B.V. All rights reserved.

Please cite this article as: Mattonai, M., Journal of Analytical and Applied Pyrolysis (2017), http://dx.doi.org/10.1016/j.jaap.2017.07.019
M. Mattonai et al. Journal of Analytical and Applied Pyrolysis xxx (xxxx) xxxxxx

Polyphenols are a fundamental category among the extractives, since added. Pyrolysis was carried out at 550 C for 12 s. Injector was
they are well-known high-value chemicals with interesting biological maintained at 280 C and injection was performed in split mode with a
activities [1921]. There is a growing interest in the use of polyphenols split ratio of 10:1. Chromatographic separation was achieved with an
from food processing wastes to produce cosmetic materials. Skin ab- HP 5MS column 30 m 0.25 mm, lm thickness 0.25 m (Agilent
sorption of polyphenols is a key parameter that must be considered Technologies, USA) coupled with a deactivated silica pre-column
when dealing with emulsions preparation, since polyphenols should not 2 m 0.32 mm (Agilent Technologies, USA) and using helium as car-
reach the vascular layer. Low-molecular weight and low-polarity rier gas (1 mL/min). The following temperature program was used for
polyphenols are the one that show high skin absorption rates. Lipids are the chromatographic run: 50 C isothermal for 1 min, 10 C/min up to
another fundamental compound category in this eld, since their pre- 100 C, 100 C isothermal for 2 min, 4 C/min up to 280 C, 280 C
sence can modulate the absorption rates of polyphenols [2224]. isothermal for 30 min. Transfer line to the mass detector was kept at
In the present work, hazelnut shell and cuticle have been studied 300 C. The mass spectrometer was operated in EI positive mode
with Py-GC/MS using in-situ derivatisation with hexamethyldisilazane (70 eV, ion source temperature 230 C, quadrupole temperature
(HMDS). Pyrolytic proles have been used to perform semi-quantitative 150 C, m/z range 50600). Reproducibility of this method has already
calculations. Using relative integrated areas, an estimation of the ho- been estimated to be under 10% by previous studies [26].
locellulose-to-lignin ratio (H/L) was obtained for the two fractions. The
results were compared with those obtained by a traditional method 2.4. Compositional analysis
based on wet chemistry. Solid-liquid extraction procedure was also
performed on the samples, and the extracts were analysed by HPLC- Cellulose, hemicellulose, lignin, moisture and ash contents of shell
DAD to obtain information on the composition of the phenolic fraction. and cuticle of hazelnut were determined by the ocial standard NREL
methods [2729].
2. Materials and methods
2.5. HPLC-DAD
2.1. Reagents
All HPLC analyses were carried out using an Ascentis Express RP-
Gallic acid (89%, Fluka), protocatechuic acid (97%, Fluka), va- Amide column 100 mm x 2.1 mm, particle size 2.7 m (Supelco), with
nillic acid (97%, Aldrich), naringin (95%, Sigma-Aldrich) and quer- an Ascentis Express RP-Amide guard column 5 mm x 2.1 mm, particle
citrin (> 99%, Sigma-Aldrich) were used as standard reference mate- size 2.7 m (Supelco). The HPLC-DAD system consisted of a PU-2089
rials for the HPLC analyses. Hexamethyldisilazane (99.9%, Sigma- quaternary pump (Jasco International Co.) equipped with a degasser,
Aldrich) was used as a derivatising agent in all pyrolysis experiments. an AS 950 auto-sampler (Jasco International Co.) and a GECKO 2000
Water (Carlo Erba), ethanol (ACS grade, Fluka) and acetonitrile (HPLC column oven (Amchro GmbH). Detection was carried out by using an
grade, Sigma-Aldrich) were used as extraction solvents and mobile MD-2010 diode-array detector (Jasco International Co.), which oper-
phases for HPLC analyses. Formic acid (98%, J. T. Baker) was used as ates in the range 200650 nm with a resolution of 1 nm. Injection vo-
mobile phase modier. lume was 10 L. Eluents were water (A) and acetonitrile (B), both with
0.3% (v/v) formic acid. Mobile phase ow rate was 0.4 mL/min, and
2.2. Samples and extraction procedure for HPLC-DAD analysis column oven temperature was 40 C. The following gradient was used
for all experiments: 03.75 min at 100% (A); 3.7519.50 min from
Hazelnut (Corylus Avellana L. Tonda Gentile Romana cultivar) shell 100% to 89% (A); 19.5027.75 min from 89% to 79% (A);
and cuticle were provided as waste materials from a hazelnut proces- 27.7544.25 min from 79% to 60% (A); 44.2550.25 min from 60% to
sing plant in central Italy (Viterbo, Rome). The extraction was per- 37% (A); 50.2551 min from 37% to 0% (A), held for 1.5 min;
formed following a procedure reported in the literature [25]. Briey, 52.5054 min from 0% to 100% (A), held for 10 min.
100 mg of shell and cuticle were weighted in two vials. 1 mL of an
ethanol/water mixture (4:1 v/v) was added to each vial. The vials were 2.6. Data interpretation
sonicated at 40 C for 10 min using a Sonorex Super 10 P ultrasonic
bath (Bandelin, Germany). After sonication, the vials were centrifuged Py-GC/MS chromatograms were analysed using the Automated
and the surnatant was retrieved. The extraction was repeated twice. A Mass spectra Deconvolution and Identication System by NIST (AMDIS,
blank sample was prepared with the same procedure by using 100 L of version 2.71). Identication of the pyrolysis products was based upon
distilled water instead of the solid sample. their retention times and their mass spectra. The identication of the
Two dierent procedures were adopted for the purication of the compounds was done also on the basis of the already published works
shell and cuticle extracts. For the shell and blank samples, 200 L of the on HPLC-DAD [30,31] and Py-GC/MS [18,32,33] data. Mass spectral
extracts were ltered on a centrifuge PTFE lter and dried under a reference libraries (Wiley and NIST/EPA/NIH) were also used. Semi-
gentle stream of nitrogen. The ltrates were re-dissolved in 200 L of quantitative calculations were performed on the pyrograms, by in-
water and injected in the HPLC system. The cuticle extract couldnt be tegrating the chromatographic peaks corresponding to identied com-
completely re-dissolved in water, therefore 100 L of extract were pounds and converting the obtained areas into relative percentages.
added to 200 L of water and ltered. Filtered solutions were directly Identication of the compounds in the HPLC-DAD chromatograms was
analysed by HPLC. performed by comparison of retention times and UVvis absorption
spectra of the reference commercial standards.
2.3. Py(HMDS)-GC/MS
3. Results and discussion
Pyrolysis-GC/MS experiments were carried out using an EGA/PY-
3030D micro-furnace pyrolyser (Frontier Lab, Japan), coupled to a 3.1. Py(HMDS)-GC/MS of whole shell and cuticle
6890-gas chromatograph with a split/splitless injector and a 5973
Agilent Mass Selective Detector (Agilent Technologies, USA). When a Fig. 1 shows the pyrograms obtained for hazelnut shell and cuticle
solid sample was analysed, 100 g were weighted and directly put into fractions. As can be noted, the use of HMDS as a derivatising agent
a stainless-steel cup with 5 L of HMDS. When the cuticle extract was generated narrow and symmetric peaks. More than 70 pyrolysis pro-
analysed, 50 L of solution were put in the cup. The solvent was eva- ducts were identied, which is a higher number than those found in
porated under a gentle stream of nitrogen, and 5 L of HMDS were previous literature studies [8], and Table 1 lists the assignments,

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M. Mattonai et al. Journal of Analytical and Applied Pyrolysis xxx (xxxx) xxxxxx

Fig. 1. Pyrograms of hazelnut shell (a) and cuticle


(b) obtained by Py(HMDS)-GC/MS. Peaks are num-
bered according to the assignments reported in
Table 1.

together with the main m/z values of their mass spectra. Table 1 also place on pyrolysis products that already lost the alkyl chain. These
reports which substrate among holocellulose (H) and lignin (L) was secondary reactions mainly aect the methoxyl groups on the aromatic
initially hypothesised to be the origin for each compound. Hypotheses ring. Catechol-type products (#39, 47, 50, 74) can be formed by loss of
were based on literature results. Most of the compounds reported in the a CH3 group followed by reaction with an H% radical. This reaction can
pyrogram of the shell fraction have been identied. Only few com- be described as a demethylation [36]. Finally, the oxidation of hydroxyl
pounds were not identied, but the m/z values of the corresponding groups by loss of hydrogen generates carbonyl compounds (#53, 62,
mass spectra are reported for completeness. 71).
Molecules with two or three carbon atoms (#1, 2, 5, 14, 26, 29) The chromatographic prole of the cuticle fraction is signicantly
showed intense peaks at short retention times. Single-carbon atom dierent than that of the shell. The most intense peaks at long retention
pyrolysis products are likely to have formed as well, but were not re- times were assigned to palmitic, linoleic and oleic acids (#76-78). Oleic
vealed with the chosen analysis method. These compounds are formed and linoleic acid co-elute, but the presence of both compounds was
from both holocellulose [34,35] and lignin [36] by various reactions, conrmed by extraction of the main m/z signals of their mass spectra
including fragmentation, elimination, secondary pyrolysis and re- (337 and 339, respectively). While fatty acids peaks were the most
arrangements. The main pyrolysis products of holocellulose are origi- abundant in the cuticle pyrogram, they were not detected in the shell
nated by thermal degradation of the monomers, which can be con- pyrogram. This result shows that the content of lignocellulose in the
sidered as dehydrated monosaccharides [37]. Anhydrosugars (#38, 43, cuticle is lower than that of the shell.
57, 58, 64, 66, 67) are formed from rearrangement of the monomers The presence of a dominating lipid component in the cuticle is not
with formation of a CeOeC bond after cleavage of the polysaccharide surprising, as fatty acids are known to be the most abundant component
chain. The loss of water molecules from the monomers generates pyrans of the edible part of hazelnuts. Our results are in agreement with the
(#16, 17, 23, 28, 31, 36, 51, 59) and furans (#2, 8, 10, 32). Dehy- literature showing that oleic acid is the most abundant fatty acid in the
dration of the monomers in their linear form can lead to the formation kernel, followed by linoleic and palmitic acids [38]. Calculation of the
of CeC bonds, generating hydroxybenzenes (#35, 42, 54, 61) [18,32] integrated area ratio for the two peaks provided a result of 8.9, which is
and cyclopentenones (#7, 13, 20, 24, 25, 37, 45, 46). Pyrolysis products also in agreement with the literature (9.0). It is possible that, during the
such as furans and cyclopentenones with less than six carbon atoms are roasting process, a small portion of the kernel is detached from the main
obtained when both dehydration and elimination reactions take place body along with the cuticle, causing fatty acids to be present as major
on the same monomer. components in the cuticle pyrogram.
Whole lignin monomers were found in the pyrograms (#70, 72, 76). The intense peak attributed to 1,3,5-trihydroxybenzene (#65) is
Thermal degradation of lignin was observed mainly aecting the alkyl another peculiarity of the cuticle pyrogram, since this isomer has never
chains of the monomers. These chains can either lose their hydroxyl been found as a pyrolysis product of lignocellulose. Literature research
group, generating long-chain products (#48, 52, 56, 63, 69, 73), or they showed that 1,3,5-trihydroxybenzene can be obtained from the pyr-
can lose part of the chain itself, generating short-chain products (#6, olysis of two dierent vegetal substrates. The rst one is cutan [39,40],
21, 27, 33, 34, 40, 41, 44, 49, 55, 60). Secondary reactions can take which is a complex polymer found in the cuticle of some plants. This

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M. Mattonai et al. Journal of Analytical and Applied Pyrolysis xxx (xxxx) xxxxxx

Table 1
Pyrolysis products which have been identied from the pyrograms of shell and cuticle fractions of hazelnut. For each compound, the source, class and main m/z values of mass spectrums
are reported. H = holocellulose; L = lignin; SM = Small molecules; Fur = Furans; ShCh = Short chains; Cyp = Cyclopentenones; Pyr = Pyrans; Hyb = hydroxybenzenes;
Ahs = Anhydrosugars; Dem = Demethylated; LoCh = Long chains; Carb = Carbonyls; M = Monomers; FA = Fatty acids; Oth = Others. Numbers refer to the peaks in the pyrograms
shown in Fig. 1.

# Name m/z Origin Class

1 1,2-dihydroxyethane (2TMS) 191, 147, 133, 103, 73 H/L SM


2 2-hydroxymethylfuran (TMS) 170, 155, 142, 125, 111, 81, 75 H Fur
3 phenol (TMS) 166, 151, 73 L Oth
4 2-hydroxypropanoic acid (2TMS) 219, 191, 147, 133, 117, 73 H/L SM
5 hydroxyacetic acid (2TMS) 205, 177, 161, 147, 133, 73 H/L SM
6 guaiacol 124, 109, 95, 81 L ShCh
7 1-hydroxy-1-cyclopenten-3-one (TMS) 169, 155, 127, 111, 101, 81, 73 H Cyp
8 3-hydroxymethylfuran (TMS) 170, 155, 142, 127, 111, 81 H Fur
9 o-cresol (TMS) 180, 165, 149, 135, 103, 91 L Oth
10 2-furancarboxylic acid (TMS) 184, 169, 125, 95, 73 H Fur
11 unknown I 167, 152 H Oth
12 m-cresol (TMS) 180, 165, 135, 105, 91 L Oth
13 2-hydroxy-1-cyclopenten-3-one (TMS) 155, 111, 81, 75, 73 H Cyp
14 3-hydroxypropanoic acid (2 TMS) 219, 177, 147, 73 H SM
15 p-cresol (TMS) 180, 165, 146, 117, 91, 75 L Oth
16 3-hydroxy-(2H)-pyran-2-one (TMS) 169, 151, 125, 95, 85, 75 H Pyr
17 3-hydroxy-(4H)-pyran-4-one (TMS) 184, 169, 147, 95, 75 H Pyr
18 unknown II 159, 131, 115, 101, 85, 73, 59 H Oth
19 unknown III 188, 171, 158, 145, 129, 115, 103, 85, 73, 59 H Oth
20 E-2,3-dihydroxy-cyclopent-2-enone (TMS) 171, 143, 101, 75 H Cyp
21 methyl-guaiacol 138, 123, 107, 95, 77, 67 L ShCh
22 1,2-dihydroxybenzene (TMS) 182, 167, 151, 136, 91, 75 H Hyb
23 5-hydroxy-2H-pyran-4(3H)-one (TMS) 186, 171, 143, 129, 101, 75, 73 H Pyr
24 2-hydroxymethyl-3-methy-2-cyclopentenone (TMS) 198, 183, 147, 117, 73 H Cyp
25 1-methy-2-hydroxy-1-cyclopenten-3-one (TMS) 184, 169, 97, 73 H Cyp
26 Dihydroxyacetone (2 TMS) 219, 189, 147, 129, 103, 73 H SM
27 guaiacol (TMS) 196, 181, 166, 151, 136 L ShCh
28 3-hydroxy-6-methyl-(2H)-pyran-2-one (TMS) 198, 183, 168, 139, 109, 75 H Pyr
29 glycerol (3 TMS) 218, 205, 147, 133, 117, 103, 73 H SM
30 unknown VI 173, 145, 131, 116, 101, 85, 73 H Oth
31 2-methyl-3-hydroxy-(4H)-pyran-4-one (TMS) 183, 153 H Pyr
32 5-hydroxymethyl-2-furaldehyde (TMS) 198, 183, 169, 139, 109, 73 H Fur
33 vinyl-guaiacol 150, 135, 107, 77 L ShCh
34 4-methylguaiacol (TMS) 210, 195, 180, 165 L ShCh
35 1,2-dihydroxybenzene (2TMS) 254, 239, 73 H Hyb
36 2-hydroxymethyl-2,3-dihydropyran-4-one (TMS) 200, 185, 170, 142, 73 H Pyr
37 Z-2,3-dihydroxy-cyclopent-2-enone (2TMS) 258, 243, 230, 204, 169, 147, 73 H Cyp
38 1,4:3,6-dianhydro--D-glucopyranose (TMS) 186, 170, 155, 129, 103, 73 H Ahs
39 4-methylcatechol (2TMS) 268, 253, 180, 149, 105, 73 L Dem
40 4-ethylguaiacol (TMS) 224, 209, 194, 179 L ShCh
41 syringol (TMS) 226, 211, 196, 181, 153 L ShCh
42 1,4-dihydroxybenzene (2TMS) 254, 239, 223 H Hyb
43 arabinofuranose (4TMS) 230, 217, 147, 103, 73 H Ahs
44 4-vinylguaiacol (TMS) 222, 207, 192, 177, 162, 73 L ShCh
45 3-hydroxy-2-hydroxymethyl-2-cyclopentenone (2TMS) 272, 257 H Cyp
46 E-2,3-dihydroxy-cyclopent-2-enone (2TMS) 258, 243, 147, 73 H Cyp
47 4-ethylcatechol (2TMS) 282, 267, 193, 179, 147, 73 L Dem
48 eugenol (TMS) 236, 221, 206, 179, 149, 103, 73 L LoCh
49 4-methylsyringol (TMS) 240, 225, 210, 195, 167 L ShCh
50 3-methoxy-1,2-benzenediol (2TMS) 284, 269, 254, 153 L Dem
51 3,5-dihydroxy-2-methyl-(4H)-pyran-4-one (2TMS) 286, 271, 199, 128, 73 H Pyr
52 Z-isoeugenol (TMS) 236, 221, 206,179, 103, 73 L LoCh
53 vanillin (TMS) 224, 209, 194, 147, 73 L Carb
54 1,2,3-trihydroxybenzene (3TMS) 342, 327, 239, 211, 179, 133, 73 H Hyb
55 4-ethylsyringol (TMS) 254, 239, 224, 209 L ShCh
56 E-isoeugenol (TMS) 236, 221, 206, 179, 103, 73 L LoCh
57 1,4-anydro-D-galactopyranose (2TMS) 243, 217, 155, 129, 116, 101, 73 H Ahs
58 1,6-anydro-D-galactopyranose (2TMS) 218, 204, 189, 161, 145, 73 H Ahs
59 2-hydroxymethyl-5-hydroxy-2,3-dihydro-(4H)-pyran-4-one (2TMS) 288, 273, 245, 217, 183, 169, 155, 129, 101, 73 H Pyr
60 4-vinylsyringol (TMS) 252, 237, 222, 179, 149, 73 L ShCh
61 1,2,4-trihydroxybenzene (3TMS) 342, 327, 254, 239, 147, 73 H Hyb
62 acetovanillone (TMS) 238, 223, 208, 193, 165, 137, 109 L Carb
63 E-propenylsyringol (TMS) 266, 251, 236, 205 L LoCh
64 1,6-anydro-beta-D-glucopyranose (2TMS at position 2 and 4) 245, 217, 204, 191, 155, 129, 116, 101, 73 H Ahs
65 1,3,5-trihydroxybenzene (3 TMS) 342, 327, 268, 253, 147, 133 Hyb
66 1,4-anydro-D-galactopyranose (3TMS) 332, 243, 217, 204, 191, 157, 129, 117, 103, 73 H Ahs
67 1,6-anydro--D-glucopyranose (3TMS) 333, 243, 217, 204, 191, 147, 129, 103, 73 H Ahs
68 1,4-anhydro-D-glucopyranose (3TMS) 332, 243, 217, 204, 191, 157, 129, 117, 103, 73 H Ahs
69 vanillylpropanol (2TMS) 326, 311, 296, 236, 221, 206, 179, 149, 73 L LoCh
70 Z-coniferyl alcohol (2 TMS) 252, 235, 221, 204, 181, 162, 131, 119, 103, 73 L M
71 coniferylaldehyde (TMS) 250, 235, 220, 192, 177, 166, 151, 89, 73 L Carb
(continued on next page)

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M. Mattonai et al. Journal of Analytical and Applied Pyrolysis xxx (xxxx) xxxxxx

Table 1 (continued)

# Name m/z Origin Class

72 E-coniferyl alcohol(2 TMS) 324, 309, 293, 235, 219, 204, 147, 131, 103, 73 L M
73 syringylpropanol (2TMS) 356, 341, 326, 240, 236, 225, 210, 179, 163, 73 L LoCh
74 3,4-dihydroxy cinnamyl alcohol (3TMS) 382, 355, 293, 205, 179, 147, 73 L Dem
75 Palmitic acid (TMS) 328, 313, 285, 269, 21, 145, 132, 117, 73 FA
76 E-synapyl alcohol (2TMS) 354, 339, 323, 293, 265, 234, 204, 73 L M
77 Linoleic acid (TMS) 352, 337, 262, 220, 145, 129, 117, 73 FA
78 Oleic acid (TMS) 354, 339, 309, 264, 222, 199, 145, 129, 117, 73 FA

polymer is constituted by hydroxybenzene moieties that form ester included in the calculation. Glycerol (#29) was excluded from the
bonds with long-chain aliphatic carboxylic acids (1531 carbon atoms). calculation as well, since its presence could be due to the thermal de-
Thermal degradation of cutans is known to cause the cleavage of the gradation of triacylglycerols that are present along with free fatty acids
ester bonds, generating fatty acids which are revealed by the GC system in the kernel [13]. Finally, 1,3,5-trihydroxybenzene (#65) was also
when a derivatising agent is present [39]. However, since no fatty acids excluded from the calculation, since its origins are unknown.
with more than 18 carbon atoms were found in the pyrogram, cutan The shell sample provided a value of H/L = 1.1, which is similar to
was not considered to be a possible origin for 1,3,5-trihydroxybenzene. those reported in the literature for the Py(HMDS)-GC/MS analysis of
The second possible substrate is condensed tannins, which are a more lignocellulosic materials [44]. The main categories of products reported
widely diused family of vegetal compounds. Condensed tannins are in the pyrogram are hydroxybenzenes and anhydrosugars for the car-
obtained from the polymerization of catechin and epicatechin, and their bohydrate fraction, and short-chain compounds and monomers for the
pyrolysis generates aromatic species [41,42]. lignin fraction. On the other hand, the cuticle sample provided an H/L
To obtain semi-quantitative information, all identied compounds value of 4.3, which is unusually high for this kind of sample. This value
were grouped into categories, according to their pyrolytic formation is probably due to an overestimation of the carbohydrate fraction, since
mechanisms and their molecular structure. Compounds originating hydroxybenzene compounds showed a total area percentage of more
from holocellulose were divided into ve categories: pyrans (Pyr), than 37%, while the area percentages of all other categories are lower
furans (Fur), cyclopentenones (Cyp), hydroxybenzenes (Hyb) and an- than 17%. This result agrees with the hypothesis that condensed tan-
hydrosugars (Ahs). On the other hand, compounds originating from nins are present in the cuticle sample, since their pyrolysis products
lignin were divided into six categories: short-chain compounds (ShCh), would increase the hydroxybenzenes category. To verify if the H/L was
long-chain compounds (LoCh), demethylated compounds (Dem), car- being overestimated, compositional analysis was carried out on both
bonyl compounds (Carb) and whole monomers (M). Small molecules samples.
(SM) can be generated from both polymers. Unknown compounds and
compounds not belonging to any of the previous categories were
grouped in the others (Oth) category. After category attributions, 3.2. Compositional analysis
peak areas were determined by integration. Areas of peaks belonging to
the same compound categories were grouped and the total area was The compositional data of shell and cuticle are reported in Table 2.
normalized to 100%. The obtained results are reported in Fig. 2. Note H/L values, also reported in Table 2, were obtained by dividing the
that this data processing method provides a way of comparing samples total cellulose and hemicellulose content for the lignin content. Com-
with similar pyrolysate compositions, and it is not capable of providing positional values for the extractives, moisture and ash contents are in
quantitative results. This data processing method has already been agreement with those found in the literature [45].
applied to obtain information on wood degradation [43]. Carbohydrates and lignin are the most important components in the
Holocellulose-to-lignin ratios (H/L) were calculated for the two shell fraction, as one would expect from a lignocellulosic material.
samples by dividing the total percentage area of holocellulose pyrolysis Moreover, the H/L value obtained with this method agrees with that
products by the total area of lignin pyrolysis products. Small molecules, obtained by the analytical pyrolysis technique. This implies that the
which can be produced by both holocellulose and lignin, were not identied pyrolysis products are representative of the composition of
the starting sample.

Fig. 2. Percentage composition of each product ca-


tegory for shell and cuticle samples.

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M. Mattonai et al. Journal of Analytical and Applied Pyrolysis xxx (xxxx) xxxxxx

Table 2
Results of the compositional analysis of shell and cuticle performed with NREL ocial procedures. Values are expressed as weight percentages of the total sample. Holocellulose-to-lignin
ratios are also reported. The proteinaceous fraction is included in Other.

Sample Cellulose (%) Hemicellulose (%) Lignin (%) Extractives (%) Moisture (%) Ash (%) Other (%) H/L

Shell 22.9 23.6 37.6 1.4 5.1 1.4 8.0 1.2


Cuticle 8.9 4.2 33.2 36.2 8.1 1.5 7.9 0.4

Extractives are the main components of the cuticle fraction, and Table 3
they are signicantly higher than the total carbohydrate content. These List of identied compounds from the HPLC-DAD chromatograms of shell and cuticle. The
wavelengths of maximum absorption (max) are reported for each compound.
results could explain the low peak intensities recorded for lig-
nocellulose pyrolysis products in the cuticle pyrogram. Moreover, these # Name max (nm)
data imply that pyrolysis products of the extractives fraction could be
present in the cuticle pyrograms, and therefore the pyrolysate is not 1 Gallic acid 271
2 Protocatechuic acid 259, 295
only representative of the lignocellulosic matrix. Compositional ana-
3 Vanillic acid 259, 291
lysis results show that shell and cuticle have very dierent exploitation 4 Catechin/Epicatechin 280
potentials, and their exploitation will require very dierent strategies. 5 Naringin 283, 331
The low percentage of carbohydrates obtained from the wet chemical 6 Quercitrin 255, 347
analysis gave an H/L value of 0.4 for the cuticle fraction. This value is 7 Procyanidins 280

in strong disagreement with the one obtained from the pyrograms. The
dierence in the H/L values obtained for the cuticle could be due to the
reported in the literature allowed us to attribute these peaks to ca-
high content of extractable compounds in the sample. For this reason,
techin, epicatechin and procyanidins [5154]. These phenolic com-
we carried out a more in-depth analysis of the extractive fraction by
pounds are commonly found in foodstus such as grapes and tea
HPLC-DAD.
[5557]. Procyanidins are oligomeric forms of catechin and epica-
techin, and the chromatographic system is not capable of resolving their
3.3. Analysis of cuticle and shell extracts elution, resulting in a very broad peak. Retention time and absorption
spectra for these peaks are in agreement with those found in the lit-
The HPLC-DAD proles obtained for the extractive fractions are erature [31]. The presence of catechins in hazelnut hull, both as
reported in Fig. 3, while a list of the identied compounds is reported in monomers and oligomers, has already been reported [47,58].
Table 3. At low retention times, phenolic acids such as gallic, proto- To obtain additional information on the behaviour of extractives in
catechuic and vanillic acid were found in the chromatograms. At high analytical pyrolysis, Py-GC/MS was performed on the cuticle extracts.
retention times, the avonoids naringin and quercetin were identied. The obtained pyrogram is reported in Fig. 4. Two of the peaks were
Identication of both phenolic acids and avonoids was performed by assigned to previously undetected compounds, which were found in
comparison with the chromatogram of a standard solution of the ana- higher abundance in the extracts. The peaks marked with Gal and
lytes of interest. These compounds are known members of the poly- Cat could be attribute to the per-silylated form of gallic acid and
phenols family. They are present in both the kernel and the other catechin, respectively. The main peaks in the pyrogram were attributed
components of the hazelnut seed [25,46,47], but can also be found in to palmitic, linoleic and oleic acid (#75, 77, 78), which were extracted
other foodstus such as wine, olive oil, cocoa and tea leaves [4850]. along with the phenolic compounds but could not be revealed by the
Gallic acid, protocatechuic acid and naringin were found in both shell HPLC-DAD system. The other main peaks were attributed to hydro-
and cuticle. Vanillic acid was only found in the shell extract, while xybenzene compounds (#35, 54, 61, 65) and glycerol (#29). This result
quercitrin was only found in the cuticle extract. shows that most of the hydroxybenzenes are produced from the pyr-
While phenolic acids and avonoids were identied in both chro- olysis of both cellulose and polyphenols, conrming the hypothesis that
matograms, the cuticle extracts showed two additional peaks in the the overestimation of the H/L ratio derives from an incorrect compound
range 1720 min, and a very broad and intense peak in the range origin assignment. Moreover, another intense peak was assigned to 4-
3040 min. All these signals showed absorption spectra with a max- methylcatechol (#39), which was treated as a pyrolysis product of
imum around 280 nm. Comparison of the absorption spectra with those lignin. This result implies that it would be very dicult to nd an

Fig. 3. HPLC-DAD chromatograms of the shell (a)


and cuticle (b) samples at 275 nm. Identied com-
pounds are numbered according to Table 3.

6
M. Mattonai et al. Journal of Analytical and Applied Pyrolysis xxx (xxxx) xxxxxx

Fig. 4. Pyrogram of hazelnut shell extracts obtained


by Py(HMDS)GC/MS. Peaks are numbered according
to assignments reported in Table 1. The peaks
marked with Gal and Cat are attributed to the
per-silylated forms of gallic acid and catechin, re-
spectively.

alternative way to estimate the H/L ratio from the pyrograms. A de- Author contributions
tailed characterisation of the phenolic fraction and its pyrolysis pro-
ducts would be required to know which compounds should be excluded The manuscript was written through contributions of all authors. All
from the calculation. authors have given approval to the nal version of the manuscript.

4. Conclusions Acknowledgements

In the present work, hazelnut shell and cuticle were analysed by Py- This work was partially supported by PRA 2016 project La chimica
GC/MS, compositional analysis and HPLC-DAD to obtain a complete analitica per la conoscenza di materiali e tecniche nellarte moderna e
characterization and insights into possible re-evaluation strategies. contemporanea (PRA-2016_13) funded by University of Pisa.
Pyrolysis data showed the characteristic signals of lignocellulosic ma-
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