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Journal of Analytical and Applied Pyrolysis57 (2001) 45–55
A study of variability of suberin compositionin cork from Quercus suber L. using thermally
assisted transmethylation GC–MS
Maria Filomena Santos Bento a,b,*, H. Pereira c,M.A. Cunha b,d, A.M.C. Moutinho e, K.J. van den Berg f,
J.J. Boon f
a Instituto Superior de Engenharia de Lisboa, R. Emıdio Na6arro, 1900 Lisbon, Portugalb Centro de Fısica Molecular da UTL, Complexo I, IST, A6. Ro6isco Pais, 1000 Lisbon, Portugal
c Centro de Estudos Florestais, ISA, UTL, Tapada da Ajuda, 1300 Lisbon, Portugald Dep. de Fısica, Instituto Superior Tecnico, UTL., A6. Ro6isco Pais, 1000 Lisbon, Portugal
e Dep. de Fısica, Fac. de Ciencias e Tecnologia, CeFiTec, UNL., 2825 Monte de Caparica, Portugalf Unit for Mass Spectrometry of Macromolecular Systems,
FOM Institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam, The Netherlands
Received 4 January 2000; received in revised form 29 March 2000; accepted 25 April 2000
Abstract
The chemical composition of suberin in cork from Quercus suber L. was determined onvirgin and reproduction cork from different sites and trees in Portugal. Extractive-freesamples were analyzed by pyrolysis-gas chromatography/mass spectrometry in the presenceof tetramethylammonium hydroxide (TMAH), which provides in situ hydrolysis and(trans)methylation of hydroxyl and carboxyl groups of the suberin monomers. The averagechemical monomeric composition of suberin on virgin and reproduction cork is, respectively,alkanoic acids 5.1, 3.3%; a,v-diacids 11.2, 10.1%; v-hydroxyacids 45.0, 48.1%; alkanols 1.9,1.8%; 9,10-epoxy-18-hydroxyoctadecanoic acid 5.9, 5.8%; 9,10-epoxyoctadecanodioic acid4.8, 3.6%; 9,10,18-trihydroxyoctadecanoic acid 7.5, 10.4%; 9,10-dihydroxyoctadecanedioic6.1, 6.8%; ferulic acid 5.8, 4.5% and glycerol 4.3, 4.3%. In general, larger variability wasfound for the suberin monomers in virgin cork. Total content of v-hydroxyacids or of C18or C22 compounds is however similar for all samples. © 2001 Elsevier Science B.V. All rightsreserved.
www.elsevier.com/locate/jaap
* Corresponding author.E-mail address: [email protected] (M.F. Santos Bento).
0165-2370/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S0165-2370(00)00093-0
46 M.F. Santos Bento et al. / J. Anal. Appl. Pyrolysis 57 (2001) 46–55
Keywords: Index-cork; Quercus suber L.; Suberin; Thermally assisted methylation; Pyrolysis massspectrometry
1. Introduction
Cork from Quercus suber L. has peculiar properties such as high elasticity andlow permeability. These should result, at least partially, from its specific chemicalcomposition and especially from suberin, since it is the main component of cellwalls, where it amounts to approximately 40% [1]. The other chemical componentsof cork are lignin (22%), polysaccharides (18%) and extractives (15%) [1].
Suberin has been considered as a polymer containing both phenolic and aliphaticcomponents [2,3]. Recently, Graca and Pereira [4,5] proposed a glyceridic polyesterstructure for cork suberin. Several wet chemical techniques have been used for thedepolymerization of suberin by cleavage of the ester bonds and for analysis of itsmonomeric subunits. Using transmethylation, Arno et al. [6] and Holloway [7]found as cork suberin monomers ester and/or ether derivatives from alkanols,saturated alkanoic acids, saturated and unsaturated a,v-alkanedioic acids, satu-rated and unsaturated v-hydroxyalkanoic acids, epoxyoctadecanedioic acid, mono-hydroxy-epoxyoctadecanoic acid, dihydroxyoctadecanedioic acid andtrihydroxyoctadecanoic acid.
Recently, Bento et al. [8] used an on-line thermally assisted trans-methylationGC–MS technique to investigate the monomeric building blocks of cork suberinand report basically the same classes of compounds. The procedure is based on thefollowing: a tetramethylammonium hydroxide (TMAH) solution was added to thesample prior to pyrolysis; as the resulting mixture was heated on the pyro probe,methylation of carboxylic and hydroxylic groups occurred in combination withtransesterification and transetherification during the pyrolysis.
This method was employed here to study the variability of suberin compositionon virgin and reproduction cork from different trees and sites. Virgin cork is thefirst cork produced by the cork-oak and it is removed when the tree is approxi-mately 25–30 years old; subsequent cork extractions (reproduction cork) are madeevery 9 years [1].
2. Materials and methods
2.1. Samples and sample preparation
Cork was removed from Q. suber L. trees of the following sites located within themain production areas in Portugal:
Site 1, Coruche (virgin cork samples C10, C32 and C87);Site 2, Extremoz (virgin cork samples E75, E118 and E151);Site 3, Chamusca (reproduction cork samples CH1D and CH2C);
47M.F. Santos Bento et al. / J. Anal. Appl. Pyrolysis 57 (2001) 46–55
Site 4, Companhia das Lezırias, Benavente. (reproduction cork samples CL1Aand CL2A);Site 5, Azaruja (reproduction cork samples AZ1D and AZ2C).The samples were milled in a knife mill, classified by wire screening, and the
granulometric fraction 40–60 meshes used for analysis. An extractive-free samplewas prepared by successive soxhlet extraction with dichloromethane, ethanol andwater.
The extractives that were removed amounted to 5.0–7.2, 2.7–7.3 and 2.2–5.3%,respectively, for the three solvents, representing a total extractives content of11.1–18.0%. These values were within the range found for cork [1].
For the thermally assisted transmethylation experiments, 5–10 ml of a suspensionof a very small quantity of sample in water were applied to a ferromagneticanalytical wire and dried under reduced pressure. A drop of 2.5% TMAH solutionin methanol was added to the sample and dried using the same procedure. The wirewas inserted into a glass liner, flushed with argon to remove air and placed in theFOM3LX Curie-point pyrolysis unit (designed and constructed at FOM-AMOLF[9]).
2.2. Thermally assisted transmethylation GC–MS
The ferromagnetic wire was inductively heated to the Curie point (610°C), during4 s, by a high frequency field in a coil placed around the wire. The pyrolysischamber was kept at 180°C to avoid condensation of pyrolysis products on theglass liner.
The methylated products were flushed with He (inlet pressure, 50 kPa) to aCPSIL-5 CB fused-silica capillary column (25 m×0.32 mm i.d., 0.25 mm filmthickness; Chrompack International, Middelburg, the Netherlands) in a Hewlett–Packard (Palo Alto, CA, USA) HP 5890 gas chromatograph (series 2) coupleddirectly to the ion source of a JEOL (Tokyo, Japan) DX-303 double focusing (E/Bconfiguration) mass spectrometer. Helium was used as the carrier gas at a flow rateof approximately 2 ml s−1. The GC oven was kept at 45°C during pyrolysis. Twominutes later, it was gradually heated at 6°C min−1. Up to 220°C and then at 4°Cmin−1 up to 360°C. Such oven temperature program allowed resolving the peaks ofinterest in each group. The GC–MS interface and the ion source were at 280 and200°C, respectively. Ions were generated by electron impact ionization (70 eV) inthe ion source, accelerated to 3 keV, mass separated and postaccelerated to 10 keVbefore detection. The mass spectrometer was scanned from m/z 40–500 Da, with acycle time of 1 s. The mass spectra were processed with a JEOL MP-7000 datasystem.
Identification was based on the interpretation of fragmentation patterns andsupported by mass spectral data from mass spectral libraries [10] and by compari-son with published spectra.
The relative abundance of the compounds was calculated from the peak areas inthe total ion gas chromatogram. The statistically significance of mean for eachcomponent and chemical class for the different sites and for both types of cork wasevaluated using the t-test.
48 M.F. Santos Bento et al. / J. Anal. Appl. Pyrolysis 57 (2001) 46–55
3. Results and discussion
The extractive-free cork samples were completely pyrolyzed to volatile com-pounds. In the conditions used, the cell wall polysaccharides were degraded to smallmolecules without identification significance, but the fragments derived from ligninand suberin could be found and identified. The total ion current chromatograms(TIC) of the samples showed peaks with a very low abundance in the lowerretention times range, which were identified mainly as methylated phenolic com-pounds derived from lignin. The peaks in the higher retention times range had agreat abundance and could be assigned to suberin as esters and/or ethers of thesuberinic subunits, as previously reported [8]. These represented on average 84% ofthe total chromatogram area. An example of the chromatograms obtained wasgiven in Fig. 1 for one sample of reproduction cork (CH1D). All samples showedsimilar chromatograms that differed only on the relative abundances of somecompounds. These results again confirm the aptitude of TMAH-py-GC–MS for thestudy of cork suberin composition [8].
The classes of compounds found for virgin cork and reproduction cork are listedin Table 1 with their percentage of the total integrated area of suberin monomers.All the samples yielded long chain aliphatic monomers, glycerol and ferulic acid,identical to those previously found on a similar thermally assisted hydrolysis andmethylation (THM) experiment with cork [8]. They also corresponded to thecomposition assessed by wet-lab transmethylation [7,11].
The most important components are the v-hydroxyacids, representing 42.6–49.0% of the total monomers for virgin cork and 43.2–55.0% for reproductioncork. Unsubstituted a,v-diacids have a significant contribution with 6.6–17.0%(virgin cork) and 6.6–16.3% (reproduction cork) of the total monomers. The9,10-epoxy and dihydroxy acids also represent a relevant proportion of the totalmonomers: 9,10-epoxy-18-hydroxyoctadecanoic acid, 4.1–8.3% (virgin cork) 4.8–7.0% (reproduction cork); 9,10-epoxyoctadecanodioic acid, 2.3–9.5% (virgin cork)2.4–5.0% (reproduction cork); 9,10,18-trihydroxyoctadecanoic acid, 3.5–11.9%(virgin cork), 4.1–14.4% (reproduction cork) and 9,10-dihydroxyoctadecanodioicacid, 3.6–9.5 (virgin cork), 3.6–9.0% (reproduction cork).
Glycerol also belongs to the suberin structure as proven recently by Graca andPereira [4,5]. Glycerol ranged from 3.4–6.2 and 2.3–5.8% of the total monomersfor virgin and reproduction cork, respectively. Ferulic acid, the only phenoliccompound detected, corresponds to 4.6–7.6% (virgin cork) and to 3.4–5.2%(reproduction cork) of the total monomers. Ferulic acid should also be associatedto the suberin structure [5,12].
Homologous series with carbon chain lengths between C16 and C26 were foundfor each chemical family. The relative proportions of the series components of1-alkanols, alkanoic acids, a,v-diacids and v-hydroxyacids are shown in Table 2.The 18:1 structure was very abundant in a,v-diacids and v-hydroxyacids, but it wasnot found among the 1-alkanols and only as vestigial quantities for the alkanoicacids. For 1-alkanols and alkanoic acids structures, the most important were 22:0and 24:0; for the a,v-diacids it was 18:1, followed by 22:0 and 16:0 and forv-hydroxyacids 18:1, and 22:0.
49M.F. Santos Bento et al. / J. Anal. Appl. Pyrolysis 57 (2001) 46–55
Tab
le1
Mon
omer
icco
mpo
siti
onof
sube
rin
bych
emic
alfa
mili
esan
dm
ain
com
pone
nts
(%of
tota
lm
onom
ers
dete
rmin
edas
met
hyl
este
rs/e
ther
s)fr
omvi
rgin
and
repr
oduc
tion
cork
sam
ples
afte
rm
ethy
lati
onw
ith
TM
AH
a
Vir
gin
cork
Rep
rodu
ctio
nco
rk
E75
E11
8E
151
Mea
nA
Z1D
AZ
2CC
L1A
CL
2AC
H1D
CH
2CM
ean
C10
C32
C87
1.7
2.4
2.0
1.9
1.7
2.2
1.6
1.0
2.3
2.0
1.8
1-A
lkan
ols
1.0
1.3
3.2
3.4
5.1
3.5
3.4
3.4
3.1
3.3
5.1
3.3
Alk
anoi
cac
ids
3.3
3.8
6.3
5.8
6.1
4.2
4.3
Gly
cero
l2.
34.
25.
35.
72.
95.
84.
04.
34.
13.
86.
23.
44.
65.
85.
24.
74.
85.
03.
46.
84.
05.
84.
55.
4F
erul
icac
id4.
67.
66.
66.
05.
95.
96.
16.
44.
95.
54.
87.
05.
84.
18.
34.
59,
10-E
poxy
-18-
hydr
oxyo
ctad
ecan
oic
acid
9.5
3.0
3.9
4.8
4.2
5.0
2.4
3.4
3.5
3.1
3.6
2.3
7.0
2.8
9,10
-Epo
xyoc
tade
cano
dioi
cac
id8.
77.
44.
18.
812
.014
.412
.33.
510
.59,
10,1
8-T
rihy
drox
yoct
adec
anoi
cac
id10
.310
.95.
83.
811
.99.
54.
27.
16.
14.
36.
59.
08.
98.
53.
66.
87.
29,
10-D
ihyd
roxy
octa
deca
nodi
oic
acid
3.6
4.8
5.5
5.8
7.3
4.7
4.5
4.4
5.4
8.0
3.9
2.9
5.1
Satu
rate
da,
v-d
iaci
ds5.
66.
06.
96.
43.
74.
95.
39.
04.
44.
23.
95.
92.
75.
110
.14.
42.
5U
nsat
urat
eda,
v-d
iaci
ds14
.46.
610
.511
.216
.39.
28.
78.
411
.36.
610
.1)
17.0
10.5
8.2
(Tot
ala,
v-d
iaci
ds32
.131
.029
.432
.133
.532
.425
.130
.129
.8Sa
tura
ted
v-h
ydro
xyac
ids
30.4
31.4
31.3
30.7
30.7
Uns
atur
ated
v-h
ydro
xyac
ids
14.8
14.4
14.0
22.9
14.1
12.5
13.5
18.1
25.2
17.7
18.3
11.6
12.4
12.4
46.9
45.0
52.3
46.2
46.0
45.9
43.2
42.6
55.0
43.8
48.1
)(T
otal
v-h
ydro
xyac
ids
45.1
49.0
42.9
TR
TR
TR
ND
TR
ND
1.2
1.0
9,10
,20-
Tri
hydr
oxye
icos
anod
ecan
oic
acid
0.5
TR
TR
TR
TR
TR
1.0
0.9
TR
TR
TR
TR
TR
Tr
TR
Tr
9,10
-Dih
ydro
xyei
cosa
nodi
oic
acid
1.8
1.2
0.7
0.5
aT
R,
trac
e;N
D,
not
dete
cted
.
50 M.F. Santos Bento et al. / J. Anal. Appl. Pyrolysis 57 (2001) 46–55
Tab
le2
Rel
ativ
epr
opor
tion
ofm
onom
ers
inea
chho
mol
ogou
sse
ries
(alk
anoi
cac
ids,
a,v
-dia
cids
,1-
alka
nols
and
v-h
ydro
xyac
ids)
a
Vir
gin
cork
Rep
rodu
ctio
nco
rk
E15
1M
ean
AZ
1DA
Z2C
CL
1AC
10C
L2A
CH
1DC
H2C
Mea
nC
32C
87E
75E
118
2.6
0.8
Tr
1.7
TR
TR
1.7
ND
TR
ND
TR
TR
Tr
ND
Dod
ecan
oic
acid
2.5
3.1
3.5
2.5
5.1
4.8
4.4
5.3
3.3
3.9
4.5
2.8
Hex
adec
anoi
cac
id3.
1N
DT
R0.
80.
01.
6T
R2.
1T
R1.
0T
R1.
31.
2O
ctad
ecan
oic
acid
0.7
2.5
TR
53.1
53.9
63.3
56.7
66.9
55.6
53.7
70.5
72.5
56.5
62.6
57.9
53.2
56.6
Doc
osan
oic
acid
38.8
38.8
30.6
37.7
26.8
34.8
40.4
22.0
25.8
29.8
29.9
39.9
Tet
raco
sano
icac
id39
.041
.1T
R1.
0N
DT
RT
RN
DN
D1.
7T
R3.
1T
RT
RH
exac
osan
oic
acid
1.0
TR
2.3
1.1
0.0
0.7
1.2
1.5
1.4
TR
1.7
TR
1.0
TR
TR
1.4
Oct
adec
-9-e
noic
acid
13.4
14.1
16.8
15.6
12.9
19.4
17.1
Hex
adec
aned
ioic
acid
25.1
19.6
17.8
14.5
12.0
13.7
16.2
3.8
6.4
5.1
4.4
7.2
3.4
6.6
5.6
5.9
5.9
5.4
Eic
osan
edio
icac
id13
.35.
34.
430
.218
.734
.931
.220
.728
.628
.530
.222
.128
.226
.420
.733
.838
.2D
ocos
aned
ioic
acid
0.8
3.1
2.0
3.0
Tet
raco
sane
dioi
c3.
5T
RT
R1.
7T
R1.
71.
06.
53.
83.
647
.045
.153
.848
.447
.946
.952
.544
.540
.831
.048
.4O
ctad
ec-9
-ene
dioi
cac
id55
.859
.342
.4T
RT
R1.
5T
RT
RT
RN
DE
icos
-9-e
nedi
oic
acid
TR
TR
TR
ND
ND
TR
ND
TR
5.6
TR
7.2
7.3
TR
4.7
5.1
10.7
1-E
icos
anol
5.0
17.8
5.0
TR
5.2
26.6
43.3
49.1
34.6
35.5
35.2
32.6
45.6
39.9
25.5
35.7
57.0
17.6
21.3
1-D
ocos
anol
45.4
51.9
45.2
45.4
45.1
54.4
44.1
64.7
42.5
45.3
46.1
1-T
etra
cosa
nol
45.2
61.9
43.0
3.6
6.2
5.4
8.0
19.2
12.2
15.0
TR
11.2
21.3
13.2
TR
15.5
15.7
1-H
exac
osan
ol16
-Hyd
roxy
hexa
deca
noic
acid
1.7
1.8
1.8
3.1
1.7
1.5
2.4
3.0
3.3
2.5
2.4
1.3
1.6
2.1
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
TR
18-H
ydro
xyoc
tade
cano
icac
idT
RT
R57
.353
.661
.154
.343
.456
.157
.263
.246
.243
.151
.549
.151
.153
.122
-Hyd
roxy
doco
sano
icac
id5.
312
.59.
111
.013
.54.
78.
17.
124
-Hyd
roxy
tetr
acos
anoi
cac
id8.
912
.311
.119
.315
.910
.9T
RT
RT
RT
RT
RT
R0.
5T
RT
R0.
8T
R26
-Hyd
roxy
hexa
cosa
noic
acid
TR
TR
1.0
18-H
ydro
xyoc
tade
c-9-
enoi
cac
id31
.631
.930
.743
.730
.527
.229
.441
.845
.936
.437
.327
.028
.329
.2
aT
R,
trac
e;N
D,
not
dete
cted
.
51M.F. Santos Bento et al. / J. Anal. Appl. Pyrolysis 57 (2001) 46–55
The distribution of monomers relative to the chain length is illustrated in Fig. 2.For both types of cork, the major contribution comes from components with 18chain carbon atoms. A significant abundance exists also for 22 chain carbon atomsbut the other chain lengths have only minor participation.
It should be noted that almost all the contribution for the C18 components comefrom six suberinic acids: C18:1 diacid and v-hydroxyacid, 9,10-epoxy-18-hydroxy-octadecanoic acid, 9,10-epoxyoctadecanodioic acid, 9,10,18-trihydroxyoctadecanoicacid and 9,10-dihydroxyoctadecanodioic acid. The results show a large variabilityfor each one of these compounds, as indicated in Table 3. The corresponding meanvalues have relatively high coefficients of variation, ranging from 16 to 60% invirgin cork and from 15 to 43% for reproduction cork. However, when considering
Fig. 1. Total ion chromatogram of reproduction cork (sample CH1D). The compounds have beenmethylated on all carboxylic and hydroxylic groups unless stated otherwise. (FA) alkanoic acids; (DIA)a,v-diacids; (v-OH) v-hydroxyacids; (1) 11-metoxy-18-hydroxyoctadec-10-enoic acid* , unmethylated in18-OH; (2) 10-metoxy-18-hydroxyoctadec-9-enoic acid* , unmethylated in 18-OH; (3) 9,10-epoxy-18-hy-droxyoctadecanoic acid; (4) 11-metoxyoctadec-10-enodioic diacidc; (5) 9-methyl-10-metoxy-18-hydroxy-octadec-9-enoic acid*, unmethylated in 18-OH; (6) 10-metoxyoctadec-9-enodioic diacidc; (7)9,10,18-trihydroxyoctadecanoic acid; (8) 9,10-epoxyoctadecanodioic diacid; (9) 9,10,18-trihydroxyoc-tadecanoic acid, unmethylated in one OH; (10) 9,10-dihydroxyoctadecanodioic acid; (11) 9,10,20-trihy-droxyeicosanoic acid; (12) 9,10- dihydroxyeicosanodioic acid. *, Artifacts from compound (3); c,artifacts from compound (8) (Bento et al., unpublished results).
52 M.F. Santos Bento et al. / J. Anal. Appl. Pyrolysis 57 (2001) 46–55
Fig. 2. Chain length distribution of the suberin monomers for virgin cork and reproduction cork. Site1 (Coruche), C10, C32, and C87; Site 2 (Extremoz): E75, E118, and E151; Site 3 (Chamusca), CH1D,and CH2C; Site 4 (Companhia das Lezırias, Benavente), CL1A, and CL2A; and Site 5 (Azaruja), AZ1D,and AZ2C.
the total content in C18 components, the variability decreases and the coefficient ofvariation of the mean is only 7% for both types of cork. Considering thebiosynthetic mechanism proposed by Holloway and Deas [13] for these compoundsfrom the corresponding 9,10-unsaturated alkanoic acids, as shown on Fig. 3, it maybe suggested that the various steps have different efficiencies, which account for thelarge variability shown by the relative proportion of the individual components. Itcould be hypothesized that in the beginning of the biosynthetic process, thereexisted only the octadec-9-enoic acid with a relative abundance that did not differsignificantly between the samples. During the biosynthetic process, this acid origi-
53M.F. Santos Bento et al. / J. Anal. Appl. Pyrolysis 57 (2001) 46–55
Table 3Coefficients of variation of the mean (%) for the C18 suberinic acids involved in the biosyntheticmechanism referred in Fig. 3
Reproduction corkVirgin cork
4318-Hydroxyoctadec-9-enoic acid 3616 309,10-Epoxy-18-hydroxyoctadecanoic acid20 159,10-Epoxyoctadecanodioic acid
25609,10,18-Trihydroxyoctadecanoic acid3549Octadec-9-enedioic acid
9,10-Dihydroxyoctadecanodioic acid 3553Total monomers 7 7
Fig. 3. Biosynthetic mechanism proposed by Holloway and Deas [10].
54 M.F. Santos Bento et al. / J. Anal. Appl. Pyrolysis 57 (2001) 46–55
nates from different C18 compounds according to Fig. 3 and its abundance shouldbe very low at the end of the referred process, as confirmed by our results.
The chemical composition of suberin showed some differences between samplesfor both chemical families (Table 1) and for monomers (Table 2). The range ofvariation is in general higher for virgin cork. However, the variation for the totalcomponents of the major homologous series, the v-hydroxyacids, is smaller. Thesame occurs for the C18 and C22 compounds, which represent together more than75% of the total monomers. Probably, as discussed for C18 compounds, thedifferent relative amounts of the single components could be related to specificconditions within the biosynthetic process.
Considering differences between types of cork, e.g. virgin cork and reproductioncork, they were statistically significant only for alkanoic acid content (P=0.002).Similarly, the influence of site proved significant only for alkanoic acids (P=0.038).Since alkanoic acids are not within the major monomeric families in suberin, theseresults suggest that the variability in the composition of suberin mainly occursbetween individual trees. A larger sampling would be however required for a betterunderstanding of the sources of variation.
4. Conclusions
The application of TMAH-Py-GC–MS technique to cork samples proved to beadequate to study the variability of suberin monomeric composition. Suberindepolymerization occurred in all cases and most peaks in the chromatograms wereidentified as suberin derived esters or ethers.
The relative proportions of the individual monomers show a large variabilitybetween samples. In general, greater differences are found for virgin cork. Totalcontent of the main components v-hydroxyacids or of C18 or C22 compounds ishowever similar for all samples.
Acknowledgements
We thank Fernando Lopes, research contract AIR-CT92-0135 and Centro deEstudos Florestais for the sampling of cork and preparation of samples, as well asJos Pureveen for the technical assistance. This work is part of the scientific programof Line 2 of Centro de Fısica Molecular (IST). The authors gratefully acknowledgeFOM and Dutch Organization Scientific Research (NWO) for their financialsupport.
References
[1] H. Pereira, Wood Sci. Technol. 22 (1988) 211.[2] D.S. Van Fleet, Bot. Rev. 27 (1961) 165.[3] P.E. Kolattukudy, K. Kronman, A.J. Poulose, Plant Physiol. 55 (1975) 567.
55M.F. Santos Bento et al. / J. Anal. Appl. Pyrolysis 57 (2001) 46–55
[4] J. Graca, H. Pereira, Holzforschung 51 (1997) 225.[5] J. Graca, H. Pereira, Holzforschung (1998) in press.[6] M. Arno, M. Serra, E. Seoane, Anales de Quim. 77C (1981) 82.[7] P.J. Holloway, Phytochemistry 22 (1983) 495.[8] M.F. Bento, H. Pereira, M.A. Cunha, A.M.C. Moutinho, K.J. van den Berg, J.J. Boon, Phytochem.
Anal. 9 (1998) 75.[9] J.J. Boon, A.D. Pouwels, G.B. Eijkel, Biochem. Soc. Trans. 15 (1987) 170.
[10] S.R. Heller, G.W. Milne, EPA/NIH Mass Spectral Data Base, US Government Printing Office,Washington, DC, 1978.
[11] J. Graca, H. Pereira, Phytochem. Anal. 11 (2000) 45.[12] J. Graca, H. Pereira, J. Wood Chem. Technol. 18 (1999) 207.[13] P.J. Holloway, A.H.B. Deas, Phytochemistry 12 (1973) 1721.
.