A study of variability of suberin composition in cork from Quercus suber L. using thermally assisted...

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.


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.


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

.


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

.


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).


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-


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].


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

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A study of variability of suberin composition in cork from Quercus suber L. using thermally assisted...

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