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Headspace-SPME Analysis of the Sapwood andHeartwood of Picea Abies, Pinus Sylvestris and LarixDeciduaAnna Wajs a , Andrey Pranovich b , Markku Reunanen b , Stefan Willför b & BjarneHolmbom ba Technical University of Lodz Faculty of Biotechnology and Food Sciences, Institute ofGeneral Food Chemistry, Stefanowskiego 4/10, 90-924, Lodz, Polandb Abo Akademi, University, Process Chemistry Centre, c/o Laboratory of Wood and PaperChemistry, 20500, Turku/Åbo, Finland
Available online: 28 Nov 2011
To cite this article: Anna Wajs, Andrey Pranovich, Markku Reunanen, Stefan Willför & Bjarne Holmbom (2007):Headspace-SPME Analysis of the Sapwood and Heartwood of Picea Abies, Pinus Sylvestris and Larix Decidua , Journal ofEssential Oil Research, 19:2, 125-133
To link to this article: http://dx.doi.org/10.1080/10412905.2007.9699244
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P. abies, P. sylvestris and L. decidua
Vol. 19, March/April 2007 Journal of Essential Oil Research/125
Received: November 2005
Revised: January 2006
Accepted: March 2006
Headspace-SPME Analysis of the Sapwood and Heartwood of Picea Abies, Pinus Sylvestris
and Larix Decidua
Anna Wajs* Technical University of Lodz, Faculty of Biotechnology and Food Sciences, Institute of General Food Chemistry,
Stefanowskiego 4/10, 90-924 Lodz, Poland
Andrey Pranovich, Markku Reunanen, Stefan Willför and Bjarne HolmbomÅbo Akademi, University, Process Chemistry Centre, c/o Laboratory of Wood and Paper Chemistry, 20500 Turku/Åbo,
Finland
Abstract
Solid-phase microextraction (SPME) combined with GC and GC/MS was used for analysis of the wood volatiles of Norway spruce (Picea abies L.), Scots pine (Pinus sylvestris L.), and European larch (Larix decidua Mill.). More than 160 compounds were extracted and identified from spruce, pine, and larch stemwood. Differences in the quantitative and qualitative composition of the volatiles from the different conifer species were found. The volatile composition was specific for each species. Only small differences in the volatiles from different wood tissues, i.e. sapwood and heartwood, were found.
Key Word Index
Picea abies, Pinus sylvestris, Larix decidua, Pinaceae, SPME.
1041-2905/07/0002-0125$14.00/0—© 2007 Allured Publishing Corp.
J. Essent. Oil Res., 19, 125–133 (March/April 2007)
*Address for correspondence
Introduction
The volatile extractives in wood tissues of different tree species are unique and may be used for species identification. Terpenes are abundant and diverse in conifers, and play a com-plex, vital role in the relationship between plants and insects. Signals for sexual reproduction (pheromones, kairmones), for defence against herbivores (allomones), or to attract natural predators of herbivores (synomones) are conveyed through volatile terpenoids (1,2). The role of terpenoids in plants has been frequently discussed in the literature (3,4).
There are several methods available for determining ter-pene composition. A decade ago, a simple, fast, solvent-free sampling technique called solid-phase microextraction (SPME) was introduced (5). This method has been used extensively e.g. in trace analysis of volatiles in environmental samples (6-8). In most cases, SPME sampling has considerable advantages over other commonly used methods, which can be time-consuming or require more complicated techniques and larger amounts of sample. However, the sensitivity of SPME towards external factors (i.e. small differences in vial shape, sampling time, and condition of the fiber) still complicates the use of this method in quantitative analysis (5). Nevertheless, SPME has proved to be a viable alternative method for the determination of volatiles when the amount of sample is limited (8). Previous
studies have shown that headspace-SPME (HS-SPME) is a convenient method for determination of volatiles from wood (9,10). Moreover, the headspace analysis (HS-GC) of pine terpenes was more suitable for quantification of highly volatile compounds than the conventional hydrodistillation (11). Thus the headspace-SPME method was now applied to study the volatiles released from wood of Picea abies (Norway spruce), Pinus sylvestris (Scots pine), and Larix decidua (European larch).
The aim of the present work was to obtain a better know-ledge of wood volatiles of industrially important trees, by analyzing these compounds using HS-SPME which, to our knowledge, has not yet been investigated. Moreover, there is no information about wood volatiles of European larch. Although some studies on the chemical composition of wood volatiles from spruce and pine have been published (3,4,12-18) there is only one publication that has examined pine volatiles with the SPME method, however, in much less detail (9).
Nevertheless, several studies on volatiles from different parts, that is needles, bark, and wood of spruce (9,19-21), pine (11,15,22-26) and larch (27,28) have been conducted.
This paper deals with the composition of the volatiles released from different wood tissues, i.e. sapwood and heart-wood, of Norway spruce, Scots pine and European larch. We
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compared both the qualitative and quantitative composition of volatiles obtained by HS-SPME and GC analysis.
Experimental
Wood material: Three Norway spruce, three Scots pine, and three European larch trees were felled. Stemwood samples were stored in tight plastic bags in a freezer (-24°C) until needed. Two trees of Norway spruce were felled in August and one in October 2003 in South-west Finland. The number of growth rings at 1.5 m for the spruce trees were: 50, 50, and 70, respec-tively. One tree of Scots pine was felled in November 2003, and two in January 2004 in South-west Finland. The numbers of growth rings at 1.5 m were: 55, 58, and 60, respectively. All European larch trees were felled in May 2003 in France, and they all had 48 growth rings at 1.5 m height.
SPME sampling: The SPME fibres (65 µm Stableflex DVB/CAR/PDMS) and the holder were obtained from Supelco Ltd., (Bellefonte, PA, USA). The fibres were first conditioned according to the manufacturer’s instructions. For each extraction, 0.8 g of well-defined wood pieces (20 x 10 x 0.36 mm size) were cut using a blade microtome (Leitz Wetzlar, Austria) and immediately placed in a 4mL glass vial with a silicone septum coated with a Teflon film. The sample was kept for 30 min in a water bath at 60°C to achieve partition equilibrium between the sample and the air in the vial. Then, the SPME fibre was exposed to the headspace in the vial to absorb the analytes. After 30 min exposure time, the fiber was retracted into the needle and introduced into the GC injector for desorption and analysis of the volatiles. Three SPME analyses were performed in parallel for each wood sample.
Chromatographic analysis: The released volatiles were analyzed by GC and GC/MS. The capillary GC-FID analyses were performed using a Varian 3400 model gas chromatograph. H was used as carrier gas (1 mL/min). An HP-5 capillary column (30 m x 0.32 mm, 0.25 µm film thickness) was used for compound separation. The column oven temperature was programmed as follows: starting temperature 50°C (0.5 min), than 50–250°C at a 4°C/min heating rate after which it was held for 10 min and finally increased to 290°C at 10°C/min. For the hydrodistilled samples, the oven temperature program was 50°C (0.5 min) to 270°C at 4°C/min and finally to 290°C at 4°C/min and held isothermally for 5 min. Injector and detector temperatures were 230°C and 270°C, respectively.
The relative composition of each SPME sample was calcu-lated from the GC peak area by normalization, without using correction factors. Relative retention indices were determined using C8-C32 n-alkanes as external retention time refer-ences. Mixtures of n-alkanes (Sigma Aldrich Chemie Gmbh, Steinheim, Germany) were injected into the GC immediately after analysis of wood sample. This procedure makes it pos-sible to calculate retention indices (RI) of organic compounds recorded on chromatograms and compared to literature data (29). Experimental and literature RI’s were in close agreement. Identifications of compounds without Adams RI were based on their GC-retention behavior (17,30,31). All compounds were identified by GC/MS analysis. The GC/MS analyses were per-formed using an HP 6890-5973 GC-MSD instrument equipped with an HP-5 (20 m x 0.25 mm, 0.25 µm) or an HP-1 (15 m
x 0.25 mm, 0.25 µm) column. He was used as carrier gas (1.0 mL/min). The oven temperature program was 50°C (0.5 min), 50°–250°C at 4°C/min (10 min), 250°–290°C at 10°C/min. The injector temperature was 240°C and the MS ionization mode was electron impact (EI) at 70 eV electron energy. The components separated in the above conditions were identified with the Wiley 275.1 and NIST 98.1 mass spectral libraries, by comparison to literature data, and according to fragmentation patterns. Mean values were obtained from analysis of three trees (Tables I – III). Each wood sample was analyzed three times in parallel.
Results and Discussion
Headspace solid-phase microextraction: The efficiency of SPME for analysis of volatile compounds depends both on the properties of the fiber coating and on experimental condi-tions (5). The most suitable HS-SPME sampling conditions for volatiles from stemwood tissues were investigated in a previous study (10). Briefly, the sampling conditions investigated were: 0.8 g 20 x 10 x 0.36 mm sapwood and heartwood slices and 30 min equilibration and exposure time, which were sufficient to reach partition equilibrium at 60ºC. In these conditions, the SPME method was very repeatable and showed the highest sensitivity. Almost identical chromatograms were observed with only small variability (< 5%) in peak areas. The two-phase coated fiber, which included a carbon sorbent layer (Carboxen), was more suitable for wood volatiles than other tested coatings. Moreover PDMS, which is a non-polar phase, extracts non-polar terpenes very well, and the DVB porous microsphere phase increases the adsorption of small organic molecules (5). These fibers and SPME conditions allowed extraction of more than 100 volatile compounds, most of which were identified.
Typical SPME-GC chromatograms for the volatiles re-leased from spruce and pine sapwood are shown in Figure 1. Identified components could be grouped by their volatility (see Table I – III). It was possible to identify 96-99% of the spruce, 98-99% of the pine, and 94-98% of the larch volatiles that were eluted in the GC analysis.
All the main compounds were identified in all three trees of each species. In addition, there were several compounds occurring in trace amounts only (<0.05%) in one or two tree samples. These components are listed in Tables I – III and their amounts are reported as “trace amount” (t), or as “not identified or found as traces” (0-t). These between-tree dif-ferences in the volatile composition within the same species may be caused by variation in the age of the trees, or growing conditions, even though the material originated from one location (6,26,31).
Monoterpenes released from Norway spruce, Scots pine, and European larch sapwood and heartwood: Of the total GC-eluted compounds, 64-86% were the most in spruce sapwood and 78-85% in heartwood. The corresponding values in pine sapwood were 89-90% and in heartwood 94-98%, whereas in larch they were 71-84% in sapwood and 81-90% in heartwood (Table IV).
α-Pinene, β-pinene, β-phellandrene, limonene, myrcene were predominant volatiles for spruce and larch. These com-pounds are the main of pine wood but the most abundant con-
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Tab
le I.
N
orw
ay s
pru
ce
Sco
ts p
ine
Eu
rop
ean
larc
h
RI l
it.*
R
I exp
.**
Co
mp
ou
nd
s S
apw
oo
d (
%)
Hea
rtw
oo
d (
%)
Sap
wo
od
(%
) H
eart
wo
od
(%
) S
apw
oo
d (
%)
Hea
rtw
oo
d (
%)
800
800
hexa
nal
≤
0.1
0.1
– 0.
3 0
– t
0 –
t 0.
6 –
2.2
t
888
884
sant
ene
≤
0.1
0 –
0.3
89
9 90
1 he
ptan
al
≤
0.1
t
926
925
tric
ycle
ne
0.2
– 0.
3 0.
2 –
0.3
0.1
≤
0.1
0.1
0.1
– 0.
293
1 92
6 α-
thuj
ene
0.1
– 0.
2 0
– 0.
1
≤ 0.
2
t
t
t93
9 93
6 α-
pine
ne
38.7
–
46.4
46
.7 –
54
.2
25.0
–
47.5
31
.5
– 41
..2
41.6
–
47.2
51
.6 –
65
.795
1 94
8 α-
fenc
hene
0
– 0.
2
0 –
0.5
0 –
0.1
953
949
cam
phen
e 0.
5 –
0.6
0.6
– 2.
1
≤ 0.
4
≤ 0.
5 0.
1 –
0.5
≤
0.6
957
952
thuj
a-2,
4(10
)-di
ene
tr
≤
0.
1
t
t
1 95
5 (E
)-2-
hept
enal
≤
0.
1 0
– t
961
961
benz
alde
hyde
≤
0.
1 0
– 0.
1
≤ 0.
1
0.
1 ≤
0.
3 0.
2 –
0.4
967
963
verb
enen
e
0.
2 –
0.3
0.1
– 0.
2
976
966
sabi
nene
0.
3 –
1.1
≤
0.2
0.6
– 0.
9
≤ 0.
5
980
978
β-pi
nene
10
.3 –
27
.8
0.8
– 16
.9
1.1
– 17
.8
≤
7.7
10.3
–
21.0
11
.4 –
21
.399
1 98
9 m
yrce
ne
1.4
– 4.
4 0.
1 –
0.2
1.7
– 2.
1 0.
2 –
0.6
1.5
– 1.
6 0.
5 –
1.3
1001
99
9 δ-
2-ca
rene
≤ 0.
1 0.
4 –
1.2
10
05
999
α-ph
ella
ndre
ne
0.1
– 0.
3 ≤
0.
1 0.
1 –
0.5
≤
0.2
0.2
– 0.
3 0.
1 –
0.2
1011
10
10
δ-3-
care
ne
0.1
– 5.
0 ≤
6.
2 35
.8
– 36
.9
40.5
–
47.9
≤
1.
2 0.
1 –
1.8
1018
10
19
α-te
rpin
ene
0.1
– 0.
8 0.
1 –
0.3
0.2
0.
2 –
0.3
≤
1.0
0.4
– 0.
810
22
1022
o-
cym
ene
0.1
– 0.
3 0.
1 –
0.3
10
26
1023
p-
cym
ene
0.5
0.8
– 1.
8 0.
3 –
0.8
0.6
– 1.
1 0.
3 –
0.5
0.6
– 0.
910
31
1029
β-
phel
land
rene
2 +
lim
onen
e2 6.
7 –
7.6
7.3
– 13
.7
1.1
– 3.
9 1.
2 –
3.6
8.5
– 11
.4
4.8
– 8.
710
33
1 1,
8-ci
neol
e
t
0
– 0.
1
1040
10
36
(Z)-
β-oc
imen
e
t
0 –
t
t
t
t
0 –
0.1
1050
10
46
(E)-
β-oc
imen
e 0
<
0.1
0 –
t
t
≤
0.6
10
62
1056
γ-
terp
inen
e 0.
1 –
0.4
≤
0.2
0.4
– 0.
5
≤ 0.
6 0.
1 –
0.3
0.1
– 0.
53
1057
(E
)-2-
octe
nal
0 –
0.1
0 –
t10
68
1064
ci
s-sa
bine
ne h
ydra
te
≤
0.1
0 –
0.2
t
0
– t
10
82
1085
m
-cym
enen
e
0
.2
≤
0.2
10
88
1087
te
rpin
olen
e 0.
7 –
1.7
0.5
– 2.
9 4.
3 –
4.5
2.2
– 3.
7 0.
4 –
0.6
≤
0.4
1089
10
91
p-cy
men
ene
0
– t
t
t
0 –
0.1
0 –
t11
02
1101
no
nana
l ≤
0.
1 0.
1 –
0.2
t
≤ 0.
1 0.
2 –
0.3
≤
0.1
1111
11
09
p-m
enth
a-1,
3,8-
trie
ne
0 –
t
0 –
t
≤ 0.
1
1112
11
12
α-fe
ncho
l
t
1117
11
20
β-fe
ncho
l
0.1
– 0.
7 0
– t
≤
0.1
0
– t
1125
11
23
α-ca
mph
olen
e al
dehy
de
0
– t
t
0
– t
1140
11
43
cis-
verb
enol
0
– 0.
1 0
– 0.
1
1140
11
45
tran
s-sa
bino
l
t
0 –
t11
43
1146
ca
mph
or
0 –
t 0
– 0.
2
1144
11
42
tran
s-ve
rben
ol
t
0
– t
11
48
1152
ca
mph
ene
hydr
ate
0
– 0.
8
0 –
t
0 –
t11
60
1159
pi
noca
mph
one
0
– 0.
1
1165
11
65
born
eol
0.
2 –
0.4
≤ 0.
1
1177
11
77
terp
inen
-4-o
l ≤
0.
1 0.
2 –
0.3
t
0.
1 –
0.2
t
t
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Tab
le I.
co
nti
nu
ed
N
orw
ay s
pru
ce
Sco
ts p
ine
Eu
rop
ean
larc
h
RI l
it.*
R
I exp
.**
Co
mp
ou
nd
s S
apw
oo
d (
%)
Hea
rtw
oo
d (
%)
Sap
wo
od
(%
) H
eart
wo
od
(%
) S
apw
oo
d (
%)
Hea
rtw
oo
d (
%)
1180
11
80
m-c
ymen
-8-o
l
t
0 –
t
1189
11
90
α-te
rpin
eol
0.1
– 0.
7 ≤
1.
6
≤ 0.
1
≤ 0.
4
0 –
0.1
1195
11
93
met
hyl c
havi
col
0.1
– 0.
8 ≤
3.
6 0.
1 –
0.3
0.1
– 0.
5
t
≤
0.1
1204
12
06
deca
nal
≤
1.0
0.2
– 1.
1
≤ 0.
6 0.
1 –
0.4
0.1
≤
0.1
1204
12
06
verb
enon
e 0
– t
0 –
0.5
12
35
1233
m
ethy
l thy
mol
≤
0.
1 ≤
0.
2
t
t
0.
1 –
0.2
≤
0.2
1244
1
met
hyl c
arva
crol
0
– t
0 –
0.1
≤
0.1
0 –
t1
1260
3,
4-di
met
hoxy
tolu
ene
≤
0.
1 ≤
0.
1
1285
12
86
born
yl a
ceta
te
0.3
– 1.
5 ≤
0.
1
0.
4 –
0.6
0.3
– 0.
512
83(8
5)
1284
bo
rnyl
ace
tate
+ (
E)-
anet
hole
≤ 0.
1 0
– t
12
91
1291
(E
,Z)-
2,4-
deca
dien
al
t
12
92
1 tr
ans-
verb
enyl
ace
tate
t
t
1297
13
00
tran
s-pi
noca
rvyl
ace
tate
t
0 –
t
1314
13
14
(E,E
)-2,
4-de
cadi
enal
t
tota
l ide
ntifi
ed [%
] 63
.6
86
.2
78.4
84.5
89
.3
96
.2
94.2
98.1
71
.1
84
.3
80.8
89.8
t=tr
ace
(<0.
1%);
RI l
it* -
Ret
entio
n in
dexe
s fr
om li
tera
ture
(A
dam
s, 1
995)
giv
en fo
r a
sem
ipol
ar c
olum
n; R
I exp
** -
Exp
erim
enta
l ret
entio
n in
dexe
s gi
ven
for
a se
mip
olar
col
umn;
Am
ount
- n
orm
aliz
ed p
eak
area
with
out u
sing
cor
rect
ion
fact
ors;
1 id
entifi
ed o
nly
by G
C/M
S; 2
com
poun
ds s
epar
ated
on
HP
-1 c
olum
n; 3
iden
tified
by
GC
/MS
and
GC
-ret
entio
n be
havi
or fr
om li
tera
ture
(17
,30,
31)
1339
13
38
δ-el
emen
e
0
– t
≤
0.
1
t13
40
1339
te
rpin
en-4
-yl a
ceta
te
0 –
0.3
0 –
t
1350
13
48
α-te
rpin
yl a
ceta
te
≤
1.5
0 –
0.6
0 –
0.4
0 –
0.3
1351
13
50
α-lo
ngip
inen
e +
α-c
ubeb
ene
0.
9 –
2.0
1.1
– 2.
0 0
– t
0 –
0.3
0.2
– 0.
3 0.
2 –
0.3
1354
13
51
citr
onel
lyl a
ceta
te
0 –
0.1
0 –
t13
68
1351
cy
clos
ativ
ene
≤
0.1
0 –
0.2
0 –
t 0
– 0.
1
1372
13
69
α-yl
ange
ne
0.2
– 0.
8 0.
9 –
1.5
0 –
0.1
0 –
0.1
13
73
1369
lo
ngic
ycle
ne
0.1
– 1.
5 0.
1 –
0.4
0.1
– 0.
2 0
.113
73
1373
is
oled
ene
0.4
– 0.
5 0
.413
76
1374
α-
copa
ene
0.3
– 0.
4 0.
3 –
0.5
t
≤ 0.
5 0.
1 –
0.3
0.1
– 0.
313
80
1380
da
ucen
e
≤
0.
1 0.
1 –
0.2
3 13
80
sativ
ene
≤
0.
1 ≤
0.
4 0
– 0.
4 0
– t
13
87
1 is
olon
gifo
lene
0
– t
0 –
t
1390
13
89
β-cu
bebe
ne
0 –
0.1
t
≤
0.
2
t13
91
1390
β-
elem
ene
0
– 0.
1 0
– t
≤
0.1
≤
0.2
1397
13
92
1,7-
di-e
pi-α
-ced
rene
≤
0.
4 0
.1
1398
13
95
β-lo
ngip
inen
e 0
.1
0 –
t
≤
0.
4 ≤
0.
114
02
1401
α-
long
ifole
ne
1.
5 –
2.9
2.3
– 3.
0 0
– 0.
3 0
– 0.
1 1.
2 –
2.4
1.5
– 1.
6
Tab
le II
.
N
orw
ay s
pru
ce
Sco
ts p
ine
Eu
rop
ean
larc
h
RI l
it.*
R
I exp
.**
Co
mp
ou
nd
s S
apw
oo
d (
%)
Hea
rtw
oo
d (
%)
Sap
wo
od
(%
) H
eart
wo
od
(%
) S
apw
oo
d (
%)
Hea
rtw
oo
d (
%)
Dow
nloa
ded
by [
Uni
vers
ity o
f Sa
skat
chew
an L
ibra
ry]
at 1
1:19
18
May
201
2
P. abies, P. sylvestris and L. decidua
Vol. 19, March/April 2007 Journal of Essential Oil Research/129
Tab
le II
. co
nti
nu
ed
N
orw
ay s
pru
ce
Sco
ts p
ine
Eu
rop
ean
larc
h
RI l
it.*
R
I exp
.**
Co
mp
ou
nd
s S
apw
oo
d (
%)
Hea
rtw
oo
d (
%)
Sap
wo
od
(%
) H
eart
wo
od
(%
) S
apw
oo
d (
%)
Hea
rtw
oo
d (
%)
1400
1
tetr
adec
ane
0 –
t 0
– t
1401
14
02
met
hyl e
ugen
ol
0.1
– 0.
3
≤ 0.
1
1404
14
03
isoc
aryo
phyl
lene
1.
4 –
3.8
1.8
– 4.
7
1409
14
06
α-ce
dren
e ≤
0.
1 ≤
0.
4
≤
1.
4 ≤
0.
114
18
1412
β-
cedr
ene
0.2
– 0.
6 0.
2 –
0.6
0.1
– 0.
4 ≤
0.
214
18
1420
β-
cary
ophy
llene
0
– 0.
1 0
– 0.
114
32
1427
β-
gurju
nene
0
.1
≤
0.6
0 –
0.1
0 –
0.1
0.1
– 0.
4 0.
1 –
0.3
1433
14
33
γ-el
emen
e
0.
2 –
0.3
0.1
– 0.
214
36
1433
tr
ans-
α-be
rgam
oten
e
0.2
– 0.
3 ≤
0.
2
≤
0.
2 ≤
0.
114
39
1 ar
omad
endr
ene
0 –
t 0
– t
14
43
1444
(Z
)-β-
farn
esen
e 0
.2
0.1
– 0.
3
0.
3 –
2.2
0.2
– 0.
714
47
1449
α-
him
acha
lene
0
– 0.
2 0
– 0.
114
54
1449
α-
neo-
clov
ene
≤
0.1
0.1
– 0.
2
1454
14
55
α-hu
mul
ene
0 –
0.2
0 –
t
1458
14
58
(E)-
β-fa
rnes
ene
0.1
– 0.
4 ≤
0.
1
1460
14
59
cis-
muu
rola
-4(1
4),5
-die
ne
0 –
t 0
– t
0 –
0.1
0 –
0.1
14
67
1464
9-
epi- β
-car
yoph
ylle
ne
≤
0.1
t
1477
14
74
γ-m
uuro
lene
≤
0.
2 0.
1 –
1.2
0 –
4.0
0 –
0.6
14
80
1478
ge
rmac
rene
D
≤
15.1
0.
1 –
2.2
≤
0.1
0 –
t 3.
7 –
6.0
1.8
– 2.
714
80
1 γ-
curc
umen
e 0
– t
0 –
0.1
14
83
1480
ar
-cur
cum
ene
0 –
0.3
≤
1.4
≤
0.4
≤
0.1
1490
14
88
cis-
β-gu
aien
e
0
– 1.
6 0
– 0.
1
1491
14
92
vale
ncen
e 0.
1 –
0.3
0.1
– 0.
3 0
– t
0 –
0.1
0.1
– 0.
4 ≤
0.
314
93
1497
ep
i-cub
ebol
≤
0.
3 0
– 0.
3 0
– t
0 –
0.2
14
94
1498
α-
selin
ene
≤
0.2
0
– 0.
8 0
– t
1495
14
98
zing
iber
ene
≤
0.2
0.1
– 0.
314
99
1502
α-
muu
role
ne
≤
0.3
≤
0.3
0 –
0.4
0 –
1.2
≤
0.6
≤
0.6
1500
15
06
tran
s-β-
guai
ene
0 –
2.1
0 –
t
1504
15
07
(Z)-
α-bi
sabo
lene
0
– 0.
1 0
– t
0 –
0.8
0.1
– 0.
315
09
1510
α-
bisa
bole
ne
0.2
– 0.
3 ≤
0.
2
0.
4 –
1.9
≤
0.9
1512
15
13
β-cu
rcum
ene
0.4
– 0.
8 ≤
0.
4
1513
15
14
γ-ca
dine
ne
≤
0.2
≤
0.2
15
14
1516
cu
bebo
l
0
– 0.
4 0
– 0.
315
15
1517
(Z
)-γ-
bisa
bole
ne
0.1
– 0.
9 0.
2 –
0.7
0.1
– 1.
1 ≤
1.
215
24
1526
δ-
cadi
nene
1.
1 –
1.9
0.4
– 2.
3 0
– 0.
3 0
– 0.
8 0.
1 –
3.0
0.1
– 0.
415
24
1527
β-
sesq
uiph
ella
ndre
ne
0 –
0.8
0 –
0.7
1532
15
33
cadi
na-1
,4-d
iene
0
– t
0 –
1.8
0 –
t 0
– t
15
33
1535
(E
)-γ-
bisa
bole
ne
0 –
0.5
0 –
0.3
0.1
– 0.
3 0.
1 –
2.1
1538
15
40
α-ca
dine
ne
≤
0.1
≤
0.1
0 –
t 0
– t
0.1
– 0.
3 ≤
0.
115
42
1541
se
lina-
3,7(
11)-
dien
e
0.
1 –
0.2
≤
0.2
1542
15
42
α-ca
laco
rene
≤
0.
2 ≤
0.
2 0
– t
0 –
t
≤
0.2
1545
15
43
cis-
sesq
uisa
bine
ne h
ydra
te
≤
0.2
≤
0.1
1556
15
46
germ
acre
ne B
t
t
1.
8 –
2.2
0.9
– 1.
015
63
1563
β-
cala
cole
rene
t
0 –
t
t
t
Dow
nloa
ded
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ity o
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an L
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201
2
Wajs et al.
130/Journal of Essential Oil Research Vol. 19, March/April 2007
Tab
le II
. co
nti
nu
ed
N
orw
ay s
pru
ce
Sco
ts p
ine
Eu
rop
ean
larc
h
RI l
it.*
R
I exp
.**
Co
mp
ou
nd
s S
apw
oo
d (
%)
Hea
rtw
oo
d (
%)
Sap
wo
od
(%
) H
eart
wo
od
(%
) S
apw
oo
d (
%)
Hea
rtw
oo
d (
%)
1564
15
72
(E)-
nero
lidol
t
t
≤
0.1
1574
15
79
germ
acre
ne D
-4-o
l 0
– t
0 –
t 0
– t
0 –
t ≤
0.
4 0
– t
1585
15
83
glee
nol
≤
0.1
0 –
0.1
15
92
1 lo
ngib
orne
ol
0 –
t 0
– t
16
11
1602
ep
i-ced
rol
0 –
0.1
0 –
t16
14
1 1,
10-d
i-epi
-cub
enol
0 –
t
1627
16
30
1-ep
i-cub
enol
0
.1
0 –
0.1
0 –
t 0
– t
t
t16
40
1640
ep
i-α-c
adin
ol=
T c
adin
ol
≤
0.2
≤
0.1
1642
16
41
cube
nol
t
0
– 0.
1
0 –
t
1653
16
57
α-ca
dino
l
t
0 –
t 0
– t
0 –
t
1674
16
73
cada
lene
0
– t
t
t
1683
16
80
α-bi
sabo
lol
0 –
t 0
– t
t
t16
86
1 ep
i-α-b
isab
olol
t
t
1700
1
hept
adec
ane
t
t17
75
1774
14
-hyd
roxy
-α-m
uuro
lene
0
– 0.
1 0
– 0.
2
tota
l ide
ntifi
ed [%
] 11
.7 –
34
.1
12.3
–
18.9
2.
0 –
7.6
0.8
– 4.
3 11
.7 –
22
.3
7.6
– 13
.9
t=tr
ace
(<0.
1%);
RI l
it* -
ret
entio
n in
dexe
s fr
om li
tera
ture
(A
dam
s, 1
995)
giv
en fo
r a
sem
ipol
ar c
olum
n; R
I exp
** -
exp
erim
enta
l ret
entio
n in
dexe
s gi
ven
for
a se
mip
olar
col
umn;
Am
ount
- n
orm
aliz
ed p
eak
area
with
out u
sing
cor
rect
ion
fact
ors;
1 id
entifi
ed o
nly
by G
C/M
S; 3
iden
tified
by
GC
/MS
and
GC
-ret
entio
n be
havi
or fr
om li
tera
ture
(17
,30,
31);
Tab
le II
I.
N
orw
ay s
pru
ce
Sco
ts p
ine
Eu
rop
ean
larc
h
RI l
it.*
R
I exp
.**
Co
mp
ou
nd
s S
apw
oo
d (
%)
Hea
rtw
oo
d (
%)
Sap
wo
od
(%
) H
eart
wo
od
(%
) S
apw
oo
d (
%)
Hea
rtw
oo
d (
%)
1
hexa
deca
nal
t
1 18
62
pent
adec
anoi
c ac
id
0 –
t 0
– 0.
2
0 –
t
1894
1
rimue
ne
t 0
– t
19
00
1899
no
nade
cane
0
– t
0 –
t
1929
19
29
cem
bren
e
t
t
0
– t
0 –
t19
41
1952
pi
mar
adie
ne
t ≤
0.
1
≤ 0.
4
t
19
59
1956
ne
ocem
bren
e ≤
0.
3
t
1 9-
hexa
dece
noic
aci
d
t
0 –
t
1 19
62
hexa
deca
noic
aci
d ≤
0.
4 ≤
0.
7
≤ 0.
3 0.
1 –
0.2
t
t
1
isop
imar
adie
ne
t
t
t
t
1983
19
77
man
oyl o
xide
≤
0.
5 ≤
1.
1
t
t
2010
20
16
13-e
pi-m
anoy
l oxi
de +
eic
osan
e 0
– t
0 –
t
1 20
36
palu
stra
dien
e 0
– t
0 –
t
t
t
t
t1
2071
is
opro
pyl h
exad
ecan
oate
t
≤
0.1
t
t
t
t
1 20
53
thun
berg
ol
0 –
t 0
– t
t ≤
0.
120
54
2055
ab
ieta
-8,1
1,13
-trie
ne
0 –
t 0
– t
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ity o
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18
May
201
2
P. abies, P. sylvestris and L. decidua
Vol. 19, March/April 2007 Journal of Essential Oil Research/131
Tab
le II
I. co
nti
nu
ed
N
orw
ay s
pru
ce
Sco
ts p
ine
Eu
rop
ean
larc
h
RI l
it.*
R
I exp
.**
Co
mp
ou
nd
s S
apw
oo
d (
%)
Hea
rtw
oo
d (
%)
Sap
wo
od
(%
) H
eart
wo
od
(%
) S
apw
oo
d (
%)
Hea
rtw
oo
d (
%)
2056
20
62
man
ool
0 –
t 0
– 0.
1
≤
0.
2 ≤
0.
120
80
2083
ab
ieta
-7,1
3-di
ene
0 –
t 0
– t
≤
0.1
t
t
t21
00
1 he
neic
osan
e 0
– t
0 –
t
1 21
14
δ-13
-(tr
ans)
-neo
abie
nol
0 –
t 0
– t
1
2156
ci
s-ab
ieno
l 0.
1 –
0.3
≤ 0.
2
0.
1
1 21
75
pim
aral
0
– t
0 –
t
≤ 0.
2
t
22
00
1 do
cosa
ne
t
t
t 0
– t
t 0
– t
tota
l ide
ntifi
ed [%
] 0.
4 –
0.9
0.9
– 1.
4 0.
4 –
0.5
0.2
– 0.
3 0.
04 –
0.
2 0.
02 –
0.
1
t=tr
ace
(<0.
1%);
RI l
it* -
Ret
entio
n in
dexe
s fr
om li
tera
ture
(A
dam
s, 1
995)
giv
en fo
r a
sem
ipol
ar c
olum
n; R
I exp
** -
Exp
erim
enta
l ret
entio
n in
dexe
s gi
ven
for
a se
mip
olar
col
umn;
Am
ount
- n
orm
aliz
ed p
eak
area
with
out u
sing
cor
rect
ion
fact
ors;
1 id
entifi
ed o
nly
by G
C/M
S
stituent of this tree was δ-3-carene (Table I). These results are in accordance with earlier headspace studies of spruce (7,10,20,32), and pine (11,17,25). The identified main components in our HS-SPME analysis were the predominant volatiles also in the extractives or oils of spruce (3,4,16,18,19,33,34), pine (4,22,23) and larch (27,28,35). Additionally, the analyzed pines belonged to a high δ-3-carene chemotype, although this volatile pattern was earlier determined in needles (8,21,25).
Many monoterpenes identified in this study: santene, tri-cyclene, α-pinene, camphene, sabinene, β-pinene, myrcene, α-phellandrene, δ-3-carene, p-cymene, β-phellandrene, limonene, 1,8-cineole, (Z)- and (E)-β-ocimene, γ-terpinene, terpinolene, trans- and cis-verbenol, borneol, terpinen-4-ol, α-terpineol, verbenone, and bornyl acetate are typical volatiles of spruce, pine, and larch and have been identified before as constituents of conifer wood extractives (1,4,16,17,22,23,34). Some monoterpenoids identified in this study: α-fenchene, α-fenchol, α-campholene aldehyde, and methyl thymol have previously been found only in spruce bark (19), in different parts of pine species (30), or in twigs of different larch species (28,36). Verbenol, which has been reported as a sex attractant (18), was also identified in our spruce and pine wood, but not in larch. Most of the compounds listed in Table I were also previously found in spruce stemwood (10), but there are a few new compounds for SPME-sampled spruce wood constituents: α-fenchene, E-(β)-ocimene, p-cymenene, camphor, and methyl carvacrol. The composition of monoterpenes and related bio-genic compounds differs greatly, not only in wood but also in the leaf oils among different conifer species. Even within dif-ferent parts of a conifer tree, the amounts and composition of the monoterpenes can differ considerably (4). This variability of composition can be attributed to environmental factors, as well as to heredity (26,31). Furthermore, the terpene pattern of volatiles is mainly genetically controlled (31). Keeping these possible variations in mind, it can be concluded that no significant differences were found in the headspace volatiles between spruce sapwood and heartwood.
Most pine constituents identified in this study have been reported before in review articles (22,23), but a few new ones, which constituted ≤ 0.6% of all volatiles, were found in this study. These were: thuja-2,4(10)-diene, benzaldehyde, verbenene, p-cymenene, nonanal, p-mentha-1,3,8-triene, m-cymen-8-ol, methyl chavicol, decanal, 3,4-dimethoxytoluene, and trans-verbenyl acetate.
For larch, mainly some new aldehydes, i.e. hexanal, hep-tanal, benzaldehyde, (E)-2-octenal, nonanal, decanal, and two decadienals, were identified together with some other compounds: Z-(β)-ocimene, methyl thymol, trans-sabinol and methyl chavicol. Despite the fact that these components are new for Norway spruce, Scots pine, and European larch wood, they have been found in other pine or larch species (30,31,36,37).
Spruce, pine, and larch sapwood and heartwood also contained non-terpenoids such as aldehydes: hexanal, hep-tanal, (E)-2-octenal, nonanal, decanal, isomers of decadienal, and aromatic derivatives such as 3,4-dimethoxytoluene. The aldehydes, produced from unsaturated fatty acids, are precur-sors for straight-chain esters and have earlier been found in TMP-turpentine (18).
Dow
nloa
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Wajs et al.
132/Journal of Essential Oil Research Vol. 19, March/April 2007
The amount of monoterpenes sometimes varies greatly within species. The largest variations were conspicuous in the content of β-pinene. It was present in amounts between 0.8% and 27.8% in spruce heartwood and sapwood, respectively.
All the main monoterpenes occurred in both stemwood tissues in all three species. Differences in composition between the sapwood and heartwood were noticed only for compounds that constituted ≤ 0.8% of all volatiles in wood. Thus, the fol-lowing components were characteristic for spruce sapwood: α-fenchene and p-mentha-1,3,8-triene, while p-cymenene, α-fenchol, α-campholene aldehyde, camphene hydrate, pi-nocamphone, and borneol were found only in heartwood. The differences in the headspace volatiles from pine sapwood and heartwood was found to be less considerable than from spruce. Only camphene hydrate and borneol differed in heartwood and sapwood. Moreover, larch sapwood was characterized by decadienal isomers, while α-fenchol, camphene hydrate and α-terpineol occurred only in the heartwood.
Sesquiterpenes released from Norway spruce, Scots pine, and European larch sapwood and heartwood: The moderately volatile components found were mainly sesquiter-penes in nature (Table II). Of the total GC-eluted compounds, 12-34% were found in spruce sapwood and 12-19% in heartwood. The corresponding values in pine sapwood were 2.0-7.6%, and in heartwood 0.8-4%, whereas in larch they were 12-22% in the sapwood and 8-14% in the heartwood.
Only a few studies have been conducted on the composition of volatile sesquiterpenes in spruce, pine, and especially larch wood. The main compounds found in spruce were α-longifolene, isocaryophyllene, and δ-cadinene, which is in accordance with earlier studies (1,7). These compounds have also been found in oleoresin of Norway spruce (32) and have been reported to constitute 13 volume-% of so-called TMP-turpentine (18). The identified sesquiterpenes were also previously identified in spruce wood (10).
The compound groups of muurolenes and cadinenes were predominant in pine wood, which is in accordance with other studies (12-14,22,23). Earlier studies on the terpene compo-sition of P. sylvestris populations showed that the sesquiter-penes in pine may be divided into two groups, one including compounds of the cadinene type, and another consisting of longifolene and related (12-14). The major sesquiterpenes of pine wood and also of Swedish sulphate turpentine are α-, γ-, and ε-muurolene (4).
The predominant sesquiterpenes of European larch stem-wood were germacrene D and, as for spruce, α-longifolene and δ-cadinene. These compounds have also earlier been reported for larch (27,28).
The quantitative and qualitative composition of volatile sesquiterpenes of spruce, pine, and larch wood were similar. Only germacrene D was found in one of the spruce sapwood samples in large amounts (15%), while in the other spruce trees it occurred in the range of 0-2.2% of all volatiles. The large amount of germacrene D found may have been caused by wounding of the tree (18).
The qualitative within-species composition of sesquiter-penes found in the headspace of the sapwood and heartwood of spruce, pine, and larch were similar. A few differences occurred;
α-selinene and (E)-nerolidol occurred only in spruce sapwood, δ-elemene only in pine sapwood, while 1,10-di-epicubenol and cubenol occurred only in heartwood. α-Calacorene was characteristic for larch heartwood.
Semi-volatiles released from Norway spruce, Scots pine, and European larch sapwood and heartwood: Using SPME, it was possible to isolate and identify trace amounts of semi-volatiles, such as diterpenoids, fatty acids, higher alkanes, and other compounds (Table III). These compounds consti-tuted 0.02-1.4% of the total amount of compounds isolated and eluted on GC.
In earlier studies, cis-abienol and thunbergol, which were present in our spruce wood samples, were reported to be pre-dominant in the fraction of free diterpene alcohols of Norway spruce sapwood and heartwood (31). Bicyclic diterpene alcohols of the labdane type have also been reported in twigs and bark of Norway spruce (38), but we now found only δ-13-(trans)-neoabienol in trace amounts. Of the diterpene aldehydes, only pimaral was present in notable amounts in the extractives of spruce and pine. Derivatives of diterpene acids constituted a significant part of wood volatiles (39). However, none one of them was now found in the free form.
Many of the now identified semi-volatiles from spruce wood are known as lipophilic non-volatile compounds that are part of spruce rosin or TMP-turpentine (18,38,39). Nonetheless, these compounds have also been identified in previous studies as semi-volatile extractives (4,10,15).
Diterpene compounds; isomers of cembrene, palustradiene, abietatriene, and pimaral, earlier identified in spruce wood (10), was found in this study also in pine and larch stemwood. These semi-volatiles are common also for other coniferous tree species (30,31).
Pentadecanoic and octadecanoic acid, and isopropyl hexadecanoate occurred in the SPME samples in very small amounts.
The semi-volatile components occurred in trace amounts only. Thus there were no significant quantitative and qualitative differences between the compositions of these compounds in sapwood and heartwood. However, cis-abienol occurred only in spruce sapwood, pentadecanoic acid only in pine heartwood, and hexadecanal only in larch sapwood.
The motive of the present study was to achieve a direct and fast analytical method to determine the profile of the fragrant volatiles in wood of coniferous tree.
It is known that the applicability of SPME for terpene extraction is intimately correlated to the parameters of sam-pling time, temperature, conditions, and type of fiber (5). The optimization of the HS-SPME procedure for wood samples was reported in our previous publication (10).
Solid-phase microextraction combined with GC and GC/MS is a useful technique for analysis of volatile organic compounds in Norway spruce, Scots pine, and European larch, sapwood and heartwood. The quantitative and qualitative compositions of volatiles are specific for each tree species.
Altogether, more than 160 volatile and semi-volatile com-pounds of different classes were identified in the three species: Picea abies, Pinus sylvestris, and Larix decidua. Figure 1 shows representative chromatograms of volatiles of Norway spruce
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P. abies, P. sylvestris and L. decidua
Vol. 19, March/April 2007 Journal of Essential Oil Research/133
(upper) and Scot pine (lower) sapwood, extracted by SPME. The most abundant groups consisted of the terpene compounds monoterpenes (C10H16) and sesquiterpenes (C15H24), as well as some of their oxidized derivatives. The SPME method also allowed extraction of small amounts of semi-volatiles, for ex-ample diterpenes, higher alkanes, and fatty acids.
The volatile composition was similar in sapwood and heartwood of each species and the main volatiles also occurred in both wood tissues of all three analyzed tree species. Small differences were noticed for a few minor compounds, which were found only in the sapwood or in the heartwood of the different species.
All the main compounds were identified as predominant in all three trees of each species. Nonetheless, there were several compounds that occurred in small amounts only in one or two tree samples. These between-tree differences in the composi-tion of the volatiles within species may be caused by variation in the chemotypes, age of tree, or growing conditions, even though the trees originated from one growth location.
Acknowledgments
The authors wish to thank Jarl Hemming for guidance with the analytical equipment. This work was funded by the European Com-mission “Marie Curie Training Site’’ project (HPMT-CT-2001-00297). This work is also part of the activities at the Åbo Akademi Process Chemistry Centre, within the Finnish Centre of Excellence Programme (2000-2005) by the Academy of Finland.
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