Isolation of Eucalyptus Essential Oil for GC-MS Analysis
by Extraction with Subcritical Water
M. M. J i m 6 n e z - C a r m o n a / M. D. L u q u e de Castro*
Analytical Chemistry Division, Faculty of Sciences, University of Cdrdoba, 14004 Cdrdoba, Spain
Key Words
Gas chromatography-mass spectrometry
Subcritical-water extraction
Hydrodistillation
Eucalyptus essential oil
Summary
A method based on subcritical-water extraction (SWE)
before detection and identification by GC FID and
GC-MS, respectively, has been developed for the isola-
tion of eucalyptus essential oil. The proposed method
has been compared with another based on, hydro-
distillation, a conventional extraction procedure. Fresh
eucalyptus leaves (4 g) w e r e e:~tracted with water at
50bar, 150~ and 2 m L m i n for 20rain. Hydro-
distillation was performed on the same amount of leaves
with 3 6 0 m L water for 3 h. After each extraction the
compounds were removed from the aqueous extract by a
single extraction with hexane (5 mL), detected by G C -
FID, and identified by electron impact (EI) MS. The
SWE-based method is clearly quicker and more effi-
cient than the hydrodistillation-based method, because
yields from the former for 11 of the 13 major com-
pounds in the extract are higher after 20-rain extraction
than after hydrodistillation for 3 h. This enables sub-
stantial cost savings of both energy and raw material.
Introduction
The isolation of essential oils from natural products is an
important task because of the widespread use of these
compounds. The methods of isolation most commonly
used are mainly based on solvent extraction [1-3] or
steam distillation [4, 5]. Losses of some volatile com-
pounds, low extraction efficiency, degradation of com-
pounds, and large amounts of toxic solvent waste are
disadvantages of these methods of extraction. This
578 Chromatographia Vol. 50, No. 9/10, November 1999
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variety of drawbacks has led to a search for new and
more rational alternatives for the isolation of essential
oils [6]. Thus, supercritical CO2 extraction has emerged
as a promising trend for the isolation of essential oils
from plants [7, 8]; the technique enables rapid extraction
and the use of low working temperatures (thus avoiding
losses and degradation of volatile and thermolabile
compounds, respectively), automation is feasible, and
solvent-free extracts are obtained. The coextraction of
cuticular waxes and lipids, the need for sample drying
before extraction, and the high acquisition and main-
tenance cost of the equipment do, however, make the
search for new alternatives mandatory.
Subcritical-water extraction (SWE), a technique based
on the use of water as extractant, at temperatures be-
tween 100 and 374 ~ and pressures sufficiently high to
maintain the liquid state, is emerging as a powerful
alternative for extraction of solid samples [9]. Thus,
subcritical water has been used to extract pollutants with
a wide range of polarities from environmental samples
[10-12], and for the extraction of pesticides [13] and
polycyclic aromatic hydrocarbons [14] from soils. The
use of subcritical-water extraction as an alternative to
conventional and supercritical CO2-extraction-based
methods for essential oils has been proposed very re-
cently [15-17], encouraging the performance of in-
depth studies in the field.
The aim of this research was to test the suitability of
subcritical-water extraction as a method for the isolation
of eucalyptus essential oil. SWE applied before G C -
FID or G C - M S for the detection or identification, re-
spectively, of the extracted compounds has been com-
pared with hydrodistillation, to demonstrate its possibi-
lities as an alternative method of extraction.
Experimental
Apparatus
Subcritical-water extraction was performed by use of
the assembly shown in Figure 1. A Shimadzu LC 10AD
pump with digital flow-rate and readouts was used to
Original
 ~ IIPP
i ~ ] TC
VR
Water Oven
Vial
Figure1
Schematic diagram of the SWE apparatus. HPP, high pressure
pump; PH, preheater; EC, extraction cell; TC. temperature con-
troller; CS, coolingsystem;VR, variablerestrictor.
impel the water used as extractant through the system.
The extractor used (a prototype designed and patented
by Salvador and Merchfin [ 18]) consisted of a 14.5-mL
extraction cell (Keystone Scientific, Bellefonte, PA,
USA) closed at both ends with screw caps enabling the
circulation of the extractant. The screw caps also con-
tained stainless steel filter plates (2/~m thickness and 1/4
inch i. d.) to ensure the plant material remained in the
extraction chamber. This chamber, with a stainless steel
preheater, is located in an oven designed to work up to
300 ~ and controlled by means of a Toho TC-22 tem-
perature controller. A cooling loop (cooled with water at
room temperature) manufactured from a 1-m length o f
stainless-steel tubing was used to cool the fluid from the
oven to a temperature close to 25 ~ thus avoiding
losses of volatile compounds as a result of the elevated
temperature. The outlet of this coil was coupled to a
home-made variable restrictor; this was used to control
the pressure in the system, to maintain the extractant in
liquid state. A simple laboratory Quickfit apparatus
comprising a 2000-mL steam generator flask, a distilla-
tion flask, a condenser, and a receiving vessel were used
to perform the hydrodistillation.
Extracts were analyzed by means of a Varian Star 3400
gas chromatograph equipped with a flame ionization
detector (FID). Finally, a Fisons VG Autospec (Micro-
mass Instruments) mass spectrometer was used to iden-
tify the compounds in the extracts.
Chemical and Materials
Eucalyptus (Eucaliptus globulus) leaves were collected
from a wild population growing in the South of Chile
(Concepcidn). Stock standard solution (4800/~g mL -1)
n-nonane (Sigma, St. Louis, MO, USA) was prepared in
HPLC-grade hexane (Scharlau, Barcelona, Spain).
NaC1 and Na2SO4 (Merck, Darmstadt, Germany) and
HPLC-grade hexane were used as demulsifier, drying
agent, and extractant, respectively, in the liquid extrac-
tion of the aqueous extracts. Twice-distilled degassed
water purified through a Milli-Q deionizing unit (Mil-
lipore) was used as extractant.
Original ChromatographiaVol.50, No. 9/10,November1999
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Subcritical-Water Extraction
Degassed Milli-Q water stored in a reservoir was
pumped into the oven where it reached the preheater and
passed through the extraction chamber containing fresh
eucalyptus leaves (4.0 g) and a glass-wool plug inserted
at the outlet end to prevent the frit being plugged. The
aqueous extract was cooled to 25 ~ in the cooling loop
and, after passage through a variable restrictor, collected
in a vial. In kinetic experiments a 75-min extraction was
performed under optimum working conditions with the
vial being changed every 5 min. To perform liquid-
liquid extraction, hexane (5 mL) and n-nonane stock
solution (70 #L), used as extractant and internal stan-
dard, respectively, were added to each extract in a se-
parating funnel, as was NaC1 (ca 1 g) to facilitate
breakdown of emulsion. The hexane layer was then se-
parated and dried with anhydrous sodium sulfate
(0.25 g) before GC analysis.
Hydrodistillation
Eucalyptus leaves (4.0 g) were placed in the steam gen-
erator flask and MilliQ water (360mL, the amount
equivalent to the optimum flow-rate in subcritical-water
extraction) was added. Hydrodistillation was performed
for 3 h, after which the aqueous extract (83 mL) was
isolated.
Chromatographic Separation and Detection
The hexane extracts (1/~L) obtained after either sub-
critical-water extraction plus liquid-liquid extraction or
hydrodistillation plus liquid-liquid extraction, were in-
jected into an SGL-5 fused-silica capillary column
(25 m x 0.25 mm i.d., 0.25/~m film thickness). The
carrier gas (helium) column head pressure was ca
0.7 bar (10 psig). The detector temperature was 300 ~
The oven temperature was 50 ~ for 2 min after injec-
tion then increased to 200 ~ at 3 ~ min -1.
Mass Spectrometric Identification
Hexane extract fractions from subcritical-water extrac-
tion under optimum conditions and from hydrodistilla-
tion were collected individually for identification of the
components by mass spectrometry. Electron impact (EI;
electron energy 70 eV) mass spectra of the compounds
were obtained by means of a Fisons VG Autospec by
direct injection of the final extract solution (1/~L); the
ionization chamber temperature was 250 ~
Results and Discussion
Optimization of Conditions
The experimental conditions were optimized to max-
imize the yield of essential oil in a time as short as
possible. Conditions affecting subcritical-water extrac-
579
tion, liquid-liquid extraction, and chromatographic de- Table I. Optimization of conditions.
tection, were considered. The ranges over which the
effects o f the variables were studied, and the optimum Condition Range studied Optimum
value
values found, are shown in Table I. The tmivariate
m e t h o d was used in all instances.
Subcritical-waterextraction
Temperature(~ 50-200 150
Conditions used for Subcritical-Water Extraction Pressure (bar) 30-100 50
Flow-rate (mL min-1) 0.5-3.0 2.0
Conditions affecting the properties o f the leaching agent
Liquid-liquidextraction
Extractantvolume (mL) 2.5-15.0 5.0
(temperature, pressure, and flow-rate) were studied to Number of extraction steps 1-2 1
establish the optimum conditions for extraction o f 4.0 g Chromatography
eucalyptus leaves in 20 min. Carrier gas - Helium
Injectionvolume (#L) - 1
Column - SGL-5
Effect of the Temperature of the Extraction Chamber Split ratio 0 1:4 1:4
Carrier gas flow-rate(mL min-1) 0.5 1.0 0.8
The temperature o f the extraction chamber is the key Rate of temperature gradient (~min-1) 3.0-5.0 3.0
variable when subcritical water is used as extractant. Its
influence was studied in-depth between 50 and 200 ~
at 50 ~ intervals, at a constant flow-rate o f 2 m L min -1
and a pressure o f ca 50 bar. The chromatograms re- Extractant Volume
vealed that the yield o f all the compounds present in the
extract increased with increasing temperature up to Volumes o f hexane between 2.5 and 15 m L were in-
150 ~ and then dropped as the temperature was in- vestigated for removal o f the compounds from the aqu-
creased to 200 ~ especially for compounds appearing eous extract and preconcentration before GC analysis.
at early retention times, for example the major com- Although use o f 2.5 m L resulted in the highest peaks,
pound, eucalyptol, probably because o f thermal de- 5 m L was selected as optimum because the organic layer
gradation ofthermolabile compounds. Because the best could be more easily separated from the aqueous extract.
yield was obtained at 150 ~ this temperature was se-
lected as optimum. Number of Extraction Steps

Effect of Pressure A second extraction with hexane (5 mL) was performed

The effect o f pressure at a constant flow-rate o f 2 m L
min -1 and a temperature o f 175 ~ was tested by manu-
ally modifying the diameter o f the variable restrictor.
This variable proved to have little influence on the yield
i f the leaching agent was in the liquid state. Thus, as
foreseeable, pressures from 30 to 100bar gave rise to
chromatograms with no significant differences. A pres-
sure o f ca 50 bar was selected as optimum for further
experiments, because this was easily set by means o f the
restrictor.
Effect of Flow-Rate
The flow-rate was studied in the range 0.5-3 m L min -1
at a constant temperature o f 125 ~ and a pressure o f ca
50 bar. The yield increased with increasing flow-rate up
to 2 m L min -1. For some compounds (especially those
with early retention times) the yield obtained at 3 m L
min -1 was slightly higher; it was, however, clearly lower
for others and gave rise to additional and undesirable
peaks corresponding to degradation products. Thus
2 m L rain -1 was selected as optimum flow-rate.
to ensure no compounds remained in the aqueous ex-
tract after the first extraction. An almost flat chromato-
gram (containing only a negligible peak corresponding
to eucalyptol) was obtained after injection o f the second
extract. Thus, a single 5-mL extraction with hexane was
sufficient for total removal o f the compounds from the
aqueous extract before GC analysis.
Conditions used for Chromatography
The results obtained after optimization o f the carrier gas,
injection volume, and type o f column are summarized in
Table I. The split radio, the carrier gas flow-rate and the
temperature program were also studied to obtain a good
separation o f the compounds as rapidly as possible.
Split Ratio
Splitless injection gave rise to off-scale peaks, making it
mandatory to work in split mode. O f the different ratios
tested, a value o f 1:4 seemed to furnish the highest peaks.
Carrier Flow-Rate

Conditions used for Liquid-Liquid Extraction A helium flow-rate o f ca 0.8 m L min -1 (measured at the
column outlet) was provided b y a column head pressure
Extractant volume and n u m b e r o f extraction steps were o f ca 0.7 bar (10 psig). This resulted in well-resolved
studied. chromatographic peaks.

580 Chromatographia Vol. 50, No. 9/10, November 1999 Original

 250 five injections o f one the organic extracts obtained by
SWE o f 4.0 g eucalyptus under optimum conditions.
The average RSD o f the chromatographic step for the
~N 200 -

thirteen major compounds was 1.62 %. To study the

precision o f the overall process, five samples o f eu-
3150 11 .4 calyptus leaves (4.0 g) were subjected to SWE under
o
4
optimum conditions and the hexane layer obtained after
loo liquid-liquid extraction was analyzed by GC. The aver-
9
age RSD o f the overall process for the thirteen major
10
compounds was 5.96 %.
-~ 50
3 1~ 5

0
o 1- 7

o 20 4O
Time(min)
Figure 2
Kinetic curves obtained for SWE under optimum working condi-
tions for ~-pinene (1),/~-pinene (2), fl-myrcene (3), eucalyptol (4),
1-isopropyl-4-methyl-3-cyclohexen-l-ol (5), linalyl propanoate
(6), 3,7-dimethyl-2,6-octadien-l-o! _(7), 2-hydroxycineo! acetate
(8), unidentified (9), tricyclo[7.3.0 6 -8 .0 1-9 ]-2,7,7 r ,lO-tetramethyl-
9-dodecane (10), unidentified (11), unidentified (12), and bicyclo
[4.4.0] [2-2-hydroxypropyl]-6-methyl- 1-decene (13).
Table II. Comparison of compound/internal standard peak area
ratio for GC of extracts obtained by hydrodistillation and by SWE
under the optimum working conditions.
60
Kinetics of the Leaching Process
The kinetics o f SWE under optimum conditions for the
major compounds in eucalyptus leaves are shown in
Figure 2. The rate o f extraction for each c o m p o u n d can
be quantitatively inferred from the plot. As a con-
sequence o f the high temperature selected as optimum,
the kinetics o f the SWE are quite rapid for many o f the
compounds. Thus complete extraction is achieved
within the first 15 min for all the compounds except
linalyl propanoate (for which the kinetic curve was
anomalous) and an unidentified compound denoted
peak 12 in the chromatogram.

Compound Hydro SWE Comparison of S W E and Hydrodistillation

istillation
The methods based on subcritical-water extraction and
c~-Pinene 2.324 9.784 hydrodistillation for isolation o f eucalyptus essential oil
/~-Pinene 0.108 0.641 were compared in terms o f time, efficiency, and cost.
/~-Myrcene 0.164 0.941 The chromatograms obtained after SWE (A) and hy-
Eucalyptol 18.566 46.923
1-Isopropyl-4-methyl-3-cyclohexen-1-ol 0.229 0.765 drodistillation (B) under optimum conditions, and the
Linalylpropanoate 1.113 4.716 peaks identified, are shown in Figure 3. One o f the most
3,7-Dimethyl-2,6-octadien-1-ol 0.396 0.823 significant advantages o f the SWE m e t h o d is its speed.
2-Hydroxycineolacetate 0.266 0.440 Thus, extraction for 20 rain furnishes higher yields (as
Unidentified 1.606 2.085
Tricyclo [7.3.06-8.01-9]-2,7,7',10- 0.464 0.932 inferred from the chromatograms) than are obtained
tetramethyl-9-dodecaene after 3 h hydrodistillation. Thus the analysis time re-
Unidentified 1.606 2.085 quired for the overall process is clearly reduced by the
Unidentified 4.149 0.375 proposed m e t h o d (1 h for SWE compared with 4 h for
Bicyclo [4.4.0][2-2-hydroxypropyl]- 0.832 0.249
6-methyl-l-decene hydrodistillation). The proposed method is clearly more
efficient than hydrodistillation, as can be inferred by
comparison o f the chromatograms obtained from both
methods under optimum conditions SWE gives higher
Temperature Programming peaks for eleven o f the thirteen compounds. (The two
last peaks only are slightly higher after hydrodistilla-
A temperature program comprising an initial tempera- tion.) The yields obtained for each compound from both
ture o f 50 ~ held for 2 rain after injection, then in- methods under optimum conditions (expressed as com-
creased to 200 ~ at 3 ~ min -] provided separation o f the pound/standard internal peak area ratio) are compared
major compounds in 45 min. in Table II, which again demonstrates the greater effi-
ciency o f subcritical-water extraction. Finally, the cost
Precision of the S W E - C h r o m a t o g r a p h i c M e t h o d o f extraction is clearly advantageous for the proposed
method. The energy cost required to perform hydro-
The precision (expressed as RSD) was studied sepa- distillation was ca twenty times that required for SWE.
rately for the chromatography and for the overall pro- This feature is o f fundamental importance to the poten-
cess (extraction and detection). The precision o f the tial future implementation o f this technique on an in-
chromatographic step was calculated by performing dustrial scale as a real alternative to hydrodistillation.

Original Chromatographia Vol. 50, No. 9/10, November 1999 5 81

 Conclusions
(A) T h e m e t h o d p r o p o s e d for the isolation o f e u c a l y p t u s
essential oil, s u b c r i t i c a l - w a t e r e x t r a c t i o n c o m b i n e d
with G C - F I D and G C - M S for the d e t e r m i n a t i o n and
identification o f the c o m p o u n d s , is quicker, cheaper,
75
and m o r e effective than hydrodistillation, enables sub-
stantial savings o f b o t h acquisition a n d m a i n t e n a n c e
costs c o m p a r e d with m e t h o d s b a s e d on supercritical
CO2 extraction, and has e n v i r o n m e n t a l a d v a n t a g e s o v e r
solvent extraction. T h e s e reasons, t o g e t h e r with the h i g h
c 50
p r e c i s i o n o f the method, m a k e S W E a c o m p e t i t i v e al-
ternative to established e x t r a c t i o n m e t h o d s for isolation
11 o f essential oil f r o m such plants.

25- 13 Acknowledgment

I111, , ,li Financial support f r o m S p a i n ' s C o m i s i d n Inter-

ministerial de C i e n c i a y T e c n o l o g i a is gratefully ac-

i. ,1 t, k n o w l e d g e d (Project P B 9 5 - 1 2 6 5 ) .

~o 4'o
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pinene, 2=/~-pinene, 3=/Lmyrcene, 4=eucalyptol, 5 = 1-iso- [18] US Patent 5400642 (1995).
propyl-4-methyl-3-cyclohexen- 1-ol, 6 = linalyl propanoate, 7 = 3,7-
dimethyl-2,6-octadien- 1-ol, 8 = 2-hydroxicineol acetate, 9 = uni-
dentified, lO=trieyelo [7.3.0 6 - 8 .0 1 9 ]-2,7,7 p,lO-tetramethyl-9-do- Received: Mar 30, 1999
decaene, 11 = unidentified, 12 = unidentified, 13 = bicyclo Revised manuscript
[4.4.0][2-2-hydroxypropyl]-6-methyl-l-decene. received: May 10, 1999
Accepted: Jun 10, 1999

582 Chromatographia Vol. 50, No. 9/10, November 1999 Original