J.

of Supercritical Fluids 55 (2011) 944–948

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

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

Optimization of process variables for essential oil components from

Satureja hortensis by supercritical fluid extraction using Box-Behnken
experimental design
Mostafa Khajeh ∗
Department of Chemistry, University of Zabol, P.O. Box 98615-538, Mofateh Street, Zabol, Iran

a r t i c l e i n f o a b s t r a c t

Article history: The oil and extracts of Satureja hortensis cultivated in Iran were extracted using supercritical carbon
Received 23 August 2010 dioxide and hydrodistillation method. The oil and extracts were analyzed by GC-FID and GC/MS. The
Received in revised form 10 October 2010 compounds were identified according to their retention indices and mass spectra (EI, 70 eV). The effects
Accepted 11 October 2010
of various parameters such as pressure, temperature, percent of modifier (methanol) and extraction time,
were investigated by a fractional factorial design (24-1 ) to determine the significant parameters and their
Keywords:
interactions. The results showed that the pressure, temperature and percent of modifier are significant
Satureja hortensis
(p < 0.05), but the extraction time was found to be insignificant. The response surface methodology (RSM),
Supercritical carbon dioxide
Box-Behnken design
based on Box-Behnken design was employed to obtain the optimum conditions of the significant param-
Hydrodistillation eters (pressure, temperature and percent of modifier). The optimal conditions could be obtained at a
pressure of 35.0 MPa, temperature of 72.6 ◦ C, and 8.6% (v/v) for methanol. The main extracted components
using SFE were ␥-Terpinene (35.5%), Thymol (18.2%) and Carvacrol (29.7%).
© 2010 Elsevier B.V. All rights reserved.

1. Introduction that consume smaller quantities of organic solvent due to of the

Satureja hortensis is an annual, herbaceous plant belonging to the
Labiatae of family. It is known as summer savory, native to southern
Europe and naturalized in parts of North America. The main com-
ponents of the essential oils of this plant are the phenols, carvacrol
and thymol, plus p-cymene, b-caryophyllene, linalool and other
terpenoids [1]. The green leaves and herbaceous parts of stems of
S. hortensis were used fresh and dried as flavoring agents in sea-
soning, stews, meat dishes, poultry, sausages and vegetables. The
essential oils and oleoresin were used in the food industry. More-
over, the essential oils that have been used in the perfume industry,
either alone or with other essential oils [1,2]. As a medicinal plant,
S. hortensis has been traditionally used as a stimulant, stomachic,
carminative, expectorant and aphrodisiac. The essential oils of this
plant have demonstrated antimicrobial and antidiarrheic activity
due to of the phenols in the oil [1–5].
The traditional procedures for the extraction of plant materials
are include hydrodistillation and organic solvent extraction using
percolation, maceration or soxhlet techniques. These methods have
distinct drawbacks such as time-consuming and labour intensive
operations, handling of large volumes of hazardous solvents. More-
over, there is increasing interest in alternative extraction methods
∗ Tel.: +98 542 2232961; fax: +98 542 2226765.
E-mail address: m khajeh@uoz.ac.ir
rising solvent acquisition and disposal costs and regulatory restric-
tions [6,7].
Supercritical fluids have been shown to exhibit several advan-
tages in the extraction of essential oils from plants. The combined
liquid-like solvating capabilities and gas-like transport proper-
ties of supercritical fluids make them particularly suitable for the
extraction of diffusion-controlled matrices such as plant tissues
[7]. Moreover, the solvent strength of a supercritical fluid can
be easily turned by simply changing the applied pressure and
temperature [7]. Carbon dioxide (CO2 ), the most commonly used
supercritical fluid, has the advantages of being non-flammable,
fairly non-toxic, cost effective and easily removed from the extract
following decompression [7,8].
The Box-Behnken is a second-order multivariate technique
based on three-level incomplete factorial designs that received a
wide application for assessment of critical experimental condi-
tions, that is, maximum or minimum of response function. The
number of experiments (N) needed for the development of Box-
Behnken matrix is defined as N = 2k (k − 1) + C0 , where (k) is the
factor number and (C0 ) is the replicate number of the central point
[9–14].
In this work, we have used SFE for the extraction of oil and
extracts of S. hortensis. The aim of the work is to analyze the
influence of different parameters such as pressure, temperature,
and percent of modifier and extraction time on the supercritical
fluid extraction of S. hortensis. A Box-Behnken experimental design

0896-8446/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.supflu.2010.10.017
 M. Khajeh / J. of Supercritical Fluids 55 (2011) 944–948 945

method was used to investigate the effects of various parameters Table 1

Experimental design the results obtained in function of the oil and extracts yield
on the SFE performance.
(Y%).

No. P (MPa) M% T (◦ C) t (min) Y%

2. Experimental
1 10.1 0.0 35 5 3.5
2 30.3 0.0 35 15 4.6
2.1. Plant material
3 10.1 5.0 35 15 4.7
4 30.3 5.0 35 5 4.9
The plant materials were collected from Zabol, Iran in April 2010. 5 10.1 0.0 55 15 4.1
A voucher specimen has been deposited in the national herbar- 6 30.3 0.0 55 5 4.7
ium of Iran (TARI). The aerial parts of the plants were air-dried 7 10.1 5.0 55 5 4.9
8 30.3 5.0 55 15 5.51
at the ambient temperature (about 40 ◦ C) and then were stored
9 20.2 2.5 45 10 4.6
at 4 ◦ C prior to extractions. The moisture content of the plant was 10 20.2 2.5 45 10 4.5
6.4 ± 0.05% (Karl Fischer). Immediately prior to SFE, the sample was
grilled in a blender to get particles powder with diameters in the
range of about 700–800 ␮m. helium, was adjusted to a linear velocity of 30 cm s−1 . The SFE sam-
ple (1 ␮l) was injected into the GC (without any further dilution)
2.2. Reagents using the splitless mode. The GC/MS analysis was carried out on
a Varian 3800 equipped to a DB-5 column with the same char-
HPLC grade dichloromethane and methanol were purchased acteristics as the one used in GC. The transfer line temperature
from Merck (Darmstadt, Germany). Carbon dioxide (99.99% purity), was 270 ◦ C. The ionization energy was 70 eV with a scan time of
contained in a cylinder with an eductor tube, was obtained from 1 s and mass range of 40–300 amu. The percentages of compounds
Sabalan Co. (Tehran, Iran). were calculated by area normalization method, without consid-
ering response factors. The components of oil and extracts were
2.3. Hydrodistillation identified by comparison of their mass spectra with those of a com-
puter library (Wiley and NIST) or with authentic compounds. Data
The plant (40 g of dried material) was submitted to hydrodistil- obtained were confirmed by comparison of their retention indices,
lation for 4 h, using a Clevenger-type apparatus, according to the either with those of authentic compounds or with the data, which
European Pharmacopoeia [15]. The volatile distillate was collected published in the literature [16].
over anhydrous sodium sulfate and refrigerated prior to analysis.
The yield of the oil was 1.5% (w/w), based on dry plant weight. 3. Results and discussion

2.4. Supercritical fluid extraction 3.1. Factorial design

A Suprex MPS/225 system (Pittsburgh, PA) in the SFE mode was
used for all of the extractions. The extraction vessel was a 10 ml
stainless steel vessel. The SFEs were carried out at pressures of
30.3–40.5 MPa, temperatures of 55–75 ◦ C and percent of modifier
(methanol) of 5–10% (v/v). A Duraflow manual variable restric-
tor (Suprex) was used in the SFE system to collect the extracted
analytes. In order to prevent sample plugging, the restrict point
was electrically warmed. The SFE flow rate through the Duraflow
restrictor was about 0.3–0.4 ml min−1 (compressed). Plant powder
(1.0 g) was mixed well with 2 mm diameter glass beads, and was
then charged into the 10 ml extraction vessel. The extracted oil and
extracts were collected in dichloromethane in a 5.0 ml volumetric
flask. The extraction vessel was a 10 ml stainless steel vessel. For all
the modifier studies, methanol was spiked directly into the extrac-
tion vessel with charged sample before extraction. To determine
the percent recovery, bubbling of the sample solution was done
using argon gas in order to evaporate the solution. Then the weight
of oil and extracts was measured. Finally the oil and extracts yield
(Y) was calculated from the following equation:
g 
1
Y% = × 100
g2
where g1 is weight of oil and extracts and g2 is weight of dry plant.
The following factors were evaluated: pressure, temperature,
and time and modifier volume. A two-level fractional factorial
design (24-1 ) with two replicates of center point was performed
in order to determine the main factors of the extraction process.
Table 1 shows the experimental design and the results, which
derived from each run. The significance of the effects was checked
by analysis of the variance (ANOVA) and Pareto chart (Fig. 1). The
standardized effect of the independent variables and their inter-
action on the dependent variable was investigated by Pareto chart
(Fig. 1), which detects the main influence of the independent vari-
ables and interactions with their relative significance on the oil and
extracts extraction. A positive value for the estimated effect indi-
cates an increase in the extraction yield if the variable increases to
its high level. A negative value indicates that a better extraction
(1)

2.5. GC and GC/MS analysis

GC analyses were carrying out using a Hewlett-Packard 7890A

series II gas chromatograph equipped with a FID and a DB-5
fused silica column (30 m × 0.53 mm i.d., film thickness 1.5 ␮m).
Oven temperature was programmed at 50 ◦ C for 1 min, and then
increased to 260 ◦ C at a rate of 4 ◦ C/min. Injector and detector tem- Fig. 1. Pareto chart of main effects obtained from 24-1 fractional factorial designs.
peratures were 260 ◦ C and 270 ◦ C, respectively. The carrier gas, The vertical line defines the 95% confidence interval [A = P, B = M%, C = T and D = t].
946 M. Khajeh / J. of Supercritical Fluids 55 (2011) 944–948

Table 2 was reduced to 15 using a Box-Behnken experimental design. The

Design matrix in the Box-Behnken model, observed response and predicted values.
results of this limited number of experiments provided a statistical
Trial no. T P M% Observed, Ya a (%) Predicted, Yp (%) Error (%) model that was used to identify trends in high yield for the extrac-
1 55 30.3 7.5 5.8 5.84 −0.65 tion process. The matrix, oil and extracts yield (Y%) were presented
2 75 30.3 7.5 8.1 8.01 1.08 in Table 2. Triplicate extractions were carried out for each run. The
3 55 40.5 7.5 7.5 7.59 −1.17 relative standard deviation (RSD%) of the three replicate extraction
4 75 40.5 7.5 8.7 8.66 0.43 was less than 0.3%. The equations below illustrate the relationship
5 55 35.4 5.0 7.4 7.3 1.35
of the three variables (i.e. P, T, M%) and Y.
6 75 35.4 5.0 7.8 7.83 −0.32
7 55 35.4 10.0 5.9 5.88 0.42
Y = −40.6 + 1.61(T ) + 0.09(P) − 1.51(M%) − 0.0081(T )2
8 75 35.4 10.0 8.5 8.6 −1.18
9 65 30.3 5.0 7.8 7.86 −0.80 − 0.0001(P) − 0.0413(M%)2 − 5.0 × 10−4 (T )(P)
10 65 40.5 5.0 8.6 8.61 −0.15
11 65 30.3 10.0 7.1 7.09 0.18 + 0.022(T )(M%) + 0.0018(P)(M%) (3)
12 65 40.5 10.0 8.8 8.74 0.71
13 65 35.4 7.5 8.5 8.47 0.39
14 65 35.4 7.5 8.7 8.47 2.68 The critical points in the surface response are found by solv-
15 65 35.4 7.5 8.2 8.47 −3.25
ing these equation systems for the condition of ı(Y)/ı(P) = 0,
a
Average of triplicate extraction. ı(Y)/ı(T) = 0 and ı(Y)/ı(M %)= 0. The method for calculating these
critical points has been published elsewhere [19]. The calculated
yield is obtained at low levels of the variables [17]. The inter- values for the critical points are as follows: P = 35.0 MPa, T = 72.6 ◦ C
pretation of this chart demonstrates that the factors pressure and and M% = 8.6. The summary of the analysis of variance (ANOVA) is
modifier volume are highly significant. An increase in pressure and shown in Table 3. The ANOVA of regression model demonstrates
modifier volume increases the oil and extracts yield. The tempera- that the model is highly significant. The goodness of the model
ture is a less significant factor. The factor time has an insignificant can be checked by the determination coefficient (R2 ). The value of
effect. The positive value for the effect of the pressure, temperature adjusted R2 (0.962) indicating that only 3.8% of the total variations
and modifier volume indicated that in the studied levels, the extrac- was not explained by the model. The value of R2 (0.986) indicates
tion efficiency increased with increase of these factors [17]. Results good relation between the experimental and predicted values of
of the fractional factorial design demonstrated that the variables the response. The lack-of-fit measures the failure of the model to
(pressure, temperature and modifier volume) in the studied levels represent data in the experimental domain at points which are not
required a final optimization. Therefore, a Box-Behnken design was included in the regression. The non-significant value of lack-of-fit
carried out. These results are similar to previous work [18]. (>0.05) revealed that the quadratic model is statistically significant
for the response.
3.2. Box-Behnken design The ANOVA of the regression model showed that the quadratic
model was significant, as was evident from the Fisher’s F-test
The significant variables like pressure (P), temperature (T) and (Fmodel = 40.37) with a very low probability value (p), as sug-
percent of modifier (M%) were selected as the critical variables and gested by Yetilmezsoy et al. [20]. Moreover, the calculated F
designated as A, B and C, respectively. The low, middle, and high lev- value was founded to be higher than the tabulated F value
els of each variable were designated as −1, 0, and +1, respectively. (F␣, df, (n−df+1) = F0.05, 9, 5 = Ftab = 4.77) at the 5% level, indicating that
The design of real experiments is given in Table 2. the computed Fisher’s variance ratio at this level was large enough
In a system involving three significant independent variables A, to justify a very high degree of adequacy of the quadratic model
B, and C the mathematical relationship of the response on these and also to show that the treatment combinations were highly
variables can be approximated by the quadratic (second degree) significant, as similarly reported by other [20].
polynomial equation as follow: Results were assessed with different descriptive statistic such
as chi-square test and Mallow’s Cp statistic to reflect the statistical
Y = ˇ0 + ˇ1 A + ˇ2 B + ˇ3 C + ˇ12 AB + ˇ13 AC + ˇ23 BC significance of the quadratic model. The chi-square (2 ) test (Eq.
+ ˇ11 A2 + ˇ22 B2 + ˇ33 C 2 (2) (4)) was also performed to check whether there was a significant
difference between the expected responses (Ei ) and the observed
where Y = estimate response; ˇ0 = constant; ˇ1 , ˇ2 and ˇ3 = linear data (Oi ).
coefficients; ˇ12 , ˇ13 and ˇ23 = interaction coefficients between the
three factors; ˇ11 , ˇ22 and ˇ33 = quadratic coefficients. 
n
(Oi − Ei )2
2cal = (4)
A multiple regression analysis was performed to obtain the Ei
coefficients and the equation, which can be used to estimate the i=1

response. A Box-Behnken experimental design was used in this
study. It is important to note that all interactions higher than sec-
ond order have been neglected in Eq. (2). A total of 15 experiments
were needed to estimate of the model.
The calculated chi-square (2cal = 0.021) was found to be less
than the tabulated chi-square value , represent that there was no
Table 3
ANOVA analysis for oil and extracts extraction.

3.3. Optimization of the experimental conditions Source Sum of Degree of Mean square F-value P
squares (SS) freedom (MSS)
The optimization step of SFE method was carried out using a Regression 12.655 9 1.406 40.37 <0.0001
Box-Behnken experimental design. Several variables that could be Linear 3.048 3 1.016 29.17 0.001
potentially affected on the oil and extracts yield were chosen such Square 2.568 3 0.856 24.57 0.002
Interaction 1.715 3 0.572 16.41 0.005
as: pressure (P), temperature (T) and percent of modifier (M%). Residual 0.174 5 0.035
Other parameters implicated in the extraction of oil and extracts Lack-of-fit 0.048 3 0.016 0.25 0.858
were kept constant, namely the extraction time was 15 min. Pure error 0.127 2 0.063
The number of experiments needed to investigate the before Adjust-R2 0.962
R2 0.986
noted three parameters at three levels would be 27 (33 ). Thus, this
 M. Khajeh / J. of Supercritical Fluids 55 (2011) 944–948 947

the independent variables and their interactions on the dependent

variables, 3D response surface plots for the measured responses
were formed based on the model equation (Eq. (3)) in this study.
Since the regression model has three independent variables, one
variable was held at constant at the central level for each plot. Fig. 2
shows the 3D response surfaces as the functions of two variables
at the center level of other variables, respectively.
The common belief among analytical chemists is that the max-
imum solubility of a solute in the SFE is achieved at the maximum
fluid density. The influence of pressure on the composition of oil and
extracts shown; the recovery was increased with an increase in the
pressure. This result was predictable due to raising the extraction
pressure, leads to a higher fluid density, which increases the solu-
bility of the oil and extracts, as similar to previous work [22]. The
variation of temperature during the SFE affects the density and the
volatility of the oil and extracts from the plant matrix. By increas-
ing the temperature, the volatilities of the oil and extracts increases
but the SFE density decreases. In this study, raising temperature
drastically increases the concentrations of content in the oil and
extracts composition due to of increasing the volatilities of the oil
and extracts [22].
Due to the limited solubility of polar organic compounds in
the SFE, quantitative extraction of these compounds with pure
supercritical carbon dioxide is impossible. Modifier is added to an
extraction process mainly for two reasons [23]: (i) to increase the
polarity of the SFE in order to improve the solubility of the oil and
extracts; and (ii) to facilitate desorption of oil and extracts from
the plant matrix. The polar modifier molecules accelerate desorp-
tion processes by competing with the oil and extracts for the active
binding sites; as well as by disrupting matrix structures. In the
present work the methanol was used as modifier, and it enhanced
the solubility of solutes in SFE and thus, the efficiency of extraction
increased.
Fig. 2. Response surface diagrams showing the effects of the mutual interactions
between two independent variables (a) pressure and percent of modifier, (b) tem- 3.4. GC/MS results
perature and percent of modifier, (c) pressure and temperature.
The results of GC/MS analyses of oil and extracts obtained by
statistically significant difference between the observed data and SFE and hydrodistillation were given in Table 4. The table shows the
the expected responses. The 2 test concluded with 95% certainly composition of volatile constituents in the extract, which are minor
that the quadratic model provided a satisfactory fit to the experi-
mental data. Table 4
Dawson and Martinez-Dawson [21] have reported that Mallow’s Satureja hortensis oils and extracts obtained by SFE and hydrodistillation.
Cp statistic (Eq. (5)) can be used to determine terms which can be
No. Name of compound R.I.a SFEb (%) Hydrodistillation (%)
omitted from the response surface model. For a response surface
model including all terms, Cp = p, where p is the number of variables 1 ␣-Thujene 931 0.7 1.9
2 ␣-Pinene 939 0.6 0.8
in the regression model including the intercept term. For response
3 Camphene 952 0.1 0.6
surface models with omitted terms Cp ∼ p shows a good model with 4 1-Octene-3-ol 959 – 2.5
little bias, and Cp ≤ p shows a very good prediction model. The goal 5 ␤-Pinene 984 0.4 0.9
is to remove terms from the response surface model until a min- 6 Myrecene 999 1.5 1.7
imum Cp value near p is obtained. If Cp > p, this shows that too 7 ␣-Phellandrene 1006 0.5 0.3
8 ␦-3-Carene 1017 – 1.6
many terms have been removed or some remaining terms are not 9 ␣-Terpinene 1021 3.5 4.7
necessary [20,21]. 10 p-Cymene 1027 4.1 3.8
 SS  11 Limonene 1033 0.8 1.1
e
Cp = + 2p − n (5) 12 (Z)-␤-Ocimene 1035 – 2.1
MSSe 13 Cis-Sabinene hydrate 1055 – 1.4
14 ␥-Terpinene 1060 35.5 22.5
where SSe is error sum of squares, MSSe is error variance of the esti-
15 Terpinolene 1080 – 4.5
mate, p is the number of variables in the regression model including 16 Trans-Sabinene hydrate 1083 – 1.9
the intercept term and n is number of experiment in Table 2. In 17 Linalool 1104 1.2 1.7
this study Mallow’s Cp statistic (Cp = 9.97) showed the third condi- 18 Trans-z-Caren-4-ol 1143 – 1.5
tion (Cp ≤ p and p = 10), indicating a very good prediction model, as 19 Terpinene-4-ol 1176 0.2 1.8
20 Thymol 1290 18.2 15.5
similarly reported by other [20].
21 Carvacrol 1299 29.7 21.5
Yetilmezsoy et al. [20] have reported that three dimensional 22 Carvacrol acetate 1374 – 2.5
(3D) response surface plots as a function of two factors, maintain- 23 ␤-Caryophyllene 1413 0.4 0.6
ing all other factors at fixed levels are helpful in understanding 24 ␤-Bisabolene 1502 0.5 0.8
both the main and their interaction effects of these two factors. a
Kovats retention indices on DB-5 column.
b
Thus, in order to gain a better understanding of the influences of Percent of component based on the area normalization (P%).
948 M. Khajeh / J. of Supercritical Fluids 55 (2011) 944–948
constituents of the products. The SFE procedure was compared with
the traditional hydrodistillation method. Table 4 reports the oil and
extracts of SFE and hydrodistillation method. This results show that
different compounds can be extracted from S. hortensis using SFE
and hydrodistillation procedure. The percent of ␥-Terpinene, Thy-
mol and Carvacrol in the SFE was higher than hydrodistillation. The
SFE is more selective than hydrodistillation. Furthermore, the SFE
is less tedious and minimizes the risk of compound degradation
due to heat. In addition, the SFE presents some advantages over
hydrodistillation such as extraction rate.
4. Conclusion
In this study, the SFE of S. hortensis was studied. The response
surface methodology (RSM) has several advantages compared to
the classical methods in which one variable at a time technique
is used. Firstly, RSM gives a large amount of information from a
small number of experiments. Indeed, classical methods are time
consuming and a large number of experiments are required to
explain the behavior of a system. Secondly, in RSM it is possible
to observe the interaction effect of the independent parameters
on the response. The model equation easily clarifies effects for
binary combination of the independent parameters [24]. This study
showed that RSM was a convenient approach to optimize the best
culture conditions for obtained maximum oil and extracts of the
plant. The application of a Box-Behnken design became possible,
fast, economical and efficient way of an optimization strategy of the
proposed method. The SFE procedure developed for the extraction
of oil and extracts required to P = 35.0 MPa, T = 72.6 ◦ C and M% = 8.6.
The SFE of S. hortensis was studied, and the results were
compared with essential oil composition obtained by hydrodistil-
lation. The SFE procedure offers many important advantages over
hydrodistillation. The SFE required shorter extraction time (15 min
vs. 4 h for hydrodistillation). Energy cost was higher for perform-
ing hydrodistillation than SFE. The flexibility in the manipulation of
the variables involved in the SFE process allows one to optimize the
experimental conditions considering the selectivity of a substance
or classes of substances of interest. The selectivity of SFE allowed
one to maximize the concentration of the selected compounds.
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