Trends in Analytical Chemistry 118 (2019) 182e193

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Trends in Analytical Chemistry

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Supercritical fluid extraction of essential oils

Mohammad Yousefi a, Mehdi Rahimi-Nasrabadi b, c, *, Seied Mahdi Pourmortazavi d,
Marcin Wysokowski e, Teofil Jesionowski e, Hermann Ehrlich f, Somayeh Mirsadeghi g
a
Student Research Committee, Department of Food Science and Technology, Faculty of Nutrition and Food Science,
Tabriz University of Medical Sciences, Tabriz, Iran
b
Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
c
Faculty of Pharmacy, Baqiyatallah University of Medical Sciences, Tehran, Iran
d
Faculty of Chemistry and Chemical Engineering, Malek Ashtar University of Technology, Tehran, Iran
e
Faculty of Chemical Technology, Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland
f
Institute of Electronics and Sensor Materials, TU Bergakademie Freiberg, Freiberg, Germany
g
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences,
1411713137, Tehran, Iran

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

Article history: The main purpose of this study was to investigate the extraction of essential oils from plants by the
Available online 31 May 2019 supercritical fluid extraction technique. However, other interesting information, regarding the process
optimization, is also discussed. Therefore, logical steps were identified to completely describe the pro-
Keywords: cess. These steps were divided into three parts: (1) characterization of essential oils; (2) properties
Supercritical fluid attributed to the supercritical fluid, including supercritical fluid extraction, parameters affecting the
Essential oil
procedure such as temperature, pressure, particle size, modifier, flow rate, and water, the importance of
Extraction
fractionation, types of collection methods, the antimicrobial and antioxidant activities of essential oils
Experimental designs
Antimicrobial
extracted by supercritical fluid, advantages and drawbacks of this method in comparison with other
Antioxidant extraction techniques; and (3) the use of experimental designs, including screening, optimizing and
modeling, in the process of supercritical fluid extraction of essential oils.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction as a medicinal plant, and has powerful antifungal and antibacterial

Essential oils (EOs) account for only a small portion of plants'
constitution; however, they determine important characteristics
of aromatic plants. EOs are known because of their complex
composition, having a few to several hundred components,
particularly hydrocarbons (terpenes and sesquiterpenes) and
oxygenated compounds (aldehydes, acids, alcohols, ketones, ox-
ides, phenols, acetals, lactones, ethers and esters). Both of these
groups determine the odor and flavor characteristics of EOs [1].
Beside their aromatic features, EOs have antioxidant and antimi-
crobial properties and myriad health benefits, leading to many
applications in the pharmaceutical, food, cosmetic, and fragrance
industries. For example, oregano (Origanum vulgare) is a herba-
ceous plant grown in the Mediterranean regions, which is utilized
* Corresponding author. Faculty of Pharmacy, Baqiyatallah University of Medical
Sciences, Tehran, Iran.
E-mail address: rahiminasrabadi@gmail.com (M. Rahimi-Nasrabadi).
effects [2]. Besides, antioxidant properties of the EO of Origanum
vulgare have been reported by Mechergui, Coelho, Serra, Lamine,
Boukhchina and Khouja [3]. The main components of this EO
identified by those authors were thymol (18e32%), p-cymene
(36e46%), g-terpinene (12e24%), and carvacrol (2e15%). They also
measured the ability to scavenge 2,2-diphenyl-1-picrylhydrazil
(DPPH) radicals, obtaining values in the range 59e80 mg/L. All of
these properties have motivated researchers to develop new
techniques for the extraction of EOs.
Supercritical fluid extraction (SFE) has become the most widely
used method for extracting and isolating EOs from aromatic plants.
This technique provides effective and quick extraction, requires
only moderate temperatures, eliminates clean-up steps, and avoids
the use of harmful organic solvents. Carbon dioxide (CO2) is an ideal
solvent for extracting and isolating EOs from plants, being non-
explosive, non-toxic, readily available, and easily eliminated from
extracted products [4]. Zermane, Meniai and Barth [5] presented
experimental data concerning the supercritical CO2 (SC-CO2)
extraction of EO from Algerian rosemary leaves. They obtained a

https://doi.org/10.1016/j.trac.2019.05.038
0165-9936/© 2019 Elsevier B.V. All rights reserved.
 M. Yousefi et al. / Trends in Analytical Chemistry 118 (2019) 182e193
yield of 0.95e3.52 g EO per gram of dry rosemary. Moreover, gas
chromatography (GC) and gas chromatographyemass spectrom-
etry (GC-MS) analyses revealed that camphor (at 48.89%wt.) was
the main compound present in the EO.
In general, the efficiency of SFE is dependent on a number of
parameters, including flow rate, pressure, temperature, extraction
time, etc. In situations such as this, where many variables may in-
fluence the result, the modeling and optimization of processing
parameters by techniques such as response surface methodology
(RSM) is a successful way to improve the process efficacy.
The aim of this study was to make a comprehensive investiga-
tion of the capacity of supercritical carbon dioxide for extracting a
wide spectrum of EOs. For more on this topic, see Pourmortazavi
and Hajimirsadeghi [1] and Fornari, Vicente, Va
zquez, García-Risco
and Reglero [6]. With respect to these latter cites, our study has
gone a step further, showing established experimental designs
pertaining the SFE of EOs as well as the antioxidant and antimi-
crobial quality of EOs obtained by SFE method.
2. Essential oils
EOs, sometimes called ethereal oils, are secondary metabolites
manufactured by various plants and herbs. They are characterized
as natural, volatile, complex, liquid substances with a strong aroma.
EOs are sometimes called volatile oils; nevertheless, Coelho, Cris-
tino, Matos, Rauter, Nobre, Mendes, Barroso, Mainar, Urieta and
Fareleira [7] designate the oil obtained by SFE as volatile oil, to
discriminate it from EO, which by definition is produced by
hydrodistillation (HD) and steam distillation (SD). EOs are mainly
composed of lipophilic constituents, containing the volatile aroma
components of the vegetal matter, which are involved in the plants'
defense system. EOs represent a small part of plants' composition
(5% of vegetal dry matter) and consist of hydrocarbons of mono-
terpenes, sesquiterpenes, and their oxygenated fractions such as
aldehydes, ketones, phenols, acids, alcohols, ethers, esters, etc. [1].
EOs are used as food flavors, preservatives, and cosmetic, cleaning,
insecticide, herbicide, and pharmaceutical products [8]. Several
other benefits of EOs, including their ability to act as stimulators of
the central nervous system, as well as antioxidant, anti-
inflammatory, antibacterial, antiviral and antifungal properties,
have been recognized. They have long been used in traditional
medicine as diuretics, digestives, sedatives, expectorants, etc.;
likewise they are marketed in the form of extracts, infusions, and
tablets. The biological activity and organoleptic properties of EOs
are dependent on their respective compositions. The quantity of
active substance in an EO ranges from high (nearly 80e90% w/w of
d-limonene present in orange EO) to trace quantities [6]. The
components and characteristics of ginger, lavender, and basil EOs
are given in the following examples.
Ginger (Zingiber officinale Roscoe) is a plant in the family Zin-
giberaceae, used in products such as pickles, jams, chutneys, and
beverages. Its rhizome is a prevalent item in Chinese folk medicine
[9]. Several species of Zingiber have been recognized to contain
many bioactive substances, including the shogaols and gingerols,
which exhibit antibacterial activity [10]. Mesomo, Corazza, Ndiaye,
Dalla Santa, Cardozo and de Paula Scheer [11] evaluated the
chemical composition and antibacterial activity of the EO obtained
from ginger (Zingiber officinale R.) roots. The main compounds
found in the EO were b-sesquiphellandrene, a-zingiberene, b-
bisabolene, geranial, a-farnesene, and b-eudesmol. They also
demonstrated the strong inhibitive effect of ginger EO against
Pseudomonas aeroginosa and a weaker inhibitive effect against
Salmonella typhimurium and Shigella flexneri.
Lavender (Lavandula angustifolia) has a well-known EO used in
the food, fragrance, aromatherapy, and pharmaceutical industries,
183
owing to its unique chemical composition. Lavender EO has been
reported to have more than a hundred components including
linalool, lavandulyl acetate, linalyl acetate, camphor, and 1,8-
cineole [12]. One of the most important features of lavender EO is
its antioxidant activity, which has attracted the interest of many
researchers [13]. Lavender EO is applied in food manufacturing as a
flavoring agent for beverages, candy, ice cream, baked goods, and
chewing gum [14] and as an antioxidant ingredient to protect food
products from loss of labile compounds, oxidative rancidity, and the
formation of off-flavors. The pleasing aroma of lavender EO is suited
to use in perfumes, soaps, colognes, skin lotion, and other cos-
metics. Recently, this EO has been proven to have beneficial effects
on the central nervous system [15]. Moreover, lavender EO has
commercial relevance in the pharmaceutical industry due to its
therapeutic properties such as antiseptic, anticonvulsive, anti-
inflammatory, antiviral, antidepressant, and antibacterial activity
[16]. The properties of several EOs are summarized in Table 1.
3. Supercritical fluid extraction
A SF emerges if temperature and pressure are incremented
above critical points determined for a specific liquid or gas. In the
supercritical region, the demarcation surface between liquid and
gas disappears, and a homogeneous fluid emerges [17]. Supercrit-
ical fluids (SFs) demonstrate a diffusivity and density between
liquid and gas. The density of SFs, in contrast to liquids, changes
when the pressure and temperature values are changed, so that a
small increase in pressure may lead to an enormous increase in
fluid density, followed by a change in the solvating power of the SF
[18]. This phenomenon makes it possible to extract targeted com-
ponents from a multicomponent mixture. Therefore, selectivity is
the main advantage of SFE. Besides, the mild conditions used to
obtain natural extracts can mitigate problems associated with the
thermal decomposition of components [19]. This method likewise
facilitates the recovery of solvent, because of the volatility of the SF
that can be used to obtain active extracts. It enables the attainment
of high extraction yields in short times without producing toxic
waste. The high solubility, selectivity, and rates of mass transfer
make SFE an attractive extraction process [18]. More details con-
cerning the SFE can be found at studies of, de Castro, Valcarcel and
Tena [20], and Mantell, Casas, Rodríguez and de la Ossa [21].
Currently, for various practical reasons, more than 90% of SFE
processes are performed using CO2. In addition to having a fairly
low critical temperature (32
C) and pressure (7.4 MPa), CO2 is
moderately non-flammable, non-explosive, non-toxic, available at
low cost and high purity, and easily removable from extracts.
Besides, CO2 has low surface tension and viscosity, but two or
three times the diffusivity of other fluids [22]. In the supercritical
region, CO2 exhibits a polarity similar to pentane, and is thus
suitable for the extraction of lipophilic substances. The main
disadvantage of CO2 is that it lacks the polarity required to extract
polar substances [23].
SFE includes both off-line and on-line techniques. The complete
process of off-line SFE is well described by Fornari, Vicente,
V
azquez, García-Risco and Reglero [6] (Fig. 1). The raw matter is put
into a basket, placed inside an extraction vessel (EV). SF is contin-
uously loaded from the bottom of EV; at the end of EV, SF con-
taining extracted solutes flows through a depressurization valve (V)
to the separator (S1), where extracts are separated from the
gaseous fluid owing to the lower pressure and collected in a col-
lector. The collected extracts can then be introduced into an
analytical instrument to measure the yield of extraction. Some SFE
instruments contain more than one separator; the purpose is to
fractionate the extract by setting different pressures and temper-
atures in the separators. At the last separator, the fluid reaches the
184 M. Yousefi et al. / Trends in Analytical Chemistry 118 (2019) 182e193

Table 1
The characteristics of certain essential oils extracted from plants.

Vegetal matter Botanical name Main components Functions Reference

Leaves of laurel Laurus nobilis Linalool,1.8-cineole, terpineol acetate, methyl Botanical fungicide [49]
eugenol, linalyl acetate, sabinene, eugenol, b-
pinene, a-terpineol
Horseradish tree Moringa oleifera 1,2-Benzenedicarboxylic acid, nonacosane, Antihypertensive, antifungal, antiulcer, [32]
mono (2-ethylhexyl) ester, heptacosane, and b- antitumor, antioxidant, and anticancer
Amyrin, activities
Flowering plant Leptocarpha rivularis a-Thujone, b-caryophyllene, caryophyllene Anticancer, hypoglycemic, and antioxidant [33]
oxide, and caryophyllene oxide effects
Rosemary Rosmarinus officinalis L. Phenolic diterpenes (carnosol, carnosic acid, Antioxidant and antimicrobial activities [39]
rosmanol, rosmaridiphenol, and
rosmariquinone), flavones (apigenin,
genkwanin hesperetin, and cirsimaritin) and
phenolic acids (rosmarinic acid and caffeic acid)
Sage Salvia officinalis L. Olean-18-ene, manool, carnoso, viridiflorol, and Antioxidant, antibacterial, hypoglycemic, anti- [24]
lupeol inflammatory, fungistatic, virustatic, astringent,
antihydrotic eupeptic, and antidiabetic
properties

chromatography (GC) columns. On-line coupling eliminates sample

loss and handling, and decreases the analysis time; it also enables
the flow of a large quantity of extracts through the extraction
vessel, and can protect samples from air and light. The main diffi-
culty is the need for experts to handle the system.

4. Differences between supercritical fluid extraction and

other extraction methods

The three most commonly applied techniques to extract EOs are

Fig. 1. A schematic diagram of off-line supercritical fluid extraction.
recirculation system pressure (usually around 4e6 MPa). After
flowing through a filter (F), the gaseous fluid is liquefied (HE1) and
kept in a supplier tank (ST). When the fluid exits this tank, it is
pumped (P1) and heated (HE2) to the preferred extraction tem-
perature and pressure. Precooling of the fluid is commonly required
(HE3) prior to pumping, to avoid pump cavitation. If a co-solvent
such as methanol is employed, a supplementary pump is required
(P2). Generally, the co-solvent is mixed with the fluid before
introduction to HE2. The whole system is more complex than those
used in classic methods. Because in opposite of many traditional
extraction techniques which are mainly based on two factors of
temperature and time, the pressure factor also needs to be adjusted
in the SFE method. In fact, it can be said that regulating the pressure
has led to the complexity of SFE, since the pressure of CO2 not only
should be adjusted in extraction vessel, but also in many sections of
the system to enable the system preserves CO2 near its supercritical
state. In off-line method, the extract can be further analyzed by
independent chromatography methods. Although, the separation
of extraction and analyzing parts leads to a little increase in time
but it is easier to find optimum conditions for extraction in an off-
line mode due to the independence of analysis step from the
extraction.
The on-line technique directly combines an SFE instrument with
analytical devices such as liquid chromatography (LC) or gas
Soxhlet, HD and SFE. Soxhlet is one of the oldest extraction pro-
cedures. It has been the standard extraction process for over 100
years, even after failing to demonstrate many ideal extraction
properties. The Soxhlet procedure suffers from a variety of draw-
backs. For instance, the average time of a Soxhlet extraction is from
1 to 72 h; the solutes extracted by this method are usually obtained
in high volume and in diluted form, and thus need to be concen-
trated before analysis; and in many cases the extraction is not se-
lective owing to the co-existence of interfering compounds, which
may further complicate the analysis of the targeted analytes.
Perhaps the greatest disadvantage of this method is the need for
expensive, toxic, and high-purity organic solvents such as methy-
lene chloride, acetone, acetonitrile, etc. Also, the high temperatures
used in a Soxhlet procedure may cause the decomposition of
thermolabile and volatile compounds.
The spectrum of analytes extractable by Soxhlet depends on the
solvent type. For instance, hexane is a solvent of high molecular
weight (greater than that of CO2); it can therefore dissolve heavy
molecular components or long chain alkanes (fatty acids, fatty acid
sterols, methyl esters, etc.) more easily than SC-CO2, but obviously
it will have a lower content of volatile compounds. The Soxhlet
technique does not need much capital investment, and in many
cases it produces a higher yield of EO extraction than SFE or HD
[24,25]. However, it is not a selective method and is not considered
an ideal method to extract analytes of this type.
Conventional distillation methods include hydrodistillation
(HD) and steam distillation (SD). Distillation has hitherto been the
most extensively used and cheapest method of EO extraction;
however, it may cause chemical changes involving the oxidation of
certain components. Also, during HD, extensive hydrolysis and
thermal degradation can be induced, possibly resulting in the for-
mation of off-odor and the removal of some components. For
example, it has been shown [26] that out of fifty compounds
identified in Cupressus sempervirens EO extracted by SFE (ac-
counting for 98% of the total EO), only eight compounds were
 M. Yousefi et al. / Trends in Analytical Chemistry 118 (2019) 182e193
identified in the EO extracted by HD. Also, the main constituents of
the latter were a-pinene, myrcene, piperonal, and trans-4,5-epoxy-
(E)-2-docenal, while the main components of the SFE extract were
manoyl oxide, trans-totarol, a-acoradiene, and cis-a-santalol.
In the HD method, a substantial amount of fresh or dried leaves
is placed in a chamber in contact with water. The water is heated to
boiling point to produce steam. The steam temperature is increased
to break down the leaf structures, which hold the EO. Since heating
is carried out in the presence of water, the water vapor pressure
rises, as does the vapor pressure of the EO. The EO is consequently
carried with the steam via a tube into the condensation chamber.
The hydrophobic EO forms on the surface of the water, and is then
decanted. This is a time-consuming process, requiring several
hours. To reduce the duration of the process, to reduce losses of
major polar compounds, to save energy, and to limit possible
modifications of the natural components of EO caused by oxidation,
steam distillation (SD) can be employed instead of HD. This involves
forcing the steam through the plant materials. However, an overall
disadvantage of distillation is its typically low extraction yield
(0.001% to nearly 4%) [27,28].
Significant criteria used to compare different extraction
methods include the extract content and the yield of extraction. A
good example of such a comparison is the study of Zhao and Zhang
[29], who extracted the EO of Eucalyptus leaves with various
extraction systems. The yields obtained by Soxhlet, SFE and HD
were 7.9%, 4.78% and 3.77% respectively. The results of GC-MS
demonstrated that the dominant component was 1,8-cineole. The
chemical compositions of the EO were markedly different in the
three cases. The oil extracted by HD contained only volatile com-
pounds, whereas the oil from Soxhlet and SFE comprised both
volatile and higher-molecular-weight substances. Nevertheless, the
contents of 1,8-ceneole in the SFE and Soxhlet extracts were 46.19%
and 29.85% respectively. Some examples showing the comparison
between the SFE and traditional extraction methods to extract EO
from plants are given in Table 2.
Another factor which can be used to compare SFE with other
extraction techniques is the antimicrobial and antioxidant activity
of the extracted EO. It may seem at first glance that the EO extracted
by the SFE technique will have better antimicrobial and antioxidant
activity, but in this case there are conflicting results, particularly
between SFE and HD. Bagheri, Manap and Solati [30], after the
extracting the EO from Piper nigrum L. by SFE (40
C, 30 MPa, 2 mL/
min flow rate, 40 min) and HD, performed antioxidant activity tests
based on DPPH. These tests gave EC50 (Half maximal effective
concentration) values of 103.28 and 316.27 mg/mL for the EO ob-
tained by SFE and HD respectively. However, in another study, the
minimum inhibitory concentration (MIC) values of Aquilaria
crassna EO against Staphylococcus aureus and Candida albicans were
lower for the EO extracted by HD than for the EO extracted by SFE
[31]. The reason is that many antioxidant and antimicrobially active
substances are typically classified as volatile compounds, and some
studies have demonstrated a higher content of volatile components
in EOs extracted by HD compared with SFE [29]. Hence, the dif-
ferences in the quantities of volatile constituents of EOs can affect
their antimicrobial and antioxidant activity.
In addition to technical differences between SFE and classical
extraction methods, the use of SF is considered as a “green alter-
native” for traditional extraction techniques. From the environ-
mental and health point of view, the method of SFE, due to the
application of mainly CO2, possess a high safety for human and
environment compared to methods such as soxhlet; because in
opposite of extraction with organic solvents, which always are
faced with issues such as solvent elimination and leaving residues
in the environment, these problems are seen rarely in SFE
technique.
185
5. Supercritical fluid extraction of essential oils
Why is SFE a suitable method to extract EOs? The answer to this
question relates to the nature of EOs and SC-CO2. As was noted in
previous sections, CO2 has a polarity analogous to pentane, hence is
suitable for extracting lipophilic substances. Considering the lipo-
philic properties of EOs, it is clear that SFE by CO2 would appear to
be the best-suited method for the extraction of EOs.
5.1. Parameters affecting the operating conditions
Most studies on SFE of EOs include investigation of the effect on
the yield of extraction of such parameters as temperature, pressure,
flow rate of fluids, size of sample, modifiers, and fractionation. The
correct configuration of these parameters has a significant impact
on extraction yield, reducing sample losses, saving energy and time,
and leading to precise results in subsequent analyses. In the next
few paragraphs, the role of these parameters will be addressed.
5.1.1. Effects of temperature
The density of CO2 decreases on an increase in temperature
when the pressure is constant, and as a result, the solvating power
of the SF is reduced. Conversely, the solute vapor pressure (vola-
tility) increases with temperature, enhancing its solubility in the
SC-CO2. In other words, higher temperatures have a dual and
contradictory effect on EO extraction. Thus, the impact of temper-
ature in the case of specific EOs is difficult to forecast. For instance,
Zhao and Zhang [32] indicated that an increase in temperature
from 40 to 60
C caused a significant increase in the yield of
extraction from Moringa oleifera leaves, while a further increase
from 60
C to 80
C resulted in only a small increase in the yield. This
effect may be explained by the fact that at a higher temperature,
although the density of the fluid decreases, the solubility of the EO
may still increase. Hence, the extraction yield under various tem-
peratures depends on a complex balance between the density of
SC-CO2 and the volatility of the EOs under the given conditions.
Also, substances such as EOs are considered to be thermolabile
compounds, thus higher temperatures might degrade their com-
ponents. As a result, it is preferable to use temperature values in the
range 35e50
C; on the other hand, the temperature should be near
the critical point of CO2 and as low as feasible without degradation.
Uquiche, Cirano and Millao [33] used a temperature range of
38e52
C to extract the EO from Leptocarpha rivularis. They found
that the yield of extraction increased from 3.27 to 5.05 g EO/100 g
dry substance when the temperature was increased at 15 MPa
constant pressure.
5.1.2. Effects of pressure
To comprehend the behavior of solute in SF and subsequently to
execute a successful analytical SFE, four parameters should be
taken into consideration: (1) the threshold pressure, which means
the pressure at which the solute partitions within the SF; (2) the
pressure at which the solute exhibits maximum solubility; (3) the
fractionation pressure, which corresponds to the pressure region
between the threshold pressure and maximum solubility pressure;
and (4) knowledge of physical characteristics of the solute, espe-
cially melting point (the majority of solutes dissolve well in their
liquid form, i.e. above their melting point).
One of the reasons for the use of SFE to extract EOs is that a high
yield can be obtained in a low pressure range (9e12 MPa) [34].
Nonetheless, higher pressures are applied to take advantage of the
impact of compression on vegetal matter, which improves mass
transfer and the release of EOs from the plant matrix. For example,
Glisic, Ivanovic, Ristic and Skala [24] demonstrated that, at tem-
peratures of 40 and 50
C, the yield of EO extraction from Salvia
 186
Table 2
The comparison of between Supercritical fluid extraction and traditional extraction methods for extracting essential oils from plants.

Plant Extraction Supercritical Temperature Pressure Flow rate Weight of Time Extraction yield Main compounds extracted in EO Reference
method fluid plant (g) of EO

M. Yousefi et al. / Trends in Analytical Chemistry 118 (2019) 182e193

Rosmarinus officinalis SFEa CO2 40
C 17.24 MPa 126.2 ± 2 mL/min 25 180 min 2.53% Camphor, eucalyptol, and b- [82]
caryophyllene
b
HD e e e e 50 90 min 2.35% Eucalyptol, camphor, borneol acetate,
and b-caryophyllene
c
SD e e e e 50 90 min 2.2% a-Pinene, octenol, eucalyptol, camphor,
and g-cadinene
Bunium persicum SFE CO2 12
C 8.3 MPa e 590 12 h 27.5% g-Terpinene, cumin aldehyde, and g- [83]
terpinen,
HD e e e e 100 8h 2.8% g-Terpinene, cumin aldehyde, a-
terpinen, and g-terpinen


Boswellia serrata SFE CO2 45 C e 2 L/min 500 0.31 ± 0.07% a-Pinene, a-thujene, b-pinene, and pino [84]
carveol
HD e e e e 300 3h 8.18 ± 0.15% a-Pinene, a-thujene, b-pinene, and pino
carveol
SD e e e e 300 3h 2.7% a-Pinene, a-thujene, b-pinene, and pino
carveol


Piper auritum SFE CO2 50 C 17.24 MPa 126.24 ± 20.83 mL/min 25 180 min 3.09 ± 0.12% 13 compounds identified (main [85]
compound, Safrole)
SD e e e e 100 2h 1.58 ± 0.05% 18 compounds identified (main
compound, Safrole)
Syzygium aromaticum SFE CO2 40
C 220 MPa 1.99 g/min 18 80 min 1.08% 29.84 ± 0.24% Eugenol [86]
Soxhlet e e e e 10 6h 1.90% 5.67 ± 0.11% Eugenol
a
Supercritical fluid extraction.
b
Hydrodistillation.
c
Steam distillation.
 M. Yousefi et al. / Trends in Analytical Chemistry 118 (2019) 182e193
officinalis by an SFE process can be increased by raising the pres-
sure. However, high pressures caused the co-extraction of com-
pounds along with the EO. As a general rule, the higher is the
pressure, the greater is the solvent power and the lower is the
extraction selectivity. Hence, when high pressures are employed, a
fractionation system containing at least two separators is needed to
isolate the EO from the other co-extracted materials. Rahimi-
Nasrabadi, Gholivand, Vatanara, Pourmohamadian, Najafabadi
and Batooli [35] observed that, considering the percentages of 1,8-
cineole and a-pinene, the best results of EO extraction from Euca-
lyptus oleosa were obtained at 10 MPa compared with 20 MPa and
30 MPa.
5.1.3. Effects of sample size and grinding
The matrix of plants has a significant effect on the quality of
extracts. Several factors related to the vegetal matrix, including
particle size, surface area, shape, porosity, etc., will affect the SFE
results. The effect of the sample matrix on an SFE process can be
interpreted at three stages: (1) EO desorption from the matrix; (2)
diffusion of fluid in the plant matrix; and (3) analyte solubilization
by the SF. EOs are usually found in intracellular spaces to a greater
degree than on the plant cell surface. Therefore, to attain sufficient
contact with the fluid, pretreatment to disrupt the cells (grinding,
comminuting) is essential, so that the efficiency of SFE is enhanced
by decreasing the mass transfer resistance. Certainly, particle size
significantly affects the process time. In general, reducing the par-
ticle size of a solid matrix results in a higher surface area, lower
mass transfer resistance, and more efficient extraction. However,
excessive grinding may delay the extraction owing to re-adsorption
of the solute on the matrix surface and a drop of pressure in the
extraction chamber. For instance, Aleksovski and Sovova [36]
demonstrated that in SFE of EO from small particles of sage
leaves, the EO was readily accessible to the SC-CO2 even under
moderate conditions (25e50
C and 9e13 MPa).
In another study of the impact of particle size on SFE perfor-
mance, Mhemdi, Rodier, Kechaou and Fages [37] showed that
decreasing the particle size of coriander seeds from 0.9 mm to
0.5 mm significantly improved the EO extraction ratio from 31% to
85%. Also, the effective diffusivity was measured at 1013 m2/s for
the 0.5 mm particle size, while for a diameter of 0.9 mm it was ten
times smaller (0.1  1013 m2/s).
5.1.4. Effects of modifier
Modifiers are co-solvents that enhance the efficiency of SFE by
increasing the solubility of solutes and extending the variety of
extractable compounds. Some common modifiers are methanol,
ethanol, 2-propanol, dichloromethane, and acetonitrile. Since SC-
CO2 is fairly nonpolar, the addition of polar modifiers is necessary to
improve the solubility of higher-weight and more polar molecules.
In addition, the use of modifiers decreases the temperature
required for the process [38]. However, modifiers transform CO2
from the supercritical to the subcritical state and introduce diffi-
culties such as the need for separation of the modifiers from the SF
after the extraction.
The main components of EOs can be assigned to two families of
hydrocarbons: phenylpropanoids and terpenoids formed by two
(monoterpene), three (sesquiterpene), or four (diterpene) isoprene
units. Both the phenylpropanoid and terpenoid families consist of
phenolic compounds, which exhibit antioxidant activity. Therefore,
in the case of addition of modifiers, the targeted substances are
antioxidants.
An interesting example in this regard is provided by the study of
Irmak, Solakyildirim, Hesenov and Erbatur [39]. They investigated
the antioxidant capacity of the EO extracted from rosemary by SC-
CO2. Their procedure consisted of three steps. First, a pressure of
187
10 MPa was applied at 40
C, and after maintenance of these con-
ditions for one hour, the extract was collected for nearly three
hours. Then the pressure value was increased to 20.6 MPa at 40
C,
and extract collection again began after one hour and continued for
approximately three hours. In the third step, 5% ethanol was added
to the remaining solid substance and the extractor was pressurized
up to 19.3 MPa at 55
C. After maintenance of these conditions for
one hour, extract collection was again carried out for three hours.
The components of the extracts were determined by GC-MS. All
extracts were found to be rich in verbenone, camphor, borneol, 1,8-
cineole, linalool, caryophyllene, and a-pinene. The use of the
ethanol modifier in the third step enabled the extraction of some
ethyl esters of acids, such as linoleic acid ethyl ester, which were
not detected in extracts 1 and 2. However, the natural presence of
linoleic ethyl ester is not very common, it could have been a reac-
tion with ethanol during extraction [40].
Leal, Maia, Carmello, Catharino, Eberlin and Meireles [41]
investigated the SFE of basil using water as modifier at different
concentrations (1%, 10% and 20%). They concluded that the yield of
extraction increases as the percentage of modifier is increased;
there was also an increase in the content of phenolic acids in the
extracted products.
5.1.5. Effects of flow rate
The velocity of the SF flowing through the plant cells has a
direct impact on the efficiency of extraction. The fluid speed is
strongly dependent on the cell geometry and the flow rate. A
decrease in the flow rate results in a reduced linear velocity. The
flow rate can be easily adjusted using a new restrictor with a
different internal diameter. A restrictor or a backpressure regu-
lator (BPR) is positioned after the extraction vessel. It allows the
depressurization of the SF and decoupling of pressure and flow to
control the pressure independently of the SF flow rate. Concerning
the effect of the flow rate on the extraction yield of EOs and their
components, Pourmortazavi and Hajimirsadeghi [1] reported that
reducing the flow rate favors an increase in the yield. However,
the impact of flow rate is more complicated than this. The effect of
flow rate on the extraction rate was well interpreted by Kumoro
and Hasan [42]. They set up an SFE process at 10 MPa and 40
C,
with a CO2 flow rate varying between 0.5 and 4 mL/min, to extract
andrographolide from Andrographis paniculata. As the flow rate
increased, the yield of extraction reached a maximum value, but
then decreased. This can be explained as a trade-off between a
thermodynamic equilibrium state and the mass transfer process
[43]. The mass transfer resistance limits the quantity of analytes
transported into the fluid at low flow rates, and the SC-CO2 exits
into the extraction vessel unsaturated. As the fluid flow rate is
increased, the mass transfer resistance falls until the exiting fluid
is saturated; consequently, the equilibrium is obtained as the
maximum yield is reached. The additional increase in the fluid
flow rate reduces the residence time, deviating the system from
the equilibrium, hence the fluid will exit into the extraction vessel
unsaturated in spite of the high mass transfer rate [43]. The reason
is that the quantity of fluid which is in excess of the quantity
required to penetrate into the cells of the leaves easily bypasses
the extractable leaves [43].
5.1.6. Effects of water and moisture content of plants
Water used as a modifier, or the moisture content of vegetal
matter, have a bilateral role in SFE. Water can help the extraction by
opening pores, swelling the matrix, and permitting the SF to better
access the analytes. For example, in the case of Origanum vulgare L.
it has been reported that the incorporation of water led to
improvement in the degree of extraction of EOs and a decrease in
the yield of waxy materials.
188 M. Yousefi et al. / Trends in Analytical Chemistry 118 (2019) 182e193

In another work, Ivanovic, Ristic and Skala [44] employed SC-

Reference
CO2 to extract the EO from Helichrysum italicum flowers having
different moisture contents of 10.5% and 28.4% (the conditions

[87]

[88]
[89]

[90]

[91]
applied were 10e20 MPa, 40
C and 60
C). Their findings showed
that the higher moisture content resulted in increased extraction

Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Candida albicans

yield of sesquiterpenes.

Escherichia coli, Staphylococcus aureus, Candida albicans, and Candida glabrata

Water enables higher recoveries of relatively polar species,
despite the fact that water is only 0.3e0.35% soluble in SC-CO2 [45].
However, an excess quantity, whether as a modifier or as the
moisture content of the plant matrix, may reduce the SFE recovery
of lower polar substances. Also, water-insoluble substances pre-
cipitate on the matrix surface, since water acts as a barrier in
transferring analytes to the SF [46]. Water can also cause restrictor
plugging owing to depressurization of the fluid. Water removal is
usually carried out by drying in an oven, freeze-drying or adding

Cryptococcus neoformans and Candida albicans

drying agents [47].

Microorganisms inhibited by the essential oil

Escherichia coli, and Pseudomonas aeruginosa

6. Antimicrobial function of essential oils extracted via

Some Aspergillus and Candida strains

supercritical fluid extraction

Plant-based antimicrobial substances are of interest since anti-

biotic resistance is becoming a serious concern to the public,
particularly in terms of nosocomial infections and food-borne dis-

Enterococcus faecali,
eases. Antimicrobial properties are among the inherent characteris-
tics of EOs. The presence of a wide spectrum of components
exhibiting antimicrobial properties, such as borneol, cinnamalde-
hyde, carvacrol, eugenol, thymol, linalool, menthol, vanillin, etc., has
caused EOs to be used as preservatives against pathogenic microor-
ganisms [48]. Therefore, the quality of extraction is one of the most
crucial factors affecting the antimicrobial properties of EOs. In many
Main constituents of the essential oil extracted

studies, SFE has been introduced as a pioneering technique for

linalool, camphor, linalyl acetate, and borneol
1,8-Cineole, cis-chrysanthenol, trans-thujone,
obtaining a high-quality EO with minimal losses of its constituents.

spathulenol, b-eudesmol, a-eudesmol, and

Dillapiol, neophytadiene, phytol, and (Z)-

muurolene, abietadien, and trans-totarol

a-Phellandrene, b-phellandrene, elemol,
Thus, the antimicrobial traits of EOs are well preserved.

Sandaracopimara-8(14)-15-diene, g-
De Corato, Maccioni, Trupo and Di Sanzo [49] studied the anti-
fungal function of laurel (Laurus nobilis) EO obtained by SC-CO2
The conditions used to extract some essential oils with supercritical CO2 and their antimicrobial activity.

against post-harvest spoilage fungi under in vivo and in vitro con-

ditions. First, they extracted the EO by the SFE method at 11 MPa,
40
C, and 24 kg/h CO2 flow rate; it was then subjected to GC
borneol, and myrtenol

analysis. The results showed that laurel oil had high contents
(10%) of 1,8-cineole, methyl eugenol, linalool, and terpineol ace-
tate, and low contents (<10%) of eugenol, linalyl acetate, b-pinene,
falcarinol

squalene

sabinene, and a-terpineol. They used the EO in vitro in concentra-

tions in the range 200e1000 mg/mL against Monilinia laxa, Botrytis
cinerea, and Penicillium digitatum. M. laxa and B. cinerea were
completely inhibited by the use of EO at both the lowest and
Supercritical fluid extraction conditions

highest concentrations. P. digitatum was only partially inhibited

even at the highest concentration. For the in vivo tests, the EO was
sprayed at concentrations of 1, 2, and 3 mg/mL onto the skin of
peaches, oranges, kiwifruits and lemons that had been artificially
45 C, 14 MPa, and 2 mL/min

inoculated with the aforementioned organisms. Strong antifungal

activity was recorded on peaches and kiwifruits when the EO was
sprayed before inoculation at 3 mg/mL. The same activity was also
9 MPa and 40 C

40
C and 9 MPa

50
C and 9 MPa

40
C and 9 MPa

found on peaches when the EO was sprayed after inoculation (76%

decay inhibition).
Ye, Dai and Hu [50] devised a study to evaluate the efficacy of
the EO of Allium cepa against food-borne and food spoilage path-

ogens, including Bacillus subtilis, Escherichia coli, Staphylococcus

aureus, Saccharomyces cerevisiae, Rhodotorula glutinis, Candida tro-
Lavandula angustifolia

picalis, Monascus purpureus, Aspergillus niger, and Aspergillus ter-

Juniperus oxycedrus
Essential oil source

Stachys yemenensis
Tanacetum vulgare
Apium nodiflorum

reus. They successfully extracted the EO with SC-CO2 at 20.6 MPa

and 40.6
C, in a time of 260 min and with a CO2 flow rate of 22 L/h.
The results of in vitro assays demonstrated MIC and MBC values of
0.18e1.80 mg/mL and 0.54e3.6 mg/mL respectively. Data on a
Table 3

number of studies related to the extraction of EOs and their anti-

microbial properties are given in Table 3.
 M. Yousefi et al. / Trends in Analytical Chemistry 118 (2019) 182e193 189

7. Antioxidant function of essential oils extracted by

Reference
supercritical fluid extraction

[92]

[13]

[93]
[94]

[95]
There is growing interest in the use of natural antioxidants from
plant sources owing to the carcinogenic properties of synthetic
antioxidants. This interest has driven researchers to extract such
bioactive compounds from raw vegetal matter. Biologically, anti-

Maximum scavenging activity (DPPH assaya) of

EC50 value (DPPH) of 32.01 mg/mL and 50.90 mg/

90.5% at 2 mg/mL (used concentrations, 0.05
oxidant compounds are described as materials which can delay or

ranging from 87.1 ± 0.23% to 92.0 ± 0.34%

Radical scavenging activities (DPPH assay)
inhibit the development of oxidation, when existing at lower

IC50b value (DPPH) of 2.36 ± 0.18 mg/mL

ethylbenzothiazoline-6-sulphonic acid)
concentration values than the oxidation analyte. There is no defi-
nition that limits the antioxidant function to a particular group of

EC50c value (DPPH) of 0.06 g/L

substances or to a definite mechanism. EOs have long been known

mL to ABTS (2,20 -azino-bis (3-

to exhibit antioxidant activity due to their terpenoid and phenolic
components [51]. However, the quality of these compounds is

Antioxidant activity
greatly influenced by their extraction methods. SC-CO2 has been
suggested as a promising alternative for the extraction of EOs
because of two properties: (1) the hydrophobic nature of CO2,

e2 mg/mL)
which is compatible with EOs; and (2) the low temperature used in
the SFE method, which is suitable for the extraction of many
components, especially thermolabile antioxidants. However, talk-
ing about the antioxidant activity of EO obtained by SFE is feasible
when fractionation does not take place. Because, as mentioned by
Vicente, García-Risco, Fornari and Reglero [52] and Ivanovi
c, Ðilas,

a-Pinene, 1,8-cineole, camphor, and verbenone

Main constituents of the essential oil extracted

acid, (Z,Z,Z)-9,12,15-octadecatrienoic acid, and

Hexadecanoic acid, (Z,Z)-9,12-octadecadienoic

r-Cymene, g-terpinene, thymol carvacrol, and

Jadranin, Vajs, Babovic, Petrovi

c and Zi zovic [53], fractionating the

hentriacontane, and 4,6-cholestadien-3-ol

extracts allows to have an antioxidant fraction and the EO.
Danh, Triet, Zhao, Mammucari and Foster [54] employed SFE
under conditions of 53.4
C, 20.72 MPa and 56.3 min to attain an

Heptacosane, stigmast-5-en-3-ol,
extraction yield of 13.2% for EO from Lavandula angustifolia. Four

Eugenol and methyl eugenol

main compounds, namely linalool, linalyl acetate, borneol, and
camphor, constituted around 80% of the total determined com-
pounds. A DPPH assay of the EO showed its antioxidant activity

Half maximal inhibitory concentration (concentration of the essential oil which shows 50% of its inhibition effect).
pentadecanoic acid

Half maximal effective concentration (concentration of the essential oil which shows 50% of its maximal effect).
to be significantly affected by time and pressure, but not
by temperature. In contrast, the ABTS [2,20 -azinobis-(3-

b-bisabolene
ethylbenzothiazoline-6-sulphonic acid)] scavenging capacity of
the oil obtained by SFE under various conditions did not display
The conditions used to extract some essential oils with supercritical CO2 and their antioxidant activity.

any significant difference. The results indicated that SFE con-

An antioxidant assay indicating the transfer of electron that creates a violet solution in ethanol.
ducted at high pressure and with a long extraction time can
produce lavender oil with high antioxidant activity and high
yield. It was found that extraction with lower parameter values
Supercritical fluid extraction conditions

313e333 K, 20e30 MPa, and 3 mL/min

(36.6
C, 10 MPa, and 73.6 min) can also produce a high yield of
45e65
C, 15e35 MPa, and 1.0 mL/min

EO, but the antioxidant activity (DPPH) is then low.

Chen, Mei, Jin, Kim, Yang and Tu [55] successfully extracted the
EO from flowers of tea (Camellia sinensis). The optimum extraction
40
C, 9 MPa, and 1.1 kg/h
37.6
C, 35 MPa, and 8 L/h

conditions were 50
C, 30 MPa, and 100 min. Based on GC-MS re-
sults, 59 compounds categorized as alkanes (45.4%), esters (10.5%),
313 K and 35.0 MPa

ketones (7.1%), terpenes (3.7%), aldehydes (3.7%), acids (2.1%), al-

cohols (1.6%), ethers (1.3%), and others (10.3%) were identified in
the EO of tea flowers. Moreover, the oil of tea flowers exhibited
somewhat stronger DPPH activity than the EOs of peppermint and
geranium, although its antioxidant activity was not stronger than
that of EO of clove, tert-butylhydroquinone, butylated hydrox-
yanisole or ascorbic acid. Table 4 presents certain data related to the
conditions used for SFE of EOs and their antioxidant activity.
Nine different species of Turkish plants

8. Experimental designs for supercritical fluid extraction

Experimental designs are used to optimize the operating con-

ditions of various procedures, and to improve the performance of
Anoectochilus roxburghii

chromatographic separation, in addition to obtaining high extrac-

Essential oil source

tion efficiency. Thus, experimental design can be regarded as an

Satureja montana

Patrinia villosa
Pimenta dioica

approach to solving problems systematically. Also, it is used to

gather and to analyze data for achieving information-rich results.
Optimum and valid results with less time, effort, and resources are
Table 4

the initial goals of applying experimental designs in analytical

a
b
c

processes.
190 M. Yousefi et al. / Trends in Analytical Chemistry 118 (2019) 182e193

Extraction by means of SF depends on certain intrinsic proper- 8.2.1. Taguchi design

ties of the SF, such as pressure and temperature, and some extrinsic The main purpose of a Taguchi design is to produce more robust
properties such as sample matrix, various environmental factors, or optimized output. The Taguchi approach utilizes an orthogonal
and interactions with the analysts. In other words, a large number array to determine experimental runs. A Taguchi orthogonal array
of variables must be carefully identified and explored in an SFE design is a kind of fractional factorial design. For example, if we
process. Therefore, the choice of an appropriate experimental have 4 factors in 3 levels, the Taguchi design proposes 9 runs (L9).
design is crucial. In general, a successful experimental design pro- Orthogonal arrays are balanced to make sure all factor levels are
cess consists of screening, optimizing, and modeling. considered equally. Thus, factors can be assessed independently of
each other in spite of the fractionality of the design. In this design,
the main effects along with two-factor interactions are considered,
8.1. Screening
but higher-order interactions are assumed to be nonexistent [60].
Analysis of variance (ANOVA), signal-to-noise analysis, and range
SFE can be affected by a huge number of parameters, but some
analysis ratio are the main analysis techniques for interpreting the
have dominant impacts on the extraction. The exploration of these
results of the designs.
principal factors is highlighted in the screening step, the first sub-
Ansari and Goodarznia [61] considered the five factors of tem-
stantial stage in the SFE experimental design process, where two-level
perature, pressure, mean particle size of sample, flow rate of SC-
full factorial, two-level fractional factorial, and PlacketteBurman de-
CO2, and dynamic extraction time to optimize the extraction of EO
signs are often utilized.
from Mentha spicata L. leaves using the Taguchi methodology. An
A two-level full factorial design includes all variables and their
L16 design was chosen, and ANOVA was applied to the experimental
levels, hence it may be valuable for screening when a small number
data. The results, at optimum conditions (45
C, 9 MPa, 500 mm size
of factors need to be studied. This design can be represented by 2n,
of feed powder, 5 mL/s flow rate, and 120 min dynamic time), gave
where n is the number of factors. Danh, Mammucari, Truong and
a maximum yield of 2.032%.
Foster [56] employed a two-level full factorial design to investigate
Based on the four factors of pressure, temperature, dynamic
the most important factor influencing the yield of EO extracted
time, and modifier volume, a four-level orthogonal test, L16 (44) was
from roots of Vetiveria zizanioides using SC-CO2. Multiple regression
selected by Xiao, Chen and Xu [62] to attain a higher yield of EO
analysis demonstrated that pressure had a major linear impact on
extraction from Marchantia convolute. The maximum yield of 4.69%
the oil extraction yield, while time and temperature had a smaller
was obtained from SFE at temperature 35
C, pressure 15 MPa, dy-
impact.
namic time 35 min, and modifier volume 40 mL.
In cases where the number of factors is high, the full factorial
In another study, performed by Barghamadi, Mehrdad, Sefidkon,
design is not utilized, owing to the required increase in experi-
Yamini and Khajeh [63], a three-level orthogonal array design with
mental runs. In these situations, a fraction of a full factorial
an L9 (34) matrix was selected to investigate the optimum values of
design is often utilized to obtain the desired information. A
temperature, pressure, modifier volume, and extraction time for
fractional factorial design with two levels can analyze f factors at
SFE of EO from Achillea millefolium L. The results of ANOVA gave an
two levels in 2nev runs (v ¼ 1,2,3, …,k), where n is the number of
extraction yield of 3.6% at 45
C, 30 MPa, 500 mL methanol, and
factors, v is the degree of fractionation. Ghasemi, Raofie and
10 min dynamic time.
Najafi [57] used a half fractional factorial design (2n1) to
discover the main factors affecting SFE of EO from Myrtus com-
8.2.2. Central composite design (CCD)
munis L. leaves. The factors selected in this experiment were
A CCD usually entails a full factorial or fractional factorial design
temperature, pressure, static and dynamic extraction time, and
with two levels, at least one central point, and additional axial or
modifier volume. It was found that pressure, volume of modifier,
star points. Both linear and quadric models can be used in this
and the interaction of dynamic extraction time and pressure
design. There are three types of CCD designs: central composite
were the most effective parameters.
inscribed (CCI), central composite face-centered (CCF), and central
The PlacketteBurman design is a widely used design to screen
composite circumscribed (CCC) [64]. CCC and CCI designs have
process variables. It is also a fractional factorial design containing
special characteristics and are rotatable. A design is considered to
two levels. A maximum of n ¼ Ne1 factors can be tested with N
be rotatable if, upon rotating the points about the central point, the
runs, where N is a multiple of 4. This design was applied by Wang
distribution moments of the design remain unchanged. CCD re-
and Wang [58] to evaluate the influence of seven factors: SC-CO2
quires a number of experiments given by N ¼ 2K þ 2k þ C0, where k
flow rate, material size, extracting pressure, extracting tempera-
is the number of factors and C0 is the number of replicates of the
ture, extracting time, separating temperature, and separating
central point.
pressure. The regression analysis indicated that extracting pressure,
A CCD was applied in a study of Danh, Truong, Mammucari and
extracting temperature, and extracting time were the most effec-
Foster [65] to evaluate the influences of temperature, pressure, and
tive factors for SFE of EO and oleoresin from anise.
concentration of added ethanol on vetiver EO extraction yield. The
Many studies omit the screening steps in the SFE process,
design included 17 runs with eight factorial points, two for ethanol
assuming that the temperature and pressure of the fluid, volume of
concentration, two for temperature, and five central point replica-
added modifier, and extraction time are normally the most
tions. The operating conditions were applied at 5 levels, except for
important factors affecting SFE performance [1,59].
pressure, which was tested at three levels owing to technical limi-
tations. The optimum conditions were determined to be 50
C,
8.2. Optimizing 19 MPa, and 15% ethanol; these produced an extraction yield of 5.9%.
Bagheri, Manap and Solati [66] conducted a study to optimize
After screening and identification of the most important factors the SFE of Piper nigrum L. EO using RSM. To obtain the maximum
in a process, these factors need to be optimized. Optimization is a yield, experiments were conducted using a three-factor CCD un-
practice of experimental design, serving to confirm the optimal der following conditions: temperature 40e50
C, pressure
conditions. Three types of designdTaguchi designs, central com- 15e30 MPa, dynamic extraction time 40e80 min. Also, a second-
posite designs (CCD) and BoxeBehnken designs (BBD)dare usually order polynomial regression model, representing the extraction
employed to optimize the SFE procedure. yield as a function of the SFE variables, produced a good fit with a
 M. Yousefi et al. / Trends in Analytical Chemistry 118 (2019) 182e193
high coefficient of determination (R2 > 0.985). The results showed
that the best extraction yield (2.16%) was obtained at 50
C,
30 MPa, and 80 min.
8.2.3. BoxeBehnken design (BBD)
This design includes a fractional factorial design formed by
combining factorials with the incomplete block design in such a
way as to appear as a rotatable or nearly rotatable design. A
BoxeBehnken design does not have any points at the vertices
formed by the lower and upper limits for each variable. BBD re-
quires a number of experiments given by N ¼ 2k(ke1) þ C0, where k
is the number of factors and C0 is the number of central points. The
significant impact of BBD on the response is usually tested by
ANOVA; also the optimal response can be found using a regression
model along with the calculation of model derivatives. Compared
with CCD, the BBD design gives smaller run numbers when the
number of factors is increased to more than 4.
Ghoreishi, Kamali, Ghaziaskar and Dadkhah [67] investigated
the optimization of SFE of linalyl acetate from lavender using a
BoxeBehnken design. They evaluated the three independent
effective variables (temperature, pressure, and number of
repetitions  extraction static time) to find the optimum conditions
to maximize the dependent variables (response) of linalyl acetate
and overall extraction yield. ANOVA proved the suitability of the
fitted quadratic model, and the R2 value for the extraction yield was
98.6%. Also, the lack-of-fit or adequacy test was significant at p-
values < 0.05, indicating the adequacy of the selected model.
In another study, Khajeh [68] extracted the EO of Diplotaenia
cachrydifolia using the SFE method. First, the impacts of different
parametersdincluding temperature, pressure, modifier volume,
and dynamic and static extraction timesdwere screened using a
fractional factorial design (252). This showed that both extraction
times had no impact on the process. Finally, the BBD was applied to
obtain the optimum values of the effective parameters. The optimal
conditions were found to be 65.6
C, 30.2 MPa, and a modifier
volume of 258.4 mL.
Xia, You, Li, Sun and Suo [69] used single-factor experimental
designs, with one independent variable (factor) and three levels, to
evaluate the effects of extracting pressure, extracting temperature,
extracting time, and the volume of modifier added. Then, a three-
variable, three-level BBD was applied to optimize the SFE condi-
tions with the aim of achieving the highest recovery of EO from
Zanthoxylum bungeanum. The optimum conditions obtained were
as follows: extraction temperature 41.19
C, extraction pressure
29.28 MPa, and quantity of modifier 10.94%. The findings indicated
that the maximum extraction yield of EO was 21.85 ± 0.23% under
the proposed settings.
8.3. Modeling of supercritical fluid extraction process
The importance of modeling in SFE is due to the difficulty of
predicting the experimental concentration profiles of analytes in
solid and fluid phases in an extraction process. Nevertheless, the
concentration profiles are easily anticipated, using SFE models,
when their parameters have been correctly fitted to the experi-
mental data. Therefore, a validated model reduces time, enabling
prediction of SFE behavior before carrying out the experiment [70].
The available models to describe the SFE of solutes such as EOs from
solid matrices are categorized into three groups: (1) empirical
models; (2) models based on heat transfer analogy; and (3) models
based on differential mass balance equations.
8.3.1. Empirical models
Empirical models of SFE usually show the direct dependence of
extraction yield or solubility of analytes in SC-CO2 on the SFE
191
parameters without presenting a complete equation. However,
they have no physical meaning. A major criticism of such modeling
is the “black box” nature of this method (providing little details of
its output), lack of extensibility to new versions of a scenario, and
the insatiable need for development data; consequently it is best
used as a compromise in conditions where a theoretical modeling
framework is not available.
In studies related to the screening, optimizing, and modeling of
EO extraction by SFs, RSM is usually used to analyze the data. In
general, RSM consists of a collection of statistical and mathematical
techniques which match experimental data to empirical models
[57]. The models used by RSM for SFE processes are usually second-
order polynomial models, which follow the equation: yield ¼ b0 þ
P P PP
bi xi þ bii x2 i þ bij xi xj þ ε, where b0 is a constant, bi , bii , and
bij are linear, quadratic, and interactive coefficients respectively, xi
and xj are independent variables in coded form, and ε denotes the
error term [71]. RSM is a valuable tool to analyze relationships
between factors (independent variables) and measured responses.
For example, the influences of four factors, namely temperature,
pressure, particle size, and dynamic extraction time, on the SFE
yield of EO from Cleome coluteoides were studied using five
different levels [71]. The RSM, based on CCD, showed that the
experimental data were adequately fitted to the quadratic poly-
nomial equation. ANOVA results showed that all factors signifi-
cantly affected the extraction yield. For example, the 3D graphic
surface showed the positive and negative effects of pressure and
temperature, respectively, on the yield of extraction. The empirical
model proposed was as follows:
Yield ¼ 0:51 þ 0:073x1 þ 0:019x2 þ 0:043x3 þ 0:071x4
 0:014x21 þ 0:0008229x22  0:027x23  0:016x24
þ 0:003563x1 x2  0:014x1 x3  0:014x1 x4
þ 0:005188x2 x3 þ 0:006813x2 x4  0:021x3 x4 ;
where x1 denotes pressure, x2 temperature, x3 particle size, and x4
dynamic extraction time. Some instances of applications of RSM are
given in Table 5.
8.3.2. Models based on heat transfer analogy and differential mass
balance equations
Models based on heat transfer analogy assume one variable
parameter, namely the effective diffusivity in the solid phase pores
[72]. By contrast, differential mass balance equation models enable
the description of all principal aspects of the process, including the
mass transfer resistances, the phase equilibrium (equal to the sol-
ubility of the analyte in the SF), and the flow pattern (the direction
of the SF passing through the matrix). Also, models based on mass
balance equations and models based on heat transfer analogy can
be considered as phenomenological models.
In the model based on heat transfer analogy, SFE is considered as
a heat transfer phenomenon. All particles of the plant are assumed
spherical, hence equations for the cooling of hot spheres in a cool
medium are applied to describe the concentration profile into the
particles as a function of time. In this model, the interactions be-
tween particles may be neglected [73]. Generally, models based on
differential mass balance equations produce better extraction
curves than those based on heat transfer analogy, perhaps because
the effect of the mass transfer resistance in the fluid phase is
satisfactorily described by the mass balance equations via mass
transfer coefficients or adjustable parameters [74].
The mass transfer resistance (internal mass transfer) is repre-
sented by three models, including diffusion, shrinking core, and
intact and broken cells (IBC) models. The so-called diffusion model
expresses the desorption-dissolution-diffusion (DDD) hypothesis.
 192 M. Yousefi et al. / Trends in Analytical Chemistry 118 (2019) 182e193

This hypothesis assumes that a part of the solute is adsorbed on the

Reference

matrix and the rest is dissolved in the SF within the inner pores of

[100]
the matrix. The desorption model includes three steps: external
[96]

[97]

[98]

[99]
mass transfer by convection via a static film of SF next to the par-
ticle, axial dispersion of dissolved analytes in the SF phase, and
R2 value

internal mass transfer by diffusion within the solid particle [75].

0.972

0.998

0.986

0.990

0.975
This model has been shown to produce a good fit in describing SFE
of EOs from Calendula officinalis [74].
Yield ¼ 3:811  0:147x1 þ 0:672x2  0:127x21

Yield ¼ 4:268 þ 0:735x1 þ 1:428x2 þ 0:238x21

þ 0:086x1 x2 þ 0:095x1 x3  0:12x2 x3

Yield ¼  8:40463 þ 0:19797x1 þ 0:07730x2
In contrast to the DDD hypothesis, the shrinking core model
Yield ¼ 0:638857 þ 0:09575x1  0:02768x2

assumes that a solute is preserved in the matrix pores by capillary

 0:016159x1 x3  0:021519x3 x4

þ 0:00171x1 x2 þ 0:00046 Tx1 x4

Yield ¼ 3:30 þ 1:22x1 þ 0:27x2 þ 0:22x3
 0:017155x24  1:012181x1 x2

or mechanical forces, such that the pore space is separated into an

 0:00314 7x23  0:00021x24

 0:60x21 þ 0:051x22  0:23x23

þ 0:07245x3 þ þ 0:04686x4
þ 1:057504x3 þ 0:102769x4
 0:017476x21  1:029750x22

inner core filled completely with condensed solute and an outer

 0:00448x21  0:00061x22
area containing the solute in SF, divided by a moving boundary [76].
 0:092x22 þ 0:085x1 x2

 0:342x22 þ 0:202x1 x2

This model is also known as a quasi-steady-state model due to the

assumption of no axial dispersion in a fixed bed [77]. The
assumption that the pores of the solid matrix must be filled with
solute has limited the application of this model to EO extraction,
Empirical model

because the content of EOs in vegetal matter is typically not suffi-

cient to fill the pores [78]. Some studies have used this model to
represent the extraction mechanism of EOs from Cinnamomum
camphora and linaloolifera fujita [79] and nutmeg [80] by SC-CO2.
The IBC hypothesis assumes that mild pretreatment such as
35
C, 21 MPa, 3.778 g/min and 160 min

grinding causes the surface of particles to be broken, while there

are intact cells in the interior. An external mass transfer coefficient
48.5
C, 10.8 MPa, 120 min static

controls transfer of the solute from the broken cells to the SF,
while an internal mass transfer (diffusive) coefficient controls
time, 24 min dynamic time
65
C, 5.5 MPa, and 35 min

transfer of the solute within the intact cells. This model has more
commonly been applied in studies related to SFE of EOs. The IBC
Optimized conditions

313 K and 30 MPa

328 K and 30 MPa

model has been successfully used for the extraction of EOs from
Persea indica [81].

9. Conclusion

Today, the extraction of EOs, due to their countless benefits, is

attracting a greater level of attention than ever before. However,
CCD [31 runs (24 factorial points, 8 axial points,

CCD [16 runs (23 factorial points, 6 axial points,

the sensitivity of many of the components of EOs limits the possi-

Full plan [18 runs (32 runs for each variable]

Full plan [18 runs (32 runs for each variable]

bility of extraction in extreme conditions. SFE, which enables the

The use of response surface methodology (RSM) in supercritical fluid extraction of essential oils.

use of milder temperatures and shorter times, has been introduced

as an advanced technique to extract EOs of high quality with
minimum losses of their components. Preservation of the major
constituents of EOs is a key advantage of SFE over other traditional
extraction methods. Many properties of EOs, such as their antimi-
and 7 central points)]

and 2 central points)]

crobial, antioxidant and anti-inflammatory activity, are dependent

BBD [15 runs (33)]
Optimizing design

on their components. It is therefore necessary to design an

appropriate and accurate SFE process. For this reason, much effort
has been made to screen and optimize the SFE conditions. Several
types of designs, including fractional factorial, PlacketteBurman,
CCD, BBD, etc., have been used with the aim of maximizing the
EO extraction yield.
P(x1 ), T(x2 ), and DTi(x3 ),

References
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Screened variables

T, P, FR, and DTi

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