J.

of Supercritical Fluids 37 (2006) 342–349

Comparison of the solubility of ␤-carotene in supercritical CO2 based

on a binary and a multicomponent complex system
Marleny D.A. Saldaña a , Li Sun a , Selma E. Guigard b , Feral Temelli a,∗
a Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alta., Canada T6G 2P5
b Department of Civil and Environmental Engineering, University of Alberta, Edmonton, Alta., Canada T6G 2P5

Received 10 August 2005; received in revised form 1 December 2005; accepted 5 January 2006

Abstract
Carotenoids such as ␤-carotene are gaining interest in the food industry due to their nutritional and antioxidant properties. Understanding the
solubility behavior of carotenoids in supercritical CO2 (SC CO2 ) is fundamental for any industrial supercritical process application and design.
Solubility of ␤-carotene was measured in both a binary and a multicomponent complex system. Solubility of ␤-carotene in the binary system was
measured using a quartz crystal microbalance technique at temperatures of 40 and 50 ◦ C and pressures ranging from 120 to 200 bar. Solubility of
␤-carotene in the multicomponent complex system was determined from dynamic extraction experiments using a laboratory-scale supercritical
extraction system. Carotenoids were extracted from freeze-dried carrots with SC CO2 at temperatures of 40 and 50 ◦ C and pressures ranging
from 120 to 327 bar. ␤-Carotene solubility values for the binary system measured herein and reported in the literature are of the order of 10−7
mole fraction while the solubility values for the multicomponent complex system (␤-carotene extracted from carrots with SC CO2 ) under the
same conditions are 5–10 times smaller. Solubility in both systems increased with temperature and pressure. The difference in the solubility values
obtained using both systems is mainly due to the matrix effects of the multicomponent complex system such as the cell structure and the interactions
of ␤-carotene with other components such as carbohydrates in the carrot matrix.
© 2006 Elsevier B.V. All rights reserved.

Keywords: ␤-Carotene; Carotenoids; Carrots; Piezoelectric quartz crystal; Solubility; Supercritical fluid extraction

1. Introduction critical temperature of CO2 is beneficial for thermally labile

The demand for “natural” food colorants such as carotenoids
is growing due to consumer concerns for food safety and quality.
Carotenoids also possess provitamin A activity and antioxidant
activity, which could lead to reduced cancer risk [1]. Carrots are
a major source of carotenoids, containing 60–80%, 10–40% and
1–5% ␤-carotene, ␣-carotene and lutein, respectively [2].
To date, two principal extraction methods have been used to
extract carotenoids from carrots: (i) traditional solvent extraction
(TSE) [3] and, (ii) supercritical fluid extraction (SFE) [4–8].
TSE requires long extraction times, consumes large amounts of
organic solvents and there may be solvent residues left in the
extracted products after the solvent removal step. SFE on the
other hand uses supercritical carbon dioxide (SC CO2 ), which
reduces potential for oxidation of carotenoids and there is no
solvent residue left in the extracted product. In addition, the low
∗ Corresponding author. Tel.: +1 780 492 3829; fax: +1 780 492 8914.
E-mail address: Feral.Temelli@ualberta.ca (F. Temelli).
carotenoid, resulting in minimal carotenoids degradation during
extraction.
Understanding the solubility behavior of ␤-carotene in SC
CO2 is essential for developing different applications at an indus-
trial scale. To date, solubility data reported in the literature for
pure ␤-carotene in SC CO2 show large discrepancies, with sol-
ubilities ranging over more than one order of magnitude. This
discrepancy can be, in part, attributed to the different experi-
mental techniques used to measure ␤-carotene solubility in SC
CO2 , such as the dynamic [9–11] or circulation [12] methods.
Some of these techniques present deficiencies in sample collec-
tion especially for solutes of low solubility such as ␤-carotene.
Correct solubility measurements rely on equilibrium conditions
being attained but this is difficult to confirm with some tech-
niques [13]. The piezoelectric quartz crystal or quartz crystal
microbalance (QCM) technique can be used for measuring sol-
ubilities in situ for compounds that exhibit low solubilities or
that are thermally labile. However, the QCM technique is very
sensitive and may sometimes be subject to electrical or mechan-
ical interferences.

0896-8446/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.supflu.2006.01.010
 M.D.A. Saldaña et al. / J. of Supercritical Fluids 37 (2006) 342–349
Sangbin and Mi [6] used dynamic extractions to deter-
mine solubility of ␤-carotene from freeze-dried carrots (Dancus
carota L.) by SC CO2 and mixtures of CO2 and ethanol or
methanol at temperatures of 40 and 60 ◦ C and at pressures
of 138–276 bar. The highest solubility achieved was 4.90 and
0.60 ␮g/g CO2 for ␤- and ␣-carotene, respectively, at 40 ◦ C and
276 bar. Other studies [7,8] have investigated the extraction of
␤-carotene from carrots using SC CO2 ; however, no solubility
data were reported.
The main objective of this study was to compare the sol-
ubility of ␤-carotene in SC CO2 obtained using two different
techniques. First, a piezoelectric quartz crystal or QCM tech-
nique was used to determine the solubility of pure ␤-carotene
in a binary system (␤-carotene + SC CO2 ). The second method
was based on the dynamic extraction of ␤-carotene from car-
rots using SC CO2 . The solubility measured using this second
method is representative of the solubility of ␤-carotene in a mul-
ticomponent complex system (␤-carotene + carrot + SC CO2 ). In
addition, the extent of degradation of ␤-carotene during solubil-
ity measurements was also evaluated.
2. Materials and methods
2.1. Materials
␤-Carotene (99.9% purity), mixed isomers ∼2:1 ␤-:␣-
carotene (95% purity) and lutein (∼70% purity) standards were
purchased from Fluka–Sigma Aldrich Co. (St. Louis, MO).
Supercritical fluid chromatography grade CO2 (99.99% purity),
CO2 bone dry (99.8% purity) and nitrogen (99.95% purity)
were purchased from Praxair Canada Inc. (Mississauga, ON,
Canada). Solvents such as hexane, acetone, methanol, chloro-
form, dichloromethane and acetonitrile, all HPLC grade, were
obtained from Fisher Scientific (Fair Lawn, NJ). Polished quartz
crystals (9 MHz AT-cut) with wrap-around gold-plated elec-
trodes were purchased from International Crystal Manufacturing
Co., Inc. (Oklahoma City, OK). The properties of the crystal
are: area of 1.96 × 10−5 m2 , density of 2649 kg/m3 and stiffness
of 2.90 × 1010 N/m2 . A Research Quartz Crystal Microbalance
(RQCM) was purchased from Maxtek (Santa Fe Springs, CA)
and was used as both the oscillator and frequency counter.
Carrots, Apache variety, were provided by I&S Food Ser-
vices Ltd. (Edmonton, AB, Canada). The carrots were washed
and chopped into 5 mm cubes at room temperature with a food
chopper. After being well mixed, the carrots were freeze-dried
for 5 days until a moisture content of ca. 0.8% was reached.
The cubes were then ground by a Quadro Comil grinder and
sieved until a desired particle size distribution (0.5–1 mm) was
achieved. All samples were vacuum packed in several moisture
and O2 -barrier bags and stored at −18 ◦ C in dark until use in
extractions.
2.2. Solubility measurements using the QCM technique
2.2.1. QCM and high-pressure apparatus
The apparatus (Fig. 1) consists of two ISCO 500D syringe
pumps from ISCO Inc. (Lincoln, NE), an Isotemp 3013 refrig-
343
Fig. 1. High-pressure QCM apparatus. 1: CO2 cylinder; 2: filter; 3: valve; 4:
cooler; 5: ISCO syringe pumps; 6: check valve; 7: pressure transducer; 8: pres-
sure relief valve; 9: extraction vessel; 10: quartz crystal holder; 11: quartz crystal;
12: spring-loaded contacts; 13: thermistor; 14: water bath; 15: digital tempera-
ture indicator; 16: thermometer; 17: vent; 18: RQCM; 19: computer with data
acquisition system.
erated circulating water bath and a heated water bath from
Fisher Scientific (Pittsburgh, PA), and a stainless steel high-
pressure vessel from Frontier Valve International (Edmonton,
AB, Canada). The temperature in the high-pressure vessel was
registered by a YSI 406 thermistor (Dayton, OH) and a ther-
mometer in the water bath indicated the temperature of the sys-
tem. A pressure transducer (Omega, Stamford, CT), connected
to the inlet tube, monitored the pressure of the high-pressure ves-
sel. Data from the pump (pressure and flow rate), the thermistor
and the pressure transducer were collected by a LabViewTM data
acquisition system.
2.2.2. Loading β-carotene on the crystal and solubility
determination
␤-Carotene was dissolved in chloroform to obtain a solu-
tion of known concentration (10 mg/mL). An aliquot (1 ␮L) was
placed on the quartz crystal electrode area. To obtain a uni-
formly distributed layer of ␤-carotene, the solvent was slowly
evaporated under gentle nitrogen flow. The crystal frequency
was recorded before and after loading to ensure that the fre-
quency shift was in agreement with the amount of solute placed
on the crystal. After loading, the crystal was placed in the high-
pressure vessel, which was in turn connected to the pumps and
placed into a heated water bath. After 30 min, the inlet valve was
opened and CO2 was introduced. The pressure transducer and
thermistor monitored the pressure and temperature in the vessel,
respectively. The frequency of the quartz crystal was recorded
every 10 s throughout the experiment. The experiment was con-
tinued until the frequency reading remained constant (±5 Hz).
At least two replicates were performed for each experimental
condition. Experiments were conducted at temperatures of 40
and 50 ◦ C and pressures ranging from 120 to 200 bar.
2.2.3. Theory
Using the QCM technique for solubility measurements in
supercritical fluids (SCFs) involves placing a known mass of a
solute on the surface of the electrode area. As the solute dis-
solves, the mass change on the surface of the crystal results in
344 M.D.A. Saldaña et al. / J. of Supercritical Fluids 37 (2006) 342–349
a frequency change according to the well established Sauerbrey
equation (Eq. (1)) [14]. The mass change (m, kg) due to solute
deposition onto the crystal can be calculated by the change in
frequency (F, Hz) and by knowing the other constants such as
the resonant frequency of unloaded crystal (F0 , Hz), the active
vibrating area (A, m2 ), the quartz crystal density (ρq , kg/m3 ),
and the quartz crystal stiffness (µq , N/m2 ). This mass (m) was
then used to calculate the solubility of ␤-carotene in SC CO2 in
the binary system.
2F 2 m
F = − √0
A µq ρ q
2.3. Solubility measurement using dynamic extraction of
carrots
2.3.1. Supercritical fluid extraction
A laboratory-scale SFE system equipped with a 300 mL
extraction cell (Newport Scientific Inc., Jessup, MD) was used
as described previously [15]. The extraction temperature was
monitored by a thermocouple immersed in the centre of the
extractor. A stainless steel basket (15 cm × 13 mm i.d.) was fit-
ted into the extraction vessel for easy loading and unloading of
the dried carrot sample (2 g). CO2 was compressed to the desired
pressure using a diaphragm compressor and the extraction pres-
sure was controlled by a back-pressure regulator. CO2 flow rate
(1.2 L/min, measured at ambient conditions) was controlled by
a micrometering valve. Each extraction run was continued for
8 h. Extract fractions were collected every 60 min in side-armed
glass tubes attached to the depressurization valve, which were
held in a refrigerated circulating bath at −20 ◦ C and weighed.
The extracted material was transferred into a pre-weighed glass
vial by washing the collection tubes with hexane, which was
then evaporated under gentle nitrogen flow. The samples were
weighed and stored at −18 ◦ C until HPLC analysis. At least two
replicates were carried out for all the experiments. Experiments
were conducted at temperatures of 40 and 50 ◦ C and pressures
ranging from 120 to 327 bar.
2.3.2. Soxhlet extraction
Soxhlet extraction of freeze-dried carrots was carried out to
determine the initial amount of carotenoids present in the car-
rots. Two grams of freeze-dried carrots and 50 mL hexane were
placed in a Soxhlet apparatus and refluxed for 4 h. The hex-
ane was removed and the extract was dried under a gentle flow
of nitrogen. Then, the dried sample was diluted using 5 mL
methanol:dichloromethane (1:1 v/v) and 100 ␮L of this solu-
tion was mixed with 400 ␮L methanol:dicholoromathane prior
to HPLC injection. Three replications were performed with a
standard deviation of less than 6%.
2.3.3. High performance liquid chromatography (HPLC)
analysis
Carotenoids identification and quantification in the extract
samples were performed according to Mendes et al. [11]
using an HPLC system (Shimadzu Scientific Instruments Inc.,
Columbia, MD) equipped with a SupelcosilTM LC-18 column
(15 cm × 4.6 cm × 5 ␮m) (Supelco Inc., Bellefonte, PA). Sam-
ples (50 ␮L) dissolved in methanol/dichloromethane (1:1 v/v)
were injected. The mobile phase consisted of methanol with
10% (v/v) acetonitrile at a flow rate of 1 mL/min at isocratic con-
ditions. The separated lutein, ␣-carotene and ␤-carotene peaks
were monitored by a UV detector at 450 nm. ␤-Carotene, ␣-
carotene and lutein standards were used for identification and
quantification based on their respective calibration curves.
In an effort to determine the ease of degradation of
carotenoids, a separate experiment was conducted where the
carrot extract obtained by SFE was subjected to heat treatment
(1)
followed by HPLC analysis. Two different samples collected
after SFE (0.050 and 0.042 g) were diluted in 10 mL hexane. An
aliquot (1 mL) was placed in a tube and heated at 70 ◦ C for ∼2 h.
Once the solvent was evaporated, 1 mL methanol:methylene
chloride (45:55 v/v) was added followed by HPLC analysis.
The chromatographic conditions used for the separation and
identification of isomers of carotenoids were adopted from
Chen et al. [2] and Chen and Tang [16], who employed a
mobile phase of methanol:methylene chloride (99:1 v/v) with
methanol:methylene chloride (45:55 v/v) as sample solvent and
a polymeric Vydac 201TP54 column (Separations Group, Hes-
peria, CA). A total of eight carotenoids were identified in
the heat-treated carrot extracts: trans lutein, 9-cis-lutein, 13-
cis-␣-carotene, 13,15-␤-carotene, trans ␣-carotene, 15-cis-␣-
carotene, trans ␤-carotene and 13-cis-␤-carotene, which agree
with the retention times presented by Chen and Tang [16].
2.4. Statistical analysis
All experiments were carried out in duplicate and means
were reported. Analysis of variance of results was performed
using the general linear model (GLM) procedure of SAS Sta-
tistical Software, version 8 (SAS Institute Inc., Cary, NC). The
model consisted of the main effects of pressure and temperature
and their interaction effect. Multiple comparison of means was
carried out using Tukey’s Studentized Range Test at a level of
significance of α = 0.05.
3. Results and discussion
3.1. Pure β-carotene solubility by the QCM technique
For each QCM experiment, the resonant frequency (F0 ) of the
9 MHz quartz crystal was measured. The average resonant fre-
quency was 8,993,155 Hz with a standard deviation of ±77 Hz
based on five measurements. Such low standard deviation was
observed in all the resonant frequency measurements for five
different crystals.
Table 1 shows the results of frequency change due to the
loading of 1 ␮L ␤-carotene solution on the surface of the quartz
crystal. The overall frequency shift (F) was used in the Sauer-
brey equation (Eq. (1)) to calculate the total mass change (m)
on the surface of the electrode. This mass was then compared
to the mass that was loaded onto the QCM with the syringe as
shown in Table 1. The mass placed on the crystal as calculated
by the Sauerbrey equation (Eq. (1)) was in accordance with the
 M.D.A. Saldaña et al. / J. of Supercritical Fluids 37 (2006) 342–349 345

Table 1
Mass calculation using the Sauerbrey equation
Mass based on solution volume loaded Mass based on Sauerbrey equation

Volume (mL) 1.0 × 10−3 F0 (Hz) 8993155

Concentration (mg/mL) 10.0 F (Hz) −9894.0
Theoretical mass (mg) 1.0 × 10−2 Sauerbrey mass (mg) 1.05 × 10−2

Error between mass values determined by two techniques = 5.0%.

Fig. 2. Frequency and pressure change in a solubility experiment of ␤-carotene

at 150 bar and 40 ◦ C.
mass delivered by the syringe. The small error (5%) between the
two methods was typical for the numerous runs performed and
was within experimental error.
Fig. 2 depicts the frequency and pressure change versus time
during a ␤-carotene solubility experiment at 40 ◦ C and 150 bar.
For the first 10 min, the frequency reading was at 25 ◦ C and
10 bar. Then, the temperature was increased to 40 ◦ C and at
20 min the pressure was increased to 150 bar. This pressuriza-
tion leads to a frequency drop, which can be attributed to: (i) the
change of CO2 properties and state, and (ii) to the CO2 adsorp-
tion onto the quartz crystal and the surface of the solute. The
frequency then begins to increase due to the dissolution and dif-
fusion of ␤-carotene into SC CO2 , resulting in a loss of mass
from the crystal surface. From 20 to 80 min, dissolution is rapid
due to a large driving force [17]. As equilibrium is approached,
less solute dissolves and the frequency stabilizes.
Solubility data for ␤-carotene in SC CO2 obtained in this
study at 40 and 50 ◦ C and different pressures are presented in
Fig. 3. Solubility of ␤-carotene at 40 ◦ C and different pressures in the binary
system (␤-carotene + SC CO2 ).
Table 2. Fig. 3 compares the solubility data from Table 2 to those
reported in the literature. A large discrepancy between solubil-
ity data can be observed at higher pressures. For example, at
120 bar, the solubility varies from 0.1 to 4.3 × 10−7 while at
200 bar, this variation is from 0.2 to 6.7 × 10−7 . This discrep-
ancy can be in part attributed to the differences in the purities
of the ␤-carotene and CO2 used and to the different experimen-
tal techniques used by the different researchers to obtain the
reported solubilities. Sakaki [9], for example, used the dynamic
technique while Škerget et al. [18] used the static technique and
Cygnarowicz et al. [12] used the circulation technique. More-
over, higher solubility values were obtained in this study for
all the pressures investigated when compared to the literature
data [9–12,18–21] (Fig. 3). Lower solubility values reported in
the literature might be attributed to the limitations of the differ-
ent experimental techniques such as the lack of a high-pressure
window on the extraction vessel to observe equilibrium, lack

Table 2
Solubility of ␤-carotene in SC CO2 in binary and complex systems at different conditions
Pressure (bar) Temperature (◦ C) Density (g/mL) Solubility (mole fraction)

Binary systema Complex systemb

120 40 0.718 4.3 × 10−7 ± 0.4 2.4 × 10−8 ± 1.1

150 40 0.780 5.8 × 10−7 ± 0.5 5.6 × 10−8 ± 2.9
200 40 0.840 6.7 × 10−7 ± 0.1 13 × 10−8 ± 3.7
120 50 0.585 7.2 × 10−7 ± 1.4 7.0 × 10−8 ± 0.8
150 50 0.700 7.5 × 10−7 ± 0.3 13 × 10−8 ± 0.6
200 50 0.784 9.4 × 10−7 ± 0.9 24 × 10−8 ± 5.0
a Solubility of pure ␤-carotene in SC CO2 by QCM (mean ± standard deviation).
b Solubility determined by dynamic extraction from carrots with SC CO2 (mean ± standard deviation).
346 M.D.A. Saldaña et al. / J. of Supercritical Fluids 37 (2006) 342–349
of attainment of equilibrium, ␤-carotene loss during depressur-
ization, and ␤-carotene loss due to precipitation inside valves
and tubing. Although, precipitated ␤-carotene can be recovered
by washing the tubes with an appropriate solvent; this step is
not implemented in all studies. Another possible source of error
might be off-line sampling protocols, which involve collection
of the ␤-carotene in a cooled flask or tube, containing a sol-
vent or sorbent trap followed by gravimetric, chromatographic or
spectrophotometric analysis. With on-line sampling, solubility is
measured, prior to the depressurization, using chromatographic
or spectrophotometric analysis [22], eliminating ␤-carotene loss
and additional sample handling. However, saturation of the UV
detectors at high concentrations should not be overlooked when
on-line sampling is used [11].
On the other hand, with the QCM technique, some of these
sources of error and ␤-carotene losses could be avoided, result-
ing in higher solubility values. However, this technique also
has some drawbacks: the preparation and loading of the sam-
ple onto the crystal can be very challenging in addition to the
system being highly sensitive to small changes in electrical cur-
rent, any air flow or motion in the surroundings and any other
interference occurring in the same room or even next door. As
well, the form in which ␤-carotene crystallizes on the quartz
crystal upon evaporation of the solvent might influence its solu-
bility. For example, Sakaki [9] noted that amorphous ␤-carotene
impregnated within alumina beads was more soluble in SC CO2
than crystalline ␤-carotene. In this study, the morphology of the
␤-carotene deposited on quartz crystal was examined by scan-
ning electron microscopy (SEM). The crystal morphology was
uniformly irregular orthorhombic, similar to that observed by
Cocero and Ferrero [23]. This crystal morphology of ␤-carotene
as well as the even spreading of the solution on the electrode sur-
face might contribute to the variation of the mass reading from
the QCM. All of the above-mentioned factors might contribute to
the somewhat larger standard deviations under some conditions
(Fig. 3), especially for a compound of low solubility (×10−7 )
such as ␤-carotene.
Analysis of variance results showed a significant (p ≤ 0.5)
pressure and temperature effect but their interaction was not
significant (p > 0.5). The solubility of ␤-carotene in SC CO2
in the binary system increased (p ≤ 0.5) with temperature from
40 to 50 ◦ C (Table 2). Solubility increased significantly (p ≤ 0.5)
with pressure from 150 to 200 bar; however, solubility at 150 bar
was similar (p > 0.5) to that at 120 bar as well as that at 200 bar.
3.2. Solubility of β-carotene in the multicomponent
complex system by dynamic extraction of carrots
Dynamic extraction of carrots was used to determine the
solubility of ␤-carotene in a multicomponent complex system
(carrot + SC CO2 ). Dynamic extraction data (in particular, the
mass of carotenoids extracted as a function of the mass of SC
CO2 ) can be used to determine the solubility provided that two
conditions are met: (i) that sufficient ␤-carotene is present to
saturate SC CO2 and reach the solubility limit, and (ii) that
the SC CO2 flow rate is sufficiently low to allow equilibrium
to be reached. In order to ensure that the first condition was
satisfied, the amount of ␤-carotene present in the freeze-dried
carrot sample was compared to an estimate of the amount of
␤-carotene needed to satisfy the solubility limit. Based on the
initial ␤-carotene content of the freeze-dried carrots (1832 ␮g ␤-
carotene/g dry carrot, as determined by Soxhlet extraction) and
assuming a solubility of the order of 10−6 mole fraction (based
on the highest level of binary solubility obtained by QCM in this
study, Table 2), the calculation reveals that sufficient ␤-carotene
exists to saturate approximately 300 g of CO2 . Thus, the slope of
the initial linear portion of the dynamic extraction curve is used
for solubility calculations. The second condition of low flow rate
must also be met in order to use dynamic extraction data to cal-
culate solubility. Dynamic extraction techniques use flow rates
that vary from 0.04 to 0.4 L/min in the literature [9,11,21,24].
However, Subra et al. [8] extracted ␤-carotene from 1 to 2.5 g
freeze-dried carrots and studied the effect of higher flow rates
(0.4 and 1.2 L/min). Subra et al. [8] observed higher yields of
␤-carotene at a flow rate of 1.2 L/min. Sun and Temelli [25] also
evaluated the effect of flow rate (0.5 and 1.0 L/min) on the extrac-
tion of ␤-carotene with SC CO2 + canola oil as a co-solvent. The
solubility controlled region of the extraction curves for the two
flow rates tested overlapped in a single line, indicating that sol-
ubility limit was reached. Dunford et al. [26] also found that the
solubility controlled region of the extraction curves for mackerel
oil using SC CO2 overlapped in a single line, indicating that the
flow rate in the range of 0.5–1.4 L/min had no effect on solu-
bility. Roy et al. [27] reported the extraction rates of oil from
tomato seeds with SC CO2 and observed that using CO2 flow
rates of 0.8–3.3 L/min resulted in a similar slope in the initial
part of the extraction curve. Therefore, the flow rate of 1.2 L/min
was chosen for this study and it was assumed that equilibrium
was indeed reached.
Fig. 4 shows the amounts of ␤-carotene extracted using SC
CO2 at temperatures of 40 and 50 ◦ C and different pressures
as a function of the mass of CO2 used. The extraction yields
of ␤-carotene obtained at the end of 8 h extraction increased
with temperature and pressure. Solubility of ␤-carotene was cal-
culated from the initial linear portion of the extraction curves
as reported in Table 2. Solubility of ␤-carotene in the com-
plex system ranged from 2.4 to 24 × 10−8 mole fraction. Sim-
ilar to the binary system, the solubility of ␤-carotene in the
complex system was also significantly (p ≤ 0.05) affected by
temperature and pressure but the pressure × temperature inter-
action effect was not significant (p > 0.05). Solubility increased
(p ≤ 0.05) with pressure from 150 to 200 bar, but solubilities at
120 and 150 bar were similar (p > 0.05). As well, the solubility
increased significantly (p ≤ 0.05) with temperature from 40 to
50 ◦ C.
Fig. 5 shows the comparison of extraction curves of ␣-,
␤-carotene and lutein at 40 and 50 ◦ C and different pressures
after 8 h. Higher amounts of ␤-carotene followed by ␣-carotene
and lutein were obtained. The quantities of ␣- and ␤-carotene
extracted by SC CO2 were one order of magnitude higher than
those of lutein. This difference is a reflection of the concentra-
tions of these carotenoids in the carrots. These results suggest
the possibility that lutein can be separated easily from the extract
by changing operating conditions.
 M.D.A. Saldaña et al. / J. of Supercritical Fluids 37 (2006) 342–349
Fig. 4. Extraction of ␤-carotene from carrots at different pressures and (A) at
40 ◦ C and (B) at 50 ◦ C.
As with ␤-carotene, the solubilities of ␣-carotene and lutein
can be calculated from the dynamic extraction data presented in
Fig. 5. This calculation yields solubility values of the order of
10−8 mole fraction for ␣-carotene and 10−9 mole fraction for
lutein. The polar nature of lutein results in a lower solubility
in SC CO2 . Both data should be used with caution since it is
unknown if sufficient ␣-carotene or lutein is present to satisfy
solubility. Scarce (if any) solubility data are currently available
in the literature for either of these compounds.
The extent of degradation of ␤-carotene through oxidation or
isomerization during SFE is a controversial topic in the literature,
especially when reporting solubility data for ␤-carotene. Chang
and Randolph [28], for example, reported that ␤-carotene was
oxidized by CO2 , producing epoxide compounds. However, Jay
et al. [29] did not find any evidence of ␤-carotene oxidation using
CO2 as a solvent and Cocero et al. [30] affirmed that this reac-
tion is not possible from a chemical view point. Isomerization
of ␤-carotene (forming mainly cis isomers) during SFE [31] and
solubility measurements [32] were also reported. Besides, sol-
vent traps such as hexane used to collect the extracted material
have also been reported to cause degradation through isomer-
ization [11]. There are also reports that some isomers are more
347
Fig. 5. Extraction of carotenoids from carrots at different pressures and (A) at
40 ◦ C and (B) at 50 ◦ C. 1: ␤-carotene, 2: ␣-carotene, 3: lutein.
soluble than others, affecting the solubility behavior, for exam-
ple, cis isomers of ␤-carotene from a matrix of algae are found
to be more soluble than trans isomers of ␤-carotene in SC CO2
[24].
In this study, isomerization of trans-␤-carotene to cis-␤-
carotene was not observed, even for samples that were cov-
ered with aluminum foil and stored for more than 6 months
at 4 ◦ C prior to HPLC analysis. Furthermore, deliberate iso-
merization of ␤-carotene was attempted in SC CO2 extracts.
Isomerization was not possible at temperatures less than 70 ◦ C
but was observed at 70 ◦ C in approximately 2 h. Fig. 6A
shows a typical HPLC chromatogram of a carrot extract
obtained after SFE before isomerization in which three main
peaks are identified (compounds a, e and g) for lutein, ␣-
carotene and ␤-carotene at 4, 13.4 and 15.3 min, respectively.
A small quantity of lutein (∼0.35 ␮g/g) was present while
␣- and ␤-carotene were in appreciable amounts (∼16.27 and
∼33.39 ␮g/g, respectively). In Fig. 6B, eight major peaks were
identified (compounds a–h) at 4, 5.9, 11.9, 13.2, 14.5, 16.1,
16.7 and 19.1 min, respectively. Compounds f, h and d were the
main isomers formed while compound a increased considerably
348 M.D.A. Saldaña et al. / J. of Supercritical Fluids 37 (2006) 342–349
Fig. 6. Typical HPLC chromatograms of carrot extracts from SC CO2 extraction:
(A) before isomerization: (a) trans-lutein, (e) trans-␣-carotene, (g) trans-␤-
carotene, and (B) after isomerization at high temperature: (a) trans-lutein, (b)
9-cis-lutein, (c) 13-cis-␣-carotene, (d) 13,15-␤-carotene, (e) trans-␣-carotene,
(f) 15-cis-␣-carotene, (g) trans-␤-carotene and (h) 13-cis-␤-carotene.
after isomerization. A total of eight carotenoids, trans-lutein
(a, ∼2.73 ␮g/g), 9-cis-lutein (b, ∼0.51 ␮g/g), 13-cis-␣-carotene
(c, ∼0.02 ␮g/g), 13,15-␤-carotene (d, ∼2.42 ␮g/g), trans-␣-
carotene (e, ∼17.93 ␮g/g), 15-cis-␣-carotene (f, ∼5.47 ␮g/g),
trans-␤-carotene (g, ∼18.95 ␮g/g) and 13-cis-␤-carotene (h,
∼1.99 ␮g/g), were found in the carrot extract following heat
treatment. To avoid degradation of ␤-carotene during the extrac-
tion process, analysis and storage, exposure of the samples to
oxygen, heat and light should be avoided or minimized. Exper-
imental precautions can minimize sample degradation such as
the use of nitrogen for drying the sample, the use of dark colored
vials or covering sample vials with aluminum foil for storage,
the use of a fresh batch of sample for each single data point to
avoid accumulation of ␤-carotene isomers [22] and maintaining
the collection flasks at a low temperature.
3.3. Comparison of β-carotene solubilities obtained by
QCM and SFE of carrots
The solubility of ␤-carotene determined by dynamic extrac-
tion of carrots was compared to the solubility determined by the
QCM method. This comparison is essential for a better under-
standing of the effects of the solid matrix of natural materials,
the effects of the location of carotenoids within the matrix and
the effects of interactions of carotenoids with other matrix com-
ponents on the solubility and extraction of carotenoids from
natural materials such as carrots. Table 2 shows that the sol-
ubility in the binary system was 4.3 × 10−7 mole fraction at
120 bar and 40 ◦ C while the solubility based on the extraction of
the multicomponent complex system was 2.4 × 10−8 mole frac-
tion. Similar trends of an overall increase in solubility with both
temperature and pressure were observed for both systems. There
was a 5–10 times difference between the solubilities obtained
for the two systems. This difference can be attributed to the size
of the carrot particles and the extent of the rupture of the cellular
structure in which the ␤-carotene is present freely or complexed
with other components. As reported by Holland et al. [33], car-
rots mainly contain carbohydrates (6 g/100 g of carrot) some
of which is in the form of fiber (2.4 g/100 g of carrot). Freeze-
dried carrots are multicomponent mixtures where the presence
of other compounds such as carbohydrates may affect the sol-
ubility behavior of the ␤-carotene, which is in part not freely
available for solubilization in SC CO2 .
4. Conclusions
The simple QCM static technique provided in situ binary
solubility data for ␤-carotene in SC CO2 . The QCM technique
allows the diffusion, dissolution and kinetics of the process to be
monitored, thus ensuring that equilibrium is reached. Further-
more, some errors are eliminated with this method, as there is no
need for sample collection after the process. Solubility values
for the binary system were in the range of 4.3–9.4 × 10−7 mole
fraction while solubility values for the multicomponent com-
plex system (␤-carotene extracted from carrots with SC CO2 ) at
the same process conditions were in the range of 2.4–24 × 10−8
mole fraction. This difference can be attributed to the formation
of complexes of ␤-carotene with other components in the car-
rot matrix and to the free availability of ␤-carotene within the
internal cell structure for solubilization into the SC CO2 . Pre-
liminary solubility data for ␣-carotene and lutein of the order of
10−8 and 10−9 (mole fraction), respectively, were also obtained
for the complex multicomponent system; however, more exper-
iments are needed to ensure that sufficient carotenoid is present
to satisfy solubility.
Acknowledgements
The authors would like to acknowledge the Natural Sci-
ences and Engineering Council of Canada (NSERC), the Alberta
Agricultural Research Institute (AARI), Alberta Crop Industry
Development Fund (ACIDF), AVAC Ltd. and AFMNet-Canada
for funding this project.
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