J.

of Supercritical Fluids 47 (2008) 259–269

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

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

Precipitation of ␤-carotene and PHBV and co-precipitation

from SEDS technique using supercritical CO2
Elton Franceschi a , Alana M. De Cesaro a , Mirian Feiten a , Sandra R.S. Ferreira b , Cláudio Dariva c ,
Marcos H. Kunita d , Adley F. Rubira d , Edvani C. Muniz d , Marcos L. Corazza a , J. Vladimir Oliveira a,∗
a Department of Food Engineering, URI-Campus de Erechim, Av. Sete de Setembro, 1621 Erechim, RS 99700-000, Brazil
b EQA-CTC/UFSC, Chemical Engineering and Food Engineering Department, Federal University of Santa Catarina, C.P. 476, CEP 88040-900, Florianópolis, SC, Brazil
c Instituto de Pesquisa e Tecnologia-ITP, Programa de Mestrado em Engenharia de Processos PEP/UNIT, Campus Farolândia, Av. Murilo Dantas, 300 Aracaju, SE 49032-490, Brazil
d Department of Chemistry, Universidade Estadual de Maringá, Av. Colombo 5790, 87020-900 Maringá, PR, Brazil

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

Article history: The objective of this work was to investigate the application of the solution enhanced dispersion by
Received 15 April 2008 supercritical fluids (SEDS) technique for the precipitation of pure ␤-carotene and copolymer poly(3-
Received in revised form 31 July 2008 hydroxybutirate-co-hydroxyvalerate) (PHBV), as well as some encapsulation tests of the solute in the
Accepted 1 August 2008
biopolymer. The following parameters were investigated in the precipitation of pure ␤-carotene: pressure
(8.0 and 12.0 MPa), anti-solvent flow rate (20–40 mL/min) and concentration of ␤-carotene in an organic
Keywords:
dichloromethane solution (4 and 8 mg/mL). For pure ␤-carotene, the results showed that the mean particle
Encapsulation
size varied from 3.8 to 246.8 ␮m, depending on the processing conditions. The morphology of ␤-carotene
SEDS technique
Fluid phase behavior
was modified from plate-like to leaf-like particles, as verified by micrographs of scanning electronic
Carbon dioxide microscopy (SEM). For the PHBV precipitation, the SEM micrographs showed that for all experimental
␤-Carotene conditions the morphology of polymer was different from the unprocessed material. The precipitated
PHBV polymer presented a quasi-spherical shape with interconnected particles in the sub-micrometric range
(particle size in the range of 278–570 nm), while the unprocessed material was composed of films and
large blocks. The co-precipitation tests showed that the best ratio of ␤-carotene to PHBV in solution was
1:3 (w/w), which resulted in approximately 80% of encapsulation. Fluid phase behavior of the ternary sys-
tems CO2 + dichloromethane + ␤-carotene and CO2 + dichloromethane + PHBV was also investigated with
the aim of elucidating the region of the phase diagram in which the precipitation occurs. Phase equilib-
rium data were measured in the temperature range of 303–343 K, with CO2 compositions ranging from 40
to 90 wt% for ␤-carotene, and from 30 to 90 wt% for PHBV. Vapor–liquid and also solid–vapor–liquid phase
transitions were observed in the phase equilibrium study. It was observed that the presence of ␤-carotene
or PHBV in the ternary mixture had a little influence on the fluid phase behavior of the systems.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction ucts have little or negligible pigmentation, provitamin-A activity

Incorporation of bioactive compounds such as vitamins, probi-
otics, bioactive peptides and antioxidants into polymeric matrices
provide a simple way to develop novel functional foods that
may have physiological benefits or reduce risks of diseases [1].
Carotenoids, considered nutraceutical compounds, are the most
common group of pigments in nature. The main role of carotenoids
in human diet is to function as vitamin A precursors and its direct
use as antioxidants [2–4,5]. Distinct from synthetic carotenoids, the
natural ones are more easily oxidized [6] and their oxidation prod-
∗ Corresponding author. Fax: +55 54 35209090.
E-mail address: vladimir@uricer.edu.br (J.V. Oliveira).
and singlet oxygen quenching activity [7].
The application properties and the color strength of pigments
are strongly dependent on their physical properties such as particle
size, particle size distribution and morphology, as well as the route
that pigments are obtained [8]. It should be emphasized, however,
that carotenoid particles with much reduced size are still more sen-
sitive to degradation due to the high superficial area that is peculiar
of particles with micro or nanometric size. Thus, it is desirable the
development of preservation techniques to avoid degradation and
to increase the dissolution rate of these compounds in water, as
carotenoids are lipossoluble. The stability of carotenoids against
oxidation can be considerably increased through their encapsula-
tion in biopolymers and even an increase in their dissolution rate
in water can be achieved [4].

0896-8446/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.supflu.2008.08.002
260 E. Franceschi et al. / J. of Supercritical Fluids 47 (2008) 259–269
There is a range of polymers that can be employed to encap-
sulate bioactive compounds, due to their biocompatibility and
biodegradability. Several biopolymers were already used for encap-
sulate pharmaceutical compounds [9,10], proteins [11–13], and
carotenoids [4,14]. The polymer used for encapsulation plays a cru-
cial role, as the characteristics of polymer used determine how
the encapsulated solute will be released. The delivery can occur
in two ways: diffusion release or degradation release. The first
mechanism takes place when the incorporated compound passes
through the polymer pores or through the polymer chain. Degra-
dation release occurs when a polymer degrades within the food
matrix, as a result of natural biological process such as hydroly-
sis. In this case, the polymer degradation is strongly dependent
on the chemical structure and molecular mass of the polymer
[10].
Polyhydoxyalkanoates (PHAs) are polyesters produced by
microorganisms under unbalanced growth conditions. PHA is
generally biodegradable, with good biocompatibility, being an
attractive polymer for use as encapsulating agent [15]. The most
common type of PHA is poly(3-hydroxibutirate) (PHB). However,
PHB is stiff and brittle thus restricting its range of application. On
the other hand, PHB copolymers with 3-hydroxyvalerate (PHBV)
are less stiff, tougher and crystalline [16]. The use of poly(3-
hydroxybutirate-co-hydroxyvalerate) (PHBV) in biomedical field
has increased mainly due to the fact that it is possible to pre-
pare an appropriate controlled drug delivery system that gradually
degrades in the body [15]. Thus, the copolymer PHBV can also be
used as encapsulation medium for the protection and controlled
delivery of carotenoids in foods.
The particle formation and encapsulation using traditional
techniques (spray-drying, coacervation, freeze-drying, interfacial
polymerization, etc.) can suffer from some drawbacks like the poor
control of particle size and morphology, degradation of thermo sen-
sitive compounds, low encapsulation efficiency, and/or low yield.
In this sense, several supercritical fluid-based techniques,
employing mainly carbon dioxide, have been proposed to explore
the solvent strength, high diffusivity and near-zero interfacial ten-
sion that are peculiar in the vicinity of the critical point [17]. The use
of supercritical or near critical fluids as solvents or anti-solvents in
the particles precipitation/encapsulation was demonstrate by sev-
eral researchers as a useful means to the modification of material
properties such as particle size, size distribution and morphology.
Another advantage of such techniques is the efficient separation of
the solvent and anti-solvent of the particles after precipitation, pre-
venting organic solvent residues in the final product and permitting
reutilization of solvent and anti-solvent [18].
Independently of the technique employed for solid materials
recrystallization (SAS, SEDS, GAS, ASES, PCA, etc.), the phase behav-
ior of the involved system plays a crucial role in understanding
the precipitation mechanism, such as jet breakup, nucleation and
growth kinetics, and mass transfer, as well as to determine the most
satisfactory operating conditions during the precipitation [19–22].
In this context, the objective of this work was to investigate
the effect of processing parameters (pressure, flow rate of solu-
tion and anti-solvent, initial concentration of the solid in solution
and mass ratio between ␤-carotene and PHBV) on the precipita-
tion of pure ␤-carotene and PHBV and co-precipitation of them
using the solution enhanced dispersion by supercritical fluid (SEDS)
technique, focusing on particle size, particle size distribution, mor-
phology and encapsulation percentage. The phase behavior of the
systems solvent/solutes/anti-solvent was also investigated to sit-
uate the operating point in the phase diagrams, and helping the
interpretation of the results. The morphology of precipitated pow-
ders was characterized by scanning electronic microscopy (SEM)
and the particle size and size distribution was calculated employing
the software Size Meter (version 1.1). The encapsulation percentage
was verified by UV–vis spectrophotometry.
2. Experimental
2.1. Materials
trans-␤-Carotene, with a purity of 95% and mean particle size of
5 ␮m, was purchased from Sigma–Aldrich (USA). Dichloromethane
(DCM—99.5%) was purchased from Merck (Germany), carbon diox-
ide (99.9% in liquid phase) was supplied by White Martins S.A.,
and the co-polymer PHBV, with a fibrous aspect, was kindly sup-
plied by the PHB Industrial S.A. All materials were used as received.
The polymer average molecular weight (MW) and polydispersity
were found to be 1.96 × 105 g gmol−1 and 1.85, respectively, as mea-
sured by GPC using a calibration curve obtained from polystyrene
standards.
2.2. High-pressure phase equilibrium apparatus and
experimental procedure
Phase behavior experiments of ternary systems were con-
ducted employing the static synthetic method in a high-pressure
variable-volume view cell. A detailed description of the experimen-
tal apparatus and procedure can be found elsewhere [23–25].
2.3. Experimental design
A full factorial 23 experimental design was adopted to orga-
nize the experiments and to evaluate the main effects of process
parameters on the precipitation of ␤-carotene and PHBV from
dichloromethane by the SEDS technique. In this work, it was inves-
tigated the influence of precipitation pressure (P), concentration
of ␤-carotene or PHBV in the solution (C) and anti-solvent flow
rate (q̇CO2 ). The temperature, nozzle diameter and solution flow
rate were maintained constant at 313 K, 100 ␮m and 1.0 mL/min,
respectively. The variables ranges were selected based on pre-
liminary precipitation tests, on previous results of the group,
on the apparatus limitations, and on the fluid phase behavior
of the ternary systems (CO2 + dichloromethane + ␤-carotene and
CO2 + dichloromethane + PHBV). The pressure range adopted was
8–12 MPa, which means pressure values near and above the mix-
ture critical point. The values of solute concentration were from
4 to 8 mg/mL for ␤-carotene and from 10 to 40 mg/mL for PHBV
at room temperature, allowing the operation with concentrated
and diluted solutions. Regarding the anti-solvent flow rate, the
selected range was from 20 to 40 mL/min with the aim of increas-
ing the turbulence inside the precipitation chamber (PC), and
to promote a more intense mixing between the solution and
anti-solvent.
The main effects of above-mentioned variables on particle size
and particle size distribution were evaluated using the commer-
cial software Statistica® 6.0, adopting a confidence level of 95%
(p < 0.05). In this work, it was defined the particle length as the
parameter to compute the characteristic size of the precipitated ␤-
carotene, whereas the diameter was the obvious choice for PHBV
particles.
2.4. Precipitation apparatus and procedure
Fig. 1 presents a schematic diagram of the experimental appara-
tus employed for ␤-carotene, PHBV and combined carotene/PHBV
precipitation [26]. Briefly it consists of a cylindrical vessel, with
an internal volume of 60 mL and internal diameter of 4 cm,
 E. Franceschi et al. / J. of Supercritical Fluids 47 (2008) 259–269
Fig. 1. Schematic diagram of the precipitation apparatus. CV—check-valve; V1, V2, V3 and V4—ball valve; V5, V6 and V7—needle valve; BPR—back pressure regulator;
PT—pressure transducer; PI—pressure indicator; TI—temperature indicator; TC—temperature control; PC—precipitation chamber; SC—separation chamber; CT—capillary
tube.
which was used as PC; two syringe pumps for CO2 displace-
ment (ISCO, Model 500D), operated independently by a set of
ball valves—V1 to V4 (Swagelok, Model SS-83KS4), and a digital
HPLC liquid pump (Acuflow, Series III) used for organic solution
delivery.
The organic solution was sprayed into the precipitation chamber
through a silica capillary fusing tube, with an internal diame-
ter of 100 ␮m, connected to a polyetheretherketone tubing (Peek
Tubing, Upchurch Scientific). This arrangement was linked to a
BPR (back pressure regulator, GO-Regulator, Series BP-66, Model
1A11QEQ151) and to a tee (T) connector (Swagelok), to link the
anti-solvent and the solution flows.
During the experiments, the temperature into the precipitation
chamber was kept constant at 313 K by an ultrathermostatic bath
(Nova Ética, Model 521/2D), while the pressure was varied and con-
trolled by two needle valves (HIP, Model 15-11AF1). The first valve
(V5) controlled the anti-solvent flow rate, while the other one con-
trolled the depressurization. A second vessel (SC) connected after
valve V6 was used to keep the stream that leaves the precipitation
chamber at a relatively low pressure (about 4 MPa) to prevent the
blockage of valve V6. A system for powder collection was located
in the bottom of the precipitation chamber, and was composed by
sintherized metal filter (superficial porosity of 1.0 ␮m) as a support
to the polytetrafluorethylene membrane filter linked to a high den-
sity polyethylene support (Millipore, model FGLP with a porosity
of 0.22 ␮m).
The experimental procedure started with CO2 filling the precip-
itation chamber up to the desired pressure. The anti-solvent flow
rate was controlled by setting V5 and V6 valves, and monitored
by the syringe pump. When the temperature, pressure and anti-
solvent flow rate were stabilized, the organic solution was added
through the capillary tubing. The pressure for solution spray into
the precipitator was controlled by BPR manipulation and monitored
by the liquid pump. The solution volume added to the chamber
261
was 20 mL, which enable the collection of sufficient amount of pre-
cipitated powder for analysis. After the solution addition, around
800 mL of CO2 was continuous flowed in order to dry the precipi-
tated particles inside the precipitation chamber. The precipitation
chamber was slowly depressurized to atmospheric pressure and
particles were collected and stored at appropriate conditions for
subsequent analysis and characterization.
2.5. Analysis and characterization
Precipitated particles were analyzed by a Shimadzu model SS-
550 Superscan scanning electron microscope (SEM) to determine
particle morphology and shape. Particle size was measured by the
Size Meter software (version 1.1), using at least 500 particles for
each experiment, while the Statistica® 6.0 software was used to
perform the statistics of particle size distribution.
The encapsulation percentage was verified by an UV–vis spec-
trophotometer. Initially, a sample of co-precipitated ␤-carotene
and PHBV was weighed. It was assumed that the ratio between
␤-carotene and PHBV remained constant after the precipitation.
The co-precipitated sample was suspended in ethanol and main-
tained under sonication for 5 min to extract the ␤-carotene not
encapsulated. Then, the resulting suspension was filtered and dried
under controlled temperature (323 K) and vacuum for 24 h. After-
wards, the dried powder was dissolved in dichloromethane and
the absorbance of ␤-carotene was measured at 450 nm. Comparing
the results with a pattern curve of absorbance vs. concentration of
␤-carotene in the solvent, the amount of ␤-carotene encapsulated
was evaluated by a straightforward calculation of the encapsulation
percentage by the expression:
␤-carotene encapsulated (%)
mass of ␤-carotene encapsulated
= × 100 (1)
total mass of ␤-carotene in the initial sample
262 E. Franceschi et al. / J. of Supercritical Fluids 47 (2008) 259–269

Table 1
Experimental phase equilibrium data for the system CO2 (1) + dichloromethane(2) + ␤-carotene(3)

T (K) P (MPa)  a (MPa) Transition type

(CO2 -free) ␤-carotene concentration in DCM solution at room temperature: 4 mg/mL

w1 = 0.401
303 4.01 0.01 BP
313 4.97 0.01 BP
323 5.79 0.02 BP
333 6.79 0.04 BP
343 7.67 0.03 BP

w1 = 0.601
303 4.90 0.03 BP
313 6.02 0.04 BP
323 6.96 0.06 BP
333 7.96 0.01 BP
343 9.32 0.05 BP

w1 = 0.800
303 5.42b 0.01 BP
313 6.96b 0.02 BP
323 8.16b 0.02 BP
333 9.26b 0.01 BP
343 10.01b 0.01 DP

w1 = 0.900
303 6.54b 0.06 BP
313 7.63b 0.03 BP
323 8.40b 0.04 DP

(CO2 -free) ␤-carotene concentration in DCM solution at room temperature: 8 mg/mL

w1 = 0.301
303 3.54 0.06 BP
313 4.02 0.03 BP
323 4.66 0.01 BP
333 5.41 0.03 BP
343 6.16 0.01 BP

w1 = 0.501
303 4.49 0.03 BP
313 5.38 0.04 BP
323 6.40 0.01 BP
333 7.26 0.03 BP
343 8.43 0.05 BP

w1 = 0.701
303 5.67b 0.04 BP
313 6.62b 0.03 BP
323 7.57b 0.02 BP
333 8.61b 0.03 BP
343 9.89b 0.02 BP

w1 = 0.900
303 6.29b 0.01 BP
313 7.74b 0.01 BP
323 8.09b 0.03 DP

w1 , CO2 mass fraction; DP, dew point; BP, bubble point; P, pressure; T, temperature.
a Standard deviation.
b Phase transition with the presence of solid ␤-carotene.

3. Results and discussions
3.1. Fluid phase behavior
In this work the fluid phase behavior of ternary sys-
tems CO2 + dichloromethane + ␤-carotene and CO2 + dichloro-
methane + PHBV was investigated. Solutions of ␤-carotene and
PHBV in dichloromethane were prepared at the same concentra-
tions used in the precipitation experiments. The CO2 mass fraction
was varied from 0.4 to 0.9 for ␤-carotene solution and from 0.3 to 0.9
for PHBV solution. The range of temperature investigated was from
303 to 343 K. Tables 1 and 2 show the experimental results for the
fluid phase behavior of ternary systems CO2 + dichloromethane + ␤-
carotene and CO2 + dichloromethane + PHBV, respectively, for the
five isotherms investigated. It can be observed from these tables
that vapor–liquid equilibria occurred, with bubble and dew points,
for both systems studied.
As can be seen in Tables 1 and 2, starting from a certain CO2
concentration, phase transitions for all isotherms occur with the
existence of a solid phase. For the ␤-carotene ternary system
(Table 1), the presence of a solid phase during vapor–liquid phase
transition started from CO2 mass fraction of 0.800 for the more
diluted solution (4 mg/mL), and from 0.701 for the concentrated
one (8 mg/mL). For the PHBV ternary system (Table 2), the PHBV
rich-solid phase started from a CO2 mass fraction lower than that
verified for the ternary system with ␤-carotene. For the diluted
PHBV solution, the vapor–liquid phase transition with the pres-
ence of solid PHBV started from CO2 mass fraction of 0.439, while
 E. Franceschi et al. / J. of Supercritical Fluids 47 (2008) 259–269 263

Table 2
Experimental phase equilibrium data for the system CO2 (1) + dichloromethane(2) + PHBV(3)

T (K) P (MPa)  (MPa) Transition type

(CO2 -free) PHBV concentration in DCM solution at room temperature: 10 mg/mL

w1 = 0.303
303 3.63 0.01 BP
313 4.36 0.02 BP
323 4.98 0.05 BP
333 5.85 0.04 BP
343 6.47 0.01 BP

w1 = 0.439
303 4.08a 0.05 BP
313 5.00a 0.04 BP
323 6.00a 0.03 BP
333 6.91a 0.04 BP
343 7.86a 0.01 BP

w1 = 0.601
303 4.95a 0.06 BP
313 5.98a 0.02 BP
323 6.93a 0.04 BP
333 8.12a 0.01 BP
343 9.21a 0.03 BP

w1 = 0.700
303 5.41a 0.02 BP
313 6.38a 0.01 BP
323 7.54a 0.03 BP
333 8.74a 0.04 BP
343 9.81a 0.03 BP

w1 = 0.800
303 5.63a 0.04 BP
313 7.02a 0.04 BP
323 8.06a 0.02 BP
333 9.26a 0.03 BP
343 9.94a 0.02 DP

w1 = 0.900
303 6.40a 0.02 BP
313 77.9a 0.03 BP
323 8.49a 0.01 DP

(CO2 -free) PHBV concentration in DCM solution at room temperature: 40 mg/mL

w1 = 0.303
303 3.43 0.03 BP
313 4.10 0.05 BP
323 4.87 0.02 BP
333 5.65 0.03 BP
343 6.29 0.01 BP

w1 = 0.402
303 4.11a 0.01 BP
313 4.98a 0.03 BP

w1 = 0.402
323 5.72a 0.02 BP
333 6.45a 0.02 BP
343 7.66a 0.01 BP

w1 = 0.501
303 4.51a 0.02 BP
313 5.31a 0.05 BP
323 6.45a 0.03 BP
333 7.39a 0.03 BP
343 8.29a 0.03 BP

w1 = 0.601
303 4.87a 0.01 BP
313 5.90a 0.02 BP
323 6.96a 0.01 BP
333 7.89a 0.03 BP
343 9.30a 0.04 BP

w1 = 0.700
303 5.43a 0.02 BP
313 6.60a 0.03 BP
323 7.63a 0.02 BP
333 8.88a 0.03 BP
343 9.86a 0.04 BP
264 E. Franceschi et al. / J. of Supercritical Fluids 47 (2008) 259–269

Table 2 (Continued )

T (K) P (MPa)  (MPa) Transition type

w1 = 0.799
303 5.97a 0.01 BP
313 7.04a 0.02 BP

w1 = 0.799
323 7.98a 0.02 BP
333 9.13a 0.02 BP
343 9.81a 0.01 DP

w1 = 0.900
303 6.62a 0.01 BP
313 7.57a 0.01 BP
323 8.37a 0.03 DP

w1 , CO2 mass fraction; DP, dew point; BP, bubble point; P, pressure; T, temperature.
a Phase transition with the presence of solid PHBV.

for the more concentrated solution at CO2 mass fraction of 0.402. where the solid phase can be considered as pure solute. When phase
As discussed in the literature, this fact is due to the co-solvent transitions occurred with solutes completely dissolved in the low
(acts enhancing the solute solubility) and anti-solvent (reduces concentrated solutions, pressure values were not influenced due to
the solute solubility) effect of CO2 in the mixture [19,21]. At low the very low solutes concentration in the mixture.
CO2 concentrations, it acts as a co-solvent and, at high concen-
trations as an anti-solvent. The limit concentration in which CO2
3.2. ˇ-Carotene precipitation
starts to behave as anti-solvent depends on the solution concen-
tration. For diluted solutions high amounts of CO2 are necessary
The variables studied in the ␤-carotene precipitation included
to induce the formation of a solid phase. On the other hand, for
the precipitation pressure, ␤-carotene concentration in the organic
concentrated solutions, the amount of CO2 needed for a solid
solution, and anti-solvent flow rate. The experimental runs and
phase formation may be much lower due to the solution saturation
results of particle size and particle size distribution are summarized
proximity.
in Table 3.
To check possible modifications in the phase diagram due
It can be noted from Table 3 that, in general, a reduction or an
to the addition of a solid compound to the binary system
increase in the particle size were observed: starting from original
CO2 + dichloromethane, two solute concentrations were tested
␤-carotene with mean particle size of 4.6 ␮m, the mean particle
in the organic solution. Results presented in Figs. 2 and 3
size of precipitated ␤-carotene varied from few microns (3.8 ␮m)
provide a comparison between the ternary system CO2 + dichloro-
to larger sizes (246.8 ␮m). The size distribution was characterized
methane + ␤-carotene and CO2 + dichloromethane + PHBV, respec-
by the variation coefficient (VC) as very distinct particle size was
tively, for two solute concentrations in the organic solution at 313 K
observed among runs. Variation coefficients in the range of 23–57%
with the binary system CO2 + dichloromethane [27].
around the mean particle size were observed in the experiments.
As can be observed from these figures, the presence of the solid
From replicate measurements shown in Table 3, runs 9–11, one can
(␤-carotene or PHBV) has negligible influence on the fluid phase
see that a good reproducibility regarding mean particle size was
behavior. This fact can be explained as phase transitions occurred
achieved.
in the presence of solid solutes, for the concentrated solutions,
The statistical analysis adopting 95% of confidence level
(p < 0.05) revealed that precipitation pressure and anti-solvent flow

Fig. 2. Experimental data at 313 K for the ternary system CO2 + dichloromethane + ␤- Fig. 3. Experimental data at 313 K for the ternary system CO2 + dichloro-
carotene at (CO2 -free) ␤-carotene concentration of 4 mg/mL and 8 mg/mL at room methane + PHBV at (CO2 -free) polymer concentration of 10 mg/mL and 40 mg/mL
temperature. Experimental binary system data from the literature [27]. at room temperature. Experimental binary system data from the literature [27].
 E. Franceschi et al. / J. of Supercritical Fluids 47 (2008) 259–269 265

Table 3 was observed as the pressure is increased. The effect of precipita-

Factorial experimental design (23 ) of ␤-carotene precipitation from a DCM/␤-
tion pressure on the particle size and morphology of precipitated
carotene solution using CO2 by the SEDS technique and experimental results of mean
particle size (PS) and variation coefficient (VC) ␤-carotene is illustrated in Fig. 4 through SEM micrographs of
experimental conditions 6 (Fig. 4A) and 8 (Fig. 4B).
Run C (mg/mL) P (MPa) q̇CO2 (mL/min) PS (␮m) VCa (%)
The pressure effect on particle size of precipitated ␤-carotene
1 4 8.0 20 6.2 53 found in this work is in agreement with those obtained by Miguel
2 8 8.0 20 29.1 33 et al. [2] that investigated the precipitation of lycopene from
3 4 12.0 20 246.8 57
dichloromethane solutions using carbon dioxide as anti-solvent.
4 8 12.0 20 186.0 42
5 4 8.0 40 4.6 24 In that work, the authors estimated the maximum supersatura-
6 8 8.0 40 3.8 42 tion that could be achieved in lycopene precipitation, and that the
7 4 12.0 40 42.8 24 supersaturation decreased as pressure increased above the mixture
8 8 12.0 40 124.7 44
critical point due to a decrease of solute concentration in the mix-
9 6 10.0 30 88.3 24
10 6 10.0 30 71.5 23
ture. Considering that the solubility of lycopene and ␤-carotene in
11 6 10.0 30 66.5 23 CO2 are similar and also due to the similar chemical structure, one
could also expect that the results obtained for lycopene should be
Precipitation temperature 313 K, solution rate 1 mL/min, nozzle diameter 100 ␮m.
C, solution concentration; P, precipitation pressure; q̇CO2 , CO2 flow rate; PS, particle applied to ␤-carotene [2].
size. In this sense, pressure effects can be explained in terms of solu-
a VC is defined as the ratio between the standard deviation and the mean particle
bility, since the supersaturation is defined by the ratio between the
size. solute concentration in the solvent + anti-solvent mixture and the
saturation concentration of the solute in the mixture. As the sol-
rate present a significant effect on particle size of ␤-carotene vent + anti-solvent mixture stands above the mixture critical point,
precipitated, whereas the solute concentration did not present a an increase in pressure leads to a decrease in solute concentration
significant effect. Pressure was the variable that more strongly in the mixture, hence reducing the supersaturation. This decrease
influenced the particle size. An increase in pressure leads to an in supersaturation causes a decrease in the nucleation rate, leading
increase in the mean particle size, while the anti-solvent flow rate to a larger growth of ␤-carotene precipitated particles, increasing
had an opposite effect, with a decrease in the mean particle size the particle size and modifying their morphology from plate-like
due to a raise in the anti-solvent flow rate. (Fig. 4A) to leaf-like (Fig. 4B) particles.
Though the solubility of CO2 in the organic solvent is diminished
3.3. Pressure effect as pressure is decreased from 12 to 8 MPa the mass transfer rates
of the CO2 in the solution droplets generated by the spray and in
Experiments for ␤-carotene precipitation were carried out by the organic solvent in the CO2 bulk phase are enhanced, leading to
changing the precipitation pressure from 8 to 12 MPa. This pressure higher nucleation rates and smaller particles.
range combined with a fixed temperature (313 K) condition, result
in a single supercritical phase where the organic solvent and the 3.4. Anti-solvent flow rate effect
anti-solvent are completely miscible, as can be seen in Fig. 2. As
commented above, as the pressure are increased, the mean particle The anti-solvent flow rate was investigated in the range of
size is also enhanced, as shown in Table 3 for runs 1 and 3 and, for 20–40 mL/min. According to statistical analysis, an increase in this
runs 6 and 8. variable induces a decrease in mean particle size and variation coef-
From runs 1 to 3, the pressure was increased and the solution ficient. However, this effect is less pronounced when compared to
concentration and anti-solvent flow rate was maintained at the the pressure influence. The effect of anti-solvent flow rate was eval-
lowest values. Comparing these runs, the mean particle size was uated by comparing runs 1 and 5 and runs 4 and 8. In runs 1 and 5
increased from 6.2 to 246.8 ␮m and the variation coefficient was the anti-solvent flow rate was varied maintaining the pressure and
also increased from 53 to 57%. When the pressure was increased, solution concentration at the lowest values (8 MPa and 4 mg/mL,
with the solution concentration and anti-solvent flow rate in the respectively). In run 1 the mean particle size obtained was 6.2 ␮m
upper limits (runs 6 and 8) the mean particle size was also increased with a variation coefficient of 53%, while in run 5 the mean particle
from 3.8 to 124.7 ␮m and the VC enhanced from 42 to 43%. The size decreased to 4.6 ␮m with a variation coefficient of 24%.
morphology of ␤-carotene precipitated also changed with pressure, When the anti-solvent flow rate increased with pressure and
where a modification of particles from plate-like to a leaf-like form solution concentration at the highest values (runs 4 and 8) the

Fig. 4. SEM micrographs of the precipitated ␤-carotene at 8.0 MPa (A—run 6) and 12.0 MPa (B—run 8). Solution concentration of 8 mg/mL and anti-solvent flow rate of
40 mL/min.
266 E. Franceschi et al. / J. of Supercritical Fluids 47 (2008) 259–269

Fig. 5. SEM micrographs of the precipitated ␤-carotene at anti-solvent flow rate of 20 mL/min (A—run 1), and 40 mL/min (B—run 5). Pressure of 8.0 MPa and solution
concentration of 4 mg/mL.

mean particle size was reduced from 186 to 124.7 ␮m with a slight
increase in the variation coefficient of the mean particle size from
42 to 43%. However, keeping constant the anti-solvent flow rate at
the lowest or at the highest value and increasing simultaneously
the pressure and the solution concentration, a notable increase in
particle size was observed. The particle morphology of precipitated
␤-carotene was not modified by the anti-solvent flow rate. SEM
micrographs of runs 1 (Fig. 5A) and 5 (Fig. 5B) presented in Fig. 5
illustrate the influence of anti-solvent flow rate on particle size of
precipitated ␤-carotene.
From this figure it can be noted that the precipitated particles
show agglomeration, mainly when the anti-solvent flow rate was
increased to 40 mL/min. This fact can be attributed to the high inlet
velocity of the anti-solvent that causes intense collisions of the par-
ticles and thus the formation of agglomerates. Reduction of particle
size with the increase of the anti-solvent flow rate is probably due
to hydrodynamics aspects. The high velocity of CO2 causes a pro-
nounced turbulence inside the precipitation chamber leading to an
increase in the kinetic energy of the atomizing CO2 . Thus, the jet
break up is intensified increasing the interaction of anti-solvent and
solution, and generating fine droplets. In addition, the mass transfer
rates between CO2 and the organic solvent are increased by the high
surface area generated; thus the CO2 diffuses more rapidly into the
droplet and the solvent evaporates from droplets instantaneously
causing accelerated supersaturation and nucleation.
The reproducibility of the precipitated ␤-carotene morphology
can be verified in Fig. 6, from SEM micrographs of runs 9 (Fig. 6A),
10 (Fig. 6B) and 11 (Fig. 6C) (Table 3) indicating that the methodol-
ogy and the experimental unit presented a good reproducibility on
morphology of precipitated ␤-carotene particles. For the purpose
of illustration, Fig. 7 depicts a histogram of PSD for run 6, which
provided the smallest particle size, and from where one can see
an approximate Gaussian distribution pattern, a common feature
found in many works in the literature.

Fig. 6. SEM micrographs of the precipitated ␤-carotene from runs 9 (A), 10 (B) and 11 (C) at pressure of 10 MPa, solution concentration of 6 mg/mL and anti-solvent flow rate
of 30 mL/min.
 E. Franceschi et al. / J. of Supercritical Fluids 47 (2008) 259–269 267

Table 4
Factorial experimental design (23 ) of PHBV (MW = 1.96 × 105 g gmol−1 and polydis-
persity = 1.85) precipitation DCM/PHBV solution using CO2 by the SEDS technique
and experimental results of mean particle size (PS) and variation coefficient (VC)

Run C (mg/mL) P (bar) q̇CO2 (mL/min) PS (nm) VCa (%)

1 10 8.0 20 487 21
2 40 8.0 20 505 24
3 10 12.0 20 329 21
4 40 12.0 20 – –
5 10 8.0 40 370 21
6 40 8.0 40 570 28
7 10 12.0 40 278 22
8 40 12.0 40 563 31
9 25 10.0 30 330 24
10 25 10.0 30 324 22
11 25 10.0 30 314 21

Precipitation temperature 313 K, solution rate 1 mL/min, nozzle diameter 100 ␮m.
C, solution concentration; P, precipitation pressure; q̇CO2 , CO2 flow rate; PS, particle
size.
a VC is defined as the ratio between the standard deviation and the mean particle

size.
Fig. 7. Particle size distribution of ␤-carotene precipitated at solution concentration
of 8 mg/mL, pressure of 8 MPa and anti-solvent flow rate of 40 mL/min.

3.5. PHBV precipitation
The same variables of ␤-carotene precipitation were inves-
tigated in the PHBV studies: precipitation pressure, solution
concentration and anti-solvent flow rate. The range of precipita-
tion pressure and anti-solvent flow rate was the same as the ones
for ␤-carotene precipitation; however the solution concentration
was investigated in distinct values due to the higher solubil-
ity of the polymer than that of ␤-carotene in dichloromethane.
Table 4 presents the experimental runs and results regarding par-
ticle size and size distribution of PHBV precipitation, where one
can observe from the replicate measurements a good experimental
reproducibility.
From Table 4 it can be noted that the particle size of precipi-
tated PHBV was in the sub-micrometric range, varying from 278
to 570 nm while the VC varied from 21 to 31%. One can note that
fibers, not particles, were produced in run 4. In all other precipi-
tation experiments quasi-spherical interconnected particles were
obtained, differently from the unprocessed PHBV that is formed by
fibers. According to statistical analysis at 95% of confidence level
(p < 0.05), only the solution concentration had a significant and
positive effect on particle size.
3.6. Solution concentration effect
The effect of the solution concentration on particle size and
morphology of PHBV precipitation was evaluated in the range of
10–40 mg/mL. Other variables (pressure and anti-solvent flow rate)
were firstly maintained at the lowest values (runs 1 and 2 of Table 4).
An increase in the solution concentration in this case resulted in
an increase in mean particle size from 487 to 505 nm and a small
change in the variation coefficient (21–24%). Runs 7 and 8 of Table 4
present the effect of the solution concentration at the higher values
of pressure and anti-solvent flow rate. In these cases, the increase
of the solution concentration resulted in a more pronounced raise
in particle size, from 278 to 563 nm, and in the variation coeffi-
cient, from 22 to 31%. The PHBV particles produced in these cases
were almost spherical shape and interconnected, as can be seen
from SEM micrographs of experiments 7 (Fig. 8A) and 8 (Fig. 8B) in
Fig. 8.
As can be noted in Fig. 8 in concentrated solutions (micrograph
B in Fig. 8) show that besides almost spherical particles there are
also fibers and more agglomerated particles were observed than
for the diluted solution (micrograph A in Fig. 8). These results
are in agreement with those obtained by Costa et al. [16] in
the precipitation of PHBV. The formation of fibers and agglom-
erated particles is characteristic of concentrated solutions due to
the high viscosity of these solutions. The high viscosity implies
in an increase in shear forces thus hindering the diffusion of
CO2 into droplets generated by the jet break up. Thus, evap-
oration of the organic solvent from droplets by CO2 action is
more affected, causing a decrease in the mass transfer rate, and
accordingly in the precipitation kinetics, generating larger parti-
cles.

Fig. 8. SEM micrographs of the precipitated PHBV at solution concentration of 10 mg/mL (A—run 7), and of 40 mg/mL (B—run 8). Pressure of 12.0 MPa and anti-solvent flow
rate of 40 mL/min.
268 E. Franceschi et al. / J. of Supercritical Fluids 47 (2008) 259–269
Fig. 9. SEM micrographs of the co-precipitated ␤-carotene and PHBV with mass ratio of 1:2 (A), 1:3 (B) and 1:4 (C) at 8.0 MPa and anti-solvent flow rate of 40 mL/min.
3.7. Encapsulation of ˇ-carotene into PHBV
Preliminary tests of ␤-carotene encapsulation into PHBV were
carried out by co-precipitation using the SEDS technique. The main
idea for co-precipitation is that the particles of the material to be
encapsulated (core) are smaller than those of the encapsulating
material (shell). In the precipitation of pure ␤-carotene and PHBV,
no experimental conditions presented such a result. Nevertheless,
experimental condition 6 in the ␤-carotene and in the PHBV precip-
itation steps (Tables 3 and 4) that led, respectively, to the smallest
and highest particle sizes of these materials was chosen to accom-
plish the co-precipitation experiments. Pressure and anti-solvent
flow rate were maintained at 8.0 MPa and 40 mL/min, respectively,
varying the mass ratio between ␤-carotene and PHBV in the organic
solution.
The carotene to polymer mass ratio investigated was 1:2, 1:3 and
1:4. Fig. 9 presents SEM micrographs of the co-precipitation experi-
ments for ␤-carotene to PHBV mass ratio of 1:2, 1:3 and 1:4. Results
showed that for the mass ratio of 1:2, the encapsulated carotene
was around 5%, indicating that the amount of polymer was not suf-
ficient to effectively encapsulate the carotene. For the mass ratio
of 1:3, the encapsulation percentage was close to 80%, suggesting
that ␤-carotene was precipitated earlier than the polymer, acting
as seed particles, for subsequent covering of the latter. For the mass
ratio of 1:4, it seems that ␤-carotene particles remained adsorbed
on the polymer surface, indicating that the polymer could precipi-
tate early due to its higher concentration, and then the ␤-carotene
stick on its surface.
A more comprehensive study on the PHBV precipitation and
co-precipitation of ␤-carotene and PHBV, assessing the effects of
process parameters is underway within our research group and will
be the subject of a next report.
4. Conclusions
This work investigated the precipitation of pure ␤-carotene
and PHBV from dichloromethane solutions by the SEDS tech-
nique with carbon dioxide as anti-solvent. Phase equilibrium
data involving CO2 , dichloromethane and the solutes (␤-carotene
and PHBV) were measured. It was verified that the addi-
tion of solutes to the binary system consisting of the organic
solvent and CO2 had negligible influence on transition pres-
sures.
Precipitation of pure ␤-carotene presented particles with mean
size in the range from 3.8 to 246.8 ␮m and variation coefficient
from 23 and 57% around the mean particle size. For the variables
investigated, (precipitation pressure, solution concentration and
anti-solvent flow rate), pressure and anti-solvent flow rate pre-
sented influence on particle size and morphology with a stronger
effect of precipitation pressure. The precipitation pressure present
a positive effect on the mean particle size of ␤-carotene, whereas
the anti-solvent flow rate showed a negative effect on this variable.
The ␤-carotene morphology changed from plate-like to leaf-like
particles.
For PHBV precipitation, sub-micrometric almost spherical parti-
cles, were generated except for the highest polymer concentration
in the solution where fibers were produced. The mean particle size
of PHBV precipitated varied from 278 to 570 nm with variation
coefficient between 21 and 31%. A raise in solution concentration
led to and increase in particle size. The anti-solvent flow rate and
precipitation pressure were not significant on PHBV particle size
precipitated at 95% of confidence. The co-precipitation tests indi-
cated that encapsulation of ␤-carotene in PHBV up to 80% were
achieved at mass ratio of 1:3 (␤-carotene to polymer) in the organic
solution.
 E. Franceschi et al. / J. of Supercritical Fluids 47 (2008) 259–269
Acknowledgments
The authors thank CNPq and CAPES for the financial support and
scholarships.
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