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J. of Supercritical Fluids 47 (2008) 259–269

J. of Supercritical Fluids 47 (2008) 259–269 Contents lists available at ScienceDirect The Journal of Supercritical

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

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

Fluids journal homepage: www.elsevier.com/locate/supflu Precipitation of -carotene and PHBV and co-precipitation

Precipitation of -carotene and PHBV and co-precipitation from SEDS technique using supercritical CO 2

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

article

info

Article history:

Received 15 April 2008 Received in revised form 31 July 2008 Accepted 1 August 2008

Keywords:

Encapsulation SEDS technique Fluid phase behavior Carbon dioxide -Carotene PHBV

abstract

The objective of this work was to investigate the application of the solution enhanced dispersion by supercritical fluids (SEDS) technique for the precipitation of pure -carotene and copolymer poly(3- hydroxybutirate-co-hydroxyvalerate) (PHBV), as well as some encapsulation tests of the solute in the 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 dichloromethane solution (4 and 8 mg/mL). For pure -carotene, the results showed that the mean particle size varied from 3.8 to 246.8 m, depending on the processing conditions. The morphology of -carotene was modified from plate-like to leaf-like particles, as verified by micrographs of scanning electronic microscopy (SEM). For the PHBV precipitation, the SEM micrographs showed that for all experimental conditions the morphology of polymer was different from the unprocessed material. The precipitated 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 CO 2 + dichloromethane + -carotene and CO 2 + 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 CO 2 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

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).

0896-8446/$ – see front matter © 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.supflu.2008.08.002

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

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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 × 10 5 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 2 3 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 (q˙ CO 2 ). 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 (CO 2 + dichloromethane + -carotene and CO 2 + 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

261

et al. / J. of Supercritical Fluids 47 (2008) 259–269 261 Fig. 1. Schematic diagram of

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 CO 2 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 CO 2 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 BPRmanipulation andmonitored by the liquid pump. The solution volume added to the chamber

was 20 mL, which enable the collection of sufficient amount of pre- cipitated powder for analysis. After the solution addition, around

800 mL of CO 2 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

total mass of -carotene in the initial sample × 100

(1)

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E. Franceschi et al. / J. of Supercritical Fluids 47 (2008) 259–269

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

T (K)

P (MPa)

a (MPa)

Transition type

(CO 2 -free) -carotene concentration in DCM solution at room temperature: 4 mg/mL w 1 = 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

w 1 = 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

w 1 = 0.800

303

5.42 b

0.01

BP

313

6.96 b

0.02

BP

323

8.16 b

0.02

BP

333

9.26 b

0.01

BP

343

10.01 b

0.01

DP

w 1 = 0.900

303

6.54 b

0.06

BP

313

7.63 b

0.03

BP

323

8.40 b

0.04

DP

(CO 2 -free) -carotene concentration in DCM solution at room temperature: 8 mg/mL w 1 = 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

w 1 = 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

w 1 = 0.701

303

5.67 b

0.04

BP

313

6.62 b

0.03

BP

323

7.57 b

0.02

BP

333

8.61 b

0.03

BP

343

9.89 b

0.02

BP

w 1 = 0.900

303

6.29 b

0.01

BP

313

7.74 b

0.01

BP

323

8.09 b

0.03

DP

w 1 , CO 2 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

five isotherms investigated. It can be observed from these tables that vapor–liquid equilibria occurred, with bubble and dew points,

3.1.

Fluid phase behavior

for both systems studied.

In this work the fluid phase behavior of ternary sys- tems CO 2 + dichloromethane + -carotene and CO 2 + 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 CO 2 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 CO 2 + dichloromethane + - carotene and CO 2 + dichloromethane + PHBV, respectively, for the

As can be seen in Tables 1 and 2, starting from a certain CO 2 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 CO 2 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 CO 2 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 CO 2 mass fraction of 0.439, while

E. Franceschi et al. / J. of Supercritical Fluids 47 (2008) 259–269

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

263

T (K)

P (MPa)

(MPa)

Transition type

(CO 2 -free) PHBV concentration in DCM solution at room temperature: 10 mg/mL w 1 = 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

w 1 = 0.439

303

4.08 a

0.05

BP

313

5.00 a

0.04

BP

323

6.00 a

0.03

BP

333

6.91 a

0.04

BP

343

7.86 a

0.01

BP

w 1 = 0.601

303

4.95 a

0.06

BP

313

5.98 a

0.02

BP

323

6.93 a

0.04

BP

333

8.12 a

0.01

BP

343

9.21 a

0.03

BP

w 1 = 0.700

303

5.41 a

0.02

BP

313

6.38 a

0.01

BP

323

7.54 a

0.03

BP

333

8.74 a

0.04

BP

343

9.81 a

0.03

BP

w 1 = 0.800

303

5.63 a

0.04

BP

313

7.02 a

0.04

BP

323

8.06 a

0.02

BP

333

9.26 a

0.03

BP

343

9.94 a

0.02

DP

w 1 = 0.900

303

6.40 a

0.02

BP

313

77.9 a

0.03

BP

323

8.49 a

0.01

DP

(CO 2 -free) PHBV concentration in DCM solution at room temperature: 40 mg/mL w 1 = 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

w 1 = 0.402

303

4.11 a

0.01

BP

313

4.98 a

0.03

BP

w 1 = 0.402

323

5.72 a

0.02

BP

333

6.45 a

0.02

BP

343

7.66 a

0.01

BP

w 1 = 0.501

303

4.51 a

0.02

BP

313

5.31 a

0.05

BP

323

6.45 a

0.03

BP

333

7.39 a

0.03

BP

343

8.29 a

0.03

BP

w 1 = 0.601

303

4.87 a

0.01

BP

313

5.90 a

0.02

BP

323

6.96 a

0.01

BP

333

7.89 a

0.03

BP

343

9.30 a

0.04

BP

w 1 = 0.700

303

5.43 a

0.02

BP

313

6.60 a

0.03

BP

323

7.63 a

0.02

BP

333

8.88 a

0.03

BP

343

9.86 a

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

w 1 = 0.799

303

5.97 a

0.01

BP

313

7.04 a

0.02

BP

w 1 = 0.799

323

7.98 a

0.02

BP

333

9.13 a

0.02

BP

343

9.81 a

0.01

DP

w 1 = 0.900

303

6.62 a

0.01

BP

313

7.57 a

0.01

BP

323

8.37 a

0.03

DP

w 1 , CO 2 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 CO 2 mass fraction of 0.402. As discussed in the literature, this fact is due to the co-solvent (acts enhancing the solute solubility) and anti-solvent (reduces the solute solubility) effect of CO 2 in the mixture [19,21]. At low CO 2 concentrations, it acts as a co-solvent and, at high concen- trations as an anti-solvent. The limit concentration in which CO 2 starts to behave as anti-solvent depends on the solution concen- tration. For diluted solutions high amounts of CO 2 are necessary to induce the formation of a solid phase. On the other hand, for concentrated solutions, the amount of CO 2 needed for a solid phase formation may be much lower due to the solution saturation proximity. To check possible modifications in the phase diagram due to the addition of a solid compound to the binary system CO 2 + dichloromethane, two solute concentrations were tested in the organic solution. Results presented in Figs. 2 and 3 provide a comparison between the ternary system CO 2 + dichloro- methane + -carotene and CO 2 + dichloromethane + PHBV, respec- tively, for two solute concentrations in the organic solution at 313 K with the binary system CO 2 + dichloromethane [27]. As can be observed from these figures, the presence of the solid

( -carotene or PHBV) has negligible influence on the fluid phase behavior. This fact can be explained as phase transitions occurred in the presence of solid solutes, for the concentrated solutions,

presence of solid solutes, for the concentrated solutions, Fig. 2. Experimental data at 313 K for

Fig. 2. Experimental data at 313 K for the ternary system CO 2 + dichloromethane + - carotene at (CO 2 -free) -carotene concentration of 4 mg/mL and 8 mg/mL at room temperature. Experimental binary system data from the literature [27].

where the solid phase can be considered as pure solute.When phase transitions occurred with solutes completely dissolved in the low concentrated solutions, pressure values were not influenced due to the very low solutes concentration in the mixture.

3.2. ˇ-Carotene precipitation

The variables studied in the -carotene precipitation included the precipitation pressure, -carotene concentration in the organic solution, and anti-solvent flow rate. The experimental runs and results of particle size and particle size distribution are summarized in Table 3. It can be noted from Table 3 that, in general, a reduction or an increase in the particle size were observed: starting from original -carotene with mean particle size of 4.6 m, the mean particle size of precipitated -carotene varied from few microns (3.8 m) to larger sizes (246.8 m). The size distribution was characterized by the variation coefficient (VC) as very distinct particle size was observed among runs. Variation coefficients in the range of 23–57% around the mean particle size were observed in the experiments. From replicate measurements shown in Table 3, runs 9–11, one can see that a good reproducibility regarding mean particle size was achieved. The statistical analysis adopting 95% of confidence level (p < 0.05) revealed that precipitation pressure and anti-solvent flow

revealed that precipitation pressure and anti-solvent flow Fig. 3. Experimental data at 313 K for the

Fig. 3. Experimental data at 313 K for the ternary system CO 2 + dichloro- methane + PHBV at (CO 2 -free) polymer concentration of 10 mg/mL and 40 mg/mL 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 Factorial experimental design (2 3 ) of -carotene precipitation from a DCM/ - carotene solution using CO 2 by the SEDS technique and experimental results of mean particle size (PS) and variation coefficient (VC)

Run

C (mg/mL)

P (MPa)

q˙ CO 2 (mL/min)

PS ( m)

VC a (%)

1

4

8.0

20

6.2

53

2

8

8.0

20

29.1

33

3

4

12.0

20

246.8

57

4

8

12.0

20

186.0

42

5

4

8.0

40

4.6

24

6

8

8.0

40

3.8

42

7

4

12.0

40

42.8

24

8

8

12.0

40

124.7

44

9

6

10.0

30

88.3

24

10 6

10.0

30

71.5

23

11 6

10.0

30

66.5

23

Precipitation temperature 313 K, solution rate 1 mL/min, nozzle diameter 100 m. C, solution concentration; P, precipitation pressure; q˙ CO 2 , CO 2 flow rate; PS, particle size.

a VC is defined as the ratio between the standard deviation and the mean particle size.

rate present a significant effect on particle size of -carotene precipitated, whereas the solute concentration did not present a significant effect. Pressure was the variable that more strongly influenced the particle size. An increase in pressure leads to an increase in the mean particle size, while the anti-solvent flow rate had an opposite effect, with a decrease in the mean particle size due to a raise in the anti-solvent flow rate.

3.3. Pressure effect

Experiments for -carotene precipitation were carried out by changing the precipitation pressure from 8 to 12 MPa. This pressure range combined with a fixed temperature (313 K) condition, result in a single supercritical phase where the organic solvent and the anti-solvent are completely miscible, as can be seen in Fig. 2. As commented above, as the pressure are increased, the mean particle size is also enhanced, as shown in Table 3 for runs 1 and 3 and, for runs 6 and 8. From runs 1 to 3, the pressure was increased and the solution concentration and anti-solvent flow rate was maintained at the lowest values. Comparing these runs, the mean particle size was increased from 6.2 to 246.8 m and the variation coefficient was also increased from 53 to 57%. When the pressure was increased, with the solution concentration and anti-solvent flow rate in the upper limits (runs 6 and 8) themean particle size was also increased from 3.8 to 124.7 m and the VC enhanced from 42 to 43%. The morphology of -carotene precipitated also changed with pressure, where a modification of particles from plate-like to a leaf-like form

was observed as the pressure is increased. The effect of precipita- tion pressure on the particle size and morphology of precipitated -carotene is illustrated in Fig. 4 through SEM micrographs of experimental conditions 6 (Fig. 4A) and 8 (Fig. 4B). The pressure effect on particle size of precipitated -carotene found in this work is in agreement with those obtained by Miguel et al. [2] that investigated the precipitation of lycopene from dichloromethane solutions using carbon dioxide as anti-solvent. In that work, the authors estimated the maximum supersatura- tion that could be achieved in lycopene precipitation, and that the supersaturation decreased as pressure increased above the mixture critical point due to a decrease of solute concentration in the mix- ture. Considering that the solubility of lycopene and -carotene in CO 2 are similar and also due to the similar chemical structure, one could also expect that the results obtained for lycopene should be applied to -carotene [2]. In this sense, pressure effects can be explained in terms of solu- bility, since the supersaturation is defined by the ratio between the solute concentration in the solvent + anti-solvent mixture and the saturation concentration of the solute in the mixture. As the sol- vent + anti-solvent mixture stands above the mixture critical point, an increase in pressure leads to a decrease in solute concentration in the mixture, hence reducing the supersaturation. This decrease in supersaturation causes a decrease in the nucleation rate, leading to a larger growth of -carotene precipitated particles, increasing the particle size and modifying their morphology from plate-like (Fig. 4A) to leaf-like (Fig. 4B) particles. Though the solubility of CO 2 in the organic solvent is diminished as pressure is decreased from 12 to 8 MPa the mass transfer rates of the CO 2 in the solution droplets generated by the spray and in the organic solvent in the CO 2 bulk phase are enhanced, leading to higher nucleation rates and smaller particles.

3.4. Anti-solvent flow rate effect

The anti-solvent flow rate was investigated in the range of 20–40 mL/min. According to statistical analysis, an increase in this variable induces a decrease in mean particle size and variation coef- ficient. However, this effect is less pronounced when compared to the pressure influence. The effect of anti-solvent flow rate was eval- uated by comparing runs 1 and 5 and runs 4 and 8. In runs 1 and 5 the anti-solvent flow rate was varied maintaining the pressure and solution concentration at the lowest values (8 MPa and 4 mg/mL, respectively). In run 1 the mean particle size obtained was 6.2 m with a variation coefficient of 53%, while in run 5 the mean particle size decreased to 4.6 m with a variation coefficient of 24%. When the anti-solvent flow rate increased with pressure and solution concentration at the highest values (runs 4 and 8) the

concentration at the highest values (runs 4 and 8) the Fig. 4. SEM micrographs of 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

et al. / J. of Supercritical Fluids 47 (2008) 259–269 Fig. 5. SEM micrographs of the

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 CO 2 causes a pro-

nounced turbulence inside the precipitation chamber leading to an increase in the kinetic energy of the atomizing CO 2 . 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 CO 2 and the organic solvent are increased by the high surface area generated; thus the CO 2 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. 6 B) and 11 ( Fig. 6 C) ( 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.

a common feature found in many works in the literature. Fig. 6. SEM micrographs of the

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

et al. / J. of Supercritical Fluids 47 (2008) 259–269 267 Fig. 7. Particle size distribution

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.

Table 4 Factorial experimental design (2 3 ) of PHBV (MW = 1.96 × 10 5 g gmol 1 and polydis- persity = 1.85) precipitation DCM/PHBV solution using CO 2 by the SEDS technique and experimental results of mean particle size (PS) and variation coefficient (VC)

Run

C (mg/mL)

P (bar)

q˙ CO 2 (mL/min)

PS (nm)

VC a (%)

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; q˙ CO 2 , CO 2 flow rate; PS, particle size.

a VC is defined as the ratio between the standard deviation and the mean particle size.

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 firstlymaintained 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

CO 2 into droplets generated by the jet break up. Thus, evap- oration of the organic solvent from droplets by CO 2 action is more affected, causing a decrease in the mass transfer rate, and accordingly in the precipitation kinetics, generating larger parti- cles.

the precipitation kinetics, generating larger parti- cles. Fig. 8. SEM micrographs of the precipitated PHBV at

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.

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E. Franceschi et al. / J. of Supercritical Fluids 47 (2008) 259–269

et al. / J. of Supercritical Fluids 47 (2008) 259–269 Fig. 9. SEM micrographs of the

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 polymermass 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 CO 2 , 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 CO 2 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.

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269

Acknowledgments

The authors thank CNPq and CAPES for the financial support and scholarships.

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