Journal of Food Engineering 88 (2008) 381387

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Journal of Food Engineering

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Fractionation of sh oil with supercritical carbon dioxide

Ana Paula Antunes Corra a, Camila Arantes Peixoto a, Lireny Aparecida Guaraldo Goncalves b,
Fernando Antonio Cabral a,*
a
Departamento de Engenharia de Alimentos, Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, 13083-862 Campinas, SP, Brazil
b
Departamento de Tecnologia de Alimentos, Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, 13083-862 Campinas, SP, Brazil

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

Article history: In order to verify the possibility of using supercritical CO2 (SC-CO2) to concentrate polyunsaturated x3
Received 2 July 2007 fatty acids in the form of natural sh oil, experimental phase equilibrium data were measured for a CO2
Received in revised form 3 January 2008 sh oil system. Phase equilibrium was measured at temperatures from 301 to 323 K and pressures from
Accepted 25 February 2008
7.8 to 29.4 MPa, using the dynamic method. The oil solubility and fatty acid composition of the extracts
Available online 5 March 2008
were measured and these data used to verify CO2 selectivity. The best operational conditions to fraction-
ate the oil were 7.8 MPa and 301.15 K, although in all the conditions analyzed, eicosapentaenoic acid
Keywords:
(EPA) could not be fractionalized. The equilibrium for a mixture of 50% sh oil and 50% babassu (Attalea
Fish oil
Phase equilibrium
funifera) fat was measured in order to understand how the fatty acid composition inuenced the fraction-
Supercritical carbon dioxide ation. It was observed that the solubility of the mixture was an average of the individual solubility values,
x3 Fatty acids and the composition of the extracts could be calculated from the individual solubility of each component.
2008 Elsevier Ltd. All rights reserved.

1. Introduction distillation, since relatively high temperatures are required for

There is commercial interest in obtaining polyunsaturated fatty
acids, in particular EPA (eicosapentaenoic acid) and DHA (docosa-
hexaenoic acid). These x3 fatty acids are an important role in
the prevention of a lot of human diseases, such as in lowering
blood cholesterol and thus preventing heart diseases. Today it is
known that x3 fatty acids are essential for normal growth and
development and may play an important role in the prevention
and treatment of coronary artery disease, hypertension, arthritis,
others inammatory and autoimmune disorders, and cancer. Stud-
ies with newborns indicate that DHA is essential for the normal
functional development of the retina and brain, particularly in pre-
mature infants. Several sources of information suggest that man
evolved on a diet with a ratio of x6 to x3 fatty acids of 1
whereas today this ratio is 10:1 to 2025:1, indicating that wes-
tern diets are decient in x3 fatty acids (Simopoulos, 1991). The
main dietary sources of EPA and DHA are sh, such as sardines,
mackerels and anchovies. Thus much research has been done on
EPA and DHA fractionation from sh oil due to potential applica-
tions in the food and pharmaceutical industries.
Decomposition or degradation of thermolabile compounds can-
not be avoided in a conventional separation method, e.g., vacuum
* Corresponding author. Tel.: +55 19 3521 4030; fax: +55 19 3521 4027.
E-mail address: cabral@fea.unicamp.br (F.A. Cabral).
these processes. A supercritical uid separation process using car-
bon dioxide as the solvent offers potential advantages because it is
non-ammable, non-toxic and inexpensive, and can be used under
mild operational conditions. Some researches on the supercritical
extraction of sh oil and mainly of fractionation of EPA and DHA
as fatty acid ethyl esters from sh oils are shown in Table 1, such
as the SC-CO2 extraction of oil from sardine (Ltisse et al., 2006);
the inuence of moisture from the matrix (Atlantic Mackerel) on
the oil yield and on the changes in sarcoplasmatic proteins of ma-
trix after SC-CO2 extraction (Dunford et al., 1997); the SC-CO2 frac-
tionation of EPA and DHA as fatty acid ethyl esters (Perretti et al.,
2007; Jachmanin et al., 2007; Gironi and Maschietti, 2006; Jaubert
et al., 2001; Riha and Brunner, 1999; Espinosa et al., 2002; Alkio
et al., 2000) and the phase equilibrium for SC-CO2 and sh oil,
including free fatty acids, cholesterol, wax esters, di- and triacyl-
glycerols were carried out (Borch-Jensen and Mollerup, 1997) in
order to supply information on the deodorization of sh oil by
stripping. The measurement show that sh oil may be deodorized
to remove the free fatty acids, cholesterol and most of the wax es-
ters at temperatures above 353 K and pressures at 350500 bar.
Increasing the temperature at constant pressure or decreasing
pressure at constant temperature increase the selectivities of CO2.
Studies have indicated that from a nutritional point of view, the
ingestion of fatty acid esters leads to intestinal problems (El-Bou-
stani et al., 1987; Lawson and Hughes, 1988; Hamazaki et al.,

0260-8774/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2008.02.025
382 A.P.A. Corra et al. / Journal of Food Engineering 88 (2008) 381387

Table 1
Some researches of SC-CO2 extraction of sh oil and of fractionation of EPA and DHA

Material Process T (C) P Analysis Observation Ref.

(bar)
Sardine heads SC-CO2 extraction of oil 50 100 Oil yield, EPA and DHA composition Better results at 75 C 300 bar (a)
80 350
Atlantic Mackerel SC-CO2 extraction of oil 35 345 Oil yield, changes in protein. 3.864% of Better results at 10.2% of moisture (b)
moisture
Commercial mixture of sh SC-CO2 fractionation of 40 100 EPA and DHA EPA/DHA ratio x3/x6 Possibility to obtain EPA and a (c)
oil fatty acid ethyl esters. fatty acid ethyl esters in a 60 300 ratio solvent mass ow rate inuence DHA and EPA/DHA ratio for
64%(EPA+DHA) three stages column market purpose
Fatty acid ethyl esters from SC-CO2 extraction of ethyl 40 86.3 Ethyl esters composition solubility, Increase of DHA content from (d)
Hake liver oil esters 70 180.4 selectivity of CO2 for EPA and DHA 17.5% to 55%
Fatty acid ethyl esters from a SC-CO2 extraction of ethyl 42 101 Thermodynamic modeling process Better results were obtained at (e)
mixture of sardine, esters 70 172 simulation 70 C 167 MPa
anchovis and mackerel oil
(95.7%) EPA and (93.6%) DHA High pressure variable- 30 47 Vaporliquid equilibria data for CO2 EPA For each system, 70 equilibrium (f)
ethyl esters volume view cell 80 242 ethyl ester and for CO2 DHA ethyl ester pressure (dew or bubble) were
thermodynamic modeling measurement
13 different fatty acid ethyl High pressure variable- 40 90 Thermodynamic modeling vaporliquid An equation of state is sufcient (g)
ester mixtures derived volume view cell 80 250 equilibria data for CO2 ethyl ester to correlate phase equilibrium
from sardine oil mixtures
Complex mixtures of sh oil Simulations models for Thermodynamic modeling. Simulation A group contribution equation of (h)
fatty acid ethyl esters process units state (GC-EOS) was used.
Fatty acid ethyl esters Supercritical uid 65 145 Concentration DHA ester concentrates up to 95% (i)
mixture from tuna oil chromatography purity were obtained
Fish oil from the sand eel High pressure variable- 20 60 Vaporliquid equilibria data for free fatty The sh oil may be deodorized at (j)
(Ammodytes lancea) volume view cell 120 650 acid, cholesterol and triacylglycerols temperature above 353 K and
selectivity of CO2 pressure at 350500 bar

(a) Ltisse et al., 2006; (b) Dunford et al., 1997; (c) Perretti et al., 2007; (d) Jachmanin et al., 2007; (e) Gironi and Maschietti, 2006; (f) Jaubert et al., 2001; (g) Riha and
Brunner, 1999; (h) Espinosa et al., 2002; (i) Alkio et al., 2000 (j) Borch-Jensen and Mollerup, 1997.

1982), so is preferable the ingestion of EPA and DHA from sh oil at
the natural form instead of fatty acid esters form. In sh oil, the
EPA and DHA are preferentially located in the middle carbon of
the glycerol backbone, so it is not possible to obtain sh oil prod-
ucts with very high EPA and DHA concentration if the oil is frac-
tionated as triacylglycerols, due to this, no studies were found in
the literature presenting the phase equilibrium involving CO2
and triacylglycerols, aiming at the fractionation of the x3 fatty
acids, EPA and DHA, but would be interesting for fractionation sh
oil with low x3 content. In this work was studied the fraction-
ation of triacylglycerols from sh oil using the SC-CO2 as a function
of the temperature and pressure. The fatty acid composition of SC-
CO2 extract were measured, which aim was to obtain fractions en-
riched on x3 fatty acid, specially the EPA and the DHA. The fatty
acid composition of sh oil and of SC-CO2 extracts was used to cal-
culate the separation factor that indicate the CO2 selectivity and
verity the possibility of fractionation.
2. Materials and methods
2.1. Material
Fish Oil (Ropufa 30 x3 Food Oil, Lot. M005875, Derbyshire,
Ukraine) with more than 25% of (DHA, EPA, DPA), was donated
by Roche (So Paulo, SP, Brazil). The samples were stored at
18 C and liqueed for use in the phase equilibrium experiments.
Liquid carbon dioxide, with a purity of 99% was acquired from
White Martins (Campinas, So Paulo, Brazil).
2.2. Apparatus and procedure
The experimental apparatus used in this work is shown in Fig. 1.
It consisted of a 100 mL equilibrium cell (stainless steel AISI 316,
Suprilab, Campinas, Brazil) (vii) immersed in a water bath con-
trolled by a heater (Suprilab, Campinas, Brazil) to within 0.1 C.

Manometer Manometer

v vi Flow
meter
x
iii
vii

Extractor
HP pump viii
Extract
CO2 cilinder

CO2 tank

i iv Solvent
ix
Refrigeration ii
bath
Peristaltic pump
Constant-temperature water bath

Fig. 1. Experimental system: CO2 cylinder (i), refrigeration bath (ii), high pressure pump (iii), CO2 tank (iv), manometers (v,vi), equilibrium cell (vii), Erlenmeyer ask (viii),
peristaltic pump to clean the system (ix).
 A.P.A. Corra et al. / Journal of Food Engineering 88 (2008) 381387 383

The CO2 from the supply tank (i) was cooled to a liquid state
(refrigerated bath model 12101-30, Cole Parmer Instrument
Company, Vernon Hills, IL, USA) (ii) and compressed into the
equilibrium cell by a high-pressure pump (Model AA100S, Eldex
Laboratories Inc., Napa, CA, USA) (iii).
The experimental conditions were 301 to 323 K and 7.8 to
29.4 MPa. The solubility was measured two or three times and
the average solubility value calculated, the values being obtained
with about 4% standard deviation. In a typical experimental run,
10 g of sh oil, adhered to glass balls with an average diameter
of 3 mm, were placed in the equilibrium cell. The SC-CO2 owed
through the extractor at the ow rate where equilibrium was
reached. The experiments were continued until a 0.5 g sample of
sh oil extract had been collected at the exit to the equilibrium
cell. The CO2 ow rate was maintained at approximately 13 mL/s
(at 0.93 bar and 298 K). This requirement was established based
on preliminary tests, whose methodology has been discussed in
detail elsewhere (Rodrigues et al., 2008). It was determined in
the experiments that a ow rate lesser than 25 mL/min was been
suitable to obtain the saturation of triacylglycerols in the SC-CO2
extracts. So as to guarantee the equilibrium, a ow rate of 13 mL/
s was used in all the experiments.
2.3. Solubility
The solubility of the oil was calculated as the ratio between the
amount of oil obtained in the extract and the carbon dioxide mass
used for this purpose.
containing 18% EPA and 12% DHA was the standard adopted. Quan-
tication of the original sample was carried out using C23:0 FR/
100 mg from Nu-Check as the internal standard.
The methyl esters were prepared according to the American Oil
Chemists Society (1997) with some modications: petroleum
ether was used instead of heptane and the ester fraction was sep-
arated using a micropipette instead of a separating funnel for oper-
ational reasons which did not alter the result. The samples were
analyzed in duplicate and their mean standard deviations were
about 3%.
2.5. Separation factor
The selectivity of CO2 in the separation of each compound (i)
was calculated using the separation factor bI, which shows how
many times the lighter phase is more concentrated than the hea-
vier phase at equilibrium in terms of each fatty acid (i) in the oil,
on a CO2 free basis. It was calculated as follows:
mass fraction of component  i in lighter phase on a CO2 free basis
bi
mass fraction of component  i in heaver phase on a CO2 free basis
2
where the mass fraction of all the components in the heavier phase
was xed at its original concentration in the sh oil. This was pos-
sible because the mass of the oil contents in the extracts was signif-
icantly smaller than the mass in the heavier phase.
3. Results and discussion

 mass of oil
Y 1
mass of CO2 3.1. Characterization of the sh oil and its solubility in supercritical
CO2
where Y is the mass fraction of oil in the light phase, calculated
from the mass of oil collected and the total mass of CO2 which Table 3 shows the fatty acid composition of the sh oil, where
owed. 78% of the total was identied and 31% of this amount represented

2.4. Gas chromatographic analyses

Quantication of the fatty acids in the sh oil and its extracts Table 3
Fish oil fatty acid composition
was carried out by gas chromatography using an Agilent-6850, ser-
ies CG system (split/splitless). The fatty acid methyl esters were Fatty acid Area (%)
separated using an Agilent DB-Wax column (10 m  0.100 mm Myristic M C14:0 6.13
i.d.  0.20 lm of lm). As shown in Table 2, three chromatographic Palmitic P C16:0 15.9
conditions (A, B, C) were used during the experiments to determine Palmitoleic Po C16:1 7.02
Stearic S C18:0 1.24
the fatty acid compositions. These modications were carried out
Oleic w11 O C18:1 x11 3.52
when the chromatographic column presented problems in peak Oleic w9 O C18:1 x9 10.9
separation. c-Linolenic Ln C18:3 2.64
The fatty acid composition of the original sh oil and the Eicosapentaenoic acid E C20:5 x3; EPA 12.4
Docosapentaenoic acid Do C22:5 x3 1.94
extracts was obtained as an area percentage and identied by
Docosahexaenoic acid D C22:6 x3; DHA 16.7
comparison with a standard: votag from Ativus Farmacutica, Minority compounds (%) 21.6

Table 2
Conditions adopted to determine the fatty acid composition

Analytical method
A B C
Stripping gas Helium Helium Helium
Sample volume 1 lL 1 lL 1 lL
Split ratio 1:41. 2 1:30 1:30
Column ow rate 3.3 lL/s 5.0 lL/s 5.0 lL/s
Linear velocity 0.28 m/s 0.30 m/s 0.30 m/s
Detector temperature 593.15 K 593.15 K 593.15 K
Injector temperature 573.15 K 573.15 K 573.15 K
Oven temperature and heating time (s)
Initial 413.15 K60 s 413.15 K60 s
Rise 413.15 a 513.15 K 600 s 413.15 a 513.15 K 600 s 433.15 a 530.15 K 2400 s
Final 530.15 K540 s 530.15 K1140 s
384 A.P.A. Corra et al. / Journal of Food Engineering 88 (2008) 381387

Table 4 313.15 K at 29.4 MPa and from 313.15 to 323.15 K at 24.5 MPa
Fish oil solubility in SC-CO2 showed no variation in the values for solubility. The highest values
Temperature (K) Pressure (MPa) CO2 density (kg/m3) Solubility (g/kg) for solubility found in this study were about 7.0 g oil/kg CO2 at
303.15 29.4 946 7.0 0.3 29.4 MPa and the different temperatures, and higher values can
313.15 19.6 837 2.8 0.1 be expected at higher pressures. For Atlantic Mackerel oil, the high-
24.5 877 3.5 0.1 est solubility observed was 14.21 g/kg CO2 at 34.5 MPa and
29.4 907 7.1 0.3 308.15 K (Temelli et al., 1995).
323.15 14.7 694 2.0 0.1
19.6 780 2.3 0.1
The behavior of the solubility values obtained in this study was
24.5 831 3.6 0.1 consistent, considering that the solubility of a solute is directly
29.4 867 6.6 0.2 dependent on the density of the solvent, and that increments in
306.15 7.8 574 0.45 0.02 pressure at constant temperature promote an increase in the den-
301.15 7.8 733 0.52 0.03
sity of the solvent. In the pressure range studied, the solubility did
not show a crossover point for retrograde behavior. Knowledge of
the solubility of the sample under study is important to estimate
by EPA, DPA and DHA, which are the fatty acids of major interest. the economic viability of the process, due to the fact that the great-
The composition obtained is a typical composition for marine sh er the value for solubility, will lesser the consumption of solvent. In
oils such as cod-liver oil, and anchovy oil which contains about 14 general, the solubility of oils can be estimated by correlations as a
31% of DHA and EPA (AOCS, 1997). proposed by Del Valle and Aguilera (1998).
Table 4 shows the solubility of the oil in SC-CO2 as a function of
temperature and pressure or as a function of temperature and den- 3.2. Fractionation of sh oil with SC-CO2
sity. Since the light phase of the equilibrium mixture for the sys-
tems under study was only slightly concentrated in the solute, Tables 5 and 6 show the fatty acid composition of sh oil (ori-
the density was considered to be that of pure CO2 and they were ginal) and of its extracts, under different operational conditions
calculated by an empirical equation (Huang et al., 1985) which of temperature and pressure, and Table 7 shows the values of sep-
reproduces the experimental values of IUPAC (Angus et al., 1976). aration factor bi, which were calculated from these compositions.
The results show that the solubility of the sh oil increased with The bi values indicated that the triacylglycerols with short chain
increase in pressure at constant temperature, the effect of temper- fatty acids tend to migrate into the lighter phase, which is rich in
ature being less meaningful. With an increase from 313.15 to CO2, inasmuch as compounds with lesser molar mass are more
323.15 K at 19.6 MPa, the solubility decreased by about 18%, and soluble in SC-CO2. For the fatty acids C16:0 and C16:1 and for
the same increase in temperature at 29.4 MPa resulted in a de- C22:5 n-3 and C22:6 n-3, which are only different in the degree
crease in solubility of only about 7%. An increase from 303.15 to of unsaturation, it was observed that the values for bi (Table 7)

Table 5
Fatty acid composition of the sh oil and its extract (%) at equilibrium

Component Original 1 29.4 MPa 323.15 K 24.5 MPa 323.15 K 19.65 MPa 323.15 K 24.5 MPa 313.15 K Original 2 7.8 MPa 301.15 K
C14:0 6.00 8.39 8.55 9.33 8.51 6.13 9.91
C16:0 15.4 17.3 17.7 17.9 17.4 15.9 17.4
C16:1 6.99 8.96 9.10 9.59 9.02 7.04 9.45
C18:0 1.35 1.65 1.62 1.68 1.64 1.24 1.56
C18:1 n-11 3.39 3.04 3.03 1.74 2.04 3.52 2.79
C18:1 n-9 12.7 13.5 10.9 11.2 11.0 10.9 10.9
C18:3 2.74 3.01 2.98 3.03 3.15 2.64 2.93
C20:5 n-3; EPA 12.6 12.7 12.6 12.4 13.0 12.4 11.7
C22:5 n-3 2.07 1.54 1.54 1.46 1.61 1.94 1.27
C22:6 n-3; DHA 15.5 13.0 12.9 12.4 13.4 16.7 11.2
Total 78.7 83.0 81.0 80.7 80.8 78.5 79.1
Minority compounds 21.3 17.0 19.1 19.3 19.2 21.6 20.9

Average standard deviation was a maximum of 3%.

Table 6
Fatty acid composition of the sh oil and its extract (%) at equilibrium

Component Original 3 29.4 MPa 303.15 K 19.6 MPa 313.15 K 29.4 MPa 313.15 K 14.7 MPa 323.15 K 7.8 MPa 306.15 K
C14:0 5.78 7.56 7.77 7.36 6.5 6.15
C16:0 14.9 15.7 17.0 15.7 15.5 15.5
C16:1 6.75 8.21 8.53 8.03 7.31 7.04
C18:0 1.20 1.32 1.37 1.26 1.15 1.14
C18:1 n-11 3.32 1.68 2.30 1.76 2.42 2.12
C18:1 n-9 12.5 10.3 11.0 10.2 12.5 12.5
C18:3 2.58 2.94 2.81 2.80 2.50 2.39
C20:5 n-3; EPA 12.2 12.6 12.3 12.1 11.6 11.4
C22:5 n-3 2.10 1.70 1.58 1.78 1.94 1.96
C22:6 n-3; DHA 16.0 13.3 13.1 12.8 14.2 14.4
Total 77.2 75.3 77.8 73.8 75.6 74.5
Minority compounds 22.8 24.7 22.2 26.2 24.5 25.5

Average standard deviation was a maximum of 3%.

 A.P.A. Corra et al. / Journal of Food Engineering 88 (2008) 381387 385

Table 7
Selectivity of SC-CO2 for the sh oil in terms of its fatty acids (calculated as the Separation Factor (bI)

T (K) P (MPa) Separation factor bI

C14:0 C16:0 C16:1 C18:0 C18:1 n-11 C18:1 n-9 C18:3 C20:5 n-3 EPA C22:5 n-3 C22:6 n-3 DHA
303.15 29.4 1.31 1.06 1.22 1.10 0.51 0.83 1.14 1.04 0.81 0.83
313.15 19.6 1.34 1.14 1.26 1.14 0.69 0.88 1.19 1.01 0.75 0.82
24.5 1.42 1.13 1.29 1.21 0.60 0.87 1.15 1.04 0.78 0.86
29.4 1.27 1.06 1.19 1.05 0.53 0.82 1.09 0.99 0.85 0.80
323.15 14.7 1.12 1.04 1.08 0.96 0.73 1.00 0.97 0.95 0.92 0.89
19.6 1.56 1.17 1.37 1.24 0.51 0.88 1.11 0.99 0.71 0.79
24.5 1.43 1.15 1.30 1.20 0.89 0.86 1.09 1.00 0.74 0.83
29.4 1.40 1.12 1.28 1.22 0.90 1.06 1.09 1.01 0.75 0.83
306.15 7.8 1.06 1.04 1.04 0.95 0.64 1.00 0.92 0.93 0.93 0.90
301.15 7.8 1.61 1.09 1.34 1.26 0.79 0.99 1.11 0.94 0.65 0.67

for C16:1 were higher than for C16, and similarly, were higher for tionation process for these compounds. In the present study with
C22:6 than for C22:5, indicating afnity by the CO2 for the condi- SC-CO2 at 301.15 K and 7.8 MPa, a bI = 0.94 was obtained for EPA
tion of unsaturation. However, the reverse behavior was observed and bI = 0.67 for DHA (6% and 33% of enrichment in the heavier
when the fatty acids C18:0, C18:1 and C18:3 were compared. phase).
For all the operational conditions studied, except at 7.8 MPa, the For a better analysis of the fractionation, one sample of crude
bi values obtained for EPA was practically equal to unity, indicating sh oil was placed in the equilibrium cell and 40% of the mass of
the difculty of fractionation of triacylglyecerols containing this the sample was extracted. Table 8 shows the fatty acid composi-
x3 fatty acid. tion of both the extract and rafnate on a CO2 free basis and the ra-
Under the operational conditions ranging from 306.15 to tio between the compounds of extract and rafnate. The results
329.15 K and from 7.8 to 29.4 MPa the bi values for DHA varied show an extract richer in short chain fatty acids (C14 and C16)
from 0.80 to 0.90. Interesting behavior was found under the condi- increasing from 22% to 28% and poorer in DHA, decreasing from
tion of 301.15 K and 7.8 MPa, where bI was 1.61 for C14:0 and 0.67 16.7 to 11%, but it can be observed that the concentrations of
for DHA (33% less in the lighter phase than in the heavier phase), EPA in the extract and in the rafnate were almost the same. From
indicating a better possibility of fractionation under these condi- the original sh oil with 31% of x3, two fractions were obtained,
tion. These results indicate that the CO2 is more selective in the one with 23.5 and the other with 36.1% of x3.
zone near of its critical point to fractionate the triacylglycerols of
sh oil. However, the solubility of 0.52 g/kg CO2 obtained for the 3.3. Fractionation of a mixture of 50% sh oil with babassu fat using
sh oil at 301.15 K and 7.8 MPa was much lower that the solubility SC-CO2
under the other operational conditions (Table 4). The low value for
solubility can limit the process because of the high consumption of In order to better visualize how the fatty acid composition of
solvent. the triacylglycerols, the molar mass and the degrees of unsatura-
Borch-Jensen and Mollerup (1997) measured the phase equilib- tion inuenced the selectivity of the solvent, a mixture containing
ria for sub and SC-CO2 and sh oil from sand eel (Ammodytes equal parts of sh oil and babassu fat was prepared, and the frac-
lancea) which was consisting of triacylglycerols, free fatty acids, tionation analyzed. The possibility of babassu fat to inuence into
vitamins and cholesterol, amongst other components. The equilib- the equilibrium and in the fractionation of sh oil was also ana-
rium data were presented for the different compounds of the crude lyzed. Babassu fat was chosen because it contains a differentiated
sh oil according to the length of the carbon chain, however, with- fatty acid composition in comparison with the sh oil, being com-
out differentiate them as to the degree of unsaturation. Under all posed of approximately 80% of saturated fatty acids (C8:0 to C18:0)
the operational conditions analyzed, the values for the partition and 20% of unsaturated C18:1 and C18:2 (AOCS, 1997; Gioielli
coefcient on a solvent free basis (equivalent to bi as dened in this et al., 1998). Table 9 shows the composition of the sample used
study) showed decreasing values with increasing molar mass, for in this study and Table 10 shows the composition in fatty acids
the same class of compounds. Thus, for 20.01 MPa and 313.2 K, of the mixture of sh oil and babassu fat (in equal parts) and the
the values for bi of the free fatty acids were 52.9 for C:14 (fatty acid
with 14 carbon atoms); 9.65 for C16 and 5.21 for C22. The values of
bi for the triacylglycerols were 1.93 for triacylglycerols with 46 car-
bon atoms and 0.21 for the triacylglycerol with 66 carbons. The Table 8
partition coefcient obtained for the C14 fatty acid was to the or- Fatty acid composition (mass%) of sh oil fractionated at 301.15 K and 7.8 MPa
der of ve times higher.
Fatty acids Original (2) Extract Rafnate Extract/Rafnate
It can be observed that there is an increase in the values for bi
C14:0 6.13 9.91 3.58 2.76
with a reduction in carbon chain length. For example, at
C16:0 15.9 17.4 14.2 1.22
19.6 MPa and 313.1 K, the value for bi was 1.34 for C14:0 and C16:1 7.04 9.45 5.02 1.88
1.14 for C16:0. In other words, under the same operational condi- C18:0 1.24 1.56 0.98 1.59
tions of temperature and pressure, the CO2 was more selective for C18:1 x 11 3.52 2.79 2.23 1.25
C14:0 than for C16:0. However, fatty acids in the form of triacylgly- C18:1x 9 10.9 10.9 10.4 1.04
C18:3 2.64 2.93 2.26 1.30
cerols have complex associations. In the case of free fatty acids, the C20:5 n-3; EPA 12.4 11.7 12.3 0.95
difference between the bi values for myristic (C14) and palmitic C22:5 n-3 1.94 1.27 2.51 0.51
(C16) acids is higher according to reports in the literature (Bor- C22:6 n-3; DHA 16.7 11.2 21.3 0.53
ch-Jensen and Mollerup, 1997). However, this did not occur in this Total 78.5 79.1 74.8
Minority compounds 21.6 20.9 25.2
study, probably because they were associated to polyunsaturated
fatty acids in molecules of triacylglycerols, complicating the frac- Rafnate Oil that remained in the equilibrium cell after extraction.
386 A.P.A. Corra et al. / Journal of Food Engineering 88 (2008) 381387

Table 9 Table 11
Fatty acid composition of babassu fat Fractionation of babassu fat with SC-CO2 at 301.15 K and 7.8 MPa

Fatty acid Mass percentage in Babassu fat Fatty acid Original Extract bi
AOCS Gioielli et al., 1998 This study C8:0 Cp 6.46 7.94 1.22
C10:0 C 5.26 6.21 1.18
C8:0 Cp 2.67.3 5.5 6.5
C12:0 L 42.97 45.89 1.06
C10:0 C 1.27.6 6.1 5.3
C14:0 M 15.67 15.09 0.96
C12:0 L 40.055.0 34.1 43.0
C16:0 P 8.76 7.70 0.88
C14:0 M 11.027.0 19.2 15.7
C18:0 S 3.77 3.21 0.85
C16:0 P 5.211.0 10.6 8.8
C18:1 O 15.54 12.88 0.83
C18:0 S 1.87.4 4.3 3.8
Others 1.54 1.06 0.69
C18:1 O 9.020.0 17.1 15.5
Others 1.46.6 3.1 1.4 Solubility (g/kg CO2) 4.3 0.2

composition of the extracts obtained in phase equilibrium with SC-

CO2 and their respective values for solubility.
In agreement with the bI values shown in Table 10, the results
indicated that in this case, fractionation should be possible, espe-
cially at 301.15 K and 7.8 MPa, where a value for bi of 2.34 was ob-
tained for C12:0, 1.37 for C14:0, 0.22 for EPA and 0.16 for DHA.
SC-CO2 was more effective in fractionating oils with distinctive
compositions in triacylglycerols. Comparing Table 10 with Table 4,
it can also be observed that the solubility of the mixture of sh oil
and babassu fat (1:1) was higher than the solubility of the sh oil,
due to the content of short chain fatty acids (lower molar mass)
from the babassu fat. This behavior can be veried in the literature
(Soares et al., 2007).
In order to verify if the babassu fat show some inuence on the
separation of the compounds of interest, the solubility of the fatty
acids EPA and DHA in the extracts of sh oil and in the mixture of
sh oil and babassu fat (1:1) were analyzed under the operational
conditions of 7.8 MPa and 301.15 K. The extracts obtained under
these distinctive situations resulted in triacylglycerols containing
11.7 (Table 5) and 1.45% (Table 10) of EPA, corresponding to values
for solubility of 0.061 and 0.032 g (EPA)/kgCO2. In an analogous
way, they contained 11.24% and 1.32% of DHA, with solubility val-
ues corresponding to 0.058 and 0.029 g (DHA)/kgCO2, conrming
that the solubility values for the triacylglycerols containing EPA
and DHA, reduced to half the original value in the mixture (sh
oil and babassu fat). This behavior indicates that the babassu fat
did not promote a change in the fractionation of the sh oil.
3.4. Fractionation of babassu fat with SC-CO2
The solubility of babassu fat in SC-CO2 and the fatty acid com-
position of its extracts were measured at 301.15 K and 7.8 MPa
and the results are presented in Table 11. It can be veried that
the bI values for each (i) fatty acid decreased with increase in size
of the fatty acid carbon chain. Short chain compounds like C8:0,
C10:0 and C12:0, hence of lower molecular mass, tended to mi-
grate to the lighter phase, whereas compounds with relatively
longer chains, like C14:0, C16:0, C18:0 and C18, tended to stay in
the heavier phase.
4. Conclusions
Under the operational conditions studied, supercritical CO2 was
little effective in fractionating sh oil in the triacylglycerol form.
Eicosapentaenoic acid (EPA; C20:5 n-3) was the compound show-
ing the greatest resistance to fractionation, its separation factors
bi being practically unity under all the operational conditions stud-
ied. The difculty in fractionation can be attributed to the complex
structure of the sh oil, composed of many triacylglycerols.
At 7.8 MPa and 301.15 K (zone of compressed liquid), interest-
ing data was obtained, where the values for bi for myristic acid
(C14:0) and for DHA were 1.64 and 0.67, respectively. These values
for bi indicate the possibility of fractionation under these
conditions.
SC-CO2 was able to fractionate the babassu fat, the calculated bI
values being different from unity. Supercritical CO2 can fractionate
oils, depending on the composition of triacylglycerols in the sam-
ple. Oils that exhibit distinct triacylglycerol fractions in terms of
the fatty acid composition and mass percentage seem to be easy
to fractionate. The triacylglycerol composition is determinant in
predicting the possibility of fractionating the oil with supercritical
CO2.
Considering that the x3 fatty acid composition of the sh oil
used in this study was high, almost 1/3 of the total fatty acids, it
was highly probable that the fatty acids of interest would be pres-

Table 10
Fractionating data for the mixture of sh oil and babassu fat

Fatty acid Operational conditions 19.6 MPa 323.15 K 7.8 MPa 306.15 K 7.8 MPa 301.15 K
Composition of the mixture (%) Composition of the extracts and bi
% bi % bi % bi
C8:0 3.10 5.09 1.64 3.20 1.03 7.10 2.3
C10:0 2.59 4.31 1.66 2.84 1.1 6.66 2.6
C12:0 21.09 39.51 1.87 24.82 1.18 49.28 2.3
C14:0 10.60 15.50 1.46 11.04 1.04 14.50 1.4
C16:0 12.14 10.07 0.83 11.21 0.92 6.80 0.56
C16:1 3.54 2.17 0.61 3.11 0.88 1.17 0.33
C18:0 2.43 3.08 1.27 2.11 0.87 2.05 0.84
C18:1 n-9 12.08 8.59 0.71 10.64 0.88 5.38 0.45
C18:3 1.35 0.65 0.48 1.13 0.84 0.36 0.27
C20:5 n-3; EPA 6.50 2.84 0.44 5.64 0.87 1.45 0.22
C22:5 n-3 1.18 0.34 0.29 1.13 0.96 0.36 0.31
C22:6 n-3; DHA 8.32 2.81 0.34 7.24 0.87 1.32 0.16
Solubility (g/kg) 6.95 0.22 2.34 0.07 2.23 0.07
 A.P.A. Corra et al. / Journal of Food Engineering 88 (2008) 381387
ent in almost all the triglyceride molecules, as evidenced by the
difculty in fractionating them. Thus it is possible that it could
be used to enrich sh oil containing a lower x3 fatty acid
composition.
Acknowledgments
The Grant for this research was supported by Fapesp (process
01/09957-9) and CNPq. Corra, A.P. is grateful to CNPq for the mas-
ters degree Grant and to Roche Pharmaceutical (Brazil) for supply-
ing the sh oil.
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