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J.

of Supercritical Fluids 40 (2007) 349353

Supercritical carbon dioxide fractionation of fish oil fatty acid ethyl esters
G. Perretti a, , A. Motori b , E. Bravi a , F. Favati c , L. Montanari a , P. Fantozzi a
aDepartment of Economic and Food Sciences, Division of Food Technology and Biotechnology, University of Perugia,
Via S. Costanzo n.c.n., I-06126 Perugia, Italy
b Department of Applied Chemistry and Materials Science, University of Bologna, Viale Risorgimento 2, I-40136 Bologna, Italy
c Department of Biology DBAF, University of Basilicata, Viale dellAteneo Lucano, I-85100 Potenza, Italy

Abstract
Fractionation of fish oil fatty acid ethyl esters was investigated with the aim of obtaining a lipid fraction enriched in -3 fatty acids and with a
suitable EPA/DHA ratio. The results obtained highlight the possibility of modifying the original fatty acid ethyl esters concentration by optimizing
the extraction conditions in terms of pressure, temperature, and supercritical carbon dioxide flow rate. Supercritical fluid fractionation (SFF) appears
to be a useful processing technique for changing the composition of lipids in order to obtain high value functional products. The use of proper
fractionation temperatures and pressures along the column influenced the solvent-to-feed ratio to obtain fractions with suitable composition for
market requirements.
2006 Elsevier B.V. All rights reserved.

Keywords: Supercritical CO2 ; Fractionation; Fish oil fatty acid ethyl esters; -3

1. Introduction and, in particular, the correct ratio between -3 and -6 fatty


acids is very important. The use of a dietary supplement con-
Interest in -3 fatty acids (e.g. C 18:3 alpha-linolenic taining essential fatty acids in a correct ratio provides the body
acidALA, C 20:5 eicosapentaenoic acidEPA, C 22:6 with useful assistance in dealing with a large number of sys-
docosahexaenoic acidDHA) began several years ago and now temic and cutaneous disorders, thanks to an anti-inflammatory,
there is a wide scientific literature supporting their positive role vasodilating and anti-platelet aggregation action. Furthermore,
for human health, because they can intervene in the prevention with regard to cholesterol prevention, a higher concentration of
and modulation of certain diseases that are common in the West- EPA is required in comparison with DHA, while the opposite
ern civilization; researches have shown that -3s can reduce the relative concentration is required during pregnancy or for retina
risk of heart disease and high blood pressure, prevent blood clots, protection (DHA > EPA) [1,5].
protect against cancer and even alleviate depression [1,2]. Fur- Several authors investigated SuperCritical carbon dioxide
thermore, DHA, found in the cell membrane of various tissues (SC-CO2 ) extraction of oils rich in polyunsaturated fatty acids
throughout the body, can represent about 36% of its total fatty (PUFAs) and this technology, having a negligible environmen-
acid content, and can be found in high concentration especially tal impact, could also represent a potential tool to change the
in the retina, brain and sperm [3]. However, in industrialized relative concentration of the various lipid moieties [610].
societies the intake of these important acids has changed in the Eissenbach [6] and Nilsson [1113] studied the fatty acid
last decades. An increased consumption of vegetable oils rich in ethyl esters (FAEE) behavior using batch-continuous equipment
-6 fatty acids and a reduced consumption of foodstuffs (fish, at high temperatures (70100 C), thus decreasing the relative
flaxseed, algae) rich in -3 fatty acids has determined a signifi- densities and influencing the solvent-to-feed ratios to exploit
cant change in the -6 to -3 fatty acids ratio in the human diet, the possibility to enrich the fractions in EPA or DHA in abso-
with negative effects on consumers health. Fish oil is the main lute values [14]. The use of fractionation temperatures of 40 C
source of -3 fatty acids, therefore, the fatty acids composition (bottom) to 60 C (top) along the column, at 100300 bar, can
of fish oil is very important for its functional properties [2,4] influence this ratio to obtain fractions collected from the col-
umn (FCFC) with proper composition in terms of EPA + DHA
Corresponding author. and EPA/DHA ratio for market requirements, and taking into
E-mail address: perretti@unipg.it (G. Perretti). account the yield and energy use.

0896-8446/$ see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.supflu.2006.07.020
350 G. Perretti et al. / J. of Supercritical Fluids 40 (2007) 349353

In this research, starting from commercially available fish Table 1


oil the use of supercritical fluid fractionation (SFF) was investi- Effect of 10 kg/h solvent mass flow rate on fractions recovery at different pres-
sures (g FAEE)
gated with the aim to concentrate the -3 FAEEs and reach high
EPA and DHA concentrations with suitable EPA/DHA ratios. FAEEs fed Pressure (bar)
A batch-continuous fractionation study was conducted and gas- 150 300
chromatographic methods were employed in order to establish
Input (g of FAEEs) 450
how different operating conditions could modify the FAEE com-
position to better satisfy the consumer needs. Output (g of FAEEs)
Separator 030 min 310.97 413.35
Separator 3060 min 103.81 12.35
2. Material and methods Separator 60120 min 4.28 1.94
FCFC 0 0
Column cleaning 1.05 0
The study was conducted working on a commercial mix-
Total 420.11 427.64
ture of fish oil fatty acid ethyl esters containing 74% PUFA,
consisting of 64% EPA + DHA with a 1.6 EPA/DHA ratio, and FCFC: fraction collected from the column.
1% d-alpha tocopheryl acetate. The initial peroxide number was
12 mequiv. O2 /kg, and the initial free acidity was 0.6%.
At set time intervals the resulting products of the fractionation
The FAEE mixture density was 0.900 g/mL and for each frac-
runs were collected from the separator and cyclonic separators
tionation run 450 g of FAEE were loaded onto the column at
and pooled; samples were also collected at the bottom of the
three different column heights using a feeding syringe pump
column at the end of each run for subsequent analyses and after
once the equipment reached the set pressure and temperature
12 h of spontaneous cleaning. The chromatographic analyses
conditions. The experiments were conducted using a pilot plant
were carried out using a 9001 GC (Chrompack, Milan, Italy),
(Muller Extract Company GmbH, Koburg, Germany) equipped
equipped with a split/splitless injection system, a CP-Wax 58 CB
with a three stages column (total length 3 m and 3 cm i.d.)
(Chrompack) capillary column (25 m 0.25 mm i.d., 0.2 m)
having an internal volume of 2.0 L and packed with stainless
and a flame-ionization detector [15].
steel Raschig rings (10 mm 10 mm); each column stage was
individually thermostated. CO2 was fed at the bottom of the col-
umn and recovery of the FAEE was achieved in the separation 3. Results and discussion
section of the plant, consisting of a 1-L cylindrical separator
followed by two cyclonic separators, all set at 30 C and 60 bar The results obtained highlight the different behavior of fatty
(Fig. 1). acids as well as that of the different moieties when submitted to
The 2-h runs were conducted using different pressures (100, fractionation with SC-CO2 at different conditions.
140, 150 and 300 bar) and different liquid CO2 flow rates (2.5, Since the aim of the present research was to study the possible
3.5, 5 and 10 kg/h), keeping the temperatures of the three column enrichment in EPA and DHA of the FAEEs fish oil mixture, the
sections at 40, 50 and 60 C, respectively, starting from the bot- mass balance of each fractionation run was assessed taking into
tom. This determined a density gradient in the solvent stream, account all the recovered fractions, while the FA composition
with SC-CO2 densities varying along the column (e.g. ranging was determined for the fractions collected at the bottom of the
from 0.584 to 0.782 g/mL when working at 150 bar). column.

Fig. 1. Muller SC-CO2 pilot plant scheme of equipment.


G. Perretti et al. / J. of Supercritical Fluids 40 (2007) 349353 351

Table 2 Table 3
Effect of column loading height on the fish oil FAEEs fractions recovery and Fatty acids composition of the fed fish oil FAEEs and of the FCFCs obtained
composition (pressure 150 bar, CO2 flow rate 5 kg/h) with different column loading heights when working at 150 bar and with a 5 kg/h
CO2 flow rate
Collected fractions FAEEs Bottom Intermediate Top
fed feed feed feed FAEE (%) FAEEs fed FCFC

Input (g of FAEEs) 450 Bottom feed Intermediate feed Top feed

Output (g of FAEEs) C16:0 6.59 0.36 0.29


Separator 030 min 164.95 185.05 222.55 C16:1 1.66 0.04 0.02
Separator 3060 min 67.07 67.59 68.73 C17:0 0.03 0.00 0.00
Separator 60120 min 121.59 146.48 111.00 C17:1 0.13 0.10 0.00
FCFC 39.5 13.13 7.62 C18:0 2.89 1.16 0.84
Column cleaning 35.17 14.21 16.91 C18:1 8.69 1.94 1.16
C18:2 0.94 0.25 0.18
Total 428.28 426.46 426.81 C20:0 0.64 0.26 0.71
C18:3 0.57 0.39 0.22
Fed and FCFC composition C20:1 1.12 1.29 0.79
EPA + DHA (% FAEE) 64 82 81 C22:0 0.00 0.15 0.24
EPA/DHA (ratio) 1.61 0.65 0.33 C18:4 5.13 1.03 0.61
C22:1 2.13 0.83 0.54
FCFC: fraction collected from the column.
C20:4 (-6) 0.52 1.36 1.96
C20:4 (-3) 0.84 0.42 1.33
The use of the 10 kg/h CO2 flow rate resulted to be too high, C20:5 39.55 32.39 20.15
and all the material was dragged out of the column both at 150 C22:4 0.09 0.45 1.15
and 300 bar (Table 1). C22:5 (-6) 0.53 2.56 8.72
C22:5 (-3) 1.79 3.61 4.69
Loading of FAEEs at the bottom of the column caused a lower
C22:6 24.54 49.57 61.12
FAEEs extraction in comparison with top and intermediate load-
ing, with a resultant FCFC being about 2.5 times higher in weight FCFC: fraction collected from the column.
(Table 2). The EPA + DHA concentration in the FCFC FAEEs
for the analyzed samples was over 80%, while the EPA/DHA showed always a substantial increase. Somehow contrasting
ratio decreased below 1, indicating a preferential extraction of results were obtained for the C20 fatty acid family; anyway
DHA over EPA. The residual amount of FCFC FAEEs obtained the concentration of the C20 PUFA of interest resulted to be
with the top loading was not analyzed because the yield was too always higher loading the fish oil at the intermediate column
low for commercial purposes. point (Table 3).
The chemical analyses showed that the injection point of the Increasing the solvent pressure from 100 to 150 bar
FAAES mixture onto the column influenced the composition of (0.5820.782 g/mL density at the bottom of the column at 40 C)
the FCFC. In particular, when comparing bottom and interme- resulted in a drop of the residual fraction of material into the
diate loading, the latter injection mode caused an appreciable column at the end of the run from 80% to 9% (Table 4). An
decrease of the C18 fatty acid concentration in the fraction col- intermediate effect was obtained at 140 bar (the flow rate at
lected from the column, while the C22 PUFAs concentration 100 bar was kept at 3.5 kg CO2 /h because at this CO2 compres-

Table 4
Effect of the extraction pressure on the fish oil FAEEs fractions recovery and composition
Collected fractions FAEEs fed Pressure (bar)

100a , = 0.582 g/ml 140b , = 0.763 g/ml 150b , = 0.782 g/ml

Input (g of FAEEs) 450

Output (g of FAEEs)
Separator 030 min 0.93 45.87 164.95
Separator 3060 min 1.80 64.57 67.07
Separator 60120 min 3.10 112.44 121.59
FCFC 371.49 187.03 39.50
Column cleaning 25.80 16.85 35.17
Total 403.12 426.76 428.28

Fed and FCFC composition


EPA + DHA (% FAEE) 64 65 72 82
EPA/DHA (ratio) 1.61 1.69 1.21 0.65

FCFC: fraction collected from the column.


a CO flow rate 3.5 kg/h.
2
b CO flow rate 5 kg/h.
2
352 G. Perretti et al. / J. of Supercritical Fluids 40 (2007) 349353

Table 5
Fatty acids composition of the fed fish oil FAEEs and of the FCFCs obtained using different extraction pressures (100, 140 and 150 bar)
FAEE (%) FAEEs fed FCFC

100a , = 0.582 g/ml 140b , = 0.763 g/ml 150b , = 0.782 g/ml

C16:0 6.59 5.37 2.18 0.36


C16:1 1.66 1.19 0.48 0.04
C17:0 0.03 0.00 0.00 0.00
C17:1 0.13 0.00 0.00 0.10
C18:0 2.89 2.47 2.20 1.16
C18:1 8.69 5.87 3.76 1.94
C18:2 0.94 0.71 0.52 0.25
C20:0 0.64 1.03 1.26 0.26
C18:3 0.57 0.82 0.81 0.39
C20:1 1.12 0.15 0.25 1.29
C22:0 0.00 0.00 0.00 0.15
C18:4 5.13 4.26 2.90 1.03
C22:1 2.13 1.71 1.78 0.83
C20:4 (-6) 0.52 0.94 1.08 1.36
C20:4 (-3) 0.84 0.17 0.18 0.42
C20:5 39.55 40.75 39.44 32.39
C22:4 0.09 0.10 0.11 0.45
C22:5 (-6) 0.53 0.48 0.80 2.56
C22:5 (-3) 1.79 1.84 2.66 3.61
C22:6 24.54 24.18 32.55 49.57

FCFC: fraction collected from the column.


a CO flow rate 3.5 kg/h.
2
b CO flow rate 5 kg/h.
2

sion level the system showed instability for higher solvent flow drop of the FCFC from 44% to 9% of the initial load (Table 6).
rates). The concentration of C16 FAEEs in the FCFC was reduced to
Moreover, the increased working pressure caused a reduc- 0% at the highest flow rates. C18 FAEEs were also reduced,
tion of the C16 and C18 FAEEs concentration in the FCFC, while the changes in C20s were less evident. The concentration
along with a noticeable increase of the C22 PUFAs, while C20 of C22s increased with the increase in the flow rate (Table 7).
FAEEs did not vary their overall concentration. For monounsatu-
rated and saturated fatty acids the values obtained highlighted an Table 7
opposite behavior, being their total percentage higher at 100 bar Fatty acids composition of the fed fish oil FAEEs and of the FCFCs collected
(Table 5). working at 150 bar and different solvent flow rates
At the column working pressure of 150 bar, increasing the sol- FAEE (%) FAEEs fed FCFC
vent mass flow rate from 2.5 to 5 kg/h, resulted in a remarkable
Flow rate (kg CO2 /h)
2.5 4 5
Table 6 C16:0 6.59 4.05 0.33 0.36
Effect of the solvent mass flow rate on the fish oil FAEEs fractions recovery and C16:1 1.66 1.03 0.05 0.04
composition for experiments conducted at 150 bar C17:0 0.03 0.00 0.00 0.00
Collected fractions FAEEs fed Flow rate (kg CO2 /h) C17:1 0.13 0.00 0.00 0.10
C18:0 2.89 2.34 0.96 1.16
2.5 4 5 C18:1 8.69 4.83 1.46 1.94
C18:2 0.94 0.18 0.2 0.25
Input (g of FAEEs) 450
C20:0 0.64 1.13 1.15 0.26
C18:3 0.57 0.84 0.23 0.39
Output (g of FAEEs)
C20:1 1.12 0.20 0.23 1.29
Separator 030 min 63.43 146.63 164.95
C22:0 0.00 0.00 0.00 0.15
Separator 3060 min 61.66 73.33 67.07
C18:4 5.13 3.77 0.89 1.03
Separator 60120 min 78.89 144.32 121.59
C22:1 2.13 1.8 1.39 0.83
FCFC 197.36 48.15 39.5
C20:4 (-6) 0.52 0.67 1.19 1.36
Column cleaning 23 15.33 35.17
C20:4 (-3) 0.84 0.00 0.39 0.42
Total 424.34 427.76 428.28 C20:5 39.55 40.31 29.6 32.39
C22:4 0.09 0.00 0.25 0.45
Fed and FCFC composition C22:5 (-6) 0.53 0.66 2.43 2.56
EPA + DHA (% FAEE) 64 68 79 82 C22:5 (-3) 1.79 2.24 4.12 3.61
EPA/DHA (ratio) 1.61 1.48 0.60 0.65 C22:6 24.54 27.26 49.05 49.57

FCFC: fraction collected from the column. FCFC: fraction collected from the column.
G. Perretti et al. / J. of Supercritical Fluids 40 (2007) 349353 353

Table 8 moreover, the study of blending the different fractions obtained


Omega-3 to -6 PUFA ratio in the FCFC when working at different column could be exploited. Finally, a further optimization of the extrac-
loading heights (a), different solvent pressures (b), different solvent mass flow
rates (c)
tion operative conditions could be considered. During prepa-
ration, the urea adduction could be proposed to decrease the
(a) Column loada amount of saturated fatty acids in the starting material, before
Bottom Intermediate supercritical fractionation [16]. Furthermore, in an industrial
3/6 20.71 8.06
application perspective, the study of continuous-countercurrent
concentration will eventually be developed during the scale-up
(b) Pressure (bar) of the process.
100b , = 0.582 g/ml 140c , = 0.763 g/ml 150c , = 0.782 g/ml
Finally, the nutritional effects of FAEEs have not yet been
completely defined. It is still to be determined if the consumption
3/6 31.81 31.52 20.71 of FAEEs or of the acyl-glyceryl form of fatty acids (glycerides)
(c) CO2 flow rated (kg/h)
is to be preferred from a nutritional point of view. Because of
this, the process setup should take into account all the new inputs
2.5 4 5 concerning the nutritional values of -3s.
3/6 46.12 21.83 20.71
a Pressure 150 bar, CO2 flow rate 5 kg/h.
References
b CO2 flow rate 3.5 kg/h.
c CO2 flow rate 5 kg/h. [1] E. Shahar, A.R. Folsom, S.L. Melnick, M.S. Tockman, G.W. Comstock,
d Pressure 150 bar. V. Gennaro, M.W. Higgins, P.D. Sorlie, W.J. Ko, M. Szklo, Dietary n-3
polyunsaturated fatty acids and smoking-related chronic obstructive pul-
monary disease, N. Engl. J. Med. 331 (1994) 228233.
The values of the -3/-6 ratio assessed for the fractions [2] C. Von Schacky, P. Angerer, W. Kothny, K. Theisen, H. Mudra, Effect
collected from the column and obtained by SFF at different oper- of dietary omega-3 fatty acids on coronary atherosclerosis: a random-
ation conditions are reported in Table 8. ized, double-blind placebo-controlled trial, Ann. Intern. Med. 130 (1999)
The results highlight the possibility of modifying the original 554562.
[3] W.E. Connor, Importance of n-3 fatty acids in health and disease, Am. J.
concentration of the FAEE by optimizing the extraction condi-
Clin. Nutr. 71 (2000) 171S175S.
tions in terms of pressure, temperature, and flow rate of SC-CO2 . [4] P.J. Nestel, Fish oil and cardiovascular disease: lipids and arterial function,
The overall effect exerted for C16, C18 and C22 chain length Am. J. Clin. Nutr. 71 (2000) 228S231S.
fatty acids is evident, while the effect exerted for the different [5] M. Maes, A. Christophe, E. Bosmans, A. Lin, H. Neels, In humans, serum
C20s is more variable changing the extraction conditions. polyunsaturated fatty acid levels predict the response of pro-inflammatory
cytokines to psychological stress, Biol. Psychiatry 47 (10) (2000) 910.
SFF appears to be a useful processing technique for changing
[6] W. Eisenbach, Supercritical fluid extraction: a film demonstration, Ber.
the composition of lipids in order to obtain high value func- Bunsenges. Phys. Chem. 88 (1984) 882887.
tional products: a pressure increase (at 5 kg/h flow rate) and [7] S.S.H. Rizvi, R.R. Chao, Y.J. Liaw, In Supercritical Fluid Extraction and
flow rate increase (at 150 bar pressure), both determined a higher Chromatography: Techniques and Applications, ACS Washington, USA,
EPA + DHA concentration and decreased the EPA/DHA ratio. 366 (1988) p. 89.
[8] F. Temelli, E. Leblanc, L. Fu, Supercritical carbon dioxide extraction of oil
No analytical results concerning the tocopherol composition
from Atlantic Mackerel (Scomber scombrus) and protein functionality, J.
of the fractions are available. However, the darker color of the Food Sci. 60 (1995) 703706.
residual fraction found in the column at the end of run, in com- [9] G. Perretti, E. Miniati, L. Montanari, P. Fantozzi, Improving the value of
parison both with the starting material and with the extracted rice by-products by SFE, J. Supercritical Fluids 26 (2003) 6371.
fractions, shows the general concentration effect exerted on polar [10] G. Perretti, E. Bravi, L. Montanari, P. Fantozzi, Proceedings of the Sixth
International Symposium on Supercritical Fluids, Versailles (F), 2003, pp.
moieties, which are only tocopherols in the starting material.
2934.
[11] W.B. Nilsson, E.J. Gauglitz, J.K. Hudson, V.F. Stout, J. Spinelli, Fraction-
4. Conclusions ation of menhaden oil ethyl esters using supercritical fluid CO2 , J. Am. Oil
Chem. Soc. 65 (1988) 109117.
SFF proved to be a useful processing technique for changing [12] W.B. Nilsson, E.J. Gauglitz, J.K. Hudson, F.M. Teeny, G.J. Flick, R. Mar-
tin E. In Advances in Seafood Biochemistry: Composition and Quality.
the lipid composition in order to obtain high value functional Technomic Publishing Co., Inc., Lancaster, PA, 1992, p. 151168.
products. [13] W.B. Nilsson, E.J. Gauglitz, J.K. Hudson, Supercritical fluid fractionation
The results of this study confirmed the possibility of modify- of fish oil esters using incremental pressure programming and a temperature
ing the original FAEEs concentration by using SC-CO2 and opti- gradient, J. Am. Oil Chem. Soc. 66 (1989) 15961600.
[14] W.B. Nilsson, J.W. In King, G.R. List (Eds.), Supercritical Fluid Technol-
mizing the fractionation process in terms of pressure, tempera-
ogy in Oil and Lipid Chemistry, American Oil Chemists Society, Cham-
ture, and flow rate of this selective solvent as already reported paign, IL, 1996, pp. 180209.
in literature. Moreover, the results highlight the possibility to [15] L. Cossignani, M.S. Simonetti, A. Neri, P. Damiani, Structural analysis
exploit different relatively low temperatures and pressures to of triacylglycerol fraction and some of its sub-fractions from an eicos-
obtain suitable EPA + DHA and EPA/DHA ratios for market pur- apentaenoic acid docosahexaenoic acid dietary supplement, Z. Lebensm.
-Unters. Forsch. 208 (1999) 203207.
poses, and with interesting yields and use of energy.
[16] H. Schlenk, Progress in the Chemistry of Fats and Other Lipids, vol. 2,
Future process developments are possible. One of the most Pergamon Press, NY, 1954, pp. 243267.
likely is the fractionated separation of the extracted fractions;