phase equilibria: supercritical Co 2 and Fish oil Components This section provides a brief summary of solubility measurements and modelling

of solubility and phase equilibria for fish oil components in supercritical CO2. Fish

oils are a complex mixture of lipid components belonging to several lipid classes, including acylglycerols, fatty acids, fatty acid esters, sterols, tocopherols, and hydro-carbons. Successful isolation using supercritical processes requires reliable infor-mation on the solubility behavior of the solutes of interest as affected by operating for prediction of solubility in CO2 at a given temperature and density. The Chrastil correlation can only be used for the vapor-phase concentration of solutes and gives no information on the liquid-phase composition. Equation of state models, such as the Peng-Robinson [92, 93], Soave-Redlich-Kwong [94], excess function (g^E) [95], group contribution [96], and lattice model equations of state (EOS) [97], have been shown to provide the most rigorous method for predicting phase equilibrium behavior.

The Chrastil correlation [91] for estimating lipid solubilities in SCFs takes the form: for the solubility of several fish oil lipid components in CO2 [98–101].

Although, binary lipid/CO2 systems have been studied extensively, multi-component data are relatively scarce. In such multimulti-component mixtures, complex intermolecular interactions may lead to significant deviations from pure component behavior of more complex multicomponent systems. However, researchers should exercise caution when making solubility measurements, interpreting the data and, ultimately, designing separation processes based on this information. Temelli and Güçlü-Üstündag˘ [102] report several discrepancies between reported solubility data for lipid/CO2 systems from different laboratories. An example of such discrepancies is given in Figure 5.8 for some selected fatty acids in CO2 [103–107]. Impurities, sample degradation, isomeric purity, and limitations to experimental methods are all contributing factors to the reported data variations.

Solute vapor pressures and solute-solvent and solute-solute intermolecular inter-actions govern solubility behavior. In binary systems of a particular homologous

(all solubilities and densities are in units of g·l^–1 unless otherwise stated)

lipid Component

k ± standard

p/mpa ref.

Fatty acids

Myristic acid, C_14:0 6.42 ± 0.33 –9300 ± 1727 –10.2 ± 5.9 98 Palmitic acid, C_16:0 7.00 ± 0.39 –12029 ± 1043 –7.0 ± 4.1 98

Stearic acid, C_18:0 5.81 ± 0.54 –15890 ± 741 12.0 ± 3.7 98

Oleic acid, C_18:1 7.92 ± 0.37 –3982 ± 691 –38.1 ± 2.3 98

Linoleic acid, C18:2 9.71 ± 0.90 –5211 ± 1626 –46.3 ± 5.3 98 triglycerides

Triolein 10.28 ± 0.66 –2057 ± 480 –61.5 ± 4.6 98

ethyl esters

Stearic acid 5.80 ± 0.50 –2446 ± 857 –26.7 ± 3.9 98

Oleic acid 7.78 ± 0.34 –1947 ± 503 –40.9 ± 2.7 98

Linoleic acid 7.17 ± 0.63 –2193 ± 896 –36.2 ± 4.4 98

EPA 8.62 ± 0.17 2473 ± 262 –45.2 ± 1.2 98

DHA 7.76 ± 0.32 –1784 ± 529 –42.1 ± 2.5 98

hydrocarbons

Squalene^a 6.54 ± 0.06 –3936.6 ± 155 –28.24 ± 0.7 313–333;

10–30 99

minor Components

Vitamin A^a 5.07 ± 0.44 –3072 ± 339 –21.7 ± 2.12 313–353;

20–35

100^b

Vitamin A palmitate^a 7.66 0 –49.2 333;

12.5–30 101

β-carotene^a 8.63 ± 0.61 –11576 ± 461 –23.3 ± 3.04 313–353;

20–35

100^b

Fish oils

Cod liver oil^a 10.91 ± 0.18 –4078 ± 122 –59.2 ± 0.98 313–333;

20–30

99^b

Spiny dogfish liver oil^a 9.97 0 –65.4 333; 20–30 101

Orange roughy oil^a 7.79 0 –50.6 333; 20–30 101

a Solubilities are in g·kg^-1 and densities are in kg·m^–3; ^bDerived from data presented in this reference.

series, where intermolecular interactions are similar, molecular weight and vapor pres-sures determine component solubilities. For example, in Figure 5.8, the solubility of fatty acids increases with decreasing molecular weight (chain length). Of the systems reported in the literature, fatty acid esters have the highest vapor pressures, followed by fatty acids. Vapor pressures of the glyceride lipid class follow the trend mono-glycerides > diglycerides > triglycerides [98]. Esterification with a C1 or C2 alcohol substantially increases the solubility of fatty acids in CO2 because the polar acid group is converted to a less polar ester group [108]. For fatty acids of the same chain length, melting points decrease with increasing degree of unsaturation (Figure 5.8). In this instance, solubility is affected by the physical state of the compound.

Johannsen and Brunner [100] measured the solubilities of fat-soluble vitamins in supercritical CO2 in the temperature range of 313 to 353 K and pressure range of 20 to 35 MPa. The solubility for both β-carotene (provitamin A) and vitamin A increased with increasing pressure. Over the temperature range studied, vitamin A shows retrograde condensation behavior (solubility decreases with increasing temperature) up to around 30 MPa (Figure 5.9). At higher pressures, the solubility curves of vitamin A exhibit a crossover point and the system exhibits nonretrograde behavior. The solubility of vitamin A palmitate has been measured by Catchpole et al. [99] at 333 K and 12.5 to 30.0 MPa. The solubility is compared to that of the vitamin A free acid in Figure 5.9. The decrease in polarity of the ester over the free acid is counterbalanced by the large increase in molecular weight, leading to a modest decrease in solubility.

Mollerup et al. [109–111] carried out a series of phase equilibria measurements for fish oil fatty acid ethyl esters (FAEEs) of the sand eel with CO2. Measurements were obtained using the crude fish oil FAEEs (283 to 343 K, 2 to 22 MPa), urea- fractionated fish oil FAEEs (313 to 343 K, 1.6 to 25 MPa), and ω3-rich fish oil FAEEs (313 to 343 K, 8 to 26 MPa). The initial FAEE oil compositions are given

Solubility/g kg–1 of CO2

5 10 15 20

FIgure 5.8 Comparison of fatty acid solubilities in CO2 from various literature sources.

(Lines have been drawn to aid the eye.)

in Table 5.7. The K-values, on a CO₂ free basis, for some selected ω3-FAEEs are given in Figure 5.10 at 313 K and 343 K as a function of pressure. The K-values depend strongly on temperature, pressure, and mixture composition. High selectivi-ties (higher relative differences between K-values) and low solubilities were observed at low pressures, whereas at high pressures the selectivity was low but the solubility was high. The crude FAEE mixture was more soluble than the urea-fractionated and ω3-rich mixtures (which had similar solubilities) because the crude mixture con-tained a large amount of saturated and monounsaturated FAEEs of medium chain length (C14–C18). For the crude fish oil esters, the K-values were found to vary with chain length but not specifically the degree of unsaturation and position of double bonds. The authors reported that solubilities increase with increasing temperature, and Figure 5.10 shows that the selectivities at 343 K are greater than those at 313 K. The selectivities are largest in the ω3-rich system because the number of components and the amount of medium-chain-length material has decreased. Using urea-fractionation as a preconcentration step under the conditions investigated, the authors determined that optimum separation conditions in terms of selectivity and solubility were in the range of 16 to 18 MPa at 343 K (corresponding to CO2 densities of 550 to 615 kg m^–3).

Catchpole and von Kamp [92] studied the phase equilibria of the system squalene/CO2 and shark liver oil/CO2 over the range 313 to 333 K and 10 to 25 MPa.

The shark liver oil consisted of around 50% squalene and 50% of a mixture of TAGs and DAGEs. There was also 0.5% by mass pristane. The TAG/DAGE mixture has been assumed to be a single pseudocomponent, with a hypothetical carbon number of 54. The phase equilibria data for squalene/CO2 at 313 K and 333 K are shown in Figure 5.11. The amount of oil dissolved in the vapor phase increases with increasing pressure and decreasing temperature, as does the amount of CO2 dissolved in the oil phase. The experimental data for the binary systems squalene/CO2 and C54-TAG/CO2 were modelled using the Peng-Robinson EOS with the usual mixing rules [92]. The Peng-Robinson EOS was also used to predict phase equilibrium and separation

Pressure/MPa Vitamin A: 353 K

Vitamin A palmitate; 333 K

Solubility/g kg–1 of CO2

15 20

15

10

5

0 20 25 30 35

FIgure 5.9 Solubility of vitamin A and vitamin A palmitate in CO2.

table 5.7

the Initial Faee Fish oil Compositions used in the studies of mollerup et al. [109–111]

Crude Fish oil urea-fractionated ω3-rich

Component Weight (%) of Faee

10:0 0.4

12:0 0.2

14:0 7.5 0.8

14:1 ω5 0.5 0.2

15:0 0.5

15:1 ω5 0.2 0.1

16:0 18.5

16:1 ω7 12.4 0.5

16:2 1.4 2.3

16:3 ω3 0.6 1.6 0.3

16:4 ω3 0.8 2.9 0.2

18:0 2.2 0.03 0.5

18:1 ω9 10.2 0.6

18:1 ω7 2.3

18:2 ω6 2.9 1.2

18:3 ω6 0.4 0.8

18:3 ω3 1.3 0.9

18:4 ω3 3.8 12.7 2.3

20:0 0.2

20:1 ω9 4.2 0.6

20:2 ω6 0.3 0.5

20:3 ω3 0.2 0.1

20:4 ω3 0.7 1.0 2.9

20:5 ω3 10.0 35.9 52.5

22:1 ω11 6.5 0.3

22:1 ω9 1.0 0.1

21:5 ω3 0.4 1.5

22:5 ω3 0.5 1.1 2.5

22:6 ω3 9.6 33.2 36.1

factors for the ternary system C₅₄-TAG/squalene/CO₂. The predicted vapor and liquid mole fractions of squalene in the ternary system are shown for selected tem-peratures and pressures in Figure 5.12. The liquid mole fractions equate to discrete mass fractions of squalene on a CO2 free basis ranging from 0 to 1. It is interesting to note that the equilibrium relationship is almost linear, as shown by the regression lines in Figure 5.12. The mass fraction of CO2 dissolved in the liquid phase at a given temperature and pressure stays almost constant even when the squalene mass fraction varies from 0 to 1 (on a CO₂ -free basis). The predicted vapor and liquid-phase mass fractions of squalene for the same system are given on a CO₂-free basis in Figure 5.13. The K-values for squalene are also shown. The selectivity toward squalene is best at low pressure and low mass fraction of squalene. The solubility of TAGs decreases more sharply with decreasing pressure than squalene, and so the increase in selectivity is to be expected. The K-value decreases as the temperature and vapor-phase density increase, although it is still sufficiently high at 333 K and 25.0 MPa to enable separation of squalene and C₅₄-TAG.

Ruivo et al. [112] measured the phase equilibria of the ternary system methyl oleate/squalene/CO2 over the range 313 to 343 K and 11 to 21 MPa. Four different

Original Fish Oil

Pressure/MPa Pressure/MPa Pressure/MPa

Pressure/MPa Pressure/MPa

Pressure/MPa

K-values at 343 K(CO2 free basis)K-values at 313 K(CO2 free

12 14 16 18 20 22 10

Urea-fractionated Fish Oil ω3-Rich Fish Oil C16:3 ω3

FIgure 5.10 Partition coefficients (K-values) on a CO2-free basis for whole sand eel oil, urea-fractionated sand eel oil, and ω3-rich sand eel oil [109–111]. (Reprinted from Fluid Phase Equilibria, 161, 169, ©1999. With permission from Elsevier.)

Liquid 0.725

26 24 22 20 18 16 14 12 10 8

0.750 0.775 0.800 0.825 0.995 0.996 0.997 0.998 0.999 1.000 Vapour

313 K 333 K

Mole Fraction of CO2

Pressure/MPa

FIgure 5.11 Liquid and vapor mole fractions for the squalene–CO2 system [92].

Liquid Phase Mole Fraction Squalene 0.00 0.05 0.10 0.15 0.20 0.25 0.0040

0.0035 0.0030 0.0025 0.0020 0.0015 0.0010 0.0005 0.0000

Vapor Phase Mole Fraction Squalene

250 Bar, 333 K 200 Bar, 313 K 200 Bar, 333 K 125 Bar, 313 K

FIgure 5.12 Liquid and vapor phase mole fraction of squalene at selected temperatures and pressures [92]. Points: Peng-Robinson equation of state predictions; lines: linear regressions.

(Reprinted with permission from Industrial and Engineering Chemistry Research, 36, 3762.

©1997 American Chemical Society.)

feed compositions were used containing 0.1079, 0.3350, 0.6447, and 0.8779 mole fractions of squalene. The selectivity of a fluid can be quantified in terms of the separation factor, α, which in this example is given by:

α = ⋅ respectively. The selectivity of CO2 toward methyl oleate over a range of pressures is demonstrated in Figure 5.14a as a function of temperature (initial squalene feed mole fraction of 0.6447) and Figure 5.14b as a function of squalene feed concentration at 313 K. Figure 5.14 shows that CO2 is highly selective for methyl oleate, with sepa-ration factors ranging from 2 to 8. The sepa is highly selective for methyl oleate, with sepa-ration factor decreases with decreas-ing temperature and with increasdecreas-ing pressure, which results in a higher loading, giving greater throughput at the expense of selectivity. An increase in solubility with temperature at fixed density is advantageous for packed column fractionation, which requires a density difference between the supercritical and oil phases to be large enough to prevent flooding.

Addition of cosolvents, such as ethanol, can enhance the solubility of solutes in supercritical CO2. Cosolvents have also been shown to act as entrainers. The entrainer effect has been defined as a phenomenon in which the solvent power of a fluid is increased by the addition of cosolvents, whilst the selectivity of that fluid is maintained or enhanced [113]. In many studies, the enhanced solubilities have been attributed to solute-cosolvent interactions, such as hydrogen bonding or dipole-dipole

Mass Fraction Squalene in the Liquid VLE

K Factors 125 Bar, 313 K 250 Bar, 333 K 125 Bar, 313 K 250 Bar, 333 K

Mass Fraction Squalene in the Vapour

Equilibrium Coefficient K

0.0 0.2 0.4 0.6 0.8 1.0

FIgure 5.13 Mass fraction in the liquid and vapor phase and K-values for squalene on a CO2-free basis [92]. (Reprinted with permission from Industrial and Engineering Chemistry Research, 36, 3762. ©1997 American Chemical Society.)

interactions. Specific intermolecular interactions between cosolvents and solutes can enhance the solubility of those specific components, which can be particularly ben-eficial for improving separation selectivities.

Catchpole et al. [99] measured the solubility of squalene, orange roughy oil, cod liver oil, and spiny dogfish liver oil in supercritical CO2 and CO2/ethanol mixtures at 313 to 333 K and 20 to 30 MPa. Ethanol mass concentrations up to 12% (on a solute-free basis) were used. Catchpole and coworkers found that ethanol substan-tially increased the solubility of all fish oil components studied (Figure 5.15). At 333 K, the authors correlated the increase in solubility due to the addition of ethanol using the following equation:

Temperature 313 K Mole Fraction of Squalene in Feed

Pressure/MPa

(a) (b)

Pressure/MPa

Separation Factor, α

Separation Factor, α

10 12 14 16 18 20 22 10 12 14 16 18 20 22

FIgure 5.14 The separation factors for methyl oleate and squalene in CO2 [112]. (Reprinted from Journal of Supercritical Fluids, 29, 77, ©2004. With permission from Elsevier.)

0 100

10

1 2 4

Mass % Ethanol (solute free basis) Squalene

Solubility (ethanol free basis)/g kg–1 of CO2

Orange Roughy Oil Cod Liver Oil Spiny Dogfish Liver Oil

6 8 10 12 14

FIgure 5.15 Enhancement of fish oil component solubilities as a function of ethanol cosolvent concentration at 333 K and 20 MPa [99]. (Reprinted with permission from Journal of Chemical and Engineering Data, 43, 1091. ©1998 American Chemical Society.)

where S is the enhanced solubility, S₀ is the solubility in pure CO2, k is a constant, and X is the mass percent of ethanol. The k constants for squalene, orange roughy oil, and spiny dogfish liver oil are 0.14, 0.15, and 0.21, respectively.

The solubility enhancement of squalene in CO₂ has also been investigated using n-hexane, toluene, and ethanol in the ranges of 303.2 to 313.2 K and 9 to 10 MPa [114]. The dependence of solubility on entrainer concentration was described by a parabolic function. The authors characterized the initial slope of this function for each solute-entrainer pair with a single number, termed the entrainer efficiency, E.

The E value was related to the similarities of the molecular structure and polarity of the entrainer and the solute. The E values followed the trend n-hexane (3.6) >

toluene (2.4) > ethanol (1.5), indicating the order of solubility enhancement at fixed temperature, pressure, and entrainer concentrations.

Nilsson et al. [115] investigated the effects of adding 5% ethanol as a cosolvent on the K-values and selectivity of menhaden oil fatty acid esters at 333 K and 12.5 MPa.

It was noted that K-values increased with increasing number of double bonds for a given chain length. Addition of ethanol increased the K-values for all fatty acid esters, regardless of chain length or degree of unsaturation. The ratio of the K-values for CO2-ethanol and pure CO2 ranged from around 1.5 for C14 esters to 3.1 for C22 esters, demonstrating that the solubility enhancement achieved by the addition of ethanol increases with increasing chain length. The fluid selectivities, defined here as the ratio of the K-values for the C_14:0 ester to those of the other mixture compo-nents, decreased for all fatty acid esters upon the addition of ethanol. K-values for the fatty acid esters in pure CO2 were also measured at 333 K and 13.1 MPa. The measured partition coefficients in pure CO2 under these conditions yielded K-values very similar to those obtained using CO₂-ethanol at 333 K and 12.5 MPa. They concluded that ethanol serves no useful purpose as a cosolvent with CO₂ for the concentration of EPA and DHA from fatty acid ester mixtures.

Although the use of cosolvents may be beneficial in terms of increasing solu-bility, the complex nature of fish oils means their application may be limited in terms of selectivity enhancement. Their use should be considered carefully because it can increase the complexity of process design. An increase in solvent loading may result in the coextraction of undesirable components. Also, using cosolvents can affect mass transfer, greatly increase processing costs, and can potentially induce degradation of the desired extract. For a more detailed discussion of binary/CO₂ and multicomponent/CO2 lipid systems, the reader is directed to the critical and in-depth reviews of Temelli and Güçlü-Üstündag˘ [98, 102]. Several articles containing comprehensive tabulated data on lipid/CO2 systems studied are also available in the literature [98, 116–118].

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