Supercritical Fluids and the Food Industry

N.L. Rozzi and R.K. Singh

Dept. of Food Science and Technology University of Georgia Athens,GA 30602-7610 Direct inquiries to author Singh (E-mail: rsingh@arches.uga.edu)

Abstract: This paper reviews the use of supercritical fluids in various aspects of the food industry, and is divided into six different areas: modeling of supercritical fluids, separation of extracted material, supercritical carbon dioxide as a solvent for extraction, supercritical fluids and analytical uses, and supercritical fluids and novel methods of food processing. An assortment of solutes are covered in the extraction section of the paper, including antineoplastic agents and lipids. The analytical methods section covers supercritical fluid chromatography, pesticide detection, and lipid analysis. The novel methods section discusses supercritical fluid extrusion and a new method of eliminating hexane from soybean oil. Keywords: Supercritical Fluid, Carbon Dioxide, Review

Introduction
Supercritical fluid extraction utilizes the ability of certain chemicals to become excellent solvents for certain solutes under a combination of temperature and pressure. The solvent becomes supercritical when it is raised above its critical point for both temperature and pressure (Table 1). In the case of carbon dioxide, the critical point is at 31.06 C and 7.386 MPa. CO2 is the solvent of choice for use in supercritical fluid extraction because it is, GRAS, nonflammable, noncorrosive and inexpensive (Rizvi and others 1994). In addition, CO2 has a low critical temperature, which can help prevent thermal degradation of food components when they are being extracted. The problem with most of the fluids listed (Table 1) besides CO2 is that they are either difficult to handle and/or obtain in a pure form. The advantages of supercritical fluid extractions are:

Table 1Critical points of selected gasses Fluid Hydrogen Neon Nitrogen Argon Oxygen Methane Krypton Carbon Tetrafluoride Ethylene Xenon Carbon Dioxide Ethane Acetylene Nitrous Oxide Chlorodifluoromethane Propane
Rizvi and others 1994

Critical Temperature (K) 33.25 44.40 126.24 150.66 154.58 190.55 209.46 227.6 282.35 289.7 304.17 305.34 308.70 309.15 369.27 369.85

Critical Pressure (MPa) 1.297 2.6545 3.398 4.860 5.043 4.595 5.49 3.74 5.040 5.87 7.386 4.871 6.247 7.285 4.967 4.247

1. Supercritical fluids have a higher diffusion coefficient and lower viscosity than liquids. 2. Absence of surface tension allows for their rapid penetration into the pores of heterogeneous matrices, which helps enhance extraction efficiencies. 3. Selectivity during extraction may be manipulated by varying the conditions of temperature and pressure affecting the solubility of the various components in the supercritical fluid. 4. Supercritical fluid extraction does not leave a chemical residue. 5. Supercritical fluid extractions can use carbon dioxide gas, which can be recycled and used again as part of the unit operation (Rizvi and others 1994). Supercritical carbon dioxide has been researched for potential applications in many different fields including Food/ Agriculture, Analytical/Supercritical Fluid Chromatography, and the Petrochemical/Chemical Industries. Figure 1, presents the breakdown of research areas of recent research articles (1999-2000). These articles were determined by performing a search on Current Contents for articles that contain the terms carbon dioxide and supercritical fluid, and resulted in 264 articles being obtained. From Figure 1 it can be seen that 32% of the research articles were from the Food and Agriculture research areas and an additional 4% of the research articles were in Pesticide research. Combined, this is 12% greater than the next largest area (Petrochemical). To further expand on this literature search, a second search was performed on Food Science and Technology Abstracts using the same terms as the previous search. In addition, the timeframe for the search was increased from 1999-2000, to 1995 to 2000. This search resulted in 105 articles being produced. The results of this search are displayed in Figures 2 and 2a. Figure 2a is a breakdown of topics found in the Analysis section of Figure 2. Based on these searches, this paper discusses Modeling of Supercritical Fluids, Separation of Extracted Materials, Extraction from Botanical Samples, Extraction of Lipids, Supercritical Fluids and Analytical Uses, and Supercritical Fluids and Novel Methods of Food Processing.

2002 Institute of Food Technologists

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modeling supercritical fluid extractions is to model the process itself. Sovova (1994) developed a model based on a mass balance of the process to describe the extraction of natural products with supercritical fluids. This model takes into account the porosity of the raw material from which the target compound is being extracted, the density of the solid phase and the length of time used to extract the target compound. Although each method is vastly different from each other, both provide important information about the extraction process.
Solubility Parameter

The solubility parameter is the solvating capability of a compressed gas as described by the Hildebrand Solubility Parameter. The Hildebrand Solubility Parameter is a rough measure of the ability of a solvent to dissolve a solute. Equation 1 relates the solubility parameter to the density of both the gas and the liquid by:

Figure 1Results of Current Contents search using supercritical fluids and carbon dioxide as search terms (19992000)

(1)

Modeling of Supercritical Fluids

The solubility of substances in supercritical fluids has been described by many different methods. Two examples of these methods will be included in this paper. One method utilizes: solubility parameters and the other is based on process modeling. Each of these methods has their benefits and drawbacks. Solubility parameters describe the solubility of the substance in supercritical CO2 under laboratory conditions and not during conventional extraction processes. In addition, the solubility parameter is also influenced by the equation of state, which is used to calculate some variables needed for the solubility parameter. Two equations of state which can be used are the Peng-Robinson Equation of State and the Sanchez-Lacombe Model. The other approach to

where d is the solubility parameter, Pc is the critical pressure, r is the gas density and rliq is the density of the liquid (Rizvi and others 1994). From this equation, the effect of pressure on the solubility parameter can be seen. When the pressure is at atmospheric pressure, the solubility parameter is zero. As the pressure increases the solubility parameter increases, with great changes taking place as the critical pressure is reached (Rizvi and others 1994). Although this equation can be used with some success to describe the solubility of some solutes in supercritical fluids, it does not work in all cases and must be used with careful attention being paid to the original work of Hildebrand and the warnings given there. Some of these warnings are that it is dangerous to employ the solubility parameters for esters, ketones, alcohols and other polar liquids. This should warn people against using the Hildebrand solubility parameter as the final word and any assumptions made using the solubility parameters should be

Figure 2Results of Food Science and Technology Abstracts search using supercritical fluids and carbon dioxide as search terms (1995-2000) 34

Figure 2(a)Distribution of target analytes from analysis section of figure 2

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Supercritical fluids and the food industry . . .

backed up with experimental evidence (McHugh 1994). Allada (1984) proposed a new equation to define the solubility parameter (Equation 2). This equation uses the, internal energy of the gas relative to isothermally expanded ideal gas state as the cohesive energy of the gas, (Allada 1984). mixing rules. In addition, and are equation specific constants. The values for the Peng-Robinson equation of state are 0.414214 and 2.414214, respectively (Smith 1996).

(8) (2) Equation 8 can be displayed as:

In Equation (2) d r is the reduced solubility parameter, R is the universal gas constant, C1 is a conversion factor to make the units cancel, E is the internal energy of the gas at temperature T and pressure P. Pr and T r are the reduced pressure and reduced temperature respectively. The reduced pressure and reduced temperature are related to (P/Pc) and (T/Tc) where P and T are the current pressure and temperature, and Pc and Tc are the critical pressure and temperatures respectively. Z is the compressibility factor and E* is the internal energy of the gas, isothermally expanded to zero pressure where intermolecular separation is infinite and intermolecular cohesive force is zero, (Allada 1984). The values for the compressibility factor and the (E* - E)/TC and Z can be calculated by using equations (3 to 6) (3) (4)

(9) where

(10)

(11)

(5)

(6)

where H is the enthalpy of the supercritical fluid and H* is the enthalpy of the ideal gas at the same temperature (Allada 1984). The values for the (H* H)/Tc and Z data are obtained from the book, Chemical Process Principles, Part Two, Thermodynamics, by Hougen and Watson (1947). This equation works well for prediction of the solubility of a substance in different supercritical fluids at different pressures. This equation (Equation 2) does not, however, work well for changes in temperature. To account for temperature effects, it would be necessary to include a solvent-solute interaction represented by a cross-virial coefficient (Allada 1984).
Peng-Robinson Equations of State

am is the attraction parameter of the mixture, bm is the mixtures van der Waals co-volume, X is the mole fraction in the liquid or supercritical fluid phase, and i and j represent the different components of the mixture. aij comes from the mixing rules presented by van der Waals, which utilizes only one interaction parameter (Rizvi and others 1994). Care should be taken when using these mixing rules though (Equations 10 and 11) since the mixing rules are for cubic equations of state. Cubic equations of state only account for dispersion forces and not for hydrogen bonding, which could be occurring during the extraction. This possible hydrogen bonding will prevent random distribution of the solute in the solvent, thus causing the need for a different set of mixing rules (McHugh 1994). One different set of mixing rule equations which can be used are equations (12) and (13). These equations incorporate two new parameters into the mixing rule. These parameters are dij and hij which represent the interaction and size parameters respectively. These data parameters are obtained by regressing the P-x data and fitting it to the equation of state.
(12)

(13) The addition of these two parameters greatly improve the fit of the data for some solutes, but they still need to be investigated for use with other solvent-solute combinations (McHugh 1994).
Sanchez-Lacombe Model

Equations of state can be used to describe the behavior of solutes being dissolved in supercritical fluids. A favored equation to describe this action is the Peng-Robinson equation (Smith 1996):

(7) where Z is the compressibility factor, P is the pressure, T is the temperature and R is the Universal Gas Constant. This equation is used to describe the behavior of pure species. It can be modified to describe the behavior of mixtures which results in Equation 8, where a and b are mixing values which are related to aI and bI by

Another equation of state is the Sanchez-Lacombe equation of state (14), which is based on the lattice-fluid theory, which assumes that the polymer has a flexible liquid structure. This limits the use of this equation to polymers which are noncrystalline, not cross-linked or slightly crosslinked, and above their glass transition temperature. This equation has been used to model the solubility of supercritical CO2 in solid, amorphous polymers or molten polymers. In addition, it also describes the subsequent swelling of the polymer as the supercritical fluid dissolves in the polymer. Three assumptions need to be made to use the Sanchez-Lacombe

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equation. These assumptions are: (1) The solubility of the gas in the polymer represents equilibrium data. (2) The solubility of the polymer in the high-pressure gas phase is essentially zero. (3) The amorphous polymer above its glass transition temperature can be modeled as a liquid. (24) Using the values for x and y solved in the mass balances, the curves describing the extraction process can be determined by the following: (25) (14) where rr, Pr, and Tr, are the reduced density, pressure and temperature. r represents the number of lattice sites represented by a molecule. As compared with the Peng-Robinson equation of state, which has only two mixing rules, the Sanchez-Lacombe equation has three adjustable characteristics (Kriszka 1988). The variables in this equation are: (15) (16) where e(t) is the mass extracted relative to the mass of the solutefree solid phase, xo is the concentration related to the solute-free solid phase, H is the height of the bed, and x(t,h) is the concentration related to the solute-free solid phase as a function of time ( t) and height (h). This modeling system can be used to describe the extraction of natural products using supercritical CO2 (Sovova 1994). This model has been successfully applied by Mira and others (1996), Perrut and others (1997), Coelho and others (1997), and King and others (1997) in modeling the extraction of natural products from plant sources.

Separation of Extracted Material

(17) (18) (19) (20) (21) (22) where: = R = = V* = M = Extraction is the most common use for supercritical fluids. The process is simple, with the major process parameters being temperature, pressure and flow rate of the supercritical fluid. Figure 3 presents a basic flow diagram for supercritical fluid extraction, and Figure 4 illustrates a similar system that is designed for fractionation experiments. The most important aspect of this flow diagram is the separation of the extracted material from the supercritical fluid. This section discusses multiple methods for the separation of extracted material from supercritical fluids.

Internal energy per mer Universal gas constant Closed-packed volume of a mer Closed pack volume of the mixture Molecular weight

The values for CO2 of r*, T*, and P* are 1.510 (g/cm3), 305 (K), and 5670 (atm) respectively (Kiszka 1988).
Process Modeling

The process of extracting natural products by supercritical fluid extraction can be modeled using the model developed by Sovova (1994). This model is based on the following assumptions: (1) The solvent flows axially through a bed of raw material in a cylindrical extractor. (2) The solvent is solute-free upon entering the extractor. (3) The solute is partially exposed to the solvent due to previous milling (Sovova 1994). The mass balance for the process is described by Equations (23) and (24) below, where is the solvent density, is the porosity, y is the solvent-phase concentration related to the solute-free solvent, x is the concentration related to solute-free solid phase, U is the superficial velocity of the solvent, h is the axial coordinate, s is the density of the solid phase, t is time, and J(x,y) is the mass transfer rate. (23)
36

Figure 3Flow diagram of a supercritical fluid extraction system

Figure 4Flow diagram of a supercritical fluid fractionation system

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Supercritical fluids and the food industry . . .

There are multiple methods that can be utilized to separate the solute from the supercritical fluid, which is acting as the solvent. There are three basic concepts that can be used to accomplish this. The first is to change the temperature or pressure so that the solvent capability of the supercritical fluid changes. The other is to wash the solute out of the supercritical fluid using a solvent that can strip the solute from the supercritical fluid. In addition to the separation of the solute from the supercritical fluid, it is possible to separate multiple solutes within the supercritical fluid using a packed column. The simplest method to remove the solute from a supercritical fluid is to change the pressure of the supercritical fluid so that either the fluid is no longer supercritical or that the solute is no longer soluble in the supercritical fluid. Snow and others (1997), Barth and others (1995), and Sugiyama and Saito (1988), all utilized this method to remove their respective solutes from the supercritical fluid. In all three cases the individuals performing the study routed the solute/solvent combination through a restriction valve and into a collection vessel. Inside the collection vessel, the pressure was reduced to room pressure, causing the supercritical fluid to return to its gas state, and the solvent to be released into the collection vessel. Inside the collection vessel, there can be a solvent to receive the solute, so that further analysis can be performed. Other times the vessel may be left empty so that the solute can crystallize upon leaving the supercritical fluid. Another variation of the previous method to remove the solute from the supercritical fluid is to change the temperature of the supercritical fluid. This may cause the fluid to leave the supercritical region, but it allows for the pressure of the fluid to be maintained. This could save money by allowing for the pressurized fluid to be recirculated, without having to repressurize it. This means that less money would have to be spent on the energy to pressurize the fluid. Lancas and others. (1990) and Vilegas and others (1994) both utilized this method to remove their respective solutes from the supercritical fluid. This method simply involves the removal of the solute by dropping the temperature of the solvent until the solute is no longer soluble and precipitates from the supercritical fluid. This is also referred to as a cryogenic trap. Once the extraction has been completed, the system can be depressurized and the extract can be removed from the sample chamber. The solute can also be removed from the supercritical fluid by washing it from the solvent. This allows for the process to be maintained at a constant pressure much as the cryogen trap did, but it also allows for the removal of the solute from the process without having to shut the process down. An example of this type of removal is used in the decaffeination of green coffee beans to remove the caffeine from the supercritical CO2 (McHugh 1994). If more than one chemical is extracted by the supercritical fluid, column chromatography can be used, utilizing the supercritical fluid as the mobile phase. As with HPLC, both normal and reversed phase chromatography can be utilized to separate the solutes depending on their polarity. The retention times on these columns can be correlated to the solubility of the solute in the supercritical fluid (Sakaki 1994). McHugh and Krukonis (1994) and Sakaki and others (1994) used column chromatography to separate multiple solutes from supercritical fluids. purposes, and flavor and fragrance purposes. Some companies have even begun to market botanical extracts obtained by supercritical CO2 extraction. One company in particular, Flavex Naturextrackte, Rehlingen, Germany, specializes exclusively in SFE produced products and markets them internationally. Table 2 includes a brief list of different botanicals, which have been investigated since 1997. Separation of Antineoplastic Agents: Antineoplastic agents can be extracted from plant materials in addition to flavors and essential oils. Krukonis and others (1979) performed a brief extraction study on plants which were confirmed by the National Cancer Institute (NCI) to contain antineoplastic agents. One example of these species is maytansine which is contained in materials B628318, B628259, and B628201. The materials that were used in the study are listed in Table 3 (McHugh and Krukonis 1994). In this experiment approximately 20 grams of plant material was extracted using 200 L of supercritical CO2. This was compared with the NCI protocol that utilized a Soxhlet extraction with 95% ethanol. The samples were then analyzed in vitro for bioactivity using 9KB leukemia cells. The results of this comparison are displayed in Table 4 and are expressed in terms of the ED50 value. This value is defined as the extract concentration in which 50% of the cell growth is inhibited, and is expressed in mg/l. The lower the ED50 value, the more cytotoxic the extract. From the results in Table 4, it can be seen that all of the extracts exhibited cytotoxic results. In addition, 2 samples exhibited an ED 50 value less than that of the Soxhlet extraction (McHugh and Krukonis 1994). Due to the small amount of raw material provided and the lack of any pure material that could be used for solubility testing, this study serves mainly as a screening study though, that indicates the potential for using supercritical fluids to isolate antineoplastic agents. If more materials were provided for in this study, determination of ideal extraction conditions, or a method to fractionate the extracts could have been developed. To prove this point, a second extraction of sample B638318 was performed. This time the extract was split into 2 fractions, fraction 1 consisted of the materials which were extracted by the first 20 L of CO2 and fraction 2 used the remaining 180 L of CO2. After analysis of both these samples, it was found that the second fraction yielded a much lower ED50 value (0.03 vs 0.19 obtained in fraction 1) (McHugh and Krukonis 1994). Annato: Bixin, a pigment used for food coloring obtained from the seeds of annatto (Bixa orellana L.) was extracted using supercritical carbon dioxide. A range of pressures (40.41 MPa to 60.62 MPa) and temperatures (40 and 80 C) were tested. In addition, 5 % chloroform, 10% chloroform, 5 % methanol, 10 % methanol, and 4% acetonitrile containing 0.05% trifluoroacetic acid were used as co-solvents in the extraction process. After analysis by HPLC, it was found that the optimum conditions for extraction of bixin was 60.62 MPa and 40 C with 4% acetonitrile containing 0.05% trifluoroacetic acid as the co-solvent (Anderson and others 1997). Cloudberry Seed: Cloudberry seeds produce oil that is low in saturated fats, and contains relatively high amounts of tocopherols and carotenoids. The extraction of Cloudberry seed oil by supercritical fluid extraction was compared to that of a Soxhlet extraction using diethyl ether as the solvent. The range of extraction conditions consisted of 9 to 30 MPa at either 40 C or 60 C. The extracts were analyzed by GC-FID and HPLC for their fatty acid and carotenoid/ tocopherol content respectively. There was no significant difference in the fatty acid composition of any of the extracts; the carotenoid content did not increase at pressures over 15 MPa, and the amount of tocopherols extracted decreased with increasing pressure (Manninen and others 1997). Grape Seeds: Phenolic compounds and lipids were extracted

Supercritical Carbon Dioxide as a Solvent for Extraction

Supercritical Fluid Extraction of Botanical Samples

Supercritical fluids have been used to extract a wide range of analytes from botanical samples. These analytes range from essential oils to phytochemicals, and can include lipid extraction. These extracts have been used for analytical purposes, supplementation

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from white grape seeds and analyzed for phenolics (Palma and Taylor 1999) and for the extracts antimicrobial activity (Palma and others 1999). The optimum extraction conditions for phenolics was determined to be CO 2 at a density of 0.95 g/ml and 55 C with a co-solvent of 10% methanol with the most influential variable on the extraction being the CO 2 density, followed by the nature of the co-solvent, percentage of co-solvent, and extraction temperature (Palma and Taylor 1999). In addition, it was found that the extract could be split into two fractions; those compounds obtained by pure CO2 and those compounds obtained with CO 2 and a co-solvent. The first fraction contained mainly fatty acids and sterols, while the second fraction contained phenolic compounds. The first fraction was found to have a high degree of antimicrobial activity while the second fraction tested positive for antifungal activity (Palma and others 1999) Paprika: Lipids, carotenoids and tocopherols were extracted from paprika and analyzed by HPLC. Vesper and Nitz (1997) compared the use of supercritical CO2 at 35 MPa and 50 C to the use of hexane and found that hexane had slightly higher carotenoid content while the extracts obtained by SFE had a higher water content (Vesper and Nitz 1997a). After storage studies of these extracts at 40 C, it was found that the samples extracted by SFE had reduced fatty acid stability compared to the samples obtained by hexane extraction (Vesper and Nitz 1997b). Jaren-Galan and others (1999) and Skerget and others (1998) analyzed the effects of temperature and pressure on the composition of the pa-

Table 2Summary of publications on extraction methods from plant and botanical samples using supercritical fluid extraction (Pub. Year >1994) Sample Annato Black Pepper Buriti Caraway Seeds Limonene Carrot Chamomile Cloudberry Seed Analyte(s) trans-Bixin Essential Oil Carotenoids Lipids Carvone Carotenes Flavinoids Lipids, -carotene, Tocopherols Pressure (MPa)/ T (C) 40 to 60/40 20 to 32/45-65 20-30/40-55 7.5-300/32-75 60.6/40 20.2/40 9-30/40-60 Analytical Instrument HPLC GC-FID GC-FID Spectrophotometry GC-FID HPLC GC-MS GC-FID, HPLC Reference Anderson and others 1997 Tipsrisukond and others 1998 Ferreira and others 1999 De Franca and others 1999 Baysal and Starmans 1999 Chandra and Nair 1997 Scalia, Giuffreda and Pallado 1999 Manninen and Kallio 1997 Manninen and Laakso 1997a Manninen and Laakso 1997b Manninen and others 1997 Della Porta and others 1998 Illes and others 2000 Simandi and others 1999 Poiana, Sicari and Mincione 1998 Palma and others 1999 Palma and Taylor 1999 Palma and others 2000 Reverchon and Della Porta 1995 Dauksas and others 1998 Sass-Kiss and others 1998 Sass-Kiss and others 1998a Skerget and others 1998 Jaren, Nienaber and Schwartz 1999 Vesper and Nitz 1997b Vesper and Nitz 1997a Illes and others 1999 Ammann and others 1999 Aleksovski and others 1999 Ibanez and others 2000 Le Floch and others 1998 Simandi and others 1998 Ibanez and others 1999 Senorans and others 2000 Lopez-Sebastian and others 1998 Coelho-Luiz and others 1997 Bauman and others 1999 Tena and others 1997 Ronyai and others 1999 Bauman and others 1999 Pino, Garcia and Martinez 1999 Della and others 1998

Clove Bud Coriander Seed Fennel Seeds Grapefruit Grape Seeds

Essential Oil Essential Oil Fennel Oil Limonene and Terpines Polyphenolic compounds

8-20/50-50 20-30/35 8.1/40 8-25/40 45.6/35

GC-MS GC-FID GC-MS GC-FID GC-FID, HPLC

Lavender Lovage Onion Paprika

Essential Oil Essential Oil Essential Oil Lipids, carotenoids, and tocopherols

9/ 48 8-30/10-50 10-30/45-65 35/50 15/40 13.7-41.3/40

GC-MS GC-FID GC-FID HPLC, GC-FID

Peppermint Olive Products Oregano Rosemary

Essential Oil Tocopherols Phenol Compounds Essential Oil Antioxidants Essential Oils

6.5-10/25-40 35/50 33.4/100 8-30/40 10-16/37-47 10-40/60

GC-MS SFC-FID Electrospray-MS GC-FID GC-MS

Sage Spearmint Leaf Star Anise 38

Essential Oils Antioxidants Essential Oil Essential Oil

10-40/60 27.6/60 8-20/50

GC_MS GC-MS GC-MS

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Table 3Plant materials analyzed for anticancer agents NCI Designation Specific Name B628201 B628259 B628318 B634635 B638786 B805592 B806512 Variety Celastraceae Celastraceae Celastraceae Caryophyllaceae Verbenaceae Moraceae Annonaceae Origin Tanzania Kenya India Michigan Panama Peru Peru Plant Material B628259 B628318 B634635 B638786 B805592 B806512 Table 4Cytoxicity of extracts as measured by ED50 values (mg extract/l C2H5OH) Supercritical Carbon Dioxide Extraction 30.0 0.25 24.0 4.0 21.0 14.0 Ambient Temperature Ethanol Extraction 11.0 0.11 43.0 >100.0 126.0 6.2

Maytenus senegalensis Maytenus senegalensis Maytenus senegalensis Gypsophilia Citharexylum caudatum Maquire calophylla Rollinia papilliionela

Reprinted with permission from McHugh and Krukonis 1994.

Reprinted with permission from McHugh and Krukonis 1994.

prika extract. It was found that the selectivity of CO2 is higher at lower temperatures (40 C) and that the optimum pressure for extracting aromatic compounds is 15 MPa and coloring compounds is 40 MPa (Skerget and others 1998). After further analysis, it was determined that the pigments isolated at lower pressures consisted mainly of -carotene. Extracts obtained at higher pressures contained a greater proportion of capsorubin, capsanthin, zeaxanthin, and -cryptoxanthin and little -carotene. It was further determined that a two stage extraction could be used to extract a majority of the compounds present (200% compared to reference). This extraction first consisted of an extraction at 40 C and 13.8 MPa followed by an extraction at 40 C and 41.3 MPa (Manuel and others 1999). Rosemary: Antioxidants from rosemary were extracted and dearomatized by supercritical CO2 for use in natural supplements. The extraction of rosemary oil was initially modeled by Coelho, and others (1997) using the model developed by Sovova (1994). Ibanez and others (1999) continued this work by utilizing fractionation to further separate the essential oil extracted from the rosemary plants into two different fractions. These fractions had different compositions and antioxidant activities (Ibanez and others 1999). Lopez-Sebastian and others (1998) utilized supercritical CO2 remove the rosemary aroma from the extracts obtained by solvent extraction. This produces an extract which is more suitable for use in food products. In addition, this process also removes almost 100% of the residual solvent left from the extraction process (Lopez-Sebastian 1998).
Supercritical Fluid Extraction of Lipids

Supercritical carbon dioxide has been utilized to extract lipids from an assortment of matrices. Some of these extractions have been used to analyze the fat content of different food products. Other extractions have been used to obtain pure lipid extracts or to produce products that contain a reduced amount of certain lipids or other compounds such as cholesterol. Table 5 presents a list of some current articles in the field. Dairy Products: Dairy products have been subjected to supercritical fluid extraction to fractionate lipids and isolate vitamins for quantification. Berg and others (2000), and Turner and Mathiasson (2000) utilized supercritical CO2 to remove vitamin A and E from powdered milk, fluid milk and meat. The extraction conditions for both studies were 80 C and 37 MPa. Berg and others (2000) furthered this research by using Hydromatrix as a water absorbent to reduce the adverse effects of the water component of milk on the extraction process. This allowed all of the vitamin A and E to be extracted from a 0.5 gram sample in 80 minutes, shorter than conventional methods. In addition to vitamins, the distribution of polychlorinated biphenyls (PCBs) in the different milk fat fractions was studied using supercritical fluid extraction. Ramos and others (2000) utilized supercritical CO2 to fractionate

the milk fat into 4 different fractions. These factions were shortchain triglycerides (SCT), medium-chain triglycerides (MCT), longchain triglycerides (LCT) and cholesterol. From this research it was found that PCBs can be found predominately in the SCT, MCT and cholesterol fractions of the milk fat (Ramos and others 2000). Professor S.S.H. Rizvi has been a virtual front runner in the use of supercritical fluids to fractionate milk fats. In 1992 he published an article on the fluid liquid equilibria of anhydrous milkfat with supercritical CO 2 (Yu and others 1992). Following this research, Shukla and others (1994) studied the effect of using milk fat obtained by supercritical fluid extraction on the physicochemical and rheological properties of butter. This research demonstrated the ability to use supercritical CO2 to produce butter with specific properties such as reduced moisture content, elevated melting points, and lower cholesterol. This research was expanded from butter to milk fat which can be used in other products by Bhaskar and others (1998). Meats: Supercritical fluid extraction has been utilized to extract lipids from a variety of meats, including pork and beef. These methods have been developed for analytical purposes, and are discussed in detail in analytical section of this paper. Nuts and Seeds: Lipids have been extracted from a series of different types of nuts and oil seeds. Some examples of these are pecans and rice bran. A reduction in the lipid content of pecans can increase their storage life (Alexander, Brusewitz and Maness 1997). From this study it was determined that the optimum extraction conditions for pecan oil was 66.8 MPa and 75 C. The kinetics of this extraction was also determined to be limited by the solubility of pecan oil in supercritical CO 2 for the initial part of the extraction, while it was diffusion limited for the second half of the extraction (Alexander and others 1997). During SFE the possibility of breakage of the pecan kernels increases with increased moisture content of the kernels (Bellmer and Brusewitz 1999). This effect was increased when a short depressurization time was used. A reduction in the moisture content of the pecans to a final moisture content range of 3.5% to 11.0% before lipid extraction can reduce the amount of kernel breakage (Bellmer and Brusewitz 1999). Rice bran is a by-product of rice milling that contains 15 to 20% oil by weight which could be of value to the food and pharmaceutical industry (Shen and others 1996). This oil has been extracted by SFE. Shen and others (1996) reported that rice bran oil was extracted with subcritical and supercritical CO2 at a temperature range of 0 to 60 C and pressures of 17 to 31 MPa. This research was furthered by Shen and others. (1997) by the addition of fractionation to separate water and free fatty acids from the extract. Kuk and Dowd (1998) continued this research by comparing the quality of oil extracted by supercritical fluid extraction as compared to that of hexane extraction. It was found that at temperatures of 80 C the quality of oil extracted by supercritical fluid

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extraction was far superior to that of oil obtained by hexane extraction (Kuk and Dowd 1998). Seaweed and Fungi. -3 fatty acids were extracted from brown seaweed, Sargassum hemiphyllum (Turn.) C., and fungi, Cunninghamella echinulata and Pythium irregulare, by SFE for potential use in functional foods and nutraceuticals. Chung, and others (1998) reported that they were able to obtain yields of total and individual -3 fatty acids that were significantly greater than those obtained by Soxhlet extraction by using extraction conditions of 37.9 MPa and 50 C. They also found that the concentration of total polyunsaturated fatty acids increased significantly and total saturated fatty acids decreased significantly with increasing pressure and solvent density as measured by the ratio of saturated to unsaturated fatty acids. Supercritical CO2 was used to extract fungal oil containing -linolenic acid from Cunninghamella echinulata with similar results (Certik and Horenitzky 1999). It was found that supercritical CO2 extraction at 30 MPa and 50 C yielded greater than 110% of the -linolenic acid that was obtained by chemical extraction. Walker and others (1999) also extracted -3 fatty acids using SFE, but they had the aid of surfactants which will be discussed later in this article.
Supercritical Fluids and Analytical Uses

Supercritical carbon dioxide has been utilized in multiple methods of analysis. Supercritical carbon dioxide is used as either an extraction medium, as in rapid analyses for fat content, or as a mobile phase, as in supercritical fluid chromatography. This section covers the use of supercritical fluids in the detection of fat content, pesticide residues, and supercritical fluid chromatography. Rapid Analysis for Fat Content: Supercritical fluids have been used to determine the fat content of numerous products ranging from beef to oil seeds and vegetables. Taylor and others (1997) compared traditional methods of analysis for fat content to newer methods developed with supercritical fluid extraction. For the analysis of fats content in soybeans, sunflower, safflower, cottonseed, rapeseed and ground beef, it was found that supercritical fluid extraction yielded higher recoveries than those obtained by

Table 5Summary of Publications on Lipid Extraction Studies Using Supercritical Fluid Extraction (Pub. Year >1995) Sample Brown Seaweed Butter Oil Corn Bran Cotton Seed Cunninghamella echinulata Ground Beef Milk Fat Analyte(s) Fatty Acid Composition Cholesterol Ferulate phytosterol esters Fatty Acid Composition Fatty Acid Composition Fatty Acid Composition Lipid Profile Vitamins Solid Fat content, PCBs Pressure (MPa)/ T (C) 24.1-37.9/40-50 10-27.6/40-70 13.8-69/40-80 51.7-62/100 20-35/40-70 38/80 6.9-17.2/40-60 Analytical Instrument GC-FID GC-FID SFC GC-FID Gravametric GC-FID GC-FID GC-FID HPLC Pulsed NMR DSC Reference Cheung, Leung and Ang 1998 Mohamed, Neves, and Kieckbusch 1998 Taylor and King 2000 Taylor, Eller and King 1997 Certik and Horenitzky 1999 King and others 1996 Taylor, Eller and King 1997 Bhaskar and others 1998 Ramos, Hernandez and Gonzalez 2000 Turner and Mathiasson 2000 Berg and others 2000 Manganiello, Rios, and Valcarcel 2000 Andersson, Demirbuker and Blomberg 1997 Alexander, Brusewitz and Maness 1997 Li, Bellmer and Brusewitz 1999 Palazoglu and Balaban 1998

Oat Bran Pecan

Digalactosyldiacylglycerols Fatty acid composition and product breakage Texture and Sensory Attributes Cholesterol/Fat Content Texture and Sensory Attributes Fatty Acid Composition Fatty Acid Composition Tocopherol Content Fatty Acid Composition -tocopherol sterols Moisture Fatty Acid Composition Fatty Acid Composition Fatty Acid Composition

40/50-70 41.3-66.8/45-75

SFC GC-FID

Pistachio

20.7-34.5/50-70

Pork

7.3-34/50-150

Pythium irregulare Rapeseed Rice Bran

13.7-27.5/40-60 51.7/100 17-31/0-60 48.2-62/70-100 51.7-62/100 51.7-62/100 51.7-62/100

Instron Universal Testing Machine and Sensory Panel HPLC TLC Sensory Panel Hunter Colorimeter GC-FID HPLC GC-FID GC-MS HPLC GC-FID GC-FID GC-FID

Lin and others 1999 Berg and others 1997

Walker, Cochran and Hulbert 1999 Bruhl and Matthaus 1999 Shen and others 1996 Shen and others 1997 Kuk and Dowd 1998 Taylor, Eller and King 1997 Taylor, Eller and King 1997 Bruhl and Matthaus 1999 Taylor, Eller and King 1997 Bruhl and Matthaus 1999

Safflower Soybean Sunflower

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Supercritical fluids and the food industry . . .

the AOCS approved methods (Taylor and others 1997). Analysis of the lipid content of dairy products has also seen some advances in the past year. Dioniosi and others (1999) compared supercritical fluid extraction to Rse-Gottlieb solvent extraction. Their research demonstrated a similarity between the two extraction methods while the supercritical fluid extraction method was faster and free of organic solvents. Manganiello and others (2000) applied the use of piezoelectric detection to supercritical fluid extraction. The use of an in-line piezoelectric detector is able to measure the change in weight of the sample during the extraction process. This allows for more accurate determination of the final weight of the sample after all of the fat has been extracted (total fat). In addition, it can allow for more rapid determination of the total fat by determining the point when the steady state mass has been reached without having to re-extract the sample multiple times to confirm that the steady state mass has been reached (Manganiello and others 2000). Rapid Analysis for Pesticides in Foods: Pesticide residues are a concern among consumers throughout the United States and other countries. Currently the methods of analyzing food products and other substances such as contaminated soil and water involve the use of organic solvents such as hexane and dichloromethane to extract the pesticides from the sample matrix. Once the pesticides have been extracted from the sample matrix, the samples must be cleaned to remove any unwanted compounds, such as lipids, which may interfere GC analysis of the sample for any pesticides present. The most common method for cleaning is solid phase extraction (van der Hoff and van Zoonen 1999). Supercritical fluid extraction provides an alternative to using organic solvents for the extraction of pesticides from their sample matrix. Some of the advantages which supercritical fluid extraction provides over the traditional methods of pesticide extraction are that the extraction can be performed in less time, and utilizes less solvent volume. In addition, supercritical fluid extraction can be tailored to the solute of interest by changing the temperature and pressure of the extraction process. Supercritical fluid extraction can also be tailored for pesticides that contain more polar groups by the addition of polar modifiers to the CO2 such as methanol (Camel 1998). The use of supercritical CO2 for the extraction of pesticide residues has been reviewed extensively in other articles and so it will be only covered briefly in this article. Three reviews, which stand out in the recent years are: Lehotay (1997), Camel (1998), and van der Hoff and van Zoonen (1999). These articles cover the analysis of pesticides in food, water, soil and animal tissue in depth along with the benefits and drawbacks of supercritical fluid extraction in the analysis of pesticide resides. Since the release of the previously mentioned review articles, more contributions to the body of scientific knowledge have been published. These articles range from the determination of fenpyroximate in apples (Halvorsen and others 2000) to the extraction/ analysis of atrazine and other triazine herbicides from eggs (Pensabene 2000). Solvents for Chromatography: Supercritical CO2 has been utilized as a mobile phase in supercritical fluid chromatography since the early 1980s when it was developed for use as a replacement/ complement to gas chromatography (Poole 2000). Using supercritical fluids as a mobile phase for chromatography provides some advantages over conventional GC or HPLC. The solvent power of the supercritical fluid can be manipulated by changing the temperature and/ or the pressure (density) of the mobile phase. In the case of carbon dioxide, when it is at lower densities, it is similar to that of n-hexane and at higher densities it is similar to that of dichloromethane (Brunner 1994). In fact, just as the composition of the mobile phase can be varied during the separation of compounds during HPLC, the density of the mobile

Figure 5Flow diagram of a supercritical fluid chromagraphy system

phase can be varied in supercritical fluid chromatography. By varying the density, the user is in fact varying the degree to which the mobile phase is a nonpolar solvent. A polar co-solvent can also be added to the mobile phase which can allow for the separation of more polar compounds as compared to those which can be separated by carbon dioxide alone (Brunner 1994). A supercritical fluid chromatograph consists of the same basic components as HPLC and GC. Both HPLC and supercritical fluid chromatography systems have a minimum of one pump with the possibility to upgrade to a system that can handle gradients, an injection apparatus, a column for separation, a detector and possibly a part for collection of different analyte fractions (Figure 5). The key differences between the two systems are the fact that the pump on the supercritical fluid chromatography system on average could handle increased pressures, and the column on the supercritical fluid chromatography system has to be heated to elevate the temperature of the mobile phase above its critical point (Brunner 1994). Much like the similarities between supercritical fluid chromatography and HPLC, supercritical fluid chromatography and GC also have many similarities. Both systems can utilize capillary columns and FID detectors. In fact, this represents two different approaches to supercritical fluid chromatography, packed column and capillary column. Packed column chromatography is similar to HPLC while capillary column is similar to GC. In packed column chromatography, it is usually necessary to utilize co-solvents to elute polar compounds from the column, whereas capillary column chromatography usually uses pure CO2. The reason for using pure CO 2 is that most polar modifiers are not compatible with FIDs, eliminating the method of detection (Pyo 2000). In addition, packed column supercritical fluid chromatography has advantages over tradition normal phase HPLC in that it offers faster separations and column re-equilibration, and higher column efficiencies. Packed column supercritical fluid chromatography also offers advantages over normal phase HPLC in that it has more variables which can be manipulated so that the analysis can be better tailored to the analyte of interest (Poole 2000).

Supercritical Fluids and Novel Methods of Food Processing

Supercritical fluids have been utilized in different novel methods of food processing. Two of these new methods will be discussed in this paper. These methods are the use of supercritical CO2 to control puffing in extrusion, and the use of supercritical

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CO2 to remove excess hexane from soybean oil produced by conventional extraction techniques.
Controlled Puffing in Extrusion

Conclusions
Based on the information presented in this paper, it can be seen that supercritical fluids have been used for developing an ever-expanding niche in the food industry. Whether it is used as a solvent for extraction in analytical methods such as gravimetric determination of fat content or in large-scale extractions such as decaffeinating coffee, supercritical CO2 has proven its usefulness as a replacement for organic solvents. With increasing concerns about the use of organic solvents and their disposal, supercritical fluid extraction is gaining in popularity faster than ever before. The future looks promising for the use of supercritical fluids, with new methods of extraction constantly being developed, as with other novel uses for the food processing industry.

Rizvi and Mulvaney (1992) patented a new method to produce extruded products using supercritical CO2 to reduce the temperature and shear needed for conventional thermoplastic extrusion. Currently, the high temperatures (130 to 170 C) and shear prevents the use of thermally liable ingredients such as some flavors, colors and whey proteins (Alavi and others 1999). This method of extrusion uses supercritical CO2 as a blowing agent to facilitate the formation of the cellular structure in extrudates in place of the expansion of water upon exit of the extruder barrel in conventional extrusion. The use of supercritical fluids in the extrusion process is based on a four-step process described by Rizvi and others (1995). The steps in this process include: 1) development of a dough with gas-holding properties by mixing alone; 2) injection of supercritical CO2; 3) creation of a controlled thermodynamic instability by manipulation of the pressure and/ or temperature in the extruder; and, 4) control of the degree of cell growth during the setting of the product through appropriate die selection and post-extrusion drying and cooling processes (Rizvi and others 1995). The use of supercritical fluid extrusion was further studied by Alavi and others (1999) and Gogoi and others (2000) when the use of whey protein concentrate and egg whites in production of extruded products was studied. The results from the study by Alavi and others demonstrated that the extrudates produced by supercritical fluid extrusion had a similar bulk density and expansion ratio to steam extrudates, while the products obtained from supercritical fluid extrusion had a unique composition and uniform microcellular composition (Alavi and others 1999). Gogoi and others then determined that the addition of egg white to the feed produced a softer skin and fragile, well formed cellular structure (Gogoi and others 2000). This research can lead to the addition of heat-sensitive flavorings to extruded products after the product has been heated or while the product is being expanded by the previously mentioned method.
Removal of Hexane from Soybean Oil

Primary References for Supercritical Fluid Extraction

Allada SR. 1984. Solubility Parameters of Supercritical Fluids. Ind J Chem Process Development 23:344-348. Brunner G. 1994. Gas Extraction, An Introduction to Fundamentals of Supercritical Fluids and the Application to the Separation Processes. Springer, New York, NY. Dionisi F, Hug B, Aeschlimann JM, Houllemar A. 1999. Supercritical CO2 extraction for total fat analysis of food products. 1999. J Food Sci 64(4):612-615. Kiszka MB, Meilchen MA, McHugh MA. 1988. Modeling High-Pressure Gas-Polymer Mixtures Using the Sanchez-Lacombe Equation of State. J Applied Polymer Sci 36:583-597. Lehotay SJ. 1997. Supercritical fluid extraction of pesticidies in foods. J Chromatoraphy A 785:289-312. McHugh M, Krukonis V. 1994. Supercritical Fluid Extraction, Second Edition . Butterworth-Heinemann, Boston. Reverchon E, Della Porta G. 1995. Supercritical CO 2 Extraction and Fractionation of Lavender Essential Oil and Waxes. J Agri and Food Chem 43:1654-1658. Rizvi SSH, Mulvaney SJ. 1992. Extrusion processing with supercritical fluids. U.S. Patent 5120559. Rizvi SSH, Yu ZR, Bhaskar AR, Chidambara Raj CB. 1994. Fundamentals of Processing with Supercritical Fluids. Ch 1 in Supercritical Fluid Processing of Food and Biomaterials . Rizvi, S.S.H. p. 1-26. Bishopbriggs, Glasgow: Chapman & Hall. Sovova H. 1994. Rate of the vegetable oil extraction with supercritical CO2- I. Modeling of extraction curves. Chem Eng Sci 49(3):409-414. Valcarcel M, Tena MT. 1997. Applications of supercritical fluid extraction in food analysis. Fresenius J Anal Chem 358:561-573.

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Supercritical CO 2 has been utilized by Reverchon and others (2000) to eliminate hexane from soybean oil during its preparation for food use. The justification for this research is that hexane elimination is the step that uses the largest amount of energy during the soybean oil extraction process. In addition to the costs associated with hexane elimination, tradition methods can leave a hexane residue of up to 1000 ppm. Reverchon and others (2000) developed a method to eliminate hexane from soybean oil by passing it countercurrent to supercritical CO2 through a packed tower. These experiments were performed with soybean oil containing 10% hexane to simulate the soybean oil/ hexane mixture found after the second stage of traditional hexane elimination. It was found that the best results for this experiment were obtained at a CO2 density of 716 kg m-3. At this density the hexane residue left in the soybean oil was only 20 ppm (Reverchon and others 2000). This publication also raises some questions, which could be addressed in future research. The authors determined the range of temperature and pressures to begin the extraction process using binary phase diagrams for the solubility of hexane in supercritical CO2 and soybean oil in supercritical CO2, but not as a ternary system taking the interaction of supercritical CO2, hexane and lipids into account. The hexane could act as a nonpolar co-solvent with the CO2 to increase the solubility of some of the shorter chain fatty acids present in the soybean oil. This could cause changes in the fatty acid composition of the soybean oil, and thus alter its nutritional value or its cooking characteristics.
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Supercritical fluids and the food industry . . .

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