Components For A Supercritical Fluid Extraction Biology Essay

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There are numerous commercial extraction units available which incorporate many or all of these features; however, a system can also be easily assembled in the laboratory. Pure CO2 serves as the fluid of choice for the majority of all extractions. The purity of CO2 employed should be as high as possible.

In many cases the solvating power of supercritical CO2 at high density is insufficient to extract an analyte because the analyte is either not soluble or is strongly bound to the matrix. Therefore other fluids like N20 and CHF3 can be used in such cases. The most common modifiers include methanol, acetonitrile, toluene, and water. In many cases the modifier is pre-mixed to a specific weight percent with CO2 either in a gas cylinder, directly in a syringe pump, or in-line with both a fluid pump and a modifier pump. A recently reported extraction strategy is to perform in situ derivatizations to improve the extraction efficiencies of polar or bound analytes (Hills et al., 1991)[23]. Both syringe and piston pumps are used to compress the CO2 to the liquid state for introduction to the heated zone. The piston pump provides a constant fluid supply but the pump head and fluid transfer lines must be cooled. A syringe pump provides pulse less flow and auxiliary cooling is not required. The pump volume is, however, limited and extra time must be budgeted for periodic refilling. If the fluids are changed continuously then it creates problem with syringe pump because it becomes very difficult to clean the pump in short time. The presence of polar modifiers in the fluid especially aggravates this situation. Since SFE is normally performed at constant pressures, the less expensive piston pumps have become more popular.

The extraction vessel must withstand high pressure (5000-10,000 psi) for performing a variety of extraction ranging in volume from 0.1 ml to 50 ml. The material used for construction is generally stainless steel and it should be chemically inert. In order to provide an effective high pressure seal, extraction vessels are usually less than 3 cm in diameter. Larger vessels require multi-seal systems in order to operate at high pressures. Stainless steel frits are placed at either end to contain the sample matrix within the vessel. The seals themselves are usually Kel-F or Teflon. Silicone rubber seals are not used as they swell and may dissolve in some SFs. The vessel is usually housed in a temperature controlled oven but a heated tube will also suffice for conversion of CO2 from the pumped liquid to the supercritical fluid state. The vessel outlet flow restrictor may be fabricated from deactivated fused silica or metal. Both fixed and variable restrictors are currently employed. With a fixed restrictor greater fluid density is achieved by an increased pumping rate. The inner diameter of the restrictor regulates the flow and back pressure. The design of the restrictor (e.g. linear, tapered, etc.) dictates the decompressed flow-density dependence.

A variable restrictor affords an adjustable flow wherein pressure and flow are decoupled. A fixed restrictor is more likely to plug and is considerably more fragile. Different decompressed flow rates can be expected with different restrictors given the same pumping rate. Both fixed and variable restrictors in commercial systems are usually heated to minimize plugging.

Accumulation or trapping of any extractables can be off-line or on-line. In the off-line configuration depressurization of the CO2 may be either onto an inert solid surface, directly into a small volume of solvent, or onto a solid sorbent. The solid surface is cryogenically cooled by the expanding extraction fluid and by another external source (e.g. CO2 or liquid N2). Typical surfaces which have been used are glass vials (flasks), stainless steel plate or beads, glass beads, and deactivated fused silica. After trapping on the cryogenically cooled surface, the analytes may be rinsed from the surface with an appropriate liquid for further analysis. Alternately, the analyte may be re-dissolved by higher density CO2 or if the analyte is volatile thermal desorption can be used. The liquid trap may be the mechanically simplest way to trap an analyte. The restrictor is simply placed in a vial of liquid solvent.

The analyte is trapped in the solvent, while the decompressed fluid vents to the atmosphere. The liquid solvent trap must be compatible with the analytes of interest and also with the extraction fluid when modifiers are used. Methanol is frequently used as a solvent for polar analytes; while, dichloromethane is most frequently used with nonpolar analytes. When CO2and N20 is used as extraction fluids there can be a great deal of cooling associated with the decompression of the fluid. While cooling of the solvent may prevent rapid evaporation of the collection solvent, it is possible for the collection fluid to freeze and for small pieces of ice to clog the restrictor tip. For this reason, the restrictor is often heated above 100°C in some cases. The flow of compressed fluid for liquid trapping is usually maintained at less than 1 ml/min because upon decompression of the fluid approximately 500 ml/rain of gas is produced. This large volume of gas can cause violent bubbling of the liquid collection solvent and lead to analyte loss via aerosol formation. For this reason newer versions of liquid traps employ a partially sealed container and/or baffles. In spite of these problems, volatile analytes have been quantitatively recovered. For example, decane is retained in 3 ml of CH2Cl2 during a 15 minute extraction using approximately 1 ml/min of supercritical CO2 (Hawthorne et al., 1989)[24]. The third type of trapping system used in off-line SFE is a solid phase sorbent, which most often is chromatographic packing material. The packing material provides two trapping mechanisms--cryogenic trapping and absorption. The trap is cryogenically cooled, again either by the expanding fluid or by another source. The analytes are trapped and then rinsed from the packing material with a small volume of organic solvent. Typical (liquid) compressed flow rates are higher than solvent or inert surface trapping and can range from 1-4 ml/min.

In the on-line SFE/GC configuration, trapping directly either onto the head of a GC column, inside a heated split GC injector, or on a cold trap external to the GC column is performed. If the extracted analytes have high vapor pressures naturally the GC column should be cooled to sub-zero temperature, in order to narrowly focus the extract. Obviously one must ensure that the extracted portion does not cause the column to overload and plug. For SFE/SFC the outlet restrictor (Lee and Markides, 1990) [17] is inserted into a low dead volume accumulator tee, Fig. 2.

. Figure 2- Schematic diagram of a combined supercritical fluid extractor

During the extraction the tee is vented to atmosphere and the extract is trapped in the tee. After extraction the valve is switched and supercritical fluid is introduced through the side arm of the tee to transfer the sample to the chromatography column. Both off-line and on-line SFE modes have advantages, however, the off-line mode tolerates larger extract samples. It frees chromatographic and spectrometric instrumentation for other analyses while sample preparation is being accomplished. The collected analytes are available for analysis on multiple instruments. The on-line mode affords the greatest analyte concentration with minimal loss of volatiles which lowers the limit of detection. Sample manipulations are minimized thereby providing the extractable protection from air, water and light. One disadvantage of on-line trapping is that the small sample required may not be representative of the bulk. Injection size is not as easily controlled in the on-line mode; therefore, column overload is of higher potential.

Strategies of supercritical fluid extraction

SFE can be accomplished by using either a static, dynamic, or coupled static/dynamic mode. A static extraction refers to one where a fixed amount of fluid is used to interact with the analyte/matrix. Normally, the extraction vessel containing the matrix is pressurized with the chosen fluid at a certain temperature. The high diffusivity of the supercritical fluid is then utilized to access the matrix/analyte. Static on-line extraction methods are limited in that a high pressure gas must be monitored, in situ, or introduced into an analytical system. One obvious way to monitor the system is via a high pressure flow cell so that the solvated components can be monitored spectrometrically. Figure 3 shows such a system which consists of a controller, a pump, a simple valving scheme and an associated spectrometric detector.

Figure 3 - Static extraction with spectrometric monitoring

The valve allows the system to be pressurized and then isolated from the pump, thereby creating a closed system. As shown, the extracted analytes must migrate to the flow cell in a diffusion limited process. The distance that the extract must travel can be decreased dramatically by building the flow cell directly into the housing of the extraction chamber. The recent development of high pressure recirculation pumps allows for the spectrometric device to be placed some distance away from the extraction chamber. Additionally, the mechanical mixing provided by the recirculation pump ensures homogeneity of the SF phase so that precise sampling can be achieved. One of the experimental problems encountered with a static extraction is that it may not be exhaustive because the SF may become completely saturated with analyte. Since only a portion of the fluid is assayed, the limit of detection for any extractables may be higher than if trapping and concentration of the extract had been carried out. There may be an additional problem in that co-extraction of components of the matrix may occur along with the analyte.

A dynamic extraction employs fresh supercritical fluid which is continuously passed over and/or through the sample matrix. A dynamic extraction can be more exhaustive than a static one because fresh SF is always in contact with the sample. However, impurities in the supercritical fluid become a concern when using large amounts of fluid during an extraction. The contaminants in the supercritical fluid will ultimately arrive at the collection device, become concentrated, and may interfere with the extract analysis. Another experimental problem with a dynamic extraction relative to a static extraction is enhanced matrix mobility (i.e. more SF should remove more extractable matrix components). Finally, a combination of initially a static period followed by a dynamic one in order to move extractable to the trap is gaining popularity especially for situations where analyte must diffuse to the matrix surface in order to be extracted. The extraction starts in strictly a static mode. There is no net flow through the system. When the extraction has proceeded for a given amount of time the system is put into a dynamic mode by the switching of valves. Fresh SF enters the vessel replacing the original SF which has exited through the restrictor. Dynamic extraction is the most easily modeled of the three modes (Stahl et al., 1988) [25]. Figure 4 shows the theoretical extraction profile of an analyte from a solid matrix using a dynamic system.

Figure 4 - Extraction profile for dynamic mode. Region I - Solubility dependent region. Region II- Intermediate region (bulk analyte is almost removed). Region III-Diffusion limited region

The y-axis represents the amount of analyte recovered from the system. The x-axis represents the amount of fluid or time used during the extraction. The extraction profile can be divided into three distinct regions. The initial extraction of material occurs relatively fast and is dependent upon the solubility of the bulk analyte in the SF (Region I). During this portion of the extraction the analyte is simply purged from the extraction vessel. The limiting factors in region I are the solubility of the analyte in the SF, the rate at which SF passes through the system, and the amount of dead volume in the extraction vessel and associated tubing. Region II is an intermediate region where the extraction process is enthalpically controlled (i.e. analyte--matrix interaction) and therefore shows a slower rate of extraction. Region III represents that portion of the extraction where the process is truly diffusion limited. The diffusion phenomenon is brought about by the limited mobility of an analyte within a matrix (such as polymer additive in a polymer bead or a natural product within animal tissue).

Figure 4 can also describe the extraction profile for a static (or equilibrium) extraction. The x-axis now represents time instead of volume of SF which is passed through the system. Region I, II, and III still represent washing out, matrix-analyte disruption and diffusion limited processes. As time increases, however, equilibrium between analyte in the SF and analyte on the matrix is established. The diffusion limited process is no longer in one direction as it is in the dynamic case. More specifically during a dynamic extraction fluid is constantly moving any extracted analyte away from the matrix so diffusion back into or onto the matrix does not occur. The static mode, however, suffers from the fact that the equilibrium between the sample matrix and the supercritical fluid may not be favourable. Therefore, exhaustive static extraction can not be expected to occur in all cases. In practice the static mode is used most frequently in combination with a dynamic mode, as described previously. Figure 5 shows the extraction profile of several static/dynamic cycles.

Figure 5 - Extraction profile for static/dynamic mode. Region I - Initial static extraction (no movement of analyte through the system). Region II - Dynamic extraction (analyte being removed from the system). Region III - Second static extraction. Region IV -Dynamic extraction. Region V - Static extraction. Region VI-Dynamic Extraction

Region I represents a true static extraction. There is no net flow through the system so the amount of analyte recovered (y-axis) is zero. Any analyte removed from the sample matrix is contained within the extraction vessel and the concentration of bulk analyte increases with time as described in the previous extraction profile. Region II is a washing out phase. Valves have been switched so that there is now a net flow through the system. The analyte which has built up in the extraction vessel quickly washes out and enthalpic and diffusion limited processes follow. By returning the system to static mode (Region III) SF is conserved while the bulk analyte concentration again continues to rise within the extraction vessel. Further dynamic/static extraction cycles are then carried out. The method can be seen to be advantageous if the SF is in limited quantity or costly. The time required to perform an exhaustive static/dynamic extraction, however, may be greater than that of a totally dynamic extraction.

Extraction with supercritical fluids

Supercritical extraction has been applied to a large number of solid matrices. The desired product can be either the extract or the extracted solid itself. The advantage of using supercritical fluids in extraction is the ease of separation of the extracted solute from the supercritical fluid solvent by simple expansion. In addition, supercritical fluids have liquid like densities but superior mass transfer characteristics compared to liquid solvents due to their high diffusion and very low surface tension that enables easy penetration into the porous structure of the solid matrix to release the solute (Montanes et al., 2007) [6]. Extraction of soluble species (solutes) from solid matrices takes place through four different mechanisms. If there are no interactions between the solute and the solid phase, the process is simple dissolution of the solute in a suitable solvent that does not dissolve the solid matrix (Brunner, 2005) [2]. If there are interactions between the solid and the solute, then the extraction process is termed as desorption and the adsorption isotherm of the solute on the solid in presence of the solvent determines the equilibrium. Most solids extraction processes, such as activated carbon regeneration, fall in this category. A third mechanism is swelling of the solid phase by the solvent accompanied by extraction of the entrapped solute through the first two mechanisms, such as extraction of pigments or residual solvents from polymeric matrices. The fourth mechanism is reactive extraction where the insoluble solute reacts with the solvent and the reaction products are soluble hence extractable, such as extraction of lignin from cellulose. Extraction is always followed by another separation process where the extracted solute is separated from the solvent (Dixton and Johnson, 1997) [4]. Another important aspect in supercritical extraction relates to solvent/solute interactions. Normally the interactions between the solid and the solute determine the ease of extraction, i.e., the strength of the adsorption isotherm is determined by interactions between the adsorbent and the adsorbate. However, when supercritical fluids are used, interactions between the

solvent and the solute affect the adsorption characteristics due to large negative partial molar volumes and partial molar enthalpies in supercritical fluids (Dixton and Johnson, 1997; Montanes et al., 2007)[4,6]. The thermodynamic parameters that govern the extraction are found to be temperature, pressure, the adsorption equilibrium constant and the solubility of the organic in supercritical fluid. Similar to the retrograde behavior of solubility in supercritical fluids, the adsorption equilibrium constants can either decrease or increase for an increase in temperature at isobaric conditions. This is primarily due to the large negative partial molar properties of the supercritical fluids. In addition to the above factors, the rate parameters like the external mass transfer resistances, the axial dispersion in the fluid phase, and the effective diffusion of the organics in the pores also play a crucial role in the desorption process. A thorough understanding of these governing parameters is important in the modeling of supercritical fluid extraction process and in the design, development and future scale-up of the process (Dixton and Johnson, 1997)[4].

SFE Process

A simplified process-scale SFE system is shown in Fig. 6 and a typical batch extraction proceeds as follows.

Figure 6 - A simple diagram of supercritical fluid extraction

Raw material is charged in the extraction tank which is equipped with temperature controllers and pressure valves at both ends to keep desired extraction conditions. The extraction tank is pressurized with the fluid by means of pumps, which are also needed for the circulation of the fluid in the system. From the tank the fluid and the solubilized components are transferred to the separator where the salvation power of the fluid is decreased by increasing the temperature, or more likely, decreasing the pressure of the system. The product is then collected via a valve located in the lower part of the separator(s) (Bravi et al., 2007; Brunner, 2005)[1,2].

Advantages and drawbacks of supercritical CO2

(SC-CO2): There are a large number of compounds that can be used as a fluid in supercritical techniques, but by far the most widely used is carbon dioxide. From the viewpoint of pharmaceutical, nutraceutical and food applications it is a good solvent, because it is non-toxic, non-flammable, inexpensive, easy to remove from the product and its critical temperature and pressure are relatively low (Tc=31.1°C, pc=72 bar) make it important for food and natural products sample preparation, is the ability of SFE using CO2 to be operated at low temperatures using a non-oxidant medium, which allows the extraction of thermally labile or easily oxidized compounds. It is environmental friendly and generally recognized as safe by FDA and EFSA. These properties make it suitable for extracting, for example, thermally labile and non-polar bioactive compounds but, because of its non-polar nature, it cannot be used for dissolving polar molecules. The solubility of polar compounds and the selectivity of the process can be increased by adding small quantities of other solvents, such as ethanol, in the fluid that named as co-solvent or modifier. On one hand, it decreases the processing times, increases yields and makes it possible to use milder processing conditions, but on the other, it complicates system thermodynamics and increases capital costs (Jose et al., 2007) [3]. The use of high purity SFE-grade CO2 is not required but impurity and moisture in industrial grade CO2 can accumulate and may interfere with further analytical operations (gas or liquid chromatography). Thus, an on-line fluid cleanup system may be used to remove trace contaminants. An important drawback of SC-CO2 and most of the other supercritical fluids is that predominantly, a non-polar extraction fluid, such as CO2, is used. Therefore, a logical trend to widen the application range of this technique is the study of new methods to decrease analyte polarity to make them more soluble in non-polar supercritical fluids. In this sense, chemical in situ derivatization has been applied to improve the selectivity of the extraction towards a specific group of compounds (Jose et al., 2007; Rozzi and Singh 2002)[3,5]. So the solvent power of SC-CO2 can be summarized by a few rules:

• It dissolves non-polar or slightly polar compounds.

• The solvent power for low molecular weight compounds is high and decreases with increasing molecular weight.

• SC-CO2 has high affinity with oxygenated organic compounds of medium molecular weight.

• Free fatty acids and their glycerides exhibit low solubilities.

• Pigments are even less soluble.

• Water has a low solubility (<0.5% w/w) at temperatures below 100oC.

• Proteins, polysaccharides, sugars and mineral salts are insoluble and;

• SC-CO2 is capable of separating compounds that are less volatile, have a higher molecular weight and/or are more polar as pressure increases (Brunner, 2005) [2].

Special applications of supercritical fluids to food processing: As mentioned before carbon dioxide is the most common supercritical fluid in the food industry. Due to the non-toxicity and low critical temperature, it can be used to extract thermally labile food components and the product is not contaminated with residual solvent. Further, the extract's color, composition, odor, texture are controllable and extraction by supercritical fluid carbon dioxide retains the aroma of the product. Supercritical fluid extraction provides a distinct advantage not only in the replacement but also extracts oils that are lower in iron and free fatty acid. Some application of SFE in food is mentioned below:

Removal of fat from foods: Edible oils and their components has been the target of supercritical fluid processing since the early 70s. Although triacylglycerides are only fairly soluble in SC-CO2, the advantages of organic solvent-free processing have stimulated research and development in various areas. One of these is the removal of fat from food. The process has been fully designed for commercial application, using the aforementioned standard design. The process has the advantage of producing fat-free or

fat-reduced potato chips. According to the expected taste the amount of remaining fat in the potato chips can easily be controlled (Catchpole et al., 2008; Chuang and Brunner, 2006) [7,8].

Enrichment of vitamin E from natural sources: SFE offers several advantages for the enrichment of tocochromanols over conventional techniques such as vacuum distillation, in particular a lower operating temperature. As starting material one can use various edible oils or their distillates. Most promising as feed materials are CPO and SODD (Fang et al., 2006) [9]. CPO contains several tocotrienols and tocopherols at a total concentration of approximately 500ppm. SODD may contain (after several conventional concentration steps) about 50% tocopherols. Both materials can be used for the production of enriched fractions of tocochromanols (Chuang and Brunner, 2006; Fang et al., 2006) [8,9]. Although it is possible to recover tocochromanolsdirectly from CPO, it is better to produce esters of the triglycerides in order to be able to more easily separate these compounds from the tocochromanols. In this method, the triglycerides are subject to an esterification with methanol to form fatty acid methyl esters, which are easily extractable with CO2. That means that the tocochromanols, together with other unsaponifiable matter such as squalene and sterol are enriched in the bottom phase of an extraction column. This attempt is described in more detail by. For a discussion of enriching tocochromanols, phase equilibrium data have to be considered first (Zeng et al., 2008)[10]. FFA and tocochromanols exhibit a much higher solubility in CO2 than the triglycerides. Hence, these components are enriched in the gaseous phase, expressed by a distribution coefficient being higher than one. The distribution coefficient of the triglycerides is smaller than one, whereas that for the carotenes is much smaller than one, meaning that these components stay in the liquid oil phase. Thus, tocochromanols can be extracted as the top phase product in a separation column, whereas carotenes remain in the bottom phase product together with triglycerides. For recovering the carotenes together with the tococromanols the above mentioned esterification to volatile (CO2 soluble) methyl esters makes possible to recover tocochromanols and carotenes (together with squalene and sterols) as bottom product from this natural source (Pessova and Uller, 2002)[11].When the glycerides (in case of the esterification) or the FFAs from deodorizer distillates have been removed, then there is a feed material available for obtaining enriched fractions of tocochromanols and carotenes of much higher concentration. In this feed material, tocochromanols and carotenes (in case of palm oil) are the main components and have to be separated from other unsaponifiable substances present, such as squalene and sterols. Of these compounds, squalene has the highest solubility in SC-CO2, all phytosterols have a rather low solubility in CO2 (and remain in the oil phase), and tocochromanols exhibit an intermediate solubility between the two. In a second separation step tocochromanols are separated from phytosterols. A further purification of these compounds is possible, e.g. with adsorptive orchromatographic techniques, again using supercritical fluids (Brunner, 2005) [2].

Removal of alcohol from wine and beer, and related applications: De-alcoholized wine or beer is achieved by removing ethanol from water. Distillation is well known for this purpose with the disadvantage that aroma compounds will also be removed. New techniques like membrane separation (pervaporation) emerge, and in between these is SFE with CO2 (Bravi et al., 2007) [1]. Starting from an aqueous solution with about 10% (w/w) ethanol, ethanol can be removed by SC-CO2 in a stripping column. The rate of ethanol removal depends strongly on temperature. Reducing the alcohol content to values well below 0.5% (w/w) requires about 2.5h at 45°C under non-optimized conditions. Much shorter times for the ethanol removal can be obtained if flow rates and mass transfer equipment are carefully selected. With the information available in the literature, for instance from, a column for dealcoholizing aqueous solutions can be designed. Recovery of aroma compounds is achieved by a side column in which a separation from ethanol is carried out (Brunner, 2005; Bravi et al., 2007)[2,1]. A related process that can be mentioned is the recovery of absolute alcohol. Many studies were carried out at conditions of complete miscibility of ethanol and CO2 in order to get a high solubility of ethanol in the vapor phase. At these conditions, anhydrous ethanol cannot be produced. However, ethanol can be concentrated above azeotropic composition whenever the pressure in the ternary mixture CO2 + ethanol + water is below the critical pressure of the binary mixture CO2+ethanol (Bravi et al., 2007)[2].

Encapsulation of liquids for engineering solid products: A liquid product can be entrapped by adsorption onto solid particles (liquid at the outside of solid particles), by agglomeration (liquid in the free volumes between the solid particles), or by impregnation (liquid within the pore system of the solid particles). Microspheres or larger capsules can be formed, totally encapsulating the liquid. The solid material provides a coating for the liquid inside. Such particulate products can be achieved by means of supercritical fluid processing (Pessova and Uller, 2002) [11]. An example is the so called concentrated powder form-process, wherein CO2 is mixed (dissolved) in the liquid feed by static mixing. The CO2-liquid feed mixture is then sprayed into a spray chamber at ambient conditions together with the substrate material. The CO2 is suddenly released from the liquid, and the liquid forms small droplets. During the spraying process, solid substrate and liquid droplets are intensively mixed and combined to a solid particulate product of the type described above. The product is finally removed from the chamber as a free flowing powder and separated from the outgoing gas stream by a cyclone. With this type of process, a wide variety of solid substrates can be applied to uptake liquids of different kind and up to about 90% (Patrick et al., 2005)[12]. As advantages can be claimed the easier handling and storage, prevention of oxidation processes, and easier dosage. Solid products can also be formed under high pressure conditions. As an example for such a type of process, the encapsulation or adsorption of tocopherol acetate on silica gel. Here, about 50% of tocopherol acetate can be incorporated onto the silica gel without apparent change of morphology and flow properties of the powder. The powder with 50% loading is still free flowing. The amount which can be adsorbed at high pressures is comparable to that of normal pressure. Only at very high densities, the equilibrium loading decreases. In the experiments the autoclave was used to saturate the SC-CO2 current with tocopherol acetate, and the density of the solvent was changed in the nozzle where the loaded SC-CO2 phase was fed to the absorber. This adsorption at high pressures makes possible the direct product formation in the supercritical fluid, with the advantageous effect that the supercritical solvent can easily be recycled without substantial compression (Brunner, 2005)[2].

Extraction and characterization of functional compounds: Nowadays, the growing interest in the so called functional foods has raised the demand of new functional ingredients that can be used by the food industry. These functional ingredients are preferred to have natural origin and to have been obtained using environmentally clean extraction techniques. As expected, the complexity of the natural ingredients with biological activity is very high; this fact has lead to the development of new methodologies to extract and characterize them. In order to preserve the activity of such ingredients and to prevent changes in the chemical composition of the functional compounds and/or mixture of compounds, sample preparation techniques based on the use of compressed fluids have been widely developed. SFE has been used to obtain extracts with antioxidant activity from microalgae; by using the combination of SFE and HPLC with both DAD ESIMS (Wang et al., 2006)[ 13]. Several functional compounds were identified corresponding to different carotenoids along with chlorophyll a and some chlorophyll degradation products. These compounds could be associated to the biological activity of such extracts (Franceschie al., 2008)[14]. Supercritical CO2 has also been used to extract and characterize antimicrobial compounds and food preservatives from microalgae. Carotenoids are a group of compounds of great importance to human health since they can act, e.g., as potent antioxidants; however, due to their chemical characteristics they are easily degraded by temperature or oxygen, so, the use of SFE has been suggested to minimize risks of activity lost being thus applied to the extraction of carotenoids from different matrices (Juan et al., 2006)[15]. In this application, a vegetable oil was also used as co-solvent showing an improvement in the extraction yield as well as in the stability of the pigment. In both cases, the use of oils as co-extracting agents presents an important drawback that is the elimination of oil. It helps to improve the extraction but the extract is a mixture of the extracted components of the oil and the "pure" extract. In general terms, the use of SFE allows the analysis of essential oil preserving its integrity, without the formation of off flavors that could interfere in the characterization of the sample. As it was told before in order to widen the range of application of SFE to relatively polar compounds, small amounts of modifiers (≤15%) are added to carbon dioxide allowing the extraction of more polar substances (Pessova and Uller, 2002)[11]. Other examples of the extraction of valuable compounds from foods using SFE are the isolation of cholesterol from cattle brains and fat-soluble vitamins from parmigiano regiano cheese. The main problem with cattle brains, as well as many other raw food matrices is its high content in water. Water can interfere in the extraction process in two ways: lixiviation or acting as co-solvent thus interfering in the reproducibility of the extraction procedure. In order to avoid this situation, the most common strategy is drying or freeze drying the sample prior to extraction although some authors prefer to mix the sample with a water absorbent inside the extraction cell, for example magnesium sulphate (Mantell et al., 2008)[16]. Another problem related to the extraction of real food samples is their variable fat content which can also interfere in the extraction of the target compounds due to a co-extraction of the fat at the selected extraction conditions. Two main approaches have been used to overcome this problem, the first one uses a fat retainer, mainly basic alumina, neutral alumina, florisil or silica, placed in a separate chamber downstream the extraction thimble, while the other one uses the fat retainer inside the extraction cell (Jose et al., 2007)[3].

Application of SFE in food safety: At present, food safety includes many different issues such as detection of frauds, adulterations and contaminations. Among these topics, detection of food pollutants is important not only for consumers but also for administrations, control laboratories, and regulatory agencies. In order to protect consumers' health, regulations establish strict limits to the presence of pollutants in foods that must be carefully observed and determined. Generally, the analysis of food pollutants is linked to long extraction and cleanup procedures commonly based on the use of, e.g., soxhlet and/or saponification. These procedures are laborious and time consuming and, besides, usually employ large volumes of toxic organic solvents. With the objective of reducing both, the sample preparation time and the massive use of organic solvents, techniques based on compressed fluids such as SFE have been developed. One of the main areas of application of SFE in the last few years has been in food pollutants analysis, mainly pesticide residues and environmental pollutants (Rozzi and Singh 2002) [5]. Several methods has been developed for the analysis of multiple pesticides (organochlorine, organophosphorus, organonitrogen and pyrethroid) in potatoes, tomatoes, apples, lettuces and honey with a single cleanup step using supercritical CO2 modified with 10% of acetonitrile. Similar study have been carried out for the analysis of multiresidues of pesticides, using SFE as a cleanup step, in cereals, fish muscle, vegetable canned soups, vegetables or infant and diet foods (Jose et al., 2007)[3]. A common characteristic of this study is the extremely high selectivity of SFE in the isolation of the low polarity pesticides; this fact makes SFE probably the technique of choice to isolate pesticides from low fat food. As mentioned in the introduction, to correctly asses the concentration of an analyte in a food sample, a quantitative recovery should be obtained that will mostly depend on the recovery of the analytes and not on the extraction itself. To improve the recovery of the pollutants, a common strategy is the use of solid traps. These traps consist on a solid phase compatible with the analyte and are flushed away with a compatible solvent. The trapping step is very important in SFE method development not only because its effect in the quantitative recovery of the analytes but also because an extra selectivity can easily be introduced, especially in the case of solid-phase trapping, avoiding the use of further post-extraction clean up. Supercritical carbon dioxide extraction can advantageously be used to extract non-polar pollutants, such as PAH from foods. Different extraction and cleanup methods have been used, but the extracting conditions turned to be very similar (around 300 bar and 100oC) to optimize the PAH extraction (Jose et al., 2007) [3].

SFE and analytical uses: SC-CO2 has been utilized in multiple methods of analysis. It is used as either an extraction medium, as in rapid analyses for fat content, or as a mobile phase, as in supercritical fluid chromatography. Accordingly, the use of supercritical fluids in the detection of fat content, pesticide residues, and supercritical fluid chromatography as well as some analytical applications are highlighted below:

Rapid analysis for fat content: SFE have been used to determine the fat content of numerous products ranging from beef to oil seeds and vegetables. 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 the AOCS approved methods. 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 (Rozzi and Singh 2002)[5].

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 (Cortes, 2009)[17]. The most common method for cleaning is solid phase extraction. 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 (Cortes, 2009) [5,17].