INTRODUCTION:

Supercritical Fluid Extraction (SFE) is the process of separating one component (the extractant) from another (the matrix) using supercritical fluids as the extracting solvent. Extraction is usually from a solid matrix, but can also be from liquids. SFE can be used as a sample preparation step for analytical purposes, or on a larger scale to either strip unwanted material from a product (e.g. decaffeination) or collect a desired product (e.g. essential oils). SCF technology is making in-roads in several pharmaceutical industrial operations including crystallization, medium for particle design and engineering, particle size reduction, preparation of drug delivery systems, coating, and product sterilization. It has also been shown to be a viable option in the formulation of particulate drug delivery systems, such as micro particles and nanoparticles, liposomes, and inclusion complexes, which control drug delivery and/or enhance the drug stability. Carbon dioxide (CO2) is the most used supercritical fluid, sometimes modified by co-solvents such as ethanol or methanol. Extraction conditions for supercritical CO2 are above the critical temperature of 31°C and critical pressure of 74 bar.

Addition of modifiers may slightly alter this. The discussion below will mainly refer to extraction with CO2, except where specified.

ADVANTAGES:

Environmental improvement and reduced product contamination:

SFE is an alternative to liquid extraction using solvents such as hexane or dichloromethane. There will always be some residual solvent left in the extract and matrix, and there is always some level of environmental contamination from their use. In contrast, carbon dioxide is easy to remove simply by reducing the pressure, leaving almost no trace, and it is also environmentally benign. The use of SFE with CO2 is approved by the Soil Association for organic products. The CO2 used is largely a by product of industrial processes or brewing, and its use in SFE does not cause any extra emissions.^{1, 2}

Selectivity:

The properties of a supercritical fluid can be altered by varying the pressure and temperature, allowing selective extraction. For example, volatile oils can be extracted from a plant with low pressures (100 bar), whereas liquid extraction would also remove lipids. Lipids can be removed using pure CO2 at higher pressures, and then phospholipids can be removed by adding ethanol to the solvent.³

Speed:

Extraction is a diffusion-based process, with the solvent required to diffuse into the matrix, and the extracted material to diffuse out of the matrix into the solvent. Diffusivities are much faster in supercritical fluids than in liquids, and therefore extraction can occur faster. Also, there is no surface tension and viscosities are much lower than in liquids, so the solvent can penetrate into small pores within the matrix inaccessible to liquids.

PRECINCTS:

The requirement for high pressures increases the cost compared to conventional liquid extraction, so SFE will only be used where there are significant advantages. Carbon dioxide itself is non-polar, and has somewhat limited dissolving power, so cannot always be used as a solvent on its own, particularly for polar solutes. The use of modifiers increases the range of materials which can be extracted. Food grade modifiers such as ethanol can often be used, and can also help in the collection of the extracted material, but reduces some of the benefits of using a solvent which is gaseous at room temperature.

METHODS:

The system must contain a pump for the CO2, a pressure cell to contain the sample, a means of maintaining pressure in the system and a collecting vessel. The liquid is pumped to a heating zone, where it is heated to supercritical conditions. It then passes into the extraction vessel, where it rapidly diffuses into the solid matrix and dissolves the material to be extracted. The dissolved material is swept from the extraction cell into a separator at lower pressure, and the extracted material settles out. The CO2 can then be cooled, re-compressed and recycled, or discharged to atmosphere.

Pumps:

Carbon dioxide is usually pumped as a liquid, usually below 5°C and a pressure of about 50 bar. The solvent is pumped as a liquid as it is then almost incompressible. As a supercritical fluid, much of the pump stroke will be "used up" in compressing the fluid, rather than pumping it. For small scale extractions (up to a few grams / minute), reciprocating CO2 pumps or syringe pumps are often used. For larger scale extractions, diaphragm pumps are most common. The pump heads will usually require cooling, and the CO2 will also be cooled before entering the pump.

Pressure vessels:

Pressure vessels can range from simple tubing to more sophisticated purpose built vessels with quick release fittings. The pressure requirement is at least 74 bar, and most extractions are conducted at under 350 bar. However, sometimes higher pressures will be needed, such as extraction of vegetable oils, where pressures of 800 bar are sometimes required for complete miscibility of the two phases.⁴

The vessel must be equipped with a means of heating. It can be placed inside an oven for small vessels, or an oil or electrically heated jacket for larger vessels. Care must be taken if rubber seals are used on the vessel, as the CO₂ may dissolve in the rubber, causing swelling, and the rubber will rupture on depressurization.

Figure 1. Schematic diagram of SFE apparatus

Pressure maintenance:

The pressure in the system must be maintained from the pump right through the pressure vessel. In smaller systems (up to about 10 mL / min) a simple restrictor can be used. This can be either a capillary tube cut to length, or a needle valve which can be adjusted to maintain pressure at different flow rates. In larger systems a back pressure regulator will be used, which maintains pressure upstream of the regulator by means of a spring, compressed air, or electronically driven valve. Whichever is used, heating must be supplied, as the adiabatic expansion of the CO₂ results in significant cooling. This is problematic if water or other extracted material is present in the sample, as this may freeze in the restrictor or valve and cause blockages.

Collection:

The supercritical solvent is passed into a vessel at lower pressure than the extraction vessel. The density, and hence dissolving power, of supercritical fluids varies sharply with pressure, and hence the solubility in the lower density CO₂ is much lower, and the material precipitates for collection. It is possible to fractionate the dissolved material using a series of vessels at reducing pressure. The CO₂ can be recycled or depressurized to atmospheric pressure and vented. For analytical SFE, the pressure is usually dropped to atmospheric, and the now gaseous carbon dioxide bubbled through a solvent to trap the precipitated components.

Heating and cooling:

This is an important aspect. The fluid is cooled before pumping to maintain liquid conditions, then heated after pressurization. As the fluid is expanded into the separator, heat must be provided to prevent excessive cooling. For small scale extractions, such as for analytical purposes, it is usually sufficient to pre-heat the fluid in a length of tubing inside the oven containing the extraction cell. The restrictor can be electrically heated, or even heated with a hairdryer. For larger systems, the energy required during each stage of the process can be calculated using the thermodynamic properties of the supercritical fluid.⁵

SIMPLE MODEL OF SFE:

There are two essential steps to SFE, transport (by diffusion or otherwise) from with the solid particles to the surface, and dissolution in the supercritical fluid. Other factors, such as diffusion into the particle by the SF and reversible release such as desorption from an active site are sometimes significant, but not dealt with in detail here. Figure 2 shows the stages during extraction from a spherical particle where at the start of the extraction the level of extractant is equal across the whole sphere (Fig. 2a). As extraction commences, material is initially extracted from the edge of the sphere, and the concentration in the center is unchanged (Fig 2b). As the extraction progresses, the concentration in the center of the sphere drops as the extractant diffuses towards the edge of the sphere shows in figure 2c.⁶

The relative rates of diffusion and dissolution are illustrated by two extreme cases in Figure 3. Figure 3a shows a case where dissolution is fast relative to diffusion. The material is carried away from the edge faster than it can diffuse from the center, so the concentration at the edge drops to zero. The material is carried away as fast as it arrives at the surface, and the extraction is completely diffusion limited. Here the rate of extraction can be increased by increasing diffusion rate, for example raising the temperature, but not by increasing the flow rate of the solvent. Figure 3b shows a case where solubility is low relative to diffusion. The extractant is able to diffuse to the edge faster than it can be carried away by the solvent, and the concentration profile is flat. In this case, the extraction rate can be increased by increasing the rate of dissolution, for example by increasing flow rate of the solvent.

The extraction curve of % recovery against time can be used to elucidate the type of extraction occurring. Figure 4(a) shows a typical diffusion controlled curve. The extraction is initially rapid, until the concentration at the surface drops to zero, and the rate then becomes much slower. The % extracted eventually approaches 100%. Figure 4(b) shows a curve for a solubility limited extraction. The extraction rate is almost constant, and only flattens off towards the end of the extraction. Figure 4(c) shows a curve where there are significant matrix effects, where there is some sort of reversible interaction with the matrix, such as desorption from an active site. The recovery flattens off, and if the 100% value is not known, then it is hard to tell that extraction is less than complete.

OPTIMIZATION:

The optimum will depend on the purpose of the extraction. For an analytical extraction to determine, say, antioxidant content of a polymer, then the essential factors are complete extraction in the shortest time. However, for production of an essential oil extract from a plant, then quantity of CO2 used will be a significant cost, and "complete" extraction not required, a yield of 70 - 80% perhaps being sufficient to provide economic returns. In another case, the selectivity may be more important, and a reduced rate of extraction will be preferable if it provides greater discrimination. Therefore few comments can be made which are universally applicable. However, some general principles are outlined below.

Maximizing diffusion:

This can be achieved by increasing the temperature, swelling the matrix, or reducing the particle size. Matrix swelling can sometimes be increased by increasing the pressure of the solvent, and by adding modifiers to the solvent. Some polymers and elastomers in particular are swelled dramatically by CO2, with diffusion being increased by several orders of magnitude in some cases.⁷

Figure 2. Concentration profiles during a typical SFE extraction

Maximizing solubility:

Generally, higher pressure will increase solubility. The effect of temperature is less certain, as close to the critical point, increasing the temperature causes decreases in density, and hence dissolving power. At pressures well above the critical pressure, solubility is likely to increase with temperature. Addition of low levels of modifiers (sometimes called entrainers), such as methanol and ethanol, can also significantly increase solubility, particularly of more polar compounds.⁸

Optimizing flow rate:

The flow rate of CO2 should be measured in terms of mass flow rather than by volume because the density of the CO2 changes according to the temperature both before entering the pump heads and during compression. Coriolis flow meters are best used to achieve such flow confirmation. To maximize the rate of extraction, the flow rate should be high enough for the extraction to be completely diffusion limited (but this will be very wasteful of solvent). However, to minimize the amount of solvent used, the extraction should be completely solubility limited (which will take a very long time). Flow rate must therefore be determined depending on the competing factors of time and solvent costs, and also capital costs of pumps, heaters and heat exchangers. The optimum flow rate will probably be somewhere in the region where both solubility and diffusion are significant factors.

Pharmaceutical applications of SCF:

1) Micro particles and Nanoparticles: - Drug and polymeric micro particles have been prepared using SCFs as solvents and antisolvents. Krukonis first used RESS to prepare 5- to 100-µm particles of an array of solutes including lovastatin, polyhydroxy-acids, and mevinolin. RESS process employing CO₂ was used to produce poly (lactic acid) (PLA) particles of lovastatin and naproxen. A GAS process was used to produce clonidine-PLA microparticles. In this process, PLA and clonidine were dissolved in methylene chloride, and the mixture was expanded by supercritical carbon dioxide to precipitate polymeric drug particles⁹.

SCF technology is now claimed to be useful in producing particles in the range of 5 to 2,000 nm. This patent covers a process that rapidly expands a solution of the compound and phospholipid surface modifiers in a liquefied-gas into an aqueous medium, which may contain the phospholipid. Expanding into an aqueous medium prevents particle agglomeration and particle growth, thereby producing particles of a narrow size distribution¹⁰. However, if the final product is a dry powder, this process requires an additional step to remove the aqueous phase. Intimate mixture under pressure of the polymer material with a core material before or after SCF salvation of the polymer, followed by an abrupt release of pressure, leads to an efficient solidification of the polymeric material around the core material. This technique was used to microencapsulate infectious Bursal Disease virus vaccine in a polycaprolactone or a poly (lactic-co-glycolic acid) (PLGA) matrix ¹¹

2) Micro porous Foam: -Using SCF technique, Hile et al prepared porous PLGA foams capable of releasing an angiogenic agent, basic fibroblast growth factor (bFGF), for tissue engineering applications. These foams sustained the release of the growth factor. In this technique, a homogenous water-in-oil emulsion consisting of an aqueous protein phase and an organic polymer solution was prepared first. This emulsion was filled in a longitudinally sectioned and easily separable stainless steel mold. The mold was then placed into a pressure cell and pressurized with CO₂ at 80 bars and 35°C. The pressure was maintained for 24 hours to saturate the polymer with CO₂ for the extraction of methylene chloride. Finally, the set-up was depressurized for 10 to12 seconds, creating micro porous foam ¹².

3) Liposome: -Liposomes are useful drug carriers in delivering conventional as well as macromolecular therapeutic agents. Conventional methods suffer from scale-up issues, especially for hydrophilic compounds. In addition, conventional methods require a high amount of toxic organic solvents. These problems can be overcome by using SCF processing. Fredereksen et al developed a laboratory scale method for preparation of small liposomes encapsulating a solution of FITC-dextran, a water-soluble compound using supercritical carbon dioxide as a solvent for lipids. In this method, phospholipid and cholesterol were dissolved in supercritical carbon dioxide in a high-pressure unit, and this phase was expanded with an aqueous solution containing FITC in a low-pressure unit. This method used 15 times less organic solvent to get the same encapsulation efficiency as conventional techniques. The length and inner diameter of the encapsulation capillary influenced the encapsulation volume, the encapsulation efficiency, and the average size of the liposomes. Using the SCF process, liposomes, designated as critical fluid liposomes (CFL), encapsulating hydrophobic drugs, such as taxoids, camptothecins, doxorubicin, vincristine, and cisplatin, were prepared. Also; stable paclitaxel liposomes with a size of 150 to 250 nm were obtained. Aphios Company’s patent (US Patent No. 5,776,486) on SuperFluidsTM CFL describes a method and apparatus useful for the nanoencapsulation of paclitaxel and campothecin in aqueous liposome formulations called TaxosomesTM and CamposomesTM, respectively. These formulations are claimed to be more effective against tumors in animals compared to commercial formulations ^{10, 13}.

4) Inclusion complexes: - Inclusion complexes with cyclodextrins. For many nonpolar drugs, previously established inclusion complex preparation methods involved the use of organic solvents that were associated with high residual solvent concentration in the inclusion complexes. Earlier, cyclodextrins were used for the entrapment of volatile aromatic compounds after supercritical extraction. Based on this principle, Van Hees et al employed supercritical fluids for producing piroxicam and ß-cyclodextrin inclusion complexes. Inclusion complexes were obtained by exposing the physical mixture of piroxicam-ß-cyclodextrin (1:2.5 mol-mol) to supercritical CO₂ and depressurizing this mixture within 15 seconds. Greater than 98.5% of inclusion was achieved after 6 hours of contact with supercritical CO₂ at 15 MPa and 150°C ¹⁴.

5) Solid Dispersions: -SCF techniques can be applied to the preparation of solvent-free solid dispersion dosage forms to enhance the solubility of poorly soluble compounds. Traditional methods suffer from the use of mechanical forces and excess organic solvents. A solid dispersion of carbamazepine in polyethylene glycol 4000 (PEG4000) increased the rate and extent of dissolution of carbamazepine. In this method, a precipitation vessel was loaded with solution of carbamazepine and PEG4000 in acetone, which was expanded with supercritical CO₂ from the bottom of the vessel to obtain solvent-free particles ¹⁵.

6) Powders of Macromolecules: -Processing conditions with supercritical CO2 are benign for processing macromolecules, such as peptides, proteins, and nucleic acids. Debenedetti0 et al used an antisolvent method to form microparticles of insulin and catalase. Protein solutions in hydroethanolic mixture (20:80) were allowed to enter a chamber cocurrently with supercritical CO₂. The SCF expanded and entrained the liquid solvent, precipitating sub micron protein particles. Because proteins and peptides are very polar in nature, techniques such as RESS cannot be used often. Also, widely used supercritical antisolvent processing methods expose proteins to potentially denaturing environments, including organic and supercritical nonaqueous solvents, high pressure, and shearing forces, which can unfold proteins, such as insulin, lysozyme, and trypsin, to various degrees. This led to the development of a method, wherein the use of the organic solvents is completely eliminated to sobtain fully active insulin particles of dimensions, 1.5-500 µm. In this invention, insulin was allowed to equilibrate with supercritical CO₂ for a predetermined time, and the contents were decompressed rapidly through a nozzle to obtain insulin powder. Plasmid DNA particles can also be prepared using SCFs. An aqueous buffer (pH 8) solution of 6.9 KB plasmid DNA and mannitol was dispersed in supercritical CO₂ and a polar organic solvent using a three-channel coaxial nozzle. The organic solvent acts as a precipitating agent and as a modifier, enabling nonpolar CO₂ to remove the water. The high dispersion in the jet at the nozzle outlet facilitated rapid formation of dry particles of small size. Upon reconstitution in water, this plasmid DNA recovered 80% of its original super coiled state. Such macromolecule powders can possibly be used for inhalation therapies ^16,
17.

7) Coating: -SCFs can be used to coat the drug particles with a single or multiple layers of polymers or lipids. A novel SCF coating process that does not use organic solvents has been developed to coat solid particles (from 20 nm to 100 µm) with coating materials, such as lipids, biodegradable polyester, or polyanhydride polymers. An active substance in the form of a solid particle or an inert porous solid particle containing active substance can be coated using this approach. The coating is performed using a solution of a coating material in SCF, which is used at temperature and pressure conditions that do not solubilize the particles being coated.

8) Product Sterilization: - In addition to drug delivery system preparation, SCF technology can also be used for other purposes, such as product sterilization. It has been suggested that high-pressure CO₂ exhibits microbicidal activity by penetrating into the microbes, thereby lowering their internal pH to a lethal level. The use of supercritical CO₂ for sterilizing PLGA microspheres (1, 7, and 20 µm) is described in US Patent No. 6,149,864. The authors indicated that complete sterilization can be achieved with supercritical CO₂ in 30 minutes at 205 bar and 34°C ¹¹.

9) Particulate Dosage Forms: -Some gases at certain pressures cause swelling of polymers likpolypropylene, polyethylene, and ethylene-vinyl acetate co-polymer and ethylene ethyl acrylate copolymer or drug carriers, and allow migration of active material in polymer matrix to give diffusion-controlled drug delivery systems. This specific behavior can be exploited for various purposes replacing the traditional techniques like Spray-drying, solvent evaporation and freeze-drying. This approach can be utilized as a solvent-free approach to develop novel, controlled-release dosage forms and deposit thermo labile materials such as peptide drugs into the polymers¹¹.

IN PHARMACEUTICAL INDUSTRIES:

1) Medium for Crystallization: -To generate high purity polymorphs, even with some morphological viz. high degree of Enantiomeric enrichment. SF technology appears to be a potential modality. Moreover, size and shape of the polymorph can be manipulated by controlling temperature and/or pressure during processing while degree of crystallization can be improved by manipulating the rate of crystallization and high degree of crystallinity. Better candidate in metered dose inhaler compared with conventionally crystallized and micronized drug ¹¹.

2) Solubilization of pharmaceuticals: -RESS technology has been used. Most of pharmaceutical compounds below 60 c and 300 bars showed a considerable higher solubility. In many a process of solubilization of polar or non-volatile compounds a limited solubility in SC CO2 is fails to form a homogenous solution under practical conditions. To aid the solubilization in such cases the CO2-philic solubilizers are being developed which rather the SC CO2 insoluble substances and make them solubilize in SC CO₂¹¹.

3) Extraction and Purification: -Supercritical fluid extraction technique could be utilized to separate impurities mainly organic complexes from the pharmaceuticals. Methods developed by Zoel are now widely used in industry as in caffeine production and Isolation of Taxol from the bark of the Taxus brevifolia in which SC CO2 is used. Purification via SCF technology gives a better alternative to all conventional purification methods as it is almost automated, quick, high yielding, SCF methods are also reported for the extraction of bryostatins, natural products, production of fat free products ¹¹.

4) Medium for Polymerization and Polymer Processing: -Supercritical fluids mainly SC CO2 is rapidly becoming an alternative solvent for polymerization. Solubility plays a very important role in the synthesis of polymers.

Mainly two processes used

1) Step growth: SC CO2 has been reported very yielding in the production of polycarbonates, polymides, polyesters, polypyrrols, polyphenoxides and silica gels.

2) Chain growth: free radical polymerization of styrenics, armlets and methacrylates, cationic polymerization of isobutylene.

Supercritical CO2 in polymerization is increased plasticization because of CO2. The highly plasticized state of polymers is also results in increased polymerization rates by the enhanced diffusion of monomer into the polymer¹⁸.

Figure 3. Concentration profiles for (a) diffusion limited and (b) solubility limited extraction

5) As a Supercritical Bio-catalyst: -Randolph et al primarily found the enzyme alkaline phosphates active in a batch reaction system yhat employed SC CO2 as solvent. In the comparison SC CO2 as the adverse effect of pressure was less profound in case of compressed propane and ethane. Nakamura et al studied the acidolysis of trioline with stearic acid in SC CO2 by using Lipase as a bio-catalyst¹⁹.

6) Micronization of Pharmaceuticals: -The RESS process has been shown to be capable of forming micron-sized particals. Krukonics, first extensively studied RESS in micronization of a wide variety of materials, including pharmaceuticals, biologicals, and polymers¹³. He produced uniform submicron powder of estradiol. Loth and Hemgesberg studied the micronization of phenacetin by RESS and compared with jet-milled phenacetin. The main limitation of RESS is the inability to process those materials which are insoluble or very less soluble in the SCF. So for this materials the SAS process has been successfully used to produced micron sized particles like insuline, bovine liver catalase, lysozyme, trypsin, methylprednisolone and hydrocortisone acetate. Insuline were in two crystalline forms; spheroidal (smaller than 1 micron) and needle (5 micron). ASES process has been studied for the preparation of a range of steroids for pulmonary delivery ²⁰.

Figure 4. Extraction Profile for Different Types of Extraction

CONCLUSION:

SFE has been suggested as the method of choice for thermo labile compounds extraction. It has been used for developing an ever-expanding niche in the Pharma industry whether it is used as a solvent for extraction or analyses. Now a day, SFE is really needed as advance method which can provide fast, reliable, clean and cheap methods for routine analysis. The special properties of SEFs bring certain advantages to chemical separation technique. Several applications have been fully developed and commercialized which include food and flavouring, pharmaceutical industry, environmental protection for volatile and lipid soluble compounds, extraction of high value oils, extraction of natural aromas, recovery of aromas form fruits, meat and fish, isolation of lipid soluble compounds. Also in order to understand the mechanism of SFE, molding consideration of SCFs and main criteria for SCF techniques are focused in this review.

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Received on 31.01.2009 Modified on 28.02.2009

Research J. Pharm. and Tech. 2(1): Jan.-Mar. 2009; Page 65-71