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Supercritical Fluids and Their Applications REVIEW

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Supercritical Fluids and Their Applications in Biotechnology and Related Areas John R. Williams,1,* Anthony A. Clifford,2 and Salim H. R. Al-Saidi1 Abstract This article serves as an overview, introducing the currently popular area of supercritical fluids (SFs) and their uses in biotechnology and related areas. It covers the fundamentals of supercritical science and moves on to the biotechnological and associated applications of these fluids. Subject areas covered include pure substances as supercritical fluids, the properties of supercritical fluids, organic cosolvents, solubility, and the following applications: extraction, chromatography, reactions, particle production, deposition, and the drying of biological specimens. Within each application, and where possible, the basic principles of the technique are given, as well as a description of the history, instrumentation, methodology, uses, problems encountered, and advantages over the traditional, nonsupercritical methods. Index Entries: Supercritical fluids; supercritical fluid extraction; supercritical fluid chromatography; rapid expansion of supercritical solutions; supercritical antisolvent; particles from gas-saturated solution; solution enhanced dispersion by supercritical fluids; critical point drying.

1. Introduction Put simply, a supercritical fluid (SF) is any substance above its critical point. Over the last 20 yr, there has been renewed interest from analytical and process chemists in SFs owing to their unique properties and relatively low environmental impact. Greatest attention has been given to the extraction and separation of organic compounds. SFs have also been used successfully for particle production, as reaction media, and for the destruction of toxic waste. Supercritical carbon dioxide has been the most widely used SF, mainly because it is cheap, relatively nontoxic, and has convenient critical values. However, superheated and supercritical water has received much attention recently. This review, unlike others, concentrates on the use of SFs in biotechnology and related areas. Consequently, it is aimed at workers in biomaterials science, natural products chemistry, forensic science, environmental monitoring, food science, and analytical, process, clinical, and medicinal chemistry. Furthermore, it serves as an introduction to

SFs for newcomers to this field, but more experienced workers will find it useful too.

2. Pure Substances as Supercritical Fluids Cagniard de la Tour showed in 1822 that there is a critical temperature above which a single substance can only exist as a fluid and not as either a liquid or gas. He heated substances, present as both liquid and vapor, in a sealed cannon, which he rocked back and forth and discovered that, at a certain temperature, the splashing ceased. Later, he constructed a glass apparatus in which the phenomenon could be more directly observed. These phenomena can be put into context by reference to Fig. 1, which is a phase diagram of a single substance. The diagram is schematic, the pressure axis is nonlinear, and the solid phase at high temperatures occurs at very high pressures. Further solid phases and also liquid crystal phases can also occur on a phase diagram. The areas where the substance exists as a single solid, liquid, or gas phase are labeled, as is the triple point where the three phases

*Author to whom all correspondence and reprint requests should be adressed: Department of Chemistry, College of Science, Sultan Qaboos University. Phone: 00 968 515483. Fax: 00 968 513415. E-mail: [email protected]. 1Department of Chemistry, College of Science, Sultan Qaboos University, PO Box 36, Al-Khod 123, Sultanate of Oman. 2School of Chemistry, University of Leeds, Leeds LS2 9JT, UK. Molecular Biotechnology 2002 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2002/22:3/263–286/$20.00

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Fig. 1. The phase diagram of a single substance.

coexist. The curves represent coexistence between two of the phases. If we move upward along the gas–liquid coexistence curve, which is a plot of vapor pressure vs temperature, both temperature and pressure increase. The liquid becomes less dense, because of thermal expansion, and the gas becomes more dense as the pressure rises. At the critical point, the densities of the two phases become identical, the distinction between the gas and the liquid disappears, and the curve comes to an end at the critical point. The substance is now described as a fluid. The critical point has pressure and temperature coordinates on the phase diagram, which are referred to as the critical temperature, Tc, and the critical pressure, Pc, and which have particular values for particular substances, as shown by example in Table 1 (1). In recent years, fluids have been exploited above their critical temperatures and pressures, and the term supercritical fluids has been used to describe these media. The greatest advantages of SFs are realized when they are used not too far above (say within 100 K of) their critical temperatures. Nitrogen gas in a cylinder is a fluid, but is


not usually considered as an SF, but more often described by an older term as a permanent gas. The region for SFs is the hatched area in Fig. 1. It has been shown to include a region a little below the critical pressure, as processes at these conditions are sometimes included in discussions as “supercritical.” Lower pressures are important in practice also because these conditions are relevant to separation stages in supercritical processes. There are no phase boundaries below and to the left of the supercritical region in Fig. 1, and behavior does not change dramatically on moving out of the hatched area in these directions. The liquid region to the left of the supercritical region has many of the characteristics of SFs and is exploited in a similar way. For this reason some people prefer the term near-critical fluids and the adjective subcritical is also used. The term supercritical fluid has, however, gained currency, is convenient, and is not problematic provided the definition is not too rigid. SFs exhibit important characteristics, such as compressibility, homogeneity, and a continuous change from gas-like to liquid-like properties. These properties are characteristic of conditions inside the

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Supercritical Fluids and Their Applications Table 1 Substances Useful as Supercritical Fluids Substance

Ammonia Carbon dioxide Ethane Ethene Fluoroform Nitrous oxide Propane Water Xenon

Critical Temperature Tc(K)

Critical Pressure Pc(bar)

406 304 305 282 299 310 370 647 290

114 74 49 50 49 72 43 221 58

Parameters from Reid et al. (1).

hatched area in Fig. 1 and, to different degrees, in the area around it. Table 1 shows the critical parameters of some of the important compounds useful as SFs. One compound, carbon dioxide, has so far been the most widely used, because of its convenient critical temperature, cheapness, chemical stability, nonflammability, stability in radioactive applications, and nontoxicity. Large amounts of carbon dioxide released accidentally could constitute a working hazard, given its tendency to blanket the ground, but hazard detectors are available. It is an environmentally friendly substitute for organic solvents. The carbon dioxide is obtained in large quantities as a by-product of fermentation, combustion, and ammonia synthesis and would be released into the atmosphere sooner rather than later, if it were not used as a supercritical fluid. Its polar character as a solvent is intermediate between a truly nonpolar solvent, such as hexane, and weakly polar solvents. Because the molecule is nonpolar, it is often classified as a nonpolar solvent, but it has some limited affinity with polar solutes because of its large molecular quadrupole. To improve its affinity with polar molecules further, carbon dioxide is sometimes modified with polar entrainers (see Subheading 4). However, pure carbon dioxide can be used for many organic solute molecules even if they have some polar character. It has a particular affinity for fluori-


265 nated compounds and is useful for working with fluorinated metal complexes and fluoropolymers. Carbon dioxide is not such a good solvent for hydrocarbon polymers and other hydrocarbons of high molar mass. Ethane, ethene, and propane become alternatives for these compounds, although they have the disadvantages of being hazardous because of flammability and of being somewhat less friendly to the environment. However, small residues of lower hydrocarbons in foodstuffs and pharmaceuticals are not generally considered a problem. Water has good environmental and other advantages, although its critical parameters are much less convenient (Table 1) and it gives rise to corrosion problems. Supercritical water is being used, at a research level, as a medium for the oxidative destruction of toxic waste (2). There is a particular interest in both supercritical and nearcritical water because of the behavior of its polarity. Ammonia has similar behavior, is often considered and discussed, but not often used. Many halocarbons have the disadvantage of cost or of being environmentally bad. Xenon is expensive, but is useful for small-scale experiments involving spectroscopy, because of its transparency in the infrared region (3).

3. Properties of Supercritical Fluids Although often pursued in practice for environmental reasons, the more fundamental interest in SFs arises because they can have properties intermediate between those of typical gases and liquids. Compared with liquids, densities and viscosities are less and diffusivities greater. Furthermore, properties are controllable by both pressure and temperature and the extra degree of freedom, compared with a liquid, can mean that more than one property can be optimized. Any advantage has to be weighed against the cost of the apparatus and the inconvenience of the higher pressures needed. Consequently, SFs are exploited in particular areas. An SF changes from gas-like to liquid-like as the pressure is increased, and its thermodynamic properties change in the same way. Close to the critical temperature, this change occurs rapidly over a small pressure range. The most familiar property is the density, and its behavior is illus-

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Fig. 2. Density–pressure isotherms for carbon dioxide.

trated in Fig. 2 (4). This shows three density– pressure isotherms and at the lowest temperature, 6 K above the critical temperature, the density change is seen to increase rapidly at around the critical pressure. As the temperature is raised, the change is less dramatic and moves to higher pressures. One consequence is that it is difficult to control the density near the critical temperature and, as many effects are correlated with the density, control of experiments and processes can be difficult. Other properties, such as enthalpy, also show these dramatic changes near the critical temperature. The behavior of density, as well as all other thermodynamic functions, as a function of pressure and temperature can be predicted by an equation of state. Some of these have an analytical form, but the most accurate equations are complex numerical forms that have been obtained by intelligent fitting of a wide range of thermodynamic data, such as is carried out at the International Union of Pure and Applied Chemistry Thermodynamic Tables Project Centre at Imperial College in London. They have carried out a study for a number of gases suitable as SFs. For carbon dioxide, a recent equation of state is that published by Span and Wagner (5). At low pressures, below 1 atm, the (dynamic) viscosity, η, of a gas is approximately constant, but MOLECULAR BIOTECHNOLOGY

thereafter rises with pressure in a similar way to density, ρ. However, the dependencies of density and viscosity on pressure at constant temperature are not conformal. A comprehensive correlation for the viscosity of carbon dioxide has been published (6). Table 2 shows typical values for the density and viscosity of a gas, SF, and liquid, taking carbon dioxide as an example. Using the example given, the viscosity of an SF is much closer to that of a gas than that of a liquid. Thus, pressure drops across chromatographic columns and through supercritical extraction and other processes are less than for the equivalent liquid processes. Diffusion coefficients, also shown in Table 2 for naphthalene in carbon dioxide, are higher in an SF than in a liquid. They are approximately inversely proportional to the fluid density (7). The advantage shown in the table is seen not to be so great, and the main diffusional advantage lies in the fact that typical supercritical solvents have lighter molecules than those of typical liquid solvents. The diffusion coefficient for naphthalene in a typical liquid would be about 1 × 10-9 m2 s-1. Thus, diffusion coefficients in SF experiments and processes are typically an order of magnitude higher than in a liquid medium. This has advantages in band-narrowing in chromatography and faster transport in extraction. However, diffusion Volume 22, 2002

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Table 2 Typical Values of Density, Viscosity, and the Diffusion Coefficient for Naphthalene Using Carbon Dioxide as an Example CO2 Density (4) ρ(kg m-3) Gas, 313 K, 1 bar Supercritical, 313 K, 100 bar Liquid, 300 K, 500 bar

2 632 1029

coefficients tend to zero at the critical point and fall in the critical region around it. At high concentrations, this can cause chromatographic bandbroadening near the critical density (8). Although the SI unit of pressure is the pascal (Pa), it is rarely used in the field of SFs because of the high pressures involved. A more appropriate unit is the megapascal (MPa). Furthermore, no one pressure unit predominates; a wide variety are used interchangeably throughout the world. To help clear the confusion, the following may be of use: 1 atm ⴝ 1.0132 bar ⴝ 0.10132 MPa ⴝ14.696 psi ⴝ1.0332 kg cm-2.

4. Cosolvents The solvent characteristics of a fluid can be modified by adding a cosolvent (also known as an entrainer or modifier), and this has been most commonly done with carbon dioxide. As this molecule is nonpolar, it is classified as a nonpolar solvent, although it has some limited affinity with polar solutes because of its large molecular quadrupole. Thus, pure carbon dioxide can be used for many large organic solute molecules, even if they have some polar character. For the extraction and chromatography of more polar molecules, it is common to add polar cosolvents, such as the lower alcohols. Cosolvents can also be added to develop other characteristics. They can impart increased or decreased polarity, aromaticity, chirality, and the ability to further complex metal–organic compounds. Just as carbon dioxide is the most popular substance for use as an SF, it is also the substance to which cosolvents are most freMOLECULAR BIOTECHNOLOGY

Naphthalene in CO2 Viscosity (5) η (µPa s) 16 17 133

Diffusion Coeff. (6) D (m2 s-1) 5.1 x 10-6 1.4 x 10-8 8.7 x 10-9

quently added. This is because cosolvents are seen as a way of making use of this desirable compound in circumstances where it is not the best solvent. For example, in the case of carbon dioxide, methanol is added to increase polarity, aliphatic hydrocarbons to decrease it, toluene to impart aromaticity, [R]-2-butanol to add chirality, and tributyl phosphate to enhance the solvation of metal complexes. They are often added in 5% or 10% amounts by volume, but sometimes much more, say, 50%. They can have significant effects when added in small quantities and, in these cases, it may be the effect on surface processes rather than solvent character that is important. For example, the cosolvent may be effective in extraction by adsorbing onto surface sites, preventing the readsorption of a compound being extracted. Similarly, in chromatography, the cosolvent may cap active or unbonded sites on a stationary phase, preventing tailing of chromatographic peaks. A comprehensive review of cosolvents has been made by Page et al. (9). Some compounds, which are commonly used as cosolvents, are listed with their critical parameters in Table 3. It is important to be aware of the cosolvent–fluid phase diagram to ensure that the solvent is in one phase. For example, for methanol–carbon dioxide at 50°C there is only one phase above 95.5 bar whatever the composition, but below this pressure, two phases can occur. Above this pressure, the character of the medium depends on the proportions of cosolvent and fluid substance. If the proportion of cosolvent is low, the mixture will have the characteristics of an SF, but if it is high, the medium will be liquid-like. Volume 22, 2002

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Fig. 3. The behavior of solubility in a supercritical fluid shown schematically.

5. Solubility in a Supercritical Fluid The behavior, at constant temperature, of the solubility of a substance in an SF, in terms of mole fraction, is illustrated schematically in Fig. 3. When the pressure is above but close to zero, only the solute is present as vapor and the mole fraction of the solute is unity. There is then an initial fall almost to zero at very low pressures as the solvent is added and the solute is diluted without being much solvated. After staying close to zero, there is then a rise in solubility at around the critical density of the fluid, that is, when the density is increasing rapidly with pressure. This rise is due to solvation originating from attractive forces between the solvent and solute molecules. Thereafter, the solubility may exhibit a fall, represented by the dashed line. If this occurs, it is because at higher pressures the system is becoming compressed and repulsive solute–solvent interactions are important. The solute can be said to be “squeezed out” of the solvent. Alternatively, a rise may occur, as represented by the dotted line. This happens if there is a critical line present at high pressures at the temperature of the isotherm and the solubility will rise toward it. The rising type of curve is a feature of smaller more volatile molecules and higher temperatures and vice versa. All situations between the two curves occur.


Correlation of SF solubility data is not straightforward. All the features shown in Fig. 3 can be reproduced qualitatively by any equation of state. For quantitative fitting, more refined equations of state are useful in certain regions and, of these, the Peng–Robinson has been the most widely used. However, even this equation is not successful in fitting all the data at all pressures and tem5æeratures. A further problem is that the parameters necessary for using the equation of state are not always available. Thus, often empirical approaches are used (10).

6. Applications of Supercritical Fluids in Biotechnology and Related Areas The SF techniques that have found use in biotechnology and related areas are described as follows and their advantages, above the general ones of less pollution in the working and general environment and less solvent disposal costs, are also given.

6.1. Extraction Supercritical fluid extraction (SFE) uses an SF to remove soluble substances from insoluble matrices. SFs have attractive properties for extraction (see Subheading 3), because they penetrate a sample faster than liquid solvents; SFs have diffu-

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Supercritical Fluids and Their Applications Table 3 Substances Useful as Cosolvents in Carbon Dioxide with Their Critical Parameters Substance

Critical Temperature Tc(K)

Acetic acid Acetone Acetonitrile Benzene 2-Butanol Chloroform Dichloromethane Diethyl ether Ethanol Hexane Methanol 1-Propanol 2-Propanol Toluene Tributyl phosphate

593 508 546 562 536 536 510 467 514 508 513 537 508 592 742

Critical Pressure Pc(bar) 58 47 48 49 42 54 63 36 61 30 81 51 48 41 24

Data from ref. 1.

sion coefficients midway between gases and liquids. They transport extracted material from the sample faster; SFs have viscosities similar to those of gases. They also dissolve solutes from a sample matrix; SFs have solvating powers approaching those of liquids. Another advantage of SFE is less solvent residues in products. The basic concept of SFE is to use a relatively cheap and safe material for the extraction of organic compounds from a matrix in place of conventional solvent extraction, cutting down on manipulation and avoiding the problems associated with the use and disposal of organic solvents. Although a number of substances are considered as potentially useful for SFE, in practice, the one of choice is carbon dioxide for the reasons given earlier (see Subheading 2). At first, SFE was used as a bench-top model by the petroleum industry prior to scale-up. The earliest reference found to true SFE was the 1940 patent by Pilat and Godlewicz (11) describing the fractionation of mineral oils using supercritical carbon dioxide. Earlier processes had used a combination of sub- and supercritical fluids to carry


269 out extractions. A detailed account of the early industrial applications of SFE has been given by McHugh and Krukonis (4). Analytical-scale SFE appeared much later. The earliest reference found dates back only to 1976, when Stahl and Schilz (12) coupled SFE to thin layer chromatography for the recovery and separation of several substances of differing polarities. There are four scales of SFE apparatus, based on the volume of the sample-holding cell: analytical (1–24 mL), bench (200–500 mL), pilot (1–50 L), and production (350+ L). All sizes of SFE apparatus, regardless of complexity and cost, share the same basic components: a source of extraction fluid, one or more pumps, a sample cell, an oven, a back-pressure regulator (BPR), and a collection device. On an analytical-scale apparatus (Fig. 4), the solvent delivery system consists of a pump to deliver liquid carbon dioxide and, optionally, a pump to supply cosolvent. The oven is used to keep the cell contents above the critical temperature of the extraction fluid. An equilibration coil situated before the sample cell is included to help mixing of carbon dioxide and cosolvent and aid thermal equilibration of the extraction fluid with the insides of the oven. The cell, usually a hollow stainless-steel cylinder, is housed in the oven and contains the sample to be extracted. It has a frit at both ends to prevent insoluble material leaving the cell, but allowing soluble substances to pass through unhindered. The BPR serves to keep the pressure in the system above the critical pressure of the extraction fluid. It is, typically, a length of stainless steel or fused silica capillary (50 µm id), known as a restrictor, or a mechanical or electronic needle valve. The silica restrictor is usually connected by a graphite ferrule to a union attached to a length of 1/16-in. stainless-steel tube coming from the sample cell. The BPR is heated (with a hairdryer or in an oven) to reduce the frequency of blockages by, for example, the formation of ice. Finally, a collection system is required to trap extracted material. On an analytical scale, it is usually a solid trap or a small glass collector containing a few cubic centimeters of organic solvent.

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Fig. 4. Schematic diagram of an analytical-scale supercritical fluid extractor. (1) Source of carbon dioxide, (2) carbon dioxide pump, (3) chiller unit, (4) cosolvent reservoir, (5) cosolvent pump, (6) oven, (7) equilibration coil, (8) cell, (9) back-pressure regulator, and (10) collector.

On larger-scale apparatus (Fig. 5), the oven is dispensed with in favor of water-heated vessels for the sample cell and collectors (often termed separators). The pumps are larger and capable of pumping up to 100 L h -1 on a pilot-scale apparatus. Needle valves, either manual or mechanical, are favored as the BPR. Collectors or separators tend to be titanium vessels, typically 200 mL, on a pilot apparatus. There may be more than one separator to enable fractionation. The titanium sample cell has a volume of up to 50 L, which reduces on insertion of the sample-containing basket. As an illustration, a sample cell of volume 1 L reduces to 600 mL when the basket is added. A flow meter is usually attached to the extractor to monitor carbon dioxide delivery. Liquid carbon dioxide from a cylinder is housed in a chilled vessel, which acts as a reservoir. All sizes of extraction system can be either manually or computer-controlled. Largerscale apparatus should be sited away from general work areas and offices, because of the noise from the chiller unit and the high-pressure hazard. During an extraction, carbon dioxide and, optionally, cosolvent are pumped at set flow rates through a cell containing the sample. Soluble components of the sample are dissolved and removed from the cell by the fluid. On an analytical scale, the extracted materials pass through the BPR and are depressurized into a collector typically containMOLECULAR BIOTECHNOLOGY

Williams et al. ing a few cubic centimeters of organic solvent. The contents of the collector are evaporated to dryness or adjusted to a known volume prior to analysis by, for example, supercritical fluid chromatography (SFC). An alternative way of collecting the extract is to depressurize it onto a packed trap. The solutes are then rinsed from the trap with an appropriate solvent into a small vial, ready for analysis or evaporation to dryness. On a larger scale, the extracted material is passed through one or a series of separators, where it collects for processing later. The depressurized gaseous carbon dioxide can be recycled for use again. SFE can be categorized as either off-line or on-line. Off-line SFE is when the extraction and analysis steps are done separately with the analytical instrument remote from the extractor. On-line is when the extract is fed directly into an analytical instrument. SFE can also be classed as dynamic or static. In dynamic SFE, the SF is pumped continually through the cell containing the sample. In the static mode, the sample is bathed in SF, and there is no flow of fluid to or from the cell during the extraction. Sometimes, both types of SFE are performed on the same sample at different times during an extraction (13). Bartle et al. (14–16) and others (17–20) have produced mathematical models of SFE that predict many of the features of extraction. Extraction was the first commercial use of SFs, and examples include the extraction of hops (4) and the decaffeination of coffee (21). Since then, SFE has been applied to many biotechnological and related areas, such as the extraction of organic pollutants from environmental samples (22 –28), food (29–31), and animal tissue (32); the isolation of natural products from plant (33–48) and animal (49) samples; the removal of fat from food (50,51); the extraction of medicinal drugs from pharmaceutical preparations (52,53); and the isolation of drugs of abuse from human hair (54,55) and plant material (56). Table 4 summarizes some recent publications of biotechnological and related interest involving SFE (57–73). More than 400 research papers have been produced on the extraction of a wide range of natural products, including high-value pharmaceutical Volume 22, 2002


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Table 4 Some Recent SFE Publications of Biotechnological and Related Interest (In All Cases, the Solute was Extracted from the Sample) Solute Drugs of abuse Grease Herbicides Microcystins Morphine Nicarbazin Nitrosamines Organochlorine pesticides Polycyclic aromatic hydrocarbons Petroleum hydrocarbons Polyphenolic compounds Sesquiterpene lactone ß-Carotene Sterols Sulfonamides Testosterones Vitamin E

Sample

Ref.

Human hair Sheepskins Food crops Cyanobacteria Blood Poultry feed, eggs, and muscle tissue Cigarettes Chinese herbal medicine Biological tissues Soil Grape seeds Tanacetum parthenium (feverfew) Paprika Plant lipid mixtures Chicken liver Bovine urine Pharmaceutical preparations

(57) (58) (59) (60) (61) (62) (63) (64) (65) (66) (67) (68) (69) (70) (71) (72) (73)

Fig. 5. Schematic diagram of a pilot-scale supercritical fluid extractor. (1) Source of carbon dioxide, (2) saturator, (3) cosolvent reservoir, (4) cosolvent pump, (5) carbon dioxide reservoir, (6) carbon dioxide pump, (7) cell, and (8) separators.

precursors (74). This is still an area of intense publication activity and has been the subject of a recent book (75). Fractionation of liquid mixtures (75,76) can be achieved by countercurrent extraction (77), and this can be improved by imposing a temperature gradient on the column, which causes MOLECULAR BIOTECHNOLOGY

refluxing to occur (78). It is largely applied to natural products, such as essential oils and lipid products, and can be used to concentrate substances prior to chromatography. The advantage of using an SF is that countercurrent extraction with reflux can be carried out in one unit. Volume 22, 2002

272 SFs can be used in environmental clean-up methods, including soil remediation (79), for the extraction of both toxic organic and metallic contaminants. This treatment is beneficial compared to liquid solvent extraction because effectively no toxic residue is left in the treated soil. Because relatively low temperatures are generally used in SFE, soils are not burned as in thermal desorption. Soil remediation is limited to relatively small volumes of soil and batch, not continuous, operation (80). SFE is facing stiff competition from other, newer methods of sample pretreatment, such as microwave-assisted solvent extraction (81) and accelerated or pressurized solvent extraction (82). An example of the latter alternative is superheated water extraction (SWE). This technique uses heated (above 100°C), pressurized water, but the temperature is less than the critical value. Under these conditions, the polarity of water decreases, and it is able to solubilize more organic compounds than water at 25°C and 1 atm. A comparison of SFE using carbon dioxide with the SWE of clove buds found that SWE recovered similar amounts of the valuable oxygenated compounds as SFE (83). In contrast, a different study found that steam distillation was more effective than SWE and SFE at extracting peppermint oil from leaves of peppermint plants (84). Clearly, SWE is in its infancy and more research must be done to ascertain suitable applications. Supercritical water is not used for the extraction of biomaterial, because the high temperatures involved can destroy or denature the solute and/or sample. The most successful applications of SFE have been for relatively nonpolar compounds. This is unsurprising because most extractions have been conducted with carbon dioxide, itself a relatively nonpolar molecule. Consequently, some polar compounds have presented problems (85), but efforts have been made to make SFE viable (86). SFE has been reviewed recently by Luque de Castro and Jiménez-Carmona (87). In-depth SFE methods and protocols of biotechnological interest, conducted on analytical and larger scales, are available (88).


Williams et al. 6.2. Chromatography SFC can be defined as the separation of organic compounds using an SF as the mobile phase. There is interest in the technique because the rapid diffusivity and low viscosity of an SF compared to a liquid should allow a more efficient separation of components in a solution than high performance liquid chromatography (HPLC). Furthermore, sensitive general detectors, like the flame ionization detector (FID), can be exploited. For certain applications, SFC can be an ecofriendly alternative to HPLC, which uses moderate volumes of toxic organic solvents, and a more versatile rival to gas chromatography (GC), which is limited to volatile organic compounds. Another advantage of SFC can be little or no solvent residues in products. The use of SFs as chromatographic mobile phases was first suggested in 1958 (89,90), but it was four years later that their use for this purpose was demonstrated (91). A superb account of the history of SFC is given by Lee and Markides (92). Unsurprisingly, carbon dioxide is the most common mobile phase in SFC. Its low critical temperature can allow the separation of thermally sensitive compounds, but supercritical carbon dioxide is not very polar, limiting its use as a solvent. To overcome this, carbon dioxide can be modified with polar organic solvents such as methanol, but the flammability of the organic solvent renders the FID redundant. Chromatography with SFs has been used with packed and capillary columns. The use of capillary columns has mostly died out, because of the relatively long analysis times (arising from very low mobile phase flow rates) and relatively poor precision (due to the injection of very small volumes of sample). Compatibility with the FID means that packed column SFC with carbon dioxide can be used for nonpolar solutes that would be difficult to detect by HPLC. The technique is relatively easy to couple to other instruments, for example, a Fourier transform infrared spectrometer (93). Chromatography with SFs has been performed on analytical (94) and preparative (95,96) scales.

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Supercritical Fluids and Their Applications The instrumentation used for SFC is similar to SFE apparatus (see Subheading 6.1.), but there are important differences. A column containing stationary phase replaces the sample-holding cell. Furthermore, SFC systems include an injector just before the column and a detector between the column and the BPR (Fig. 6). The mobile phase is initially pumped as a liquid until it reaches the oven, where it becomes an SF. The oven houses the body of the injector and the column, and keeps them above the critical temperature of the substance used as the mobile phase. The sample (in liquid solvent) is injected into the mobile phase and passes on to the column, where its constituents are separated. A guard or precolumn (not shown in Fig. 6) is usually included just before the main, expensive analytical or preparative column to protect it from unwanted material from the injected sample, mobile phase, and instrument. From the column, the isolated components pass into the detector (still under considerable pressure) before entering the BPR and on to waste or collection. Here, the depressurized fluid becomes a gas (carbon dioxide) and, if cosolvent is used, a liquid. On a preparative scale, the instrument components are generally scaled-up, for example, the use of preparative columns instead of analytical ones. A wide range of compounds of biotechnological and related interest have been separated and/ or analyzed by SFC. Examples include cholesterol (97), bile acids (98,99), ecdysteroids (100), azadirachtin (101), acidic drugs (102), and basic drugs (102). It is worth noting that many of these analyses can be performed satisfactorily by GC and/or HPLC. More recently, SFC has been applied to the separation and analysis of pesticides in soil (103), chiral drug enantiomers (104), and vitamins (105). As an illustration, Fig. 7 shows an example of an SF chromatogram for the separation of three vitamin B compounds. Supercritical fluid chromatography has not achieved the wide use anticipated in the 1980s owing to competition from HPLC and GC, but still has a role for certain specialized applications, for example, chiral (106) and petrochemical separations (106), and SFC-


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Fig. 6. Basic instrument used for supercritical fluid chromatography. (1) Source of carbon dioxide, (2) carbon dioxide pump, (3) chiller unit, (4) cosolvent reservoir, (5) cosolvent pump, (6) oven, (7) equilibration coil, (8) injection valve, (9) column, (10) detector, and (11) back-pressure regulator.

mass spectrometry (103). In addition, efficient simulated bed units are available (107) and SFC can be used to determine diffusion coefficients by the Taylor–Aris peak broadening technique (108). The use of SFs in separation science was the subject of a recent review by Smith (109), who gives a sober account of the events that took place, particularly during the 1980s and 1990s, in SF separation science. A relatively recent area worthy of note is superheated water chromatography; the use of superheated water as the mobile phase for reversed-phase chromatography. The separation and analysis of several compound classes, including parabens (110,111), barbiturates (110,111), and amino acids (112), have been reported. Detailed SFC procedures, mostly of pharmaceutical interest, are published (88).

6.3. Chemical Reactions An area of SF technology currently being studied with vigor is the use of an SF as an active participant in a reaction or as the solvent for the reactants, catalysts, and products. Reactions of interest include those involving enzymes (113), derivatizations (114), and homogeneous (115) and heterogeneous catalysis (116). Some chemical reactions in SFs are currently in production (117).

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Fig. 7. Chromatogram of a mixture of vitamins using water-modified supercritical carbon dioxide as the mobile phase. Peaks: (1) vitamin B1, (2) vitamin B6, and (3) vitamin B2. Separation conditions: 150°C; pressure programming, 27.58 - 34.47 MPa (rate, 0.276 MPa min-1); column, 100 mm x 2 mm id packed column (Nucleosil diol). [Reprinted from Pyo, D. (2000) Separation of vitamins by supercritical fluid chromatography with water-modified carbon dioxide as the mobile phase, J. Biochem. Biophs. Methods 43, 113–123, with permission from Elsevier Science.]

The use of SFs for chemical reactions dates back to the 19th century. In 1857 (the earliest example found), Daubrée studied the reactions of supercritical water with glass and minerals at 400°C (118). For more information on this, and other early SF-based reactions, an excellent review of the history of chemical reactions in SFs has been produced by Jessop and Leitner (119). There is interest in this area because SFs can homogenize a reaction mixture, diffusion is more rapid for diffusion-controlled reactions than in a liquid (Table 2), they can incorporate controlled phase separation of products, and, especially in the critical region, they can be used to control the distribution of products. One promising applica-


Williams et al. tion for this last mentioned use of SFs is the production of purer medicine. Changes in the SF temperature and pressure alter the composition of products formed in chemical reactions. This is particularly important with pharmaceuticals, as some reactions produce two chiral forms of the same product. These can have different actions inside the human body, giving medicines unexpected side effects if the unwanted compound is not removed. The use of thalidomide by pregnant women is a classic example here. The exploitation of an SF allows fine-tuning of the synthesis to produce very little of the unnecessary, sometimes dangerous, version. The basic apparatus required to perform SFbased reactions is similar to an SFE system (Fig. 4), but the extraction sample cell is replaced with a reaction vessel containing a magnetic bar to stir the reaction (Fig. 8). The reaction vessel sits on a magnetic stirrer plate and the reactants are placed inside the vessel. The medium to be used as the SF, for example, carbon dioxide, is pumped into the vessel and the system is pressurized to the desired level. The reactants are stirred and the temperature is increased to the required value. The reaction is then left to progress for a set time. After the set time, stirring stops and the system is depressurized to atmospheric pressure. The products and any remaining reactants are removed from the reaction vessel and collector for processing. Detailed SF reaction procedures are available (88). Some examples of reactions involving supercritical carbon dioxide are now described. A useful bioapplication of a chemical reaction occurring in a supercritical medium is the staining of fingerprints on cheques and banknotes using ninhydrin (120). Supercritical carbon dioxide is used as the solvent instead of ozone-depleting CFC113. The ninhydrin reacts with amino acids present in sweat to give the strong purple color familiar when it is used as a stain for protein. The procedure is described in detail in a recent book (121). Another published example of the use of an SF in a chemical reaction was the study of the interesterification of edible palm oil by stearic acid in supercritical carbon dioxide (122). The SF served to extract the

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Fig. 8. Apparatus for reactions in supercritical fluids. (1) Carbon dioxide cylinder, (2) high-pressure pump, (3) cooling jacket, (4) valve, (5) preheating coil, (6) pressure gauge, (7) valve (6-way), (8) high-pressure cell, (9) magnetic bar, (10) magnetic stirrer, (11) filter, (12) air bath, (13) pressure controller, (14) backpressure regulator, and (15) collector.

oil and deliver it to a column packed with enzymes that functioned as a catalyst for the interesterification. Birnbaum et al. (123) reported the oxidation of cyclohexene using molecular oxygen catalyzed by halogenated iron porphyrins in supercritical carbon dioxide. Their results demonstrated that supercritical carbon dioxide served well as a solvent replacement for methylene chloride in the above reaction. Fixed bed hydrogenation of organic compounds in supercritical carbon dioxide has been studied by Arunajatesan and coworkers (124). Although they considered the palladium–carbon catalyzed hydrogenation of cyclohexene to cyclohexane, it may be possible to apply their method and apparatus to hydrogenations of a more biotechnological nature. Some reactions are combined with SFE. An example is the lipase-catalyzed hydrolysis of canola oil in supercritical carbon dioxide, studied as a model reaction to develop an on-line extraction–reaction process to extract oil from oilseeds and convert it to other valuable products (125). They concluded that the process showed great potential. Most of the work has taken place in supercritical carbon dioxide, but recently there has been much interest in supercritical and superheated water, most famously for the destruction of toxic waste (2). Some modern examples of reactions (all hydrolysis) in supercritical and/or superheated water now follow. Sasaki and coworkers


275 (126) proposed a new method to hydrolyze cellulose rapidly in supercritical water to recover glucose, fructose, and oligomers. They found that hydrolysis product yields were higher in supercritical water than in subcritical water. In a different study, the hydrolysis and oxidation of thiodiglycol in sub- and supercritical water has been studied by Lachance et al. (127). Their results suggested that under supercritical conditions, thiodiglycol degradation to carbon dioxide, water, elemental sulfur, and sulfates occurred within a few seconds. The data indicated that for the hydrolysis of thiodiglycol, a first-order model was suitable. Another example is the kinetics of the hydrolysis of ethyl acetate in sub- and supercritical water, which has been investigated as a model reaction (128). The ester hydrolysis proceeded selectively to the expected acid and alcohol without involving catalysts. The authors concluded that the dominating mechanism in supercritical water was, in all probability, the uncatalyzed nucleophilic attack of water. Supercritical water oxidation is another application of SFs in chemical reactions (129,130). Inserting oxygen into supercritical water causes oxidation, and reaction rates are very high. This is because a lot of substances are completely miscible with water under these conditions and, therefore, the limitations of a two-phase system are not present. New products can be obtained that are impossible to produce under normal conditions. Destruction of organic substances with over 95% decomposition can be achieved within minutes. As an illustration, Jin and coworkers (131) reported the rapid oxidation of food wastes with hydrogen peroxide in supercritical water. They found that high total organic carbon decompositions of up to 97.5% were obtained within 3 min at 420°C for carrots and within 5 min at 450°C for beef suet with a sufficient oxygen supply. A disadvantage to the use of supercritical water is corrosion of the apparatus by the fluid, particularly in an acidic medium containing anions. This corrosion reduces the lifetime of reactor components and is potentially dangerous, if stress corrosion cracking occurs (132). It has been recorded

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276 that no material can withstand every conceivable acidic high-temperature solution (133). The area of organic chemical reactions in supercritical water has lately been the subject of a 171-reference review by Savage (134). Very recently, sub- and supercritical methanol have been used as reaction media for the transesterification of rapeseed oil to its methyl esters. It was found that the uncatalyzed supercritical methanol process required a shorter reaction time and a simpler purification procedure than the conventional method (135). A kinetic study revealed that the conversion rate of rapeseed oil to its methyl esters increased dramatically in the supercritical state (136).

6.4. Particle Production Particle size and particle size distribution are key performance factors in the use of different organic and inorganic materials. The use of SFs for particle formation in the micron range with a narrow size distribution is of interest to the pharmaceutical industry, who want to exploit the technology for inhalation sprays and controlledrelease drugs. Particle formation involving SFs can be carried out by four different techniques: supercritical antisolvent (SAS) precipitation (also known as the gas antisolvent or GAS process), the rapid expansion of a supercritical solution (RESS), the particles from gas-saturated solution (PGSS) process, and finally, solution enhanced dispersion by supercritical fluids (SEDS). The origins of the four methods are found in the 19th century. Precipitation from a depressurizing supercritical solution was first observed in 1879 by Hannay and Hogarth (137), but it was not until Krukonis (138) reported his results in 1984 that interest was shown in using the technique commercially as a replacement for crushing, grinding, ball milling, and precipitation from solution. An advantage of this approach is the absence of degradation by heating during the alternative milling process. McHugh and Krukonis (4) give a good account of the historical development of SF-based particle production.


Williams et al. 6.4.1. SAS The SAS technique exploits the antisolvent properties of SFs, as opposed to their solvating power, which is often too low for practical purposes. As an illustration, carbon dioxide does not exert any detectable solubility for some high-molecular-weight compounds, such as polysaccharides, regardless of the pressure and temperature conditions used. However, it can be solubilized to any extent in many organic solvents. The amount of carbon dioxide dissolved in the liquid is an increasing function of pressure, so that the properties of the organic solvents can be strongly modified by carbon dioxide addition up to a point where the mixture is no longer able to keep the high-molecular-weight compound in solution. At this point, a complete precipitation of the compound of interest can be obtained by a further, but small, increase in pressure. A knowledge of the expansion curve vs pressure, and of the precipitation pressure at the selected temperature, is necessary for performing supercritical antisolvent precipitation work. Both of them depend on the SF, the organic solvent, the solute, and the temperature chosen. On the other hand, the concentration of solute in the starting organic solution affects the shape and dimensions of the precipitate obtained. The sample is dissolved and saturated in a liquid solvent. The resulting solution meets the SF in one of two ways; either the sample solution is sprayed into the SF or the SF is sprayed into the liquid (Fig. 9). The SF causes a decrease in the solvating power of the liquid solvent because of volume expansion. This begins precipitation of the solute. Separation of powdered product from the solvent can be achieved by filtration and drying or after the powdering step, the SF can remove the liquid solvent from the powdered material. The technique was the subject of a 1999 review by Reverchon (139) that included references up to 1998. Some later examples of SAS or GAS now follow. Lecithin has attracted interest because of its use in the food and pharmaceutical industries. Weber et al. (140) recovered lecithin (85+%) from egg yolk extracts by GAS crystallization. They

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Fig. 9. Schematic diagram of an SAS system. (A) carbon dioxide reservoir, (E) expansion vessel, (F) flow indicator, (L) filter, (P) carbon dioxide pump, (PI) pressure indicators, (R) flow meter, (S) precipitation vessel, (TIC) temperature controller, (Vr) one-way valve, (WH) heat exchanger, (WR) chiller, and (Vm) metering valve.

concluded that their procedure showed large-scale promise. Magnan and coworkers (141) successfully micronized soy lecithin by precipitation with a compressed fluid antisolvent based on the use of supercritical carbon dioxide. They found that increasing solute concentration and solution flow rate had a marked effect on particle size, but pressure did not. Regarding other solutes, Reverchon and coworkers (142) processed various biopolymers by semicontinuous SAS based on the use of supercritical carbon dioxide to evaluate the possibility of producing nano- and micro-particles of controlled size and distribution. They concluded, as others working with polymers have done and in contrast to the lecithin work, that the particle size of the biopolymers showed only a limited dependence on the process parameters. A detailed SAS procedure has been described recently by Bertucco and Pallado (143).

6.4.2. RESS The RESS process involves first dissolving a solute in an SF. The equilibrium between solute and SF is reached at temperatures well below the


melting point of the solute so that the two phases are the solid phase (almost pure solute) and the supercritical phase (in which the solute is partially dissolved). The supercritical phase is then depressurized causing the SF to expand and allowing the precipitation of uniform, fine, solid solute particles. In a typical experiment, a solution of the compound of interest is first made in supercritical carbon dioxide at 300 bar and 50°C and the solution is passed, after preheating, into a vessel (atomizer) maintained at 70 bar through a nozzle of 25 µm diameter (Fig. 10). The drop in pressure is sufficient to precipitate the material, which is formed as fine particles (for example, 1 µm mean diameter), with a narrow size distribution. As an illustration, Fig. 11 shows the production by RESS of uniform fine particles of ß-estradiol from nonuniform starting material. A comprehensive list of RESS references up to and including the year 1999, containing ones for pharmaceuticals and other biomaterials, has been given by Marr and Gamse (80). In addition, a thorough RESS method has been published by Alessi and coworkers (144).

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Fig. 10. Schematic diagram of a RESS pilot plant. (R) carbon dioxide reservoir, (P) carbon dioxide pump, (E) extraction cell, (H) prenozzle heater, (M) microtonization chamber (in which microtonized particles are made after passing through a nozzle), and (V) valve.

6.4.3. PGSS In the PGSS process (145,146), the binary system (solute and SF) at a given temperature and pressure, is unstable, originating a two-phase system. At temperatures higher than the melting point of the solute, these two phases are liquids: the first phase is the SF saturated with the solute, the second one is constituted by the organic solute saturated with the SF. The solute-rich phase is then depressurized through a nozzle, causing precipitation of the solute (Fig. 12). The solute particles are separated from the depressurized gas by the use of a cyclone and an electric filter. Fractionation on the basis of particle size occurs; the coarse fraction is found in the spray chamber, the medium fraction is situated in the cyclone, and the fines are deposited in the electric filter. 6.4.4. SEDS Solution enhanced dispersion by SFs is a relatively new technique, first appearing in 1995 in a doctoral thesis (147) and then as a patent one year later (148). The basic principle is to keep biomolecules in an aqueous solution before the formation of particles, then use supercritical carbon dioxide to extract the aqueous phase from the product.


Fig. 11. Photomicrograph of (A) virgin β-estradiol and (B) β-estradiol following RESS. [From Supercritical Fluid Extraction by Mark McHugh and Val Krukonis. Reprinted by permission of ButterworthHeinemann.]

The method involves the use of HPLC pumps to deliver carbon dioxide and a solution containing the solute of interest into a crystallization vessel. Carbon dioxide is furnished from a pressurized cylinder, chilled, and pumped into the ves-

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Fig. 12. Schematic diagram of the PGSS process. [Reprinted from Chemical Engineering and Processing, Marr, R., and Gase, T. (2000). Use of supercritical fluids for different processes inducing new developments—a review, Chem. Engring. and Proc. 39, 19–28, with permission from Elsevier Science.

sel. At the same time, the solution of solute is also pumped into the vessel through a custom-built nozzle (149). The vessel is put inside an oven, which allows thermal control. An electronic BPR is used to control pressure. An enhanced description of the apparatus and its operating procedure can be found elsewhere (147). Solution enhanced dispersion by SF is used for the controlled particle formation of biological materials and has been applied to proteins, peptide, antibodies, and plasmid DNA (150).

6.4.5. Choice of Method At present, there is some debate over the role and future of each of the methods. Some authors (143) have commented that the RESS technique is no more than a potential, owing to the extremely low solubility of the compounds of interest in SFs, even at pressures as high as 500 bar. On the other hand, the PGSS process often requires temperatures too high for the stability of the drug. On the basis of different authors’ experiences, it is believed that the SAS technique, which can be per-


formed at pressures usually lower than 100 bar, is the only one likely to become of practical interest as an alternative to traditional technologies currently used for the production of fine biocompatible particles. Furthermore, this method is becoming increasingly popular. In contrast, others (144) believe that the processes complement one another. Solubility is the key to choosing the correct method for the desired application. If the solubility of the material of interest in the SF is higher than a few milligrams per gram of solvent, the RESS process can be used, but if the solubility is lower, the SAS process is preferred. In the case of low melting point and relatively thermally stable materials, PGSS can be used. Finally, SEDS has promise for the controlled particle formation of biological macromolecules, which are insoluble in supercritical carbon dioxide and many organic solvents, and hence unsuitable for treatment by SAS, RESS, and PGSS. Three (SAS, RESS, and PGSS) of the four methods were reviewed recently by Marr and Gamse (80) as part of a larger overview.

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280 6.5. Deposition There is an increasing need for improved methods to effectively deliver fluids into matrices for reacting, extracting, or depositing other materials. Existing methods, using liquid solvents, do not always produce a uniform distribution or penetrate deep enough into the sample. One strategy to improve the process is to change the properties of the solvent to increase diffusivity or reduce viscosity. Only limited changes are possible with the usual liquid solvents. One approach to impregnating materials, such as wood, with chemical agents is to use SFs as carriers (151,152). SFs have diffusivities that are intermediate between liquids and gases, and viscosities similar to gases (Table 2). A number of these fluids have the ability to solubilize materials at levels that can approach those of liquid carriers. A conventional SFE apparatus can be used for the purpose (Fig. 4) and the experimental procedure is similar to extraction. However, reversing the direction of flow periodically helps give an even distribution of the deposited material. At the end of the experiment, the pressure is released rapidly. The drop in pressure results in solute deposition within the sample. A procedure for the deposition of a biocide into wood-based material has been described in detail recently using carbon dioxide or carbon dioxide– methanol as the SF (153).

6.6. Drying of Biological Specimens Critical point drying (CPD) can be used to dry virtually all specimens, particularly prior to examination with a scanning electron microscope. It is the most widely used method for this purpose, because an SF lacks surface tension, improving penetration and avoiding distortion of delicate components during drying. The basic idea behind CPD is that a liquid housed in a sealed cell will expand and, at the same time, evaporate when exposed to a temperature increase. The molecules in the liquid experience an increase in their kinetic energy. Consequently, more leave the liquid and enter the gas phase. As a result of this, the liquid density decreases and the gas density increases. Eventu-


Williams et al. ally, both densities become the same, the liquid mensicus disappears and the surface tension is zero. The critical point of water is too high (374°C and 217.7 atm) for biological specimens; they would be cooked and destroyed under these conditions. It is necessary, therefore, to replace water with a transitional solvent, such as carbon dioxide, that has a critical point appropriate for biological samples. Maintaining a specimen in the transitional solvent at, or above, its critical point, while gradually venting off the solvent, results in a dried specimen that has not been subjected to the harmful effects of surface tension forces. Critical point drying is an established technique (154), appearing in the literature as early as the 1950s (155). It compares favorably to rival techniques (156) and an in-depth CPD method has been described recently by Bray (157). This review gives only a brief introduction to SFs. Much more detailed texts are available, for example, those by McHugh and Krukonis (4), Berger (106), Taylor (158), Smith and Hawthorne (159), Jessop and Leitner (160), and Clifford (161). In 1999, a whole issue of Chemical Reviews was dedicated to supercritical fluids (162).

7. Conclusions Supercritical fluids have been applied across a wide spectrum of biotechnological and other areas. Not all uses of SFs enjoy equal popularity at any given time. In the 1980s and 1990s, extraction and chromatography with carbon dioxide attracted much attention. Now reactions and materials research are coming to the fore. The former in particular is predicted to receive a lot of study in the near future. In addition, interest in SFC has waned and it looks destined to remain a niche technique. However, SAS precipitation and the use of superheated and supercritical water for extraction, chromatography, and reactions are being investigated more. The field of SFs is not without problems; specialist equipment is expensive and much glassware and organic solvents can be purchased for the price of one SF system. Although the relatively high cost of the apparatus is off-putting, as time goes by, the costs may go down due to tech-

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Supercritical Fluids and Their Applications nological advances. More efforts have to be made to overcome difficulties, such as the problem of corrosion when using hot, pressurized water. However, SFs look set to remain an area of intense research interest for years to come. Furthermore, one can envisage a time in the future when the use of sub- and supercritical carbon dioxide and water becomes very important in laboratory work, with organic solvent use considerably reduced.

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Supercritical Fluids and Their Applications 57. Brewer, W. E., Galipo, R. C., Sellers, K. W., and Morgan, S. L. (2001) Analysis of cocaine, benzoylecgonine, codeine, and morphine in hair by supercritical fluid extraction with carbon dioxide modified with methanol. Anal. Chem. 73, 2371–2376. 58. Marsal, A., Celma, P. J., Cot, J., and Cequier, M. (2000) Supercritical CO2 extraction as a clean degreasing process in the leather industry. J. Supercrit. Fluids 16, 217–223. 59. Lanças, F. M., Rissato, S. R., and Galhiane, M. S. (1999) Determination of 2,4-D and Dicamba in food crops by MEKC. Chromatographia 50, 35–40. 60. Pyo, D., Park, K., Shin, H., and Moon, M. (1999) Extraction of microcystins from cyanobacteria by acetic acid modified supercritical CO2. Chromatographia 49, 539–542. 61. Allen, D. L., Scott, K. S., and Oliver, J. S. (1999) Comparison of solid-phase extraction and supercritical fluid extraction for the analysis of morphine in whole blood. J. Anal. Toxicol. 23, 216–218. 62. Matabudul, D. K., Crosby, N. T., and Sumar, S. (1999) A new and rapid method for the determination of nicarbazin residues in poultry feed, eggs and muscle tissue using supercritical fluid extraction and high performance liquid chromatography. Analyst 124, 499–502. 63. Song, S. and Ashley, D. L. (1999) Supercritical fluid extraction and gas chromatography/mass spectrometry for the analysis of tobacco-specific nitrosamines in cigarettes. Anal. Chem. 71, 1303–1308. 64. Ling, Y.-C., Teng, H.-C., and Cartwright, C. (1999) Supercritical fluid extraction and clean-up of organochlorine pesticides in Chinese herbal medicine. J. Chromatogr. A 835, 145–157. 65. Ali, M. Y. and Cole, R. B. (1998) SFE plus C18 lipid cleanup method for selective extraction and GC/MS quantitation of polycyclic aromatic hydrocarbons in biological tissues. Anal. Chem. 70, 3242–3248. 66. Morselli, L., Setti, L., Iannuccilli, A., Maly, S., Dinelli, G., and Quattroni, G. (1999) Supercritical fluid extraction for the determination of petroleum hydrocarbons in soil. J. Chromatogr. A 845, 357–363. 67. Palma, M. and Taylor, L. T. (1999) Extraction of polyphenolic compounds from grape seeds with near critical carbon dioxide. J. Chromatogr. A 849, 117–124. 68. Kery, A., Ronyai, E., Simandi, B., et al. (1999) Recovery of a bioactive sesquiterpene lactone from Tanacetum parthenium by extraction with supercritical carbon dioxide. Chromatographia 49, 503–508. 69. Weathers, R. M., Beckholt, D. A., Lavella, A. L., and Danielson, N. D. (1999) Comparison of acetals as in situ modifiers for the supercritical fluid extraction of ß-carotene from paprika with carbon dioxide. J. Liq. Chrom. Rel. Technol. 22, 241–252. 70. Snyder, J. M., King, J. W., Taylor, S. L., and Neese, A. L. (1999) Concentration of phytosterols for analysis by supercritical fluid extraction. JAOCS 76, 717–721.


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