VII.

Supercritical Fluid Chromatography & Extraction

CHM 614/714

Introduction
During the past 3 decades, two new techniques that are based upon the use of supercritical fluids have been developed and promise to play an important role in the analysis of environmental, biomedical, and food samples. These new methods are supercritical fluid chromatography (SFC) and supercritical fluid extraction (SFE). Instruments for both of these techniques have been commercialized during the last decade and their use appears to be growing rapidly in the analytical community. In this chapter, we describe the theory, instrumentation, and applications of both of these methods.

Supercritical fluids

Relatively new technique as an instrumental method First suggested by Lovelock in 1958 First commercial instruments

1981 packed columns 1985 capillary columns

As a chromatographic method, it fits somewhere between LC and GC

Offers advantages of both

Supercritical fluid advantages

As a chromatographic method:

Can use low temperatures for separation Can use packed or capillary columns Pressure and polarity of mobile phase can be tuned for optimum separation

As an extraction method:

Can also be tuned Removal of extracting solvent is very easy

Supercritical fluid disadvantages

More expensive equipment Increased method development time more factors to control means more to be optimized Methods are not as developed as GC or LC not as many applications yet.

Properties of supercritical fluids The critical point is the end of the vapor pressure curve. The temperature at the critical point is called the critical temperature, Tc. The pressure at the critical point is called the critical pressure, Pc. At temperature above the critical temperature, a substance can not be liquefied no matter how great the pressure.

At temperature above the critical temperature the molecules have so much kinetic energy that no amount of pressure is enough to hold them in contact with each other. At the critical point and beyond, there is no longer any difference between liquid and gas. The densities of liquid and gas are the same. At the critical point and beyond, a substance is still fluid. Because it is neither a gas nor a liquid it is referred to as a supercritical fluid. Supercritical fluids have low viscosities like gases but are dense as liquids, which are characteristics of good solvents. Supercritical fluids are sometimes classified as a fifth state of matter (plasma is the fourth).

Table 1 compares certain properties of supercritical fluids to those of typical gases and liquids. Supercritical fluids have densities, viscosities, and other properties that are intermediate between those of the substances in their gaseous and liquid states.

Table 1. Comparison of properties of supercritical fluids with liquids and gases (all of the data are order-of-magnitude only)

This means that it is possible to develop methods that offer: (a) Pressures and flows similar to GC based methods due to low viscosity. (b) Efficiencies similar to HPLC methods due to low diffusion coefficient. Table 2 lists properties of four of perhaps two dozen compounds that have been employed as mobile phases in supercritical fluid chromatography. [Note]: The critical temperatures and pressures at these temperatures are well within the operating conditions of ordinary HPLC. An important property of supercritical fluids is their remarkable ability to dissolve large nonvolatile molecules, which is related to their high densities (0.2 to 0.5 g/cm3).

Table 2. Properties of some supercritical fluids*

For example, supercritical carbon dioxide readily dissolves n-alkanes containing from 5 to over 30 carbon atoms, di-n-alkylphthalates in which the alkyl groups contain 4 to 16 carbon atoms, and various polycyclic aromatic hydrocarbons made up of several rings. It is perhaps noteworthy that certain important industrial processes are based upon the high solubility of organic species in supercritical carbon dioxide. For example, this medium has been employed for extracting caffeine from coffee beans to give decaffeinated coffee and for extracting nicotine from cigarette tobacco.

The solubility of material in a supercritical fluid tends to increase with density of the fluid (at constant temperature). Since density increases with pressure, solubility tends to increase with pressure. The relationship with temperature is a little more complicated. At constant density, solubility will increase with temperature. However, close to the critical point, the density can drop sharply with a slight increase in temperature. Therefore, close to the critical temperature, solubility often drops with increasing temperature, then rises again. In addition, there is no surface tension in a supercritical fluid, as there is no liquid/gas phase boundary. By changing the pressure and temperature of the fluid, the properties can be tuned to be more liquid- or more gas-like.

A second important property of many supercritical fluids is that analytes dissolved in them can be easily recovered by simply allowing the solutions to equilibrate with the atmosphere at relatively low temperatures. Thus, an analyte dissolved in supercritical carbon dioxide, the most commonly used solvent, can be recovered by simply reducing the pressure and allowing the fluid to evaporate under ambient laboratory conditions. This property is particularly important with thermally unstable analytes. Another advantage of many supercritical fluids is that they are inexpensive, innocuous, and nontoxic substances that can be allowed to evaporate into the atmosphere with no harmful environmental effects. Supercritical fluid carbon dioxide is especially attractive for extractions and chromatography. Finally, supercritical fluids have the advantage of solute diffusivities that are an order of magnitude higher and viscosities that are an order of magnitude lower than their liquids. As we shall show, these last two advantages are important in both chromatography and extractions with supercritical fluids.

Supercritical Fluid Chromatography (SFC)

SFC is a hybrid of gas and liquid chromatography that combines some of the best features of each.
* M.D. Palmieri, J. Chem. Educ., 1988, 65, A254; 1989, 66, A141. * P.R. Griffiths, Anal. Chem., 1988, 60, 593A. * R.D. Smith, B.W. Wright, and C.R. Yonker, Anal. Chem., 1988, 60,1323A. * M.L. Lee and K.E. Markides, Science,1987,235,1342. * For a monograph on the subject, see Supercritical Fluid Chromatography, R.M. Smith, Ed. London: The Royal Society of Chemistry 1988. C.M. White, Modern Supercritical Fluid Chromatography. Heidelberg, FRG: Alfred Huethig Verlag, 1988.

This technique is an important third kind of column chromatography that is beginning to find use in many industrial, regulatory, and academic laboratories. In 1985, several instrument manufacturers began to offer equipment specifically designed for supercritical fluid chromatography, and since that time, the use of such equipment has expanded at a rapid pace.

Supercritical fluid chromatography is of importance because it permits the separation and determination of a group of compounds that are not conveniently handled by either gas or liquid chromatography. These compounds (1) are either nonvolatile or thermally labile so that gas chromatographic procedures are inapplicable, and (2) contain no functional groups that make possible detection by the spectroscopic or electrochemical techniques employed in liquid chromatography. It was estimated that up to 25% of all separation problems faced by chemists today involve mixtures containing such intractable species*. * T.L. Chester, J. Chromatogr. Sci., 1986, 24, 226.

In SFC, we can control: (a) Pressure of mobile phase (b) Temperature (c) Nature of solvent (e.g., polarity index) (d) Type of stationary phase

Instrumentation and Operating Variables

As mentioned earlier, the pressures and temperatures required for creating supercritical fluids derived from several common gases and liquids lie well within the operating limits of ordinary HPLC equipment.

Figure 1. Schematic of an instrument for supercritical fluid chromatography.

As shown in Figure 1, instruments for supercritical fluid chromatography are similar to those for LC system. However, there are two important differences between the SFC and LC. First, a thermostated column oven, similar to that used in gas chromatography, is required to provide precise temperature control of the mobile phase; second, a restrictor, or back-pressure device is used to maintain the pressure in the column at a desired level and to convert the eluent from a supercritical fluid to a gas for transfer to the detector. A typical restrictor for a 50- or 100-m open-tubular column consists of a 2- to 10-cm length of 5- to l0-m capillary tubing attached directly to the end of the column. Alternatively, the restrictor may be an integral part of the column formed by drawing down the end of the column in a flame. The former permits the use of interchangeable restrictors having different inside diameters, thus providing a range of flow rates at any given pumping pressure. Either GC or LC detectors can be used, which is based on the sample and pressure. The major difference over GC and LC is the ability of SFC to modify pressure or solvent nature during a run.

Effect of Pressure
Pressure changes in supercritical chromatography have a pronounced effect on the capacity factor k'. This effect is a consequence of the increase in density of the mobile phase with increases in pressure. Such density increases cause a rise in solvent power of the mobile phase, which in turn shortens the elution time for solutes. As an example of the effect of pressure, it is found that when the CO2 pressure in a packed column is increased from 70 to 90 atm, the elution time for hexadecane decreases from about 25 to 5 min. This effect is general and produces results that are analogous to those obtained with gradient elution in gas and liquid chromatography. Figure 2 illustrates the improvement in chromatograms realized by pressure programming. The most common pressure profile used in supercritical fluid chromatography is often constant (isobaric) for a given length of time followed by a linear or asymptotic increase to a final pressure.

Figure 2. Effect of pressure programming in supercritical fluid chromatography.

Stationary Phases
Both open-tubular and packed columns are used for SFC, although currently the former are favored. Open tubular columns are similar to the fused-silica columns used in gas chromatography with internal coatings of bonded and cross-linked siloxanes of various types. Column lengths are often 10 or 20 m and inside diameters are 50 or 100 m. Film thicknesses vary from 0.05 to 1 m. Packed columns, similar to those used in partition liquid chromatography, are also employed in SFC. These columns vary in diameter from 0.5 mm or less to 4.6 mm with particle diameters ranging from 3 to 10 m. The coatings are similar to those used in partition HPLC.

Mobile Phases
The most widely used mobile phase for supercritical fluid chromatography is carbon dioxide. It is an excellent solvent for a variety of nonpolar organic molecules. In addition, it transmits ultraviolet light, is odorless, nontoxic, readily available, and remarkably inexpensive when compared with other chromatographic mobile phases.

Carbon dioxide's critical temperature of 31C and its critical pressure of 72.9 atm permit a wide selection of temperatures and pressures without exceeding the operating limits of modern HPLC equipment. In some applications, particularly with polar analytes, polar organic modifiers such as methanol are introduced in small concentrations (~1%) to modify values for analytes. A number of other substances have served as mobile phases for supercritical chromatography, including ethane, butane, nitrous oxide, dichlorodifluoromethane, diethyl ether, ammonia, and tetrahydrofuran.

Detectors
A major advantage of SFC over HPLC is that the flame ionization detector of gas chromatography can be employed. This detector exhibits a general response to organic compounds, is highly sensitive, and is largely trouble free. Mass spectrometers are also more easily adapted as detectors for SFC than HPLC. Several of the detectors used in liquid chromatography find their uses in SFC as well, including ultraviolet and infrared absorption, fluorescence, thermionic, and flame photometric detectors.

Comparison of SFC to Other Types of Chromatography

The data in Tables 1 and 2 reveal that several physical properties of supercritical fluids are intermediate between gases and liquids. As a consequence, supercritical fluid chromatography combines some of the characteristics of both gas and liquid chromatography. For example, like gas chromatography, supercritical fluid chromatography is inherently faster than liquid chromatography because the lower viscosity makes possible the use of higher flow rates. Diffusion rates in supercritical fluids are intermediate between those in gases and in liquids. As a consequence, band broadening is greater in supercritical fluids than in liquids, but less than in gases. Thus, the intermediate diffusivities and viscosities of supercritical fluids should in theory result in faster separations than are achieved with liquid chromatography accompanied by lower zone spreading than is encountered in gas chromatography. Figures 3 and 4 compare the performance characteristics of a packed column when elution is performed with supercritical carbon dioxide and a conventional liquid mobile phase.

In Figure 3, it is seen that at a linear mobile-phase velocity of 0.6 cm/s, the supercritical column yields a plate height of 0.013 mm while the plate height with a liquid eluent is three times as large or 0.039 mm. Thus, a increase of separation efficiency (N) should be realized by a factor of 3 in SFC. Alternatively, there is a gain of 4 in linear velocity at a plate height corresponding to the minima in the LC curve; this gain would result in a reduction of analysis time by a factor of 4. These advantages are reflected in the two chromatograms shown in Figure 4.

Figure 3. Performance characteristics of a 5-m ODS column.

Figure 4. Comparison of chromatograms obtained by HPLC and SFC. Column: 20 cm x 4.6 mm packed with 10 m reversed-phase bonded. Analytes (1) biphenyl; (2) terphenyl. For HPLC: mobile phase, 65/35% CH3OH/H2O; flow rate, 4 mL/min; linear velocity, 0.55 cm/s; sample size, 10 L. For SFC: mobile phase, CO2; flow rate, 5.4 mL/min; linear velocity, 0.76 cm/s; sample size, 3 L.

It is worthwhile comparing the role of the mobile phase in gas, liquid, and supercritical fluid chromatography. In GC, the mobile phase serves but one purpose --- zone movement. In LC, the mobile phase provides not only transport of solute molecules but also interactions with solutes that influence selectivity factors (). In SFC, when a molecule dissolves in a supercritical medium, the process resembles volatilization but at a much lower temperature than would normally be used in gas chromatography. Thus, at a given temperature the vapor pressure for a large molecule in a supercritical fluid may be 1010 times greater than in the absence of that fluid. As a consequence, high-molecular-weight compounds, thermally unstable species, polymers, and large biological molecules can be eluted from a column at reasonably low temperatures. Interactions between solute molecules and the molecules of a supercritical fluid must occur to account for their solubility in these media. The solvent power is thus a function of the chemical composition and the density of the fluid. Therefore, in contrast to gas chromatography, the possibility exists for varying selectivity factor () by changing the mobile phase.

Range of molecular weights and sizes for various column chromatographic techniques

Applications SFC has been applied to a wide variety of materials, including natural products, drugs, foods, pesticides and herbicides, surfactants, polymers and polymer additives, fossil fuels, and explosives and propellants. Figure 5 shows the separation of a series of dimethyl polysiloxane oligomers ranging in molecular weight from 400 to 700 Da. This chromatogram was obtained by using a 10 m x 100-m inside diameter fused-silica capillary coated with a 0.25-m film of 5% phenyl polysiloxane. The mobile phase was CO2 at 140C, and the following pressure program was used: 80 atm for 20 min, then a linear gradient from 80 to 280 atm at 5 atm/min. A FID was used.

Figure 5. Separation of oligomers of dimethyl-polysiloxane by SFC.

Figure 6 illustrates the separation of polycyclic aromatic hydrocarbons extracted from a carbon black. Detection was by fluorescence excited by two different wavelengths (Note the selectivity provided by this technique). The chromatogram was obtained by using a 40-m x 50-m inside diameter capillary coated with a 0.25-m film of 50% phenyl polysiloxane. The mobile phase was pentane at 210C, and the following program was used: initial mobilephase density held at 0.07 g/mL for 24 min, then an asymptotic density program to 0.197 g/mL.

Figure 6. Portions of the supercritical fluid chromatograms of polycyclic aromatics in a carbonblack extract.

Figure 7 illustrates a separation of oligomers in a sample of the nonionic surfactant Triton X-100. Detection involved measuring the total ion current produced by chemical ionization mass spectrometry. The mobile phase was carbon dioxide containing 1% methanol by volume. The column was a 30-m capillary column that was coated with a 1-m film of 5% phenyl polysiloxane. The column pressure was increased linearly at a rate of 2.5 bar/min.
Application reviews: * T.L. Chester & J.D. Pinkston, Anal. Chem.,1990, 62, 394R. * M.D. Palmieri, J. Chem. Educ., 1989, 66, A141. * C.M. White & R.K. Houk, .HRC& CC, 1986, 9, 4.

Figure 7. Chromatograms for the nonionic surfactant Triton X-100 with total current mass spectrometric detection.

Supercritical Fluid Extraction (SFE)

Often, the analysis of complex materials requires as a preliminary step separation (or extraction) of analyte(s) from a sample matrix. Ideally, an analytical extraction method should be rapid, simple, and inexpensive; should give quantitative recovery of analytes without loss or degradation; should yield a solution of the analyte that is sufficiently concentrated to permit the final measurement to be made without the need for concentration; and should generate little or no laboratory wastes that have to be disposed of. With normal solvent extraction methods, solutes that have low extraction efficiencies require: Batch extractions. Use of large amount of solvent followed by solvent removal. Continuous extractions. Heating of sample/solute mixture. Both require heating of the solute and some venting of the solvent to the atmosphere.

Unfortunately, normal solvent extraction, frequently fail to meet several of the ideal criteria listed in the previously. They usually require several hours or more to achieve satisfactory recoveries of analytes and sometimes never do. The solvent costs are often high. The solution of the recovered analytes are often so dilute that a concentration step must follow the extraction. Analyte degradation or loss as well as atmospheric pollution may accompany this concentration step. Beginning in the mid-1980s, chemists began to explore the use of supercritical fluids for extration/separation of analytes from matrices of many samples which are interest to industry and governmental agencies because supercritical fluids could avoid many of the problems using normal organic liquid extractants.
For more detailed information about SFE, see: * S.B. Hawthorne, Anal. Chem.,1990, 62,633A. * J.L. Hedrick, L.J. Mulcahey, & L.T. Taylor, Mikrochim. Acta, 1992, 108, 115. * L.T. Taylor, Anal. Chem., 1995, 67, 364A; Supociticat Fluid Extraction and Its use in Chromatographic Sample preparation, S. A. Westwood. Ed. Boca Raton; CRC Press, 1993. * M.D. Luque de Castro, M. Valcarcel, & M.T. Tena, Analytical Superoitical Fluid Extraction. New york: Springer-Verlag, 1994.

Advantages of Supercritical Fluid Extraction

1. SFC is generally fast. The rate of mass transfer between a sample matrix and an extraction fluid is determined by the rate of diffusion of a species in the fluid and the viscosity of the fluid --the greater the diffusion rate and the lower the viscosity, the greater will be the rate of mass transfer. As we have noted earlier, both of these variables are more favorable for supercritical fluids than for typical liquid solvents. As a consequences, supercritical fluid extraction can generally be completed in 10 to 60 minutes, whereas extractions with an organic liquid may require several hours or even days. 2. The solvent strength of a supercritical fluid can be varied by changes in the pressure and to a lesser extent in the temperature. In contrast, the solvent strength of an organic liquid is essentially constant regardless of conditions. This property allows the conditions for extraction with a supercritical fluid to be optimized for a given class of analytes.

3. Many supercritical fluids are gases at ambient conditions. Thus recovery of analytes becomes simple compared to organic liquids, which must be vaporized by heating, which leads to the possibility of decompositions of thermally unstable analytes or loss of volatile analytes. In contrast, a supercritical fluid can be separated from the analyte by simply releasing pressure. Alternatively, the analyte stream can be bubbled through a small vial containing a good solvent for the analyte, which will dissolve in the small volume of solvent. 4. Some supercritical fluids are cheap, inert, and nontoxic. Thus they are readily disposed of after an extraction is completed by allowing them to evaporate into the atmosphere.

Instrumentation
The instrumentation for SFE can be relatively simple as shown in Figure 8. Instrument components include a fluid source, most commonly a tank of carbon dioxide; a syringe pump having a pressure rating of at least 400 atm and a flow rate for the pressurized fluid of at least 2 mL/min; a valve to control the flow of the critical fluid into a heated extraction cell having a capacity of a few mL; an exit valve leading to a flow restrictor that depressurizes the fluid and transfers it into a collection device. In the simplest instruments, the flow restrictor is simply a 10- to 50-cm piece of capillary tubing. In modern sophisticated commercial instruments, the restrictors are variable and controlled manually or automatically. Several instrument manufacturers offer various types of SFE apparatus.

* F. Wach, Anal. Chem., 1994, 66, 369A.

Figure 8. A typical arrangement for off-line SFC. The shutoff valve is required for static SFC but not for dynamic SFC.

A SFE system can be operated in one of two ways. In the dynamic extraction mode, the valve between the extraction cell and the restrictor remains open so that the sample is continually supplied with fresh supercritical fluid and the extracted material flows into the collection vessel where depressurization occurs. In the static extraction mode, the valve between the extraction cell and the restrictor is closed and the extraction cell is pressurized under static conditions. After a suitable period, the exit valve is opened and the cell contents are transferred through the restrictor by a dynamic flow of fluid from the pump. The dynamic mode is more widely used than the static mode.

Choices of supercritical fluids

Two dozen or more supercritical fluids have been described as extraction media, but as in SFC by far, the most widely used substance is carbon dioxide or carbon dioxide with organic modifier.

The best choice of fluid is determined by a number of variables, including polarity and solubility of the analytes and the matrix components, physical nature of the matrix, concentration of the analytes, moisture content of the sample, and kinetic considerations. * M.E.P. McNally, Anal. Chem., 1995, 67, 308A. Unfortunately, the theory of supercritical fluid extractions is currently imperfect, and final conditions in most cases must be determined empirically. Carbon dioxide has been the fluid of choice in most studies. It is an excellent solvent for nonpolar species, such as alkanes and terpenes, and a moderately good extraction medium for moderately polar species, such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls, aldehydes, esters, alcohols, organic chloropesticides, and fats. It is generally not a good extraction medium for highly polar compounds unless modified by the addition of strongly polar modifiers such as methanol. Modifiers can be introduced into the extraction system either by means of a second pump, or by injecting the modifier into the sample prior to extraction.

A variety of modifiers have been used to enhance the polarity of supercritical fluid carbon dioxide. Including several of the lowermolecular-weight alcohols, propylene carbonate, 2methoxyethanol, methylene chloride, and certain organic acids. The most common is a few percent of methanol. Figure 9 demonstrates the improved efficiency in the presence of a small amount of methanol in the extraction of various materials from soil samples.
Figure 9. Comparison of extraction efficiencies obtained by using CO2 and CO2 modified with methanol. Samples were a soil. All extractions were for 30 min. Diuron is a common herbicide that is an aromatic substituted derivative of urea. TCDD is 2,3,7,8-tetrachlorodibenzo-p-dioxin. LAS is linear alkylbenzenesulfonate detergent.

Off-line and on-line extractions

Two types of methods have been used to collect analytes after extraction: off-line and on-line. In off-line collection, which is the simpler of the two, the analytes are collected by immersing the restrictor in a few milliliters of solvent and allowing the gaseous supercritical fluid to escape into the atmosphere (see Figure 8). Analytes have also been collected on adsorbents, such as silica. The adsorbed analytes are then eluted with a small volume of a liquid solvent. In either case the extracted analytes are then identified by any of several optical, electrochemical, or chromatographic methods. In the on-line method, the effluent from the restrictor, after depressurization, is transferred directly to a chromatographic system. In most cases tire latter is a gas chromatograph or a supercritical fluid chromatograph, although occasionally a liquid chromatographic instrument has also been used. The principal advantages of an on-line system is the elimination of sample handling between the extraction and the measurement and the potential for enhanced sensitivity because no dilution of the analyte occurs.

Typical applications of SFE

The following reference lists over 100 analytical applications of supercritical fluid extractions that have appeared in the literature between 1988 and 1993. * L.T. Taylor, Supercritical Fluid Extraction. New York: Wiley, 1996. A majority of these have been for the analysis of environmental samples. Others have been for the analysis of foods, biomedical, and industrial samples of various types. Table 3 provides a few typical applications of off-line and on-line supercritical fluid extractions.

Table 3. Some Typical Applications of SFE.