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CHROMATOGRAPHY: SUPERCRITICAL FLUID | Historical Development
CHROMATOGRAPHY: SUPERCRITICAL FLUID Historical Development T. A. Berger, AccelaPure Corporation, Newark, DE, USA & 2007 Elsevier Ltd. All rights reserved.
Discovery of Supercritical Phenomena The characteristics associated with ‘supercritical’ fluids were first observed by Baron Cagniard de la Tour in 1822, when he recognized that a liquid could be converted into a gas without a phase transition, if the liquid were heated above a specific temperature. Nearly 50 years later (1869), Andrews further developed the idea of a critical point (a ‘critical temperature’ T c and a ‘critical pressure’ Pc) above which only a single phase existed. In this context, the word ‘super’ is only intended to indicate ‘above.’ A schematic of a phase diagram helps in explaining this phenomenon, as shown in Figure 1. In the figure, a line emerges from the lower left-hand corner, separating solids from gases. Crossing the line from left to right represents sublimation of the solid to a gas. At the ‘triple point,’ solid, liquid, and gas are all present. One line continues nearly vertically and
C
Critical point
Liquid A Solid
Pressure
B
E
separates the liquid and solid forms of the compound. To the left of the line only a solid exists. To the right, only a liquid exists. The line represents conditions where a solid is in equilibrium with a liquid. The other line directed diagonally toward the upper right of the figure separates the liquid form from the gas form of the compound. Above and to the left of the line only a liquid exists. Below and to the right of the line, only gas exists. Directly on the line, a gas exists in equilibrium with a liquid. This line is sometimes called the boiling line or the vapor– liquid equilibrium line (VLE). This line ends at the critical point, characteristic of each fluid and mixture of fluids. Note that above this point there is no separation of gas and liquid. Increasing the pressure of a liquid above Pc (line A–B in Figure 1), then increasing the temperature above T c (line B–C), decreasing pressure (line C–D), and the temperature (line D–E), produces a low temperature, low-pressure gas. The transition from liquid to gas occurred without an apparent phase transition (there was never a meniscus between the two phases). Moving directly from point A to point E, results in a two-phase region where part of the fluid is a gas and part is a liquid. In 1879, Hannay and Hogarth observed that ‘supercritical’ fluids could act as solvents. They dissolved inorganic salts in supercritical ethanol and re-precipitated them by decreasing the temperature. In 1906, Buchner showed that nonvolatile organic compounds were much more soluble in supercritical fluids than expected. Throughout the twentieth century, many patents appeared, particularly with respect to extraction and processing of petroleum and oils.
D
Gas Triple point Temperature Figure 1 A phase diagram indicating the temperature and pressure regions where solids, gases, and liquids exist. The dashed line indicates how a high density liquid (at point A) can be converted to a low(er) density gas by first increasing pressure (to point B), increasing temperature (to point C), decreasing pressure (to point D), and then decreasing temperature (to point E), without two phases co-existing.
Early Developments Milestones in the development of supercritical fluid chromatography (SFC) are outlined in Figure 2. At an international gas chromatography (GC) symposium, Jim Lovelock, an early pioneer proposed ‘critical-state chromatography’ to separate polar and ionic species (such as the inorganic salts of Hannay and Hogarth) using inorganic gases and capillary columns. Later in 1958, he had a letter proposing the
2 II CHROMATOGRAPHY: SUPERCRITICAL FLUID / Historical Development
1822 1961 1966 1966 1967 1969 1970 1971 1979 1981 1982 1985 1986 1988 1989 1992 1998 1999 2000
Critical point discovered First separation First chromatogram FID introduced UV−Vis introduced Binary mobile phases Pressure programming Doubts about packed cols. MS detector Capillary SFC composition gradients First commercial SFC Independent control of press, flow, comp. First chiral separation Commercial Cap. SFC First use of additives first journal (journal of supercritical fluids) Solvent density and polarity deconvoluted 2nd generation commercial hardware introduced highefficiency-packed columns Last commercial SFC withdrawn Emphasis shifts toward semi-prep/pharma Gas delivery systems change economics
Figure 2 Milestones in the development of SFC.
same notarized. He never attempted to confirm his predictions experimentally. Ernst Klesper separated porphorins with a supercritical chlorofluorocarbon mobile phase in 1961. The liquid solvent was heated in a tube in a sand bath, connected to a heated packed column. The fluid was then cooled back to room temperature before passing through a fixed restrictor. Fractions were collected for off-line analysis. The first supercritical fluid chromatograms with an inline detector were made in 1966. Klesper later produced a series of papers mostly about the separation of polymers using liquids such as pentane and mixtures, like pentane/dioxane, heated to high temperatures (such as 2601C) in a sand bath. Calvin Giddings dominated the theoretical development of what he called ‘dense gas chromatography’ throughout the later 1960s. In a 1966 article in Science, Giddings proposed an elutrophic series in which carbon dioxide was placed next to isopropanol in solvent polarity. The numbers were based on estimated constants used to calculate Hildebrand solubility parameters. If true, changes in pressure would cause huge changes in polarity of carbon dioxide. Unfortunately, it is not true. Had it been true, programming a physical parameter (pressure) would have changed the polarity of the solvent from hydrocarbon-like to alcohol-like. Programming a physical parameter is much less expensive and complicated than mixing compressible and incompressible fluids. If the fluids had followed Giddings elutrophic series, it is likely that, today, high-
Giddings series
Nile Red solvent strength Methanol Ethanol
Methanol
NH4
Pyridine 2-Propanol 40% MeOH
Ethanol
Acetonitrile
2-Propanol
Ethyl acetate CO2
20% MeOH 10% MeOH
Ethyl acetate Diethyl ether CCl4
Pentane
CCl4
Pentane CO2
Figure 3 Comparison of Giddings ‘‘elutrophic series’’ and a measured solvent strength scale. Giddings scale attempted to predict the strength of solvents in eluting compounds from a silica column using estimates which have since proven inaccurate. The higher the solvent appears on the scale, the less retained a compound should be. Note that carbon dioxide appears half-way up Giddings series, while on the right it appears at the bottom! Giddings predicted that carbon dioxide was as polar (as strong a solvent) as 2-propanol. It is NOT. Carbon dioxide is a much weaker solvent than Giddings predicted, as shown on the bottom of the scale at the right.
performance liquid chromatography (HPLC) and SFC would be on an equal footing in terms of users and instruments sold. Curiously, this elutrophic series was never challenged, causing serious misconceptions for more than 30 years. A modern elutrophic series is compared with Giddings in Figure 3, showing carbon dioxide is more like pentane in solvent polarity. This means that to significantly change polarity one must mix a low-polarity fluid (such as carbon dioxide) with a higher polarity fluid (such as methanol). Sie and Rijnders were the first use the name ‘SFC’ in 1967. Sie, Beersum, and Rjinders introduced the flame ionization detector (FID) to SFC in 1966, and Sie and Rjinders introduced the ultraviolet (UV) detector in 1967. Jentoff and Gouw demonstrated pressure programming in 1978. Gere and coworkers published a series of papers in 1979 and 1980, on composition programming, and the use of small particle diameter packed columns, which included designs to modify a commercial HPLC to an SFC.
II CHROMATOGRAPHY: SUPERCRITICAL FLUID / Historical Development 3
Caude’s group in Paris published the first Chiral separations by packed column SFC in 1985. Today, this is one of the most important applications. The first international symposium on SFC supercritical fluid extraction (SFE) was held in Pittsburg in 1987. With a change in organizers it has continued to the present. In 1991 the first official standard method, ASTM 5186-91, based on SFC was introduced. The group separation of aromatics from paraffins and olefins in diesel fuel replaced the old fluorescence indicator analysis (FIA), and allowed quantitation below 1% aromatics. It was later followed by olefins in gasoline standard method. Both were based on early work by Rawdon and Norris at Texaco in the late 1970s and early 1980s. Controversies over Packed Columns
Owing to the introduction of commercial capillary SFC instrumentation in 1985, most of the packed column effort in the late 1980s involved attempts to emulate it by ‘deactivating’ the silica support to allow use of pure carbon dioxide with pressure programming. Most workers wanted to avoid binary mixtures, in part because the elutrophic series of Giddings had never been publicly challenged, and many still thought carbon dioxide was as polar as isopropanol. It was universally believed that modifiers, such as methanol, simply increased the density of the mixture and did not noticeably change polarity. Berger and Deye measured the density of carbon dioxide–methanol mixtures and proved that even at constant density, changing the modifier concentration had a major effect on retention. Later, they used solvatochromic dyes to prove that carbon dioxide was nonpolar (meaning the Giddings elutrophic series was wrong), and that modifiers dramatically increased the polarity of binary mixtures. These findings led to renewed interest in packed columns with composition programming. Even with the use of modifiers, many polar solutes failed to elute, or eluted with poor peak shapes from the packed columns available in the late 1980s. Berger and Deye, along with Taylor’s group at Virginia Tech, published the first use of additives in the mobile phase. Additives are very polar substances, such as strong acids and bases, added to the modifier in small concentrations. During the 1990s, the emphasis of packed column SFC shifted increasingly toward small drug-like molecules, where additives were required. Additives and modifiers transformed SFC from a largely lipophylic technique to a small, polar molecule technique.
Deactivation schemes never produced columns that could elute polar test mixes using pure carbon dioxide as the mobile phase. Modifiers and additives were necessary to elute polar solutes. However, in the early 2000s, Princeton Chromatography developed a series of column packings that significantly decreased the need for additives. This was a remarkable achievement, given the great deal of activity followed by lack of success of the 1980s attempts at deactivation. In 1971, Milos Novotny had proposed that the pressure gradients in packed columns prevented generation of high efficiencies. Shortly thereafter, L.B. (Buck) Rodgers appeared to corroborate Novotny’s findings. By the late 1980s, several competing theories had emerged that claimed SFC could not generate more than 20 000 plates if pressure drops were 420 bar. In rebuttal, Berger and Wilson demonstrated packed column separations with 42 20 000 plates, using 11 columns, each 20 cm long, packed with 5 mm particles connected in series, with a pressure drop approaching 250 bar. This ended the controversy, and pointed out the need for careful instrumental design for controlling the column outlet pressure.
Capillary SFC Although Golay predicted, and Desty had demonstrated spectacular capillary GC separations in the 1960s, all such work involved nonpolar solutes. Polar solutes tended to tail badly, severely degrading efficiency. Glass columns appeared to be somewhat more ‘inert’ than metal capillaries, but were notoriously fragile. The introduction of fused-silica tubing in 1979 caused a revolution in GC because the material was both highly flexible (you could tie small knots!) and incredibly inert. Until the late 1970s, GC stationary phases were not ‘bonded’ and tended to ‘bleed’ into the carrier gas. Passing a solvent through such columns washed off the phase. Milton Lee and Novotny were major contributors to bonded-phase chemistry for GC columns. With these two innovations (fused silica and bonding), Novotny and Lee were able to publish the first description of capillary SFC in 1981. They used a syringe pump to program pressure columns 50 mm i.d. 10 m long with bonded stationary phases, an FID, and a fixed restrictor to limit flow. With the introduction of commercial capillary SFC equipment in 1985 or 1986, and the still accepted Giddings’ elutrophic series, most workers abandoned packed column SFC. Patent disputes led some to try
4 II CHROMATOGRAPHY: SUPERCRITICAL FLUID / Historical Development
to develop deactivated micropacked columns to emulate capillary columns.
Developments in the Use of Fluids Many fluids have been tried in SFC, searching for a ‘magic bullet’ (probably nonexistant!), a fluid with modest critical temperature and pressure but highly polar, capable of eluting polar solutes without modifiers or additives. Lovelock’s early proposal using liquified inorganic gases such as sulfur dioxide has been unsuccessful. The first SFC separation by Klesper used a heated chlorofluorocarbon as the mobile phase. Comparison studies showed that the freons/actons F-12 and F-116 were less polar than even carbon dioxide. F-23 (fluoroform) was slightly more polar than carbon dioxide but still required a modifier to elute even moderately polar solutes. Chlorofluorocarbons have been banned owing to their ozone depletion potential. Several replacement fluorocarbons, especially F134a (the automobile refrigerant), have recently shown potential for extending the polarity range available with a pure fluid. However, they still require the addition of modifier, are more expensive, and raise some concerns about buildup in the atmosphere. In the late 1960s, Giddings reported the separation of amino acids and other highly polar solutes using ammonia as the mobile phase. Others have been unable to repeat this work, leading some to conclude that the ammonia Giddings used contained significant water. The extreme sensitivity of some people to traces of ammonia make it impractical as a mobile phase for routine analysis or preparative separations. In the early 1980s, Klesper and coworkers used supercritical pentane either neat, or mixed with other organic solvents such as isopropanol, and 1,4dioxane at 42351C and relatively high pressures. Polymers with molecular weights 41 000 000 were eluted. However, because of the flammability and the temperature requirement today, they are rarely used. Sulfur hexafluoride has been used as a mobile phase, particularly in the study of hydrocarbons. Its main utility is its compatibility with the FID, although a combustion product is hydrofluoric acid. Fluorocarbons exhibit the same problem. Nitrous oxide has polarity characteristics similar to carbon dioxide but is also an extreme oxidizer. It should not be mixed with fuels such as organic modifiers (or even high concentrations of sample). There have been several explosions owing to the use of nitrous oxide in SFC and SFE. Nitrous oxide has little to offer compared with carbon dioxide except
for the absence of carbon–oxygen bonds, which may be helpful in certain spectroscopic methods. The use of critical and ‘near-critical’ water should be noted. Programming the temperature of water from room temperature to above 3001C results in significant changes in its polarity, which has been exploited chromatographically. While some have attempted to call this by other names, it is essentially a version of SFC. The system requires a backpressure regulator since the temperatures are above the boiling point at atmospheric pressure. Diffusion is enhanced, viscosity decreases, and retention becomes a function of temperature and pressure. Unfortunately, many compounds cannot withstand the temperatures required and decompose. Carbon dioxide remains the fluid of choice for SFC. It has a modest critical pressure and temperature, is inexpensive, readily available, safe, transparent o185 nm, compatible with the FID, and readily mixes with a wide range of solvents.
Infrastructure Development The infrastructure required to deliver carbon dioxide to chromatographs has always caused problems. The large syringe pumps used in capillary SFC provide an interesting example. If one rapidly draws fluid out of a cylinder through an eductor tube or ‘dip’ tube even a small pressure drop can result in the formation of gas bubbles in the liquid. Worse, the pump temperature can be higher than the cylinder temperature causing flash vaporization. It was not uncommon for 80–90% of the pump cylinder to be filled with gaseous carbon dioxide. To deliver fluid to the column, this gas had to be recompressed to the working pressure, which usually took considerable time and wasted most of the pump stroke length. Most manufacturers put cooling jackets on their syringe pumps, but this did not completely solve the problem. An apparent solution was the addition of 1200– 1500 psi of helium to the cylinders. This higher pressure assured that the fluid emerging from the dip tube was a liquid. Much later, Taylor and Schweighardt demonstrated that the helium partially dissolved in the carbon dioxide and actually changed its polarity. As the cylinder was used up, concentration in the liquid phase changed, and the retention drifted. Since the addition of helium increased the cost substantially, and actually degraded performance, padded tanks were largely abandoned. In the early 1980s, Gere published work where he made binary mixtures by pipetting known volumes of organic solvents into a high-pressure bomb; he then added a desired weight of fluid to compare the
II CHROMATOGRAPHY: SUPERCRITICAL FLUID / Historical Development 5
chromatographic result with mixtures generated with two high-pressure pumps. After 1990s, there was a general acceptance that modifiers did indeed change solvent strength, but most equipment in use consisted of syringe pumps operated as pressure sources. With such pumps it is impossible to generate binary mixtures or gradients (even using two pumps). Gere’s idea of premixed fluids was revived to allow the use of syringe pumps with binary mixtures. Gas supply companies began delivering premixed cylinders. However, Taylor and Schweighardt also showed that as the cylinder was used up, composition changed, and retention could change more than a factor of 2. Premixed cylinders have been abandoned in SFC but remain in use in SFE. The largest shift in gas usage over the last few years has been the introduction of gas delivery systems specifically designed for SFC. Prior to that time, gas companies sold ‘SFC-grade carbon dioxide’ in small aluminum cylinders that held B14 kg of usable carbon dioxide. The price for such cylinders was in the $200 range and was designed to be equivalent to the cost of the most expensive organic solvents (B$14L 1). Gas delivery systems can take the 15–20 bar vapor phase from a Dewar or a large bulk tank and boost the pressure while chilling the fluid for delivery to pumps. Such systems have a number of advantages: first, the fluid is distilled just before use. Only contaminants with a significant vapor pressure at 51C will distill across. The system becomes part of the infrastructure, decreasing the downtime and labor needed to keep the chromatograph operating. Lastly, the cost plummets from B$14L 1 to as little as $0.10 L 1. Many companies are installing gas delivery systems and plumbing labs to accommodate large numbers of SFCs. Commercialization
Gere’s series of papers, around 1981, drew widespread attention to SFC and resulted in Hewlett Packard introducing the first commercial instrument in 1982. It was withdrawn in 1985 or 1986 when the 1084 HPLC it was based on was replaced by the incompatible1090. With the introduction of commercial capillary SFC equipment in 1985 or 1986, and the still accepted Giddings elutrophic series, most workers abandoned packed column SFC. Patent disputes led some to try to develop micropacked columns. In 1992, several manufacturers introduced instruments primarily designed to perform packed column SFC using multiple reciprocating pumps capable of
composition programming and electronic backpressure regulators. In the 1990s, it became clear that carbon dioxide was nonpolar and incapable of dissolving polar solutes without modifiers. Syringe pumps were not compatible with binary fluids. Further, it was shown that 50 mm diameter capillaries are at least 25 times slower than 5 mm particles at the same efficiency. The last, purely capillary, commercial SFC instruments were withdrawn from the market in the mid-late 1990s. Some capillary applications, such as ethoxylates, proproxylates, isocyanates, and silicone oils were successfully transferred to packed columns. Unfortunately, others remain best suited to capillary columns. Semipreparative Developments
Klesper performed fraction collection in 1961 and Perrut demonstrated large-scale SFC using pure carbon dioxide, plus patented recycling the mobile phase in 1982. However, the development of semipreparative scale collections (0.46–2 cm i.d. columns) has been anything but smooth. During the mid-1980s, the understanding of the parameters needed for adequate fraction collection was either inadequate or incorrect. The pressure drop through the backpressure regulator results in a 500-fold increase in volume. If the modifier concentration is high enough (i.e., 3%, v/v for methanol), liquid droplets form and are entrained in a high-speed gas stream. However, attempts to simply let modifier collect in a test tube or bottle generally result in a major loss of solute owing to aerosol formation. It became clear that either a means had to be found to not make aerosols, or the aerosols needed to be trapped after generation. One approach to trap aerosols was to bubble the effluent through a column of solvent. However, one could often see the fog of an aerosol inside the bubbles. When the bubbles reached the surface, they burst and released the aerosol into the headspace. Some solutes could be trapped in this way. Particle beds will trap aerosols but require significant solvent to flush them off the bed. If modifiers were present, one needed to insure liquid modifier did not buildup in the trap and cause loss of sample owing to breakthrough. Further, each fraction collected required a separate trap. Jasco built a combined SFE–SFE and started selling it in 1984/1985. It suffered from several drawbacks in pumping and pressure control and tended to lose part of the sample as aerosol. Starting in 1992, Gilson sold a similar HPLC/analytical/ semipreparative SFC (20 mL min 1 max) but it also
6 II CHROMATOGRAPHY: SUPERCRITICAL FLUID / Historical Development
had problems with reproducible pumping and aerosol generation, which resulted in the loss of solute. It was withdrawn in the early 2000s. Prochrom/NovaSep and Thar both started selling semipreparative SFCs with 50 mL min 1 pumping systems and cyclone separators in the early to mid1990s. In 2000, Berger Instruments introduced a new kind of separator that did not form aerosols and allowed each injection to be a different sample without operator intervention. Such performance is particularly useful in library purifications. This device has helped blur the lines between the laboratory and pilot plant scale facilities. Today it is becoming common to purify grams per hour, and, in a few extreme cases, grams per minute on 20– 50 mm diameter columns. Ethyl esters of Omega-3 fatty acids have been commercially separated from cold water fish oils by packed column SFC since around 1993. The capacity had reached 35 metric tons per year in one company by 1998. The process uses pure carbon dioxide as the mobile phase with columns up to 1 m in diameter. The chromatographs were assembled from commercially available components. Today, almost all major pharmaceutical manufacturers use semipreparative SFC. The low viscosity of carbon dioxide mixtures is also changing the perceptions of column manufacturers and users. It has been repeatedly demonstrated that an SFC can employ 5 mm particles even with columns of 30– 50 mm internal diameter, dramatically increasing the throughput. Recent developments in HPLC toward much higher pressure and smaller particle sizes means that there is a less compelling case for SFC for high-speed analytical work. However, these HPLC developments do not scale well. With the emergence of the infrastructure needed for semipreparative and preparative SFC, it is becoming clear that on these larger scales, SFC is much faster and much less expensive than HPLC.
Future Developments If it is not already, SFC will be the technique of choice for chiral separations at both the analytical and semipreparative scale. There appears to be a renewing interest in study of the fluids, particularly in the role of additives, and development of new stationary phases. Semipreparative SFC is growing rapidly and this trend should continue. Dramatically higher throughput and much lower operating costs suggests that
SFC is likely to displace HPLC from most preparative applications, at least up to the ton per year scale. The low cost of carbon dioxide changes the dynamics of process optimization in drug development. There are very few production scale SFCs at present and the few that exist use pure carbon dioxide and perform batch separations on complex samples. Production-scale HPLC equipment generally involves very large columns (i.e., 0.6–1.1 m diameter) operated as part of a simulated moving bed (SMB). While there have been at least two small SFC-SMBs built, including one by Johannsen and coworkers in Hamburg, no large-scale SFC-SMB capable of significant production has ever been built. At the analytical scale, cost is a less-compelling argument. Recent improvements in HPLC suggest that most users are likely to stay with it for highspeed analysis, although technologically, it is easier to reach very high speed in SFC. SFC manufacturers are at a major disadvantage in that their present market is tiny compared with HPLC. They must make much higher relative investment to remain competitive with HPLC. A trend toward using supercriticnal and nearcritical fluids as a reaction solvent is likely to enhance the utility of SFC in the future. See also: III/Chromatography: Supercritical Fluid: Instrumentation; Large-Scale Supercritical Fluid Chromatography. III/Preparative Supercritical Fluid Chromatography. III/Supercritical Fluid Extraction-Supercritical Fluid Chromatography.
Further Reading Berger TA (1995) Packed Column SFC, RSC Chromatography Monographs Series. Cambridge: Royal Society of Chemistry, Chapters 1–3. Gere DR (1982) Supercritical fluid chromatography. Science 222: 253–259. Giddings JC, Manwaring WA and Myers MN (1966) Science 154: 146. Jannsen HG and Lou X (1999) Packed columns in SFC: Mobile and stationary phases and requirements. In: Caude M and Thiebaut D (ed.) Practical Supercritical Fluid Chromatography and Extraction, Chapter 2. Harwood Academic Press, Amsterdam. Bartle KD (1988) Theory and principles of supercritical fluid chromatography. In: Smith RM (ed.) Supercritical Fluid Chromatography, Chapter 1. RSC Chromatography Monographs Series Royal Society of Chemistry. Lee ML and Markides KE (eds) (1990) Analytical Supercritical Fluid Chromatography and Extraction, Chapter 1. Chromatography Conferences, Inc. Provo, UT.
Discovery of Supercritical Phenomena The characteristics associated with ‘supercritical’ fluids were first observed by Baron Cagniard de la Tour in 1822, when he recognized that a liquid could be converted into a gas without a phase transition, if the liquid were heated above a specific temperature. Nearly 50 years later (1869), Andrews further developed the idea of a critical point (a ‘critical temperature’ T c and a ‘critical pressure’ Pc) above which only a single phase existed. In this context, the word ‘super’ is only intended to indicate ‘above.’ A schematic of a phase diagram helps in explaining this phenomenon, as shown in Figure 1. In the figure, a line emerges from the lower left-hand corner, separating solids from gases. Crossing the line from left to right represents sublimation of the solid to a gas. At the ‘triple point,’ solid, liquid, and gas are all present. One line continues nearly vertically and
C
Critical point
Liquid A Solid
Pressure
B
E
separates the liquid and solid forms of the compound. To the left of the line only a solid exists. To the right, only a liquid exists. The line represents conditions where a solid is in equilibrium with a liquid. The other line directed diagonally toward the upper right of the figure separates the liquid form from the gas form of the compound. Above and to the left of the line only a liquid exists. Below and to the right of the line, only gas exists. Directly on the line, a gas exists in equilibrium with a liquid. This line is sometimes called the boiling line or the vapor– liquid equilibrium line (VLE). This line ends at the critical point, characteristic of each fluid and mixture of fluids. Note that above this point there is no separation of gas and liquid. Increasing the pressure of a liquid above Pc (line A–B in Figure 1), then increasing the temperature above T c (line B–C), decreasing pressure (line C–D), and the temperature (line D–E), produces a low temperature, low-pressure gas. The transition from liquid to gas occurred without an apparent phase transition (there was never a meniscus between the two phases). Moving directly from point A to point E, results in a two-phase region where part of the fluid is a gas and part is a liquid. In 1879, Hannay and Hogarth observed that ‘supercritical’ fluids could act as solvents. They dissolved inorganic salts in supercritical ethanol and re-precipitated them by decreasing the temperature. In 1906, Buchner showed that nonvolatile organic compounds were much more soluble in supercritical fluids than expected. Throughout the twentieth century, many patents appeared, particularly with respect to extraction and processing of petroleum and oils.
D
Gas Triple point Temperature Figure 1 A phase diagram indicating the temperature and pressure regions where solids, gases, and liquids exist. The dashed line indicates how a high density liquid (at point A) can be converted to a low(er) density gas by first increasing pressure (to point B), increasing temperature (to point C), decreasing pressure (to point D), and then decreasing temperature (to point E), without two phases co-existing.
Early Developments Milestones in the development of supercritical fluid chromatography (SFC) are outlined in Figure 2. At an international gas chromatography (GC) symposium, Jim Lovelock, an early pioneer proposed ‘critical-state chromatography’ to separate polar and ionic species (such as the inorganic salts of Hannay and Hogarth) using inorganic gases and capillary columns. Later in 1958, he had a letter proposing the
2 II CHROMATOGRAPHY: SUPERCRITICAL FLUID / Historical Development
1822 1961 1966 1966 1967 1969 1970 1971 1979 1981 1982 1985 1986 1988 1989 1992 1998 1999 2000
Critical point discovered First separation First chromatogram FID introduced UV−Vis introduced Binary mobile phases Pressure programming Doubts about packed cols. MS detector Capillary SFC composition gradients First commercial SFC Independent control of press, flow, comp. First chiral separation Commercial Cap. SFC First use of additives first journal (journal of supercritical fluids) Solvent density and polarity deconvoluted 2nd generation commercial hardware introduced highefficiency-packed columns Last commercial SFC withdrawn Emphasis shifts toward semi-prep/pharma Gas delivery systems change economics
Figure 2 Milestones in the development of SFC.
same notarized. He never attempted to confirm his predictions experimentally. Ernst Klesper separated porphorins with a supercritical chlorofluorocarbon mobile phase in 1961. The liquid solvent was heated in a tube in a sand bath, connected to a heated packed column. The fluid was then cooled back to room temperature before passing through a fixed restrictor. Fractions were collected for off-line analysis. The first supercritical fluid chromatograms with an inline detector were made in 1966. Klesper later produced a series of papers mostly about the separation of polymers using liquids such as pentane and mixtures, like pentane/dioxane, heated to high temperatures (such as 2601C) in a sand bath. Calvin Giddings dominated the theoretical development of what he called ‘dense gas chromatography’ throughout the later 1960s. In a 1966 article in Science, Giddings proposed an elutrophic series in which carbon dioxide was placed next to isopropanol in solvent polarity. The numbers were based on estimated constants used to calculate Hildebrand solubility parameters. If true, changes in pressure would cause huge changes in polarity of carbon dioxide. Unfortunately, it is not true. Had it been true, programming a physical parameter (pressure) would have changed the polarity of the solvent from hydrocarbon-like to alcohol-like. Programming a physical parameter is much less expensive and complicated than mixing compressible and incompressible fluids. If the fluids had followed Giddings elutrophic series, it is likely that, today, high-
Giddings series
Nile Red solvent strength Methanol Ethanol
Methanol
NH4
Pyridine 2-Propanol 40% MeOH
Ethanol
Acetonitrile
2-Propanol
Ethyl acetate CO2
20% MeOH 10% MeOH
Ethyl acetate Diethyl ether CCl4
Pentane
CCl4
Pentane CO2
Figure 3 Comparison of Giddings ‘‘elutrophic series’’ and a measured solvent strength scale. Giddings scale attempted to predict the strength of solvents in eluting compounds from a silica column using estimates which have since proven inaccurate. The higher the solvent appears on the scale, the less retained a compound should be. Note that carbon dioxide appears half-way up Giddings series, while on the right it appears at the bottom! Giddings predicted that carbon dioxide was as polar (as strong a solvent) as 2-propanol. It is NOT. Carbon dioxide is a much weaker solvent than Giddings predicted, as shown on the bottom of the scale at the right.
performance liquid chromatography (HPLC) and SFC would be on an equal footing in terms of users and instruments sold. Curiously, this elutrophic series was never challenged, causing serious misconceptions for more than 30 years. A modern elutrophic series is compared with Giddings in Figure 3, showing carbon dioxide is more like pentane in solvent polarity. This means that to significantly change polarity one must mix a low-polarity fluid (such as carbon dioxide) with a higher polarity fluid (such as methanol). Sie and Rijnders were the first use the name ‘SFC’ in 1967. Sie, Beersum, and Rjinders introduced the flame ionization detector (FID) to SFC in 1966, and Sie and Rjinders introduced the ultraviolet (UV) detector in 1967. Jentoff and Gouw demonstrated pressure programming in 1978. Gere and coworkers published a series of papers in 1979 and 1980, on composition programming, and the use of small particle diameter packed columns, which included designs to modify a commercial HPLC to an SFC.
II CHROMATOGRAPHY: SUPERCRITICAL FLUID / Historical Development 3
Caude’s group in Paris published the first Chiral separations by packed column SFC in 1985. Today, this is one of the most important applications. The first international symposium on SFC supercritical fluid extraction (SFE) was held in Pittsburg in 1987. With a change in organizers it has continued to the present. In 1991 the first official standard method, ASTM 5186-91, based on SFC was introduced. The group separation of aromatics from paraffins and olefins in diesel fuel replaced the old fluorescence indicator analysis (FIA), and allowed quantitation below 1% aromatics. It was later followed by olefins in gasoline standard method. Both were based on early work by Rawdon and Norris at Texaco in the late 1970s and early 1980s. Controversies over Packed Columns
Owing to the introduction of commercial capillary SFC instrumentation in 1985, most of the packed column effort in the late 1980s involved attempts to emulate it by ‘deactivating’ the silica support to allow use of pure carbon dioxide with pressure programming. Most workers wanted to avoid binary mixtures, in part because the elutrophic series of Giddings had never been publicly challenged, and many still thought carbon dioxide was as polar as isopropanol. It was universally believed that modifiers, such as methanol, simply increased the density of the mixture and did not noticeably change polarity. Berger and Deye measured the density of carbon dioxide–methanol mixtures and proved that even at constant density, changing the modifier concentration had a major effect on retention. Later, they used solvatochromic dyes to prove that carbon dioxide was nonpolar (meaning the Giddings elutrophic series was wrong), and that modifiers dramatically increased the polarity of binary mixtures. These findings led to renewed interest in packed columns with composition programming. Even with the use of modifiers, many polar solutes failed to elute, or eluted with poor peak shapes from the packed columns available in the late 1980s. Berger and Deye, along with Taylor’s group at Virginia Tech, published the first use of additives in the mobile phase. Additives are very polar substances, such as strong acids and bases, added to the modifier in small concentrations. During the 1990s, the emphasis of packed column SFC shifted increasingly toward small drug-like molecules, where additives were required. Additives and modifiers transformed SFC from a largely lipophylic technique to a small, polar molecule technique.
Deactivation schemes never produced columns that could elute polar test mixes using pure carbon dioxide as the mobile phase. Modifiers and additives were necessary to elute polar solutes. However, in the early 2000s, Princeton Chromatography developed a series of column packings that significantly decreased the need for additives. This was a remarkable achievement, given the great deal of activity followed by lack of success of the 1980s attempts at deactivation. In 1971, Milos Novotny had proposed that the pressure gradients in packed columns prevented generation of high efficiencies. Shortly thereafter, L.B. (Buck) Rodgers appeared to corroborate Novotny’s findings. By the late 1980s, several competing theories had emerged that claimed SFC could not generate more than 20 000 plates if pressure drops were 420 bar. In rebuttal, Berger and Wilson demonstrated packed column separations with 42 20 000 plates, using 11 columns, each 20 cm long, packed with 5 mm particles connected in series, with a pressure drop approaching 250 bar. This ended the controversy, and pointed out the need for careful instrumental design for controlling the column outlet pressure.
Capillary SFC Although Golay predicted, and Desty had demonstrated spectacular capillary GC separations in the 1960s, all such work involved nonpolar solutes. Polar solutes tended to tail badly, severely degrading efficiency. Glass columns appeared to be somewhat more ‘inert’ than metal capillaries, but were notoriously fragile. The introduction of fused-silica tubing in 1979 caused a revolution in GC because the material was both highly flexible (you could tie small knots!) and incredibly inert. Until the late 1970s, GC stationary phases were not ‘bonded’ and tended to ‘bleed’ into the carrier gas. Passing a solvent through such columns washed off the phase. Milton Lee and Novotny were major contributors to bonded-phase chemistry for GC columns. With these two innovations (fused silica and bonding), Novotny and Lee were able to publish the first description of capillary SFC in 1981. They used a syringe pump to program pressure columns 50 mm i.d. 10 m long with bonded stationary phases, an FID, and a fixed restrictor to limit flow. With the introduction of commercial capillary SFC equipment in 1985 or 1986, and the still accepted Giddings’ elutrophic series, most workers abandoned packed column SFC. Patent disputes led some to try
4 II CHROMATOGRAPHY: SUPERCRITICAL FLUID / Historical Development
to develop deactivated micropacked columns to emulate capillary columns.
Developments in the Use of Fluids Many fluids have been tried in SFC, searching for a ‘magic bullet’ (probably nonexistant!), a fluid with modest critical temperature and pressure but highly polar, capable of eluting polar solutes without modifiers or additives. Lovelock’s early proposal using liquified inorganic gases such as sulfur dioxide has been unsuccessful. The first SFC separation by Klesper used a heated chlorofluorocarbon as the mobile phase. Comparison studies showed that the freons/actons F-12 and F-116 were less polar than even carbon dioxide. F-23 (fluoroform) was slightly more polar than carbon dioxide but still required a modifier to elute even moderately polar solutes. Chlorofluorocarbons have been banned owing to their ozone depletion potential. Several replacement fluorocarbons, especially F134a (the automobile refrigerant), have recently shown potential for extending the polarity range available with a pure fluid. However, they still require the addition of modifier, are more expensive, and raise some concerns about buildup in the atmosphere. In the late 1960s, Giddings reported the separation of amino acids and other highly polar solutes using ammonia as the mobile phase. Others have been unable to repeat this work, leading some to conclude that the ammonia Giddings used contained significant water. The extreme sensitivity of some people to traces of ammonia make it impractical as a mobile phase for routine analysis or preparative separations. In the early 1980s, Klesper and coworkers used supercritical pentane either neat, or mixed with other organic solvents such as isopropanol, and 1,4dioxane at 42351C and relatively high pressures. Polymers with molecular weights 41 000 000 were eluted. However, because of the flammability and the temperature requirement today, they are rarely used. Sulfur hexafluoride has been used as a mobile phase, particularly in the study of hydrocarbons. Its main utility is its compatibility with the FID, although a combustion product is hydrofluoric acid. Fluorocarbons exhibit the same problem. Nitrous oxide has polarity characteristics similar to carbon dioxide but is also an extreme oxidizer. It should not be mixed with fuels such as organic modifiers (or even high concentrations of sample). There have been several explosions owing to the use of nitrous oxide in SFC and SFE. Nitrous oxide has little to offer compared with carbon dioxide except
for the absence of carbon–oxygen bonds, which may be helpful in certain spectroscopic methods. The use of critical and ‘near-critical’ water should be noted. Programming the temperature of water from room temperature to above 3001C results in significant changes in its polarity, which has been exploited chromatographically. While some have attempted to call this by other names, it is essentially a version of SFC. The system requires a backpressure regulator since the temperatures are above the boiling point at atmospheric pressure. Diffusion is enhanced, viscosity decreases, and retention becomes a function of temperature and pressure. Unfortunately, many compounds cannot withstand the temperatures required and decompose. Carbon dioxide remains the fluid of choice for SFC. It has a modest critical pressure and temperature, is inexpensive, readily available, safe, transparent o185 nm, compatible with the FID, and readily mixes with a wide range of solvents.
Infrastructure Development The infrastructure required to deliver carbon dioxide to chromatographs has always caused problems. The large syringe pumps used in capillary SFC provide an interesting example. If one rapidly draws fluid out of a cylinder through an eductor tube or ‘dip’ tube even a small pressure drop can result in the formation of gas bubbles in the liquid. Worse, the pump temperature can be higher than the cylinder temperature causing flash vaporization. It was not uncommon for 80–90% of the pump cylinder to be filled with gaseous carbon dioxide. To deliver fluid to the column, this gas had to be recompressed to the working pressure, which usually took considerable time and wasted most of the pump stroke length. Most manufacturers put cooling jackets on their syringe pumps, but this did not completely solve the problem. An apparent solution was the addition of 1200– 1500 psi of helium to the cylinders. This higher pressure assured that the fluid emerging from the dip tube was a liquid. Much later, Taylor and Schweighardt demonstrated that the helium partially dissolved in the carbon dioxide and actually changed its polarity. As the cylinder was used up, concentration in the liquid phase changed, and the retention drifted. Since the addition of helium increased the cost substantially, and actually degraded performance, padded tanks were largely abandoned. In the early 1980s, Gere published work where he made binary mixtures by pipetting known volumes of organic solvents into a high-pressure bomb; he then added a desired weight of fluid to compare the
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chromatographic result with mixtures generated with two high-pressure pumps. After 1990s, there was a general acceptance that modifiers did indeed change solvent strength, but most equipment in use consisted of syringe pumps operated as pressure sources. With such pumps it is impossible to generate binary mixtures or gradients (even using two pumps). Gere’s idea of premixed fluids was revived to allow the use of syringe pumps with binary mixtures. Gas supply companies began delivering premixed cylinders. However, Taylor and Schweighardt also showed that as the cylinder was used up, composition changed, and retention could change more than a factor of 2. Premixed cylinders have been abandoned in SFC but remain in use in SFE. The largest shift in gas usage over the last few years has been the introduction of gas delivery systems specifically designed for SFC. Prior to that time, gas companies sold ‘SFC-grade carbon dioxide’ in small aluminum cylinders that held B14 kg of usable carbon dioxide. The price for such cylinders was in the $200 range and was designed to be equivalent to the cost of the most expensive organic solvents (B$14L 1). Gas delivery systems can take the 15–20 bar vapor phase from a Dewar or a large bulk tank and boost the pressure while chilling the fluid for delivery to pumps. Such systems have a number of advantages: first, the fluid is distilled just before use. Only contaminants with a significant vapor pressure at 51C will distill across. The system becomes part of the infrastructure, decreasing the downtime and labor needed to keep the chromatograph operating. Lastly, the cost plummets from B$14L 1 to as little as $0.10 L 1. Many companies are installing gas delivery systems and plumbing labs to accommodate large numbers of SFCs. Commercialization
Gere’s series of papers, around 1981, drew widespread attention to SFC and resulted in Hewlett Packard introducing the first commercial instrument in 1982. It was withdrawn in 1985 or 1986 when the 1084 HPLC it was based on was replaced by the incompatible1090. With the introduction of commercial capillary SFC equipment in 1985 or 1986, and the still accepted Giddings elutrophic series, most workers abandoned packed column SFC. Patent disputes led some to try to develop micropacked columns. In 1992, several manufacturers introduced instruments primarily designed to perform packed column SFC using multiple reciprocating pumps capable of
composition programming and electronic backpressure regulators. In the 1990s, it became clear that carbon dioxide was nonpolar and incapable of dissolving polar solutes without modifiers. Syringe pumps were not compatible with binary fluids. Further, it was shown that 50 mm diameter capillaries are at least 25 times slower than 5 mm particles at the same efficiency. The last, purely capillary, commercial SFC instruments were withdrawn from the market in the mid-late 1990s. Some capillary applications, such as ethoxylates, proproxylates, isocyanates, and silicone oils were successfully transferred to packed columns. Unfortunately, others remain best suited to capillary columns. Semipreparative Developments
Klesper performed fraction collection in 1961 and Perrut demonstrated large-scale SFC using pure carbon dioxide, plus patented recycling the mobile phase in 1982. However, the development of semipreparative scale collections (0.46–2 cm i.d. columns) has been anything but smooth. During the mid-1980s, the understanding of the parameters needed for adequate fraction collection was either inadequate or incorrect. The pressure drop through the backpressure regulator results in a 500-fold increase in volume. If the modifier concentration is high enough (i.e., 3%, v/v for methanol), liquid droplets form and are entrained in a high-speed gas stream. However, attempts to simply let modifier collect in a test tube or bottle generally result in a major loss of solute owing to aerosol formation. It became clear that either a means had to be found to not make aerosols, or the aerosols needed to be trapped after generation. One approach to trap aerosols was to bubble the effluent through a column of solvent. However, one could often see the fog of an aerosol inside the bubbles. When the bubbles reached the surface, they burst and released the aerosol into the headspace. Some solutes could be trapped in this way. Particle beds will trap aerosols but require significant solvent to flush them off the bed. If modifiers were present, one needed to insure liquid modifier did not buildup in the trap and cause loss of sample owing to breakthrough. Further, each fraction collected required a separate trap. Jasco built a combined SFE–SFE and started selling it in 1984/1985. It suffered from several drawbacks in pumping and pressure control and tended to lose part of the sample as aerosol. Starting in 1992, Gilson sold a similar HPLC/analytical/ semipreparative SFC (20 mL min 1 max) but it also
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had problems with reproducible pumping and aerosol generation, which resulted in the loss of solute. It was withdrawn in the early 2000s. Prochrom/NovaSep and Thar both started selling semipreparative SFCs with 50 mL min 1 pumping systems and cyclone separators in the early to mid1990s. In 2000, Berger Instruments introduced a new kind of separator that did not form aerosols and allowed each injection to be a different sample without operator intervention. Such performance is particularly useful in library purifications. This device has helped blur the lines between the laboratory and pilot plant scale facilities. Today it is becoming common to purify grams per hour, and, in a few extreme cases, grams per minute on 20– 50 mm diameter columns. Ethyl esters of Omega-3 fatty acids have been commercially separated from cold water fish oils by packed column SFC since around 1993. The capacity had reached 35 metric tons per year in one company by 1998. The process uses pure carbon dioxide as the mobile phase with columns up to 1 m in diameter. The chromatographs were assembled from commercially available components. Today, almost all major pharmaceutical manufacturers use semipreparative SFC. The low viscosity of carbon dioxide mixtures is also changing the perceptions of column manufacturers and users. It has been repeatedly demonstrated that an SFC can employ 5 mm particles even with columns of 30– 50 mm internal diameter, dramatically increasing the throughput. Recent developments in HPLC toward much higher pressure and smaller particle sizes means that there is a less compelling case for SFC for high-speed analytical work. However, these HPLC developments do not scale well. With the emergence of the infrastructure needed for semipreparative and preparative SFC, it is becoming clear that on these larger scales, SFC is much faster and much less expensive than HPLC.
Future Developments If it is not already, SFC will be the technique of choice for chiral separations at both the analytical and semipreparative scale. There appears to be a renewing interest in study of the fluids, particularly in the role of additives, and development of new stationary phases. Semipreparative SFC is growing rapidly and this trend should continue. Dramatically higher throughput and much lower operating costs suggests that
SFC is likely to displace HPLC from most preparative applications, at least up to the ton per year scale. The low cost of carbon dioxide changes the dynamics of process optimization in drug development. There are very few production scale SFCs at present and the few that exist use pure carbon dioxide and perform batch separations on complex samples. Production-scale HPLC equipment generally involves very large columns (i.e., 0.6–1.1 m diameter) operated as part of a simulated moving bed (SMB). While there have been at least two small SFC-SMBs built, including one by Johannsen and coworkers in Hamburg, no large-scale SFC-SMB capable of significant production has ever been built. At the analytical scale, cost is a less-compelling argument. Recent improvements in HPLC suggest that most users are likely to stay with it for highspeed analysis, although technologically, it is easier to reach very high speed in SFC. SFC manufacturers are at a major disadvantage in that their present market is tiny compared with HPLC. They must make much higher relative investment to remain competitive with HPLC. A trend toward using supercriticnal and nearcritical fluids as a reaction solvent is likely to enhance the utility of SFC in the future. See also: III/Chromatography: Supercritical Fluid: Instrumentation; Large-Scale Supercritical Fluid Chromatography. III/Preparative Supercritical Fluid Chromatography. III/Supercritical Fluid Extraction-Supercritical Fluid Chromatography.
Further Reading Berger TA (1995) Packed Column SFC, RSC Chromatography Monographs Series. Cambridge: Royal Society of Chemistry, Chapters 1–3. Gere DR (1982) Supercritical fluid chromatography. Science 222: 253–259. Giddings JC, Manwaring WA and Myers MN (1966) Science 154: 146. Jannsen HG and Lou X (1999) Packed columns in SFC: Mobile and stationary phases and requirements. In: Caude M and Thiebaut D (ed.) Practical Supercritical Fluid Chromatography and Extraction, Chapter 2. Harwood Academic Press, Amsterdam. Bartle KD (1988) Theory and principles of supercritical fluid chromatography. In: Smith RM (ed.) Supercritical Fluid Chromatography, Chapter 1. RSC Chromatography Monographs Series Royal Society of Chemistry. Lee ML and Markides KE (eds) (1990) Analytical Supercritical Fluid Chromatography and Extraction, Chapter 1. Chromatography Conferences, Inc. Provo, UT.