ANALYTICAL-SCALE
SUPERCRITICAL FLUID EXTRACTION
Steven B. Hawthorne University of North Dakota Energy and Environmental Research Center Grand Forks, ND 58202
Since the development of the transistor in 1948, analytical research has led to tremendous advances in instrumental techniques for organic analysis, particularly in the areas of chromatography and spectroscopy. Although some samples are inherently ready for analysis by these techniques, most require extraction of the analytes into a liquid solvent, especially when the analyst wishes to identify and quantitate minor or trace organic components from a bulk sample matrix. It is ironic that some modern chromatographic techniques have become so highly developed that they can separate, identify, and quantitate hundreds of sample components per hour, yet analysts often rely on sample extraction procedures—such as liquid solvent extraction in a Soxhlet apparatus—that were in use when Tswett first reported chromatography in 1906. An ideal extraction method should be rapid, simple, and inexpensive to perform; yield quantitative recovery of target analytes without loss or degra0003-2700/90/0362-633A/$02.50/0 © 1990 American Chemical Society
dation; yield a sample that is immediately ready for analysis without additional concentration or class fractionation steps; and generate no additional laboratory wastes. Unfortunately, liquid solvent extractions frequently fail to meet these goals. They often require several hours or even days to perform, result in a dilute extract (which must be concentrated when trace analysis is desired), and may not result in quantitative recovery of target analytes. Recent concerns about the hazardous nature of many commonly used sol-
REPORT vents, the costs and environmental dangers of waste solvent disposal, and the emission of hazardous solvents into the atmosphere during sample concentration further support the development of alternative sample extraction methods. The limitations of conventional methods have fueled interest in the development of supercritical fluid extraction (SFE) as an alternative to extractions using liquid solvents. This REPORT describes current techniques and applications as well as the capabilities and limitations of using supercritical fluids for the analytical-scale ex-
traction and recovery of organic analytes from complex sample matrices such as environmental solids, polymeric resins, and biological tissues. Why use supercritical fluids for sample extraction? A substance that is above its critical temperature and pressure is defined as a supercritical fluid. The combined gas-like mass transfer and liquid-like solvating characteristics of supercritical fluids have led to considerable excitement for their use as mobile phases for supercritical fluid chromatography (SFC) (1), but their use for analyticalscale extraction has only recently received attention. For example, a computer search of Chemical Abstracts listings for ANALYTICAL CHEMISTRY
and selected chromatography journals revealed only two papers on SFE prior to 1986, compared with 26 articles on this technique published from 1986 to mid-1989. Supercritical fluids are sometimes considered to be "super solvents," but this is not true when their solvating power is compared with that of liquids. The solvent strengths of supercritical fluids approach those of liquid solvents only as their density is increased, and the maximum solubility of an organic compound is frequently higher in liq-
ANALYTICAL CHEMISTRY, VOL. 62, NO. 11, JUNE 1, 1990 · 633 A
REPORT uid solvents than in supercritical flu ids. Although supercritical fluids do not have any advantage over liquid sol
vents in solvating power, comparison of several other characteristics of super critical fluids with those of liquid sol
Figure 1. Effect of temperature and pressure on the Hildebrand solubility parameter for supercritical C0 2 . Tc represents critical temperature. (Adapted from Reference 2.)
vents demonstrates the potential for SFE to approach the idealized goals for analytical extraction. • SFE is fast. Mass transfer limita tions ultimately determine the rate at which an extraction can be performed. Because supercritical fluids have sol ute diffusivities an order of magnitude higher (10~4 vs. 10 - 6 cm 2 /s) and viscosi ties an order of magnitude lower (10 - 4 vs. 10~3 Ν • s/m 2 ) than liquid solvents, they have much better mass transfer characteristics. Quantitative SFEs generally are complete in 10-60 min, whereas liquid solvent extraction times can range from several hours to days. • The solvent strength of a supercri tical fluid can easily be controlled. The solvent strength of a liquid is essential ly constant regardless of extraction conditions, but the solvent strength of a supercritical fluid depends on the pressure and temperature used for the extraction, as shown in Figure 1 by the dependence of the Hildebrand solubili ty parameter on temperature and pres sure for supercritical CO2 (2). At a con stant temperature, extraction at lower pressures will favor less polar anaiytes, and extraction at higher pressures will favor more polar and higher molecular weight anaiytes. This allows an extrac tion to be optimized for a particular compound class by simply changing the pressure (and, to a lesser extent, the temperature) of the extraction. As discussed below, this same characteris tic allows class-selective extractions to be performed by extracting a single sample at different pressures. • Many supercritical fluids are gases at ambient conditions. Liquid solvent extracts need to be concentrated prior to the determination of trace organic anaiytes, a step that requires additional time and can result in the loss of more volatile anaiytes. In contrast, because supercritical fluids are gases at ambi ent conditions, concentration steps af ter SFE are greatly simplified and di rect coupling of the SFE step to chro matographic techniques is facilitated. • Supercritical fluids have addition al practical advantages. Most are rela tively inert, pure, nontoxic, and inex pensive (on a per-extraction basis). Be cause fluids such as C 0 2 and N 2 0 have relatively low critical temperatures (31 °C and 36 °C, respectively), SFE can be performed at low temperatures to extract thermally unstable com pounds. The generation of liquid waste solvents and exposure of laboratory personnel to toxic solvents also can be reduced or eliminated.
Figure 2. Schematic of an SFE system.
Experimental considerations
The supercritical fluid (red) is pumped to the extraction vessel where the anaiytes (purple) are extracted from the sample matrix (brown). The anaiytes are then swept through the flow restrictor into the collec tion device, and the depressurized supercritical fluid (now a gas, for most fluids) is vented.
A frequent misconception is that, be cause SFE and SFC both use supercri-
634 A · ANALYTICAL CHEMISTRY, VOL. 62, NO. 11, JUNE 1, 1990
tical fluids, SFE must somehow rely on subsequent analysis using SFC tech niques. Although analysis of supercriti cal fluid extracts using SFC is an op tion, SFE is an independent sample preparation method (analogous to liq uid solvent extraction), and supercriti cal fluid extracts can be analyzed by any technique that is appropriate for the extracted analytes. In addition to chromatographic methods, supercriti cal fluid extracts are frequently ana lyzed by spectroscopic, electrochemi cal, radiochemical, gravimetric, and other techniques. SFE is conceptually simple to per form (Figure 2). A pump is used to sup ply a known pressure of the extraction fluid to the extraction vessel, which is placed in a heater to maintain the ves sel at a temperature above the critical temperature of the supercritical fluid. During the extraction, the soluble ana lytes are partitioned from the bulk sample matrix into the supercritical fluid, then swept through a flow restrictor into a collection device that is normally at ambient pressure. The flu ids used for SFE are usually gases at ambient conditions and are vented from the collection device while the ex tracted analytes are retained. Several experimental variables must be considered and optimized for SFE to be successful, including the choice of supercritical fluid, pressure and tem perature conditions, extraction time, sample size, the method used to collect the extracted analytes, and the equip ment that is needed. Each of these as pects is discussed below. Choosing supercritical fluids and extraction conditions Predicting optimal extraction con ditions. Perhaps because of the histori cal development of SFE in process en gineering, the choice of analytical SFE conditions has most often been based on the pressure and temperature con ditions where the target analytes have their highest solubility in the supercri tical fluid. This condition can be ap proximated if the solubility parameter of the analyte is known and if certain correlations are used, such as one pro posed by Giddings et al. (3):
(e.g., the extraction of fats from meat products), but because they are con cerned with maximum solubilities, they become less useful when the con centrations of the target analytes are at minor and trace levels. For such sam ples, dissolving the maximum amount of the target analyte in the supercriti cal fluid is not of concern, and the ana lytes need only be soluble enough in the supercritical fluid to be transported out of the extraction vessel. King (4) suggests combining maximum solubili ty conditions with the threshold pres sure (i.e., the pressure where the ana lyte becomes significantly soluble) to determine the range of pressures (or fluid densities) that are applicable to a particular extraction. Solubility data and correlations pro vide useful information for choosing initial SFE conditions. However, deter mining optimal extraction conditions for minor and trace components has been largely empirical, for two reasons. First, analytical SFE often involves the recovery of a complex variety of ana lytes (rather than a single target ana lyte). In such cases, extractions must be optimized for groups of compounds, which complicates the prediction of op timal extraction conditions. Second, and more importantly, solu bility considerations address only part of the extraction problem. Because ex traction of an analyte depends on its distribution between the supercritical fluid and the sorptive sites on the sam ple matrix, the ability of the supercriti cal fluid to compete with the analytes for the sorptive sites may be more im portant than solubility considerations for determining optimal extraction conditions. This is indicated by the comparative extractions using CO2 and N2O, discussed below. Unfortunately, the importance of the competition for active sites between the analytes and the supercritical fluid has received very little attention in the development of analytical SFE techniques (5).
Table 1. Characteristics of representative supercritical fluids Fluid
δ = 1.25 P\n(p/pù where δ is the Hildebrand solubility pa rameter, Pc is the critical pressure of the fluid, ρ is the density of supercriti cal fluid, and p\ is the density of the fluid in its liquid state. Such correlations are quite useful when the target analytes represent a large percentage of the bulk sample
Understanding the mechanisms and interactions among the matrix surface, the analytes, and the supercritical flu id, as well as the kinetics that control these interactions, will be important to the future design of analytical SFE methods. Suitable models of extraction mechanisms should be particularly useful for designing extractions using modified supercritical fluids, because it may be possible to select modifiers that compete best with the analytes for sorptive sites on the matrix surface as well as modifiers that increase ana lyte solubility in the supercritical fluid. Choosing a supercritical fluid. The characteristics of several fluids that have been used for SFE are listed in Table I. (Note that the Hildebrand solubility parameters listed in Table I are maximum values t h a t are ap proached at very high pressures, as demonstrated in Figure 1 for CO2. Be cause of hardware considerations, SFE is normally performed at pressures that yield lower solvent strengths.) Al though fluids with a range of polarities are available to optimize an extraction based on the polarity of the target ana lyte (6), the choice of a fluid for analyti cal SFE frequently depends on practi cal considerations. Supercritical CO2 has been the choice for most SFE studies, primarily because of attractive practical charac teristics: relatively low critical tem perature and pressure, low toxicity and reactivity, and high purity at low cost. Unfortunately, supercritical CO2 does not have sufficient solvent strength at typical working pressures (80-600 atm) to quantitatively extract analytes that are quite polar. In general terms, CO2 is an excellent extraction medium for nonpolar species such as alkanes and terpenes [δ ~ 6 - 8 (cal/cm 3 ) 1/2 ]. It is rea sonably good for moderately polar spe cies, including polycyclic aromatic hy drocarbons (PAHs), polychlorinated biphenyls (PCBs), aldehydes, esters,
Critical temperature (°C)
Critical pressure (atm)
73 31 C0 2 72 N20 36 NH3 112 132 MeOH 78 240 29 38 CCIF3 Ethane 32 48 Ethylene 10 51 a Hildebrand solubility parameter in (cal/cm3)1'2 from Reference 6.
Solubility parameter9 10.7 10.6 13.2 14.4 7.8 6.6 6.6
ANALYTICAL CHEMISTRY, VOL. 62, NO. 11, JUNE 1, 1990 · 635 A
REPORT alcohols, organochlorine pesticides, and fats (δ ~ 8 - l l ) , but is less useful for more polar compounds.
The results reported in the literature and our own experiences indicate that, as rule of thumb, compounds that can
Figure 3. Comparison of the extraction efficiencies obtained using C0 2 and N20. The PAHs, chrysene and indeno[1,2,3-cd]pyrene, were extracted for 10 min from marine sediment (S). The tetrachlorodibenzo-p-dioxins (TCDDs) were extracted for 2 h from municipal incinerator fly ash (7). TCDD extractions with C0 2 are shown before and after acid treatment of the fly ash.
Figure 4. Comparison of extraction efficiencies obtained using C0 2 and C0 2 modi fied with methanol. All extractions were performed for 30 min. The dibenzo[a,/]carbazole was extracted from XAD-2 sorbent resin (9); and the diuron, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and the linear àlkylbenzenesulfonate (LAS) detergent were each extracted from soil samples ( 10-12).
636 A · ANALYTICAL CHEMISTRY, VOL. 62, NO. 11, JUNE 1, 1990
be analyzed by conventional gas chromatographic techniques can be quantitatively extracted using supercritical COz- Some additional nonpolar organics, such as triglycerides (which have vapor pressures too low to be amenable to GC), are also easily extracted with CO2. A somewhat frustrating problem arises when fairly polar analytes need to be extracted. The first choice would be a fluid with higher solvent strength, but the use of more polar fluids is severely limited by practical considerations. Supercritical ammonia would be very attractive from a solvent strength standpoint, but it is difficult to pump (it tends to dissolve pump seals), is chemically reactive, and is likely to be too dangerous for routine use. Supercritical methanol is also an excellent solvent but is less attractive because of its high critical temperature and because it is a liquid at ambient conditions, which complicates sample concentration after extraction. For some analyte/matrix combinations, extraction efficiencies can be increased using fluids that would not be expected to yield better efficiencies based on their solubility parameters. For example, N 2 0 gives more rapid extractions of some samples than can be achieved with CO9 (7, 8) as demonstrated in Figure 3 for tetrachlorodibenzo-p-dioxins (TCDDs) and PAHs. This result is surprising given the similarity of N2O and CO2 in physical properties and solubility parameters (shown in Table I). (N2O does have a small permanent dipole moment; CO2 does not.) The results of Alexandrou and Pawliszyn (7) for the SFE of TCDDs from incinerator fly ash are particularly interesting. Ν 2 0 yielded good recoveries from untreated fly ash, whereas CO2 yielded good recoveries only after the fly ash surface was modi fied by acid treatment, demonstrating that displacement of the analytes from the sorptive sites on the fly ash—and not differences in their solubility—was responsible for the increased extrac tion efficiencies demonstrated by N20. Because of the practical difficulties in using polar fluids such as ammonia for SFE, extractions of highly polar analytes have most often been done us ing CO9 containing a few percent add ed organic modifier. Modifiers can be introduced as mixed fluids in the pumping system, with the aid of a second pump, or by simply injecting the modifier as a liquid onto the sam ple before beginning the extraction. Figure 4 shows the increase in extrac tion efficiencies obtained in 30 min by adding a methanol modifier for extrac-
REPORT tions where the recovery using pure CO2 was not quantitative (9-12). In each case the extraction efficiencies in creased dramatically, and even the ion ic compound linear alkylbenzenesulfonate was quantitatively recovered. Although methanol has been the most widely used modifier, a variety of organic compounds have been used, ranging from alcohols, propylene car bonate, 2-methoxyethanol, and organic acids to methylene chloride and carbon disulfide. The selection of modifiers and their concentrations has been largely empirical because very little analyte solubility data exist for modified supercritical fluids. In addition, the competitive interactions between the modified supercritical fluid and the target analytes with the sorptive sites on the bulk matrix are poorly under stood. Clues can be gleaned from SFC separations that have used modified CO2, and a reasonable starting point would be to select a modifier that is a good solvent in its liquid state for the target analyte. However, until the ex traction mechanisms that control the distribution of the analytes between the matrix and the bulk supercritical fluid are better understood, optimiza tion of SFE methods using modified fluids will frequently require testing modifiers with different polarities and concentrations as well as determining optimal temperature and pressure con ditions. Class-selective extractions. As previously discussed, the solvent strength of a supercritical fluid can eas ily be controlled by changing the pres sure (and, to a lesser extent, the tem perature), which makes it possible to perform selective extractions with a single supercritical fluid by varying the extraction conditions. Although ob taining a pure analyte from complex matrices using SFE is unlikely, sequen tial extractions of different compound classes have been demonstrated. For example, alkanes can be extracted from urban air particulates with CO2 at 75 atm (45 CC) whereas the PAHs remain unextracted until the pressure is raised to 300 atm. By sequentially extracting the air particulates at these two pres sures, 85-90% selectivities can be achieved (13). In some cases, proper selection of ex traction conditions can also be used to extract analytes from bulk matrix ma terial that is itself soluble in the super critical fluid under different condi tions. For example, even though fat components are highly soluble in su percritical CO2 under most conditions, extracts containing organochlorine pesticides that are sufficiently fat-free to allow direct gas chromatographic
analysis have been prepared by extrac tion at a pressure under the threshold solubility pressure (~120 atm at 40 °C) of the bulk fat matrix (4,14). Class-selective extractions have also been achieved by depositing the ana lytes onto a sorbent, then eluting them with supercritical fluids (although this may more properly be called SFC than SFE). This technique has been success fully coupled with GC (SFE/GC) for the class fractionation of alkanes, alkenes, and aromatics from gasoline us ing supercritical CO2 elution of each compound class from a silica column and transferring each compound class to a capillary gas chromatograph for separation of the individual compo nents (15,16). Many other variations of class-selective SFE are possible, and a better understanding of extraction mechanisms will facilitate the develop ment of class-selective SFE methods. SFE techniques and hardware Hardware requirements. Commer cial extraction units are available for performing analytical SFE, and sys tems can easily be assembled in the laboratory. Analytical SFE is typically performed using syringe pumps that are similar or identical to those used for SFC, although less expensive alterna tives are available because SFE is nor mally performed at constant pressures without the sophisticated pressure/ density ramp controllers required for SFC. Gas compressors are also useful (e.g., for CO2), particularly for larger scale extractions (e.g., samples >50 g) for which a syringe pump may have in sufficient capacity. Extraction cells can be purchased or constructed from com mon tubing fittings, but care must be taken to ensure that all of the extrac tion system components are rated for the working pressures. The tempera ture of the extraction cell is normally controlled by placing the cell in a chro matographic oven or in a simple tube heater. SFE can be performed in either a dynamic or a static mode. For dynamic SFE, the supercritical fluid is constant ly flowing through the cell, and a flow restrictor is used to maintain pressure in the extraction vessel and allow the supercritical fluid to depressurize into the collection device. Both micrometering valves and short lengths of fused-silica tubing (~10-50-μπι i.d.) have been widely used for flow restrictors. Static SFE is performed by pres surizing the cell and extracting the sample with no outflow of the supercri tical fluid. After a set period of time, a valve (e.g., an HPLC switching valve) is opened to allow the analytes to be swept into the collection device. One
638 A · ANALYTICAL CHEMISTRY, VOL. 62, NO. 11, JUNE 1, 1990
advantage of dynamic SFE is that the supercritical fluid is constantly re newed during the extraction, but this technique requires more fluid than static SFE, particularly when extract ing large samples. Specific criteria for selecting dynamic or static modes are not yet clear, and both methods have been used for quantitative SFE of a variety of samples. Sample size considerations. Al though analytical SFE has been used with samples ranging from 1 mg to hundreds of grams, SFE of samples 1 mL should be packed full of the sample to avoid significant void volumes in the extraction system, which would increase extraction times.) Fortunately, several approach es have been reported that are capable of quantitatively retaining extracted analytes under a wide variety of SFE conditions and sample sizes. Off-line vs. on-line collection techniques. Methods that have been used to collect extracted analytes upon depressurization of the supercritical fluid fall into two general categories: "off-line" SFE (analytes are collected for subsequent analysis) and "on-line" or "coupled" SFE (analytes are direct ly transferred to a chromatographic system). Off-line SFE is inherently simpler to perform because only the ex traction step must be understood, and
the extract can be analyzed by any ap propriate method. On-line SFE re quires an understanding of both the SFE and the chromatographic condi tions, and the sample extract is not available for analysis by a different method. The principal advantages of on-line SFE are the elimination of sam ple handling between extraction and chromatographic analysis and the po tential to achieve maximum sensitivity by quantitatively transferring the ex tracted analytes into the chromato graphic column. With off-line SFE, the analytes are most often collected in a few milliliters of a liquid solvent, and the analysis of the extract is conducted as it would be for any conventional liquid solvent ex tract. The cooling of the solvent caused by the expanding supercritical fluid prevents rapid evaporation of the col lection solvent that would be expected because of the high gas flows, and even relatively volatile analytes are quanti tatively recovered (e.g., decane is quan titatively retained in 3 mL of methyl ene chloride during a 15-min extraction using ~ 1 mL/min of supercritical CO2 [12]). SFE effluents have also been col lected on sorbents such as silica or bonded-phase packings and then eluted with liquid solvents or supercritical fluids for subsequent analysis. When large quantities of bulk matrix are ex tracted (e.g., fat from meats), direct depressurization into an empty receiv ing vessel has been successful (17), but with trace analytes, trapping in empty vessels (even with cooling) has been
Table II.
less successful because of losses from aerosol formation (9). Off-line SFE has been most exten sively studied as a processing tech nique, but the use of SFE as a quantita tive analytical procedure has also been successful for a variety of analytes. All of the extractions listed in Table II yielded quantitative recovery of the target analytes, and the majority were completed in
