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The diversity of real-world matrices makes a wide range of approaches essential
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raditional sample preparation often requires more than 50% of the analyst's time and consumes large quantities of diverse hazardous organic solvents. As a result of the Environmental Protection Agency's voluntary organic solvent reduction program and the 100% phaseout of chlorofluorocarbons (by Jan. 1,1996), interest in methods that use benign solvating media such as supercritical fluids is increasing. The early 1980s saw great interest in supercritical fluid chromatography (SFC) as its usefulness was demonstrated (i). In the 1990s analytical supercritical fluid extraction (SFE) has emerged as a major technique. Because a universal extraction strategy for all analytes and matrices will never be feasible, an awareness of various extraction strategies, given the wide diversity of real-world matrices, is vital for the future success of analytical SFE. Last month, McNally reviewed recent theoretical and technological advances in SFE (2). This article will provide a sense of the strategies necessary for successful analytical SFE.
STRATEGIES FOR ANALYTICAL
SFE
pends to varying degrees on the solubility of the analyte (s) in the extracting supercritical fluid (SF), the analyte-matrix interaction, the location of the analyte within the matrix, and the porosity of the matrix. Unfortunately, having a knowledge of analyte solubility in the SF does not always enable one to predict the effectiveness of SFE for the analyte from a particular matrix. For example, caffeine is soluPhysical factors ble in SF C02, but SFE does not extract it Regardless of the extraction mode, the ability to remove analyte from a matrix de- from dry coffee beans. In early SFE studies, researchers often based extraction conditions on the particular pressure and temperature that maxiLarry T. Taylor Virginia Polytechnic Institute and State mized the solubility of neat target analytes. Although this approach is useful University 364 A
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when no co-extractives are anticipated and the analyte is present in a relatively high concentration, extraction parameters that favor maximum solvating power for the analyte may be less desirable if a dynamic extraction is being performed for minor and trace amounts of analytes and extensive co-extractives are anticipated. King (3) has suggested that the concept of threshold solubility pressure (i.e., the pressure at which an analyte becomes significantly soluble) can be useful in certain circumstances. If the goal is to perform SF fractionation, as opposed to exhaustive extraction, the threshold solubility pressure can be used for each extracted component. This method is es0003 - 2700/95/0367 -364A/S09.00/0 © 1995 American Chemical Society
pecially useful for the selective extraction of oligomers; as the density increases, higher oligomers become solvated. The physical morphology of the matrix can also have a profound influence on the efficiency of an extraction. Components from polymer powders are more efficiently extracted, for example, than components from polymer films. In general, the smaller the particle size of the substrate, the more rapid and complete the extraction, because of the shorter internal diffusion pathlengths (created by the now greater surface area) over which the solutes must travel to reach the bulk fluid phase. Because the removal of additives from polymers is determined largely by analyte diffusion through the internal volume of the sample matrix, an increase in the matrix's porosity, prompted by swelling of the matrix, will generally promote a more efficient and rapid extraction. Extractions of polymeric materials should be conducted at temperatures greater than the glass transition temperature (at which the polymer's effective surface area is increased) but lower than the melting point (at which plugging of the vessel and transfer lines by the molten polymer can occur), thus allowing the liquid polymer to mechanically move en masse from the vessel to the trap. The high temperature also enhances analyte diffusivity, which can lead to more efficient extraction. High temperatures have been found to be beneficial for release of semivolatiles, such as PCBs, from solid environmental matrices (4).
of fluid are used during an extracExtracting matrix amounts tion. These contaminants will ultimately at the collection device, become components may seemarrive concentrated, and can interfere with the analysis. For example, an extraccounterproductive, extract tion with 100 g of C0 that contains 1.0 ppb nonvolatile hydrocarbon will yield but it affords an 0.1 mg of impurity at the trap. alternative "inverse" Another problem with dynamic extraction is the increased likelihood of coapproach to extraction of matrix components. A long dynamic extraction may also cause the unconcentrating the wanted physical movement of matrix components to the trapping device as well analyte. as removal of analytes from the trap. In
vessel containing the matrix is pressurized with the chosen SF at a certain temperature. The high diffusivity of the fluid causes it to permeate the matrix and remove the analyte. Alternatively, a recirculation pump can be used to cycle the limited amount of SF through the matrix. After the extraction is completed, a valve is opened at the outlet of the cell and the analyte is swept into the trap via decompression. Typically, a static extraction is followed by several minutes of dynamic extraction to complete removal of the extracted analytes from the vessel. The static mode is often used when modifiers and derivatizing reagents are added. The liquid polar modifier or derivatizing reagent can be added to the cell prior to pressurization. A static extraction may not be exhaustive if insufficient fluid is used, but fluid contamination is seldom a problem unless the analyte is present at trace levels. Extraction modes A dynamic extraction, on the other SFE can be performed in a static, dynamic, hand, uses fresh SF that is continuously or coupled static/dynamic mode. A static passed over or through the sample maextraction takes place when a fixed trix, as hot water is in a coffee maker. Alamount of SF interacts with the analyte though a dynamic extraction is more exand the matrix, the way a tea bag interacts haustive than a static one, impurities in the with a cup of hot water. The extraction SF can cause problems when large
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spite of these problems, dynamic extraction is the favored strategy for at least 90% of applications. The combination of a static extraction period followed by a dynamic one is gaining popularity, especially for situations in which analyte must diffuse to the matrix surface to be extracted. After the static phase is over, fresh SF enters the vessel, replacing the original SF that has exited through the restrictor to the trap. The use of multiple combinations of static/ dynamic cycles increased the recovery of a drug from a crushed tablet from ~ 90% to 99% (5). Extraction scenarios
Various scenarios (no material extracted, only analyte (s) extracted, only matrix component (s) extracted, and both analyte (s) and matrix component(s) extracted) can occur during the extraction of an analyte and matrix (3). The failure to extract anything (assuming the analyte is on the matrix surface) may imply that the SF has insufficient solvating power to solubilize the analyte or to disrupt the analytematrix interaction. Because solubility of an analyte in an SF does not automatically ensure extraction of that analyte from a real-world matrix, it is best tofirstextract Analytical Chemistry, June 1, 1995 365 A
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the neat analyte from an inert matrix. If analyte extraction occurs, the failure to extract from the real-world sample under the same conditions is caused by a matrix effect. The situation in which only the analyte (s) is extracted is ideal but rarely encountered. Extraction of only matrix components) may at first seem counterproductive, but it affords an alternative, "inverse" approach (a term coined by King) to concentrate the analyte. This approach has recently been used for the analysis of very polar drugs in ointments and creams (6). A more realistic situation is one in which both analyte and (potentially interfering) matrix components are coextracted. In these cases, additional separation of the extracted material may be required prior to analysis, or a selective detector used.
Figure 1 . Theoretical dynamic extraction profile of an analyte from a solid matrix.
Extraction profiles
to a number of matrices during the past two years.
Of the three extraction modes, the dynamic mode is the most easily modeled (7). Figure 1 shows the theoretical extraction profile of an analyte from a solid matrix using a dynamic system. The extraction profile can be divided into three distinct regions. In region 1, the initial extraction of material occurs quickly and depends on the solubility of the bulk analyte in the SF. During this portion of the extraction, the solubilized analyte is purged from the extraction vessel. Quasi-equilibrium conditions govern the partition of the solute into the dense mobile fluid phase. Efficient extraction in region 1 depends on high solubility of the analyte in the SF, rapid movement of SF through the system, and a minimal amount of dead volume in the extraction vessel and associated tubing. Region 2 is an intermediate area in which the extraction process is enthalpically controlled (i.e., the analyte-matrix interaction is disrupted), resulting in a slower rate of extraction. A transition to diffusion-controlled kinetics is also taking place in this region. In region 3, the extraction process is truly diffusion limited, brought about by the limited mobility of the analyte within the sample matrix. A mathematical model, called the "hot ball" model (8), that describes this diffusion portion of the extraction profile has been successfully applied
For quantitative dynamic extractions, the number of fluid extraction vessel volumes used is more useful for describing extraction conditions than is the time of extraction. This is most apparent when extracting samples of different mass, different matrix density, orfixed-sizesamples in different-sized vessels. For example, excessive vessel void volume will require long extraction times, most of which are devoted to sweeping the sample from the vessel rather than solubilizing additional sample. If there is no sample expansion during extraction for moderately dense samples, the sample volume and vessel volume should always be closely matched. Some workers have questioned the practice of using vessel void volumes rather than mass of SF as a measure of the fluid required to complete an extraction because the contents of the vessel can be compressed during the course of the extraction, thereby changing the void volume of the vessel. If, however, one assumes that the vessel void volume equals the empty vessel volume, ample SF would always be used. For an exhaustive extraction, 15 vessel void volumes should prove ample if analyte extraction in the SF is high and if extraction is not controlled by a diffusion process.
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Mechanics
When the sample size and extraction vessel size do not match, an inert matrix should be added to the vessel tofillany extra void volume. A glass rod, Celite, Hydromatrix, or Ottawa Sand can be inserted into the vessel. The voidfillermaterial should be as inert as possible, free of any extractables, and contain nofinesto clog screens and frits. The sample should be placed within the vessel close to the trap to prevent the inert matrix from forming an impervious plug in the vessel, thereby introducing preferential flow paths through or around the matrix. Making extraction easier
The extraction of an analyte can often be made easier by adding extraneous material that either enhances the extraction of the desired analyte or inhibits the coextraction of unwanted matrix. Because it can form ice in the restrictor, end up in the trap itself, or cause problems if the analyte has more affinity for it than for COa, water often causes problems in SFE, especially in extractions of polar analytes. Although oven drying, desiccation, and freeze drying are commonly used to remove moisture, these methods increase the risk of losing volatile or thermally labile analytes. Adding a drying agent to the extraction vessel is thus preferred. Because water-saturated Na2S04 or MgS04 can plug a restrictor or extraction cell, Celite and Hydromatrix (diatomaceous
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earths that function not only as drying agents but also as dispersing agents) are more satisfactory. Alumina has been used to bind interfer ing fats from tissue samples, allowing the extraction of pesticides (9). The alumina is added directly to the sample matrix or to the extract, which is then passed through an alumina cartridge where both lipid and analyte are trapped. The analyte is then selectively removed from the alumina by rinsing with an appropriate solvent. An other annoying interference is sulfur in certainriversediments, which can plug restrictors. Adding 1-3 g of acid-washed copper granules to the outlet of the cell binds the sulfur as copper sulfide and elim inates this problem. Even in the absence of co-extractives, achieving a successful extraction is some times impossible with C02-based fluids because the analyte is either highly polar or ionic and the SF has limited solvating power. In this case analysts should con sider in situ analytical derivatization or chelation within the SFE vessel. For polar organic analytes, derivatization normally involves capping hydroxyl functionalities and converting them to either ethers or es ters, which are more soluble in supercrit ical CO2(i0). The extraction and the derivatization reaction should occur simultaneously. Hawthorne et al. (11) have studied the extraction of linear alkyl benzene sulfon ates from a municipal wastewater treat ment facility, and found that pure C0 2 and N20 with modifier content as high as 40% failed to extract anything. How ever, in situ chemical derivatization/ SFE with trimethylphenylammonium hy droxide yielded 30-40% recovery after one derivatization. This technique has also been extended to phenoxy acids and pentachlorophenol. Ion pair reagents can also be used to extract polar compounds from natural samples. An anionic analyte [A~] reacts with a cationic tetraalkylammonium ion pair reagent [NR4] to form an ion pair [ANRJ that is soluble in C02. A highly fluorinated nucleophilic alkylation reagent such as methyl iodide can also be used to form a derivative with enhanced C0 2 solubility. This approach has been used to remove sulfonated aliphatic and aro matic surfactants from sewage sludge. 368 A
When analytes do not readily derivatize, the matrix can be chemically altered. For example, Hills and Hill (10) have used a 2:1 mix of hexamethyldisilane and trimethylchlorosilane to cap active sites on an urban dust sample with trimethylsilane groups, thereby disrupting the adsorptive interactions between the polyaromatic hydrocarbon and the matrix surface. Because of their ionic nature, metal ions must be converted to neutral metal complexes. These complexes must have high stability constants, good solubility in the SF, and fast complexation kinetics (12). Lithium bis(trifluoroethyl)dithiocarbamate (Li[FDDC]) and perfluorinated β-diketones have been used as chelating agents for SFE of lanthanides and se lected divalentfirst-rowtransition met als. In the case of copper (II), the extract ing phase was C02 containing Li(FDDC),
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SFE can be performed in a static, dynamic, or coupled static/ dynamic mode. made by passing C02 through a ligand ex traction vessel. The resulting SF was passed through an aqueous solution of Cu(N03)2 or over a Cu(NC>3)2-fortified solid support. Extraction efficiencies of > 80% for copper(II) were obtained. Caution with any extraction aid is nec essary, because these agents can immobi lize or greatly alter the chemical integ rity of the analytes. For example, sensitive compounds such as fat-soluble vitamins, unsaturated fatty acids, and certain per fume components have been chemically changed in the presence of additives. In addition, the chemical aid must interact completely with the analyte, although the excellent mass transfer properties of the SF should afford complete and efficient in teraction. Matrix problems
With sufficient SF solvating power, the rate of the extraction process can be expe
dited by increasing the surface area or po rosity of the matrix by grinding the sam ple. However, analytes such as polymer additives can be volatilized by the heat generated by grinding, so it is best to grind with dry ice or liquid nitrogen. Particles smaller than 0.05 mm should be avoided because they may become compacted in the vessel, which can lead to channeling of the SF and inefficient contact between the SF and the matrix, and because the particles may be mechanically forced out of the vessel during pressurization. Matrices sometimes swell when C02 or modified C02 is introduced. This phe nomenon occurs most often with certain polymers and natural materials such as raw coal. When working with these matri ces,fillingthe extraction vessel to only 75% capacity is recommended because swelling of the matrix can cause the ves sel frits to plug. Semisolid material can also extrude into the transfer lines, caus ing plugging and mechanical transfer problems. There are also cases in which the melt ing point is reached during extraction or decreases with pressure such that the sample becomes mobile. If the analyte or co-extractives are present in trace quan tities, this particular problem is not se vere; however, if the quantity of any of these extractives becomes large enough, measures such as mixing or layering semisolid samples with an adsorbent, or smearing very thin layers of sample onto filter paper, must be taken to immobilize the sample. SFE of analytes from aqueous samples has received little attention compared with that from solid matrices, probably be cause of the mechanical difficulty of retain ing the liquid matrix in the extraction ves sel. If the analyte is present at trace levels, the volume of matrix must be unduly large to ensure obtaining quantities of ana lyte sufficient enough to measure. Direct extraction of phenols, phosphonates, and selected organic bases from aqueous solu tions has been demonstrated, but not at trace levels (13). An alternative system, in which the analyte isfirstconcentrated on a solid-phase cartridge or disk that is then subjected to SFE to remove the analyte, has been demonstrated for extracting phe nols and sulfonyl urea herbicides from water (14).
Modifier introduction Pure C0 2 is not an appropriate extraction solvent for polar analytes and retentive matrices, although it has been successfully used for extracting many priority pollutants from environmental matrices (15). In addition, pure C0 2 is often ineffective in disrupting a strong analyte-matrix interaction. Alternative fluids with greater solvating power have not proven very useful because of their extreme critical parameters and corrosive properties, although Freon-22 (CHC1F2) and fluoroform (CHF3) can be used for selected analytes and matrices {16). One popular method of enhancing the solvating power of C0 2 is to add a small amount of an organic solvent, such as methanol or toluene, to the C0 2 . When extracting several PCBs from river sediment, much higher extraction efficiency was obtained when toluene was present than when it was not. There was also a 50% reduction in the amount of C0 2 used (17). Modifiers can be directly introduced to either the fluid or the matrix. The use of premixed tanks is very popular, is convenient if a single concentration of modifier is desired, and is the method of choice if an auxiliary modifier pump is not available. Mixtures can be obtained as weightweight percent or as a mole percentage. The most obvious problem with this approach is that a large inventory of tanks is quite expensive. Problems may also result from phase separation over time (organic liquid settling on the bottom of cylinders), which could lead to nonreproducible extractions. For example, temperature variations during shipment and in the laboratory can shift the vapor-liquid equilibrium and change the composition of the liquid phase in the cylinder. In addition, the composition of a premixed cylinder is not constant as the contents are drawn down. A full cylinder ( ~ 40 lb of liquid C0 2 ) that starts out at 5% (w/w) methanol contains ~ 10% (w/w) methanol when the tank is only 20% full (18). This change is believed to be caused by shifts in the C02-methanol vapor-liquid equilibrium. Whether small changes in the quantity of modifier can significantly affect extraction recovery has not been well documented, although it is known that solid-
phase trapping efficiency is markedly affected by modifier in excess of 2% (w/w). Most analytical SFE experiments to date do not assess the effect of small changes in recovery (and ultimately precision) with subtle changes in premixed tank modifier percentage. The alternative method of generating mixed SFs involves using two pumps, one for the primary SF and the other for the modifier. This approach is similar to that routinely used in gradient HPLC, but it requires sophisticated equipment because of the relatively high compressibility of supercritical C0 2 . Compressibility is an even greater problem with a fixed restrictor if pressure or composition gradients are called for in the extraction. Complete miscibility of the SF and the liquid modi-
fier is an obvious prerequisite for reproducible analytical SFE. Most commercially available instruments mix modifier with C0 2 in the liquid phase as opposed to the SF phase. In either case, mixing is performed on a volume-volume basis by noting the modifier pump displacement. The simplest method for introducing modifier into an extraction is to simply inject a small volume of the modifier into the extraction cell. This method is particularly useful for static extractions, because the concentration of the modifier is maximized in the vessel, remains constant during the extraction, and greatly simplifies the testing of many different modifiers in a short time. It may not be very useful for dynamic SFE, however, because the modifier is rapidly purged from the cell after extraction begins. At best, a negative modifier gradient could be ex-
pected to develop during the extraction. Although this may not seem important, it is possible that the amount of time during which the sample is in contact with the modifier can influence the efficiency of the extraction for certain analyte-matrix combinations. The best way to introduce modifier depends on the matrix and the analyte. If there are strong matrix-analyte interactions that the modifier is expected to disrupt, direct introduction to the matrix may be preferred. However, if the matrix is voluminous, the cosolvent spike may not sufficiently wet the matrix, and if the matrix is dried prior to the extraction, a volatile modifier may disappear before the C0 2 is introduced. If the analyte has minimal solubility in pure C0 2 and there are minimal matrix effects, the modifier might be more effective if added to the C0 2 either on line via an HPLC micropump or from a premixed cylinder. Knipe et al. (19) have compared the use of premixed modifiers with the direct addition of modifier to the matrix during the extraction of herbicides from ~ 1.0-g samples of spiked Celite and soil. The recoveries obtained on fortified Celite and soil matrices using modified premixed tanks were generally lower than those obtained by adding modifier directly to the matrix in the extraction vessel. Knipe's group theorized that the role of modifier was to disrupt the analyte-matrix interaction. If the modifier is to compete with the analyte for sites on the matrix, the greater initial concentration of modifier prior to extraction should be more effective than a greater net amount (albeit lower concentration) of modifier introduced over the duration of the extraction. The modifier stays in the fluid phase during the extraction and therefore has less interaction with the matrix itself compared with the liquid modifier being placed directly on the sample. Surprisingly, the researchers did not observe this trend when CH2C12 was used as the modifier for a soil matrix. Method development Once the extraction vessel has been properly loaded with the sample, the various SFE parameters of fluid, density, pressure, temperature, and flow rate must be determined. Equally important are the trap Analytical Chemistry, June 1, 1995 369 A
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conditions, such as type, temperature, and rinse. The details of an actual method are usually instrument specific. The accepted protocols for restriction and trapping are debated continually (e.g., liquid vs. solid phase trap; fixed vs. variable restrictor). Because of differences in sample chemical composition, there is no universal trap ping method that will work in all situa tions (17,20, 21). For a totally new application for which there is no prior information, analysts should begin method development with the pure analyte or an analyte-fortified sample. Extract the analyte (10-100 ppm) with C0 2 from a spiked solution (< 100 mL) after evaporation of the solvent, ei ther directly from the extraction vessel or from an inert matrix using several densi ties at low temperature. If the analyte has high volatility, removing the spike solvent by evaporation prior to extraction is not wise because analyte may be removed from the matrix. If the spike solvent can not be evaporated and it is anticipated that it will act as a modifier (thereby en hancing the extraction of unwanted com ponents) , one must go to a higher concen tration of analyte in the spike solution and smaller volumes (< 5 pL) of spiked so lution. Although the use of low tempera ture (40-50 °C) affords the maximum C0 2 density, gains in analyte solubility and ac companying decreases in extraction time may be realized by experimentation at higher temperatures. In the simplest extraction case, analyte solubility in 100% C0 2 will be assessed as suming that an adequate mechanism for trapping is in place. At this point, the ana lyst must realize that finding an adequate trap is not always easy and some testing will be necessary. Modifiers to enhance the solvating power of the C0 2 should be used only as a last resort because this strategy can become more costly and result in a less efficient trapping mechanism. Extracting the same analytes from realworld samples will require somewhat dif ferent (and possibly more severe) condi tions than those required for a fortified sample. Different matrices may bind the same analyte to different extents. For ex ample, removal of fat from a corn product requires a longer extraction time than re moval of fat from a potato product. Compo nents can also be physically encapsu 370 A
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lated in real-world samples, such as antiox idants in polyalkanes, so that accessibility of the extraction fluid is impaired. As a rule of thumb, if analytes are bound to the matrix, greater solvating power or higher temperature is needed. Experimental considerations should then be explored in this order: raise supercriti cal C0 2 density, raise fluid flow rate, increase extraction temperature, and add modifier. If analytes are encapsulated and recov eries are diffusion controlled, extraction kinetics begin to dominate. In this case, the considerations should be: increase ex traction time, equilibrate with SF to mini mize consumption, increase extraction temperature, and swell sample with or ganic solvent. The order of experimenta tion in both cases is subject to the matrix and the thermal stability of the analytes.
To enhance the solvating power ofC02, add a small amount of organic solvent to the C02. The analyst must also realize that the parameters selected for one process can markedly affect other factors. For exam ple, decreasing extraction time by in creasing flow rate may have a deleterious effect on trapping. For this reason, the more rigorous approach of statistical ex perimental design (22) can be used to make trade-offs among various operat ing conditions to maximize recoveries. The interaction among various SFE param eters can also be established with statisti cally designed experiments. This ap proach is increasingly popular, although it has been applied only to the extraction step (23). Systematic multivariate optimi zation of all variables that do not yield 100% recovery should probably be used for the complete process, from extraction through recovery.
References (1) Hawthorne, S. Κ Anal. Chem. 1990, 62, 633 A. (2) McNally, Μ.Ε.Ρ.ΛΜΟ/. Chem. 1995,67, 308 A. (3) King,J. W.J. Chromatogr. Sci. 1989,27, 355. (4) Langenfeld, J. J.; Hawthorne, S. B.; Miller, D.J.;Pawliszyn,J.P.AKa/. Chem. 1993, 65,338. (5) Howard, A. L; Strode, J.T.B.; Taylor, L. T.; Shah, M. C; Ip, D. P.; Brooks, M. /. Pharm. Sci. 1994,83,1537. (6) Moore, W. N.; Taylor, L. T. /. Pharm. Biomed. Anal. 1994,12,1227. (7) Stahl, E.; Quirin, Κ W.; Gerard, D. Dense Gases for Extraction and Refining; Ashworth, M.R.F., Transi.; Springer-Verlag: Berlin, 1988; p. 176. (8) Bartle, K. D.; Clifford, Α Α.; Hawthorne, S. B., et al./. Supercrit. Fluids 1990,3, 143. (9) France, J. E.; King, J. W.; Snyder, J. M. /. Agric. Food Chem. 1991,39,1871. (10) Hills, J. W.; Hill, H. E.J. Chromatogr. Sci. 1993,31, 6. (11) Hawthorne, S. B.; Miller, D. J.; Nivens, D. E.; White, D. C. Anal. Chem. 1992, 64, 405. (12) Lin, Y.; Braver, R. D.; Laintz, K. E.; Wai, C. M.Anal. Chem. 1993, 65, 2549. (13) Hedrick, J. L; Taylor, L. T.J. High Résolut. Chromatogr. 1992,15,151. (14) Howard, A. L; Taylor, L. T.J. Chromatogr. Sci. 1992,30,374. (15) Hawthorne, S. B.; Krieger, M. S.; Miller, D. J. Anal. Chem. 1989, 61, 736. (16) Howard, A L.; Yoo, W. J.; Taylor, L T., et al./ Chromatogr. Sci. 1993, 31,2. (17) Mulcahey, L. J., Hedrick, J. L.; Taylor, L. T.Anal. Chem. 1991, 63,2225. (18) Via, J. C; Taylor, L. T.; Schweighardt, F. K.Anal. Chem. 1994, 66, 1459. (19) Knipe, C. R; Gere D. R; McNally, M.E.P. In Supercritical Fluid Technology, Bright, F. V.; McNally, M.E.P., Eds.; ACS Symposium Series 488; American Chemical Society: Washington, DC, 1992; p. 251. (20) Mulcahey, L. J.; Taylor, L. T. Anal. Chem. 1992, 64, 2352. (21) Thompson, P. G; Taylor, L. T.J. High Résolut. Chromatogr. 1994,17, 759. (22) Box, G.E.P.; Hunter, W. G.; Hunter, J. S. Statistics for Experimenters, and Introduction to Design, Data Analysis, and Model Building; John Wiley and Sons: New York, 1978. (23) Li, K; Ong, C. P.; Li, S.F.Y./ Chromatogr. Sci. 1994,32, 53.
Larry T. Taylor, professor of chemistry at Virginia Polytechnic Institute and State University, focuses on developing applications ofhyphenated techniques for identifying and quantitating components in environmental, pharmaceutical, polymer, and food matrices. Address correspondence about this article to him at the Dept. of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0212.
