Analytical scale extractions

A complete understanding of SFE and its relation to other extraction methods cannot be made without some knowledge of the basic properties of supercritical fluids and the basic principles of analytical SFE instrumentation. The purpose of this section is to give an introduction to the use of supercritical fluids in analytical-scale extractions while focusing on the application of SFE to pharmaceutical analysis. 

The delivery system can either be a syringe pump, reciprocating piston pump, or gas compressor. Both syringe and reciprocating piston pumps are available on analytical-scale commercial instrumentation. These are used for analytical-scale extractions, while gas compressors are typically used for large-scale... 

Supercritical fluid extraction (SFE) has been extensively used for the extraction of volatile components such as essential oils, flavours and aromas from plant materials on an industrial as well as an analytical scale (61). The extract thus obtained is usually analysed by GC. Off-line SFE-GC is frequently employed, but on-line SEE-GC has also been used. The direct coupling of SEE with supercritical fluid chromatography (SEC) has also been successfully caried out. Coupling SEE with SEC provides several advantages for the separation and detection of organic substances low temperatures can be used for both SEE and SEC, so they are well suited for the analysis of natural materials that contain compounds which are temperature-sensitive, such as flavours and fragrances. 

The use of SCF as extracting media and mobile phases for chromatography is now commonplace, and SFE and SFC occupy established niches in analytical chemistry. Commercially available instrumentation for SFE and SFC have been available since the mid 1980s. Basic requirements for analytical scale SFE equipment that can perform selective removal or solubilisation of target analytes or analyte classes consist of a... 

Silica gel thin-layer plates may be used to separate lipids on either a preparative or an analytical scale. They are sometimes used to fractionate the lipids into classes prior to removal from the plate and further analysis by GLC. In this case the appropriate area of silica gel is scraped off and the lipid extracted into chloroform or diethyl ether containing 1-2% methanol for simple lipids or into chloroform-methanol-water (5 5 1) for polar lipids. 

Much of the current interest in using analytical-scale SFE systems comes from the need to replace conventional liquid solvent extraction methods with sample preparation methods that are faster, more efficient, have better potential for automation, and also reduce the need for large volumes of potentially hazardous liquid solvents. The need for alternative extraction methods is emphasized by current efforts to reduce the use of methylene chloride as an extraction fluid for environmental sample preparation [158]. The potential for applying SFE to a wide variety of environmental and biological samples for both qualitative and quantitative analyses is widely described in reviews [159-161] and the references therein. Analytical-scale SFE is most often applied to relatively small samples (e.g., several grams or less). 

Analytical-scale SFE can be divided into off-line and on-line techniques. Off-line SFE refers to any method where the analytes are extracted using SFE and collected in a device independent of the chromatograph or other measurement instrument. On-line SF techniques use direct transfer of the extracted analytes to the analytical instrument, most frequently a chromatograph. While the development of such on-line SFE methods of analysis has great potential for eventual automation and for enhancing method sensitivities [159-161], the great majority of analytical SFE systems described use some form of off-line SFE followed by conventional chromatographic or spectroscopic analysis. 

Eskilsson, C.S. and Bjorklund, E., Analytical-scale microwave-assisted extraction, J. Chromatogr. A, 902, 227, 2000. 

The sample extracts that show either toxicity or no dose response on initial testing should be fractionated. An aliquot of the extract is solvent exchanged to acetonitrile, and an initial analytical scale separation is made to assess the distribution of constituents in the sample. This separation is accomplished by using a Qg reversed-phase system eluted for 45 min with a linear gradient of 0-100% acetonitrile in water. If >75% of the sample elutes after the solvent composition of 80% and 20% acetonitrile, then the fractions are isolated by preparative reversed-phase HPLC. Fraction A is eluted with 100% water fraction B is eluted with a linear mobile-phase gradient from 100% to 75% water and 25% acetonitrile fractions C, D, and E are eluted with gradients with final compositions of 50%, 75%, and 100% acetonitrile. 

Analytical methods for monitoring the compounds were developed or modified to permit the quantification of all 23 compounds of interest. As noted earlier, the compounds were initially studied in small-scale extractions by groups. This approach assured minimal interferences in the analyses conducted during the initial supercritical fluid carbon dioxide extractions. Table II summarizes the data on the recovery of organics from aqueous samples containing the compounds of interest at concentration levels listed in Table I when the sample preparation techniques and analytical methods described were used. For each experimental run, blank and spiked aqueous samples were carried through the sample prepration and analytical finish steps to ensure accurate and reproducible results. Analyses of sodium, calcium, and lead content were also conducted on selected samples by using standard atomic ab-... 

The supercritical fluid extraction of analytes from solid sorbents is controlled by a variety of factors including the affinity of the analytes for the sorbent, the tortuosity of the sorbent bed, the vapor pressure of the analytes, and the solubility and the diffusion coefficient of the analytes in the supercritical fluid. Additionally, SFE efficiencies are affected by a complex relationship between many experimental variables, several of which are listed in Table I. Although it is well established that, to a first approximation, the solvent power of a supercritical fluid is related to its density, little is known about the relative effects of many of the other controllable variables for analytical-scale SFE. A better understanding of the relative effects of controllable SFE variables will more readily allow SFE extractions to be optimized for maximum selectivity as well as maximum overall recoveries. 

Several liquids and gases can be brought into the supercritical phase. Different solvents can be selected as extraction media for use in analytical-scale SFE. Carbon dioxide is most commonly used as an SFE medium because of its desirable properties and easy handling it is relatively inexpensive and commercially available at a purity grade acceptable for most analytical applications. Another advantage of carbon dioxide is that the polarity can easily be adjusted by adding modifiers such as methanol to the supercritical fluid or the extraction vessel. 

Supercritical fluid extraction (SFE) utilizes the unique properties of supercritical fluids to facilitate the extraction of organics from solid samples. Analytical scale SFE can be configured to operate on- or off-line. In the online configuration, SFE is coupled directly to an analytical instrument, such as a gas chromatograph, SFC, or high-performance liquid chromatograph. This offers the potential for automation, but the extract is limited to analysis by the dedicated instrument. Off-line SFE, as its name implies, is a stand-alone extraction method independent of the analytical technique to be used. Off-line SFE is more flexible and easier to perform than the online methods. It allows the analyst to focus on the extraction per se, and the extract is available for analysis by different methods. This chapter focuses on off-line SFE. 

Basic experiments were carried out with an extractor in analytical scale (SFE-703, DIONEX). For a typical experiment the extraction cells (10 ml internal volume) were fully filled with a homogeneous mixture of the flame retardent and the inert MgS04 and placed into the oven chamber of the extractor. After reaching the desired extraction conditions (pressures of 250 to 500 bar and temperatures of 60, 80 or 100 °C) the samples were extracted for 45 min. The extracted components were analysed by gravimetric, spectroscopic and/or chromatographic methods (IR, GC-MSD). Further experiments were made with realistic brominated ABS composites (granulated composites) in analytical scale and also with an extraction autoclave in laboratary scale (500 ml internal volume). 

Here we steer a middle course, emphasizing analytical results extracted from the mean-field theories, in context with the lattice and scaling approaches, and explaining the relationship to macroscopic phenomena. Our treatment draws on Russel et al. (1989) in some places. 

Fahmy, T.M., Pulaitis, M.E., Johnson, D.M., McNally, M.E.P., Modifier effects in the supercritical fluid extraction of solutes from clay, soil, and plant materials. Anal. Chem., 65 (10), 1462-1469,1993. Langenfeld, J.J., Hawthorne, S.B., Miller, D.J., Pawliszyn, J., Role of modifiers for analytical scale supercritical fluid extraction of environmental samples. Anal. Chem., 66(6), 909-916,1994. Hawthorne, S.B., Methodology for off-line supercritical fluid extraction. In Supercritical Fluid Extraction and Its Use in Chromatographic Sample Preparation, Westwood S.A. (Ed.), Blackie Academic and Professional, 39-64, 1993. 

Camel, D. Tambut6, B. Caude, M. Analytical-scale supercritical fluid extraction A promising technique for the determination of pollutants in environmental matrices. J. Chromatogr., A 1993, 642 (1-2), 263 -281. 

On-line and off-line analytical scale SEE can be applied. In the former, the coupling step (i.e., the transfer and collection of extracted analytes from the SFE to the chromatographic system) is of great importance. 

Analysis of the final Am02 product is shown in Table VI. A1 and Mg were below the detectable limits for these elements. The product met specifications of >95% Am02 with less than 0.5% Pu and less than 1% of any other single contaminant. The results of the preliminary lab scale extraction chromatography tests are shown in Table VII. Again, in spite of some analytical problems, it is evident that americium was decontaminated from aluminum and magnesium. A 7M HNO3 wash step is assumed to account for the americium loss. 

J. J. Langenfeld, S. B. Hawthorne, D. J. Miller, J. Pawliszyn, Role of modifiers for analytical-scale supercritical fluid extraction of environmental samples. Anal. Chem., 66 (1994), 909-916. 

With normal-phase HPLC, oil samples were analyzed as is by simple dilution in n-hexane. A Du Pont Zorbax amino-bonded phase column, 25 cm x 0.46 cm ID, was used, with n-hexane and dichloromethane as solvents. For reversed-phase HPLC, Vydac 201TP5 columns were used (25 cm x 0.46 cm ID for analytical scale and 25 cm x 1 cm ID for preparative scale). Samples for reversed-phase HPLC were fractionated in order to remove the saturated hydrocarbons which can interfere with the separation mechanism. The samples dissolved in n-hexane were passed Baker silica solid-phase extraction cartridges. The PAH fraction was then collected by eluting with a 1 1 mixture of dichloromethane and methanol. Acetonitrile and dichloromethane were used in the HPLC gradient. 

Hawthorne SB. Analytical-scale supercritical fluid extraction. Anal Chem 1990 62 633A-642A. 

With the advent of automated analytical SFE equipment, it has become possible to rapidly ascertain what extraction or fractionation conditions would be most relevant in scaling up the process. In the United States, analytical SFE instrumentation is produced by such firms as Isco, Applied Separations, Leco, and Jasco. In Europe, analytical-scale SFC equipment is available from Berger Instruments, Thar Designs, Jasco, and Sensar. The equipment is obtained from these vendors can be, if needed, slightly modified to study the conditions that are amenable to processing nutraceuticals. King (34) has provided a interesting review of how lab-constructed equipment can be used for both analytical and process development purposes. 

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