Supercritical fluid solutions, sample preparation

Principles and Characteristics Supercritical fluid extraction uses the principles of traditional LSE. Recently SFE has become a much studied means of analytical sample preparation, particularly for the removal of analytes of interest from solid matrices prior to chromatography. SFE has also been evaluated for its potential for extraction of in-polymer additives. In SFE three interrelated factors, solubility, diffusion and matrix, influence recovery. For successful extraction, the solute must be sufficiently soluble in the SCF. The timescale for diffusion/transport depends on the shape and dimensions of the matrix particles. Mass transfer from the polymer surface to the SCF extractant is very fast because of the high diffusivity in SCFs and the layer of stagnant SCF around the solid particles is very thin. Therefore, the rate-limiting step in SFE is either... 

SP refers to a family of solid/liquid handling techniques to extract or to enrich analytes from sample matrices into an analyzable format, namely, the final analyte solution. While SP techniques are well documented, " few publications address the specific requirements for drug product preparations, most of which tend to employ the simple dilute and shoot approach. A more elaborate SP is often needed for complex sample matrices (e.g., lotions and creams). Many newer SP technologies such as solid-phase extraction (SPE), " supercritical fluid extraction (SFE), "i° pressurized fluid extraction or accelerated solvent extraction (ASE)ii"i and robotics " " are topics of numerous research papers, symposia and commercial promotion. However, for reasons discussed later, these newer developments have had little impact on the way pharmaceutical laboratories conduct their SP for drug products today. 

Before the extraction procedure may commence, the sample must be prepared in such a way that it is in a condition for extraction of the analyte(s). For analyzing sulfonamide residues in liquid samples such as milk, a pretreatment dilution step with water prior to direct fluorometric detection may be required (207). Dilution of milk with aqueous buffer (208) or sodium chloride solution (209) prior to sample cleanup has also been reported. For the analysis of honey a simple dissolution of the sample in water (210, 211) or aqueous buffer (212) is generally required. Semisolid samples such as muscle, kidney, and liver, require, however, more intensive sample pretreatment. The analyte(s) must be exposed to extracting solvents to ensure maximum extraction. The most popular approach for tissue break-up is through use of a mincing and/or homogenizing apparatus. Lyophilization (freeze-drying) of swine kidney has been carried out prior to supercritical-fluid extraction of trimethoprim residues (213). 

Although SFE and SFC share several common features, including the use of a supercritical fluid as the solvent and similar instrumentation, their goals are quite distinct. While SFE is used mainly for the sample preparation step (extraction), SFC is employed to isolate (chromatography) individual compounds present in complex samples (11 -15). Both techniques can be used in two different approaches off-line, in which the analytes and the solvent are either vented after analysis (SFC) or collected (SFE), or on-line coupled with a second technique, thus providing a multidimensional approach. Off-line methods are slow and susceptible to solute losses and contamination the on-line coupled system makes possible a decrease in the detection limits, with an improvement in quantification, while the use of valves for automation results in faster and more reproducible analyses (16). The off-line... 

While it is wonderful to be able to inject neat samples directly, sample preparation can often improve selectivity and sensitivity. If the resolution is poor, the salt content of the sample too high, or the capillary fouls, consider a sample cleanup. This can include liquid-liquid extraction, solid-phase extraction, supercritical fluid extraction, protein precipitation, or dialysis, depending on the solutes and application [38]. The final sample diluent should be a solution that is CE-Mendly. That usually means low ionic strength compared to the BGE. 

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. 

When the analytes are to be retained in a sorbent, the sample (which can be solid, semi-solid, liquid or gaseous) is inserted in the solid state into the extraction cell. Samples in the latter three forms are supported on an appropriate material in order to ensure effective attack by the supercritical fluid. Solid supports are not used for liquid, gaseous and semi-solid samples only, however. Some research work conducted so far on solid samples has involved not natural samples but synthetic ones prepared from a selected sorbent (a natural matrix where the presence of the analytes of interest was previously excluded or a synthetic support such as polyurethane foam or glass wool) with which a solution containing the analytes was homogenized. Quantitative evaporation of the analyte solvent is mandatory as any residual solvent may alter the polarity of the supercritical fluid and hence its action to an extent dependent on the particular fluid and solvent properties, and also on the amount of solvent retained. 

A key to successful chromatographic analysis lies in proper sample preparation. Ideally, it is preferred to dissolve the sample in a suitable solvent and analyze that solution directly, provided the presence of dissolved polymer does not complicate the chromatographic analysis. These cases are indeed rare. Normally, filtration or precipitation followed by final filtration is desirable to remove interferences in sample components (polymers) and higher-molecular-weight components. This approach works well when the polymers are, first, soluble, and second, can be precipitated with an antisolvent. Less soluble polymers, such as highly crystalline resins, require extraction to remove the components of interest from the resin matrix. Numerous extraction techniques (supercritical fluid extraction, solvent extraction, resin dissolution followed by antisolvent precipitation, etc.) are also available [14]. 

Supercritical Fluid Chromatography (SFQ and Supercritical Fluid Extraction (SFE) A separation technology similar to other extraction and chromatographic methods, but in which the mobile phase is actually a fluid in its supercritical fluid state. A supercritical fluid is a fluid that is held above its critical temperature and pressure, and for which no application of additioncJ pressure can result in the development of a liquid phase. Supercritical fluids are unique in that while they possess liquid-like densities, the mass transfer behavior is superior to that of liquids. Supercritical fluid chromatography remains a niche method that is applicable to pharmaceuticals and other high relative molecular mass solutes. Supercritical extraction, on the other hand, is more widely used as a sample preparation method, especially in pharmaceutical analyses, polymers, and environmental analyses. 

One area of preparative SFC that would benefit from further investigation is the sample injection technique. With the exception of on-line extraction/chromatogra-phy, the sample is usually introduced as a solution in an organic solvent aind injected onto the column by means of a loop rotary valve. The sample loop is then flushed with a high density liquid that eventually becomes the supercritical fluid mobile phase on entering the heated column. 

In order to demonstrate that scale-up can be successfully performed from lab to commercial scale, we performed the atomization of inulin (a polysaccharide extracted from chicory root) from NMP solutions (300 g/L) by antisolvent with supercritical CO2 (20 MPa, 40 °C) After the first test a lab scale (XO.l), we prepared samples in three plants 2 g in XI, 20 g in XIO, and 200 g in XlOO (80). As shown in Figure 11, the particle size distributions (by volume) are strictly the same at the three scales in the range for which we want to obtain a nondusty powder. Moreover, this work permits us to show that the fluid/substance ratio ( 50 kg/kg) can be optimized at a much lower value than generally stated in most publications (500-10,000). Extended work is now ongoing on therapeutic molecules and for smaller-sized particles on a large scale. 

---