Supercritical fluid analytical procedures

High performance liquid chromatography (HPLC) has been by far the most important method for separating chlorophylls. Open column chromatography and thin layer chromatography are still used for clean-up procedures to isolate and separate carotenoids and other lipids from chlorophylls and for preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography. The whole analysis should be as brief as possible, since each additional step is a potential source of epimers and allomers. 

Garimella et investigated the effect on trifluralin recovery of different extraction methods. A supercritical fluid extraction (SEE) procedure for the isolation of the analytes from the matrices with a commercial SEE system (Dionex Model 703)... 

If simple sample pretreatment procedures are insufficient to simplify the complex matrix often observed in process mixtures, multidimensional chromatography may be required. Manual fraction collection from one separation mode and re-injection into a second mode are impractical, so automatic collection and reinjection techniques are preferred. For example, a programmed temperature vaporizer has been used to transfer fractions of sterols such as cholesterol and stigmasterol from a reversed phase HPLC system to a gas chromatographic system.11 Interfacing gel permeation HPLC and supercritical fluid chromatography is useful for nonvolatile or thermally unstable analytes and was demonstrated to be extremely useful for separation of compounds such as pentaerythritol tetrastearate and a C36 hydrocarbon standard.12... 

Fig. 3.70. Flow scheme for the analytical procedure based on supercritical fluid extraction (SFE). Reprinted with permission from C. S. Eskilsson et al. [140],...

Current practices in analytical SFE are organized into off-line and on-line procedures, despite their common physicochemical basis. Off-line SFE, the current method in fashion, offers more flexibility with respect to extracting different sample sizes and types, as well as in the choice of the final analytical method. On-line procedures are usually combinations of SFE with ancillary techniques such as GC, LC, supercritical fluid, or gel permeation chromatography. 

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). 

Tire aqueous or organic extract obtained at this point may be a very dilute solution containing interfering compounds and making it difficult to determine trace level concentrations of the analyte(s) of interest. To reduce interferences and concentrate the analyte(s), the primary sample extract is furiher subjected to various types of sample cleanup procedures such as conventional liquid-liquid partitioning, solid-phase extraction, matrix solid-phase dispersion, online trace enrichment, liquid chromatography, online dialysis and subsequent trace enrichment, and supercritical fluid extraction. In most cases some of Urese procedures are used in combination to obtain highly purified extracts. 

Starting with a description of the analytical challenge in Chapter 19, the third part, which is devoted to analytical attitudes, proceeds with a detailed description in Chapter 20 of modern sample preparation procedures including solid-phase extraction, matrix solid-phase dispersion, use of restricted-access media, supercritical fluid extraction, and immunoaffinity cleanup. Flexible derivatization techniques including fluorescence, ultraviolet-visible, enzymatic, and photochemical derivatization procedures are presented in Chapter 21. 

Solid-phase extractions can reduce solvent consumption in analytical chemistry. For example, a standard procedure approved by the U.S. Environmental Protection Agency for the analysis of pesticides in wastewater requires 200 mL of dichloromethane for the liquid-liquid extraction of 1 L of water. The same analytes can be isolated by solid-phase extraction on C g-silica disks. The pesticides are recovered from the disks by supercritical fluid extraction with C02 that is finally vented into a small volume of hexane. This one kind of analysis can save 10s kg of CH2C12 per year.24... 

Supercritical fluid extraction (SFE) has the potential to change the analytical laboratory and its current extraction procedures dramatically. Predictions on the monetary exploitation of this technique are astronomical. Subsequently, the amount of research, development and applications in SFE is growing rapidly. 

An underlying assumption in these discussions is that SFE is a viable alternative for sample preparation procedures for a significant number of samples - even though equipment more sophisticated than traditional laboratory glassware is necessary. For example, SFE systems can be operated at temperatures up to 150 C and pressures to 600 bar using a variety of fluids. The unique characteristics of supercritical fluids which make them so attractive as solvents have been discussed fully on many occasions elsewhere (15-17) a similar discussion is outside the scope of this paper. However, in the next section we will briefly 1. explore the use of supercritical fluids from the perspective of potentially enhanced robustness and 2. outline considerations which are typically considered prior to analytical methods development and which should be employed for SFE as for any other technique. 

Metabolites of chlorobenzene in biological materials cannot be determined in routine practice because of the lack of standard methods for measuring these metabolites. Further research on supercritical fluid (SCF) extraction holds great promise for meeting the goals of quantitative, rapid, easily performed, low cost, and safe procedures for the determination of nonpolar organic analytes such as chlorobenzene in biological samples. 

To understand any extraction technique it is first necessary to discuss some underlying principles that govern all extraction procedures. The chemical properties of the analyte are important to an extraction, as are the properties of the liquid medium in which it is dissolved and the gaseous, liquid, supercritical fluid, or solid extractant used to effect a separation. Of all the relevant solute properties, five chemical properties are fundamental to understanding extraction theory vapor pressure, solubility, molecular weight, hydrophobicity, and acid dissociation. These essential properties determine the transport of chemicals in the human body, the transport of chemicals in the air water-soil environmental compartments, and the transport between immiscible phases during analytical extraction. 

In more recent years, supercritical fluid extraction has been found to be useful for the extraction of low to moderately polar compounds. The requirements in any particular case depend on the characteristics of the matrix, the drug, and the drug metabolites. The concentration range of the drug is also obviously important, as it will determine the methods selected for the separation and analytical procedures. 

The choice of the most suitable instrumental technique depends on several factors, such as the physical-chemical characteristics of analytes, the detection limits required, the level and type of interferences, the resolution needed, the identification power required, the accuracy and the precision of the quantitative determination, the availability of instrumentation and finally the cost and the time necessary per each determination. Moreover, extraction and clean-up procedures have to be suitably matched with instrumental analysis. GC coupled with Electron Capture Detection (ECD) or Mass Spectrometry (MS) has been widely applied for the determination of PCBs in organic extracts of environmental samples. In few cases the instrumentation includes the extraction step, such as an SEE system coupled with Supercritical Fluid Chromatography (SFC) or with GC (40). 

Supercritical Fluids (SFs) allow analytes to be extracted from solid samples, i.e., marine sediments, faster and more efficiently since they have lower viscosity and higher diffusivity than liquid solvents (56). CO2 is the most widely used supercritical fluid with or without a modifier, e.g. methanol and toluene. A very exhaustive discussion on the role of a modifier in the enhancement of the extraction efficiency was recently published (39). Few procedures have been described in the literature based on SFE of organic pollutants from environmental samples, including PCBs and PAHs (39, 41, 56-59). Generally, the extraction is performed... 

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