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SUPER CRITICAL FLUID EXTRACTION
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Figure 25.1 Supercritical uid extraction.
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instruments. These components include plant and animal pigments, lipids, organic material from soil and water, and inorganic compounds. If not removed, the impurities decrease the sensitivity of the detectors and columns in the analytical instrument, mask peaks, or produce extraneous peaks on chromatograms. Although some more recently developed instruments automatically remove these substances and concentrate the samples to small volumes for quantitative analysis, they are expensive. Thus most laboratories rely on other methods. These include adsorption chromatography, thin-layer chromatography (TLC), and solvent partitioning. Generally, adsorption chromatography is the method of choice to remove co-extractives from the compound in question. Because most techniques use large volumes of solvent, the solvent must be removed to obtain a working volume (e.g., 5 10 mL) that is easy to manipulate by the analyst. This is accomplished by distillation, evaporation under a stream of air or an inert gas such as nitrogen, or evaporation under reduced pressure. Once the working volume is reached, extracts can be further puri ed by one or more procedures. In addition to the use of adsorbents, many organic toxicants will distribute between two immiscible solvents (e.g., chloroform and water or hexane and acetonitrile). When shaken in a separatory funnel and then allowed to equilibrate into two original solvent layers, some of the toxicant will have transferred from the original extracting solvent into the other layer. With repeated additions (e.g., 4 to 5 volumes), mixing, and removal, most or all of the compound of interest will have been transferred, leaving many interfering compounds in the original solvent. Regardless of the separation method or combination of methods used, the toxicant will be in a large volume of solvent in relation to its amount that is removed as described. Final volumes used to identify and quantitate compounds generally range from 250 L to 10.0 mL. Recent advances in circuit miniaturization and column technology, the development of microprocessors and new concepts in instrument design have allowed sensitive measurement at the parts per billion and parts per trillion levels for many toxicants. This increased sensitivity has focused public attention on the extent of environmental pollution, because many toxic materials present in minute quantities could not be detected until technological advances reached the present state of the art. At present, most pollutants are identi ed and quanti ed by chromatography, spectroscopy, and bioassays. Once the toxicant has been extracted and separated from extraneous materials, the actual identi cation procedure can begin, although it should be remembered that the puri cation procedures are themselves often used in identi cation (e.g., peak position
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ANALYTICAL METHODS IN TOXICOLOGY
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in gas-liquid chromatography [GLC] and high-performance liquid chromatography [HPLC]). Thus no de nite line can be drawn between the two procedures.
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Chromatography. All chromatographic processes, such as TLC, GLC, HPLC, or capillary electrophoresis (CE), use a mobile and immobile phase to effect a separation of components. In TLC, the immobile phase is a thin layer of adsorbent placed on glass, resistant plastic, or berglass, and the mobile phase is the solvent. The mobile phase can be a liquid or gas, whereas the immobile phase can be a liquid or solid. Chromatographic separations are based on the interactions of these phases or surfaces. All chromatographic procedures use the differential distribution or partitioning of one or more components between the phases, based on the absorption, adsorption, ionexchange, or size exclusion properties of one of the phases. Paper Chromatography. When the introduction of paper chromatography to common laboratory use occurred in the mid-1930s, it revolutionized experimental biochemistry and toxicology. This technique is still used in laboratories that lack the expensive instruments necessary for GLC or HPLC. The stationary phase is represented by the aqueous constituent of the solvent system, which is adsorbed onto the paper; the moving phase is the organic constituents. Separation is effected by partition between the two phases as the solvent system moves over the paper. Although many variations exist, including reverse-phase paper chromatography in which the paper is treated with a hydrophobic material, ion-exchange cellulose paper, and so on, all have been superseded by equivalent systems involving thin layers of adsorbents bonded to an inert backing. Thin-Layer Chromatography. Many toxicants and their metabolites can be separated from interfering substances with TLC. In this form of chromatography, the adsorbent is spread as a thin layer (250 2000 m) on glass, resistant plastic or berglass backings. When the extract is placed near the bottom of the plate and the plate is placed in a tank containing a solvent system, the solvent migrates up the plate, and the toxicant and other constituent move with the solvent; differential rates of movement result in separation. The compounds can be scraped from the plate and eluted from the adsorbent with suitable solvents. Recent developments in TLC adsorbents allow toxicants and other materials to be quantitated at the nanogram (10 9 g) and picogram (10 12 g) levels. Column: Adsorption, Hydrophobic, Ion Exchange. A large number of adsorbents are available to the analyst. The adsorbent can be activated charcoal, aluminum oxide, Florisil, silica, silicic acid, or mixed adsorbents. The characteristics of the toxicant determine the choice of adsorbent. When choosing an adsorbent, select conditions that either bind the co-extractives to it, allowing the compound of interest to elute, and vice versa. The ef ciency of separation depends on the ow rate of solvent through the column (cartridge) and the capacity of the adsorbent to handle the extract placed on it. This amount depends on the type and quantity of adsorbent, the capacity factor (k ) and concentration of sample components, and the type and strength of the solvents used to elute the compound of interest. Many environmental samples contain a suf cient amount of interfering materials so that the analyst must prepare a column using a glass chromatography tube into which the adsorbent is added. In the most common
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