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sequence the column is packed in an organic solvent of low polarity, the sample is added in the same solvent, and the column is then developed with a sequence of solvents or solvent mixtures of increasing polarity. Such a sequence might include (in order of increasing polarity) hexane, benzene, chloroform, acetone, and methanol. Once removed, the eluate containing the toxicant is reduced to a small volume for quantitation. However, cartridge technologies are improving to allow similar concentrations of sample to be added that result in a less expensive and more rapid analysis. A number of miniaturized columns have been introduced since the early 1980s. Most contain 0.5 to 2.0 g of the adsorbent in a plastic tube with tted ends. The columns can be attached to standard Luer Lock syringes. Other companies have designed vacuum manifolds that hold the collecting device. The column is placed on the apparatus, a vacuum is applied, and the solvent is drawn through the column. Some advantages of these systems include preweighed amounts of adsorbent for uniformity, easy disposal of the co-extractives remaining in the cartridge, no breakage and decreased cost of the analysis because less solvent and adsorbent are used. Other forms of column chromatography can be used. They include ion-exchange chromatography, permeation chromatography, and af nity chromatography. Ion-exchange chromatography depends on the attraction between charged molecules and opposite charges on the ion exchanger, usually a resin. Compounds so bound are eluted by changes in pH and, because the net charge depends on the relationship between pH of the solution and the isoelectric point of the compounds, compounds of different isoelectric point can be eluted sequentially. Both ionic and anionic exchangers are available. Permeation chromatography utilizes the molecular sieve properties of porous materials. Molecules large enough to be excluded from the pores of the porous material will move through the column faster than will smaller molecules not excluded, thus separating them. Cross-linked dextrans such as Sephadex or agarose (Sepharose) are commonly used materials. Af nity chromatography is a potent tool for biologically active macromolecules but is seldom used for purifying small molecules, such as most toxicants. It depends on the af nity of an enzyme for a substrate (or substrate analogue) that has been incorporated into a column matrix or the af nity of a receptor for a ligand.
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Gas-Liquid Chromatography (GLC). GLC is used most commonly for the separation and quantitation of organic toxicants. This system consists of an injector port, oven, detector, ampli er (electrometer), and supporting electronics (Figure 25.2). Current modern gas chromatographs use a capillary column to effect separation of complex mixtures of organic molecules and has replaced, to a large extent, the packed column. Instead of coating an inert support, the stationary phase is coated onto the inside of the column. The mobile phase is an inert gas (called the carrier gas), usually helium or nitrogen that passes through the column. When a sample is injected, the injector port is at a temperature suf cient to vaporize the sample components. Based on the solubility and volatility of these components with respect to the stationary phase, the components separate and are swept through the column by the carrier gas to a detector, which responds to the concentration of each component. The detector might not respond to all components. The electronic signal produced as the component passes through the detector is ampli ed by the electrometer, and the resulting signal is sent to a recorder, computer, or electronic data-collecting device for quantitation.
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Injection Port
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Column Controls Oven Data System Gas-Liquid Chromatograph
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Figure 25.2 Gas-liquid chromatograph.
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Column Technology. Increased sensitivity and component resolution have resulted from advances in solid-state electronics and column and detector technologies. In the eld of column technology, the capillary column has revolutionized toxicant detection in complex samples. This column generally is made of fused silica 5 to 60 m in length with a very narrow inner diameter (0.23 0.75 mm) to which a thin layer (e.g., 1.0 m) of polymer is bonded. The polymer acts as the immobile or stationary phase. The carrier gas ows through the column at ow rates of l to 2 ml/min. Two types of capillary columns are used: the support-coated, open tubular (SCOT) column and the wall-coated, open tubular (WCOT) column. The SCOT column has a very ne layer of diatomaceous earth coated with liquid phase, that is deposited on the inside wall. The WCOT column is pretreated and then coated with a thin lm of liquid phase. Of the two columns, the SCOT is claimed to be more universally applicable because of large sample capacity, simplicity in connecting it to the chromatograph, and lower cost. However, for dif cult separations or highly complex mixtures, the WCOT is more ef cient and is used to a much greater extent. Many older chromatographs are not designed to accommodate capillary columns, and because of these design restrictions, manufacturers offer the wide-bore capillary column along with the ttings and valving required to adapt the columns to older instruments. These columns also can be used on current instruments. With inner diameters of 0.55 to 0.75 mm, ow rates of 5.0 to 10.0 ml/min of carrier gas can be used to affect separations of components approaching that of the narrow-bore columns. Water samples chromatographed on capillary columns routinely separate 400 to 500 compounds, as compared with 90 to 120 resolved compounds from the packed column. Detector Technology. The second advance in GLC is detector technology. Five detectors are used widely in toxicant detection: the ame ionization (FID), ame photometric (FPD), electron capture (ECD), conductivity, and nitrogen-phosphorous detectors. Other detectors have application to toxicant analysis and include the Hall conductivity detector and the photoionization detector. The FID operates on the principle of ion formation from compounds being burned in a hydrogen ame as they elute from a column. The concentrations of ions formed
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