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A very realistic possibility of increasing the efficiency and rapidity of planar chromatography is a novel category of multilayer OPLC, the long-distance OPLC (65,66), where the efficiency of the separation is increased significantly. With this technique the end of the first chromatoplate has a slit-like perforation to permit the mobile phase to migrate to a second layer. Clearly, on this basis a very long separation distance can be achieved by adding one plate to another (65).
The sample application is one of the most important steps for a successful planar chromatographic separation; this has been summarized by Kaiser et al. (67).
During the development method the applied development mode, forced-flow technique, and development distance always depends on the separation distance, as is summarized in Figure 7a and 7b in a flow chart.
IV. DETECTION AND QUANTITATION
A. Conventional Detection Modes
For the visualization of compounds, one can use physical, chemical, or biological detection methods. Physical detection methods are based on substance-specific properties. The most commonly em-
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Figure 7 Flow chart for the selection of development mode, development distance, and forced-flow techniques. a) Selection of development mode, b) Selection of development distance and forced-flow techniques.
ployed methods are the absorption or emission of electromagnetic radiation, which is measured by detectors. The ^-radiation of radioactiveiy labeled compounds can also be detected directly on the plate. These nondestructive detection methods allow subsequent isolation and can also be followed by microchemical and/or biological detection methods (68). Since physical detection methods are frequently not sufficient to establish identity, they must be complemented by specific chemical reactions (derivatization). These reactions may be carried out either before or after chromatography.
Prechromatographic derivatization can be performed either during sample preparation or on the chromatoplate at the origin. It is generally used to introduce a chromophore, leading to the formation
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of strongly absorbing or fluorescent derivatives, to increase the selectivity of the separation, enhance the sensitivity of detection, and improve the linearity. It includes oxidation, reduction, hydrolysis, halogenation, nitration, diazotization, esterification, etherification, hydrazone formation, and dansylation (68).
The primary aim of postchromatographic derivatization is the detection of the chromatographi-cally separated compounds for better visual evaluation of the chromatogram. This step generally improves the selectivity and the detection sensitivity. Postchromatographic reactions can be carried out by spraying reagents onto the chromatoplate, by dipping the layer in reagent solutions, or exposing the plate to vapors. The reagent can also be in the solvent system or in the adsorbent. In most instances, subsequent heating is necessary. Postchromatographic deri vatizations have been extensively reviewed (68-70). Recently a new method, overpressure derivatization, was reported (71) that can be carried out by pressing an absorbent polymeric pad (prewetted with derivatization reagent) on the TLC plate.
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For biological/physiological detection, the separated compounds can be transferred to the biological system. Alternatively, bioautographic analyses, reprint methods, and enzymatic tests may also be applied.
Reagents and detection methods have been summarized in a practical textbook by Jork et al. (36,69). A summary on postchromatographic derivatization in quantitative TLC for pharmaceutical applications was reported in (72).
B. Instrumental Detection Modes
Regardless of whether they are colored or colorless compounds, absorbing in the UV range can be detected by direct scanning at the wavelength of maximum absorption of the compound in the sample. Some types of compound are naturally fluorescent, while others can be converted to fluorescent derivatives through pre- and/or postchromatographic reactions; this is a highly specific and sensitive method. Fluorescence quenching is limited to compounds that readily adsorb in the wavelength range of maximum excitation of a phosphor incorporated into the stationary phase (37).
Instruments for scanning densitometry after the above treatments can be operated in the reflectance, transmission, or combined reflectance/transmission mode. Usually the sample beam is fixed and the plate is scanned by mounting it on a movable stage, controlled by a stepper motor. The geometry of the light beam of scanners can have the form of a slit or a spot (73). Different principal optical geometries are used in scanning densitometry: the single-beam method, which can be used in the reflectance, transmittance, or simultaneous mode, and the double-beam method. In the latter case the two beams can be either separated in time at the same point on the chromatoplate or separated in space and recorded simultaneously by two detectors (37).