Postchromatographic derivatization

These few questions serve to demonstrate that there must be great interest in characteri2dng chemical compounds by means of their reactive functional groups. The most important group-specific reagents for postchromatographic derivatization are listed alphabetically in Table 4 below ... 

Prechromatographic derivatization reactions re usually favored when it is desired to modify the properties of the sample to enhance stability during measurement (i.e., minimize oxidative and catalytic degradation, etc.), to improve the extraction efficiency of the substance during sample cleanup, to improve the chromatographic resolution, or to simplify the optimization of the reaction conditions [698-702]. As both pre- and postchromatographic methods enhance the sensitivity and selectivity of the detection process a choice between the two methods will usually depend on the chemistry involved, ease of optimization, and which method best overcomes matrix and reagent interferences. 

Ultraviolet-visible (UV-Vis) detection in liquid chromatography is often hampered by the poor spectral properties of the analytes at the applied analytical wavelengths. In this respect, a variety of pre- and postchromatographic derivatization reactions have been proposed for improving UV-Vis detectability. 

When using photochemical reactions, a gain in fluorescence output or electroactivity not only lowers detection limits but also contributes to confirm an analytical result. Examples are the conversion of diethylstilbestrol to a fluorescent hexahydrophenanthrene (277), and the conversion of fenbendazole to fluorescent species (278). Tlie fluorescence of photoconverted diethylstilbestrol can further be enhanced by a subsequent online postchromatographic derivatization with bisulfite to the highly fluorescent phenanthrenediol (279). Another example of photolytic derivatization is the postchromatographic conversion of penicillins and cephalosporins into electroactive species that can be detected by an amperometric detector (280). 

This type of derivatization seems promising for the detection of drugs and metabolites with phenolic groups. A recent application on the determination of chlorophenols in surface water showed that the dansyl derivatives of phenols are readily convertible to the highly fluorescent dansyl-OH and dansyl-OCHs products after postchromatographic irradiation (281). Fluorescence gain factors of up to 8000-fold were obtained for chlorophenol derivatives with a low native fluorescence. 

Fater, Z. Tasi, G. Szabady, B. Nyiredy, S. Identification of amphetamine derivatives by uni-dimensional multiple development and two-dimensional HPTLC combined with postchromatographic derivatization. J. Planar Chromatogr.-Mod. TLC 1998, 11, 225-229. 

The authors recommend using 5-10 relatively low concentrations of analyte spotted on a TLC plate. DL can be increased by using pre- or postchromatographic derivatization. ... 

The aim of every postchromatographic derivatization, which, rmlike prechromatographic reactions, should be performed as quickly as possible, is therefore principally ... 

In contrast to postchromatographic derivatization, reactions performed before the chromatography are used... 

The advantages of postchromatographic reaction methods are that the separation is unaffected by the reaction, by-products of the reaction do not interfere in the chromatogram, and all samples and standards are derivatized simultaneously and... 

Postchromatographic derivatization reactions can be classified as reversible or destructive depending on the type of interaction the reagents undergo with the separated compounds, and as selective or universal, based on the specificity of the reaction. The most common reversible methods employ iodine vapor, water, fluorescein, or pH indicators as visualizing reagents [193-95,201]. In the iodine vapor method, the dried plate is enclosed in a chamber containing a few crystals of iodine substance zones are... 

Although in situ) prechromatographic derivatization of the analytes in thin-layer chromatography (TEC) has been described in several cases, much more important are the postchromatographic reactions with various reagents. 

The aim of postchromatographic derivatization is to make the separated compounds visible at daylight or to transform them to fluorescent derivatives, thus enabling primarily the sensitivity and often also the selectivity of the detection and of the densitometric determination to be increased. 

Prechromatographic derivatization is a useful tool to change the chromatographic behavior of the sample or some of its components in order to increase their stability, improve separation, or reduce matrix effects. It is either performed as part of sample preparation or following sample application in situ on the TLC plate. Postchromatographic derivatization is a necessary step in TLC if a sample component is otherwise undetectable and if selectivity or sensitivity of detection can be improved. 

As an offline technique the TLC process consists of several individual steps. Traditionally, instruments for each step are controlled by separate more or less sophisticated software. winCATS-Planar Chromatography Manager (CAMAG, Switzerland) is the first integrated software that can communicate with all instruments involved in the TLC process. A win-CATS method file can include information about the stationary phase, samples and their components, standards and their preparation, application parameters, prechromatographic derivatization, development conditions, postchromatographic derivatization, all parameters of densitometric and spectral evaluation, and details of electronic documentation using a... 

Table 1 Reagents used for postchromatographic derivatization of different classes of compounds. 

Cimpan, G. Pre- and postchromatographic derivatization. In Planar Chromatography, Nyiredy, Sz., Ed. Springer Scientific Publisher Budapest, Hungary, 2001 410-445. 

Ethyl acetate, postchromatographic derivatization with 25% polypropylene glycol in n-hexane... 

Acetonitrile, in reverse direction with acetone, postchromatographic derivatization with 0.05% primuline in acetone-water (4 1)... 

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