Thin-layer chromatography sample preparation

Clark et al. [53] subjected primaquine to metabolic studies using microorganisms. A total of 77 microorganisms were evaluated for their ability to metabolize primaquine, of these, 23 were found to convert primaquine to one or more metabolites (thin-layer chromatography analysis). Preparative scale fermentation of primaquine with four different microorganisms resulted in the isolation of two metabolites, identified as 8-(3-carboxy-l-methylpropylamino)-6-methoxyquinoline and 8-(4-acetamido-l-methylbutylamino)-6-methoxyquinoline. The structures of the metabolites were proposed, based primarily on a comparison of the 13C NMR spectra of the acetamido metabolite and the methyl ester of the carboxy metabolite with that of primaquine. The structures of both metabolites were confirmed by direct comparison with authentic samples. 

One of trends of development of thin-layer chromatography implies that replacement of aqueous-organic eluents by micellar surfactants solution. This is reduces the toxicity, flammability, environmental contamination and cost of the mobile phases, reduce sample prepar ation in some cases. 

To obtain reliable chromatograms in the final step of the determination of the analytes by LC or GC, it is important to remove interfering signals resulting from coelution of other compounds. To this end, a variety of techniques are applied for cleanup of the sample extract. The most effective procedures for sample cleanup for PAH measurements are partitioning between M, N-dimethylformamide/water/cyclo-hexane and LC on silica and on Sephadex LH 20. Other cleanup procedures include LC on alumina or XAD-2 and preparative thin-layer chromatography. 

All previous discussion has focused on sample preparation, i.e., removal of the targeted analyte(s) from the sample matrix, isolation of the analyte(s) from other co-extracted, undesirable sample components, and transfer of the analytes into a solvent suitable for final analysis. Over the years, numerous types of analytical instruments have been employed for this final analysis step as noted in the preceding text and Tables 3 and 4. Overall, GC and LC are the most often used analytical techniques, and modern GC and LC instrumentation coupled with mass spectrometry (MS) and tandem mass spectrometry (MS/MS) detection systems are currently the analytical techniques of choice. Methods relying on spectrophotometric detection and thin-layer chromatography (TLC) are now rarely employed, except perhaps for qualitative purposes. 

Thin-layer chromatography (TLC) is one of the most popular and widely used separation techniques because of its ease of use, cost-effectiveness, high sensitivity, speed of separation, as well as its capacity to analyze multiple samples simultaneously. It has been applied to many disciplines including biochemistry [1,2], toxicology [3,4], pharmacology [5,6], environmental science [7], food science [8,9], and chemistry [10,11]. TLC can be used for separation, isolation, identification, and quantification of components in a mixture. It can also be utilized on the preparative scale to isolate an individual component. A large variety of TLC equipment is available and discussed later in this chapter. 

A good general purpose screening technique for organic explosive traces, albeit often undervalued, is thin-layer chromatography (TLC). The advantages of TLC are that it requires only Hmited capital equipment, litde sample preparation other than dissolution in a suitable solvent, and that it provides rapid results that are easily interpreted and explained [16]. 

Thin-layer chromatography (TLC), sometimes also called planar chromatography, employ a stationary phase immobilized on a glass or plastic plate and an organic mobile phase. It is a rather old technique whose application in residue analysis has been limited in the past by poor chromatographic resolution, inadequate selectivity, and insufficient sensitivity (49). This was due to inherent problems in the quality of the available stationary phase materials and in the uniformity of the layers prepared. Today, the availability of affordable, precoated plates with acceptable performance and consistency has led to the general acceptance of TLC as an efficient procedure for residue analysis (50). The method is used preferentially when analysts must process large numbers of samples in a short period of time (51). 

The quality of FAME prepared by the methods described in this unit must be examined by GC analysis. Generally, impurities in the extracted lipid samples are not removed before methylation. If the GC results are not satisfactory due to sample contamination, additional steps may be necessary to clean the sample either before or after methylation. Commonly used techniques for purifying lipid samples are thin-layer chromatography (TLC), solid phase extraction (SPE), and column chromatography. 

Chi is purified from chloroplast extracts, usually obtained from spinach leaves, by dioxane precipitation method (51) and conventional sugar column chromatography (52). For rapid and easy preparation, the method recently developed by Omata and Murata (53) is satisfactory synthetic (DEAE-) Sepharose is substituted for sugar on the column. Besides using spectroscopic criteria (52), the purity of Chi samples can be checked readily by means of silica-gel thin layer chromatography (54). Colorless contaminations in Chi... 

The identification of the major products of the photooxidation of phenothiazine upon its direct excitation does not support the idea of a N-hydroperoxy intermediate (Equation 5) (11). In fact, phenothia-zine-5-oxide (A) which has been first identified by comparing Rf values in thin layer chromatography of isolated and separately prepared samples (11) represents the best evidence of a singlet oxygen reaction (Equation 9) (12). 

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