Thin-layer chromatography preparative plates

Technique of thin-layer chromatography. Preparation of the plate. In thin-layer chromatography a variety of coating materials is available, but silica gel is most frequently used. A slurry of the adsorbent (silica gel, cellulose powder, etc.) is spread uniformly over the plate by means of one of the commercial forms of spreader, the recommended thickness of adsorbent layer being 150-250 m. After air-drying overnight, or oven-drying at 80-90 °C for about 30 minutes, it is ready for use. 

Taylor and co-workers further demonstrated the value of open-access LC/MS systems for generating a widened scope of pharmaceutical analysis applications, including (1) characterization of synthetic intermediates and target compounds (2) reaction monitoring (3) reaction optimization (4) analysis of preparative HPLC fractions and (5) analysis of thin layer chromatography (TLC) plate spots. The availability of these methods led to the increased use of LC/MS for structural analysis. The short analysis time and reliable structure confirmation resulted in the use of LC/MS as a first choice for structure characterization for synthetic chemistry applications, as well as an expanded, and perhaps, integrated role of sample generator and analyst. 

B. Apparatus (See Chromatography, Appendix IIA.) Assemble a suitable apparatus for ascending thin-layer chromatography. Prepare a slurry of chromatographic silica gel containing about 13% of calcium sulfate (1 g to each 2 mL of water) as the binder, apply a uniformly thin layer to glass plates of convenient size, dry in air for 10 min, and activate by drying at 100° for 1 h. Store the cool plates in a clean, dry place until ready for use. 

After anaerobic incubation of reaction mixture at 37°C for 24h, the alkane containing fraction was prepared by chloroform extraction. Chloroform extracts were separated on silica gel 60 thin layer chromatography (TLC) plate with hexane as a developing solvent. Radioactivity was measured by liquid scintillation counter (LS6500, Beckman). 

Instant thin layer chromatography (ITLC) plates are prepared from ground-up glass fibers impregnated with silica gel. This provides a uniform plate, readily wetted by the more polar solvents so capillary action is rapid. The plates are fragile and should be handled accordingly. 

Gas chromatography (gc) is inferior to hplc in separating abiUty. With gc, it is better to use capillary columns and the appHcation is then limited to analysis (67). Resolution by thin layer chromatography or dc is similar to Ic, and chiral stationary phases developed for Ic can be used. However, tic has not been studied as extensively as Ic and gc. Chiral plates for analysis and preparation of micro quantities have been developed (68). 

Examine by thin-layer chromatography (TLC) using a suitable TLC silica gel as the coating substance on the TLC plate. The method requires preparation of the following solutions ... 

Most of the reactions described in the following chapters were monitored by Thin Layer Chromatography (TLC) using plastic TLC plates coated with a thin layer of Merck 60 F254 silica gel. The products were detected by using an ultraviolet lamp or the TLC plates were treated with p-anisaldehyde reagent, prepared as explained below, and then heated to 120 °C to stain the spots. After visualization and measurement, the Rf values were recorded. 

In our laboratory crude preparations of aphantoxins and anatoxin-a(s) are extracted similarly except at the final stages of purification (Fig. 2). A Bio-gel P-2 column (2.2 x 80 cm) is used for aphantoxins gel filtration and a Sephadex G-15 (2.6 x 42 cm) column for ana-toxin-(s). Both toxins are eluted with 0.1 M acetic acid at 1.5 ml/ min. Fractions of aphantoxins from Bio-gel P-2 run are spotted on thin-layer chromatography plates (Silica gel-60, EM reagents) and developed according to Buckley et al. (1976) (31). The Rf values for the aphantoxins, saxitoxin and neosaxitoxin standards (Table 1) indicates that two of the aphantoxins (i.e. I and II) are similar to saxitoxin and neosaxitoxin. 

The filtrate obtained from this reaction mixture is reduced to a volume of 15.0mL in a rotating evaporator and purified by means of thin layer chromatography (TLC) preparative plates of 1-mm thickness (Kieselgel P. F. Merck) eluant diethyl ether (5% vol) in light petroleum ether. The dark brown band of the complex is eluted with dry diethyl ether (peroxide-free). 

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). 

Each column fraction is analyzed for lipid material by spotting on a 5 X 10 cm silica gel thin-layer plate and exposing it to iodine vapor as follows. Prepare seven tapered capillary tubes and use these to place a spot of each solution on the TLC plate. Put at least 10 capillary applications from a single fraction on a spot. Your final plate should then have seven spots, one for each fraction. Set the plate in an iodine chamber and allow it to remain for about 15 minutes or until some spots are yellow or red-brown. The presence of lipid in a fraction is indicated by the red-brown color. Retain all the column fractions that appear to have lipid. Each of these fractions will be analyzed in part B by thin-layer chromatography. 

Analysis of the purity of each silica gel column fraction and classification of the unknown lipid can be accomplished by thin-layer chromatography on silica gel plates. On a single plate will be spotted (1) a solution of the crude lipid extract from part A, (2) aliquots from each lipid fraction of the column (or recrystallized lipid), and (3) solutions of standard lipids (listed in the Materials section). On a 20 X 20 cm silica gel plate there is room for nine different analyses. Prepare a 1% solution of the crude lipid from part A in chloroform (10 mg/1 mL). If recrystallized lipid is to be... 

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