Plate development, thin layer chromatography

Occasionally use several drops to spot, develop, and visualize a thin layer chromatography plate. Although thin layer is very similar to column, you should read up on it as 1 do not have time to go into the complete operation. 

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. 

E. Merck cellulose thin-layer chromatography plates (available from American Scientific Products) are developed with chromatography buffer (Note 13) and visualized with sulfosalicylic acid/ferric chloride spray. The system consists of a solution of 1.0 g of sulfosal icyl ic acid (from Aldrich... 

A diagram of a typical thin-layer chromatography plate after development and spraying to locate the analytes is shown in Figure 13.2. 

The mobile phases used in the development of thin-layer chromatography plates are usually combinations of organic and aqueous solvents. Ethanol, t-amyl alcohol, acetonitrile, and methanol are commonly used organic solvents. Acetic acid is the most commonly used aqueous solvent. In normal-phase partition chromatography the polar stationary phase is developed with relatively nonpolar mo-... 

I2 Chamber—Fill the bottom of a rectangular glass chamber with I2 (solid) and cover tightly. Allow 6 to 8 hr for the chamber to saturate with I2 vapors before the thin-layer chromatography plates are developed. 

Fernando, W.P.N. Poole, C.F. Determination of kinetic parameters for precoated silica gel thin layer chromatography plates by forced flow development. J. Planar Chromatogr. 1991, 4, 278-287. 

D-enantiomers of amino acids have been frequently reported in various tissues of diverse organisms. A simple and rapid method of separating optical isomers of amino acids on a reverse-phase thin layer chromatography plate is described. Amino acids, derivatized with l-fluoro-2,4-dinitrophenyl-5-L-alanine amide, were spotted onto a reverse-phase thin layer chromatography plate. Acetonitrile in triethylamine-phosphate buffer was used as the developer. 

Figure 2 Thin layer chromatography plate (K6F - Whatman) traces of the standard extract labelled at 100,000 uic (top) and the MAP extract (bottom). The plate was eluted in a hexane propanol mixture (92 8) and did not require any developing agent given the deep coloration of the extracts. For the purpose of reproduction we have digitised the plate at 16,000,000 colours and converted the digital file into a 256-tone gray-scale one. Note that despite this dramatic graphical loss in resolution the differences between the two products are readily observed.

Novel analytical techniques such as forced-flow planar chromatography (FFPC) and optimum pressure laminar chromatography (OPLC) are other additions to ever-refined tools for separation on a preparative scale, wherein small amounts of complex mixtures may be separated more efficiently on thin-layer chromatography plates operating at fast medium-pressure development with continuous collection of mobile phase at the end of chromatographic plates (Nyredy, 20(X), 2003). 

Figure 1.1 Separation of complex standard lipid mixtures on a 20 cm x 20 cm high-performance thin-layer chromatography plate using four developments. The first development was up to a distance of 5 cm above the origin in the solvent system ethyl acetate/1-propanol/chloroform/ methanol/0.025% KCl, 25 25 25 10 9 vol./vol. The second development was up to 8 cm above the origin in the solvent system toluene/ether/ethanol/acetic acid, 60 40 1 0.23 vol./vol. The third development was to the full length (9 cm) in the solvent system hexane/diethyl ether, 94 6 vol./ vol., followed by the last development to full length in hexane. The plates were freed of solvent between developments by blowing with hot air. Reproduced with permission from Yao, J. K. and Rastetter, G. M., Microanalysis of complex tissue lipids by high-performance thin-layer chromatography, Analytical Biochemistry, 150, 111-16.

Gresser et al. (1995) combined their PPF with VP and hydroxyapatite, crosslinking this mixture with benzoyl peroxide and DMT. Samples were allowed to crosslink at 37°C for 24 hours. These hardened samples were ground to analyze the amount of PPF and VP incorporated. The ground power was run in acetonitrile on reverse-phase thin layer chromatography plates, and developed with iodine vapor or ultraviolet light. Over 90% of the PPF was found to have been crosslinked in mixtures containing between 0.33 to 2 PPF/W ratios. The fraction of VP incorporated was dependent on the ratio of the two components. The fraction of PPF or VP incorporated was independent of the amount of hydroxyapatite, benzoyl peroxide, or DMT used. The VP monomer was found to add to poly( inyl pyrrolidinone) twice as often as it added to the fumarate double bond. 

Zellmer, S., and Lasch, J. (1997). Individual variation of human plantar stratum comeum lipids, determined by automated multiple development of high performance thin layer chromatography plates. J. Chromatogr., B Biomed. Appl. 691 321-329. Zieloff, K. (1995). Automation in thin layer chromatography and good laboratory practice (GLP). Oesterr. Chem. Z. 96 120-122. 

Decant off extract and leave overnight in a deep freezer, filter and evaporate off solvent from the filtrate at room temperature. Redissolve the extract in 1 cm of chloroform and apply amounts of 1 - 10 ul to a thin-layer chromatography plate evaporating off solvent with cold air between applications. After development examine plate under short and long UV and recotd observations. 

A methanol extract of the polyurethane is applied to a thin-layer chromatography plate and developed with a mixture of chloroform, ethylacetate, ethanol, glacial acetic acid (120 33 20 7 v.v). Fluran spray reagent reveals the two diaminotoluene isomers. The intensity of the spots are evaluated (500 nm excitation 340 nm emission) using a spectrofluorimeter coupled to a thin film scanner and photomultiplier microphotometer. The method is calibrated against standard solutions of the two diaminotoluene isomers. 

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). 

The progress of the reaction may be followed by analytical thin-layer chromatography on alumina. The submitters used polygram pre-coated plastic sheets (Alox N/UV254) purchased from Macherey-Nagel, Inc. The plates were developed with 1 1 hexane-ether and stained with basic permanganate. The Rf of the product is 0.56. 

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