Thin-layer chromatographic plate

Figure 9. Infrared spectrum of pyrethrin I decomposition product isolated from thin-layer chromatographic plate...

Isolation of Sesquiterpene Lactones. The ether extract was evaporated and dissolved in 952 ethanol. Then an equal volume of 42 aqueous lead acetate was added. After 1 hour the mixture was filtered to remove precipitated chlorophyll and phenolic products and the ethanol removed under vacuum. The aqueous layer was extracted with chloroform giving a dark colored oil from which the sesquiterpenes were isolated by a combination of chromatographic procedures, i.e., LH-20 gel permeation, silica gel using both packed columns and thin layer plates. A variety of solvents were also used to purify the individual sesquiterpene lactones, e.g., benzene-acetone (1 1), ethyl acetate, chloroform-methanol (9 1). On thin layer chromatographic plates, spots were visualized by spraying with 22 aqueous KMn04 solution. 

Separated metabolites (and parent compound) are quantitated by first scanning developed thin-layer chromatographic plates to locate radioactive spots by means of a radiochromatogram scanner and then accurately measuring radioactivity by scintillation counting. Unknowns are identified by the usual chemical and spectrometric methods. 

Guittard, J. Hronowski, X.L. Costello, C.E. Direct MALDI Mass Spectrometric Analysis of Glycosphingolipids on Thin Layer Chromatographic Plates and Transfer Membranes. Rapid Commun. Mass Spectrom. 1999,13, 1838-1849. 

Indinavir sulfate may be analyzed by TLC using E. Merck silica gel 60F-254 high performance thin layer chromatographic plates, and eluted with a mobile phase of 8 1 1 (v/v/v) ethyl acetate methanol ammonium hydroxide [8], Visualization was performed by developing the plate with iodine stain, and viewing under short-wave UV (254 nm) light. When using this method, the Rf of indinavir sulfate is approximately 0.6 [8]. 

Since interferences could not be adequately eliminated with the alumina column, Gissel and Schaal (611) proposed an alternative procedure for extract purification involving use of Kieselgel thin-layer chromatographic plates, elution with trichloroethane/ethanol (8 2), and detection at 254 nm. The detection limit for three thiouracil residues in thyroid glands was in the range 0.5-1.0 ppm. 

A more comprehensive approach was reported in 1975 by Brabander and Verbeke (612). In this method, tissue samples were extracted with methanol and the acidified extract defatted with petroleum ether to be loaded onto a Dowex 50W-X8 anion-exchange resin. Following elution with aqueous methanol, the concentrated buffered extract was further defatted with diethyl ether. The sample was derivatized with 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole (NBD-Cl) to be further spotted on a silica high-performance thin-layer chromatographic plate developed in two dimensions using chloroform/ethanol and chloroform/propionic acid consecutively as eluents. Detection of the propylthiouracil, phenylthiouracil, and tapazole residues was carried out on the basis of the fluorescence induction of the NBD derivatives of the drugs with an alkaline cysteine solution. 

Khan, M.U. and Williams, J.P., Microwave-mediated methanolysis of lipids and activation of thin-layer chromatographic plates, Lipids, 1993, 28,953. 

C. F. Poole and M. T. Belay, The influence of layer thickness on the chromatographic and optical properties of high performance silica gel thin layer chromatographic plates , J. Planar Chromatogr. 4 345-359 (1991). 

Draper WM, Crosby DG. Sensitive, enzyme-catalyzed chromogenic reagent for hydrogen peroxide and other peroxygen compounds on thin-layer chromatographic plates. J Chromatogr 1981 216 413—416. 

J. Guittard, X. P. L. Hronowski, and C. E. Costello, Direct matrix-assisted laser desorption/ ionization mass spectrometric analysis of glycosphingolipids on thin layer chromatographic plates and transfer membranes, Rapid Commun. Mass Spectrom., 13 (1999) 1838-1849. 

Vovk I, Simonovska B and Vuorela H, Separation of eight selected flavan-3-ols on cellulose thin-layer chromatographic plates. J Chromatogr A 1077 188-194 (2005). 

C. Sarbu, Detection of some non-steroidal anti-inflammatory agents by thin-layer chromatographic plates coated with fluorescent mixtures, J. Chromatogr., 367 286 (1986). 

Figure 12. Relative spectral distribution of a low-pressure mercury vapor lamp with visible light cut-off filter as measured through the emission monochromator of a spectrofluorometer (2 nm slit width). (Typical of lamps used to view fluorescence/ fluorescence quenching on thin-layer chromatographic plates.)...

Scanning the developed colored spot on the thin layer chromatographic plate with the help of a photoelectric densitometer. 

A variety of precoated thin-layer chromatographic plates are available from commercial suppliers. The types described here are ones that have proven successful in the author s laboratory, but others could be equally adequate depending on the investigator s preference. 

Another type of precoated thin-layer chromatographic plate that is particularly effective for a separation of lipids is Whatman K6. With this plate, a suitable solvent would be chloroform-acetone-methanol-acetic acid-water (4.5 2/1 1.3 0.5, v/v). However, a word of caution in the use of these plates is that it is not feasible to heat activate them prior to use due to a darkening effect under elevated temperatures. Hence, sulfuric acid charring is not recommended. Nonetheless, the TNS and phosphorus sprays are satisfactory detection agents. 

Chromatographic Plates Use suitable thin-layer chromatographic plates (see Chromatography, Appendix IIA) coated with a 0.25-mm layer of chromatographic silica gel. Pretreat the plates by placing them in a chromatographic chamber saturated with ether. Remove the plates from the chamber, allow the ether to evaporate, and immerse them in a 2.5% solution of boric acid in alcohol. After about 1 min,... 

Procedure Separately apply 2 pL each of the Standard Solution and the Test Solution to a thin-layer chromatographic plate coated with a 0.25-mm layer of chromatographic silica gel. Allow the spots to dry, and develop the plate in a developing chamber containing the Developing Solvent until the solvent front has moved about 17 cm. Remove the plate from the chamber, mark the solvent front, and allow the solvent to evaporate. Spray the plate with a 1 500 solution of sodium metaperiodate, air-dry for 15 min, and spray with a 1 50 solution of 4,4 -tetramethyldiaminodiphenylmethane in a 4 1 mixture of acetone and glacial acetic acid. The principal spot obtained from the Test Solution corresponds in Rf value and color to that obtained from the Standard Solution. 

Chromatographic Plates Use Whatman LKC18 thin-layer chromatographic plates coated with a 0.20-mm layer of silica gel absorbent, or equivalent. 

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