Development of Thin-Layer Chromatograms

Solvent system for flavonoids (in-house development) Ethyl acetate Formic acid Methanol 

HPLC quality Migration distance Developing time (at 23 °C) 

All the operations described above are in general only preliminaries to the actual process of TLC, i.e. the development of the TLC plates by a liquid with the object of separating mixtures of substances. All roads lead to Rome is an old saying. The same applies to TLC, in which the results can be obtained by various kinds of development  

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Bennett, R.D. and Heftmann, E. (1966) Separation of closely related steroids by an improved technique for continuous development of thin-layer chromatograms. /. Chromatogr., 21,488-90. 

Various chambers have been used for the development of thin-layer chromatograms. In the classical cases the mobile phase migration is the consequence of the capillary forces, that is the TLC developing solvent moves forward on the dry stationary phase. 

The AMD method of Burger is also based on vertical TLC. This system for automated multiple development of thin-layer chromatograms is discussed in Chapter 11 Special Methods in TLC . This equipment falls into the top price bracket and is included here only for completeness. 

Quantitative evaluation of thin-layer chromatograms can be performed by direct, in situ visual, and indirect elution techniques. Visual evaluation involves comparison of the sizes and intensities of color or fluorescence between sample and standard zones spotted, developed, and detected on the same layer. The series of standards is chosen to have concentrations or weights that bracket those of the sample zones. After matching a sample with its closest standard, accuracy and precision are improved by respotting a more restricted series of bracketing standards with a separate sample spot between each of two standard zones. Accuracy no greater than 5-10% is possible for trained personnel using visual evaluation. The determination of myco-toxins in food samples is an example of a practical application of visual comparison of fluorescent zones. 

The first publications on the evaluation of thin-layer chromatograms by image processing appeared as early as the mid-1980s [118], These methods, also known as digital evaluation , were presented to the scientific world in the lectures of Prosek, who developed his own software for the purpose in 1991 [119], although only the enormous developments in the hardware (video cameras, computers, printers etc.) have made video evaluation of thin-layer chromatograms economically justifiable. Prosek presented his paper on the validation of quantitative TLC by video camera in 1997 [120]. 

This form fulfills all the GMP/GLP requirements for the documentation of thin-layer chromatograms, and has been found to be highly satisfactory over the years, especially in method development. It is advisable to fill out this sheet before starting to apply samples to a plate. 

Quantitative evaluation of thin-layer chromatograms can be performed by direct, in situ visual and indirect elution techniques. Visual evaluation involves comparison of the sizes and intensities of color or fluorescence between sample and standard zones spotted, developed, and detected on the same layer. The series of standards is chosen to have... 

The classical way to develop a thin-layer chromatogram is to immerse the plate with its lower edge in the developing solvent contained in a tank. Flow of the solvent by capillary action starts when the plate is inserted. 

The literature reports [a]n +23.2° (c = l, aqueous 5N hydrochloric acid). The product was analyzed by the submitters. Analysis caleulated for C5H11NO2S C, 40.25 H, 7.43 N, 9.39 S, 21.49. Found C, 40.14 H, 7.42 N, 9.50 S, 21.52. The product was homogeneous according to thin-layer chromatograms on precoated silica gel G plates purchased from Analtech, Inc., Newark, Delaware, and developed with the following two solvent systems (solvents, volume ratios of solvents in the same order) 1-butanol-acetic acid-ethyl acetate-water, 1 1 1 1, Rf 0.49 1-butanol-acetic acid-pyridine-water, 15 3 10 12, R/0.51. 

Figure 11.1a [1] shows a schematic representation of a micropreparative thin-layer chromatogram obtained on a 0.5-mm Florisil (magnesium silicate) layer prewetted with benzene of a crude extract, i.e., containing coextracted plant oil obtained from Heracleum moelendorfi fruit. The initial band of extract was washed with benzene and then separated by continuous development with ethyl acetate in benzene [1]. As seen from the fraction analysis presented in Figure 11.1b, small quantities of pure bergapten and xanthotoxin can be isolated in this maimer. 

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