Thin-layer separation

Reagents used for the visualisation of amino acids on the dried chromatogram may be applied either by spraying or dipping. Those commonly used produce intensely coloured bands with approximately 20 nmol of each amino acid for paper chromatography and 5 nmol for thin-layer separations, although smaller amounts can be detected. 

A 8] The micro injection system relies on an electromagnetic drive and injects a small volume of only 0.5 pi per injection. The valve section is heated. The injected gas pulse is separated in a thin-layer separation column. 

On tasting the narrow, horizontal segments of the area from the start to the solvent front in many thin—layer separations of pepper extracts, we have noticed that there is a component always coming above the piperine spot which has a tingling sensation on the tongue. This substance has not been identified, but could consist of isobutylamide-like compounds reported from other Piper species by Atal t al (38). 

Typical thin layer separations are performed on flat glass or plastic plates that are coated with a thin and adherent layer of particles, which constitute the stationary phase.Commercial plates come in two categories conventional [thick layers (200-250 pm) of particles having sizes of 20 pm or greater] and high-performance plates (film thickness of 100 pm and particles whose diameters are 5 pm or less). 

Typical thin-layer separations are performed on a glass plate that is coated with a thin and adherent layer of finely divided particles this layer constitutes the stationary phase. The particles are similar to those described in the discussion of adsorption, normal- and reversed-phase partition, ion-exchange, and size-exclusion column chromatography. Mobile phases are also similar to those employed in high-performance liquid chromatography. 

The amount of laetisaric acid in a sample can be readily quantified using the above extraction, thin layer separation, diazomethane derivatization and capillary gas chromatographic procedure. Calculations of the amount of laetisaric acid by bioassay and by the described physical analytical methods are in agreement. 

TLC is a good technique to use when normal-phase solvents provide optimum separation. Typical thin-layer separations are performed on glass plates that are coated with a thin layer of stationary phase. The stationary phases used in TLC encompass all modes of chromatography including adsorption, normal- and reverse-phase, ion-exchange, and size-exclusion." The equipment required is simple and inexpensive. TLC is an ideal technique for the isolation of compounds because of its simplicity. However, for TLC to be successful, the impurity and/or degradant level should be at or above 1%. Any component present below this level is very difficult to isolate on a TLC plate because of higher detection limits. 

Photographic Record of Thin-Layer Separations of Drug Extracts (A Photographic TLC Drug Atlas)... 

Photographic reproduction of thin-layer separations ha.s a large didactic advantage over mere graphic representation. The TLC photo-drug atlas has an immediate clarity of representation that facilitates the learning of TLC clrug analysis for the student. 

Exploratory thin-layer separations are commonly used as guides in selecting the best separation conditions for corresponding preparative scale separations on columns. In this connection it should be possible to establish optimum sample size as well, using thin-layer chromatography. 

Equations (10-4) and (10-4a) predict a linear dependence of logX or R i on n for samples of the general class X-i . Some experimental examples of this relationship are shown in Fig. 10-1. These include the elution of the unsubstituted aromatic hydrocarbons of carbon number n from alumina (a) and Florisil (f>), the equilibrium adsorption of the homologous carboxylic acids (C Fl2 nCOOH) on charcoalt (c), and the thin-layer separation of some methyl ester-substituted porphyrins (porph... 

In most separations we will not be interested in changing temperature deliberately, but minor fluctuations in temperature may occur unless special precautions are taken. As a result sample retention volumes or Rj, values may vary by unacceptable amounts. Table 12-3 permits the rapid estimation of the potential importance of temperature variation in this respect. For example, most adsorbents commonly used will have values of log K orlog —1 to —2. In thin-layer separations the reproduc-... 

The major decision in the design of a suitable adsorption chromatographic system is the selection of the correct solvent. Most samples can be separated on any of several, general purpose adsorbents (most often silica). Similarly, little thought is generally required in the adjustment of the remaining separation variables bed dimensions, sample size, solvent flow rate, or temperature. In many laboratories more or less standard separation schemes are used, and only the solvent is varied to meet the specific requirement of individual samples. This is especially true of thin-layer separations, where standard silica plates and fixed sample sizes are the rule. Only when such standardized procedures fail is serious attention paid to separation variables other than the solvent. 

Thin-layer separations may be analytical or preparative (up to 100 mg per 20 x 20 cm plate depending on the ease of separation). Separated lipids can be made visual with a destructive spray (for analytical purposes only) such as phosphomolybdic acid solution or with a non-destructive spray such as 2, 7 -dichlorofluorescein solution (essential for preparative purposes). Improved separations are sometimes achieved through multiple development or by operating at subambient temperature. 

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