Experimental Conditions for TLC

Resolution on the thin, fine-grained cellulose layers used in TLC is superior to that in PC and is accomplished in shorter time. As has been mentioned already, the solvents used in PC can be applied in TLC and, by and large, the same separation sequences are then obtained. The seven solvents listed below are particularly suitable for development on cellulose layers and they are adequate for most purposes  

Benzene-acetic acid-water (57 -f 28 + 15), upper phase [17, 239] this solvent is convenient for oligophenols and phenolcarboxyHc acids which migrate too rapidly in the mixtures below. 

Chloroform-acetic acid-water (50 + 45 -f 5) this is suitable for TLC of polyphenols, phenolcarboxyHc acids, flavone-aglycones and monoglycosides. The corresponding methyl ethers can be recognised through their much higher hi /-values [36 48]. It is important to apply 

Ethyl acetate-formic acid-water (66 + 14 + 20) this mixture migrates fast and is appropriate for separating both aglycones and glycoside mixtures [96]. Di- and triglycosides remain in the vicinity of the start, however. 

Butanol-acetic acid-water (40 + 10 + 50) the upper phase of this Partridge mixture yields with all glycosides excellently sharp resolution which cannot be surpassed even on polyamide or silica gel. The aglycones have, however, very high IlR/-values and the time of run is relatively long. The LR/-values are given in Table 165, column C, IV. 

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Table 109 a. Experimental condition for TLC of analgesics with narcotic activity... 

These difficulties of tailing and too small i /-values do not generally arise in the TLC of direct dyes. Moreover, the resolution of direct dyes in TLC usually surpasses that in PC. Table 133 contains experimental conditions for TLC of direct dyes, compiled from numerous publications. 

Table 173. Experimental conditions for TLC of some drugs or their extracts...

Suggestions for unifying experimental conditions in TLC have had a universal response. These standards have created a good point of departure for the method. Only when really necessary and when experimentally justified, should one deviate from these conditions. The approved conditions are summarised as follows ... 

The most favourable experimental conditions for this reaction have been worked out only in recent years TLC played a decisive role in these investigations [167, 170]. 

Teichert et al. [45] were the first to carry out TLC of amines. The hydrochlorides were dissolved in 70% alcohol and applied in 1 to 10 [xg amounts. The hi /-values and experimental conditions for the separation are summarised in Table 94. As can be seen from the table, only a partial improvement in separation is achieved by buffering silica gel G layers with a mixture of 0.2M primary potassium phosphate and 0.2 M secondary sodium phosphate (1 -h 1) or with 0.15M sodium acetate solution. 

Mixtures of basic dyes may be separated by column chromatography on polyamide powder, usiug water or ethanol-water (80 + 20) or (60 + 30) as solvents [71]. Separations are possible by PC on impregnated paper [6, 18] or acetyl-paper [70]. TLC separations are advantageously shorter than these PC procedures. Many authors have worked on the TLC of basic dyes. Experimental conditions for separations are summarised in the following Table 128. 

Chromatographic Parameter-Relationships Correlations between Kov/ and various chromatographic parameters (CGP), such as HPLC retention time and thin-layer chromatography (TLC) capacity factors, allow the experimental estimation of Kow [19]. Usually, the CGP-A ow correlation is evaluated for a calibration set of compounds with accurately known K0w values. The Kow of a new compound can then be estimated by determining its CGP under the same experimental conditions as those used for the calibration set. 

The separation between the different dansyl-can-nabinoids can be obtained by the use of the one or two-dimensional TLC. Autoradiographies showed that the major difficulty is to have a good separation between dansyl-cannabinoids and 14C dansyl breakdown products which show several spots which are formed under the experimental conditions. All cannabinoids tested show one spot except CBD which give two spots. 14C DANS-neutral cannabinoids give a satisfactory separation in a one-dimensional TLC using the cyclo-hexane/ethyl acetate (95 5) (Fig. 9) and also in a two-dimensional TLC with cyclohexane/ethyl acetate (95 5) for the first migration followed by hexane/ benzene (60 40) for the second (Fig. 10). The best separation for the 14C DANS-ll-OH-A8 and A9-THC was obtained using the cyclohexane/acetone triethylamine (70 25 5) system (Fig. 11). 

Solute identification means qualitative analysis. Various methods are used today to identify a separated substance on a thin-layer chromatographic plate. Of all chromatographic methods, thin-layer chromatography (TLC) provides a unique simultaneous separation of up to 70 samples on the same plate therefore the reproducibility of the experimental conditions is not an issue because the experimental conditions are the same for all samples. This, together with the advantage of separating compounds with very different polarity and the possibility of using different detection methods for the same spot or for adjacent spots on the same plate, is the power of TLC. 

Limited experimental data [see discussion of Ref. (22)] suggest that the A term of Eq. (5-15) for TLC separation is commonly much larger than are the second two terms (2 and 3), so that /t to a first approximation. This simplifies any discussion of the dependence of bed efficiency in TLC on separation conditions. The value of A in TLC separations appears to be given by the same relationship as for column elution separations Eq. (5-8). Thus TLC adsorbents normally fall in the particle size range, for which A (and hence H ) is predicted equal to... 

Wagner E, Sodtke C, Schweizer N, Stephan P (2006) Experimental study of nucleate boiling heat transfer under low gravity conditions using TLCs for high resolution temperature measurements. Heat Mass Transf 42 875-883... 

Hodgson et al. [88 d] have made use of two-dimensional TLC in their study of nicotine metabolism. The hR/-values and experimental conditions are seen in Table 74. Decker and Sammeck [42 b] have carried out chromatography on silica gel G, using n-butanol-ethanol-0.5 N ammonium hydroxide (66 + 17 + 17) in order to solve similar problems. In both these cases [88 d, 42 b] the layers were sprayed with a mixture of equal volumes of 2% p-aminobenzoic acid in ethanol and 0.1 M phosphate buffer, pH 7 after drying for 15 min, the plate was... 

Acid dyes are employed above all for colouring wool, polyamide fibres, paper, leather and inks. Only certain acid dyes are permitted for colouring foodstuffs and such food colorants are therefore treated in a special section below (p. 623). The difficultly soluble salts of some acid dyes are used as pigments. Most acid dyes are azo-, triarylmethane-, xanthene-, or anthraquinone derivatives. Various investigators have reported PC-separations of acid dyes in detail [12, 31, 33, 40]. Such separations can be carried out more quickly with TLC and often with superior resolution. Table 131 summarises experimental conditions of the abundant work in this field. 

The optimum method for obtaining tentative identification of a substance is to spot the sample and a series of reference compounds on the same chromatogram. In this way, mobilities of all compounds are compared under the same experimental conditions, and a match in Rp values between a sample and standard is evidence for the identity of the sample. Experimental conditions should be chosen so that the compound to be identified moves to a point near the center of the layer (Rp 0.5) and resolution between spotted standards is as good as possible. If Rp values on silica gel are higher than desired, the polarity of the mobile phase is reduced for Rp values that are too low, the polar component of the mobile phase is increased. If the spots of interest from the sample do not line up with the standards, they are either not the same as any of these compounds or their mobilities have been affected by accompanying extraneous material from the sample. If the latter is the case, sample cleanup (see Chapter 4) or two-dimensional TLC will be necessary. 

This extensive list of variables illustrates the importance of controlling and reporting as many experimental conditions as possible when describing the results of TLC so that others wanting to duplicate results will know how to proceed. All the stated conditions under which the initial results were obtained should be followed as carefully as possible for best reproducibility. 

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