Silica Gel G Layers

Reactions can also occur during chromatographic development. These can either be undesired reactions or planned derivatizations. Thus, Weicker and Brossmer [11] have reported, for example, that hexoses, pentoses and disaccharides can be ammated when ammonia-containing mobile phases are employed on silica gel G layers. On the other hand, fluorescamine or ninhydrin have been added to the... 

The amoimt of lipid applied to the plate varies depending on the ease of separation of individual lipid components in the sample. Usually 25 to 50 mg of the neutral lipid sample can be applied to a 20 X 20 cm preparative plate with the silica gel G layer thickness of 0.5 mm, whereas only about 4 mg of phospholipids can be applied on these plates. 

Chromatography. DNPH-steroid derivatives may be separated on plates of silica gel G (layer thickness, 0.25 nm) with chloroform—carbon tetrachloride (2 1) or chloroform-dioxane (47 3) [30]. The spots may be quantitated directly by spectrodensitometry in situ at 367 nm. Amounts as low as 20-40 ng per spot can be detected. A chromatogram scan of 2.5 jug each of dehydroepiandrosterone, etiocholanolone and androsterone separated with chloroform—dioxane (47 3) is shown in Fig. 4.12. 

In our laboratory Rm values were used as an expression of the lipophilic character of the molecules. Details of the chromatographic procedure have been described (9, 10). The compounds were partitioned between a nonpolar stationary phase and a polar mobile phase. The stationary nonpolar phases was obtained by impregnating a silica gel G layer with silicone DC 200 (350 cS) from Applied Science Laboratories. 

TLC. Samples and standards applied as 2 cm bands, silica gel G layer, petroleum ether mobile phase. 

Silver nitrate-impregnated silica gel has also been used successfully in the separation of some classes of compounds that contain double bonds in their structures. Thus it was used in the separation of glycerides and isomers of unsaturated fatty acid esters. The 2-D TLC on adjacent layers of silica gel G and sihca G impregnated with 5% of silver nitrate was used for development. The silica gel G layer covered a surface of 3 x 20 cm and the difference of the surface of 17 x 20 cm was covered with silica gel G impregnated with 5% AgNOs. The first direction was performed on silica gel G with petroleum ether-diethyl ether (9 1, v/v) as eluent. The second direction was carried out on the impregnated silica gel G and developed twice with the same eluent as in the first direction. After the first direction, the mixture was... 

The useful effect of impregnation is clearly seen in the various chapters of the special part (of this book). A single example must suffice here the terpene alcohols from C5 to Cgo appear more or less together on silica gel G layers. If this adsorbent is impregnated with paraffin, they... 

Fig. 17. Course of the release of water from freshly prepared silica gel G layers under...

Fig. 24. The influence of various degrees of saturation (a—d) and relative humidities (20— 79%). a test mixture, developed with methylene dichloride on a silica gel G layer without a chamber b the same in a charuber at NS c in the same chamber but at CS d in the S-chamber. Alongside are developments in the GS chamber at different relative humidities, o = Butter Yellow = Sudan Red G ...

The solv ent migration rate can be accelerated through centrifugal force. The plate and layer are rotated in a horizontal plane and solvent introduced at the axis of rotation. Rosmus, PAVLf EK and Deyl [585], who had previously studied centrifugal paper chromatography in detail, have tried out the technique with TLC. They showed that the Stahl test mixture and mixtures of 2,4-dinitrophenylhydrazones could be separated in 5—10 min on silica gel G layers of 20 cm diameter, using 500 revs/min. 

Fig. 71. Examples of ways of documenting thin-layer chromatograms, a chromatogram traced on transparent paper (transferred back to ordinary paper) h documentation by photostating c as rectangles of the same area as the planimetrically measured spot areas. Experimental details separation of bromoureides using cyclohexane-chloroform-pyridine (20 + 60 -f 5) on silica gel G layers. 8 start ...

Fig. 72. Comparison of the spot areas of increasing amounts of alkaloids at equal length of run. Curve I on paper impregnated with formamide, curve II on a silica gel G layer. The stippling density is roughly equivalent to the number of molecules...

Silica gel G and anthracene are mixed in the ratio 1 1 and pulverised in a ball mill to a grain size of 1— 5 p.m (about one third of the range of H j5-radiation ). 30 g of this powder are slurried with 80 cm of 96% ethanol and spread on to 5 20 X 20 cm glass plates as layers 0.25 mm thick, using the standard method (see p. 85). The coated plates are air dried. The customary solvents used for silica gel G layers can be used with this mixed layer too. The anthracene has little influence on the separating power of the adsorbent. 

Several authors have reported the analysis and purification by TLC, of substances which had been tritiated according to the Wilzbach procedure. Thus glycerol ethers [262], the insect hormone ecdysone [338], vitamin Dg [501] and other steroids [501, 513, 617] (see Fig. 93, p. 170) and cardiac glycosides [551] have been purified on silica gel G layers. Unsaturated fatty acids have been isolated by TLC on silver nitrate-impregnated silica gel layers [425, 628]. 

In order to obtain a better separation of monoterpenes and to eliminate the interference of evaporation during chromatography, Adhtkari [1] and Mathis [159] have developed at —15° C, using silica gel G layers and n-hexane (chamber saturation, hBf 25—85). The time of run was shorter and the hBf-values lower than under normal conditions, as Stahl [254] has been able to show (cf. Fig. 49). The mono- and sesquiterpene fractions of Daucus oils have been chromatographed also at —20° C, on silica gel H layers [255]. A separation in accordance with the influence of the C=C double bonds was accomplished with n-hexane-pentane (50 + 50) as solvent. 

The position of these substances in the chromatogram is determined by the polarity of the parent compound. Menthofuran migrates just behind guaiazulene [182 a, 247]. The terpene and sesquiterpene epoxides follow, lying in the upper part of the ester zone on silica gel G layers (Table 23) (cf. also [155]). in agreement with El-Deeb [55]. The carbonyl and alcohol oxides follow with lower hRf-values. This sequence holds even at — 9° C using Freon (Frigen 21) as solvent [255]. 

Terpene esters constitute an important compound class in the essential oils. Acetate esters are those most frequently encountered more rarely, formates, propionates, butyrates, valerates and caprylates. Experience accumulated with over 30 different esters of lower fatty acids, shows that the zones on silica gel G layers, when developing with benzene or chloroform, are about 2.5 times higher than those of the corresponding alcohols (chamber saturation). Brud and Daniewski [28] have confirmed our finding [260] for loose silica gel layers also. The largest differences in hRf-values have been observed when using normal saturation (NS). 

Table 24. Separation of terpene esters on silica gel G layers...

The intramolecular esters, the lactones, may be mentioned here. The antihelminthic agent santonin, for example, belongs to this class. A well known lactone is coumarin, the odour of which resembles dried woodruff and which is found in numerous essential oils, also as derivatives. SuND and Saccabdi [273] have succeeded in separating 5 cou-marins on silica gel G layers, using petroleum ether (50—75° C)-ethyl acetate (67 + 33) or n-hexane-ethyl acetate (72 + 29). The sequence found was coumarin, 6-methylcoumarm, dihydrocoumarin, 3-methylcoumarin, 3-ethylcoumarin. 

Korte and Vogel [140] have chromatographed lactones, lactams and thiol-actones of organic chemical interest. They used standard conditions on silica gel G layers with diisopropyl ether, ethyl acetate and isooctane alone and in mixtures. 

The ketolactone isolated from Jasminum grandiflorum by Demole and co-workers [41, 44] can be satisfactorily separated from the other compounds on silica gel G layers, using petroleum ether (80—100° C)-ethyl acetate (75 + 25) (JiEf 31). Investigations so far carried out indicate that it contains a 10-membered lactone ring. Higher membered lactone rings are found in the naturally occurring musk odorous principles like civetone, muscone etc. 

Dhont and Dijkman [47] have separated the isomeric ionones and methyhonones on air-dried silica gel G layers, developing 6 times with benzene. Whereas the lower acyclic ketones (up to about C q) occur in essential oils, the homologous products of breakdown of keto-fatty acids are no longer volatile in steam. Separation can be achieved on silica gel G layers with benzene [152, 153] or n-hexane-diethyl ether (50 + 50) [190]. 

Table 28. Separation of terpene and sesquiterpene alcohols by adsorption chromato-graphy on a silica gel G layer using chloroform...

Table 30. Separation of alcohols on a paraffin-impregnated silica gel G layer, using...

Method. The dried silica gel G layer, after cooling to room temperature, is immersed for one minute in a 5% solution of paraffin or of silicone oil DC 550, in petroleum ether (see p. 48). The petrol ether (BP 40 to 60" C) is allowed to evaporate for 15 min with the plate horizontal and the layer then used in the normal way. Impregnation by ascending treatment with the paraffin-petrol ether solution has no advantage in this case. 

Pesnelle et al. [195] have used 10% silver nitrate-silica gel G layers and cyclohexane-ethyl acetate (60 + 40) in order to separate galbanol [327], formerly believed to be a single compound, into bulnesol, guaiol and jS-eudesmol. 

Another way of characterising alcohols is through TLC after having treated them with 3,5-dinitrobenzoyl chloride (preparation, see Chapter TS, 3a). Dhont and Derooy [49] were the first to show that the dinitrobenzoate esters could be separated by using benzene-petrol ether (50 + 50) on silica gel G layers. Butter yellow (p-dimethylamino-azobenzene) was chosen as reference substance to determine the positions in the chromatogram. The following values,... 

Beaxjn [20], Gbaf and Hoppe [79) and Mehlitz and co-workers [163] have used similarly non-polar solvent combinations. Menthol and isomenthol dinitrobenzoates can be separated with hgroiu (105—120° C)-isopropyl ether (95 + 5) on silica gel G laj -ers under normal conditions [79]. The dinitrobenzoates of other terpene and sesquiterpene alcohols could be clearly differentiated by double development on 500 xm-silica gel G layers using petrol ether-isopropyl ether (94 + 6). Solvent mixtures containing twice as much ether are more suitable for TLC of the dinitrobenzoates of lower alcohols. 

Fig. 106. Separation of methoxyphenylpropane derivatives (4 (xg of each) on a silica gel G layer, using benzene. S-chamber 10 cm run, lasting 35 min spray reagent molybdophosphoric acid solution (No. 168). T Desaga test mixture 1 safrole 2 methylchavicol S myristicin 4 apiole 5 eugenol methyl ether 6 asarone 7 tetra-methoxyallylbenzene 8 elemol 9 catechol G mixture (2 [xg of each)...

Introduction of an aliphatic side chain (methyl-, ethyl-, allyl-, propenyl-,) into the nucleus influences only slightly the adsorption affinity and hence the hRf-values on normal silica gel G layers. 

Phenols may be better characterised by conversion into 3,5-dinitro-benzoates, as with terpene alcohols (p. 228). In TLC on silica gel G layers under standard conditions using butter yellow as reference substance, the following values have been found for dinitrobenzoates of eugenol 0.55 isoeugenol 0.56 phenol 0.76 a-naphthol 0.89 / -naphthol 0.93 cresols 1.00—1.05 thymol 1.42 [49]. 

Should this method also fail to provide information about the phenols or phenol isomers present, TLC of suitable coupling products may be attempted. Knappe and Rohdewald [133 a] have separated phenol, the 3 cresol and 6 xylenol isomers in this way. They chromatographed the coupling products with Fast Red AL Salt (Firm 127) on sihca gel G-potassium carbonate layers in combination with oxalic acid-silica gel G layers solvents were methylene dichloride-ethyl acetate-diethylamine (92 + 5 + 3) and chloroform-ethyl acetate-ethanol (93 + 5 + 2). 

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