Preparative layer chromatography separation

Modem planar chromatography is suitable not only for qualitative and quantitative analysis but also for preparative purposes. The separation efficiency of a thin-layer chromatographic system is independent of this intended purpose and is mainly determined by the quahty of the stationary phase, that is to say, by the applied coated layer. Therefore, progress in modem planar chromatography can be attributed not only to the development of the efficiency of the instmments but also to a large extent to the availability of high-quahty precoated layers. And today, as in the past, bulk sorbents for self production, especially of preparative layer chromatography (PLC) layers, are widely used. 

Application of Preparative Layer Chromatography for the Separation of Secondary Metabolites from Plant Tissues... 

FIGURE 12.2 Diagrammatic representation of the effect of the concentration zone in preparative separations, (a) Precoated layer without concentrating zone, (b) Precoated layer with concentrating zone. (From Nyiredy, S., Preparative layer chromatography, in Handbook of Thin Layer Chromatography, 3rd ed., Vol. 89, Sherma, J. and Fried, B., Eds., Marcel Dekker, New York, 2003, pp. 99-133. With permission.)... 

TLC of larger quantities of materials (10 to 1000 mg) on thick layers (1-5mm), for the purpose of isolating separated substances for further analysis or use, is called preparative layer chromatography (PLC). Most preparative applications are carried out on 20 x 20 silica gel or alumina plates with a layer containing a fluorescent indicator to facilitate nondestructive detection. 

Exercises in thin-layer chromatography. Separation of amino acids. Prepare solutions of DL-alanine, L-leucine and L-lysine hydrochloride by dissolving 5 mg of each separately in 0.33 ml of distilled water, measured with a graduated 1 ml pipette (leucine may require warming to effect solution). Mix one drop of each solution to provide a mixture of the three amino acids and dilute the remainder of each solution to 1 ml to give solutions of the respective amino acids. The latter will contain about 5 pg of each amino acid per pi. Apply approximately 0.5 pi of each of the solutions to a Silica Gel G plate and allow to dry in the air (i.e. until the spots are no longer visible). 

In 1970, 1 was prepared by direct glycosylation of the silylated 5-azacytosine with acylated 1-halo sugars, but the yields were very low.20 In this procedure, 1,3,5-tri-O-acetyl-2-deoxy-D-ribofuranose (9) was converted into 3,5-di-0-acetyl-2-deoxy-D-ribofuranosyl chloride (10).21 The trimethylsilyl derivative of 5-azacytosine (11),22 prepared from 4-amino-6-pyrimidine by treatment with hexamethyldisilazane, was then allowed to react with intermediate 10 in acetonitrile over 7 days to give a mixture of anomers of l-(3,5-di-(9-acetyl-2-deoxy-D-ribofuranosyl)-5-azacytosine (12) in 10% overall yield. The anomeric mixture was treated with ethanolic ammonia to remove the acetyl groups. The resulting a and p anomers were separated by a combination of fractional crystallization and preparative layer chromatography on silica gel to give pure decitabine (1) and its a-anomer 13 in 7% and 52% yield, respectively. 

Into a solution of the starting alkene (5.0 mmol) in the appropriate solvent under N2, TfjNF (5.5 mmol) in CH2CI2 was added dropwise during a period of 10-20 min with stirring. Reactions were followed by F and HNMR. After the reaction appeared to be complete, the mixture was diluted with CH2CI2, rinsed with aq NaHCO., and sat. brine, and then dried (Na2S04). The crude products were separated by column or preparative layer chromatography (silica gel). 

Mean particle size. In addition to the particle size distribution, the mean particle size determines separation efficiency. With the particle size distribution remainig unchanged, then the smaller the particle size, the better is the efficiency. However, the flow properties of the TLC system also depend on the mean particle size. For TLC separation, 12-14 pm mean particle size is usual, while smaller particles (5-6 pm) is used for HPTLC, and larger particles (18-22 pm) are used for preparative layer chromatography. 

To a solution of l,l-bis(phenylseleno)cyclopropane (0.354 g, 1.5 mmol) dissolved in anhyd THE (1 mL), stirred and kept at — 78"C, was added 2.4 M BuLi in hexane (0.63 mL, 1.5 mmol). The mixture was stirred at this temperature for 1 h and benzaldehyde (0.160 g, 1.5 mmol) dissolved in anhyd THE (1 mL) was added. The resulting mixture was stirred for 15 min at - 78"C and was then quenched with sat. aq NH4CI. The hydrolysate was allowed to warm to rt, the layers were separated, and the aqueous phase was extracted with EtjO (2 x). The combined organic layers were dried (anhyd MgS04). Purification, carried out by preparative layer chromatography (silica gel, pentane/EtjO 8 2, Rj- 0.6), gave 0.276 g (91%) of the product. 

Reductive cyclization has been used in a novel, recent synthesis of the alkaloids ( )-isoretronecanol (22) and ( )-trachelanthamidine (23) by Borch and Ho. Condensation of the dianion derived from methyl acetoacetate with Z-l,4-dichlorobut-2-ene, followed by cyclization with sodium meth-oxide yielded the cycloheptenone ester intermediate (32) (Scheme 2). Reductive amination of this ketoester with sodium cyanoborohydride and ammonium nitrate gave a mixture of the diastereoisomeric aminoesters 33 and 34. Oxidation with osmium tetroxide and periodate, followed by reductive cyclization, again using sodium cyanoborohydride, gave the two pyrrolizidine esters 35 and 36 in a ratio of 1 2 [gas-liquid chromatography (GLC) analysis]. The esters were separated by preparative layer chromatography, and lithium aluminum hydride reduction of the individual esters gave the two pyrrolizidine alkaloids 22 and 23. 

To a stirred suspension of 100 g of actively fermenting yeast and 100 g of sucrose in 500 mL of water is added 0.5 g (1.76 mmol) of methyl 2-(5-methoxycarbonyl-2-furyl)glyoxalate and the mixture is allowed to ferment at 25 "C for 2 d. The extract is diluted with 500 mL of ethyl acetate and filtered. The filtrate is separated and the aqueous layer is re-extracted with 500 mL of ethyl acetate. The combined organic layers are dried and evaporated. Preparative layer chromatography of the residue affords the (— )-(.R)-ester yield 0.28 g (55%) mp 132- 134 C [oc]d° —66.5 (c = 1. acetone). 

The best yields were obtained with (Me3SiS)2R and R = C3H6 or C4H8. In the case of R = C2H4, oligomeric compounds were formed. The reaction with SCI2 is carried out in THF followed by evaporation of the solvent and of MesSiCl and preparative TLC (Thin-layer Chromatography) separation with hexane as an eluent. 

Thin-layer chromatography (separations) can be made using either plates prepared in the laboratory or ready-prepared TLC plates. Plates with... 

Isolation procedures for Sceletium alkaloids have generally relied on column chromatography over alumina and/or silica gel for the separation and purification of the major alkaloids, with rqieated preparative-layer chromatography often necessary for separation of the minor bases. In one instance high-pressure liquid chromatography was used for purification of an alkaloid. This latter technique is likely to find increasing application in the future for isolation of the minor alkaloids of this family. 

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