Layers preparative scale

Nonionic surfactants, including EO-PO block copolymers, may be readily separated from anionic surfactants by a simple batch ion exchange method [21] analytical separation of EO-PO copolymers from other nonionic surfactants is possible by thin-layer chromatography (TLC) [22,23] and paper chromatography [24], and EO-PO copolymers may themselves be separated into narrow molecular weight fractions on a preparative scale by gel permeation chromatography (GPC) [25]. 

Chromatographic development chambers for analytical pirrposes are commercially available in several different sizes. The most commonly used ones are rectangiflar glass tanks with inner dimensions of 21 X21 X9 cm, and they can be used to develop two plates simultaneously in the preparative scale. Even bigger tanks are available for much larger plates, for preparative layer chromatography. The width of the chamber should be varied depending on the size and the number of plates to be developed. 

Silica gel plates also have been used for the separation of 16 different eye pigments of Drosophila melanogaster using two-dimensional development in nonpolar solvent systems [55]. Although not very common, two-dimensional development may be nsed in preparative scale on thick-layered plates for further analysis. 

Clark et al. [53] subjected primaquine to metabolic studies using microorganisms. A total of 77 microorganisms were evaluated for their ability to metabolize primaquine, of these, 23 were found to convert primaquine to one or more metabolites (thin-layer chromatography analysis). Preparative scale fermentation of primaquine with four different microorganisms resulted in the isolation of two metabolites, identified as 8-(3-carboxy-l-methylpropylamino)-6-methoxyquinoline and 8-(4-acetamido-l-methylbutylamino)-6-methoxyquinoline. The structures of the metabolites were proposed, based primarily on a comparison of the 13C NMR spectra of the acetamido metabolite and the methyl ester of the carboxy metabolite with that of primaquine. The structures of both metabolites were confirmed by direct comparison with authentic samples. 

Thin-layer chromatography (TLC) is one of the most popular and widely used separation techniques because of its ease of use, cost-effectiveness, high sensitivity, speed of separation, as well as its capacity to analyze multiple samples simultaneously. It has been applied to many disciplines including biochemistry [1,2], toxicology [3,4], pharmacology [5,6], environmental science [7], food science [8,9], and chemistry [10,11]. TLC can be used for separation, isolation, identification, and quantification of components in a mixture. It can also be utilized on the preparative scale to isolate an individual component. A large variety of TLC equipment is available and discussed later in this chapter. 

Although separate determination of the kinetic and thermodynamic parameters of electron transfer to transient radicals is certainly important from a fundamental point of view, the cyclic voltammetric determination of the reduction potentials and dimerization parameters may be useful to devise preparative-scale strategies. In preparative-scale electrolysis (Section 2.3) these parameters are the same as in cyclic voltammetry after replacement in equations (2.39) and (2.40) of Fv/IZT by D/52. For example, a diffusion layer thickness S = 5 x 10-2 cm is equivalent to v = 0.01 V/s. The parameters thus adapted, with no necessity of separating the kinetic and thermodynamic parameters of electron transfer, may thus be used to defined optimized preparative-scale strategies according to the principles defined and illustrated in Section 2.4. 

The more recent applications of open-column chromatography in fat-soluble vitamin assays utilize liquid-solid (adsorption) chromatography using gravity-flow glass columns dry-packed with magnesia, alumina, or silica gel. Such columns enable separations directly comparable with those obtained by thin-layer chromatography to be carried out rapidly on a preparative scale. 

The traditional procedures are similiar to analytical methods with the major differences being the use of thicker sorbent layers. These procedures are generally faster and more convenient than classical column chromatography [6,49]. Preparative scale TLC usually has decreased analyte resolution compared to analytical TLC. 

The multilayer coil separation column is prepared by winding a single piece of Teflon or Tefzel tubing around a spool-shaped column holder making multiple coiled layers between a pair of flanges. Currently, three different sets of multilayer coils are commercially available the large preparative scale (2.6-mm ID, ca. 1000 ml total capacity) the standard preparative column (1.6-mm ID, ca. 320 ml total capacity) and the analytical scale (0.85-mm ID, ca. 120 ml capacity). The optimal revolution speed of the apparatus ranges from 800 to 1200 rpm. 

Using these especially adjusted adsorbents for DCC, one can use the same sorbent and the same solvent for the column work and can transfer the TLC results to a preparative scale column operation rapidly, saving time and money. DCC materials are available corresponding with the most common thin layers silica DCC and alumina DCC. 

It was decided that the most efficient method of identification was to couple succinic anhydride with the drug substance and isolate the peak of interest by preparative-scale HPLC. A solution of the drug substance was treated with dimethylaminopyridine and succinic anhydride. The resultant solution was stirred at room temperature for 48 hours. The reaction mixture was partitioned between ethyl acetate and water, and the aqueous layer was then treated with IN HC1. The two layers were shaken well, and the aqueous layer was removed. The organic layer was then washed with water, saturated sodium chloride solution, dried with magnesium sulfate, and concentrated by evaporation to afford a clear colorless oil (1.81 g). A suitable preparative HPLC method using a volatile mobile phase of 0.1% formic acid in water/ methanol was developed, and the crude reaction mixture was purified by preparative-scale HPLC. The solution was concentrated by evaporation and the water was removed from this solution by freeze-drying to afford a white lyophilate (40 mg). 

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. 

Dry-column chromatography is a variant of preparative-scale thin-layer chromatography with similar resolution but a higher sample capacity. A glass column or Nylon tube... 

Dry-column chromatography is not a widely used today. Preparative-scale thin-layer chromatography or flash chromatography is generally preferred. Although separations are fast, the recovery of separated zones is slow and labor intensive compared with elution methods. 

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