Polar compounds, separation from crude

Chemical separations may first be accomplished by partitioning on the basis of polarity into a series of solvents from non-polar hexane to very polar compounds like methanol. Compounds may also be separated by molecular size, charge, or adsorptive characteristics, etc. Various chromatography methods are utilized, including columns, thin layer (TLC) gas-liquid (GLC), and more recently, high pressure liquid (HPLC) systems. HPLC has proven particularly useful for separations of water soluble compounds from relatively crude plant extracts. Previously, the major effort toward compound identification involved chemical tests to detect specific functional groups, whereas characterization is now usually accomplished by using a... 

In some instances, combinations of Cig and silica columns are also used for better purification of the crude extracts (431, 445). A combination of Cg, silica, and amino solid-phase extraction columns has been successfully employed to fractionate anabolic and catabolic steroid hormone residues from meat in polar and nonpolar neutral and phenolic compounds, and to purify further each fraction effectively (452). Another combination of two solid-phase extraction columns, one using a graphitized carbon black sorbent and the other Amberlite resin in the hydroxyl form, allowed neutral anabolics to be isolated and separated from acidic anabolics and their metabolites (453). A combination of basic alumina column placed in tandem with an ion-exchange column has also been applied for the purification of the crude extracts in the determination of diethylstilbestrol and zeranol (427), and estradiol and zeranol in tissues (450). 

Compared to refined vegetable oils, the compositions of crude vegetable oils and oil and fat products are more complicated. These samples contain proteins, carbohydrates, and minerals that interfere with HPLC separation and reduce the lifetime of the HPLC column. These compounds need to be largely eliminated from the extract before HPLC analysis. Saponification and heating are used to weaken sample matrices to allow the solvent to fully access all tocopherols and tocotrienols of the sample. Liquid/liquid extraction is used to remove these polar compounds from the organic solvent layer that contains tocopherols and tocotrienols. The normal-phase HPLC method is usually used for crude vegetable oils and vegetable oil products reversed-phase HPLC can be used for animal fat products. 

Thin-layer chromatography (TLC) is mainly applied in micropreparative taxoids separation [2-4]. Silica gel 6OF254 preparative plates are usually applied for this purpose. The problem of taxoids separation involves not only their similar chemical structure (e.g., paclitaxel versus cephalomannine) but also, due to different coextracted compounds usually encountered in crude yew extracts (polar compounds such as phenolics and nonpolar ones such as chlorophylls and biflavones), the separation is very difficult. The common band of paclitaxel and cephalomannine was satisfactorily resolved from an extraneous fraction in isocratic elution with ethyl acetate as a polar modifier [4] and n-heptane-dichloromethane as the solvent mixture and it was of suitable purity for high-performance liquid chromatography (HPLC) quantitative determination. 

For resorcinolic lipids, particularly those with long saturated side-chains, the use of polar solvents is important due to their amphiphilicity. The crude extracts in many cases are subjected to preliminary fractionation/purification either by solvent fractionation/partition or by application of chromatography. For prepurification of the material and its separation from polymerized phenolics, gel filtration on hydrophobic Sephadex or TSK gel is sometimes used. Silica gel is most frequently employed for the separation and/or purification of resorcinolic lipids, notably in some studies with Ononis species (12-14). The array of compounds reported appears partly attributable to methylation or acetylation reactions occurring during column chromatographic separation. An interesting approach for I the pre-purification and selective separation of resorcinolic lipid from phenolic lipids or resorcinolic lipids from impurities has recently been reported. A selective partitioning of different non-isoprenoid phenolic lipids... 

FIG. 3—Schematic diagram of chromatographic separation procedures for extraction of polar compounds from crude oil [9]. 

After synthesis and chromatographic separation of a solid compound, recrystallization is usually used as the final purification step. Suppose you wish to separate a compound A from minor impurities. First, it is necessary to find a solvent in which A is insoluble at low temperatures, but is very soluble at higher temperatures. Typically, polar or ionic compounds are soluble in polar solvents (e.g. water, acetonitrile, dichloromethane, methanol), while non-polar compounds are soluble in non-polar solvents (e.g. hexanes, toluene). The impurities may also be soluble in the same solvent or solvent mixture but, ideally, a solvent is found in which the impurities are insoluble. For the latter case, after preliminary tests to find a suitable solvent, the solvent is added to crude A and the mixture is heated. The insoluble impurities are removed by filtering the hot solution, and the filtrate is allowed to cool either at or below room temperature. As the solution cools, the solubility of A decreases and, once the solution has become saturated, A begins to crystallize from the solution. If crystallization is relatively rapid, microcrystals form. If both A and the impurities are soluble in the same solvent, the crude sample is dissolved in boiling solvent and the solution is allowed to cool slowly. Crystal growth involves the assembly of an ordered crystal lattice (Fig. 4.5 and see Chapter 6) and the aim is to exclude impurities from the lattice. 

Axenic cultures of dwarf spikerush (Eleocharis colorado-ensis) were established in 4 L aspirator bottles containing quartz sand and a synthetic culture medium. These were periodically drained and the effluent subjected to fractionation and bioassays. This crude leachate was passed through a C. cartridge to separate polar from nonpolar compounds. The nonpolar fraction was eluted from the cartridge with acetone and the solvent evaporated with gas. The polar fraction was lyophilized. Both... 

Leachate Fractionation. Crude leachate was filtered through cartridge (Sep-Pak) in order to separate polar from nonpolar compounds. Nonpolar fraction was eluded from C. cartridge with acetone, evaporated under nitrogen gas and stored at -20 C. Polar leachate was lyophilized and stored at -20 C. 

The crude product is dissolved in a minimum amount of dichloromethane and adsorbed onto 25 g of silica gel (32-63 micron) by subsequent evaporation of the dichloromethane under reduced pressure. The sample of dry, dark brown silica gel is added to the top of a column containing 500 g of silica gel (32-63 micron) with a mobile phase of hexanes. The polarity of the mobile phase is gradually increased from hexanes to 95 5 hexanes-ethyl acetate. The fractions containing the desired product (Rf = 0.26, 95 5 hexanes-ethyl acetate) are combined and concentrated under reduced pressure to yield 6.9 g of 3 as a yellow solid. Additional purification is required to remove traces of tin by-products. Compound 3 is dissolved in 70 mL of ethyl acetate and stirred over 70 mL of saturated, aqueous potassium fluoride for 24 hr. The two phases are separated and the organic phase is dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting yellow solid is recrystallized from ethyl alcohol to provide 6.1 g (48%, 2 crops) of 3 as yellow needles (Notes 17,18, and 19). 

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