Analytical Separations 2 Recrystallization

This chapter and Chapters 12 and 13 describe modem analytical separation science. First, purification procedures known as recrystallization and distillation will be described. Then the separation techniques of extraction and chromatography are discussed. This is followed by, in Chapters 12 and 13, instrumental chromatography techniques that can resolve very complicated samples and quantitate usually in one easy step. 

A total of 3 g (0.13 moles) of sodium hydride is added to a solution consisting of 10 g of 17 -hydroxy-5a-androstan-3-one (36 mmoles) in 200 ml of benzene and 10 ml of ethyl formate. The reaction mixture is allowed to stand under nitrogen for 3 days followed by dropwise addition of 10 ml of methanol to decompose the excess of sodium hydride. The solution is then diluted with 300 ml water and the layers are separated. The basic aqueous solution is extracted with ether to remove neutral material. The aqueous layer is acidified with 80 ml of 3 A hydrochloric acid and the hydroxymethylene steroid is extracted with benzene and ether. The combined organic extracts are washed with water and saturated sodium chloride solution and then dried over magnesium sulfate and concentrated. The residue, a reddish-yellow oil, crystallized from 10 ml of ether to yield 9.12 g (83%) of 17 -hydroxy-2-hydroxymethylene-5a-androstan-3-one mp 162-162.5°. Recrystallization from chloroform-ether gives an analytical sample mp 165-165.5° [a]o 53° (ethanol) 2 ° 252 mjj. (g 11,500), 307 m u (e 5,800). 

A mixture of 10 g of 4-(2, 4 -difluorophenyl)-phenol and 27.2 g of potassium carbonate is exposed to carbon dioxide at 1,300 psi and 175°C. The dark mass obtained from this car-donation is then dissolved in 300 ml of water and 200 ml of methylene chloride and the two layers separated. The water layer is then extracted with 100 ml of methylene chloride and then acidified with 2.5 N hydrochloric acid. This mixture is then filtered and the cake dried in vacuo to yield 5.32 g of the crude product. The crude product is then recrystallized from benzene-methanol. An additional crystallization of this semipure material from benzene-methanol yields analytically pure 2-hydroxy-5-(2, 4 -difluorophenyl)-benzoic acid (MP 210°-211°C). 

The preparation of metal complexes from hydroxypyridinones is usually simple and straightforward—the more difficult task is generally the synthesis of the required ligand. There may also be difficulties in separating complex from ligand, especially for the more lipophilic complex S sublimation, used in the case of A -n-hexyl, may be more successful than recrystallization. 3-Hydroxy-4-pyridinones are for the most part accessible from hydroxypyranones. Reagents developed for analytical and separation purposes (preparative methods may be traced back... 

After removal of solvent in vacuo, the yellow residue is chromatographed on silica thin layer chromatography (TLC) plates using hexane-CH2Cl2(85 15) as eluent. The major product, 2, is a nearly colorless band observed at Rf = 0.17. This band can be seen better by using an UV lamp and UV sensitized TLC plates (precoated TLC plates, silica gel 60 F-254, layer thickness 0.25 mm, Merck). This band is separated from the starting material (Rf = 0.47) and several minor bands and the products are eluted from the silica gel with CH2C12. An analytically pure sample can be obtained by recrystallization from hexane at - 20 °C. Yield 0.053-0.070 g (30-40%). 

A solution of 2-(2-amino-5-chlorophenyl)-4-cyclopropyl-l,l,l-trifluoro-3-butyn-2-ol (15.00 g, 0.0518 mol) and 41.98 g (0.259 mol) of 1,1 -carbonyldiimidazole in 250 mL of dry THF was stirred under argon at 55°C for 24 hours. The solvent was removed on a rotary evaporator and the residue was partitioned between 500 mL of ethyl acetate and 400 mL of water. The layers were separated and the aqueous phase was extracted once more with ethyl acetate. The combined ethyl acetate extracts were washed with 2 times 200 mL of 2% aqueous HCI, saturated aqueous NaHC03, and brine. Drying over MgS04, filtration, and removal of the solvent in vacuo provided 16.42 g of the title compound as a solid. Recrystallization from ethyl acetate/hexane afforded 12.97 g of analytically pure ()-6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-l,4-dihydro-2H-3,l-benzoxazin-2-one as a white crystals. Melting point 178°-180°C. 

Bromomethyl-2-cyanobiphenyl (4.6 g) was alkylated onto 2-n-butyl-4-chloro-5-(hydroxymethyl)-imidazole. For separation of the product was used a flash chromatography in 1 1 hexane/ethyl acetate over silica gel. The regioisomeric products yielded 2.53 g of the faster eluting isomer. Recrystallization from acetonitrile yielded 1.57 g of analytically pure 2-n-butyl-4-chloro-l-[2,-cyanobiphenyl-4-yl)methyl]-5-(hydroxymethyl)-imidazole, melting point 153.5 -155.5°C. 

A mixture of this material with 500 ml of toluene and 30 g of manganese dioxide was heated to reflux for IV2 hours. The manganese dioxide was separated by filtration over Celite. The filtrate was evaporated and the residue was crystallized from ether to yield 8-chloro-6-(2-fluorophenyl)-l-methyl-4H-imidazo[l,5-a][l,4]benzodiazepine, melting point 152°C to 154°C. The analytical sample was recrystallized from methylene chloride/hexane. 

Anatoxin-a (3) is a powerful neurotoxin (inhibitor of acetylcholine esterase) found in freshwater blue-green algae. The compound was required as an analytical standard and also for development of an immunoassay. It was synthesized by the 8-step sequence summarized in Scheme 29.1. The key intermediate 4 was resolved by separation of the dibenzoyl tartrates, and the remaining steps then gave both (+)- and (-)-anatoxin-a.2 The efficiency of the resolution was monitored by formation of the BocAla derivatives, which were distinguishable by NMR an ee of >98% was achieved even before recrystallization of the salts. It is also noteworthy that the published absolute configuration of the intermediate 53 was shown to be in error by X-ray crystallography. 

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