Sodium sulfate fractionation

A solution of 21 g (0.15 mole) of perbenzoic acid (Chapter 17, Section II) in 250 ml of chloroform is prepared in a 500-ml round-bottom flask. Styrene (15 g, 0.145 mole) is added, and the solution is maintained at 0° for 24 hours with frequent shaking during the first hour. At the end of the reaction period, only the slight excess of perbenzoic acid remains. The benzoic acid is extracted from the reaction mixture by washing several times with 10% sodium hydroxide solution. The solution is then washed with water and dried over anhydrous sodium sulfate. Fractional distillation gives 24-26 g (69-75%) of 1,2-epoxyethylbenzene, bp 101 /40 mm. 

New interest in the nature of the constituent serum proteins resulted from the introduction of the sodium sulfate fractionation scheme by Howe (H24, H25). This was a technical advance because, unlike ammonium sulfate, sodium sulfate need not be removed prior to nitrogen analysis, the only accurate available means at that time of determining protein. Howe (H24) observed three ranges of sodium sulfate concentration at which an increase in molarity of 0.05 in a 1 30 dilution of calf serum failed to produce the expected increase in precipitation. One of these critical concentrations occurred at 1.45-1.50 M and was thought to indicate complete globulin precipitation. 

Using this method, some investigators have obtained albumin yields which compare well with those obtained by 1.8 ilf sodium sulfate fractionation and by separation by free electrophoresis (M13). However, several other reports suggest that difficulties may be encountered with the procedure of Pillemer and Hutchinson, which probably arise from variable and uncontrollable factors owing to the high concentration of serum in the... 

To a vigorously stiiied solution of sodium ethylate, prepared from 35 mmol of sodium metal (808 mg) and 210 mL absolute ethanol, 100.0 g (0.845 mol) of phenylacetonitrile was added at -10 °C and then 90.9 g (1.241 mol) of isobutyraldehyde was slowly added at such a rate that the temperature did not rise above -5 C (approximate 120 min). The resulted solution was allowed to stand in in the refrigerator for one night. The reaction mixture was extracted with ether (3 x 300 mL) after the dilution with 300 mL of water, washed with water (5 x 500 mL) and dried over sodium sulfate. Fractionation of the dried product gave 143.7 g (purity 98%, yield 96%) of the nitrile boiling at 68 - 69 C70.3 Torr. 

The preparation of styrene oxide (phenyloxirane) may be described as an example 33 Styrene (30 g, 0.29 mole) is added to a solution of peroxybenzoic acid (42 g, 0.30 mole) in chloroform (500 ml), and the mixture is set aside at 0° for 24 h, being repeatedly shaken during the first few hours. Completion of the oxidation can be determined by titration of an aliquot part of the chloroform solution. Then the benzoic acid formed and the small excess of peroxybenzoic acid are removed by repeated extraction with 10% sodium hydroxide solution, and the organic phase is washed with water until free from alkali and then dried over sodium sulfate. Fractional distillation affords styrene oxide (24-26 g, 70-75 %) as a colorless liquid, b.p. 101°/40mm. 

Abrams, Kegeles, and Hottle (1) isolated crystalline botulinus toxin from cultures grown on a medium similar to that used by Lamanna et al. except that a tryptic digest was used rather than whole casein. The toxin was purified by acid precipitation at pH 3.5 followed by sodium sulfate fractionation at controlled pH. Considerable purification was also achieved by ethanol fractionation in the cold. The toxin was crystallized by dialysis at 4 C. of solutions containing 1% protein or... 

Liquid carboxylic acids are first freed from neutral and basic impurities by dissolving them in aqueous alkali and extracting with diethyl ether. (The pH of the solution should be at least three units above the pKg of the acid, see pK in Chapter 1). The aqueous phase is then acidified to a pH at least three units below the pK of the acid and again extracted with ether. The extract is dried with magnesium sulfate or sodium sulfate and the ether is distilled off The acid is fractionally distilled through an efficient column. It can be further purified by... 

Common impurities found in aldehydes are the corresponding alcohols, aldols and water from selfcondensation, and the corresponding acids formed by autoxidation. Acids can be removed by shaking with aqueous 10% sodium bicarbonate solution. The organic liquid is then washed with water. It is dried with anhydrous sodium sulfate or magnesium sulfate and then fractionally distilled. Water soluble aldehydes must be dissolved in a suitable solvent such as diethyl ether before being washed in this way. Further purification can be effected via the bisulfite derivative (see pp. 57 and 59) or the Schiff base formed with aniline or benzidine. Solid aldehydes can be dissolved in diethyl ether and purified as above. Alternatively, they can be steam distilled, then sublimed and crystallised from toluene or petroleum ether. 

Cyclohexene can be prepared on a large scale still more rapidly and efficiently by the distillation of cyclohexanol over silica geP or, better, activated alumina. Using a 25-mm. tube packed with 8- to 14-mesh activated alumina (Aluminum Company of America) and heated to 380-450 over a 30-cm. length, 1683 g. of cyclohexanol was dehydrated in about four hours. After separating the water, drying with sodium sulfate, and fractionating with a simple column, 1222 g. (89 per cent yield) of cyclohexene, b.p. 82-84 , was obtained. 

Bromine (128 g., 0.80 mole) is added dropwise to the well-stirred mixture over a period of 40 minutes (Note 4). After all the bromine has been added, the molten mixture is stirred at 80-85° on a steam bath for 1 hour, or until it solidifies if that happens first (Note 5). The complex is added in portions to a well-stirred mixture of 1.3 1. of cracked ice and 100 ml. of concentrated hydrochloric acid in a 2-1. beaker (Note 6). Part of the cold aqueous layer is added to the reaction flask to decompose whatever part of the reaction mixture remains there, and the resulting mixture is added to the beaker. The dark oil that settles out is extracted from the mixture with four 150-ml. portions of ether. The extracts are combined, washed consecutively with 100 ml. of water and 100 ml. of 5% aqueous sodium bicarbonate solution, dried with anhydrous sodium sulfate, and transferred to a short-necked distillation flask. The ether is removed by distillation at atmospheric pressure, and crude 3-bromo-acetophenone is stripped from a few grams of heavy dark residue by distillation at reduced pressure. The colorless distillate is carefully fractionated in a column 20 cm. long and 1.5 cm. in diameter that is filled with Carborundum or Heli-Pak filling. 4 hc combined middle fractions of constant refractive index are taken as 3-l)romoaccto])lu iu)nc weight, 94 -100 g. (70-75%) l).p. 75 76°/0.5 mm. tif 1.57,38 1.5742 m.]). 7 8° (Notes 7 and 8). 

The enantioselective determination of 2,2, 3,3, 4,6 -hexachlorobiphenyl in milk was performed by Glausch et al. (21). These authors used an achiral column for an initial separation, followed by separation of the eluent fraction on a chiral column. Fat was separated from the milk by centrifugation, mixed with sodium sulfate, washed with petroleum ether and filtered. The solvent was evaporated and the sample was purified by gel permeation chromatography (GPC) and silica gel adsorption chromatography. Achiral GC was performed on DB-5 and OV-1701 columns, while the chiral GC was performed on immobilized Chirasil-Dex. 

A perbenzoic acid solution in benzene is prepared as in Chapter 17, Section II. (This solution is approximately 1.8 A/ in perbenzoic acid.) To 67 ml (approx. 0.12 mole of perbenzoic acid) of this solution contained in an Erlenmeyer flask is added 0.10 mole of the ketone in one batch. The resulting solution is swirled at intervals and allowed to stand at room temperature for 10 days. The solution is then washed three times with 50-ml portions of saturated sodium bicarbonate solution to remove benzoic acid and unreacted peracid, and is then washed with water. The solution is dried (anhydrous sodium sulfate), the benzene is evaporated, and the residue is fractionally distilled at reduced pressure to give the ester. 

In a 200-ml round-bottom flask equipped with a magnetic stirrer and a thermometer is placed a mixture of 50 ml of di- -butyl ether and 25 ml of water. The flask is immersed in an ice bath and the mixture is cooled to 5°. In one portion is added 23.2 g (0.1 moles) of trichloroisocyanuric acid (Chapter 17, Section IV), and stirring in the ice bath is continued for 12 hours. The ice bath is removed and the mixture is stirred at room temperature for an additional 8 hours. The reaction mixture is then filtered to remove solids. The water is separated from the organic layer, which is then washed with two additional portions of water, dried with anhydrous sodium sulfate, filtered, and fractionated as above. 

The lactone (fraction boiling at 220-260°) is dissolved in a minimum amount of water and heated on a steam bath for 2 hours. The cooled solution is extracted six times with small portions of ether (or, preferably, continuously extracted with ether overnight). The ether extracts are dried (anhydrous sodium sulfate), and the ether is evaporated. The residue is recrystallized from acetonitrile giving cw-4-hydroxycyclo-hexanecarboxylic acid, mp 150-152°. 

In a 250-ml three-necked flask fitted with a magnetic stirrer, a pressure-equalizing dropping funnel, and a thermometer is placed a solution of l,4-cyclohexanediol(l 1.4g, 0.10 mole), 35 ml of chloroform, and 27 ml of dry pyridine. The solution is cooled in an ice bath to 0-5 and is maintained below 5 throughout the addition. A solution of benzoyl chloride (14 g, 0.10 mole) in 30 ml of dry chloroform is added with stirring at a rate so as to keep the temperature below 5° (approx. 40 minutes). After completion of the addition, the mixture is allowed to come to room temperature and stand overnight. The chloroform solution is washed four times with 50-mI portions of water, once with 50 ml of 5 % sulfuric acid solution, and finally with saturated sodium chloride solution. The chloroform solution is then dried (sodium sulfate), and the solvent is removed. Fractionation of the residue gives a cis and trans mixture of 4-benzoyloxycyclohexanol, bp 175-17870.2 mm, as a very viscous oil, yield about 55%. 

Methylsulfinyl carbanion (dimsyl ion) is prepared from 0.10 mole of sodium hydride in 50 ml of dimethyl sulfoxide under a nitrogen atmosphere as described in Chapter 10, Section III. The solution is diluted by the addition of 50 ml of dry THF and a small amount (1-10 mg) of triphenylmethane is added to act as an indicator. (The red color produced by triphenylmethyl carbanion is discharged when the dimsylsodium is consumed.) Acetylene (purified as described in Chapter 14, Section I) is introduced into the system with stirring through a gas inlet tube until the formation of sodium acetylide is complete, as indicated by disappearance of the red color. The gas inlet tube is replaced by a dropping funnel and a solution of 0.10 mole of the substrate in 20 ml of dry THF is added with stirring at room temperature over a period of about 1 hour. In the case of ethynylation of carbonyl compounds (given below), the solution is then cautiously treated with 6 g (0.11 mole) of ammonium chloride. The reaction mixture is then diluted with 500 ml of water, and the aqueous solution is extracted three times with 150-ml portions of ether. The ether solution is dried (sodium sulfate), the ether is removed (rotary evaporator), and the residue is fractionally distilled under reduced pressure to yield the ethynyl alcohol. 

Catalysis by PhosphorousPentoxide (7) A 500-ml round-bottom flask is charged with a mixture of 1-ethynylcyclohexanol (40 g, 0.32 mole), 250 ml of dry benzene, and 10 g of phosphorous pentoxide. (The addition of the phosphorous pentoxide may be attended by considerable heating if the benzene is not well dried no particular disadvantage is found in this case, providing provision for initial cooling is made.) A condenser is attached to the flask, and the contents are refluxed gently (steam bath) for 2 hours. The cooled solution is then decanted from the phosphorous pentoxide, washed once with bicarbonate solution, and dried (anhydrous sodium sulfate). Removal of the benzene (rotary evaporator) and fractionation of the residue affords the desired product, bp 85-88°/22 mm, 1.4892, about 25 g (61 %). 

Twenty grams of the resulting oily substances were mixed with 20 grams of silicic acid (Mal-linckrodt Chemical Co.),applied to a column 40 cm in length and 4.5 cm in diameter filled with silicic acid,and eluted with a be nzene-ecetone-methanol mixture. The initial eluate which eluted with a 1 1 0 mixture was discarded and the active fractions eluted with 1 3 0 and 1 3 0.3 mixtures were collected and concentrated to dryness in vacuo. 11 g of this crude substance was then dissolved in a small amount of ethyl acetate and applied to the same silicic acid column as above. After discarding the initial eluates by the 1 1 and 2 1 benzene-acetone mixtures, aclacinomycin 8 fractions were first eluted with the above mixtures of 1 3 and 1 5 ratio, and aclacinomycin A fractions were then eluted with the 1 5 0.5 and 1 5 1 benzene-acetone-methanol mixtures. The eluates were dried over anhydrous sodium sulfate and concentrated to dryness in vacuo. 4,8 g of crude aclacinomycin A and 3.5 g of aclacinomycin 8 were obtained as yellow powder. 

Preparation of cholesta-5,7-diene-ia,3/3-diol a solution of 500 mg of the 1,4-cyclized adduct of cholesta-5,7-dien-3/3-ol-ia,2a-epoxideand 4-phenyl-1,2,4-triazoline-3,5-dione in 40 ml of tetrahydrofuran is added dropwise under agitation to a solution of 600 mg of lithium aluminum hydride in 30 ml of THF. Then, the reaction mixture liquid Is gently refluxed and boiled for 1 hour and cooled, and a saturated aqueous solution of sodium sulfate is added to the reaction mixture to decompose excessive lithium aluminum hydride. The organic solvent layer is separated and dried, and the solvent Is distilled. The residue Is purified by chromatography using a column packed with silica gel. Fractions eluted with ether-hexane (7 3 v/v) are collected, and recrystallization from the methanol gives 400 mg of cholesta-5,7-diene-la, 3/3-diol. 

The mixture was heated to reflux for 24 hours. The chloroformic reaction mixture was washed with water, and then dried over anhydrous sodium sulfate. The chloroform was evaporated off and the oil obtained was fractionally distilled under a pressure of 0.2 mm Hg. The fraction distilling at 140°C to 160°C, being the desired product indicated above, was collected and crystallized. Yield 94 g (32% of theory) MP 7B°C (after recrystallization in petroleum ether). 

The combined extracts were washed with water, dried over anhydrous sodium sulfate and concentrated to approximately 35 ml. The solution was chromatographed over 130 g of Florisil anhydrous magnesium silicate. The column was developed with 260 ml portions of hexanes (Skellysolve B) containing increasing proportions of acetone. There was thus eluted 6a,9a-difluoro-11/3,17a,21 -trihydroxy-16a-methy 1-1,4-pregnadiene-3,20-dione-21 -acetate which was freed of solvent by evaporation of the eluate fractions. 

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