Column chromatography combination

Next we studied high temperature bromination of benzobarrelene at 150 C. NMR analysis indicated that the reaction mixture was very complex and consisted of at least ten products. After repeated column chromatography combined with fractional crystallization we have been able to separate 18 compounds (Scheme 6). Four of them were bromoalcohol compounds 18, 12, 22 and 2fl. After high temperature bromination we expected three isomeric non-rearranged products with benzobarrelene skeleton and isolated 22, 22, and 24 in yields of 34, 9.3, and 6.2 %, respectively. Because of the very close structural similarity we were not able to make a clear-cut differentiation between the stereochemistry of 22 and 24-Therefore, we carried out an X-ray analysis (ref. 9) of the isomer 22-... 

In preliminary studies, the binding of C-maculosin was noted in the cytosolic (soluble) fraction of a spotted knapweed leaf extract, and little if any in the insoluble fraction. Binding activity was reduced or destroyed by treatment of the soluble fraction with heat or proteases, suggesting that the toxin receptor is a protein. Purification of the receptor has been pursued successfully using size exclusion column chromatography combined with affinity chromatography. The affinity support consists of epoxy activated Sepharose 6B to which synthetic maculosin has been attached. Further work should reveal the role of the receptor in the process of toxin-induced symptom production. 

Accurately quantifying the amount of provitamin A carotenoids in a food product is essential for determining nutritional value of foods. The AO AC method for determination of vitamin A (974.29) and carotenoids (941.15 and 970.64) in foods utilizes open column chromatography combined with a colorimeter (vitamin A) or spectrophotometer (carotenoids) (Horwitz, 2006). It is recommended to extract with acetone-hexane followed by filtration, then remove the acetone by rinsing with water. The extracts in hexane are applied to an activated Mg02 diatomaceous earth column and eluted using acetone and hexane... 

Mixtures passed through special columns (chromatography) in the gas phase (GC) or liquid phase (LC) can be separated into their individual components and analyzed qualitatively and/or quantitatively. Both GC and LC analyzers can be directly coupled to mass spectrometers, a powerful combination that can simultaneously separate and identify components of mixtures. 

K. Yamashita, M. Motohaslii and T. Yashiki, High-performance liquid cliromatograpliic determination of phenylpropanolamine in human plasma and urine, using column switching combined with ion-pair chromatography , J. Chromatogr. 527 103-114 (1990). 

A mixture of 22 parts of 1 -ethyl-1,4-dihydro-5H-tetrazol-5-one,45 parts of 1 -bromo-2-chloro-ethane,26 parts of sodium carbonate,0.3 part of potassium iodide and 240 partsof 4-methyl-2 pentanone is stirred and refluxed overnight with water-separator. The reaction mixture is cooled, water is added and the layers are separated. The aqueous phase is extracted three times with dichloromethane. The combined organic phases are dried, filtered and evaporated. The residue is purified by column-chromatography over silica gel using trichloromethane as eluent. The pure fractions are collected and the eluent is evaporated, yielding 28.4 parts (80%) of 1-(2-chloroethyi)-4-ethyl-1,4-dihydro-5H-tetrazol-5-one as a residue. 

Raney nickel (3.5 cc) was suspended in 10 ml of ethanol and 356 mg of methyl -(5-ben2yl-oxyindolyl-3)-a -aminoacetyl-a -methylthiopropionate was added to the mixture together with 20 ml of ethanol. Then, the reaction mixture was stirred for 1 hour at room temperature and thereafter filtered to remove insoluble substances. The residue was washed with 100 ml of ethanol and 50 ml of acetone and both the filtrate and the wash liquid were combined and concentrated under reduced pressure. By column chromatography (silica gel and acetone), 210 mg of methyl -(5-hydroxyindolyl-3)-0 -acetylaminopropionate as colorless glasslike substance in the yield of 90%. 

The frozen shells were ground in a cold mortar with 50 mM sodium acetate buffer, pH 5.8, containing 10 mM EGTA and 0.2 M NaCl, then the mixture was centrifuged. The pellets were re-extracted with the same buffer, and centrifuged. All supernatants were combined, and the photoprotein was precipitated with ammonium sulfate. The photoprotein in the precipitate was purified by four steps of column chromatography at near 0°C. Due to the instability of the photoprotein, efforts were made to reduce the time required for purification. 

To a solution of 1.44 g (3 mmol) of MAD in 10 mL of toluene are added 154 mg (1 mmol) of 4-rerr-butyl-cyclohexanone (3) at — 78°C. Butyimagnesium bromide (3 mmol) in btzO is added and the reaction mixture is stirred at — 78 C for 2 h. After quenching with 1 N HCI and extraction with Et20, the combined extract is dried and concentrated. The crude product is purified by column chromatography on silica gel (Et20/hex-ane) yield 142 mg (67%) d.r. 100 0 [determined by capillary GC (column PEG-HT, 0.25 mm x 25m temp. 130"C) by comparison with authentic samples]. 

To 158 mg (1 mmol) of 2-formyl-jVJV,3-trimcthylbutanamide in 5 ml, of CH2C12 are added at 0°C 2 mL of 1.0 M (dichloro)methylaluminum (2 mmol) in hexane. The reaction mixture is stirred at 25 °C for 2 h, then quenched with 1 M HC1. After extraction with CHCI,/EtOH (3 1), the combined organic layer is dried over Na,So4 and concentrated in vacuo. The crude product is purified by silica gel column chromatography yield 106 mg (62%) d.r. [(26, 35 )/(25, 3R )] 99 1 (determined by capillary GC). 

A mixture of 1.4 g (10 mmol) of 4-chlorobenzaldehyde and 0.71 g (5 mol %) of the chiral polymer E is stirred in 10 mL of dry toluene for 15 h, under a dry nitrogen atmosphere, to form the Schiff base. After cooling to 0lC, 15 mL (15 mmol) of 1 M diethyl/inc in hexane is added and the mixture is stirred for a further 24 h at O C. 1 N HC1 is then added dropwise at O C, and the chiral polymer is removed by filtration. The polymer is washed several times with 11,0 and Et,0. The aqueous layer is separated and extracted with Et20. The combined organic layer is dried over MgS04 and concentrated under reduced pressure. The crude product is purified by column chromatography (silica gel, CHC1,) yield 1.61 g (95 %) 99 % ee [a]2,0 —23.9 (r = 4.93, benzene). 

Use of the valine derived (4S )-3-acetyl-4-isopropyl-1,3-oxazolidine (8)92, the C2-symmetric reagents (2.5,55)-l-acetyl-2,5-bissubstituted pyrrolidine 994, or the doubly deprotonated acetyl urea /V-acetyl- V..V -bis[(.S)-l-phcnylethyl]urea (10), also does not lead to sufficient induced stereoselectivity combined with acceptable chemical yield. When the acetyl urea enolate is reacted with aliphatic and aromatic aldehydes, the diastereomeric adducts (ratios ranging from 1 1 to 3 1) may be separated by column chromatography to give ultimately both enantiomers of the 3-hydroxy acids in 99% ee110. 

To a 0.02-0.05 M soln of (1 / , l R)-(2-hydroxy-l-phenelhyl)( I-arylalky I (amine 5 in CH2C12/CH,0H (2 1) at 0 C is added, in one portion, 1 equiv of Pb(OAc)4. The mixture is stirred for 2-20 min, then 5 ntL of a 15% aq solution of NaOlI is added. The aqueous layer is separated, extracted with CH,C1, and the combined organic extracts are evaporated in vacuo. Then the residue is dissolved in Ei,0 and stirred for 4-16 h wilh an equal volume of 3 N aq HC1. Subsequently, the aqueous phase is basified by the addition of Na2C02 and extracted with Et,C>. After drying over MgS04 and removal of the solvent the crude amine 6 is purified by Kugclrohr distillation or Hash column chromatography yield 46 - 65% 92- >99% ee. 

Removal of solvent from the extracts leaves a residue that is purified by dry-column chromatography.2 The residue is dissolved in 40 ml. of acetone in a 300-ml., round-bottomed flask, 30 g. of silica gel (Note 8) is added, and the acetone is removed with a rotary evaporator. The resulting solid mixture is placed on top of 360 g. of dry silica gel (Note 8) packed in flexible nylon tubing (Note 9), and the column is developed with 420 ml. of 10 1 (vjv) benzene-acetone. Approximately 150 ml. of solvent drips from the bottom of the column toward the end of development, and this eluent is collected in 25-ml. fractions and checked for product by thin layer chromatography (Note 10). The column itself is then cut into 2-cm. sections, the silica gel in each section is eluted with three 25-ml. portions of ethyl acetate, and the eluent from each section is analyzed by thin-layer chromatography (Note 10). Combination of all the product-containing fractions yields 1.2-1.5g. (40-47%) of the benzylated compound as an oil, n 1.6083 (Notes 11 and 12). 

External cooling is now discontinued, and 21.4 g. (0.12 mole) of A-bromosuccinimide [2,5-Pyrrolidinedione, 1-bromo-] (Note 5) is added. Stirring is continued until all of the solid is dissolved (ca. 1 hour). The resulting solution, which may be pale yellow, is concentrated with a rotary evaporator (bath temperature 40—45°) to a volume of about 300 ml. and extracted with five 120-ml. portions of ether. The combined ether extracts are dried over anhydrous magnesium sulfate (20-50 g.), and removal of solvent at reduced pressure provides an oil, which is purified by column chromatography on silica gel (Note 6). Pure bromo-hydrin acetate is obtained as a colorless oil in amounts of up to 26 g., a 65% yield based on famesyl acetate (Notes 7 and 8). 

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