Aqueous biphasic extraction chromatography

NEW TECHNOLOGIES FOR METAL ION SEPARATIONS POLYETHYLENE GLYCOL BASED-AQUEOUS BIPHASIC SYSTEMS AND AQUEOUS BIPHASIC EXTRACTION CHROMATOGRAPHY Robin D. Rogers and Jianhua Zhang... 

New Technologies for Metal Ion Separations Polyethylene Glycol Based-Aqueous Biphasic Systems and Aqueous Biphasic Extraction Chromatography... 

Because of the behavior of the resins, which appear to mimic aqueous biphase extraction in PEG-ABSs, this separation technique has been named aqueous biphasic extraction chromatography (ABEC). Many of the potential commercial applications for metal ion separations utilizing PEG-ABSs may reach fruition because of the ability to strip the columns with water [27]. 

V. METAL ION EXTRACTION USING AQUEOUS BIPHASIC EXTRACTION CHROMATOGRAPHY (ABEC)... 

Preparation of 26 [14] Allyl alcohol 22 (0.91 mmol) and triethylamine (1 equiv.) were dissolved in dry tetrahydrofuran (THF) (2 mL) under argon. A solution of bromo tris(2-perfluorohexylethyl)silane 23 (0.25 equiv) in THF (2 mL) was slowly added to the reaction mixture at 25 °C. The resulting mixture was stirred at 25 °C for 3 h. After removal of the solvent, the residue was purified by three-phase extraction with FC-72 (10 mL), dichloromethane (10 mL), and water (10 mL). The organic/aqueous biphase was extracted twice more wdth FC-72 (10 mL). After concentration of the combined fluorous extracts, the residue was purified by flash chromatography (hexane/diethyl ether, 50 1) to yield a colorless oil. 

A 1-L, oven-dried, round-bottomed flask equipped with a magnetic stirrer is charged with 9.92 g (27.9 mmol) of methyl R)-3-(tert-butyldiphenylsilyloxy)-2-methylpropionate and 200 mL of dry hexanes (Note 15). The solution is cooled to -78°C, and 31.5 mL (31.5 mmol) of 1 M diisobutylaluminum hydride (in hexane) (DIBAL-H) (Note 16) is added dropwise over 15 min via a syringe pump. After the addition is complete, the resultant solution is stirred at -78°C for 2 hr. The reaction is quenched by pouring the cold solution info 250 mL of saturated aqueous Rochelle s salt. Ether (300 mL) and HjO (75 mL) are added and the biphasic mixture is stirred vigorously for 1 hr (Note 17). The layers are separated and the ether layer is washed with brine. The aqueous layer is extracted with ether (2 x 50 mL) and the combined extracts are dried over Na2S04. Filtration of the solution and concentration of the filtrate under reduced pressure followed by purification of the crude product by flash chromatography (Note 18) yields 7.85 g (86%) of (R)-3-(tert-bUtyldiphenylsilyloxy)-2-methylpropanal as a white solid (Note 19). 

The reaction mixture is cooled to 0°C, one ol the septa is removed, and excess hydride is quenched by the cautious addition of 10-mL portions of 3 N aqueous hydrochloric acid solution (350 mL total) over 5 min (Note 18). The biphasic mixture is stirred at 23°C for 30 min, then poured into a 1-L separatory funnel. The aqueous layer is separated and extracted with three 150-mL portions of ether. The combined organic fractions are washed with 50 mL of 3 N aqueous hydrochloric acid solution followed by 50 mL of brine. The organic layer is dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure (Note 19). The residue is purified by flash column chromatography (230-400 mesh silica gel, 250 g, 43% ether-petroleum ether as eluent) to afford 4.35 g (90%) of analytically pure (R)-p-methylbenzenepropanol as a colorless oil (Note 20). The ee of this product is determined to be >95% (Note 21). 

An overview chapter by Hamel and Hunter presents the state of the art of research on bioseparations. Extraction processes using biphasic aqueous systems, liquid membranes, reversed-micellar systems, and membrane processes are all being actively studied. Significant advances in these topics, including predictive mathematical models, are presented in the first section. The second section includes several papers on affinity and other interaction techniques that are finding uses in protein purification. In the last section, we offer several reports that delineate advances in isolation and purification processes such as electrophoresis and chromatography. 

To a 20 mL flask was added 2.5 g of (R,S)-2-ethoxycarbonyl-3,6-dihydro-2H-pyran, followed by 7 mL of 0.2 M pH 7.5 phosphate buffer and 2 mL of Bacillus lentus protease-III solution (approximately 5% solution of the protein). The biphasic solution was stirred at room temperature (23 °C) using a magnetic stirrer. The pH was checked at 0.5 hour intervals and readjusted to 7.5 by the drop-wise addition of 1 N NaOH (approximately 7 mL were required over the complete reaction). The progress of the reaction was monitored by chiral gas chromatography. After 5 hours, the enantiomeric purity of the unreacted ester was >99% and the reaction was stopped by the addition of 10 mL of MTBE. The pH of the aqueous phase was adjusted to 8.5 and the mixture was transferred into a separatory funnel. The aqueous phase was extracted twice with 20 mL of MTBE and the combined organic layers were extracted once with saturated sodium bicarbonate (10 mL), followed by saturated sodium chloride solution (10 mL), and the organic... 

Example 1. Kinetic Resolution in a Biphasic System [20]. A solution of antibody 84G3 (500 mg, 6.67 pmol active site, 0.0086 mol%) in phosphate buffered saline (PBS pH 7.4, 87.5 mL) at room temperature vas added to a solution of ( )-45 (20 g, 77.4 mmol) in toluene (600 mL) in a Teflon tube. The mixture vas shaken at 250 rpm. under argon at the same temperature and the ee vas monitored by chiral-phase HP LG. When ee > 99% vas reached (65 h) the mixture vas cooled to —20 °G for several hours to enable the aqueous phase to freeze. The organic phase vas decanted. The frozen aqueous phase vas left to thaw and extracted vith toluene (3x3 vol. equiv.) and 1 1 EtOAc-toluene (3x3 vol. equiv.). The combined organic phase vas dried over Na2S04, concentrated in vacuo, and purifled by silica gel column chromatography (1 3 EtOAc-hexane) to afford (S)-45 (10 g, 50%). 

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