Protein extraction by reverse micelle phase in a high speed countercurrent chromatography

• Main Authors: Li Hsin-Yi, 李心怡
• Other Authors: Tiing Yu
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/87598289598830924355

碩士 === 國立交通大學 === 應用化學系 === 89 === As surfactants are added into non-polar organic solvent, they will aggregate to form reverse micelles. Reverse micelles provide micro water pools that enable solubilization of polar molecules in non-polar solutions. Extraction using reverse micelles includes two steps, i.e. forward and backward extractions. Forward extraction is a process that protein molecules in aqueous solution are extracted to surfactant-containing organic solution, and backward extraction is a process that protein molecules in reverse micelle phase are extracted into another aqueous solution. The extraction solvent was prepared by thouroughly mixing sodium bis(2-ethylhexyl)sulfosuccinate in n-hexane (or isooctane) solution with potassium chloride aqueous solution. The upper organic phase thus obtained was used as the stationary phase in a countercurrent chromatography apparatus; while the lower aqueous phase as the mobile phase. Sample solutions were prepared by dissolving cytochrome c in the mobile phase. The forward extraction was operated by delivering the sample solution through the CCC apparatus. The protein molecules were dissolved in the surfactant-containing organic phase, i.e. the stationary phase. A small volume of base was then injected into the column to extract the proteins back to the aqueous mobile phase and collected at the outlet. During the forward and backward extractions, the dilute protein solution was effectively concentrated. For example, a 40 mL of 124.23 mg/L cytochrome c solution could be obtained from a sample of 700 mL of 8.64 mg/L cytochrome c solution.