以逆向流層析儀使用含界面活性劑之1,1,1,2-tetrafluoroethane進行蛋白質的萃取和濃縮

Abstract

本實驗室已由紫外/可見光光譜觀測到陽離子界面活性劑 trioctylmethylammonium chloride (TOMAC) 可在液態環保冷媒1,1,1,2-tetrafluoroethane (R-134a) 中形成反微胞，並可溶解親水性物質。以液態R134a取代傳統反微胞萃取之有機溶劑的優點是，液態R134a在常溫常壓下為氣體，可與欲萃取物直接分離，無須額外程序即可達成濃縮效果。 本研究使用逆向流層析儀(countercourrent chromatography，CCC)，嘗試以含界面活性劑的液態R134a萃取水溶液中的蛋白質。所使用的蛋白質有 cytochrome c和bovine serum albumin，實驗結果顯示，兩者皆可被含界面活性劑的R134a從水溶液中萃取出。雖然不論將蛋白質水溶液當成是靜相或動相都可進行萃取程序，但當使用蛋白質水溶液做為動相時，樣品溶液體積可以大為增加。所測試的界面活性劑有陰離子界面活性劑bis(2-ethylhexyl)sodium sulfosuccinate (AOT)和陽離子界面活性劑 trioctylmethylammonium chloride (TOMAC)，兩者都可得到很好的萃取效率。利用此種方式，可將蛋白質高度濃縮於R134a中，但對於萃取後的收集方法我們還需作進一步的改善。

Solubilization of hydrophilic compounds using liquid 1,1,1,2-tetrafluoroethane (R134a) in the presence of surfactants was studied previously in our laboratory. Micelle formation was verified through absorption spectroscopic data. The advantage of replacing conventional hydrocarbon solvent by liquid R134a lies in the fact that the compressed liquid R134a becomes a gas at room temperatures and pressures, therefore separates from analytes of interest after extraction. Accordingly, the analytes are concentrated directly after the completion of extraction without further process. In this study, we tried to employ surfactant-containing liquid R134a to extract proteins from aqueous solutions using a countercurrent chromatography (CCC) apparatus. Two proteins, i.e., cytochrome c and bovine serum albumin, were extracted from aqueous solutions using the surfactant-containing R134a. While using the protein solution as the stationary phase in the CCC could do the extraction process, the sample volume could be greatly increased when using the protein solution as the mobile phase in the CCC. Very good extraction efficiencies were obtained either using surfactant bis(2-ethylhexyl)sodium sulfosuccinate or trioctylmethylammonium chloride. Proteins were highly concentrated in R134a. However, we need yet to improve the technique to collect protein molecules after the extraction.