- 學位論文
溫度敏感型高分子PNIPAAm於毛細管電泳之應用:中性分子分離的新穎方法
Applications of thermoresponsive PNIPAAm polymer on capillary electrophoresis: a novel approach to separate neutral compounds
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摘要
本研究分為兩部份。第一部份為高分子材料聚丙基丙烯醯胺 (poly(N-isopropyl acrylamide，PNIPAAm)以自由基聚合法合成。使用不同的鏈轉移試劑，如3-mercaptopropionic acid (MPA) 和 2-aminoethanethiol hydrochloride (AET·HCl)可在高分子鏈末端形成羧基和胺基官能基，在溶劑THF中鏈轉移試劑及IPAAm單體間的莫耳比控制合成出數種大小之高分子，並利用末端官能基滴定測得平均分子量。另外我們合成表面修飾PNIPAAm之二氧化矽奈米粒子，經掃描式電子顯微鏡觀察粒徑約 20 nm，並以FT-IR進行官能基鑑定。PNIPAAm高分子快速且可逆的轉變親疏水性可隨著水溶液溫度及濃度的改變進行調整，我們利用此性質設計一種新的分離機制，可利用來進行毛細管電泳分離中性分子。 第二部份為對於五種中性固醇類標準品，testosterone、dexamethasone、hydrocortisone 21-acetate、hydrocortisone及prednisolone，進行分離之毛細管電泳實驗。將第一部分所合成之 PNIPAAm及修飾PNIPAAm的二氧化矽奈米粒子應用於毛細管電泳中，形成在動相中之偽靜相，設計了二種毛細管電泳層析方法，卻因PNIPAAm在毛細管內的分佈不均勻和二氧化矽奈米粒子的團聚導致無法觀察分離效果。之後發展了PNIPAAm輔助微胞電動力層析方法，PNIPAAm與界面活性劑十二烷基硫酸鈉 (sodium dodecyl sulfate，SDS) 形成之微胞混合，進行電泳實驗，PNIPAAm參與了樣品在微胞與緩衝溶液間之分配，因而得到優於傳統MEKC較好的效果。再進一步將此分離模式改良以SDS微胞從進樣端帶動樣品進入含PNIPAAAm 緩衝溶液的毛細管中，使其除了能提高分離的效率，並保留傳統MEKC樣品堆積之效。使用這個新改良的方法可以有效的將原本MEKC分離不開之中性固醇類標準品分離。這種新的分離方法可以有效的調整MEKC對樣品之選擇性，並可藉由緩衝溶液濃度(離子強度)的改變得到不同之分離效果，此方法的優點為避免有機溶劑的使用及對於複雜的修飾劑選擇，提供了綠色化學的實驗基礎並降低實驗的困難度。在本研究中，我們對於此新方法的各項條件，如PNIPAAm的電性影響、分子量大小、及毛細管電泳的各項參數進行討論。
並列摘要
In this study, an effective PNIPAAm-assisted MEKC system was developed for natural steroids separation. Two parts of studies were carried out in this research. The first part is the synthesis of Poly(N-isopropyl acrylamide (PNIPAAm) by free radical polymerization. The polymerization of NIPAAm was carried out using 3-mercaptopropionic acid (MPA) and 2-aminoethanethiol hydrochloride (AET·HCl) as chain-transfer agents to form carboxyl and amine functional end group, respectively. The polymer length was controlled by the molar ratio of chain transfer agents to IPAAm in THF, and was confirmed by end group titration. The synthesized polymer was also designed to graft onto the surface of silica nanoparticles. SEM, FT-IR spectroscopy were used to characterize polymer composition, particle size and functional group on surface of nanoparticles. The fast reversible hydrophilic/hydrophobic changing properties of PNIPAAm polymer by varying temperature and salt concentration of aqueous solution was used to design for neutral steroids separation by capillary electrophoresis. In the second part, we separated five neutral steroids (testosterone, dexamethasone, hydrocortisone, hydrocortisone 21-acetate, and prednisolone) by capillary electrophoresis analysis. There are two approaches in CEC analysis. Firstly, the materials which were synthesized in first part, PNIPAAm polymer and PNIPAAm-grafted silica nanoparticles, were employeed as a pseudo-stationary phase. Results did not show good separation of these steroids in these designs due to the poor distribution of the PNIPAAm and the aggregation of silica nanoparticles in the capillary. Therefore, the second design was performed. A PNIPAAm-assisted MEKC was carried out by mixing PNIPPAm polymers and SDS micelles simultaneously. The separation efficiency of PNIPAAm-assisted MEKC was better than traditional MEKC because PNIPAAm polymer participated in the sample partition between SDS micelles and buffer environment. In order to obtain higher separation efficiency and maintain the stacking effect, a sequential mode was then used. Results indicated that an effective separation of steroids was achieved with an excellent resolution, which was better than that of traditional MEKC. The hydrophilic/hydrophobic properties of PNIPAAm polymer could be tuned by varying buffer concentration (ionic strength), which significantly changed the selectivity of MEKC for steroids separation. The new separation approach that avoided the use of organic solvent and the complicated choices of modifiers provided a tool for green chemistry and reduced complex designs of experiment for MEKC. In addition, factors that involved in steroids separation such as PNIPAAm polymer molecule weight, the end functional group of polymer and the parameters of CE condition were also discussed.
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