臺灣博碩士論文加值系統

本研究以非水解型溶膠凝膠法將二氧化鋯修飾於熔融矽毛細管內壁作為靜相。依序將前驅物zirconium isopropoxide propanol complex與zirconium chloride 的氯仿溶液填入毛細管，以梯度式升溫法從室溫升溫 300 oC 高溫反應塗佈一層於管壁並以一個小時反應時間，使烷氧基與管壁的矽氧基進行反應。將如此製備的二氧化鋯管柱，以磷酸緩衝溶液在不同的 pH 值下進行電滲流測試，由於二氧化鋯在約 pH < 6時表面電位會帶正電，在此時電滲流理應呈現逆向，但由於磷酸離子會錯合在二氧化鋯管壁上，形成一層遮蔽層使管壁表面帶負電。因此在pH 的偏低時，只觀察到二氧化鋯管柱的電滲流較空管小的趨勢，而在 pH = 3 的電滲流才呈現逆相。將此二氧化鋯管柱應用在ovalbumin (OVA)、bovine serum albumin (BSA)、conablumin (ConA) 及apo-transferrin (apoTf) 的分離。由於二氧化鋯的水解穩定度較二氧化矽高，可以在極酸或是極鹼的環境下進行偵測。由研究發現，最佳化條件為磷酸緩衝溶液 (10 mM , pH 8)，並加入 5% MeOH (v/v) 當有機修飾劑，所獲得的流析順序為 ConA > apo-Tf > OVA > BSA，而且可以進一步分離出OVA 的五種醣蛋白異構物與 BSA 的兩種構形異構物。此研究之分離機制，除了電泳效果之外，應是基於二氧化鋯奈米表面的特殊化學，在磷酸緩衝溶液中，所形成的鋯－磷酸錯合物會與分析物進行配位基交換，進而達到較為優異的分離效果。

A ZrO2 nanoparticle-coated open-tubular column was prepared through the non-hydrolysis sol-gel reaction of zirconium isopropoxide propanol complex and zirconium chloride with silanol groups of the fused-silica capillary. The condensation reaction was carried out by gradient temp. raising to 300oC to form a monolayer. EOF measurements and SEM image were used to monitor the completion of reactions. The property of ZrO2 on the inner wall of capillary was significantly affected by the running buffer. Cathodic EOF at pH between 4~6 was indicated. It can be elucidated that the negatively charge phosphate ion in the buffer absorb strongly on the surface of ZrO2 resulting in the formation of phosphate ions-capped ZrO2 NPs complex. We selected four proteins, such as conalbumin (ConA), apo-transferrin (apoTf), ovalbumin (OVA), and bovine serum albumin (BSA) as model compounds, whose molecular weights and pI values are alike to each other. They could be separated with phosphate buffer (10 mM, pH 8.0) with 5 % MeOH and an applied voltage of 20 kV. In addition, five peaks of glycoisoforms of OVA and two forms of BSA were observed under these conditions. The column was also used to separate egg-white proteins. In addition to separating OVA and ConA, it can resolve three microheterogeneities of OVA and two forms (iron-free and iron-saturated) of ConA. In comparison with the retention behavior of the analytes on the bare fused-silica column, the new column of high resolving power seems to be predominantly derived from the ligand exchange of the analytes with the phosphate adsorbed onto the ZrO2 .

摘要 I
Abstract III
目錄 V
圖目錄 IX
表目錄 XII
第一章 緒論 1
第一節 毛細管電層析介紹 1
1-1 毛細管電層析原理 1
1-1.1 文獻回顧 1
1-1.2 毛細管電層析種類 2
1-2 開管式毛細管電層析應用 4
第二節 二氧化鋯性質與在層析分離的應用 11
2-1 二氧化鋯基本性質 11
2-2 二氧化鋯的表面特性 14
2-2.1 表面酸性 14
2-2.2 包覆劑對表面的影響 18
2-2.3 表面磷酸吸附現象 18
2-3 以溶膠凝膠法製備二氧化鋯 20
2-3.1 溶膠凝膠法反應機制 21
2-3.2 反應參數 22
2-3.3 溶膠凝膠法於原子層沉積法的應用 23
2-4 二氧化鋯為層析靜相的應用 24
第三節 蛋白質分析物介紹 28
3-1 蛋白質的簡介 28
3-2 蛋白質的分析 31
第四節 研究動機 34
第二章 實驗部份 35
第一節 實驗儀器 35
第二節 實驗試藥 39
2-1 管柱製備 39
2-2 緩衝溶液 39
2-3 分析樣品 40
第三節 二氧化鋯管柱製備與性質鑑定 41
3-1 毛細管前處理 41
3-2 二氧化鋯管柱之製備 41
3-2.1 將膠體溶液以高溫縮合方式製備 41
3-2.2 以原子層沉積方式製備 43
3-3 二氧化鋯奈米溶液及管柱的性質鑑定 43
3-3.1 紫外線吸收光譜測定 43
3-3.2 穿透式電子顯微鏡鑑定 43
3-3.3 掃描式電子顯微鏡鑑定 44
第四節 毛細管電層析的實驗操作 45
4-1 樣品配置 45
4-2 電滲流的測定 46
4-3 分析樣品於毛細管電層析進行流程 46
第三章 結果與討論 47
第一節 二氧化鋯膠體粒子的製備與鑑定 47
1-1 二氧化鋯膠體粒子的製備 47
1-2 二氧化鋯膠體粒子的性質鑑定 47
1-3 穿透式電子顯微鏡鑑定 50
第二節 以二氧化鋯膠體溶液製備塗佈管柱 51
2-1 毛細管表面前處理 51
2-2 二氧化鋯膠體粒子的濃縮 51
2-3 管柱電滲流及表面性質之測定 54
第三節 以原子層沉積法方式製備塗佈管柱 59
3-1 管柱製備 59
3-2 不同條件下對管柱塗佈的影響 61
3-2.1 反應溫度對管柱電滲流的影響 61
3-2.2 塗佈層數對管柱電滲流的影響 63
3-2.3 聚合時間對管柱電滲流的影響 65
3-3 掃描式電子顯微鏡偵測 67
第四節 毛細管電層析的應用 69
4-1 動相 pH 值的影響 70
4-2 動相種類的影響 78
4-3 動相濃度的影響 80
4-4 有機修飾劑的影響 82
4-4.1 甲醇 82
4-4.2 乙睛 83
4-5 毛細管內徑的影響 87
4-6 真實樣品的分離 92
第四章 結論 95
參考文獻 97

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