二氧化鈦塗佈管柱之製備及其以毛細管電層析分析植物酚類之應用

Abstract

本研究以二氧化鈦溶膠凝膠作為開管式毛細管電層析之靜相。首先，在pH 1.5 且冰浴 (4℃) 的情況下來水解titanium isopropoxide，然後將溫度升溫至50℃，溫度維持7小時，以進行縮合反應，所得的產物為穩定且透明的膠體溶液。此膠體溶液，加入0.32 mg/ml空間穩定劑 (polyethylene glycol)，經減壓濃縮成較高濃度的膠體溶液。然後將其引入內徑為 50 μm 的毛細管中，在200℃ 下反應24小時，使毛細管管壁之矽純基與溶膠之鈦醇基進行縮合反應。經由電滲透流的測定，發覺在甲酸—Tris緩衝溶液中，管壁的等電點為pH 4.7，當pH 小於此值時，電滲透流為逆向；反之，則為正向。而在磷酸緩衝溶液下，因為磷酸分子會錯合在管壁上，所以電滲透流在pH 2.0以上恆為正向。將此二氧化鈦管柱應用於植物酚之分離，由研究發現其可用於 catechin 與 epicatechin 中性幾何異構物之分析，以分析電壓為15 kV，動相為磷酸緩衝溶液 (pH 9.0, 40 mM) 為最佳條件，平均理論板數為 3600 m-1；此管柱尚可用於分離植物酚類中的一些酸性分析物，如syringic acid、caffeic acid、salicylic acid 、p-coumaric acid 和 4-hydroxybenzoic acid。本研究選用甲酸/ Tris、EDTA 與磷酸緩衝溶液，改變 pH 值與緩衝溶液濃度等測試，發現動相會與分析物競爭管壁上之二氧化鈦而將分析物從靜相中交換出來，其分析之最佳條件為電壓 -20 kV，動相為EDTA 緩衝溶液 (pH 5.0, 40 mM)，流析順序為salicylic acid > 4-hydroxybenzoic acid > syringic acid > p-coumaric acid > caffeic acid，平均理論板數為 3.2 × 104 m-1，RSD < 4%。綜合以上，本研究所製備之二氧化鈦塗佈管柱，可成功分離植物酚的幾何異構物與植物酚酸。

In this study, titanium dioxide nanoparticles as a stationary phase in open-tubular capillary electrochromatography (CEC) was prepared. The process has been done in the following way. First, titanium isopropoxide was hydrolyzed at the pH 1.5 (4℃), then the mixture was condensed by heating at 50℃ for 7 h. A stable, clear solution was obtained. PEG 8000 (0.32 mg/ml) was then added to the TiO2 colloidal solution and concentrated under vacuum at 50℃.The high-concentration solution was introduced into the capillary column by nitrogen, then put in the oven and reacted at 200℃ for 24 h. The step made the condensation reaction of TiO2 nanoparticles with silanol groups of fused-silica capillary. EOF property was investigated in a variety of buffer solutions : /formate/Tris, phosphate and EDTA/. With formate/Tris buffer, EOF reversal at pH below 4.7 and cathodic EOF at pH above 4.7 were indicated. With phosphate and EDTA, only cathodic EOF were indicated. This is because of phosphate and EDTA buffer arosed titanium complex and yield a negative charge layer on the surface of TiO2. The CEC performance of the column was tested with plant phenols and phenolic acids. When mobile phase in phosphate (40 mM, pH 9.0), applied voltage of 15 kV, two plant phenols which are diastereomers, (+)-(2R,3S)-catechin and (−)-(2R,3R)-epicatechin, could be baseline separated. With the same column at the mobile phase of EDTA (40mM, pH 5.0), applied voltage of 20 kV, five phenolic acids, syringic acid, caffeic acid, salicylic acid, p-coumaric acid and 4-hydroxybenzoic acid could be baseline separated, the average theorical plate is 3.2 × 104 m-1 and RSD < 4% with four measurements.