Title

離子液體共價鍵結之管柱於毛細管電層析分離蛋白質的應用

Translated Titles

Capillary Electrochromatographic Separation of Proteins with Covalently Bonded Ionic Liquid Column

DOI

10.6342/NTU.2008.02235

Authors

王彗玲

Key Words

毛細管電層析 ; 離子液體 ; 蛋白質 ; capillary electrochromatographic separation ; ionic liquid ; protein

PublicationName

臺灣大學化學研究所學位論文

Volume or Term/Year and Month of Publication

2008年

Academic Degree Category

碩士

Advisor

劉春櫻

Content Language

繁體中文

Chinese Abstract

毛細管電層析是一種結合毛細管電泳與高效能液相層析,涵蓋此兩種技術優點的分離方法。本研究將離子液體共價鍵結於毛細管內壁,作為開管式管柱的靜相,應用於蛋白質之分離。管柱製備過程分別對官能基化及烷化反應的最適當溫度及時間系統化探討。由研究得知,將熔矽毛細管首先以(3-chloropropyl)trimethoxysilane (CPTMS) 於120℃進行矽烷化反應15小時,再將咪唑溶於DMF,與CPTMS的活性基於110℃進行咪唑官能基衍生化反應10小時,最後以1-bromooctane於100℃進行烷化反應10小時。上述所製備管柱在pH 3.5、50 mM的醋酸緩衝溶液中,可以使逆向電滲流達到-8.32 × 10-4 cm2 V-1s-1,RSD值為4.08% (n = 4)。將此管柱應用於分析分子量及pI值相似之蛋白質混合物 (ovalbumin、apo-transferrin、bovine serum albumin、conalbumin、rat serum albumin)。由研究發現,以磷酸緩衝溶液不同pH值 (2 ~ 10) 進行實驗時,磷酸皆易與管壁作用,而使靜相脫落,因此改用醋酸及甲酸緩衝溶液來分離蛋白質。嘗試改變緩衝溶液的pH值 (3 ~ 5)、濃度 (10 mM~100 mM),添加有機修飾劑 (甲醇、正丙醇、乙腈) 或離子液體 (1-butyl-3-methylimidazolium chloride),發現以電壓-15 kV,管柱有效長度50 cm,添加15% (v/v)甲醇於醋酸緩衝溶液 (pH 3.5, 40 mM),可出現三支吸收峰,藉由增長管柱有效長度為85 cm,分離效果明顯增加。若進而改用甲酸銨緩衝溶液 (pH 3.5, 40 mM) 為分離條件時,則可出現四支吸收峰,分析物的滯留時間依序為ovalbumin < apo-transferrin < bovine serum albumin < conablumin = rat serum albumin。此外,本研究亦比較不同碳鏈長度之咪唑陽離子靜相對分離的影響,發現分析物與咪唑鍵結管柱靜相之作用力較強,故無法流析出來;而咪唑陽離子碳鏈較短 (C4) 時,分析滯留時間較短,使分離不佳;碳鏈長度較長 (C12) 時,疏水性作用力較大,使得波峰變寬且分離時間變長,最後以C8離子液體管柱分離效果較佳。綜合以上所述,此離子液體不僅提供管柱表面的正電荷,使其具逆向電滲透流,還可減少分析物在管柱表面的吸附,且靜相具凡得瓦爾力、π-π電子作用力、氫鍵與靜電交互作用力等機制。所以本研究除了電泳分離機制外,離子液體尚提供額外的層析作用力,因而有助於性質相近的蛋白質分離。

English Abstract

To prepare ionic liquid columns, the capillary was first silanzed with (3-chloropropyl)trimethoxysilane (CPTMS). Imidazole was then subjected to be bonded covalently to the CPTMS. Finally, bonded imidazole in the column was linked with 1-bromooctane to form a positively charged diakylimidazolium layer for the generation of reversed electroosmotic flow (EOF) over a pH range of 4 to 8.5. Using phosphate as background electrolyte (BEG), the strong ion-pair interaction between diakylimidazolium and phosphate caused the bleeding of stationary phase. We selected five proteins, such as ovalbumin (OVA), apo-transferrin (apoTf), bovine serum albumin (BSA), rat serum albumin (RSA) and conalbumin (CON) as model compounds, whose molecular weights and pI values are alike to each other. Both acetate and formate buffer have been systematically studiced as mobile phase. Formate buffer gave better performance than acetate buffer. With running buffer of formate (pH 3.5, 40 mM) and effective length of 85 cm, five proteins showed four absorption peaks within 40 min with RSD ≤ 4.59% (n = 3). In this study, the retention behavior of imidazole coated column was also compared with those of the ionic liquid column. The best is the C8 substituent of imidazolium ionic liquid. High resolving power of the ionic liquid column seems to be predominantly from hydrophobic force, hydrogen bonding, π-π electron and electrostatic interactions.

Topic Category 基礎與應用科學 > 化學
理學院 > 化學研究所
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