第三章 實驗藥品、設備與方法
4.10 ChromSpher Lipids HPLC (銀離子)管柱的清洗與再生
Smith et al. (1994)提到在使用 ChromSpher Lipids HPLC (銀離子)管柱的實驗過程中，TG 的滯留時間會隨著分析樣品數的增加而逐漸的減少。他們提出假設說這是由於銀離子在緩 慢的變質(denaturation)，因為在管柱中存在著低濃度的分析樣品和溶劑且這些物質會與管柱 的固定相堅強的結合，造成管柱的分析效能降低。在本研究中也遇到了此管柱容易失效的 問題。 Christie (1988)指出此管柱能以下述方法有效的再生。先以甲醇/氰化甲烷(1/1, v/v) 流洗，再注射微量的硝酸銀氰化甲烷溶液。
Smith et al. (1994)指出 ChromSpher Lipids 管柱能用下述方法再生 2-3 次，且其管柱的效能 可完全恢復。管柱先分別以氰化甲烷、甲醇及水(HPLC-grade)，各流洗 20 min，流速 1 mL/min。然後注射硝酸銀水溶液(0.1 g in 1 mL)，直到過量的硝酸銀從管柱中被沖提出來(使 用氯化鈉溶液偵測)。再以水、甲醇、氰化甲烷同上述條件分別流洗。但我利用上述兩種方 法再生結果都不是很好，由於流洗液與移動相組成不同，必須更換溶液非常不便且造成溶 劑浪費。本研究嘗試利用丙酮(移動相組成之一)來再生管柱，在流速 1 mL/min 下以丙酮流 洗 60 min，然後注射硝酸銀丙酮/氰化甲烷(1/1, v/v)溶液(0.1 g in 1 mL)，直到過量的硝酸銀 從管柱中被沖提出來(使用氯化鈉溶液偵測)，再以丙酮流洗 60 min，接著以實驗的移動相 組成空白流洗兩次，結果發現此方法也能成功的使管柱再生且再生效果非常好，結果如圖 4-15 所示。
mv
minutes
0 5 10
50 100 150 200 250
mv
minutes
0 5 10
200 400 600 800
mv
minutes
0 5 10
50 100 150
圖 4-15 ChromSpher Lipids HPLC 管柱效能測試圖。樣品 POP、PPO 溶於二氯 甲烷，固定組成移動相 (Isocratic): 二氯甲烷/丙酮=98/2 (v/v)，流速 1 mL/min。
(a)實驗過程分析效能降低，(b)管柱完全失效，(c)再生後管柱效能。
4-15(a)
4-15(b)
4-15(c)
笫五章 結論
本研究以三個步驟合成結構脂質 OPO，(一)棕櫚酸與甘油以 Lipozyme IM 催化進行酯 化反應，反應產物甘油酯經矽膠管柱純化後得到三棕櫚酸甘油酯(純度 100%)。(二)以橄欖 油為原料經兩階段低溫溶劑結晶法純化油酸。(三)以上述三棕櫚酸甘油酯與油酸進行酸解反 應，目的是得到 1,3-dioleoyl-2-palmitoyl-glycerol 之結構脂質 OPO。結論分別陳述如下：
1. 將棕櫚酸與甘油以 Lipozyme IM 催化進行酯化反應，在下述條件 FFA 2.1 mmol、甘油 0.7 mmol、操作溫度 80℃、壓力 5 mm-Hg、酵素量 50 mg、轉速 500 rpm 下，反應 15 h 可得到酯化程度 98.8%，產物甘油酯中三酸甘油酯含量為 98.62%，經矽膠管柱純化後，
可得到 100%的三酸甘油酯，其脂肪酸組成 99%為棕櫚酸。
2. 從橄欖油為原料經兩階段低溫溶劑結晶法，可將橄欖油脂肪酸中油酸由原來之 76.76%
提昇至 96.64%，總回收率達 72.84%。
3. 在正己烷中，以 Lipozyme IM 脂解酵素催化三棕櫚酸甘油酯與油酸之酸解反應，於三棕 櫚酸甘油酯對油酸的莫耳比為 1:8，酵素量 20 mg，反應溶液體積 3 mL，三棕櫚酸甘油 酯濃度 0.1 mmol，操作溫度 50℃及反應 24 h 之反應條件下，油酸在甘油酯中之含量可 達 66.07 mol%，三酸甘油酯在甘油酯中之含量可達 97.12%。
4. 酸解反應產物三酸甘油酯 sn-2 位置脂肪酸組成分析，油酸比例為 9.26 mol%(利用猪胰臟 脂解酵素分析)及 9.21 mol% (利用 Novozym 435 分析)。
5. 以 HPLC 分析酸解產物三酸甘油酯組成，經檢量線換算後可知酸解產物三酸甘油酯中 OPO 佔 74.26 mol%，OOP 與 OOO 合佔 10.11 mol%，表示酸解反應有醯基轉移發生，
且油酸在酸解產物三酸甘油酯之 sn-2 位置佔 10.11 mol%，與利用猪胰臟脂解酵素分析 佔 9.26 mol%及利用 Novozym 435 分析佔 9.21 mol%的結果接近。
6. ChromSpher Lipids HPLC (銀離子)管柱能用丙酮與硝酸銀丙酮/氰化甲烷溶液成功的再 生 2-3 次且管柱的效能可完全恢復。
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計畫成果自評
本研究為兩年期計畫，已達成預定之目標。研究成果並已發表於美國油品化學會誌 Chen, M.L. S.R. Vali, J.Y. Lin and Y.H. Ju*, “The Synthesis of Structured Lipid 1, 3–Oleoyl-2-Palmitoylglycerol from Palm Oil”, J. Am. Oil. Chem. Soc., 81, 6, 525-532 (2004)
可供推廣之研發成果資料表
□ 可申請專利 □ 可技術移轉 日期 95 年 8 月 1 日
國科會補助計畫
計畫名稱：以棕櫚油為原料合成結構酯質 OPO 之研究 計畫主持人：朱義旭
計畫編號：NSC 93－2214－E－011－015；NSC 94－2214－E－011
－004 學門領域：化學工程
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中文：
（100~500 字）
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推廣及運用的價值
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