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  • 學位論文

分子拓印材料的製備及探討於毛細管電層析分離神經傳導物質和其結構相似化合物之可行性

Development of imprinted materials for the CEC separation of neurotransmitters and their analogues

指導教授 : 劉春櫻

摘要


本研究以左旋型式的去甲腎上腺素為模板分子經由與單體甲基丙烯酸(MAA)或衣康酸(IA)、交聯劑乙二醇二甲基丙烯酸脂(EDMA)或二乙烯基苯(DVB)聚合成具有特殊立體結構的聚合物。合成步驟為去甲腎上腺素((-)-NE, 4.03×10-5 mol), MAA或IA (4.61×10-4 mol) 溶於1.4毫升二甲基甲醯胺(DMF)中,在室溫下加入EDMA或DVB 9.54×10-4 mol和2,2’-重氮-雙-異丁腈(AIBN, 1.00×10-5 mmol),震盪使其完全溶解後,利用高壓氮氣導入毛細管內進行熱聚合(65℃, 17 min),再將所製備之整體式拓印分子聚合物管柱應用於毛細管電層析。由於IA相較於MAA,在相同比例下具有兩倍的羧酸可以形成氫鍵,形成的聚合物的分離效果優於MAA;而交聯劑DVB則因為苯環結構可以提供較大的疏水性作用力,且和分析物的苯環可以產生π-π電子吸引力而增加作用力,另由介面活性劑、環糊精、冠狀醚和具有光學活性的酒石酸酯類的添加物發現以帶負電的介面活性劑的分離效果最好,而中性物質則因為不帶電荷分離效果較差,而含有硫酸鹽取代基的環糊精則可以在負端注入的模式下,成功分離模版分子的鏡像異構物,但負端注入所得到的分析物流析順序則與正端注入相反。由於模板分子具有旋光性的立體結構,以毛細管長75 μm × 75(50) cm,高度差注入法(10 cm × 10 sec),以體積比2/2/1的磷酸鹽緩衝液(pH 4, 10 mM)/ SDS (40 mM)/ 乙腈 (ACN),分離電壓 +10kV,偵測波長210 nm下可將NE的光學異構物分離,而相同體積比的醋酸緩衝液(pH 4, 10 mM) /SDS (20 mM)/ ACN 混合物,可將EP的光學混合物分離,雖然兩者無法同時在同一系統下達到完全分離,若改以檸檬酸鹽緩衝液(pH 3, 10 mM)/SDS (40 mM)/ ACN,發現在偵測波長200 nm下可同時分離六個與模板分子相似結構的化合物,包括多巴胺(DA)、章魚胺 (OCT)、辛弗林素(SYN)和異丙腎上腺素(-)-ISO。本研究也發現高溫(30℃)可以增強分離效率,但EP和OCT例外的在低溫(10℃)時可以得到比較好的分離效果,溫度效應則是與分析物本身特性有關。此合成管柱的分離機制主要有三個,除了拓印聚合物本身的形狀選擇之外,還包含氫鍵作用力和疏水性作用力,又以疏水性作用力為主要,所以流析順序為DA < OCT < NE < SYN < EP < (-)-ISO。由於DA不含光學活性中心,故結構上和合成孔洞較不能配合而滯留時間最短,NE因為多了一個甲基提供疏水性作用力而比OCT滯留時間長,SYN少了一個氫鍵作用力但甲基的疏水性使其時間更長,由此可以證明此系統的疏水性作用力大於氫鍵,故EP的甲基和(-)-ISO的異丙基使得其流析順序在最後。另外以蛋白質分離來探討此管柱的廣用性,利用前述實驗結論的疏水性特性,改變緩衝溶液的濃度、 種類、有機修飾劑等變因,最後在磷酸緩衝液(pH 7, 10 mM)/甲醇(5%, v/v),施以電壓15kV時可以分離血紅蛋白蛋肌紅蛋白分子。而血紅蛋白的結構異構物則在有機修飾劑乙腈存在時,可以有較好解析度。肌紅白異構物則在磷酸緩衝液濃度提高到25 mM時,可以分離出七個,。硼酸緩衝液(pH 9, 10 mM)/甲醇(4/1, v/v),施以電壓15kV時可以成功分離血紅蛋白和肌紅蛋白分子,且其異構物也可以在此條件下被分離。

並列摘要


A novel molecularly imprinted polymer (MIP) monolithic column was prepared as the stationary phase for capillary electrochromatographic enantioseparation. The in situ polymerization was using (-)-norepinephrine as template and carried out at 65℃ for 17 min. The molar ratio of all the compositions was investigated to achieve the optimum condition. Two types of functional monomers, methacrylic acid (MAA) and itaconic acid (IA) were studied by separation of neurotransmitters with the addition of modifiers in the mobile phase. These additives were surfactants, cyclodextrins, crown ether, tartrate esters etc. The running buffer consisted of citrate buffer (10 mM, pH 3)/ additive/ acetonitrile in a volume ratio of 2:2:1. It was found that anionic surfactants provided better separation efficiency than the others. Moreover, cyclodextrin derivative (S-β-CD) could separate template racemic mixture successfully with negative injection mode, and the retention order was reverse by comparison with the normal mode. Employed IA instead of MAA could improve the recognition ability of the synthetic stationary phase due to more carboxylate functional groups. The retention behavior of MIP prepared from either EDMA or DVB was also investigated in detail. The resultant product from DVB demonstrated longer retention time than that from EDMA due to the additional π-π interaction force between stationary phase and analytes. Unfortunately, the results were not good. Eventually the condition of (-)-NE (4.03×10-5 mol), IA (4.61×10-4 mol), EDMA (9.54×10-4 mol) and 2,2’-azobisisobutylnitrile (AIBN, 2 mg) was selected as the optimum polymerization condition. The chiral separation for NE was achieved with phosphate buffer (10 mM, pH 4)/ SDS (40 mM)/ ACN (2/2/1, v/v/v) at 210 nm and applied voltage of +10 kV. While that for the separation for epinephrine (EP) was using acetate buffer (10 mM, pH 4). Interestingly, the enantioseparation of six structure related compounds simultaneously was achieved with citrate buffer (10 mM, pH 3)/ 40 mM SDS/ ACN (2/2/1) at 200 nm. The separation was mainly originated from the hydrophobic interaction and hydrogen bonding. The elution order was DA < OCT < NE < SYN < EP < (-)-ISO. We also found that the enantioseparation efficiency was affected significantly by elevating the temperature, except SYN and EP. The application of protein separation with the synthetic MIP column was tested. The challenge was similar hydrophobic index and pI values of hemoglobin (Hb) and myoglobin (Mb). The pH, concentration types and organic modifier of the mobile phase were studied. The optimum condition was a mobile phase of phosphate buffer (10 mM, pH 7)/ MeOH (5%, v/v), an applied voltage of +15 kV and detection at 210 nm. The isomers of myoglobin were separated at higher phosphate concentration of 25 mM without addition of organic modifier. Borate buffer (10 mM, pH 9)/ MeOH (4/1, v/v) with applied voltage of +15 kV, the separation of Hb and Mb was also achieved.

參考文獻


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