毛細管電層析新型整體式管柱之開發及微胞電動層析線上濃縮大麥中酚酸化合物之測定

The development of a novel monolithic column in capillary electrochromatography and on-line pre-concentration for the determination of phenolic acids in barley by micellar electrokinetic chromatography

王冠仁(Guan-Ren Wang)

指導教授 ： 劉春櫻

國立臺灣大學/理學院/化學研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.02313

摘要

本研究以鄰苯二甲醛與L-苯丙胺酸錯合物為反應中心，製備一以聚合物為基底的新型毛細管電層析整體式管柱。嘗試將其利用於金屬離子混合物的分離，以磷酸緩衝溶液(20 mM, pH 3.0)，溴化四丁氨(10 mM)及甲醇(15%, v/v)混合溶液為移動相，施加電壓-15 kV，在波長220 nm偵測下可以將七種重金屬離子完全分離，所得的理論板數為10,500-30,800 plates/m。本研究亦嘗試在不添加溴化四丁氨的條件下進行分離，發現分析物流析順序不同且滯留時間較短，此整體式管柱的分離行為亦與空白聚合物管柱作比較，驗證了此整體式管柱的分離性質包含了電泳行為與層析的滯留，其中層析的滯留是包含了疏水及親水作用力與配位基交換。本研究也經由移動相的選擇以探討其層析行為的改變。發現乙二胺四乙酸比磷酸緩衝溶液具有更佳的分離效率，在乙二胺四乙酸緩衝溶液(20 mM, pH 3.5)，溴化四丁氨(10 mM)的條件下，所得到的理論板數為20,300-64,100 plates/m，這說明了以較弱配位基為移動相需要低導電度溶劑來幫助分離，為探討上述系統於實際樣品利用的可行性，以台大醉月湖的湖水為基質，將重金屬離子添加於湖水中，只經由簡單過濾後，此系統即可將重金屬離子完全分離，這證明了此新型整理式管柱的確可以用於複雜基質中重金屬離子的分析。
除無機分析外，本研究也藉由簡單調控功能性聚合物單體的成分，加入3-磺酸丙基甲基丙烯酸鉀鹽作為第二個單體以避免蛋白質吸附在靜相上，所合成管柱嘗試利用於分離血色素(Hb)與肌紅蛋白(Mb)，發現以硼酸緩衝溶液(20 mM, pH 9.0)，三羥甲基氨基甲烷緩衝溶液(40 mM, pH 9.0)或者是乙二胺四乙酸緩衝溶液(10 mM, pH 9.0)作為移動相均可使血色素與肌紅蛋白達到基線分離。此分離機制可能是由L-苯丙胺酸與兩個單體所提供的π-π作用力與疏水性作用力所導致。
近年來，人們對於酚酸類化合物具有抗氧化的性質及避免相關疾病充滿了興趣。本研究利用微胞電動毛細管層析法在酸性條件下分離植物酚酸。使用毛細管管柱為70公分(有效長度為40公分)，十二烷基硫酸鈉/磷酸緩衝溶液(100/50 mM, pH 3.0)，施加電壓-15 kV與在波長214 nm偵測下可以將十種常分佈於植物中的酚酸在25分鐘內有效地分離，而所有分析物的滯留時間的相對標準偏差都在1.94%以下，所得到的理論板數為10,500-11,900 plates/m。將此建立的系統應用於大麥萃取液中所含植物酚酸的分析，吸收峰由於樣品基質的影響，改用波長280 nm的條件，即可辨識大麥中所含的七種植物酚酸，全部植物酚酸的流析順序為ferulic acid， coumaric acid， chlorogenic acid， caffeic acid， vanillic acid， p-hydroxybenzoic acid與protocatechuic acid。本研究進而以線上濃縮的技術，使用pH-junction方法，首先於毛細管柱中填滿磷酸緩衝溶液(100 mM, pH 2.5)，再以十二烷基硫酸鈉/磷酸緩衝溶液(60/50 mM, pH 2.5)為移動相，將分析物配製於硼酸緩衝溶液中(50 mM, pH 9.0)，以-15 kV進樣二十分鐘，施加電壓-15 kV與在波長280 nm偵測下，可有效完成線上濃縮的效果，所得的放大倍率在300-760倍之間，且其回歸係數(r2)均在0.997以上，所得到的理論板數為24,900-35,800 plates/m。

關鍵字

毛細管電層析 ； 整體式管柱 ； 微胞電動層析 ； 線上濃縮

並列摘要

A novel polymer-based capillary electrochromatography (CEC) monolithic column was prepared using an o-phthalaldehyde-L-phenylalanine Schiff base complex as the reactive center and a mixture of methanol and n-propanol as the porogen. This monolithic column was employed for the separation of a metal ion mixture. Tetrabutylammonium bromide (TBAB) was used as a mobile phase additive to enhance the separation efficiency of metal ions by EDTA precomplexation. Using a phosphate buffer (20 mM, pH 3.0), TBAB (10 mM), MeOH (15%, v/v) an applied voltage of -15 kV, and detection at 220 nm, the metal ion mixture was satisfactorily resolved. The theoretical plate number was between 10,500-30,800 plates/m. The separation was also carried out in the absence of TBAB, leading to a dissimilar elution order and shorter retention times. The separation behavior of the monolithic column was also compared with that of the blank polymer. The unique properties of the monolithic column might be mediated by a combination of electrophoretic behavior and chromatographic retention, involving hydrophobic and hydrophilic interactions, as well as ligand exchange. Different mobile phases have also been investigated to study their effects on retention behavior. Using EDTA was found to be superior to phosphate buffer. Under the condition of EDTA (20 mM, pH 3.5) and TBAB (10 mM), the theoretical plate number was between 20,300-64,100 plates/m. The results imply that the separation needs a low dielectric constant solvent when a weak ligand is used as the mobile phase. The established method was applied to determine the metal ions in lake water sampled from the NTU campus. The sample was analyzed directly and without any preliminary treatment; baseline separation indicated that the monolithic column was highly promising for the separation of metal ions in this complex matrix sample.
Another application of the novel column was to the separation of hemoglobin (Hb) and myoglobin (Mb). Here, note that 3-sulfopropyl methacrylate potassium salt was added as a second functional monomer for the in situ polymerization to help prevent irreversible adsorption of proteins on the stationary phase. Several kinds of buffer have been tested as the mobile phase. Baseline separation of Hb and Mb could be achieved with borate buffer (20 mM, pH 9.0), Tris buffer (40 mM, pH 9.0) or EDTA buffer (10 mM, pH 9.0) at 214 nm and 15 kV. The separation mechanism might be attributable to π-π interactions and hydrophobic forces between the proteins and the L-phenylalanine and the functional monomers.
Phenolic acids are plant metabolites that are wide spread throughout the plant kingdom. Recent interest in phenolic acids stems from their potential protective role against oxidative damage and related diseases. Micellar electrokinetic chromatography (MEKC), seldom operated at acidic conditions, was adopted for the separation of phenolic acids. Using an SDS (100 mM)/phosphate buffer (50 mM, pH 3.0), an applied voltage of -15 kV and detection at 214 nm, the baseline separation of a mixture of ten phenolic acids was satisfactorily achieved within 25 min. The relative standard deviations (RSD) of the retention times were less than 1.94%. The number of theoretical plates was 10,500-11,900 plates/m. The established method was applied for the determination of phenolic acids in barley extract. With the addition of methanol (20%, v/v), only 4-hydroxybenzoic acid was found. Many sample peaks were concealed by the large sample matrix effect. By using 280 nm as the analytical wavelength, the matrix effect can be overcome; seven peaks could then be detected. The elution was in the following order: ferulic acid, coumaric acid, chlorogenic acid, caffeic acid, vanillic acid, p-hydroxybenzoic acid and protocatechuic acid. For analysis of phenolic acids in barley, a pH-junction for on-line pre-concentration was explored. The capillary was filled with phosphate buffer (100 mM, pH 2.5). The analytes, in borate buffer (50 mM, pH 9.0), were then injected in electrokinetic mode (-15 kV, 20 min). With SDS/phosphate buffer (60/50 mM, pH 2.5) as the mobile phase, phenolic acids in barley extract could be detected at an applied voltage of -15 kV, and detection at 280 nm. A sensitivity enhancement of ~300-760-fold could be achieved and regression coefficients (r2) are above 0.997.The theoretical plate number was 24,900-35,800 plates /m.

並列關鍵字

CEC ； monolithic column ； MEKC ； on-line pre-concentration

參考文獻

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