新型態二乙烯苯−烷基甲基丙烯酸酯共聚物做為層析靜相之研究

Investigation of the Novel Divinylbenzene−Alkyl methacrylate Copolymer as Stationary Phase in Chromatography

鄭亦傑(Yi-Jie Cheng)

指導教授 ： 黃悉雅

中原大學/理學院/化學研究所/博士(2012年)

https://doi.org/10.6840/cycu201200232

摘要

摘要
在本研究中開發了數種新穎的二乙烯苯(divinylbenzene, DVB)−烷基甲基丙烯酸酯(alkyl methacrylate, AMA)共聚物以做為毛細管電層析(CEC)及奈升級液相層析(nano-LC)之靜相。
在本研究中的第一部分，首先開發一系列聚(苯乙烯−二乙烯苯−十二烷基甲基丙烯酸酯)(poly(styrene- divinylbenzene –lauryl meth- acrylate), poly(S-DVB-LMA))整體成形管柱做為毛細管電層析之靜相用以檢測五種人工合成之抗氧化劑，並經由探討靜相合成的反應時間、反應溫度及LMA-styrene比例完成管柱合成的最佳化。並藉由SEM影像、IR光譜及表面積、孔洞大小及高分子轉換率的量測完成靜相性質的鑑定。研究結果指出，LMA-styrene比例明顯影響分析物波峰訊號的對稱性。當以LMA單體取代styrene單體時可有效改善波峰對稱性及縮短五種抗氧化劑的分離時間。本研究驗證了poly(S-DVB-LMA)整體成形管柱具有優秀的熱穩定性及管柱再現性證實其做為毛細管電層析靜相之可行性。
此外，本研究以原位聚合方式製備一系列poly(DVB-AMA)整體成形管柱，並藉由探討DVB與具有不同烷基鏈長之烷基甲基丙烯酸酯單體(如butyl-、octyl-、及lauryl-methacrylate)之靜相組成對benzophenone化合物分離之影響。當benzophenone化合物之苯環結構與poly(DVB- AMA)靜相表面所帶之長且柔軟的烷基碳鏈官能基作用時。實驗結果顯示，當利用具有較長烷基碳鏈的烷基甲基丙烯酸酯單體(如LMA)增加靜相表面疏水性時，可有效降低benzophenone化合物的波峰拖尾問題，並同時減少了靜相對分析物的滯留。而這種特殊現象可能來自於分析物與靜相間的π−π作用力。當使用具有較長烷基碳鏈的烷基甲基丙烯酸酯單體時，可能限制了芳香族化合物與靜相表面的苯環作用，進而降低了π−π作用力。對照於第一部分，本研究也證實了以具有較長烷基碳鏈官能基的烷基甲基丙烯酸酯單體在poly(DVB-AMA)管柱中，可改善分析物波峰拖尾的問題。
在第三部分中，研發出一系列的poly(DVB-AMA)整體成形管柱，結合「線上前濃縮技術−毛細管電層析串聯質譜法用」以分析九種非類固醇抗發炎藥物(NSAIDs)及九種磺胺類抗生素。結果指出，在毛細管電層析系統中結合線上前濃縮技術與階段式梯度沖提方法，並探討樣品基質與移動相的沖提強度可有效改善所有分析物的偵測靈敏度(非類固醇抗發炎藥物的偵測極限為0.01–0.19g/L，磺胺類抗生素的偵測極限為0.01–0.14 g/L)。並且發現在poly(DVB-OMA)管柱末端利用簡單的抛光程序可改善在電噴灑過程中所產生的訊號不穩定現象，使波峰面積的再現性提高，相對標準偏差(RSD)由原本的46.1–60.2%降低至8.9–16.4%。
在本研究的最後一部分，評估以poly(DVB-SMA)整體成形管柱做為層析管柱並應用於奈升級液相層析系統(結合UV與MS偵測器)中之可行性，針對結構更為單純的六種多環芳香族化合物與蛋白質水解胜肽進行測試。在多環芳香族化合物的部分可證明在LC系統下以烷基甲基丙烯酸酯單體取代苯環結構的含量，仍然確實可有效降低π−π作用力改善分析物的層析行為(波峰拖尾現象)。而van Deemter曲線也證明了poly(DVB-SMA)整體成形管柱在高線性流速下仍可維持良好的管柱效能。將poly(DVB-SMA)管柱應用nano-LC−MS/MS對蛋白質水解胜肽進行分析，其波峰容量達424且成功的由牛血清蛋白水解胜肽中鑑定出46的胜肽其胜肽序列覆蓋率為73%。

關鍵字

整體成形管柱 ； 奈升級液相層析 ； 毛細管電層析

並列摘要

Abstract
Several novel divinylbenzene(DVB)-alkyl methacrylate (AMA) copolymers were developed as stationary phases in capillary electrochromatography(CEC) and nano-liquid chromatography (nano-LC).
In the first part of this study, several poly(S-DVB-LMA) monolithic columns were developed as CEC stationary phases for the analysis of five synthetic antioxidants. Polymerization procedures were optimized by vaying the reaction time, reaction temperature, and LMA-styrene ratio. These monoliths were characterized by examining their SEM images, IR spectra, surface area, pore size, conversion yield, and thermal decomposition temperature. Results showed that the LMA-styrene ratio had the most significant influence on analyte peak symmetry. This also showed that the use of LMA monomer, instead styrene monomer, can improve the peak symmetry and shorten the separation time of the five synthetic antioxidants. This study demonstrates the potentiality of poly(S-DVB-LMA) monoliths as stationary phases, because of high thermal stability and good column reproducibility.
In addition, a series of poly(DVB-AMA) monolithic columns, which were prepared by in situ polymerization of DVB and various AMA(butyl-, octyl, and lauryl-methacrylate, in same amount), were developed as CEC separation columns for benzophenone compounds. The benzene moieties in benzophenone compounds interact with the long and flexible alkyl group on the surface of the stationary phase. Results indicated that with an increase in the molecular length of the AMA (e.g. LMA), the polymeric monolith with higher hydrophobicity, can effectively reduce the peak tailing problem of benzophenones, however, a weaker retention was observed. This unusual phenomenon was likely due to the π−π interaction between the aromatic compound and the stationary phase. Using longer alkyl methacrylates as reaction monomer limited the retention of aromatic compounds on the stationary phase surface, thus the π−π interaction between them was possibly reduced. In comparison to the first part, this study demonstrated that the peak tailing problem could also be improved by the inclusion of long alkyl methacrylates to poly(DVB-AMA) columns.
In the third part, a series of poly(DVB-AMA) monolithic columns were developed as separation columns of nine common non-steroidal antiinflammatory drugs (NSAIDs) and nine common sulfonamide antibiotics for on-line concentration capillary electrochromatography coupled to mass spectrometry (MS). Results indicated than an on-line concentration step of step-gradient elution combined to this CEC system, and optimizing the difference in eluent strength between the sample matrix and mobile phase, improved the detection sensitivity of all NSAIDs and sulfonamide antibiotics (0.01–0.19g/L for NSAIDs, 0.01–0.14 g/L for sulfonamide antibiotics). Moreover, a simple polishing was done to reduce the roughness of the column end which resulted to a great improvement in the signal stability. The relative standard deviations (RSDs) of the peak areas for the unpolished and polished ends of the poly(DVB-OMA) columns (n = 5) were in the range of 46.1–60.2% and 8.9–16.4%, respectively.
For the final part of this study, the potential of poly(DVB-SMA) monoliths as separation columns for the six polycyclic aromatic compounds and peptide digests in nano-LC coupled with UV or MS was evaluated. For the six polycyclic aromatic compounds, this study proved once again that the peak tailing problem can be reduced by decreasing the benzene moieties on the stationary phase (even in an LC system where EOF is absent). The van Deemter curve indicates that the poly(DVB-SMA) column had high efficiency at high linear flow rate. This monolith, with a peak capacity of 424, successfully identified the tryptic digested BSA peptides using nano-LC−MS/MS with a 70-cm long column. In total, 46 peptides were identified with sequence coverage of 73%.

並列關鍵字

capillary electrochromatography ； nano-LC ； monolithic column

參考文獻

A. Maruška, C. Ericson, A. Vegvari, S. Hjerten, (Normal-phase) capillary chromatography using acrylic polymer-based continuous beds, J. Chromatogr. A 837(1999) 25.

A. Premstaller, H. Oberacher, W. Walcher, A.M. Timperio, L. Zolla, J.P. Chervet, N. Cavusoglu, A. van Dorsselaer, C.G. Huber, High-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic capillary columns for proteomic studies, Anal. Chem. 73(2001) 2390.

A.A.A.M. van de Goor, B.J. Wanders, F.M. Everaerts, Modified methods for off- and on-line determination of electroosmosis in capillary electrophoretic separations, J. Chromatogr. 470(1989) 95.

A.H. Que, A. Palm, A.G. Baker, M.V. Novotny, Steroid profiles determined by capillary electrochromatography, laser-induced fluorescence detection and electrospray-mass spectrometry, J. Chromatogr. A 887(2000) 379.

A.H. Que, A. Palm, A.G. Baker, M.V. Novotny, Steroid profiles determined by capillary electrochromatography, laser-induced fluorescence detection and electrospray-mass spectrometry, J. Chromatogr. A. 887(2000) 379.