探討高分子在毛細管電泳層析之應用

本研究以高分子聚異丙基丙烯醯胺 (poly(N-isopropylacrylamide)，PNIPAAm) 結合微胞電動層析法進行中性固醇物質的分離。聚異丙基丙烯醯胺具有極性(側鏈上之醯胺基)與非極性(主結構碳鏈)之特性，控制溫度變化可因聚合物結構變化而改變整體聚合物之極性。此極性改變之特性已應用為分離機制。但溫度改變可能會因種種因素，如溫度分佈不均，而影響分離效果。因此，本研究在不改變溫度下(室溫)，使用帶負電(COO-)之PNIPAAm為偽靜相(pseudostationary phase，PSP)，配合使用毛細管電動層析法(capillary electrokinetic chromatography, CEC)，進行中性固醇類分離。利用此聚合物之極性與非極性結構，與中性物質之極性官能基與非極性之主體結構相互作用，進而達到分離。實驗中所使用之中性固醇類標準品為testosterone、dexamethasone、hydrocortisone、hydrocortisone 21-acetate、prednisolone以及hydrocortison衍生物cortisone、corticosterone、6α-methyprednisolone。此外、此系統亦成功應用於尿液樣品之分析。影響此系統之參數，如緩衝溶液濃度 (離子強度)、pH值、聚合物使用濃度、聚合物分子量、聚合物電性影響、微胞濃度、線上預濃縮，在本研究中皆有討論。此方法的優點為使用簡便、減少有機溶劑的用量、降低修飾劑的選擇，及提供綠色化學的實驗基礎。其最佳分離條件：聚合物濃度為0.6 % (w/v)，分子量為1957 g/mol，BGE濃度為20 mM (檸檬酸鹽緩衝液，pH 5.0)，樣品基質為20% MeOH (40 mM醋酸緩衝液，pH 5.0)以及10 mM SDS (20 mM 檸檬酸緩衝液，pH 5.0)。另外，本研究也同時發展異丙基丙烯醯胺與甲基丙烯酸 (methacrylic acid，MAA) 、丙烯酸 (acrylic acid，AA) 或異丙基丁烯醯胺酸 (N-isopropyl-maleamic acid，NIPMMA) 以自由基聚合法合成共聚物 (copolymer)進行毛細管電泳層析 (CEC) 分離中性固醇物質。

關鍵字

異丙基丙烯醯胺；微胞電動層析；偽靜相；高鹽堆積

並列摘要

In the present study, we used a negatively (-COO-) charged polymer, Poly(N-isopropylacrylamide) [PNIPAAm], as pseudo-stationary phase (PSP) to assist a modified micellar electrokinetic chromatography for the separation of corticosteroids, which are essential steroid hormones for several physiological activities. The used thermo-responsive polymer, PNIPAAm, is previously used as separation matrix due to its hydrophobic property at elevated temperatures through a conformational change that exposes its hydrophobic backbone. This polymer contains hydrophilic amide group at its side chain, which could likely form hydrogen bonds with corticosteroids to facilitate their separation. We assess the feasibility of this hypothesis through performing the assay at ambient temperature (bellowing its lower critical solution temperature) with negatively charged PNIPAAm. Corticosteroids such as cortisone, hydrocortisone, corticosterone, and 6?-methylprednisolone, carried by SDS micelles, were partitionated between PSP and micelles to achieve an efficient separation. Improved resolution was obtained by high-salt stacking. Additionally, effect of polymer end group charges was investigated. Positively charged semitelechelic polymer resulted in poor resolution probably due to their interaction with capillary inner wall. The negatively charged polymers migrated against the reduced EOF to maintain the polymer remain in capillary that ensures the separation. Moreover, elevated temperature causing reduced resolution could partly explain the hydrogen bond effect. Several factors including polymer molecular weight, polymer concentration, and SDS micelle concentration were also discussed. The performance of the separations conducted in aqueous phase requires least amount of organic solvents. The optimal separation parameters were at a polymer concentration of 0.6% (w/v) with a molecular weight of 1957 g/mol, outlet BGE concentration of 20 mM (citrate buffer, pH 5.0), sample plug (acetate buffer 40 mM, pH 5.0), and 10 mM SDS (in inlet citrate buffer of 20 mM, pH 5.0). This newly developed system, without capillary modification, presents an efficient and user-friendly operation means for corticosteroids determination. Moreover, co-polymers composed of PNIPAAm and other polymers such as methacrylic (MAA), acrylic acid (AA), and N-isopropyl-maleamic acid (NIPMMA), were used for steroids separation by the developed system.