Proline-coated column for the capillary electrochromatographic separation of amino acids by in-column derivatization
Proline-coated column for the capillary electrochromatographic separation of amino acids by in-column derivatization
复制标题
DOI：
10.1002/elps.200406037
复制
发表时间：
2004-10-01
期刊：
影响因子：
2.9
通讯作者：
Liu, CY
中科院分区：
文献类型：
--
作者：
Lin, CC;Liu, CY
以3-三甲氧基硅丙基氯为间隔基，制备了脯氨酸涂敷毛细管柱，用于氨基酸的柱内衍生化毛细管电色谱分离。进样9种标准品混合物，包括天冬氨酸、谷氨酸、缬氨酸、苯丙氨酸、丙氨酸、异亮氨酸、亮氨酸、酪氨酸和色氨酸。以邻苯二甲醛（OPA）、OPA/2-巯基乙醇（2-ME）和OPA/N-乙酰半胱氨酸（NAC）为衍生化试剂。其中，在硼酸盐缓冲液（pH 9.5，50 mM）中的OPA（50 mm）的性能最好。异吲哚的形成可以通过紫外检测来检测。以流体静力学模式（10 cm）进行间歇式进样，程序为R（10 s）S（10 s）R（10 s）W（10 min），R、S和W为试剂、样品和等待时间。异亚丙基丙酮、苯甲醇和丙酮与色谱柱表现出一定的相互作用。使用电流监测方法代替电渗流（EVOF）的测定。由于一些未反应的硅烷醇基团，即使在低于键合基团的p/值（6.31）的酸性条件下，氧化方向也是从阳极到阴极。考察了pH值、移动的相性质和浓度以及有机改性剂对CEC分离的影响。用脯氨酸包被的柱（75（50）cm × 75 μ m ID），在磷酸盐缓冲液（pH 4.00，100 mM）中以-15 kV的施加电压进行最佳分离。所建立的方法也进行了比较，与那些柱前衍生前的分离与脯氨酸涂层柱，以及在毛细管衍生化和分离与裸露的熔融硅胶柱。
With 3-trimethoxysilylpropyl chloride as the spacer, a proline-coated capillary column was prepared for the capillary electrochromatographic (CEC) separation of amino acids by in-column derivatization. Nine standard mixtures, including aspartic acid, glutamic acid, valine, phenylalanine, alanine, isoleucine, leucine, tyrosine, and tryptophan, were injected. o-Phthalaldehyde (OPA), OPA/2-mercaptoethanol (2-ME) and OPA/N-acetylcysteine (NAC) in borate buffer were tested as the derivatizing agent. Among them, OPA (50 mm) in borate buffer (pH 9.5, 50 mM) gave the best performance. The formation of isoindole could be detected by UV detection. The sandwich-type injection was carried out in hydrostatic mode (10 cm) with the program R(10 s)S(10 s) R(10 s)W(10 min) with R, S, and W being the reagent, sample, and waiting times. Mesityl oxide, benzyl alcohol, and acetone showed some interaction with the column. A current monitoring method was used instead of the determination of the electroosmotic flow (EOF). The direction of EOF was from anode to cathode even under acidic condition lower than the p/ value (6.31) of the bonded group due to some unreacted silanol groups. Some parameters including pH, nature, and concentration of the mobile phase and the effect of organic modifier with regard to the CEC separation were investigated. With the proline-coated column (75 (50) cm x 75 mum ID) the best separation was performed in phosphate buffer (pH 4.00, 100 mM) with an applied voltage of -15 kV. The established method was also compared with those precolumn derivatized prior to the separation with proline-coated column as well as with in-capillary derivatization and separation with a bare fused-silica column.