L-异亮氨酸键合色谱固定相的制备及其性能

利用异氰酸丙基三乙氧基硅烷与L-异亮氨酸反应合成了一种新型的硅烷偶联剂,并进一步将其与硅胶反应制得键合有L-异亮氨酸的亲水色谱固定相。通过核磁共振氢谱表明亮氨酸功能化硅烷偶联剂的成功合成、元素分析表征证明亮氨酸已成功键合到硅胶表面。将其作为亲水模式下的固定相填料填装在150mm×4.6mm不锈钢色谱柱中,以一系列经典的极性小分子作为探针,考察了这些探针分子在固定相上的色谱行为。极性化合物的保留时间随着流动相中有机溶剂含量提高而逐渐增大,表现出典型的亲水保留特征。进一步研究了流动相中乙腈含量、缓冲盐pH值及缓冲盐浓度等因素对分析物在固定相上的保留的影响。在优化了相关参数后,将固定相应用于碱性化合物、水溶性维生素以及核苷类极性物质的分离当中。在等度洗脱下,5种碱性化合物、6种水溶性维生素和8种核苷类物质分别在8、18及25min内被成功分离。分离结果表明了合成的L-异亮氨酸键合亲水色谱固定相具有较好的色谱性能,在极性化合物的分离上具有良好的应用前景。

Preparation and Chromatographic Evaluation of a L-Isoleucine-Bonded Chromatographic Stationary Phase

L-Isoleucine-bonded silica stationary phase was prepared starting from L-isoleucine and 3-isocyanatopropyltriethoxysilane, and further reaction between the resulting L-isoleucine functionalized silane coupling agents and activated silica. The result of ¹H NMR spectroscopy showed that the expected L-isoleucine functionalized silane was successfully synthesized. The successful grafting was further confirmed by elemental analysis. The synthesized stationary phase was slurry-packed into stainless steel columns(150 mm×4.6 mm). A set of typical polar compounds were employed to evaluate the chromatographic behaviors on the stationary phase in hydrophilic interaction liguid chromatography(HILIC) mode. Increased retention factors were observed for these compounds with the increase of acetonitrile(ACN) content in the mobile phase, demonstrating a typical HILIC retention characteristics. Additionally, the effect of various parameters, such as ACN content, pH values and ionic strength of the mobile phase on the retention of the tested solutes were investigated. Under optimized conditions, 5 basic compounds, 6 water-soluble vitamins and 8 nucleosides were successfully separated within 8 min, 18 min and 25 min, respectively, by isocratic elution. The resulting separation chromatogram exhibits remarkable chromatographic performance, demonstrating the excellent application potential of the L-isoleucine-bonded silica stationary phase in the separation of polar compounds.