Simultaneous Separation of Water- and Fat-Soluble Vitamins by Selective Comprehensive HILIC × RPLC (High-Resolution Sampling) and Active Solvent Modulation
Simultaneous Separation of Water- and Fat-Soluble Vitamins by Selective Comprehensive HILIC × RPLC (High-Resolution Sampling) and Active Solvent Modulation
复制标题
通过选择性综合 HILIC × RPLC（高分辨率采样）和活性溶剂调制同时分离水溶性和脂溶性维生素
作者：
Stefanie Bäurer;Wenkai Guo;Stefan Polnick;M. Lämmerhofer
水溶和脂溶维生素的同时液相色谱分析具有挑战性，因为它们的极性范围很广。通常，水溶性维生素通过亲水作用层析(HILIC)分离和分析，而脂溶维生素通过反相液相色谱(RPLC)分析。将这两种保留原理结合在柱耦合或多维液相色谱方法中似乎是解决问题的合乎逻辑的结果。本文研究了一种选择性的综合HILIC×RPLC 2D-LC方法。在这种方法中，极性的水溶性维生素在2-吡啶脲混合模式相上通过HILIC梯度操作在一维(1D)中分离，而在色谱图早期部分的脂溶维生素在十个40-µL组分中被综合转移到C8核壳柱上的第二维(2D)分离。这种分离模式也称为高分辨率采样。系统地优化了一维和二维的分离条件，并用这些数据解释了混合模式柱的保留机理。一维HILIC和二维RPLC条件的溶剂不相容是由于从一维富含乙腈的馏分采样到二维RPLC，导致在进行直接馏分转移时，峰严重展宽。等度重聚焦步骤可以在一定程度上改善保留较强脂溶维生素的情况。带有专门设计的阀的活性溶剂调制，允许从2D泵到柱头的弱洗脱液旁路，并从采样回路稀释分馏样品，完全解决了问题，并提供了完美的峰形和色层效率。
The simultaneous liquid chromatographic analysis of water- and fat-soluble vitamins is challenging because of their wide polarity range. Typically, water-soluble vitamins are separated and analyzed by hydrophilic interaction chromatography (HILIC) while fat-soluble vitamins are analyzed by reversed-phase liquid chromatography (RPLC). The combination of these two retention principles in a column coupling or multidimensional liquid chromatography approach seems to be a logical consequence to solve the problem. In this work, a selective comprehensive HILIC × RPLC 2D-LC approach is investigated. In this method, the polar water-soluble vitamins are resolved in the first dimension (1D) on a 2-pyridylurea mixed-mode phase operated by a HILIC gradient and the coeluted fat-soluble vitamins in the early part of the chromatogram are comprehensively transferred in ten 40-µL fractions into a second dimension (2D) separation by RPLC on a C8 core–shell column. This mode of separation is also known as high-resolution sampling. The separations in 1D and 2D were optimized systematically and the retention mechanism on the mixed-mode column interpreted by support of these chromatographic data. The solvent incompatibility of 1D HILIC and 2D RPLC conditions due to sampling of acetonitrile-rich fractions from 1D into 2D RPLC led to severe peak broadening when a direct fraction transfer was carried out. An isocratic refocusing step could partly improve the situation for the stronger retained fat-soluble vitamins. Active solvent modulation with a specifically designed valve which allows a bypass of the weak eluent from the 2D pump to the column head and dilution of the fractionated sample from the sampling loop completely solved the problem and provided perfect peak shapes and chromatographic efficiencies.
影响因子：
4.1
作者：
Baeurer, Stefanie;Polnick, Stefan;Laemmerhofer, Michael
通讯作者：
Laemmerhofer, Michael