Université de Lyon, Institut des Sciences Analytiques, UMR 5280, CNRS, 5 rue de la Doua, 69100, Villeurbanne, France.
Université de Lyon, Institut des Sciences Analytiques, UMR 5280, CNRS, 5 rue de la Doua, 69100, Villeurbanne, France.
J Chromatogr A. 2021 Apr 12;1642:462001. doi: 10.1016/j.chroma.2021.462001. Epub 2021 Feb 18.
In two-dimensional liquid chromatography, the combination of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) is very attractive due to the complementarity of their separation mechanisms. On-line comprehensive HILIC x RPLC is well-known to give rise to a large retention space coverage when dealing with ionisable compounds. However, method development in on-line HILIC x RPLC is challenging due to the reversed solvent strength between both dimensions, which can greatly affect the peak shapes in the second RPLC dimension, and thus the separation quality and the method sensitivity. In the present contribution, we compared four strategies designed to avoid this problem: (1) flow splitting, which consists in reducing the injection volume in the second dimension (D), (2) on-line dilution with a make-up flow and (3) on-line dilution with Active Solvent Modulation (ASM), which both consist in reducing the solvent strength of the injected fractions, and (4) Total Breakthrough Strategy, which we recently proposed. Unlike the three preceding strategies, this latter one consists in injecting large volumes of strong solvent in D. The performance of each strategy was evaluated for sub-hour separations of a tryptic digest in on-line HILIC x RPLC. In this work, we considered the critical case for which the same column internal diameters (i.e. 2.1 mm here) are used in both dimensions. Peak capacity, peak shapes and peak intensities were considered for this evaluation. The highest peak capacity could be achieved with Total Breakthrough Strategy while the lowest one with on-line dilution using ASM. Peak intensities were usually higher with on-line dilution approaches (make-up flow and ASM). However, despite the presence of breakthrough, peak intensities were approximately 7-fold higher with Total Breakthrough Strategy than with flow splitting.
在二维液相色谱中，亲水作用液相色谱（HILIC）和反相液相色谱（RPLC）的组合非常有吸引力，因为它们的分离机制具有互补性。众所周知，在线综合 HILIC x RPLC 在处理可离子化化合物时会产生很大的保留空间覆盖度。然而，由于两个维度之间的溶剂强度相反，在线 HILIC x RPLC 的方法开发具有挑战性，这会极大地影响第二 RPLC 维度中的峰形，从而影响分离质量和方法灵敏度。在本研究中，我们比较了四种旨在避免该问题的策略：（1）分流，即将第二维（D）的进样体积减小；（2）在线稀释，包括用辅助流稀释和（3）在线稀释与主动溶剂调制（ASM），这两种方法都可降低进样部分的溶剂强度；（4）我们最近提出的全穿透策略。与前三种策略不同，这种方法是在 D 中注入大量强溶剂。我们在在线 HILIC x RPLC 中对小时内的胰蛋白酶消化物的亚分离评估了每种策略的性能。在这项工作中，我们考虑了在两个维度中使用相同柱内径（即这里的 2.1mm）的关键情况。我们对峰容量、峰形和峰强度进行了评估。全穿透策略可以获得最高的峰容量，而使用 ASM 的在线稀释则获得最低的峰容量。使用在线稀释方法（辅助流和 ASM）通常可以获得更高的峰强度。然而，尽管存在穿透现象，但与分流相比，全穿透策略的峰强度大约高 7 倍。
