Two-dimensional liquid chromatography (2D-LC) is becoming increasingly popular for the analysis of complex samples, which is partly due to the recent introduction of commercial 2D-LC systems. To deal with the mobile phase incompatibility between highly orthogonal retention mechanisms, such as hydrophilic interaction liquid chromatography (HILIC) and reversed-phase LC (RPLC), several strategies have been introduced over the years. One of these strategies is active solvent modulation (ASM), a valve-based approach allowing the on-line dilution of the effluent eluting from the first dimension before transfer to the second dimension. This strategy has gained a lot of attention and holds great potential, however, no clear guidelines are currently in place for its use. Therefore, this study aims to investigate how the ASM process can be optimized when using highly incompatible LC combinations, such as HILIC and RPLC, in a simplified selective comprehensive 2D-LC set-up (sHILIC x RPLC) to suggest guidelines for future users. Using a representative sample, the dilution factor (DF), the duration of the ASM phase, the filling percentage of the sample loops, and their unloading configuration are investigated and optimized. It is observed that a DF of 10 with an optimal ASM phase duration, a sample loop filling of maximum 25%, and an unloading configuration in backflush mode, result in the best peak shapes, intensities, and recoveries for early eluting compounds, while keeping the total analysis time minimal. Based on these results, some general recommendations are made that could also be applied in other 2D-LC modes, such as comprehensive 2D-LC (LC x LC), heart-cutting 2D-LC (LC–LC), and other chromatographic combinations with mobile phase incompatibility issues.

二维液相色谱 (2D-LC) 在分析复杂样品方面越来越受欢迎，部分原因是最近引入了商用 2D-LC 系统。为了处理高度正交保留机制（例如亲水相互作用液相色谱 (HILIC) 和反相液相色谱 (RPLC)）之间的流动相不相容性，多年来引入了多种策略。这些策略之一是主动溶剂调制 (ASM)，这是一种基于阀门的方法，允许在转移到第二维之前在线稀释从第一维洗脱的流出物。该策略已引起广泛关注并具有巨大潜力，但是，目前尚无明确的使用指南。所以，本研究旨在研究在简化的选择性综合 2D-LC 设置 (sHILIC x RPLC) 中使用高度不相容的 LC 组合（例如 HILIC 和 RPLC）时如何优化 ASM 过程，以为未来用户提供指南。使用代表性样品，对稀释因子 (DF)、ASM 阶段的持续时间、样品环的填充百分比及其卸载配置进行了研究和优化。据观察，DF 为 10 且具有最佳 ASM 相持续时间、最大 25% 的样品定量环填充以及反吹模式下的卸载配置可导致早期洗脱化合物的最佳峰形、强度和回收率，同时保持总分析时间最少。根据这些结果，提出了一些一般性建议，这些建议也适用于其他二维液相色谱模式，