A Protocol for Characterizing Comprehensive Two‐Dimensional Liquid Chromatography Systems

Comprehensive two-dimensional liquid chromatography (LC × LC) is an effective strategy to increase peak capacity and improve separation performance for complex samples, offering a valuable alternative to conventional one-dimensional liquid chromatography (1D-LC). However, LC × LC method development and optimization remain challenging due to the interconnection of numerous parameters within and between dimensions. Both thermodynamic factors (e.g., stationary phase, mobile phase, temperature) and kinetic factors (e.g., gradient profile, sampling rate, column geometry, flow rates) strongly influence the analytical performance. To achieve effective method optimization, two-dimensional liquid chromatography (2D-LC) instrumentation-related parameters must first be characterized, as they directly influence both method development and overall performance. In this work, we propose a practical guide (protocol) for comprehensive 2D-LC system characterization. For clarity and ease of implementation, all calculations associated with this protocol can be performed using the Characterization module of the 2D-LC Smart Calculator software, freely accessible online. The protocol usability was validated through inter-laboratory testing. The impact of instrumental features on LC × LC performance was then assessed by in silico evaluation of the theoretical LC × LC performance of the characterized systems. The results clearly demonstrate the benefits of the proposed protocol, emphasizing the importance of system-specific characterization for reliable LC × LC implementation. In addition, practical recommendations are provided to help users optimize their instruments when certain parameters are not adapted.