Recent developments in two-dimensional liquid chromatography (2D-LC) are revolutionizing the field, offering simplified method development and environmentally friendly approaches. Researchers have focused on creating user-friendly strategies and tools to overcome the barriers that have hindered widespread adoption of 2D-LC. These advancements aim to enable a more systematic approach, reducing reliance on trial-and-error experimentation and user experience. Additionally, efforts are being made to minimize the environmental impact of chromatographic methods, including the reduction of solvent consumption and the use of eco-friendly alternatives. The application of these advancements spans a wide range, from resolving complex mixtures of biomolecules to analyzing polyphenolic compounds in various samples.
In his keynote lecture at HPLC 2023, Dwight R. Stoll from Gustavus Adolphus College, USA, highlighted several key developments (1). First among these is the creation of a large and freely available database of retention data that supports the development of modelling and simulation tools, enhancing method development in 2D-LC. Second, a web-based simulator for 2D-LC has been developed, providing users with a user-friendly interface to explore different method development scenarios. Finally, generic method development strategies have been formulated to simplify decision-making during the 2D-LC method development process. These tools and strategies not only expand the application space for 2D-LC but also increase the number of users engaged in method development.
Tadeusz Górecki from the University of Waterloo, Canada, focused on promoting a greener future for high-performance liquid chromatography (HPLC) (2). By implementing multidimensional separation techniques such as LC×LC, solvent consumption can be reduced and toxic solvents can be replaced with eco-friendly alternatives such as propylene carbonate. The use of narrower columns in the second dimension, combined with proper refocusing of analytes, enables significant solvent reduction and improved sensitivity. Propylene carbonate and pure ethanol were successfully employed as green organic modifiers in 2D-LC to separate pharmaceutical compounds and polyphenolic compounds in grape juices and wines, respectively.
In the realm of automated method development for LCxLC, André de Villiers from Stellenbosch University in South Africa presented a tool developed in Matlab (3). This tool optimizes LCxLC separations by simultaneously optimizing both kinetic and selectivity parameters. By predicting peak capacity and utilizing gradient tailoring, the program calculates performance for all possible combinations of experimental parameters, ultimately obtaining optimal conditions. The program's capabilities were demonstrated through the analysis of phenolic compounds in various samples, showcasing its potential to decrease method development time and improve separation performance.
These advancements bring comprehensive 2D-LC closer to LC–high-resolution mass spectrometry (HRMS) analysis, as highlighted by Andrea Gargano from the University of Amsterdam, Netherlands (4). The development of a parallel gradient method for protein digest analysis can enable highly orthogonal separations with increased peak capacity. By employing two simple linear gradients in both dimensions, column re-equilibration is eliminated, simplifying gradient programming. The parallel gradient method demonstrated superior performance compared to one-dimensional (1D) reversed-phase LC–MS, resulting in a substantial increase in the number of identified peptides in human cell culture lysate digest.
These talks all emphasize the strides that have been made in simplifying method development and supporting a greener future in 2D-LC. The advancements pave the way for expanded application possibilities and increased engagement of researchers in the development of 2D-LC methods.
(1) Stoll, D.R. Strategies and Tools to Simplify and Support Method Development in Two-Dimensional Liquid Chromatography – A Progress Report. Presented at: HPLC 2023. June 18–22, 2023. Düsseldorf, Germany. KN25.
(2) Górecki, T. Towards Greener Future for HPLC. Presented at: HPLC 2023. June 18–22, 2023. Düsseldorf, Germany. OR35.
(3) de Villiers, A. Automated Method Development Tools to Explore the Limits of Online Comprehensive 2D-LC Combining HILIC and RP-LC. Presented at: HPLC 2023. June 18–22, 2023. Düsseldorf, Germany. OR36.
(4) Gargano, A. Bringing Comprehensive 2DLC Closer to LC–HRMS: How Parallel Gradient Methods Can be Used to Analyze Complex Protein Digest. Presented at: HPLC 2023. June 18–22, 2023. Düsseldorf, Germany. OR37.