In pursuit of full usage of the two-dimensional separation space as prescribed by Giddings , the LC×LC chromatographic community is continuously scouting for new methods that yield fully orthogonal separations. With the rich and diverse LC toolkit of available retention mechanisms, chromatographers mainly focus on improving the compatibility of orthogonal – yet incompatible – separation methods. This has spurred the development of active-modulation techniques such as stationary-phase-assisted modulation  and active-solvent modulation (ASM) . Ultimately, this angle of innovation is mainly driven by the selection of stationary -and mobile phases, as well as their underlying retention mechanisms.
Rather than fine-tuning selectivity, another branch focuses on tweaking retention factors. For LC×LC separations, this has led to the introduction of shifted gradients. Here, the second-dimension gradient is altered and adapted as a function of the first-dimension gradient program . While extremely effective, the optimization of shifted-gradient assemblies introduces additional complexity to the already more-complex method development process for comprehensive 2D-LC.
The situation of LC×LC contrasts heavily with that of GC×GC. For GC×GC, orthogonal separations are extremely difficult if not impossible due to analyte volatility. As a consequence, wrap-around effects are frequently generated, yet this is rather seen as advantage than disadvantage.
Prof. Tadeusz Gorecki (University of Waterloo, Canada) thus set to investigate what would happen if the same approach would be applied in LC×LC . The project was an international collaboration with Alshymaa Aly (Minia University, Egypt, and University of Waterloo, Canada), Prof. Andre de Villiers and Magriet Muller from Stellenbosch University in South Africa, and Bob Pirok from the CAST team at the University of Amsterdam in the Netherlands.
RPLC×RPLC separations were simulated based on experimental data using the MOREPEAKS framework (formerly PIOTR ). Predicted separations using shifted gradients and parallel gradients were compared. The results suggested that parallel gradients indeed may advantageous. To verify this assessment, optimized experimental methods were executed and the resulting separations compared.
Supported by both experimental data and theoretical simulations, the authors concluded that non-orthogonal separation mechanisms could still yield good separation methods in LC×LC.
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