Parallel gradients as alternative to shifted gradients in 2D-LC

A joint study by the University of Waterloo (Prof. Tadeusz Górecki), the Stellenbosch University (Prof. Andre de Villiers) and the University of Amsterdam (Dr. Bob Pirok) was just published in Journal of Chromatography A. In the study, parallel gradients are evaluated as alternative for shifted gradients in comprehensive 2D-LC.
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In pursuit of full usage of the two-dimensional separation space as prescribed by Giddings [1], 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 [2] and active-solvent modulation (ASM) [3]. 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 [4]. 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 [5]. 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 [6]). 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.


[1] Two-dimensional separations: concept and promise. J.C. Giddings, Anal. Chem. 1984, 56(12), 1258A–1270A, DOI: 10.1021/ac00276a003

[2] Recent Developments in Two-Dimensional Liquid Chromatography: Fundamental Improvements for Practical Applications. B.W.J. Pirok, D.R. Stoll and P.J. Schoenmakers, Anal. Chem., 2019, 91(1), 240-263, DOI: 10.1021/acs.analchem.8b04841

[3] Active Solvent Modulation: A Valve-Based Approach To Improve Separation Compatibility in Two-Dimensional Liquid Chromatography. D.R. Stoll, K. Shoykhet, P. Petersson, and S. Buckenmaier, Anal. Chem. 2017, 89(17), 9260–9267, DOI: 10.1021/acs.analchem.7b02046

[4] Optimizing separations in online comprehensive two-dimensional liquid chromatography
B.W.J. Pirok, A.F.G. Gargano and P.J. Schoenmakers, J. Sep. Sci., 2018, 41(1), 68–98, DOI: 10.1002/jssc.201700863

[5] Parallel gradients in comprehensive multidimensional liquid chromatography enhance utilization of the separation space and the degree of orthogonality when the separation mechanisms are correlated. A.A. Aly, M. Muller, A. de Villiers, B.W.J. Pirok, T. Górecki, J. Chromatogr. A, 1628, 2020, 461452, DOI: 10.1016/j.chroma.2020.461452

[6] Program for the interpretive optimization of two-dimensional resolution. B.W.J. Pirok, S. Pous-Torres, C. Ortiz-Bolsico, G. Vivó-Truyols and P.J. Schoenmakers, J. Chromatogr. A, 2016, 1450, 29–37, DOI: 10.1016/j.chroma.2016.04.061


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