Selecting Optimal Two-Dimensional Liquid Chromatography Columns

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Scientists from University College London in London, UK, created a new technique for selecting which columns to use for two-dimensional liquid chromatography (2D-LC). Their findings were published in the Journal of Chromatography A (1).

High performance liquid chromatography (HPLC) is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture | Image Credit: © Love Employee - stock.adobe.com

High performance liquid chromatography (HPLC) is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture | Image Credit: © Love Employee - stock.adobe.com

2D-LC is a process that uses two chromatographic columns with different stationary phases to diversify solute interactions with a resin; this allows for a second “dimension” to enable solute-specific separation. The sample is transferred from the first to the second-dimension column either off-line or on-line. In the off-line set-up, the fractions from the first column are isolated, preconcentrated, and injected onto the second column (2).

First introduced in 1944, when amino acid separation was performed using a thin cellulose sheet and applying two different solvents, 2D-LC can resolve heterogeneity of a sample using two different LC columns, each corresponding to a different separation mechanism (3). Developing 2D-LC methods begins with preliminary column selection, and while selecting columns that metaphorically yield orthogonal dimensions is very important, the process remains challenging.

There are various metrics for quantifying column orthogonality, but there is currently no established methodology, with no existing methods accounting for the non-homogeneity of peak band broadening across each separation dimension.

In this study, the scientists proposed a new resolution approach for preliminary column selection. This approach required the knowledge of retention behavior (such as retention factor or retention time) of components under analysis in a set of chromatographic systems. This can be gained from experimental campaigns or predicted with appropriate retention models, such as linear solvation energy relationships (LSER) or quantitative structure retention relationships (QSRR). The approach used in this study accounts for short-length scale effects and peak band broadening and can be tailored for heart-cutting and comprehensive 2D-LC modes. For this study, their own approach was compared with eight different metrics of orthogonality, including Pearson, Spearman and Kendall’s correlation coefficient, geometric surface coverage, and minimum convex hull, which are well-established in literature; further, they assessed the impact of preliminary column selection on the optimization results, when both method development and optimization of comprehensive 2D-LC are considered.

Two main outcomes were found in this experiment. First, preliminary column selection is a key step of in silico method development of comprehensive 2D-LC, where using an appropriate approach is essential. Second, the scientists’ proposed approach outperformed traditional methods for evaluating column orthogonality. With that in mind, the scientists pointed out their method’s applicability is not limited to predicting the best combinations of chromatographic columns for 2D-LC system optimization. For example, it can help evaluate the separation performance of a multidimensional chromatographic system or help select a second independent column (for an initial one-dimensional [1D] separation) to increase peak purity to meet regulatory requirements. These extensions are not considered in this work, though they could be potential research points in the future.

References

(1) Tirapelle, M.; Duanmu, F.; Chia, D. N.; et al. Method Development of Comprehensive Two-Dimensional Liquid Chromatography: A New Metric for Preliminary Column Selection. J. Chromatogr. A 2025, 1741, 465593. DOI: 10.1016/j.chroma.2024.465593

(2) Jandera, P. Column Selection for Two-Dimensional LCxLC. LCGC Europe 2007, 20 (10), 510–525.

(3) Rathore, A. S.; Auclair, J.; Bhattacharya, S.; Sarin, D. Two-Dimensional Liquid Chromatography (2D-LC): Analysis of Size-Based Heterogeneities in Monoclonal Antibody–Based Biotherapeutic Products. LCGC N. Am. 2022, 40 (1), 27–31. DOI: 10.56530/lcgc.na.cz9881a2

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