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Techniques & Tools Technology, Liquid Chromatography

Through the Maze

Most developments in column and instrument technology over the last 10–15 years have been focused on achieving higher separation efficiencies in a shorter time (UHPLC), with smaller particles and higher pressures. Although further decreases in particle size and/or increases in operating pressure are theoretically feasible, the potential gain is limited. On the other hand, samples are becoming more and more complex in terms of number of components and chemical heterogeneity, particularly in metabolomics, lipidomics and the analysis of biopharmaceuticals. Often, a single method is not enough to fully separate all components in a mixture and the use of different (orthogonal) separation mechanisms is required.

The majority of future drug approvals will likely be biologics. In particular, monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) have emerged as potent and specific anti-cancer agents. High molecular weights, numerous possible conformations, post-translational modifications and micro-heterogeneity make these therapeutic proteins challenging to analyze, and a single separation mode is often not enough to characterize all relevant properties. This increasing complexity in sample composition can ultimately only be addressed by multidimensional LC coupled to MS, a powerful separation technique that combines high separation efficiency with complementary selectivity  and in addition enables coupling of non-compatible MS chromatographic methods (HIC or IEX as the first dimension) to MS instrumentation, by using reversed-phase LC in the second dimension (1).

Increasing complexity in sample composition can ultimately only be addressed by multidimensional LC coupled to MS.

Beyond the developments in column technology and instrument hardware, a prerequisite for the widespread success of complex separations schemes such as 2D-LC is the availability of software tools that support method development and data interpretation. Before these technologies can be applied in routine use, system robustness and ease of use have to reach the same level as in current 1D-UHPLC instruments.

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  1. DR Stoll et al., J Chromatogr B, 1032, 51–60 (2016).
About the Author
Monika Dittmann

Monika Dittmann is Principal Scientist R&D at Agilent Technologies, Germany.

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