Two-dimensional liquid chromatography (2D-LC) is increasingly being used in a variety of fields due to its versatility and advantages it can provide over conventional one-dimensional separation, including improved selectivity, throughput, and resolving power. In spite of the power of these 2D-LC separations, major gaps still exist in our understanding of some fundamental aspects of the technique, which in turn limits the effectiveness of these methods, and impedes method development. In this presentation we will discuss one of these gaps – understanding dispersion of analytes in the open tubular loops that are most commonly used to collect fractions of peaks eluting from the first dimension to transfer them to the second dimension for further separation. In previous work we developed quantitative models that predict breakthrough of an analyte zone from the outlet of a sampling loop, and dispersion of the analyte zone inside the loop when it is operated in the “First-In-Last-Out” mode. These models – based on fluid dynamics simulations – were verified using experimental data collected using the small molecule uracil. In the present work we extend this work by investigating the breakthrough and dispersion behavior using much larger biomolecules, with some exceeding 1 MDa molecular weight. We will compare the results obtained from simulations and experiments, and discuss the implications of these results for development of 2D-LC methods for molecules small and large. To illustrate these points, we will use contemporary examples such as the coupling of affinity (Protein A) and size exclusion (SEC) separations for the determination of aggregation level of monoclonal antibody (mAb) in cell culture supernatant samples.

Dispersion of analytes in open tubular fraction collection loops used in two-dimensional liquid chromatography

ACS Spring 2022

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