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  • Primer
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Comprehensive two-dimensional liquid chromatography

Nature Reviews Methods Primers volume 3, Article number: 86 (2023) Cite this article

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Abstract

Despite progress in hardware and column technology, the complexity and heterogeneity of many samples still pose a challenge for state-of-the-art 1D liquid chromatography (LC). As an effective answer to these analytical questions, multidimensional comprehensive techniques have become an attractive strategy across disciplines. This Primer provides a general overview of 2D comprehensive LC (LC × LC), including hardware, software and best practices for experimentation. The underlying principles are described, alongside the requirements of separation and detection equipment. In addition, this Primer discusses data collection, processing and analysis, with an exploration of inherent challenges and possible workarounds. Selected examples are presented to show how the gain in resolution afforded by LC × LC has enabled in-depth characterization of complex non-volatile samples, such as pharmaceuticals, polymers, foods and the ‘omics. Finally, the technique progress is evaluated, with an outline of future directions for the field.

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Fig. 1: On-line LC × LC hardware setup.
Fig. 2: Configuration of the most used valve modulators.
Fig. 3: Instrumental scheme of the on-line SPE–HILIC–SPE × RP for the clean-up of peptides.
Fig. 4: An example of data visualization in the LC × LC analysis of α-casein tryptic digest separated using RP × RP.
Fig. 5: An illustrative example of spectral compression applied to LC × LC–MS data.
Fig. 6: Temperature-responsive × RPLC separation analysing mixtures of pharmaceuticals containing a main active principle (sulfamethizole) at 1 mg ml−1 and impurities at a 0.5% level.
Fig. 7: µLC × LC separation obtained at 254 nm from hemp strains.

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Authors and Affiliations

  1. Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy

    Luigi Mondello, Paola Dugo & Paola Donato

  2. Chromaleont S.r.l., Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy

    Luigi Mondello & Paola Dugo

  3. Laboratory of Foodomics, Institute of Food Science Research — CIAL (CSIC-UAM), Madrid, Spain

    Miguel Herrero

  4. Faculty of Chemistry, Applied Analytical Chemistry, University of Duisburg-Essen, Essen, Germany

    Lidia Montero & Oliver J. Schmitz

Authors
  1. Luigi Mondello
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  2. Paola Dugo
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Contributions

Introduction (L. Mondello, P. Dugo and P. Donato); Experimentation (L. Montero and O.J.S.); Results (L. Mondello, P. Dugo and P. Donato); Applications (M.H.); Reproducibility and data deposition (M.H.); Limitations and optimizations (L. Montero and O.J.S.); Outlook (L. Mondello, P. Dugo and P. Donato); Overview of the Primer (all authors).

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Correspondence to Luigi Mondello.

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Nature Reviews Methods Primers thanks Davy Guillarme and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

HPLC column database: http://www.hplccolumns.org/

Glossary

Co-elutions

Two or more compounds that are not chromatographically separated owing to retention times closer than the method resolution.

C-term

In the van Deemter equation, C-term describes the resistance to mass transfer coefficient of the analyte between the stationary and mobile phase.

Cuts

In 2D-LC practice, the term ‘cut’ refers to a fraction of the first dimension (1D) sample, which is stored in a loop system for second-dimensional (2D) separation.

Heart-cutting 2D-LC

A targeted 2D-LC technique, in which only a selected eluent fraction (single heart-cutting) or fractions (multiple heart-cutting) are collected for second-dimensional (2D) separation.

Isocratic retention factor

The ratio of the retention time of an analyte on the stationary phase to the time it spends in the mobile phase under isocratic conditions.

Off-line

In 2D-LC practice, the term off-line means that the fractions eluted from the first column are stored for subsequent reinjection onto the second column.

On-line

In 2D-LC practice, the term on-line means that the second separation is carried out in real time, in an automated fashion.

Peak capacity

The maximum number of resolvable peaks within a given analysis time.

Resolving power

In the chromatography practice, it is sometimes used to estimate the goodness of a separation between adjacent peaks.

Reversed-phase separation

Compound separation using hydrophobic interactions between the solute molecules in the mobile phase and the stationary phase.

Sample dimensionality

The number of distinct chemical classes in a sample that themselves lead to different retention mechanisms in the separation.

van Deemter curve

Describes the dependence of the height of a theoretical plate (H) on the linear flow velocity of the mobile phase (u). It is used to predict the optimum velocity at which there will be minimum variance per unit column length and hence maximum efficiency.

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Mondello, L., Dugo, P., Donato, P. et al. Comprehensive two-dimensional liquid chromatography. Nat Rev Methods Primers 3, 86 (2023). https://doi.org/10.1038/s43586-023-00269-0

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