Multidimensional LC

On a fundamental level, equilibrium and diffusion define chemical separations. Practical applications of partition coefficients to chromatographic sample preparation illustrate this point.

In this extended special feature to celebrate the 35th anniversary edition of LCGC Europe, key opinion leaders from the separation science community explore contemporary trends in separation science and identify possible future developments.

This review article summarizes the results obtained from the combined efforts of a joint academic and industrial initiative to solve the real-life challenge of determining low levels of peptide-related impurities in the presence of the related biologically-active peptide at a high concentration.

The methodologies demonstrated here open the way to fully compliant at-line monitoring of monoclonal antibody quality attributes.

This article explores potential pitfalls associated with 1D‑LC and how 2D‑LC can overcome these obstacles.

Having a mixed-mode column that can provide both achiral and chiral resolution in one-dimension (1D) would significantly improve impurity profile understanding and reduce testing workload.

In this article, I share my perspective on the trends in 2D-LC, and the developments we are likely to see in the field in the near future.

As two-dimensional liquid chromatography (2D-LC) becomes more widely used, system suitability tests (SSTs) become even more important.

This month we interview Alexandre Goyon, Senior Scientist in the Small Molecule Pharmaceutical Sciences Organization of Genentech, about his work focused on the online digestion and analysis of RNA molecules using immobilized RNase cartridges attached to a mDLC–MS system, and why the accurate sequencing of sgRNA is important.

Multidimensional liquid chromatography (MDLC) methods have revolutionized the characterization of complex drug modalities like antibodies–drug conjugates, antisense oligonucleotides, and small interfering RNA therapeutics.

Two-dimensional LC, with advanced modulation techniques, can advance the analysis of advanced polymeric materials, assisting in characterizing copolymer composition heterogeneity and identifying ingredients in complex products.

James P. Grinias is an Associate Professor in the Department of Chemistry & Biochemistry at Rowan University in Glassboro, New Jersey, and the winner of the 2022 Emerging Leader in Chromatography Award, which is presented by LCGC magazine.

Barry L. Karger and James P. Grinias are the winners of the 15th annual LCGC Lifetime Achievement and Emerging Leader in Chromatography Awards, respectively, for 2022. Here, we review their achievements.

We review different approaches and coupling strategies for analyzing monoclonal antibody aggregates with 2D-LC.

In 2D-LC, properties of the mobile phase used in first step can negatively affect the second step. We explain why this problem happens and how to avoid it.

Filip Cuyckens from Janssen R&D in Belgium spoke to LCGC Europe about recent innovative approaches he and his team developed to support drug metabolism and pharmacokinetic studies, and the inventive role that two-dimensional liquid chromatography (2D-LC) plays in his laboratory to boost sensitivity, solve recovery issues, and increase overall efficiency.

This article will discuss the benefits of 2D-LC and multiple application areas in (bio)pharmaceutical analysis, and will highlight the challenges and future outlook.

In this instalment of “GC Connections”, the advantages of multidimensional chromatography with HPLC as the first dimension and GC as the second are discussed.

Retention of peptides is strongly dependent on solvent composition in reversed-phase separations with gradient elution. In this instalment we provide tips, tricks, and suggestions for best practices to help minimize retention time variations over time.

This review article discusses the novel separation and detection strategies that are considered promising in clinical metabolomics to enhance the metabolome coverage. It includes hydrophilic interaction chromatography (HILIC), supercritical fluid chromatography (SFC), multidimensional LC approaches, as well as ion-mobility mass spectrometry (IM-MS) and data-independent acquisition (DIA) analysis methods.

The evolution of two-dimensional liquid chromatography (2D-LC) instruments along with improved software capabilities has transferred 2D-LC from the hands of experienced researchers to functioning analytical laboratories in the pharmaceutical industry. 2D-LC offers chromatographers novel solutions to problems ranging from analyzing complex samples requiring excessively large peak capacities to separating simple compounds that are difficult to resolve. Recent developments in 2D-LC and 2D-LC–MS have demonstrated the potential of this technique in practice and 2D-LC is set to become an essential tool in the pharmaceutical sector to address problems ranging from coelution, peak purity assessment, simultaneous achiral-chiral analysis, genotoxic impurities, and more.

Multidimensional chromatography, or comprehensive chromatography, is a well-established technique for the analysis of complex mixtures. However, the technique is often perceived as highly complex and difficult to put into practice for routine applications. Nonetheless, the technique provides exceptional potential for addressing challenging separations. The addition of a dilution factor allows multidimensional chromatography to provide a high level of flexibility and selectivity. The dilution effect is achieved by using a column chemistry format compatible with large flow rates, which now offers the option of large volume injection without volume or mass overload issues. This novel solution can reduce or eliminate the need to add a solvent exchange step, such as evaporation or reconstitution, which significantly reduces the most time-consuming part of the sample preparation process.

Food products are very complex mixtures containing organic and inorganic nutrients, and also xenobiotic substances that can derive from technological processes, agrochemical treatments, or packaging materials, including residues of pesticides, drugs, and toxins.

This is an exciting time for 2D-LC, as the expanding community of users works to develop creative solutions involving 2D-LC to solve analytical challenges that are very difficult or impossible to solve by conventional means.

The enhanced separation power of two-dimensional (2D) chromatography has become accessible thanks to the commercialization of dedicated two-dimensional systems. However, with great separation power comes great system complexity. All two-dimensional systems require a means for collecting and transferring fractions of the first dimension to the second dimension typically via a loop-based interface in on-line methods. It is important to collect a sufficient number of fractions to prevent loss of the first dimension resolution; that is, the sampling rate must be sufficient to prevent undersampling. Another key parameter to consider is selectivity. By coupling two selectivities that have unrelated retention mechanisms we are able to exploit the different physiochemical characteristics of the sample we wish to separate. This is the concept behind the term orthogonality. By coupling orthogonal selectivities and reducing under‑sampling, our system should be able to achieve the theoretical maximum two-dimensional peak

Some members of the separation science community are still not yet convinced of the value of comprehensive two‑dimensional liquid chromatography (LC×LC). They feel that the large increase in separation power (that is, in peak capacity: the number of component peaks that may possibly be separated) may be compromised by losses in sensitivity and robustness of the separation. However, the chairmen of HPLC 2017 will have seen a great number of abstracts come their way from scientists who want to change this perception.

Comprehensive two-dimensional liquid chromatography (LC×LC) is evolving and becoming more commonly used in practice, but there are some specific problems still present that hamper the widespread use of this technology. One key aspect is the coupling of an on-line LC×LC system to a mass spectrometer. Generally, on-line LC×LC is based on a very fast second dimension separation to achieve low cycle times. This often results in flow rates that are far above the optimum for electrospray ionization mass spectrometry (ESI-MS). This month’s “Multidimensional Matters” looks at the benefits of miniaturization in the first and second dimension for coupling with a high-resolution mass spectrometer (HRMS) and describes an environmental analysis application.

This is the second in a series of articles exploring current topics in separation science that will be addressed at the HPLC 2017 conference in Prague, Czech Republic, from 18–22 June.

Two-dimensional liquid chromatography (2D-LC) has in recent years seen an enormous evolution, and with the introduction of commercial instrumentation, the technique is no longer considered a specialist tool. One of the fields where 2D-LC is being widely adopted is in the analysis of biopharmaceuticals, including monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs). These molecules come with a structural complexity that drives state-of-the-art chromatography and mass spectrometry (MS) to its limits. Using practical examples from the authors’ laboratory complemented with background literature, the possibilities of on-line 2D-LC for the characterization of mAbs and ADCs are presented and discussed.

On-line two-dimensional liquid chromatography (2D-LC) embracing mainly comprehensive LC (LC×LC) and multiple heart-cutting LC (mLC–LC) offers new opportunities for in-depth characterization of pharmaceuticals. Reversed-phase LC × reversed-phase LC using different column chemistries and mobile phases provides good orthogonality for a wide range of applications related to small molecule drugs. Moreover, hardware configurations and software are now commercially available to perform LC×LC and mLC–LC measurements in a reproducible manner.