Multidimensional LC

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.

The use of off-line 2D-LC–MS for the characterization of HCPs and their monitoring during downstream processing

Carotenoids are a class of natural pigments, widely distributed in vegetables and fruits. A comprehensive two-dimensional liquid chromatography (LC×LC) method, based on the use of a cyano and an octodecylsilica column, placed in the first and second dimension, respectively, was applied to evaluate carotenoid composition and stability in selected overripe fruits representing the waste generated by a local food market. This research also evaluates if post-climacteric biochemical changes are linked to carotenoid degradation in the investigated fruits. A total of 22 compounds was separated into seven different chemical classes in the two-dimensional space, and identified by photodiode array (PDA) and mass spectrometry (MS) detection. The results prove that the waste generated by the large distribution of food still represent an important source of bioactives that could be used for other purposes.

Carotenoids are a class of natural pigments, widely distributed in vegetables and fruits. A comprehensive LC×LC method, based on the use of a cyano and an octodecylsilica column, placed in the first and second dimension, respectively, was applied to evaluate carotenoid composition and stability in selected overripe fruits representing the waste generated by a local food market.

The interface between the two separation dimensions is a key element of any comprehensive two‑dimensional liquid chromatography (LC×LC) system. LC×LC has typically been implemented by using one or more switching valves, equipped with either sampling loops or trap column(s). Temperature manipulation is a relatively unexplored yet promising route towards non-valve-based LC×LC. The fairly recent emergence of thermal modulation has provided a less conventional method for performing LC×LC separations. This article illustrates the variety of commonly used modulators, paying specific attention to focusing modulators.

High-resolution mass spectrometry (MS) is not readily compatible with chromatographic techniques using high-salt mobile phases. This study presents the development and use of 2D-LC–MS via an on-line desalting step for the analysis of monoclonal antibodies and antibody–drug conjugates.

Here, I will attempt to convey a few basic but critical concepts when getting started in the development of a new LCxLC method.

Protein biopharmaceuticals have seen an enormous growth in the last decade, and as a result, separation scientists are giving increased attention to methods for characterizing biopharmaceuticals. One powerful technique for analyzing proteins is two-dimensional liquid chromatography (2D LC). Gerd Vanhoenacker of the Research Institute for Chromatography (RIC) in Kortrijk, Belgium, has been conducting research into peptide mapping of therapeutic monoclonal antibodies (mAbs) using 2D LC. He recently spoke to The Column about this work.

This application note discusses the comprehensive 2D‑LC analysis of green and black tea. The purine alkaloids caffeine and theobromine, as well as the catechins catechin, epicatechin, and epigallocatechin gallate, are quantified.

Comprehensive 2D-LC enables the analysis and quantification of purine alkaloids and catechins in green and black tea. As expected, green tea contained higher amounts of the catechins epigallocatechin gallate and epicatechin than black tea.

Analyzing Host Cell Proteins Using Off-Line Two-Dimensional Liquid Chromatography–Mass Spectrometry

In this video from LCGC TV, Luigi Mondello from the University of Messina in Italy reveals his views on the prospect of mass spectrometry eliminating the need for chromatographic separations in the future.

enable authentication of Si-Wu-Tang, a separate comprehensive 2D-LC analysis of each herb contained in Si-Wu-Tang was performed.

Estrogen can have a big effect on physiological processes in the human body, even when present at trace levels. In this video, Kevin Schug from the University of Texas at Arlington, USA, talks about the advantages of using restricted access media for the on-line sample preparation of biofluids in a “trap-and-elute” LC–MS arrangement, and the potential of this approach for other applications.

This application note demonstrates how comprehensive 2D-LC can be used to resolve the complex mixture of hydrophilic phenols found in virgin olive oil and investigates differences in the phenol composition of several olive oils.

In this new video from LCGC TV, Luigi Mondello from the University of Messinia, Italy, describes the fundamental principles of 2D GC, and explains the advantages over 1D GC.

In this new video clip from LCGC TV, Dwight Stoll of Gustavus Adolphus College discusses the development of new carbon-based phases for 2D LC that are well characterized and understood.

Kelly Zhang from Genentech discusses the next steps needed for the use of 2D LC by the pharmaceutical industry.

This review illustrates the benefits of LC?C in food analysis and discusses the combination of LC modes used in the two dimensions, and the nature of the samples analyzed.

Click here to view the complete E-separation Solutions newsletter from September 18, 2014.

A novel system for fully automated comprehensive two-dimensional liquid chromatography is presented.

Agilent Technologies