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Exorbitant demands being placed on analytical separations by fields such as proteomics, natural products, and biopharmaceuticals have elicited remarkable performance gains in high performance liquid chromatography (HPLC). However, it is also true that, barring a paradigm shift in column design and instrumentation, one-dimensional (1D) LC ultimately cannot meet the separation requirements imposed by such complex samples. As a consequence of the diminishing returns encountered for high-resolution separations-analysis time increases much more dramatically than the peak capacity does-the practical limit of state-of-the-art 1D-LC separations lies in the region of peak capacities of 1000. Considering that the available peak capacity should significantly exceed the number of sample components to improve the probability of their separation (1), the challenge is clear.
Currently, the most promising means to obtain an order of magnitude increase in performance for liquid-based separations is comprehensive two-dimensional (2D) LC (LC×LC) because of the oft-cited multiplicative increase in peak capacity that is attainable. Indeed, having just returned from the 45th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2017) conference in Prague, Czech Republic, it was gratifying to see a noteworthy increase in the number of contributions involving 2D-LC. This increase seemingly indicates that multidimensional (MD) LC has successfully bridged the gap from a primarily research-based technique to one increasingly being applied to solve real-world separation problems in industry. Here, I would like to share some thoughts on the reasons underpinning this evolution, and the potential of future developments in the field that might further increase the performance and application of MDLC.
The two most-used modes of 2D-LC are online comprehensive (LC×LC) and (multiple) heartcutting ([M]HC) 2D-LC. With the availability of robust and reliable instrumentation, MHC is ideally suited for regulated environments and to meet particular separation goals, although the performance gain is not sufficient for untargeted separation of complex samples. In such cases, LC×LC, where the entire sample is subjected to separation in two different columns, is required. Although LC×LC has been around for a long time (2), widespread use of the techniques was hampered by several constraints. Developments that have played a role in overcoming these challenges include important fundamental contributions, improvements in high-speed and high-performance columns (notably ultrahigh-pressure liquid chromatography [UHPLC] and core-shell columns), and instrumentation. More recently, the availability of commercial MDLC instrumentation has arguably had the greatest impact on the growing use of the technique: In contrast to the situation only 5 years ago, where most LC×LC separations were performed using laboratory-built systems, the availability of “off-the-shelf” hardware with dedicated software has made 2D-LC accessible to many more scientists. Indeed, new applications demonstrating the power of LC×LC are being published weekly in the literature, with peak capacities up to a few thousands attained for conventional analysis times.
However, several aspects still have to be addressed before LC×LC will find more widespread use. One of these is method development, which, similar to the performance of the technique, is much more complex than in 1D-LC. Important contributions have been made in terms of kinetic (3) and selectivity (4) optimization, although much further work is required to provide the tools for nonspecialists to overcome the expertise barrier. A second, more fundamental limitation of online LC×LC has to do with the very fast 2D separations required to meet 1D sampling criteria; this speed requirement ultimately restricts the peak capacity attainable in LC×LC. While off-line (and stop-flow) LC×LC provide peak capacities on the order of tens of thousands because of the removal of 2D separation time constraints, this performance also comes at the cost of much longer analysis times. An alternative strategy is to perform LC×LC separations in space (xLC×xLC) as opposed to in time (tLC×tLC). The principal advantage of this approach is that all 2D separations are performed in parallel, which allows better performance than tLC×tLC for the fast separation of complex samples (5). Practically, the benefits of xLC×xLC have yet to be demonstrated, although researchers are actively involved in this area (6).
Despite the contemporary success of tLC×tLC, the performance of the technique remains inadequate for the most complex samples currently encountered in liquid-phase separations: protein and peptide samples, which may contain in excess of 10,000−100,000 molecular species. It is therefore not surprising that researchers are contemplating the use of a third separation dimension.
The potential of comprehensive three-dimensional (3D) LC has long been recognized, and indeed 3D size exclusion chromatography (SEC)×reversed-phase LC×capillary electrophoresis (CE) was already implemented in a landmark paper in 1995 (7), although it is only recently that renewed interest has resulted in active exploration of the further potential of this approach (8−11). Currently, much of this work is based on theoretical considerations on the best way to achieve optimal performance in 3D-LC; practical implementation remains an exceedingly complex task (which emphasizes the achievement of Jorgenson in 1995). There are different ways of achieving this goal by combining spatial and time-based separations. Studies indicate that the performance gains of tLC×tLC×tLC are relatively limited because of the decreasing performance of each dimension due to sampling constraints, as well as significant dilution (9,10). In contrast, xLC×xLC×xLC potentially provides the best performance in terms of peak production rate (peak capacity/total analysis time). However, design and analyte detection represent major challenges in any such potential system. xLC×xLC×xLC is considered a more feasible approach, since compounds may be detected as they are eluted from the third dimension (10,11). While much of the work in comprehensive 3D-LC currently remains theoretical, this approach is essential in pointing the way to practically achieve the exceedingly ambitious goal of attaining peak capacities close to 1 million.
Although not involving chromatographic separation, the role of ion mobility (IM) in combination with MDLC separations is another interesting direction for future research. In IM, gas-phase ions are separated based on their averaged collision cross sections (CCSs) through transfer across a drift tube filled with buffer gas. In addition to the utility of the technique for facilitating compound identification by MS, IM also potentially allows separation of isobaric compounds, and therefore provides a complementary separation mechanism to MS, and indeed to many chromatographic modes. The relevance of the technique to comprehensive MD separations stems from the fact that IM separations are performed in the millisecond timescale, which allows the technique to meet the sampling requirements of LC×LC separations. This, therefore, opens the options for comprehensive 3D-LC×LC×IM separations. There are also significant challenges to overcome here, primarily in terms of data analysis and representation, although recent work on LC+LC×IM confirms the promise of the approach (12).
From the above, very brief, overview, it can be concluded that
It is clear that significant challenges remain in the latter fields, but the extraordinary benefits promised by each provide the clear incentive to continue this extremely rewarding journey.
André de Villiers is an associate professor in the Department of Chemistry and Polymer Science at Stellenbosch University in Stellenbosch, South Africa.