LC on the Edge

Testing Boundaries, By Davy Guillarme
New Wave, By Koen Sandra
Keep Pushing, By Fabrice Gritti
Up the (R)Evolution!By Gert Desmet
Through the Maze, By Monika Dittmann
2D-LC Has Arrived, By James Jorgenson

Testing Boundaries

Complex biological samples demand a new generation of LC systems

A major breakthrough in LC over the past 10 years has been the commercialization of modern chromatographic systems able to work at pressures up to 1,000–1,500 bar, with low extra-column volume and dwell volume. In parallel, new columns packed with sub 2 μm fully porous particles and sub 3 µm superficially porous particles entered the market. Together, these two innovations have led to a resurgence of interest in HPLC.

Good resolution

High-throughput separations are now easily attained. A HPLC separation that used to take 20–40 minutes can be replaced by a UHPLC separation of  2–5 minutes. The next hurdle is resolution. Our samples are increasingly complex – particularly in the fields of lipidomics, metabolomics, proteomics, natural products, and biopharmaceuticals. Such samples can contain thousands of compounds, outstripping the number that can be separated by HPLC.

How can we further increase resolution – and without sacrificing speed? It’s clear to me that the solution is not to increase the upper pressure limit of current UHPLC systems. Working at pressures beyond 1,500 bar would create more problems than it solves (frictional heating). Instead, we need to develop innovative materials that allow excellent chromatographic performance without the need for extreme pressure, such as ordered chromatographic columns (pillar array), alternative particle designs (sphere-on-sphere) or monolithic supports. I suspect that monoliths, in particular, represent a very promising approach, and it’s a shame that so little work is being done in this area.

As well as improved stationary phases, a major goal is to construct chromatographic systems with no tubing between the column, injection valve and detector. In modern HPLC, tubing remains the main source of band broadening when using narrow-bore or micro-bore columns, and narrow tubes could also generate a significant pressure drop at elevated flow rate. Tubing decreases the flexibility of HPLC systems and disrupts compatibility with a wide range of column widths. Dispensing with the tubing between injector and detector would allow use of a much wider range of columns – from capillary (0.3–0.5 mm ID) to micro-bore (1mm ID), narrow bore (2.1 mm ID) and conventional (4.6 mm ID).

2D-LC is another promising strategy for complex mixtures – it drastically improves peak capacity and overall resolving power, while maintaining a reasonable analysis time. The 2D-LC instruments on the market today remain somewhat difficult for non-specialists to use for comprehensive 2D-LC, but that’s improving with efforts from providers. However, there is still no user-friendly software for data treatment and no calculator available to determine optimal conditions in the first and second dimensions of 2D-LC.

In terms of chromatographic method development, I’m always surprised that so few chromatographers use modeling software, such as DryLab or ChromSword, to develop their methods. Instead, they rely on a time-consuming trial and error approach. It’s a shame, since today’s optimization software is extremely powerful, able to simultaneously optimize gradient profile, pH and temperature based on only 12 initial experiments. Plus, method robustness (important in a quality by design environment) can be assessed without additional experiments.

Big molecule, little molecule

Today, one of the most important fields of application for liquid chromatography is the characterization of biopharmaceuticals. For this, we need columns that can analyze very large and complex molecules, such as monoclonal antibodies, which may have several hundred potential microvariants. Columns are available for performing size-exclusion chromatography, hydrophobic interaction chromatography and ion exchange chromatography, but the performance of these “historical” techniques will need to be enhanced with more innovative stationary phases; the resolving power is currently too limited compared to the complexity of the biopharmaceutical samples that need to be analyzed.

It’s not just large molecules that pose challenges for separation scientists; the analysis of small polar molecules is also difficult, as they cannot be retained under reversed-phase LC conditions and are not soluble in normal-phase LC mobile phases. Metabolomics regularly involves the analysis of polar substances, including sugars, amino acids, nucleosides, small organic acids or substances with several phosphate groups. Hydrophilic interaction chromatography (HILIC) is the best technique we have, but is still not as robust and versatile as reversed-phase LC. There is a strong demand for new analytical strategies able to deal with polar molecules, while maintaining MS compatibility. It’s not yet clear what that will look like, but it could be either: 

• an evolution of HILIC involving new stationary-phase chemistry or mobile-phase components
• the use of purely aqueous liquid chromatography
• the use of hydride silica (Type C silica)
• something entirely new...

Taking cues from MS

LC and MS are becoming increasingly inseparable. In the future, we need to exploit that relationship by developing the two techniques in tandem. The MS market is currently evolving in two different directions. In recent years, companies have commercialized easy-to-use, compact and relatively cheap MS detectors based on single-quadrupole technology. Such detectors are very well adapted to chromatographers with a limited background in LC-MS. On the other hand, there is also a need for high-performance MS systems able to deal with increasingly complex samples – devices that offer enhanced sensitivity, resolution, mass accuracy and overall performance, and are obviously dedicated to MS specialists. Obviously, these two types of MS devices can be easily hyphenated with LC and in the near future, I expect to see even more interactions between chromatographers and MS specialists, since they clearly have to learn from each other.