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Fields & Applications Liquid Chromatography, Pharma & Biopharma, Mass Spectrometry, Proteomics

Pushing the Limits of Liquid Chromatography

Expanding LC Boundaries

We must inspire creative minds to keep LC moving forward

By Mary J. Wirth, W. Brooks Fortune Distinguished Professor, Department of Chemistry, Purdue University, West Lafayette, Indiana, USA.

Today, the pharmaceutical industry is a major user of liquid chromatography (LC), where stainless steel columns offer reproducibility and sensitive UV detection. The best commercially available LC columns for small-molecule separations now give about 50 percent more plates and faster separation times compared to 20 years ago. Even such a small improvement in resolution gained through the higher plate numbers is valuable for analysis of impurities and degradation products in pharmaceuticals – and the higher speed allows for faster methods development. Notably, separation speed has improved more than resolution.

The field has achieved these advances by decreasing the diffusion distance of the analytes, either with sub-2 µm fully porous or superficially porous particles. Both of these recent advances give comparable performance, and both advances were made on a sound theoretical foundation. For example, Jim Jorgenson and his group introduced sub-2 µm particles, and Jack Kirkland and co-workers introduced the superficially porous particles. Further reductions in diffusion distances will eventually give diminishing return, which means that diffusion distance will no longer be the limit. For large proteins, the best columns still give more peak dispersion than the best instruments, so some combination of packing heterogeneity, bonded phase, fittings and frits is apparently the main limit now.

Improving drug safety

As the primary users of LC are people in the pharmaceutical industry, both in drug development and in quality control, improvements in the field have essentially made drugs safer. Protein separations are a current and growing demand, both in the pharmaceutical industry, where protein drugs are the largest growth sector, and in proteomics, which is an integral part of biomedical research. Drug targets, cancer biomarkers, and diagnostics to monitor therapy usually involve proteins, and discovering these requires better columns due to the complexity of cell lysates and blood serum.

To that end, we need to think about the future and what steps we need to take. Advances require creativity, and one cannot really organize creativity. And, above all, we need to inspire creative minds to push the limits. Thankfully, there a number of people doing such pushing! For instance, Jack Kirkland continues to explore the limits of smaller diffusion distance, as well as the role of particle size, which affects packing homogeneity; Jim Jorgenson and Ulrich Tallarek (see next article) are addressing what underlies packing homogeneity; and Gert Desmet and his colleagues are trying to make the perfect LC column by micromachining.

There are many other efforts going on in the chromatography industry that are confidential, and we need to inspire more basic research. My own group, for example, is working on improving resolution in protein separations by improving packing homogeneity and avoiding the need for frits by using monodisperse colloidal silica.

Columns: still watching and waiting

Ultimately, the limit in any chromatographic separation is having the peak width determined only by diffusion of the analyte; that is to say, the instrument, the column, and other hardware contribute negligibly. This limit is only meaningful, however, if the separation time remains reasonable, since one could technically reach the diffusion limit by making the column length absurdly long. Therefore, the goal must be to reach the diffusion limit without making separation time or sensitivity worse than what we have today. Currently, the dispersion of the best columns is still quite a bit higher than the dispersion of the best instruments, so columns need attention, including the bonded phases. It is possible that connectors and frits also contribute to the dispersion.

Mass spectrometry, particularly top-down proteomics, demands higher resolution for protein separations by LC. Further, Fourier transform MS (FTMS) adds an additional constraint in column design because the mass resolution is dictated by the Heisenberg uncertainty principle. This means that the sharpest peaks in the time domain are no longer desirable since these would lower mass resolution. Instead, the sharpest peaks in the spatial domain are needed, with flow rate controlling the peak width in the time domain.

Daring to predict

Chromatography has changed very slowly in the past; for example, plate heights have dropped by about a factor of two over the last 20 years, and so, predicting the field five years from now is more daring than predicting over 10 years. Progress occurs slowly because the largest part of the LC market is regulated, meaning that change is not readily adopted. I think in 10 years we will at least be well on the road toward diffusion limited LC of small molecules, perhaps even with commercial products. For protein separation, it is certainly my own goal to enable diffusion limited LC-MS for top-down proteomics using capillaries. We have demonstrated that these can give diffusion limited separations that are fast, and the next goal is to do this with commercial instrumentation.

Have We Really Peaked?

By James Jorgenson

Marching Ever Onward

By Gert Desmet

Marking Progress

By Monika Dittman and Fabrice Gritti

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