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From »The Analytical Scientist« (Issue 29, June 2015). (6/15). "Pushing the Limits of Liquid Chromatography. LC Specialists in Academia and Industry: Mary Wirth, Gert Desmet, James Jorgenson, Monika Dittman, and Fabrice Gritti".

Organisations Organisation Purdue University, Dept. of Chemistry
  Group Purdue University
  Organisation 2 University of North Carolina
Product Product liquid chromatography (LC)
Persons Person Wirth, Mary J. (Purdue Univ 201506 Professor at Dept of Chemistry)
  Person 2 Jorgenson, James (Univ North Carolina 201506 Professor at Dept of Chemistry)

Pushing the Limits of Liquid Chromatography

LC specialists in academia and industry – including Mary Wirth, Gert Desmet, James Jorgenson, Monika Dittman, and Fabrice Gritti – share a common and bold vision: to ensure that LC continues to be a platform for innovation rather than stuttering into stagnation. Here, our experts consider where we are, where we need to go, and how we get there.


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?
Liquid chromatography is not just an analytical tool for today, it offers a great deal for the future.

By James Jorgenson, William Rand Kenan, Jr. Distinguished Professor, department of chemistry, University of North Carolina, Chapel Hill, USA.

Compared with 20 or 30 years ago, today’s routine LC separations are much faster and have higher resolution. Columns are far better deactivated and much more base-tolerant and reliable. The biggest single benefit from these developments has been the increased productivity of the individual analyst.

I won’t mention names of individuals or vendors: it’s not who you name, it’s who you overlook and wish you had named. In reality, countless thousands of scientists, engineers and computer scientists have made important contributions to the development of LC. Identifiable important trends include the steady progress in developing and using smaller particles, the use of higher pressures, and development of instruments with decreased extra-column dispersion. Equally important are the improvements in silica support synthesis, more inert and hydrolytically stable bonded phases, and novel particle morphologies such as core-shell particles.
One of the sharpest tools in the box

The impact of LC is well recognized and it is regarded as an essential tool in the analytical toolbox. However, let’s not forget that a lot of room for improvement and development remains. For example, there is a real need – driven by proteomics as well as by protein-based pharmaceuticals – for better columns for separating intact proteins. Metabolomics is also driving the necessity for more capable stationary phases for retaining highly-polar metabolites, while using mass spectrometry (MS) compatible mobile phases. And, for the foreseeable future, nanomaterials and nanoparticles are poised to be important. Surely, LC offers much for the analysis and purification of nanoparticles; however, this may require improvements in modes such as hydrodynamic chromatography, size exclusion chromatography, or LC-related technologies, such as field-flow fractionation.

As we push the limits of LC, I would love to see more studies involving molecular dynamics simulations of analytes and their interactions with stationary and mobile phases. As people become more adept at molecular dynamics simulations, I think there is much we can learn about retention kinetics and equilibria. Smaller particles have been a steady trend in LC; and, I see no reason for that trend to stop. Also, the use of high temperatures in LC is interesting, but always must be taken on a case-by-case basis. The biggest concern is the thermal stability of the analytes. In addition, can we continue to aim for higher pressures? Why not? High pressure (unlike high temperature) tends to be benign in terms of analyte stability.

One thing is clear: we have a pressing need for new types of LC stationary phases. Classic reversed phase is wonderful, and will continue to be the workhorse method in the future, but it won’t work for everything. There are a lot of questions that need answering, such as how do we best handle highly polar small molecules (for example, metabolites)? Are there possibilities for novel alternative mobile phases or mobile phase additives? Are there practical alternatives to water for higher-polarity mobile phases?

Risky business?

How can we as a community organize a dedicated effort to further advancing LC? Well, I can’t think of anyone who would refuse more funding or collaboration for separations research. But in reality, collaborations tend to be best initiated from the bottom up by individual researchers. Furthermore, it is just as important for researchers in separations to interact with people in biochemistry, biology, materials science, engineering, synthetic chemistry, spectroscopy, and mass spectroscopy, as it is for them to interact with other people in separations. The most important thing is for grant awarding bodies to fund good people to do good research – and they shouldn’t be afraid to fund risky research. Risky research usually fails, but it is also the source of unique new approaches...

As to limiting factors, in my opinion, we can never have enough resolution or peak capacity. We should never be satisfied with the status quo. There is no end to our imagination – and that opens up our horizons to other techniques; and how we work with them or embrace them. For example, the role of MS in analysis is constantly increasing. Any development within LC must maintain compatibility with MS. Top-down proteomics of intact proteins is increasingly viable as mass spectrometers become more powerful. We need highly effective columns for separating intact proteins coupled to these new mass spectrometers. There is a long way to go before the chromatography of intact proteins measures up to the great strides taken in the MS of intact proteins.

The future is full of promise, but...

When I look at what’s changed over the past year, I am always disappointed. However, when I look at what has changed over a decade, I am always amazed. How does the incremental accumulation of 10 years’ of disappointments eventually become an exciting qualitative shift in performance? I’m sure one factor is the occasional unanticipated paradigm-shifting development; the electrospray ionization source for MS, for example.

The introduction of monolithic columns as an intelligent stationary phase morphology is permitting greater freedom in design of column architecture. One of the questions that springs to mind about such developments is: can the dimensions of the physical features of successful and efficient monolithic columns be scaled down further to yield still greater efficiencies? The main benefit so far of higher pressures and smaller particles has been greatly increased speed of analysis, with modest improvements in resolution and peak capacity. A second question then: can the benefits of high pressure be extended to longer columns with extraordinary resolving power?

It is difficult to predict what the next big new ideas will be, but a couple of things that cross my mind: will chromatography with slip-flow revolutionize separations, and will advanced 3D printing technologies permit direct fabrication of near-perfect chromatography columns? In reality, I don’t know what the great developments of the next 10 years will be, but I am certain they will happen – and will be remarkable.


Marching Ever Onward
The evidence contradicts those who say liquid chromatography has fully matured.

By Gert Desmet, department head, chemical engineering, Free University of Brussels, Belgium.

In a recent survey looking back to 2002, my research group set out to compare optimal performances of LC. The starting date was well before the arrival of sub-2- µm columns. In those days, we found that the time needed to obtain 20,000 plates took about seven minutes, which with today’s technology and techniques only takes 50 seconds (to measure a compound eluting with a retention factor of 10). And, in 2002 it took an impractically long 100 minutes to do an N=100,000 plates separation, whereas today we can do it in just over 20 minutes. It’s impressive progress that contradicts those who say that chromatography is fully matured and so doesn’t deserve further R&D.

LC has been – and continues to be – a platform for innovation. The first major breakthrough, for example, was the introduction of UHPLC inspired by the seminal work of James Jorgenson and promoted by the late Uwe Neue. The second big breakthrough was the reintroduction of core-shell particles around 2007. The latter breakthrough was somewhat serendipitous because the increase in efficiency compared to fully porous particles was much larger than could theoretically be expected, based on the reduction of the differential paths inside the particles alone.

Nothing stands still in LC. I think demands for faster and more efficient separations will continue hand in hand with the development of more efficient columns that reduce the time needed for method development – a very costly process in industry. We do need more efficient columns to support the current search for biomarkers; and, we need them for more general research, such as analyzing how the cells in our bodies are functioning and how we could cure them when something goes wrong. 2D-LC will certainly be needed to produce the required peak capacities for this type of research, but even then, the efficiency of the individual dimensions will first need to go up as well. And as biologists dig ever deeper into our bodies, aiming at single cell or even sub-cell level analysis, miniaturization of LC systems could become an important issue again.

Design for today, not the last century

Design is the limiting factor of today’s instruments. They still have the same “hi-fi tower” design as those produced in the 1970s and 1980s. This form factor leads to such high levels of extra-column band broadening that we can say that our columns have become too good for our instruments, and we (or better, the instrument manufacturers) should do something about it to bring their designs into the 21st century!

Pressure is not an issue, as it seems theoretically and practically possible to run a column up to 3000 bar or so. As a matter of fact, Ken Broeckhoven and I are running a project on 2600 bar separation using normal bore columns and up until now things are going very well – we’ve had no explosions so far! So, in terms of mechanical strength there might be no fundamental impediment. What is trickier are the compressibility effects that make it more difficult to generate a precise flow rate, but perhaps there are ways to circumvent that as well.

No one can second-guess the future, but I do believe that we should be able to operate columns up to 2000 bar without any difficulty. I also think it should be possible to automatically couple up to three 10-cm columns packed with 1.5 µm core-shell particles within an integrated system that clamps the columns directly between the injector and detector to eliminate all connecting tubing.

Finally, because of the gradual decline in chromatography training for analytical scientists, we must improve the ability of instrument software to assist the analyst with decision-making. This – and more powerful instruments – will undoubtedly take our field even further forward.


Marking Progress

Monika Dittman (Agilent Technologies) and Fabrice Gritti (Waters) offer a manufacturer point of view on the practical limits of LC.

What is the current state of play?

Monika Dittman: In 1D-HPLC, using sub-2µm particles and (U)HPLC instruments operating at over 1000 bar, the time to reach a desired plate number has been reduced roughly by a factor of 5 (30,000 plates in four minutes with 1.8 µm particles at 1000 bar compared to 20 minutes with 5 µm particles at 300 bar) in routine analysis. The gain in performance can be used to obtain higher resolution in the same separation time (typically a factor of 3–4 on plate number or a factor of 1.5 in resolution/peak capacity), depending on the separation conditions.

These gains have enabled the fast separation of complex samples, and reduced the effort and time required for method development because a lack in selectivity is counteracted by increasing efficiency.

A further increase in operating pressure along with a further decrease in particle size could provide some additional improvement in speed and/or resolution, but 1D-HPLC eventually reaches a theoretical limit. Multidimensional LC can provide much larger peak capacities in a shorter time, which offers more power for very complex samples. And although it cannot be considered a routine technology right now, commercial solutions are available and being used by an increasing number of researchers.

Fabrice Gritti: Fast 1D-LC analyses of moderately complex samples and biomolecules is possible with short (5–10 cm) and narrow-bore (2.1 mm) columns packed with sub-2 µm particles. For the same level of efficiency, the analysis times in UHPLC have decreased by a factor of 10 relative to those observed in HPLC using 15-cm long columns packed with 5 µm particles. The volume consumption of eluent is also reduced by a factor of 10. The only downside is that system pressures have increased from 100-300 in HPLC to 400-1200 bars in UHPLC. The milestone for such an achievement was the emergence in 2004 of instrument pumps capable of delivering high flow rates at pressure of 1 kbar and the preparation of high strength sub-2 µm silica particles.

For all that, UHPLC rapidly touched its limits in terms of resolution because most standard UHPLC systems cannot provide more than 20 percent of the intrinsic efficiency of short (5 cm) and narrow-bore (2.1 mm) columns packed with sub-2 µm core-shell particles. System dispersion must be reduced further without precluding detection sensitivity (by diminishing the UV cell volume and path length) and speed (by shrinking the inner diameter of the connecting tubes). The transfer of United States Pharmacopeia methods from HPLC to UHPLC is also a critical issue because pressure and frictional heating affect retention (identification), peak width (resolution), and peak area (quantification) in UHPLC.

Since 2007, the lowest reduced plate heights of 2.1 mm to 4.6 mm i.d. analytical columns (capillary columns excluded) have been reached with core-shell packing materials (h=1.4) and 2.7 µm Halo silica particles. These developments mean there is no need for ultra-high pressures but it is still necessary to optimize standard HPLC instruments to reduce band spreading.

Resolving complex sample mixtures is routine practice using online and offline 2D (time x time) LC. Some commercial instruments provide customers with the possibility of 2D analyses, which plays an important role in polymer separation, in proteomics, and in genomics. Yet, 2D-LC faces some practical difficulties in terms of column orthogonality, solvent compatibility, sensitivity loss, and MS detection rate. For instance, the digest of host cell proteins having a dynamic range of concentration from one to more than a million cannot be solved satisfactorily by 2D-LC/MS.

Hyphenated LC/MSn techniques are now a routine analysis tool in any lab around the world. LC is still indispensable to fractionate complex mixtures, while MS enables identification of all the precursor and product ions. The limits of MS detection are quantification and the scan rate.

What contributed to these advancements in LC?

MD: Calvin Giddings laid the foundation for ultra-high pressure LC back in the 1960s when he predicted that gains in resolution/time could be achieved with higher operating pressures. Then, more than 20 years ago, James Jorgenson’s group demonstrated extremely high column efficiencies when operating with 1 µm particles at very high pressures.

In 2004, Waters introduced the first commercial system that could operate at 1000 bar, together with 1.7-µm particle packed columns, which subsequently led to every major HPLC equipment vendor offering UHPLC systems with operating pressures up to 1500 bar. Although routine analyses are still performed under standard HPLC conditions, customers often buy UHPLC systems when replacing older equipment.

FG: I agree with Monika. Giddings’ basic theory of chromatography has continuously pushed manufacturers to innovate and build new instruments (higher pressure, smaller system dispersion, 2D separation systems) and smaller particles to achieve faster separation and higher resolution performances by LC. Theory is obviously a major driving force for innovation.

The success of the sub-3 µm core-shell packing materials for analyzing small molecules is due to some unexpected properties of their beds. They are more radially uniform than those packed with conventional particles. This observation was not predicted by any theory of chromatography, which explains why most column manufacturers were initially reluctant in preparing such new materials. So, innovations can also result from pure luck! In contrast, Horvath predicted the use of core-shell particles for separating heavier molecules in the early 1960s based on the reduction of the average diffusion time across giant 50 µm particles.

In any case, technical achievements are delivering faster analyses, higher analysis throughput, and solving more complex sample mixtures than the old conventional HPLC techniques. But we must not rest on our laurels; separation science still faces numerous challenges:

> The complete resolution of protein mixtures in host cells for which the dynamic range of concentration extends from 1 to 107. This requires better isolation of the least abundant from the most concentrated proteins, more accurate quantification of the detected ions, and higher MS scan rates.

>The separation of glycan-binding protein for determining biological mechanisms.

>The separation of protein isoforms that differ by a few mutations of amino acids.

>A need to increase analysis throughput of chiral candidates during the drug discovery process demanded by pharmaceutical companies.

Where are we heading?

FG: Computerized molecular dynamics studies will further refine our established, basic theories of chromatography (Schure, Tallarek, and Mountain all talk about this). These studies enable a deeper understanding of the mass transfer and retention processes in LC from a molecular scale. In the end, they will consolidate or correct our old theories, and, in practice, they will enable the new design of the structures (kinetics or mass transfer) and of the chemistries (thermodynamics or selectivity) of separation systems for targeted performance.

UHPLC systems (injection, tubes, connections, detection) and column hardware (end-fittings + frits) will need redesigning to benefit fully from short (< 5cm) narrow-bore (1-2.1 mm i.d.) columns packed with 0.5 µm particles for analyzing biomolecules at low linear velocity.

Sub-1 µm particles are currently useless for the analysis of small molecules: that said, UHPLC systems would have to withstand pressures as high as 3 kbar to deliver optimum velocity. This could become a reality in years to come. Ideally, on-column injection/detection is required to eliminate system bandspreading. Ultra-low small injection volume (< 10 nL), short and small i.d. connecting tubes (< 50 µm), small volume detection cell (< 100 nL) or on-tube detection, and improved column end-fittings need developing. Currently, we are just reaching the limits of miniaturizing “classical” UHPLC parts, operating in a range of flow rate from 0.1 to 1.0 mL/min with today’s technology.

We must push research efforts for fully integrated LC-MS separation systems using either packed micro-channels or capillary tubes (100-500 µm i.d.) as separation media. The challenge with small volume separation systems is the detection sensitivity (too short path length). New detection techniques other than UV-Vis are therefore needed. Light emitting diodes are a solution (excellent monochromaticity and noiseless signal), yet they cannot cover a wide continuous range of wavelengths. In terms of speed, a larger number of compounds will be identified if the scan time of MS detectors is reduced. Injection, separation, and MS detection devices should be integrated to minimize losses of resolution and sensitivity.

What are the limits?

MD: With modern UHPLC instruments and small particles we have come very close to the limits that can theoretically be reached in 1D LC. However, very important improvements would include ease-of-use and maintenance, cost of ownership, overall system intelligence – those things that ultimately improve efficiency and reduce cost. With multidimensional LC, the complexity of instrumentation and applications had been limiting its extended use, but this is starting to change.

FG: The current limiting factors of UHPLC techniques in terms of resolution and speed are essentially instrumental: the column hardware affects the resolution power of short columns severely for the least retained compounds, small UV cells are reducing sensitivity, and very narrow connecting tubes generate too much backpressure for the current generation of pumps.
What other technologies/techniques will affect LC?

MD: We should expect to see MS-systems that keep pace with fast separations. In addition, we need intelligent software to not only generate data but also enable their meaningful use – one of the current bottlenecks is efficient and fast handling of data produced by ultra-fast, ultra-high resolution analysis.

Where do you expect LC to be in five years?

MD: MS will become more of a routine detection method (as in GC already) and multidimensional LC will become standard for medium complex samples. In addition, multidimensional LC-MS (or even multidimensional LC-ion mobility spectrometry-MS/MS) will become standard for complex samples.

As the majority of LC users will not be separation scientists but organic chemists or biologists the big challenge will be to create intelligent software solutions that support users in instrument operation, method development and data interpretation. This will be of particular importance for the success of multi-dimensional LC to be adopted as a routine technique.

FG: In five years from now, we should expect that UHPLC coupled to MS detection will finally play a major role in most academic and industrial laboratories. The high price of UHPLC technology, the difficulty to transfer well-established methods developed for HPLC to new methods for UHPLC, and the need for integrated MS detectors (everyone needs a LC-MS detector now, not a simple LC-UV instrument) holds back progress in the instrument market. SFC-based instrumentations (SFC-MS, LC-SFC-MS or SFC-LC-MS) will become available and friendly to use given the numerous advantages of supercritical CO2 fluids (cheap and green solvent, fast mass transfer and high resolution, high analysis speed).

The boom in micro-fluidic devices will likely provide a new generation of high-resolution portable instrumentation for routine analysis. Nano-LC/MS at very high pressures should enable the use of relatively short capillary columns or micro-channels packed with sub-1 µm particles for the analysis of biological samples.

If we look further to 10 years, we may see the emergence of 3D-printing technologies capable of reproducing perfectly ordered column structures with a characteristic dimension smaller than a few µm. Fabricated from relatively cheap raw silicon material, these printed columns could well supplant the current silica-based particulate and monolith technologies.

Looking forward even further, the ultimate and unified analytical tool, which could solve any of the analysts’ needs will feature in a single apparatus selected extraction units (by either liquid or supercritical fluids), selected injection devices (head space for GC analysis of volatiles, trap beds for SFC-based extracts, standard LC injectors), selected pump devices (for GC, SFC, or LC separations), switch-column valves (for 2D analyses), and MSn detection. All these technologies are currently available but as separate units for most. The fundamental, engineering, and technical challenges required to bring them altogether are obviously immense but not out of reach in the long term. At least, research efforts towards this direction should be funded and encouraged.

From an academic viewpoint, separation scientists are becoming a rare species even close to extinction after the passing of Giddings, Horvath, and Guiochon. Chromatography is no longer considered as an indispensable science instead is seen as merely a preparative tool. Yet, it should be remembered that most improvements made in GC and LC over the last 50 years were based on sound physical chemistry, material chemistry, and engineering. Future challenges in separation sciences and their new technologies to come will not escape this rule.


[ This article has been taken from the June 2015 Issue of »The Analytical Scientist«. You can read the original version and more articles with insights into analytical topics online at (free registration required) ]

Record changed: 2016-01-10


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