A Final Word from Incognito—The Past, Present, and Future of Chromatography

Incognito’s last article takes a look at what has changed over a career in chromatography, but it predominantly focuses on what the future might hold in terms of theory, technology, and working practices.

In the past 14 years of contributing to this column, I’ve written about just about every aspect of chromatographic life. From a trip to a sushi bar that inspired a conversation about the analysis of inks, to the acetonitrile and helium crises, which almost broke the editor’s email inbox. I’ve considered questions such as “Is chromatography boring?” and “Is there a future for chromatography?”, written about the threat of the academic supremacy of the East, and discussed fraud in the laboratory. The whole gamut has been considered and I hope you have found at least some of these musings interesting or even stimulating. As I pen this last editorial, I’m caught between writing about the fantastic advances I have witnessed during my career in chromatography and what the world of the future holds for the chromatographer. Perhaps a mix of both topics is a fitting way to bow out.

The Past and the Present

The chromatographic world I entered some 35 years ago was pioneering and emergent. Conferences were exciting and literature regularly heralded highly significant new advances. We packed our own high performance liquid chromatography (HPLC) columns and to this day I can remember the recipes we used to make the silica slurries and the packing pressures and times for many of the columns we used back then. I also remember a column blank (an unpacked column) blowing off the slurry tube during a pre-wash at around 11 metric tonnes of pressure and recall it bouncing off the laboratory floor prior to being launched through the tiles in the laboratory ceiling. When column efficiency dropped as a result of bed collapse, we simply took off the end fitting, topped up the bed with a high concentration slurry, waited for the solvent to evaporate, and closed it up again—usually with a new frit fitted. Good again for another 100 or more injections.

We “built” HPLC systems from modular components, usually from different manufacturers, and joined them all together with various remote start and signal cables. The computing integrator allowed us to record chromatograms on rolls of paper and these technological wonders could integrate chromatographic peaks on the fly, which was a real advance on cutting out peaks from standard-grade chromatography chart paper and weighing them on a balance to get a quantitative result. In the earlier models, there was no option to “reintegrate” and unless one got the integration parameters just right, the analysis had to be repeated. Autosamplers brought significant freedom and the ability to not have to stand by the instrument each time an injection was required. Mind you, the complexity of some of these early systems was literally mind-boggling and I was very much on first-name terms with the instrument company engineers, who seemed to always be on-site to fix and maintain these marvels of engineering. Five micrometre particle size packing materials brought high efficiency and the possibility of reducing column lengths from 25 cm to 15 cm and the commensurate benefits in reducing chromatographic run times. Detectors were fairly rudimentary, and many UV instruments needed to be balanced or auto-zeroed between injections, and certainly over long analysis campaigns to stop the baseline wandering so much that the computing integrator chart pen ended up permanently bouncing off the end stop—and off the chart paper too! We also had very exotic refractive index, electrochemical, and fluorescence detectors too. Highly innovative, very useful for certain applications, and all of them required the skill of an artisan to get a stable baseline within a day of powering them up! LC–mass spectrometry (MS) had not been invented and the prospect of electronic storage of data was but a twinkle in the eye of the latest generation of whizz kids. Very few laboratories had computers and those that did had one or two at most for administrative or managerial use only.

The world of gas chromatography (GC) was similarly neophyte. Columns were all packed glass or stainless tubes, again mostly packed by the end user and restored after efficiency dropped by topping up with packing material. It was commonplace to have to replace the silylated glass wool plugs at the inlet end for every campaign of analysis to avoid gross peak tailing due to analyte interactions with the wool, which quickly lost its deactivation. Manual injection was the norm, and your injection technique was honed over many hundreds of injections, until qualitative peak shape was satisfactory and quantitative reproducibility assured. Flame ionization detection was also the norm, but we used nitrogen-phosphorus detection or electron-capture detectors much more frequently in those days to get the required sensitivity for various applications. I clearly remember the day we took delivery of our first GC–MS system; the excitement was palpable, and I remember the fear I felt when the vacuum system was explained as the oil-filled roughing pump started to pump down for the first time. It took me months to be truly comfortable with venting and pumping down the instrument, shaking with trepidation as I imagined the consequences of an oil backstream event, or a catastrophic vacuum loss leading to mangled blades of the very sensitive turbomolecular pump. The data system (yes, a whole computer dedicated to one instrument) ran on a turbo-pascal operating system and took around six months to be able to operate. Each spectrum was printed and manually searched against the many printed volumes of the McLafferty reference library, by either molecular ion or base peak searching. There were no autosamplers and there was certainly no automation of sample preparation.

It felt a little like the Wild West at times; we had to quickly learn how to keep a system running, perform maintenance to allow the next analytical campaign to proceed—frankly, how to be chromatographers. We have come such a long way in those 35 years, but I sometimes pine for those days and the intrepid voyages of discovery that occurred on a daily basis. However, as instruments and data systems have become more capable, robust, and reliable (one might say “industrialized”), there has been a paradigm shift in the chromatographic discipline and those who practice it. No longer do you have to think about the instrument and keeping it running, and so there is more time to consider the separation itself. Troubleshooting, development, and optimization of methods is much more commonly discussed in today’s laboratory because we can consider the separation rather than the equipment. That lack of insight into “what’s in the black box” can sometimes lead to a poorer understanding of the technique itself. Many of today’s chromatographers are users of chromatography rather than true chromatographers, but I’m not decrying that position. The demands of throughput, productivity, and capability have risen in line with the maturity of the science and the technology, and so the shift from a technique employed only by experts to one that is accessible to everyone in the analytical laboratory is perfectly natural. However, as I’ve often written in these pages, this can only be a defensible position if the level of training given to users is sufficient to allow them to recognize good data from bad.

Looking to the Future

Anyone working in an analytical laboratory today will recognize how far we have come since the “early days” of chromatography that I described earlier. But what of the future chromatography laboratory and the working lives of those within it?

One may be flippant and muse on the possibility that manufacturers may consider moving away from beige, dull blue, or cream and make instrument covers from more visually appealing colours. We might ask if HPLC system stacks will grow ever higher until even the tallest of folks in the laboratory will need a ladder to change solvents. But that would be facetious.

I do see a future chromatography world where sensitivity of detectors pushes the problem of detectability to the sample preparation phase. We simply can’t inject large enough sample volumes to detect the vanishingly low analyte concentrations unless there is significant focus on sample preconcentration, and for this I forecast significant advances in the automation of sample preparation. Why automation? The sample preparation required will become ever more complex and to attain the reproducibility required in a reasonable timeframe automation is really the only way to advance in this area.

Micro- or nanoscale chromatography has been much heralded throughout my career. I had my first conversation on nano-LC in the very early noughties and was excited about the possibilities. But nanoscale or microscale systems have never really delivered on a globally adopted scale. There are many reasons, but as the kids say on long journeys to holiday destinations, “Are we there yet?”

Undoubtedly there are advances that need to be made in system hardware, and—particularly in HPLC—extracolumn and dead volumes need to be reduced to take full advantage of the improvements to efficiency and analysis time that the ever‑decreasing particle sizes of column packings offer. Perhaps we won’t use tubular packed bed formats, with all of their thermal disadvantages, at all, and the “chip” and microfabricated array formats will win out in the end.

For the last 20 years I’ve been told that, for liquid-phase separations at least, the chromatographer’s future is bleak because the mass spectrometric detector can select the required analytes from the sample “soup” without the need for a separation. This hasn’t yet come to pass, and the ability of an MS source and mass analyzer to effectively discern, for example, enantiomers or isomers remains a significant challenge. Perhaps advances in ion source technology that can overcome suppression or enhancement effects combined with the use of high mass accuracy mass analyzers and techniques such as ion mobility spectrometry as a pre‑filter will eventually make all of us using HPLC effectively redundant.

Gas chromatography is a fairly mature technique, with good robustness and a plethora of highly sensitive mass analyzing detectors. One might think there is little room for improvement, but I would disagree. I see the future of GC focused on more advanced sample inlets that can overcome the fundamental “Achilles heel” of gas-phase separation techniques, which is sample volatilization and introduction into the analytical column. It is true that today’s GC inlet choices have expanded way beyond the standard split-splitless device, but in most laboratories this is still the default choice and internal standards are still used in many applications to account for the inherent disadvantage of losses or discrimination during sample injection. Cool on-column injection always held a lot of promise in my laboratory, as did thermal desorption, but I can’t help but feel there are areas of sample introduction technology that we have not yet fully explored, and whilst the current instrumentation is good, some inherent fallibilities remain.

Multidimensional separations are here to stay and indeed hold great promise for the future. However, to be truly game-changing, I think the reliability of the transfer mechanism (modulation) between dimensions needs to be more robust and reliable. The limitations on the physical dimensions of the second, or higher, dimension columns need to be minimized, perhaps with very highly efficient chromatographic media or devices that allow very rapid separations in the second dimension. Standardization of methodologies for various industries and application types also needs to be further considered. Software, datafile size, the ease of quantitative analysis, and the presentation and manipulation of data all need to be improved for multidimensional separations to reach global adoption.

Of course, our green credentials will also be a future focus. Think of the amount of plastic that we use in the laboratory without recycling. Think of the number of plastic disposable pipettes or pipette tips that we get through. The number of glass Winchester bottles. The volumes of solvent. The volumes of non-renewable gases. I’m aware that the “green chromatography” movement has already been established, but I do believe the future chromatographer will have environmental impact very much at the forefront of their minds. I’ll give them a starter for 10: Why don’t we recycle pipettes, pipette tips, and solvent Winchester bottles? The future will no longer offer us the option of green comparison. The fact that our environmental impact is significantly less than industry X or another of our business operations will not wash. We need to act soon and unilaterally to have a viably sustainable future.

Software used to acquire and process data has been revolutionized during my career, but I feel the future will be even more highly dependent upon developments in the digital aspects of our work. The ability to deconvolve and process huge amounts of data in reasonable timeframes will be key. We already have highly complex data systems whose features are underutilized because the average chromatography user either does not realize their potential or is not given the time and support to employ them in a compliant fashion. I don’t know many data systems that cannot take you from sample injection to final result given time and expertise, yet few of us really take advantage of these systems. Perhaps this brings us back to a discussion on training and expertise within the laboratory, but time and word count won’t allow yet another discourse on these particular topics.

I believe “industrialization” will also lead us to a future direction based on the Pareto principle. Consider how many different HPLC and GC phases are available and the number of manufacturers of these phases. Where do they fit? I recently had a conversation with a learned associate in which we mused on the possibility of the majority of separations—given an assumed level of system efficiency—that could be completed with only a handful of chromatographic phases from reliable vendors. Yes, there will always be “difficult” separations that will prove the exception to this rule, but perhaps 80% of all separations will be undertaken by the chromatography users, whilst the remaining 20% are undertaken by the chromatographers. Heck, your laboratory may even be set up this way currently. Maybe the future multidimensional system, when fully developed, may negate the need for this approach entirely. Let’s face it, a 5 × 5 array of columns in either liquid- or gas-phase techniques in the first and second dimensions, and which could be quickly screened in all of their combinations using automated method development platforms, could probably solve most of the selectivity issues that I’ve ever encountered. Method development software exists today that can help to evaluate the initial results of these screens and help with the heavy lifting of phase selection and initial solvent choice when using HPLC.

I could go on with future musings, but I’m afraid that I’ve already begged your indulgence for long enough.

So here we are then, the end of an era, and of a column in which I’ve had the privilege of sharing my thoughts and sometimes controversial viewpoints with you all, primarily to spark a reaction, which it has often succeeded in doing. I suspect it will not be much of a spoiler to admit that the opinions I have given in this column were not always my firmly held beliefs, rather a stance that I have taken to encourage debate and I hope that I have managed to achieve this.

We certainly have a long way to go, and to answer one of my own questions, chromatography certainly isn’t dead, but the future is in our own hands. We must continue fundamental research in separation science, both academically and industrially. We must respect and encourage our vendor partners to continue to push the capabilities of instrumentation, software, and consumables. We should continue to foster and develop the knowledge and skills of our younger colleagues and to promote our industry to the wider scientific and industrial world.

I leave you with the thoughts of one of my own personal heroes, the British comedian and author Spike Milligan: “A sure cure for seasickness is to sit under a tree”. Take to the boats my friends and don’t fear the sea, but push your boundaries to ensure that the next generation of chromatographers have better boats and better cures for sea sickness. I wish you all a fair wind and calm waters.

Contact Author: Incognito
Email the Editor: kjones@mjhlifesciences.com