High Impact Chromatographic Developments of the Past 60 Years

W. John Lough from the University of Sunderland, UK, in conversation with (old) friends from academia and industry to celebrate 60 years of advances in chromatography.

With the main theme of this publication being to celebrate 60 years of developments in chromatography rather than just 60 years of The Chromatographic Society, we thought it might be useful to bring together a small group of individuals, all of whom have been around long enough to have seen a good proportion of these 60 years. For the Society, John Lough (JL) (Reader in Pharmaceutical Analysis in the Sunderland Pharmacy School at the University of Sunderland, Sunderland, UK) is talking to (in order of appearance!) Wolfgang Lindner (WL) (Emeritus Professor, University of Vienna and a Chromatographic Society Martin and Jubilee Medal winner), Ted Adlard (TA) (former GC pioneer at Shell, a founding member of The Chromatographic Society, and Chairman 1970–1973, 1979–1983), Alan Handley (AH) (Senior Scientist at ICI and LGC, past President of the RSC Analytical Division [2011–2014], and The Chromatographic Society [2009–2014]), and Chris Bevan (CB) (Big Pharma chromatographer, Chromatographic Society President 2001–2007, and currently the Chromatographic Society’s Event Organizer).

John Lough: Let’s be perverse and start with the here and now. Wolfgang, it is timely that you are just back from HPLC 2016 in San Francisco. Is there anything exciting and new in separation science that we should be looking forward to in eager anticipation?

Wolfgang Lindner: I think we have to face it: high performance liquid chromatography (HPLC) in its current status is based on evolution rather than on revolution. There were always incremental improvements based on theoretical predictions but also on technological improvements in material science, engineering, software, physics, mass spectrometry (MS) technologies, etc. Also, we should not forget it takes time to appreciate a contribution as a revolution because only the field (the users) will decide whether or not it is a revolution. One can see this in very many areas. These developments are often driven by challenging applications whereby these have also changed over the years. It is quite clear that at the beginning most of the HPLC developments were driven by the analytics of small molecules and of the pharmaceutical industry: now we are shifting more and more towards large molecules and proteins (RNA, DNA). This presents challenges on several levels, including MS detection, which needs to be mastered. Although it has been around for some time, the “slip flow” phenomenon explored by Mary Wirth could offer new possibilities (1). Then we have the multidimensional LC×LC (LC×LC×LC) concepts, which continuously get better in order to substantially increase the overall peak capacity to deconvolute otherwise overlapping peaks. This, however, builds on selectivity criteria, which are in my view still one of the most important issues for the success of the HPLC technology as a whole subject. Without the mastering of selectivity issues, multidimensional LC will not become routine in the future. The engineering of the 2D-systems is already at such a high level to the point that the move is now to add further dimensions.

Last, but not least, the material science component remains an issue and there we will see new developments, but there is a longway to go. The bottom line is that there is no standstill - certain application areas will significantly advance HPLC over the coming years. These are related to life science analytics as to the various “omics” fields, biotechnology fields (proteins, antibodies, etc.), and diagnostic areas. Synthetic polymers will get tackled in order to characterize them better from a chemical point of view.

At this point it is worth noting that this year two advanced European Research Council (ERC) grants (ca. 2.5 million euros each) have been appointed to “chromatography”. One for the potential of three-dimensional chromatography (Peter Schoenmakers [2]) and one for new silica-based material using etching technologies (Gert Desmet [3]). This is a clear sign about the importance of HPLC now and in the future.

JL: Well, that’s the present and the future all neatly wrapped up (for now) on a positive note. Having said that, I can certainly support your provisos on selectivity issues. There is still a lot to do on this. Anyhow, let’s go back 60 years or so and have a think about how we got here. In the beginning there was packed column GC.

Ted Adlard: The mid-1950s saw the emergence of gas chromatography (GC) with packed columns but with capillary columns already on the agenda. There was a great deal of development work being carried out in industry (in particular petrochemical) working alongside academia. The 1960s saw major detector development with the introduction of flame ionization (FID) and electron capture (ECD) detectors, and glass capillaries started to take over from packed columns. The real game changer came with the development of the flexible fused silica columns in the late 1970s to early 1980s, which really brought robust GC technology to the masses.

Alan Handley: My career started in the GC area in the early 1970s and I had first-hand experience of the impact of some of the GC developments mentioned by Ted, but was also party to the emergence of a number of other separation technologies in my role supporting ICI’s growing polymer businesses.

Gel permeation chromatography (GPC), or size-exclusion chromatography (SEC) as it now seems to be called, required both instrumentation and column technology capable of being operated with “exotic solvents” and at high temperature - to facilitate the dissolution of the newer polymer types. This saw the major developments of both the “universal” refractive index detector and porous silica and cross linked polystyrene column packings.

Like all separation technologies, once established, we see the driver for quicker or better separations and more information-rich detection. From the 1980s onwards we saw the refinement of the column technologies - smaller diameter GPC packings, mixed bed and multipore columns (reducing the need for multiple individual pore-size column sets), the development of multidetector systems involving refractive index/viscometry and light scattering, and the associated challenges of handling and interpreting such data.

GPC has always been a very robust technique thanks in the main to the overengineering required to handle the solvents used in such systems and it is still the mainstay of the polymer industry. I did eventually succeed in building an instrument in the late 1980s to run in concentrated sulphuric acid for the characterization of some of the newer engineering polymers - this is what chromatography challenges are all about!

As ICI’s interests moved to polymer formulation I suddenly found myself swept up in the early days of HPLC, the progress of which is adequately summarized by some of the other contributors, but I still look back at my earlier experiences with a coil pump, syringe injector, and a UV detector - one of the first modular HPLC systems.

 I have seen techniques such as field-flow fractionation (FFF), supercritical fluid chromatography (SFC), capillary electrophoresis (CE), and hydrodynamic chromatography (HDC) come and go - some have seen a recent resurgence but others have stayed as niche techniques awaiting an industry need, a technology challenge (human genome), or for instrument manufacturers to reinvent them.

JL: Wolfgang, as I recollect, your early career involved using GC in investigations related to tobacco. Did your switch to being an LC researcher involve that technique offering you far more options in what you were doing or was it simply that you shifted to a different application area?

WL: Yes, I started with GC during my PhD studies at the Institute of Organic Chemistry at the University of Graz, Austria. However, shortly after I switched to the Institute of Pharmaceutical Chemistry (at the same university) - also doing GC. I very soon saw the limitations for real‑world GC applications so I became interested in HPLC, for theoretical but also practical reasons. In 1973 I bought the first HPLC machine from Hewlett-Packard (serial number 3), which was actually an instrument designed and built by Hupe and Busch. From thereon in, while I worked in GC and LC, LC became my addiction. (Later I did also capillary electrophoresis [CE] and capillary electrochromatography [CEC].) My passion has always been the application and the understanding of “selectivity in LC” and very early on I saw the potential and need, particularly when I worked for Sandoz (now Novartis) in Basel in 1973 and 1974 on a sabbatical for six months.

JL: I too was old enough to have started out working with GC. Such was the passion of practitioners of GC for their instrumentation, and one in particular that was set up in the early 1990s by The Chromatographic Society: The Pye 104 Club. It wasn’t like that for me. I used a PerkinElmer F11 and had no particular liking for it. I often had niggling thoughts around the consequences of the hydrogen or air flame on the FID not lighting or the hydrogen flame sucking back. Neither of these needs have concerned me, what was more important was that it gave me low, highly variable results when analyzing the black tars produced when following a patented industrial process. It took the advent of HPLC and a summer spent at ICI, Blackley in Manchester to sort all of this out. Using a system very similar to the one described by Alan, I was soon able to identify and quantify all the major components in every black tar I produced. My beloved 100 × 4.6 mm i.d. Corasil (40 µm pellicular) column with septum injector followed me around everywhere thereafter until 1986 when an over-zealous laboratory tidy-up was held at Beecham Pharmaceuticals while I was away attending HPLC in San Francisco!

Two very important and obvious high impact developments in the early days of HPLC were the introduction of 5-µm totally porous spherical silica particles and then of reversed‑phase HPLC using alkyl-bonded silicas, though somehow they both rather passed me by. When I moved to Edinburgh to work for Dick Wall, an important but less well known senior member of the Edinburgh chromatography team, I found that the use of 5-µm totally porous spherical silicas was already commonplace there. Then, by the time I had completed my postdocs involving the use of chiral mobile phase additives with silica and using silica to prepare immobilized chiral stationary phases (CSP) to move to the pharmaceutical industry, I found that reversed-phase HPLC was already the workhorse of the pharmaceutical analytical laboratory. (Yes - chiral LC really was difficult at that time but at least one of my chiral stationary phases [CSPs] proved to be more successful as a chiral catalyst [4]). For me, during these years as a postdoc, the most striking development was the introduction of the dual piston reciprocating pump. It was my rite of passage as a chromatographer to break my first sapphire piston rod on a spanking new instrument. I successfully installed the replacement and never made the same mistake again on the Waters pump or on the LDC Constametric III pumps I later used in industry.

JL: Moving on from the first decade or so of HPLC, the high impact of the commercial availability of successful broad spectrum chiral LC columns is undeniable. In the pharmaceutical industry it transformed the way in which chiral drugs were developed. For me, the big breakthrough was the introduction of Hermansson’s α1-acid glycoprotein CSP in 1985, commercialized by LKB as Enantiopac. At Beecham, we went from being able to separate one racemic synthetic intermediate into its enantiomers using the Pirkle Type-1A CSP to being able to achieve chiral separations for every racemic drug development candidate. Would you agree, Wolfgang, that Enantiopac was THE major breakthrough in chiral LC?

WL: As you know, in 1978 I went as a postdoc to Barry Karger and there I introduced them to chiral ligand exchange chromatography (CLCE) via the design and synthesis of a chiral ion pairing (chelating) compound being added to the mobile phase (of a reversed phase system). It was a lipophilic chiral tri-amine and not an amino acid derivative as developed by Vadim Davankov. (He patented his approach in 1968 in Russia and developed the first fully synthetic chiral stationary phase). The year 1978 was the start of my “chiral” career.

To be frank, it is very difficult to trace to one individual the success story of enantioselective chromatography (see Vadim [5]). Very early on in the game there were also S. Allenmark, G. Blaschke, S. Hara, Y. Okamoto, W. Pirkle, and so on. You are right, the AGP column of J. Hermansson became very popular quickly in the pharmaceutical area because it showed a remarkable broad spectrum of selectivity for many drugs. Many important decisions within the pharmaceutical industry may have been based on this analytical technology. From a theoretical point of view one could not extract too much of an insight on molecular recognition. It became the area of other scientists who contributed very important papers to the advancement of “chiral” chromatography. Now the protein‑based CSPs are more or less outdated - this is the way it goes.

Preparative enantioselective chromatography became extremely important in modern drug development and this was the clear limitation of the protein-type CSPs. The bottom line was that the Enantiopac column may have been for a short time THE first choice chiral column to have in the pharmaceutical industry, but it was certainly not the first chiral column on the market and described in the literature. In a broader sense and from a chemical point of view, it very soon became clear that the polysaccharide-based CSPs would win the game. However, even today, there remain niches to be tackled and there is still room for further developments.

JL: Of course it hasn’t all been about LC and GC. From time to time other techniques have risen to prominence. Overall though, would anyone subscribe to the notion that CE, CEC, and SFC have been under-achievers rather than high impact developments?

WL: The CE and CEC methods to resolve small molecules went through a hype but slowed down significantly. However, for large(er) molecules, CE techniques clearly have true advantages over LC techniques, particularly since the hyphenation with MS/MS has been elegantly solved.

So, CE and CEC have found their currently unbeatable niche. The robustness of these techniques in the course of small molecule analysis is not super strong, so it was not surprising that people shied away from these methodologies in routine laboratories.

SFC became first choice (at least judged from a large pharma company’s perspective) in preparative chromatography for medium-polar and non-polar compounds. Analytically, however, it is still not fully accepted, particularly in routine laboratories. The merits are not so convincing that people would switch from (U)HPLC to SFC. For lipidomics applications, however, SFC seems to have become the first choice of separation technique (speed and efficiency possibilities are the drivers).

JL: Chris, elsewhere in this publication you have championed the impact of the much more sophisticated data handling we now have at our disposal. However, as an awardee for novelty in capillary techniques, would you also like to say a word on behalf of CE, CEC, or SFC?

Chris Bevan: The separation of natural and unnatural oligonucleotides was routinely only achievable by micellar electrokinetic chromatography (MEKC) at the time. It was considered one of the most significant challenges to analytical chemistry ever perhaps: certainly a really tricky problem neatly solved by electrophoresis (6). Micellar electrokinetic chromatography was used to resolve diastereomers of oligonucleotides possessing several chiral unnatural phosphoramidate bridges. These materials were not resolved by conventional liquid chromatographic techniques.

Technological advances using novel nano-switching and flow management have been applied to solve capillary switching in instrumentation developed to decode the human genome (7) -arguably the biggest challenge ever to analytical chemistry! A multiplexed freeze-thaw switching principle (8) and a distribution network were used to manage flow and sample transportation.

These three techniques and their derivatives have made a particular impact on chiral resolution and on the separation of biomolecules and structurally complex molecules. Notable examples from my own experience at Glaxo were separating some rather unusual natural product‑derived compounds isolated from fermentation broths. These squalestatin compounds had complex and extraordinary chirality (eight chiral centres) and isomeric structures and were not fully separable by HPLC... at least not in our hands at the time (mid-1990s). Micellar CZE (MEKC) separated them very nicely, but of course we could not scale up this miniaturized technique so were unable to prep them out in meaningful quantities. This spurred us on to research and develop CZE–MS hyphenation to allow structural elucidation of these and other molecules that had been successfully separated by electrophoresis. In some specific cases we were able to separate preparatively using ion exchange HPLC on an ionPac column because the separation principles parallel some of the mechanisms of CE.

I would argue that the most‑significant-to-man application of CE is in the separation of proteins and DNA and RNA to elucidate the structure of the human genome.

The impact of SFC and the desire to develop and apply it comes from the ease at which the volatile fluids can be depressurized and evaporated and the high plate efficiency with which separated products can be isolated very rapidly.

Supercritical fluid extraction has been applied industrially for many years to decaffeinate tea and coffee, and it does this very well. Many would argue that SFC should have predated HPLC, but this ignores the difficulties of controlling pressures in systems and the poor solubility of many compounds in supercritical carbon dioxide, thus limiting preparative applications. It may be argued that CO2 is a “green” solvent but one should examine the whole procedure for “ecological greenness”.

JL: Chris, to turn to you again, you are also very well known in the UK for your series of prep LC meetings. Are there any truly groundbreaking developments in that area that we should be noting?

CB: Our series of BIG PREP symposia was founded initially on a desire to mix the analyst’s know-how with those of the synthetic chemist’s and allow a forum for cross exchange of ideas, technologies, and working practices.

Many of my synthetic chemistry colleagues at Hoechst
Pharmaceutical laboratories thought that it was somehow not the right way to use HPLC to isolate compounds of interest and felt that they had failed in their ability to synthesize specifically to produce pure desired products in high yields.

We were working on isomeric cis/trans penem antibiotics at the time (early-1980s) and these compounds were very difficult to synthesize pure in the desired isomeric form. I had been measuring these isomers analytically by HPLC and suggested that we could scale-up HPLC to isolate both isomers in a pure form. We could then test them for activity or toxicity and progress or reject them more quickly. Pressure from the parent company in Germany (Hoechst AG) was mounting to get pure isomers for testing so I built a large-scale HPLC system from scratch and purified both cis and trans penem isomers using a kilogram of 10-µm Spherisorb ODS in a 4-inch diameter self-built axial compression prep HPLC system. This saved a lot of time and went on to become common practice. The notion of fail fast and fail cheap changed the way in which our chemists thought, and led to high throughput screening and combinatorial chemistry making impure mixtures for activity testing.

It is curious to look back and realize that chemists were routinely using gravity column low resolution chromatography and thin-layer chromatography (TLC) to help purify their products, but they thought that HPLC was a sledgehammer to crack a nut and such sophisticated tools were the province of the analytical laboratories.

WL: Final point from me: I think the separation techniques have strongly matured over the years and so have the chromatographers. We have learnt to use the most appropriate technique for a given analytical question. There is no game anymore over which technique is the better one. I always advocated that a good analytical chemist has to know to play the piano (keyboard) of methodologies and has to know the best key to meet the right tone. This is what we need to teach. It is based on a chemical understanding of the molecules to be analyzed and the matrix it is dissolved in together with a fundamental understanding of separation sciences, including MS.

JL: You are certainly right there. One of the key (sic) agenda items for now and for the future of chromatography is to ensure that we have the trained chromatographers to do and further develop the chromatography. However, to round off the conversations I would like to return to the actual chromatography. What will we be working on and what chromatographic techniques will we be using? Indeed, to use Wolfgang’s metaphor, what tones will we be trying to find and what keys will we be using? I know that we have skipped over the past decade, and having celebrated 50 years it would seem logical to emphasize the past decade when celebrating 60 years, but the whole UHPLC vs. fused core–shell issue has been played out at ChromSoc meetings and in ChromSoc-related media for a long time now. It seems to be politically correct these days to say that they both have their merits, that they will continue to come together, and that eventually it will be all about even smaller but totally solid particles and slip flow. For me though, Waters deserve credit for their Acquity system, which, at the very least, provided the catalyst stimulus for an exciting period of growth in LC instrumentation and particle technology.

AJH: I agree with many of the comments and whilst we do now have more robust technologies available, there are still challenges out there, particularly in the area of large biological molecules
and complex matrices. This will require “breakthrough technologies” and will need the further development of both separation and detection technologies if we are going to break “the million compound separation” and provide measurement solutions “at source”.

Capillary technologies have brought much to modern society, increasing scientific understanding and fuelling product understanding and development.

A number of instrument companies are worth a mention: Hewlett Packard (now Agilent) successfully brought the chromatography community robust and fully validated GC and HPLC instrumentation when they were needed; Shandon (now Thermo) and Phase Separations (now Waters) brought us high efficiency packings; and Waters, who have brought much to the table, gave us a “paradigm shift” with the launch of UPLC, which I feel reignited many people’s interest again in chromatography.

The UK contributed much to the early development of chromatography: we had major companies who themselves pushed the boundaries of research and had chromatographers who understood. Sadly, many of these companies no longer exist, and with UK academia not specifically focusing on this subject area, I do have serious concerns as to where we will produce the next generation of chromatographers needed to take up future challenges.

However, as mentioned in Wolfgang’s opening paragraph, there is still new monies coming into chromatography, albeit in Europe, but it does demonstrate that chromatography is still, after 60 years, worthy of future investment and interest.

JL: Thanks, and thanks everyone for your thoughts. I’d say let’s reconvene in 10 years’ time to reconsider our thoughts, but that would definitely be tempting providence.

References

1. B.A. Rogers, Z. Wu, B. Wei, X. Zhang, X. Cao, O. Alabi, and M.J. Wirth, Anal. Chem.87(5), 2520–2526 (2015).
2. http://www.ti-coast.com/news-events/latest-news/385-coast-congratulates-prof-peter-schoenmakers-with-his-erc-advanced-grant.html
3. http://we.vub.ac.be/en/prof-gert-desmet-wins-%E2%82%AC25m-grant-map-body%E2%80%99s-molecules
4. S.A. Matlin, W.J. Lough, L. Chan, D.M.H. Abram, and Z. Zhou, Chem. Soc. Chem. Commun. 1038–1040 (1984).
5. V.A. Davankov, in Advances in Chromatography, J.C. Giddings, E. Grushka, J. Cazes, and P.R. Brown, Eds. (Marcel Dekker, New York, USA, 1984) Chapter 17.
6. C.D. Bevan, I.M. Mutton, and A.J. Pipe, Journal of Chromatography A636(1), 13–123 (1993).
7. H. Tan and E.S. Yeung, Anal. Chem.70(19), 4044–4053 (1998).
8. C.D. Bevan and I.M. Mutton, Journal of Chromatography A697(1–2), 541–548 (1995).
9. See also: C.H. Arnaud, Chemical & Engineering News94(24), 28–33 (2016).

John Lough is a Reader in Pharmaceutical Analysis in the Sunderland Pharmacy School at the University of Sunderland, in Sunderland, UK, and a member of the Executive Committee of The Chromatographic Society.

Wolfgang Lindner is an Emeritus Professor at the University of Vienna, Austria, and a Chromatographic Society Martin and Jubilee Medal winner.

Ted Adlard is a former GC pioneer at Shell, a founding member of The Chromatographic Society, and Chairman 1970–1973 and 1979–1983. Alan Handley is a Senior Scientist at ICI and LGC, past President of the RSC Analytical Division (2011–2014), and The Chromatographic Society (2009–2014).

Chris Bevan is a Big Pharma chromatographer, Chromatographic Society President 2001–2007, and currently the Chromatographic Society’s Event Organizer.