The 2019 Lifetime Achievement in Chromatography Award: Professor Milos V. Novotny on His Chromatographic Journey

Milos V. Novotny, the director of the Institute for Pheromone Research and the Director of the Novotny Glycoscience Laboratory at Indiana University, is the winner of the 2019 LCGC Lifetime Achievement in Chromatography Award. In this interview, he talks about how his biggest scientific developments came about, the joys of mentoring exceptional students, and his views of the future of separation science.

Milos Novotny's contributions to the field of chromatography span a wide range of activities. He was a pioneer in virtually all capillary separation techniques, starting with significant surface treatment technologies in the preparation of glass capillary columns for gas chromatography (GC) and the first credible combination of capillary GC with mass spectrometry (MS) in the late 1960s. He then continued with his conceptually important work in capillary liquid chromatography (CLC) and supercritical fluid chromatography (SFC) in the late 1970s. This was followed by seminal contributions to capillary electrophoresis (CE) and capillary electrochromatography (CEC) of peptides and carbohydrates about ten years later. He is known as a major contributor to important methodologies, based on liquid chromatography (LC), MS, and CE, in the areas of glycomics and glycoproteomics. He also made a unique contribution to separation science and society at large by designing two chromatographic columns that were an important component of miniaturized GC–MS equipment that was landed on the surface of Mars in July 1976 by the U.S. National Aeronautics and Space Administration (NASA) in the Viking Mission. Novotny was named a foreign member of two scientific academies: the Royal Society for Sciences, Sweden (1999) and the Learned Society/Academy of Czech Republic (2005). He has received more than 40 national and international awards, medals and distinctions for his work. He recently spoke to LCGC North America regarding his research, and his history in advancing chromatography and related fields.

Looking back, some of your early research in surface wettability problems in preparation of highly efficient glass gas chromatography (GC) capillaries was groundbreaking (1,2). The surface treatment techniques you reported were said to stimulate the field, which was revolutionary by making available high separating performance for biological mixtures. Some of this work led to pioneering research in early demonstration of capillary GC–MS and its applications to complex mixtures. What prompted you to investigate these problems? What was your most surprising discovery from this work?

My "love affair" with chromatography started during my undergraduate research in 1961. While the used methodology (mostly paper chromatography) was clearly primitive by today's standards, I could somehow relate to the impact that A.J.P. Martin and his colleagues made in separating and detecting the mixtures of amino acids at microscale; no wonder they received a Nobel Prize for their classy contributions to science. But it was not love at first sight. I was trying to separate components of an alkaloid mixture, and the paper chromatography was awfully slow, and the solvent systems somehow inefficient. My frustrations quickly ended when I was told about a new technique called thin-layer chromatography (TLC).

The few papers on the subject were exclusively in German, the language which I had long resisted learning during the post-war years, but TLC was, to me, chemistry at its best. I was able to play with different adsorbents, quickly optimize the solvent systems, and even come up with my own ways to visualize and detect the separated TLC spots. I became thus a sort of the local TLC expert as a second-year university student who was ready to solve everyone's problems through chromatography. It even helped me to get a paid summer job in a local research institute!

After I chose the field of biochemistry for my graduate studies, I used some established techniques of chromatography as well to purify proteins, but, most importantly, I could start appreciating the real structural complexity of biological systems. This perspective remains with me to this day, and it served me well through my entire research career. In short, I was even then an analytical chemist at heart, but my biochemistry background was important as a long-term incentive to develop better ways to separate and identify important biological compounds.

My real chance to join the mainstream of separation science came through accepting eagerly an offer of a junior scientific staff position in the Institute of Analytical Chemistry of the Czechoslovak Academy of Sciences, directed by Dr. Jaroslav Janak, one of the pioneers of GC. The institute was a small and congenial place to fully immerse myself in the fundamental aspects of chromatography, even though some of my colleagues might have been suspicious as to what a biochemist was going to do there. In retrospect, I learned a great deal from their physico-chemical insights into chromatography, but eventually managed to carve my own territory in capillary GC. I was particularly fascinated by the publications of Denis Desty from the United Kingdom, who was the first to describe glass capillaries and achieve most impressive separations, albeit only with hydrocarbons; after all, he worked for the British Petroleum Company.

Together with my colleague, Dr. Karel Tesarik, and based on Desty's description of the glass drawing machine, we constructed our own version in Brno, with some occasional sacrifice of our fingers' skin (a hot glass tube looks about the same as a cold one!). However, we were disappointed that coating a glass surface with a stationary phase was very difficult. It quickly turned my attention to the need for chemical modification of a glass surface. It turned out to be rather frustrating, trial-and-error work; without the availability of today's surface measurement techniques (not even electron scanning microscopy was available at that time); we worked empirically and in darkness trying various surface treatment procedures. For example, we were probably the first to demonstrate that high-temperature gas-phase silylation stabilized coated nonpolar stationary phases, but this treatment was clearly negative for polar coatings. During 1967, we applied some gas-phase surface corrosive procedures, and some of these provided the needed breakthrough. I still cherish the day when I saw my first chromatogram with 4000 theoretical plates per meter efficiency on a column coated with a moderately polar stationary phase.

During the fateful year of 1968, I found myself stuck in Sweden when the Soviet Union's army invaded my homeland (in August of that year). I was in Sweden to learn the basics of GC–MS, by then practiced exclusively with packed GC columns. Given that being an immigrant isn't exactly fun anywhere, I totally immersed myself in science. And there was a lot to learn. Karolinska Institute in Stockholm was world-renowned for their biomedical research, and their close connection to a major producer of GC–MS instruments (LKB-Produkter) gave me great opportunities for research. There was much excitement there about GC–MS of derivatized biological compounds, and I had a chance to meet some of the leading authorities from the USA and Western Europe. To my great surprise, some of these people already knew about our work on glass capillary columns done in Czechoslovakia. Some of these scientists seemed to endorse my ideas and the first attempts of connecting glass capillaries to the LKB 9000 instrument. The first glass drawing machine was meanwhile commercialized in Germany, and a laboratory in Stockholm bought one. I was allowed to use it occasionally. One day, during 1968, an interesting opportunity came from a totally unexpected direction: a French Government laboratory was ready to buy an LKB instrument, if it could prove its performance through resolving a complex essential oil mixture. The salesperson in France thought it entirely feasible, but the folks in Stockholm were horrified; their instrument didn't even have a splitter injection on it, which was necessary those days for capillary GC of neat samples. With the help of a machine shop, I quickly put together a simple splitter and the necessary flanges to make connections. To the later displeasure of my boss, I ripped open, during the weekend, the highly "treasured and secret" Becker-Ryhage GC–MS interface, and interchanged the dimensions of its jets to suit my experiments. Meanwhile, a loaned stainless-steel commercial capillary arrived from France, together with a chromatogram detected by the flame ionization detector. Could we match it? It turned out that I got a much prettier chromatogram than theirs, although I could not immediately understand why. The explanation came to me a week or so later, when I carefully measured the van Deemter curves with standard solutes, which differed in their curve minima due to a different column average pressure under different detection techniques. I promptly published these measurements and their theoretical justification in Chromatographia, then a new journal for rapid communication. Due to the proprietary nature of the "French sample," the chromatogram could not be included. Nevertheless, I was happy with these results, and at least a part of the scientific community noticed.

Another demonstration of the power of capillary GC–MS had to wait for a few more months, when we applied a 100-m long capillary (with over 500,000 theoretical plates) to separate the components of tobacco smoke. This was done together with Dr. Keith Bartle (an English scientist on a temporary assignment at the University of Stockholm); Keith has become a life-long friend and collaborator of both myself and my first Indiana University graduate student, Milton Lee.

In an extension of your early work, you applied glass capillary columns to the separation of complex steroid mixtures, which became one of the milestones in the development of chromatography (3,4). The original work stimulated a great deal of interest in high-resolution separations. The importance of this column work became internationally known very quickly. How was your research approach different from that of your contemporaries at that time?

With the political situation in Czechoslovakia becoming worse all the time, I had to make a major decision where I wanted to live permanently. Although I maintain fondness for Sweden and some friends I met there to this day, I thought an English-speaking country would be professionally preferable at the time. Why not America? This brought me from a cool summer in Sweden to the sweltering heat of Houston, Texas, in August of 1969. The following two years as a postdoc of Albert Zlatkis were an extremely important transition in my entire career. "Dr. Z.," as his associates affectionately called him, was definitely the most friendly and helpful supervisor that I ever had. His laboratories were full of interesting gadgets, and he wanted me to work on a wide range of projects.

When I mentioned to him my interest in separating steroids on glass capillary columns, it was enthusiastically received, but then he said, "This could be a good subject for your presentation at our next international symposium in Miami Beach the next May." I was first in a state of shock; we were already in the month of September, so how much time did we really have? But my answer was "Yes, sir." My European roots and experiences told me that you just cannot say no to your boss! Within reasonable limits, I had everything I needed for this complex task, and I was also familiar with the literature on various derivatization techniques used in making polar molecules sufficiently volatile for GC. I worked long hours, and we also worked on Saturdays. Dr. Zlatkis often ordered pizza for us on Saturdays, and sat with us discussing our research. When the Advances in Chromatography conference was approaching, we had many interesting results on the steroid project. Although I was extremely nervous about my first presentation at the top international conference, my talk went well. I was publicly congratulated by the session chair, Art Karmen, and received some nice comments from other conference participants. Clearly, capillary GC was on its way to replace packed columns in the gas-phase separations of many classes of biological compounds. From various pieces of correspondence to Al Zlatkis, and even to myself, we learned that glass capillaries and steroid separations were among the hot subjects in a number of European laboratories. During the fall of 1970, there was going to be a major chromatography conference in Dublin. Although we didn't have a budget to cover my travel expenses, I decided to go on my own. Al Zlatkis kindly gave me enough time to participate, and even arranged for me visits to some European laboratories. In a "marathon trip," I went through Paris and Zurich, then to Germany, the Netherlands, and Belgium prior to the meeting. The interest in glass capillaries and steroid separations was high almost anywhere I went on that trip. In the United States, the acceptance of these techniques was a bit slower, but these things caught up within a few years.

I owe much gratitude to Al Zlatkis for many things, but perhaps most for his insistence that I had to apply for an academic job. Although I had thought more at that time about getting a job with an instrument company, I applied to five academic institutions, and subsequently received invitations to visit three state-supported universities. The interviews opened my eyes on academia, and I never regretted my decision to accept.

At Indiana University, you developed a highly respected research program, and mentored future leaders of the field, including Jim Jorgenson and Milton Lee, among others. What was personally the most satisfying aspect of developing your own research program with such young and talented students?

Developing a research program at Indiana University and working with many incredibly talented students represent the most satisfying aspects of my overall career. Over the years, I have been blessed by interacting with some of the brightest and most serious and resourceful associates that I could imagine. The names of the former students you mention, Jim Jorgenson and Milton Lee, come first to people's minds for (a) being prominent leaders in our field; and (b) producing their own "scientific progeny." The brightest of their students will continue the academic heritage that is so urgently needed in today's science, in general, and the separation science in particular. Milton Lee was my first graduate student, starting in 1971, with Jim joining us in 1974, not to forget mentioning Vicki McGuffin, who became a faculty member at Michigan State University later, and the two bright and diligent Czech students, Jan Sudor and Lukas Zidek, who are now on the faculties of two major European Union universities. They all enhance the reputation of our research group in their own different ways.

During my first decade at IU, I tried to work in the laboratory alongside my graduate students on my own projects (such as the NASA-sponsored design of the chromatographic column for the 1975 Viking Mars Lander, or a development of new stationary phases for HPLC), but, as the research group grew larger and the equipment more complicated, I assumed more a function of just supervising their activities. I always assumed that the "contract" between a graduate student and his or her faculty supervisor is mutually a serious business. Over the years, I have learned that different students need a personalized approach pertaining to how we proceed with their scientific training, but one criterion is absolute: They must be serious and dedicated to the science we pursue.

I would be amiss not to mention the enormous contributions by a large number of postdoctoral and visiting scientists who, over the years, helped me greatly in assisting with the graduate students' research besides pursuing their own research lines. Among others, Susan Olesik (now at Ohio State University), Takao Tsuda (Nagoya, Japan), Yukio Hirata (Toyohashi, Japan), Keith Bartle (United Kingdom) and Milan Madera (Czech Republic) were particularly helpful.

Over the years, there were many outstanding graduate students who went on to get jobs in industry and in national labs. I have followed with great interest as some individuals became leaders in the instrument industry, pharmaceutical research, or biotechnology. I try to keep in touch with some of these individuals, but it is often hard due to the large numbers and diverse careers. I am always happy to see my former colleagues and friends at the international symposia such as HPLC and the Riva del Garda capillary chromatography conferences.

You and your students were some of the first to apply serious chemometrics to chromatographic problems (5,6). What were some of the greatest insights or advances that chemometric techniques allowed you to discover?

Yes, it is true that we were in this game quite early. The lion's share of credit goes to two amazingly gifted graduate students, Mike McConnell and Jerry Rhodes. I had very limited knowledge of computational techniques, but we had a great need to deal on a statistical basis with highly complex "metabolic profiles" (today conveniently renamed metabolomics). Mike was a Purdue University graduate with a substantial knowledge of computational techniques and capabilities. He first built a fully automated high-precision capillary GC system synchronizing all important instrumental functions. Mike and Jerry then proceeded with the application of non-parametric pattern recognition in the analysis of urinary volatile profiles from diabetic patients. The metabolic patterns at 95% correct classification were subjected to a feature-extraction algorithm for selection of metabolically significant components for the subsequent GC–MS identification. While these procedures may seem trivial through the eyes of today's computational technologies in the "omics era," ours were developed during the 1970s! Both Mike and Jerry went on with successful careers in industry; both now happen to live in the [San Francisco] Bay Area.

You have completed pioneering work in the areas of capillary LC and SFC. In 1978, two key papers were published (7,8), which are considered by many to be the beginning of the field of capillary LC. What intellectual steps led to the creation of this idea?

This goes back to what I might call my "post-tenure blues." The naive perception is that, once you receive your tenure at a major university, you can do any damn thing you want to do without being fired. I have seen some people in academia slow down their research activities and stop looking ahead in their fields after a positive tenure decision. This was certainly not my case. I wanted to explore the less travelled roads, in addition to where we were already strong (such as metabolic profiling and identification of the first mammalian pheromones) through the rapidly maturing technique of GC–MS. If one needed to deal with increasingly more complex polar and nonvolatile mixtures, it would require some high-resolution condensed-phase techniques. For much of my career in separations, I drew great inspiration from the theoretical insights of J. Calvin Giddings (University of Utah). Already as a young scientist in Czechoslovakia, I had almost religiously studied his 1965 book Dynamics of Chromatography and related papers. There it was: To counter the much slower solute diffusion in the liquids, you have to resort to very small, preferably spherical, particles, and small diameter columns, which eventually necessitate the use of unusually high column inlet pressures.

When Dr. Takao Tsuda, a postdoc from Japan, joined my group in 1976, we made first steps to tackle the instrumental problems of capillary LC. Our first capillary columns were drawn from thick-walled glass tubes, some empty, some filled with adsorbents. Even though our equipment was relatively primitive at that time, the results were quite encouraging. It took another decade to have capillary LC demonstrating its real strengths. Karl-Erik Karlsson, a postdoctoral associate from Sweden, was primarily responsible for this breakthrough study (see Anal. Chem. 60:1662–1665 [1988]). For more details on the development of capillary LC, see also my recent review in J. Chromatogr. A 1523: 3–16 (2017).

The development of capillary SFC is quite a different story, but it also has its connections to the observations made by Giddings and coworkers on the solvating properties of dense gasses near the critical point during the 1960s. Milton Lee, my former graduate student, and later a faculty member at Brigham Young University, spent two highly productive summers at IU when I introduced him to my thoughts on "rejuvenating" Giddings' dense gas ideas in terms of much improved capillary column technologies. Milton's and my students collaborated, and it worked; the rest is history. Capillary SFC enriched our research programs, attracted other groups into the field, and was successfully commercialized, although the further developments in SFC later took some unexpected turns.

For a while, we were faced in both capillary LC and SFC with some undue criticism from some of the then "chromatography establishment," such as, "Why bother with these tricky small columns when HPLC works so well?" This opposition for a while influenced the granting agencies, which did not buy in either. All is now totally different with the current revolution in the "omics technologies" where capillary LC–MS is one of the most essential tools. It only proves that sometimes one must withstand conservative criticisms, and do what you believe in.

What can you share with our readers regarding what you would consider to be the most important new areas of research in chromatography? And what do you see as your greatest contribution to the field?

I feel fortunate to have long witnessed the enormous development of the field of chromatography, with respect to its resolving power, and sensitivity, and selectivity of its detection techniques. Not only has it become a highly respected part of analytical chemistry and beyond, but its proliferation in other fields such as systems biology, environmental analysis, and industrial uses are truly amazing. In the development of its techniques and conceptually important applications to their current state, many dedicated scientists actively participated, and I am proud to be one of them. Most importantly, I value the seminal contributions of my best former students to virtually all capillary techniques of chromatography and electrophoresis. Then, there are still some "unsung heroes" in the industrial arena. My former students never had problems with getting good jobs and prospering, and this means a lot to me as well.

With the sophisticated tools now at hand, are we on a "comfortable plateau," expecting only incremental developments? I don't think so. As long as we don't achieve a nearly complete understanding of how biological systems work, and so long as chemists continue to synthesize and isolate novel molecules, there will be further needs for methodological advances. For example, in proteomics and glycoscience, my main areas of interest during the last two decades, I still see great needs to deal with complex native protein separations and dealing with molecular aggregates and nano-sized biological entities such as exosomes.

Closer to what we as separation scientists have been discussing at the recent symposia, multidimensional separations will undoubtedly remain an important field. The column designs may undergo substantial improvements due to the upcoming advances in microfabrication, 3-D printing, and other related technologies; there are already some promising signs in that direction. In terms of selective molecular interactions, chiral separations still remain important. I also don't believe that the ultrahigh-pressure capillary LC work, initiated by Jim Jorgenson already two decades ago, has reached its potential as yet. These are just a few directions, and I am sure my chromatography colleagues and friends could add substantially to the list.

What do I see as my greatest contribution? Perhaps in helping to bridge the gap between what was the theoretical potential of LC and its practice as far as the separation efficiency and feasibility of LC–MS are concerned. Many others made substantial contributions as well toward demonstrating what's feasible today, but after our first publications on both capillary LC and SFC, I was often seen during the first years as the one poking the "hornet's nest." It took a while to get us all there, but it has been exciting and I don't regret my resilience.

What words of advice would you share with us for young researchers just getting started, or even undergraduates considering a future career in science?

Science education is a really complex issue. I can tell you that when I came in 1969 to the United States, our society's respect for science was far greater than it is today. I believe the quality of education, in general, and science education, in particular, has recently suffered, due to lack of funding at all levels.

It served me well to start undergraduate research already toward the end of my first year, but it was a different educational system and different set of circumstances. As a faculty at IU, I had some very positive interactions with bright undergraduates; some ended up being coauthors of our publications, and a few went on to graduate school elsewhere. However, most undergraduates want some research experience only in their senior year, which is usually a bit late to accomplish anything meaningful.

As for advice to a beginning faculty member in our field: Since you know that your "tenure clock" starts ticking right away, you need to strategize concerning your long-term and short-term research goals. We are still a part of a wide and flourishing field, so this will give you plenty of opportunities to develop ideas that are sufficiently different from those of your former research advisor. I tend to say, "Don't put all your chips on the same place," but this may not be universally applicable. Due to increasing role of chromatography in other scientific fields, you may be frequently asked to collaborate with others. Don't refuse scientifically sound collaborations, but try to define your role and scientific credit in a joint project; you may be one out of ten investigators listed on a future publication! However, I remain very optimistic that there is a bright future in separation science, so that the serious scientists will find their field as rewarding as I have.

References

(1) M. Novotný and K. Tesařík, Chromatographia 1(7), 332–333 (1968).

(2) K. Tesařik and M. Novotný, Chromatographia 2(9), 384-385 (1969).

(3) M. Novotný and A. Zlatkis, J. Chromatogr. Sci. 8(6), 346–350 (1970).

(4) M. Novotný and A. Zlatkis, Chromatogr. Rev. 14(1), 1–44 (1971).

(5) M. Novotný, M.L. McConnell, and M.L. Lee, J. Agric. Food Chem. 22(5), 765–770 (1974).

(6) M.L. McConnell, G. Rhodes, U. Watson, and M. Novotný, J. Chromatogr. B: Biomed. l Sci. Appl. 162(4), 495–506 (1979).

(7) T. Tsuda and M. Novotný, Anal. Chem. 50(2), 271–275 (1978).

(8) T. Tsuda and M. Novotný, Anal. Chem. 50(4), 632–634 (1978).

ABOUT THE AWARDEE

Milos V. Novotny, the director of the Institute for Pheromone Research and the Director of the Novotny Glycoscience Laboratory at Indiana University, is the winner of the 2019 LCGC Lifetime Achievement in Chromatography Award. In this interview, he talks about how his biggest scientific developments came about, the joys of mentoring exceptional students, and his views of the future of separation science.

Milos Novotny's contributions to the field of chromatography span a wide range of activities. He was a pioneer in virtually all capillary separation techniques, starting with significant surface treatment technologies in the preparation of glass capillary columns for gas chromatography (GC) and the first credible combination of capillary GC with mass spectrometry (MS) in the late 1960s. He then continued with his conceptually important work in capillary liquid chromatography (CLC) and supercritical fluid chromatography (SFC) in the late 1970s. This was followed by seminal contributions to capillary electrophoresis (CE) and capillary electrochromatography (CEC) of peptides and carbohydrates about ten years later. He is known as a major contributor to important methodologies, based on liquid chromatography (LC), MS, and CE, in the areas of glycomics and glycoproteomics. He also made a unique contribution to separation science and society at large by designing two chromatographic columns that were an important component of miniaturized GC–MS equipment that was landed on the surface of Mars in July 1976 by the U.S. National Aeronautics and Space Administration (NASA) in the Viking Mission. Novotny was named a foreign member of two scientific academies: the Royal Society for Sciences, Sweden (1999) and the Learned Society/Academy of Czech Republic (2005). He has received more than 40 national and international awards, medals and distinctions for his work. He recently spoke to LCGC North America regarding his research, and his history in advancing chromatography and related fields.

Looking back, some of your early research in surface wettability problems in preparation of highly efficient glass gas chromatography (GC) capillaries was groundbreaking (1,2). The surface treatment techniques you reported were said to stimulate the field, which was revolutionary by making available high separating performance for biological mixtures. Some of this work led to pioneering research in early demonstration of capillary GC–MS and its applications to complex mixtures. What prompted you to investigate these problems? What was your most surprising discovery from this work?

My "love affair" with chromatography started during my undergraduate research in 1961. While the used methodology (mostly paper chromatography) was clearly primitive by today's standards, I could somehow relate to the impact that A.J.P. Martin and his colleagues made in separating and detecting the mixtures of amino acids at microscale; no wonder they received a Nobel Prize for their classy contributions to science. But it was not love at first sight. I was trying to separate components of an alkaloid mixture, and the paper chromatography was awfully slow, and the solvent systems somehow inefficient. My frustrations quickly ended when I was told about a new technique called thin-layer chromatography (TLC).

The few papers on the subject were exclusively in German, the language which I had long resisted learning during the post-war years, but TLC was, to me, chemistry at its best. I was able to play with different adsorbents, quickly optimize the solvent systems, and even come up with my own ways to visualize and detect the separated TLC spots. I became thus a sort of the local TLC expert as a second-year university student who was ready to solve everyone's problems through chromatography. It even helped me to get a paid summer job in a local research institute!

After I chose the field of biochemistry for my graduate studies, I used some established techniques of chromatography as well to purify proteins, but, most importantly, I could start appreciating the real structural complexity of biological systems. This perspective remains with me to this day, and it served me well through my entire research career. In short, I was even then an analytical chemist at heart, but my biochemistry background was important as a long-term incentive to develop better ways to separate and identify important biological compounds.

My real chance to join the mainstream of separation science came through accepting eagerly an offer of a junior scientific staff position in the Institute of Analytical Chemistry of the Czechoslovak Academy of Sciences, directed by Dr. Jaroslav Janak, one of the pioneers of GC. The institute was a small and congenial place to fully immerse myself in the fundamental aspects of chromatography, even though some of my colleagues might have been suspicious as to what a biochemist was going to do there. In retrospect, I learned a great deal from their physico-chemical insights into chromatography, but eventually managed to carve my own territory in capillary GC. I was particularly fascinated by the publications of Denis Desty from the United Kingdom, who was the first to describe glass capillaries and achieve most impressive separations, albeit only with hydrocarbons; after all, he worked for the British Petroleum Company.

Together with my colleague, Dr. Karel Tesarik, and based on Desty's description of the glass drawing machine, we constructed our own version in Brno, with some occasional sacrifice of our fingers' skin (a hot glass tube looks about the same as a cold one!). However, we were disappointed that coating a glass surface with a stationary phase was very difficult. It quickly turned my attention to the need for chemical modification of a glass surface. It turned out to be rather frustrating, trial-and-error work; without the availability of today's surface measurement techniques (not even electron scanning microscopy was available at that time); we worked empirically and in darkness trying various surface treatment procedures. For example, we were probably the first to demonstrate that high-temperature gas-phase silylation stabilized coated nonpolar stationary phases, but this treatment was clearly negative for polar coatings. During 1967, we applied some gas-phase surface corrosive procedures, and some of these provided the needed breakthrough. I still cherish the day when I saw my first chromatogram with 4000 theoretical plates per meter efficiency on a column coated with a moderately polar stationary phase.

During the fateful year of 1968, I found myself stuck in Sweden when the Soviet Union's army invaded my homeland (in August of that year). I was in Sweden to learn the basics of GC–MS, by then practiced exclusively with packed GC columns. Given that being an immigrant isn't exactly fun anywhere, I totally immersed myself in science. And there was a lot to learn. Karolinska Institute in Stockholm was world-renowned for their biomedical research, and their close connection to a major producer of GC–MS instruments (LKB-Produkter) gave me great opportunities for research. There was much excitement there about GC–MS of derivatized biological compounds, and I had a chance to meet some of the leading authorities from the USA and Western Europe. To my great surprise, some of these people already knew about our work on glass capillary columns done in Czechoslovakia. Some of these scientists seemed to endorse my ideas and the first attempts of connecting glass capillaries to the LKB 9000 instrument. The first glass drawing machine was meanwhile commercialized in Germany, and a laboratory in Stockholm bought one. I was allowed to use it occasionally. One day, during 1968, an interesting opportunity came from a totally unexpected direction: a French Government laboratory was ready to buy an LKB instrument, if it could prove its performance through resolving a complex essential oil mixture. The salesperson in France thought it entirely feasible, but the folks in Stockholm were horrified; their instrument didn't even have a splitter injection on it, which was necessary those days for capillary GC of neat samples. With the help of a machine shop, I quickly put together a simple splitter and the necessary flanges to make connections. To the later displeasure of my boss, I ripped open, during the weekend, the highly "treasured and secret" Becker-Ryhage GC–MS interface, and interchanged the dimensions of its jets to suit my experiments. Meanwhile, a loaned stainless-steel commercial capillary arrived from France, together with a chromatogram detected by the flame ionization detector. Could we match it? It turned out that I got a much prettier chromatogram than theirs, although I could not immediately understand why. The explanation came to me a week or so later, when I carefully measured the van Deemter curves with standard solutes, which differed in their curve minima due to a different column average pressure under different detection techniques. I promptly published these measurements and their theoretical justification in Chromatographia, then a new journal for rapid communication. Due to the proprietary nature of the "French sample," the chromatogram could not be included. Nevertheless, I was happy with these results, and at least a part of the scientific community noticed.

Another demonstration of the power of capillary GC–MS had to wait for a few more months, when we applied a 100-m long capillary (with over 500,000 theoretical plates) to separate the components of tobacco smoke. This was done together with Dr. Keith Bartle (an English scientist on a temporary assignment at the University of Stockholm); Keith has become a life-long friend and collaborator of both myself and my first Indiana University graduate student, Milton Lee.

In an extension of your early work, you applied glass capillary columns to the separation of complex steroid mixtures, which became one of the milestones in the development of chromatography (3,4). The original work stimulated a great deal of interest in high-resolution separations. The importance of this column work became internationally known very quickly. How was your research approach different from that of your contemporaries at that time?

With the political situation in Czechoslovakia becoming worse all the time, I had to make a major decision where I wanted to live permanently. Although I maintain fondness for Sweden and some friends I met there to this day, I thought an English-speaking country would be professionally preferable at the time. Why not America? This brought me from a cool summer in Sweden to the sweltering heat of Houston, Texas, in August of 1969. The following two years as a postdoc of Albert Zlatkis were an extremely important transition in my entire career. "Dr. Z.," as his associates affectionately called him, was definitely the most friendly and helpful supervisor that I ever had. His laboratories were full of interesting gadgets, and he wanted me to work on a wide range of projects.

When I mentioned to him my interest in separating steroids on glass capillary columns, it was enthusiastically received, but then he said, "This could be a good subject for your presentation at our next international symposium in Miami Beach the next May." I was first in a state of shock; we were already in the month of September, so how much time did we really have? But my answer was "Yes, sir." My European roots and experiences told me that you just cannot say no to your boss! Within reasonable limits, I had everything I needed for this complex task, and I was also familiar with the literature on various derivatization techniques used in making polar molecules sufficiently volatile for GC. I worked long hours, and we also worked on Saturdays. Dr. Zlatkis often ordered pizza for us on Saturdays, and sat with us discussing our research. When the Advances in Chromatography conference was approaching, we had many interesting results on the steroid project. Although I was extremely nervous about my first presentation at the top international conference, my talk went well. I was publicly congratulated by the session chair, Art Karmen, and received some nice comments from other conference participants. Clearly, capillary GC was on its way to replace packed columns in the gas-phase separations of many classes of biological compounds. From various pieces of correspondence to Al Zlatkis, and even to myself, we learned that glass capillaries and steroid separations were among the hot subjects in a number of European laboratories. During the fall of 1970, there was going to be a major chromatography conference in Dublin. Although we didn't have a budget to cover my travel expenses, I decided to go on my own. Al Zlatkis kindly gave me enough time to participate, and even arranged for me visits to some European laboratories. In a "marathon trip," I went through Paris and Zurich, then to Germany, the Netherlands, and Belgium prior to the meeting. The interest in glass capillaries and steroid separations was high almost anywhere I went on that trip. In the United States, the acceptance of these techniques was a bit slower, but these things caught up within a few years.

I owe much gratitude to Al Zlatkis for many things, but perhaps most for his insistence that I had to apply for an academic job. Although I had thought more at that time about getting a job with an instrument company, I applied to five academic institutions, and subsequently received invitations to visit three state-supported universities. The interviews opened my eyes on academia, and I never regretted my decision to accept.

At Indiana University, you developed a highly respected research program, and mentored future leaders of the field, including Jim Jorgenson and Milton Lee, among others. What was personally the most satisfying aspect of developing your own research program with such young and talented students?

Developing a research program at Indiana University and working with many incredibly talented students represent the most satisfying aspects of my overall career. Over the years, I have been blessed by interacting with some of the brightest and most serious and resourceful associates that I could imagine. The names of the former students you mention, Jim Jorgenson and Milton Lee, come first to people's minds for (a) being prominent leaders in our field; and (b) producing their own "scientific progeny." The brightest of their students will continue the academic heritage that is so urgently needed in today's science, in general, and the separation science in particular. Milton Lee was my first graduate student, starting in 1971, with Jim joining us in 1974, not to forget mentioning Vicki McGuffin, who became a faculty member at Michigan State University later, and the two bright and diligent Czech students, Jan Sudor and Lukas Zidek, who are now on the faculties of two major European Union universities. They all enhance the reputation of our research group in their own different ways.

During my first decade at IU, I tried to work in the laboratory alongside my graduate students on my own projects (such as the NASA-sponsored design of the chromatographic column for the 1975 Viking Mars Lander, or a development of new stationary phases for HPLC), but, as the research group grew larger and the equipment more complicated, I assumed more a function of just supervising their activities. I always assumed that the "contract" between a graduate student and his or her faculty supervisor is mutually a serious business. Over the years, I have learned that different students need a personalized approach pertaining to how we proceed with their scientific training, but one criterion is absolute: They must be serious and dedicated to the science we pursue.

I would be amiss not to mention the enormous contributions by a large number of postdoctoral and visiting scientists who, over the years, helped me greatly in assisting with the graduate students' research besides pursuing their own research lines. Among others, Susan Olesik (now at Ohio State University), Takao Tsuda (Nagoya, Japan), Yukio Hirata (Toyohashi, Japan), Keith Bartle (United Kingdom) and Milan Madera (Czech Republic) were particularly helpful.

Over the years, there were many outstanding graduate students who went on to get jobs in industry and in national labs. I have followed with great interest as some individuals became leaders in the instrument industry, pharmaceutical research, or biotechnology. I try to keep in touch with some of these individuals, but it is often hard due to the large numbers and diverse careers. I am always happy to see my former colleagues and friends at the international symposia such as HPLC and the Riva del Garda capillary chromatography conferences.

You and your students were some of the first to apply serious chemometrics to chromatographic problems (5,6). What were some of the greatest insights or advances that chemometric techniques allowed you to discover?

Yes, it is true that we were in this game quite early. The lion's share of credit goes to two amazingly gifted graduate students, Mike McConnell and Jerry Rhodes. I had very limited knowledge of computational techniques, but we had a great need to deal on a statistical basis with highly complex "metabolic profiles" (today conveniently renamed metabolomics). Mike was a Purdue University graduate with a substantial knowledge of computational techniques and capabilities. He first built a fully automated high-precision capillary GC system synchronizing all important instrumental functions. Mike and Jerry then proceeded with the application of non-parametric pattern recognition in the analysis of urinary volatile profiles from diabetic patients. The metabolic patterns at 95% correct classification were subjected to a feature-extraction algorithm for selection of metabolically significant components for the subsequent GC–MS identification. While these procedures may seem trivial through the eyes of today's computational technologies in the "omics era," ours were developed during the 1970s! Both Mike and Jerry went on with successful careers in industry; both now happen to live in the [San Francisco] Bay Area.

You have completed pioneering work in the areas of capillary LC and SFC. In 1978, two key papers were published (7,8), which are considered by many to be the beginning of the field of capillary LC. What intellectual steps led to the creation of this idea?

This goes back to what I might call my "post-tenure blues." The naive perception is that, once you receive your tenure at a major university, you can do any damn thing you want to do without being fired. I have seen some people in academia slow down their research activities and stop looking ahead in their fields after a positive tenure decision. This was certainly not my case. I wanted to explore the less travelled roads, in addition to where we were already strong (such as metabolic profiling and identification of the first mammalian pheromones) through the rapidly maturing technique of GC–MS. If one needed to deal with increasingly more complex polar and nonvolatile mixtures, it would require some high-resolution condensed-phase techniques. For much of my career in separations, I drew great inspiration from the theoretical insights of J. Calvin Giddings (University of Utah). Already as a young scientist in Czechoslovakia, I had almost religiously studied his 1965 book Dynamics of Chromatography and related papers. There it was: To counter the much slower solute diffusion in the liquids, you have to resort to very small, preferably spherical, particles, and small diameter columns, which eventually necessitate the use of unusually high column inlet pressures.

When Dr. Takao Tsuda, a postdoc from Japan, joined my group in 1976, we made first steps to tackle the instrumental problems of capillary LC. Our first capillary columns were drawn from thick-walled glass tubes, some empty, some filled with adsorbents. Even though our equipment was relatively primitive at that time, the results were quite encouraging. It took another decade to have capillary LC demonstrating its real strengths. Karl-Erik Karlsson, a postdoctoral associate from Sweden, was primarily responsible for this breakthrough study (see Anal. Chem. 60:1662–1665 [1988]). For more details on the development of capillary LC, see also my recent review in J. Chromatogr. A 1523: 3–16 (2017).

The development of capillary SFC is quite a different story, but it also has its connections to the observations made by Giddings and coworkers on the solvating properties of dense gasses near the critical point during the 1960s. Milton Lee, my former graduate student, and later a faculty member at Brigham Young University, spent two highly productive summers at IU when I introduced him to my thoughts on "rejuvenating" Giddings' dense gas ideas in terms of much improved capillary column technologies. Milton's and my students collaborated, and it worked; the rest is history. Capillary SFC enriched our research programs, attracted other groups into the field, and was successfully commercialized, although the further developments in SFC later took some unexpected turns.

For a while, we were faced in both capillary LC and SFC with some undue criticism from some of the then "chromatography establishment," such as, "Why bother with these tricky small columns when HPLC works so well?" This opposition for a while influenced the granting agencies, which did not buy in either. All is now totally different with the current revolution in the "omics technologies" where capillary LC–MS is one of the most essential tools. It only proves that sometimes one must withstand conservative criticisms, and do what you believe in.

What can you share with our readers regarding what you would consider to be the most important new areas of research in chromatography? And what do you see as your greatest contribution to the field?

I feel fortunate to have long witnessed the enormous development of the field of chromatography, with respect to its resolving power, and sensitivity, and selectivity of its detection techniques. Not only has it become a highly respected part of analytical chemistry and beyond, but its proliferation in other fields such as systems biology, environmental analysis, and industrial uses are truly amazing. In the development of its techniques and conceptually important applications to their current state, many dedicated scientists actively participated, and I am proud to be one of them. Most importantly, I value the seminal contributions of my best former students to virtually all capillary techniques of chromatography and electrophoresis. Then, there are still some "unsung heroes" in the industrial arena. My former students never had problems with getting good jobs and prospering, and this means a lot to me as well.

With the sophisticated tools now at hand, are we on a "comfortable plateau," expecting only incremental developments? I don't think so. As long as we don't achieve a nearly complete understanding of how biological systems work, and so long as chemists continue to synthesize and isolate novel molecules, there will be further needs for methodological advances. For example, in proteomics and glycoscience, my main areas of interest during the last two decades, I still see great needs to deal with complex native protein separations and dealing with molecular aggregates and nano-sized biological entities such as exosomes.

Closer to what we as separation scientists have been discussing at the recent symposia, multidimensional separations will undoubtedly remain an important field. The column designs may undergo substantial improvements due to the upcoming advances in microfabrication, 3-D printing, and other related technologies; there are already some promising signs in that direction. In terms of selective molecular interactions, chiral separations still remain important. I also don't believe that the ultrahigh-pressure capillary LC work, initiated by Jim Jorgenson already two decades ago, has reached its potential as yet. These are just a few directions, and I am sure my chromatography colleagues and friends could add substantially to the list.

What do I see as my greatest contribution? Perhaps in helping to bridge the gap between what was the theoretical potential of LC and its practice as far as the separation efficiency and feasibility of LC–MS are concerned. Many others made substantial contributions as well toward demonstrating what's feasible today, but after our first publications on both capillary LC and SFC, I was often seen during the first years as the one poking the "hornet's nest." It took a while to get us all there, but it has been exciting and I don't regret my resilience.

What words of advice would you share with us for young researchers just getting started, or even undergraduates considering a future career in science?

Science education is a really complex issue. I can tell you that when I came in 1969 to the United States, our society's respect for science was far greater than it is today. I believe the quality of education, in general, and science education, in particular, has recently suffered, due to lack of funding at all levels.

It served me well to start undergraduate research already toward the end of my first year, but it was a different educational system and different set of circumstances. As a faculty at IU, I had some very positive interactions with bright undergraduates; some ended up being coauthors of our publications, and a few went on to graduate school elsewhere. However, most undergraduates want some research experience only in their senior year, which is usually a bit late to accomplish anything meaningful.

As for advice to a beginning faculty member in our field: Since you know that your "tenure clock" starts ticking right away, you need to strategize concerning your long-term and short-term research goals. We are still a part of a wide and flourishing field, so this will give you plenty of opportunities to develop ideas that are sufficiently different from those of your former research advisor. I tend to say, "Don't put all your chips on the same place," but this may not be universally applicable. Due to increasing role of chromatography in other scientific fields, you may be frequently asked to collaborate with others. Don't refuse scientifically sound collaborations, but try to define your role and scientific credit in a joint project; you may be one out of ten investigators listed on a future publication! However, I remain very optimistic that there is a bright future in separation science, so that the serious scientists will find their field as rewarding as I have.

References

(1) M. Novotný and K. Tesařík, Chromatographia 1(7), 332–333 (1968).

(2) K. Tesařik and M. Novotný, Chromatographia 2(9), 384-385 (1969).

(3) M. Novotný and A. Zlatkis, J. Chromatogr. Sci. 8(6), 346–350 (1970).

(4) M. Novotný and A. Zlatkis, Chromatogr. Rev. 14(1), 1–44 (1971).

(5) M. Novotný, M.L. McConnell, and M.L. Lee, J. Agric. Food Chem. 22(5), 765–770 (1974).

(6) M.L. McConnell, G. Rhodes, U. Watson, and M. Novotný, J. Chromatogr. B: Biomed. l Sci. Appl. 162(4), 495–506 (1979).

(7) T. Tsuda and M. Novotný, Anal. Chem. 50(2), 271–275 (1978).

(8) T. Tsuda and M. Novotný, Anal. Chem. 50(4), 632–634 (1978).

ABOUT THE AWARDEE

Milos V. Novotny

Milos V. Novotny the 2019 Lifetime Achievement in Chromatography Award winner, received his undergraduate and graduate degrees in biochemistry from the University of Brno (now Masaryk University), in what is now the Czech Republic. In 1968, he emigrated to Sweden and held a position of Research Assistant Professor at the Royal Karolinska Institute in Stockholm in mass spectrometry. He then moved to the United States as a postdoctoral fellow at the University of Houston, Texas. In 1971, he was appointed an Assistant Professor of Chemistry at Indiana University; he was promoted to Associate Professor in 1974 and Full Professor in 1978. In 1988, he was named the James H. Rudy Professor of Chemistry, in 1999 a Distinguished Professor, and in 2000 became additionally the Lilly Chemistry Alumni Chair. At Indiana University, he was Director of the National Center for Glycomics and Glycoproteomics (2004–2009). He is currently the Director of the Institute for Pheromone Research and the Director of the Novotny Glycoscience Laboratory.

Jerome Workman, Jr. is the Senior Technical Editor for Spectroscopy and LCGC North America. Direct correspondence to: jerome.workman@ubm.com