Interview With Professor Emeritus Barry L. Karger

Professor Barry L. Karger, Northeastern University, has spent more than 55 years in research in separation science. In collaboration with over 225 Ph.D. students, post docs, and staff, Professor Karger has made significant contributions to high-performance liquid chromatography, high-performance capillary electrophoresis, and mass spectrometry. Further, he has consulted with many organizations, including Genentech (now Roche) and the FDA.

Throughout his career, he had a common MO: research the fundamentals and then apply this knowledge to solving problems of high impact. I had an opportunity for a nostalgic interview with Professor Karger in June 2018.

RLS: We’ve known each other for more than 50 years. How did you come to focus on separation science?

BLK: My undergraduate MIT senior thesis was on analytical separations working in the laboratory of L.B. (Buck) Rogers. Then, as a Ph.D. student at Cornell, I worked on developing packed columns for gas chromatography in the laboratory of W. Donald Cooke. I was particularly interested in the optimization of separations under time normalization. I also built my own flame ionization detector that had recently been introduced by Jim Lovelock. Upon graduation in 1963, when I was 24, I accepted the position of Assistant Professor at Northeastern University in Boston and remained there throughout my whole career. I continued to focus on the understanding and development of separation processes. I felt that one needed to know the fundamentals of any separation process in order to optimize it. Also, this strategy provided a rational basis to select the most suitable method. This is still true today.

For example, early on, I needed to focus on the relative differences of packed-column GC versus capillary GC, which was just emerging. I chose to focus on capillary GC since it was clearly higher resolving and faster.

RLS: So, you had good familiarity with GC. How did you shift your focus to HPLC?

BLK: In 1964, I met Csaba Horváth, who was then a postdoc at Harvard Medical School. He introduced me to HPLC and to István Halász. I also met Georges Guiochon of École Polytechnique in France, who shared my interest in exploring the fundamentals of HPLC. So, in the late 1960s, I refocused the attention of my group to HPLC and started exploring the principles of the LC method. With this focus, it was much easier to solve practical problems and optimize separations. Also, I quickly formed alliances with other early leaders in HPLC, including Lloyd Snyder, J.J. Kirkland, Josef Huber, and Klaus Unger. Unger was a visiting professor in my lab.

RLS: In the early days of HPLC, the commercial marketplace, as today, was very competitive, with competing designs for pumps, detectors, and columns. Yet your lab always seemed to have good instruments.

BLK: Throughout my career, I’ve been successful in providing my students with experience using state-of-the-art commercial instrumentation. This helped them get a good start when they graduated. I also greatly appreciated the support of my lab by early leaders in HPLC including Jim Waters, the founder of Waters Associates. I was honored when Jim endowed the Waters Chair at Northeastern University in 1985, which I held for over 30 years. Over the years, Waters Associates provided my lab with generous support of instruments, modules, and columns. Other companies such as Thermo Fisher Scientific, Agilent, and Beckman were also quite helpful with their support.

RLS: I recall that you coauthored a book in 1973 with Lloyd Snyder and Csaba Horváth, An Introduction to Separation Science. What was the impact?

BLK: Since GC and HPLC were growing rapidly, and other separation methods were already of high importance, Lloyd, Csaba, and I decided to write a book to explain the fundamentals and how to achieve success with chemical separations. The book became very popular and was the standard graduate text used for over 25 years.

RLS: In the mid- to late-1980s, I recall you talking about high-performance capillary electrophoresis (HPCE). What prompted that? After all, HPLC was in the exponential growth phase.

BLK: Yes, the 1980s were a golden time for HPLC and I continued research in this area. But biology was converting into a molecular science, and this was also attracting attention. The fundamentals for the large biopolymers such as proteins and nucleic acids were quite different from small-molecule HPLC. High-resolution separations of large biopolymers were possible with slab gel electrophoresis, but this was slow, cumbersome, and not very quantitative. I had experience with open-tube capillary GC, so it seemed natural to look for a capillary technology that offered speed and quantitative capability, using sensitive detectors. HPCE, as introduced by Jim Jorgenson, met these criteria, so we jumped on it. My experience with separation fundamentals helped us find and solve numerous “early-stage problems.” In 1988, we showed that CE could baseline-resolve closely related DNA molecules. A decade later, HPCE was the key enabling technology for the Human Genome Project. We were very active in that area, and, in fact, our linear polymer was used to sequence roughly 50% of the first human genome sequence.

RLS: I recall that you held the first HPCE meeting in Boston. How important are technical meetings like the HPLC and HPCE series?

BLK: I started the HPCE series in 1989 to help grow the CE field. This meeting was quite successful, but about a decade later, capillary LC was also emerging and needed attention. Hence, the meeting name changed to Microscale Bioseparations. MSB continues to this day, and there are many hot topics, including single cell analysis and exosome analysis. I also was a member of the Permanent Committee of the HPLC meeting series for over 25 years.

RLS: The Human Genome Project was an outstanding success. But, I recall that in about 2000, your lab started to focus on separations coupled to mass spectrometry. Why?

BLK: Right! With the successful sequencing of the Human Genome, it was clear the next generation of DNA sequencing would require complicated engineering efforts. Furthermore, mass spectrometry (MS) was developing rapidly with the use of electrospray ionization. MS offered a way to identify separated peaks. Methods for bioseparations and assays by HPLC and HPCE were criticized since we had some difficulty to detect coelution. UV absorbance was the primary detector for quantitative analysis. Plus, if one saw an unexpected peak, the obvious question was: What compound is that and where did it come from? Because of the small amount of sample and difficulty in collecting it for characterization, these questions were often not answered. I saw that mass spectrometry should be able to help solve some of these problems. Driving this effort was peptide mapping as a way to characterize a protein pharmaceutical, which was becoming more and more important with the rapid growth of biotechnology at this time.

RLS: How well did MS detection work out?

BLK: For HPLC, MS detection has been enormously successful, since electrospray ionization worked exceptionally well. In contrast, the lack of a good ionization interface held back CE/MS. In fact, it held back HPCE to some extent as a general method of analysis. Today, advances have been made, and, while CE-MS could still be further developed, the interfaces are much more successful.

RLS: Can one develop columns for HPCE with high plate counts that can reduce the risk of coelution?

BLK: Yes—we explored this approach early on with our entangled linear polymer capillary columns for DNA sequencing. The polymer not only provided a means of size separation, but also limited diffusion, a source of band broadening in HPCE separations. Indeed, columns were able to deliver up to 107 plates that enabled baseline separations of single base differences in 1000 or more DNA sequences with relative migration differences as small as 1.001. This was a key point in the success of HPCE in DNA sequencing.

RLS: I was just at ASMS 2018. It seems that LC/MS is the key enabling technology for ’omics research and products. Will this trend continue?

BLK: Yes. Life science research and subsequent biotech products are really very complex topics. LC/MS is quite reproducible and hence reliable. Retention behavior can be controlled to better than ±1%. Triple quadrupoles and Orbitraps with multiple reaction monitoring provide reliable quantitation, especially with stable isotope internal standards. At ASMS, mass spectrometers able to work successfully with reasonable resolution with m/z in the 80,000 range were introduced. This allows analysis of large proteins, protein complexes, and even viruses in a state close to native. The need for high-resolution separation methods for these large species, while maintaining close to native conditions, is clear and represents a significant opportunity for the separations community to develop new high-resolution columns and conditions for separation of such large species.

RLS: What about the reproducibility crisis?

BLK: Obviously, science advances require that experiments be reproducible. But this mandates that the person seeking to reproduce a reported result is capable of duplicating the complete protocol. This entails having the right experimental apparatus, reagents, and, more recently, software. Part of the problem is that the journals have pressured authors to reduce word count by condensing the experimental section of articles. Important details are often put in the supplement where they are frequently ignored. Besides the instrumental analysis, which is often automated, sample procurement and sample preparation are also critical and often under-reported. Was a representative sample taken that can be reproduced and did any losses of sample components occur before injecting into the LC or CE column? Experience shows that transferring a method from one lab to another is often a very difficult task, even when communication is good.

RLS: Where are we now and what can we expect in the future?

BLK: We are moving toward tools that will lead to a deep understanding of the details of life on a molecular level. This will involve analysis of single cells, organelles, and microbes. Things such as chemical-based communication that governs life processes will be understood and ultimately used as therapy. We are already seeing promising gene and immunotherapies for genetic defects and cancer. In addition, we are witnessing a revolution in diagnostics. Biomarkers of DNA and proteins in blood (liquid biopsy) are emerging with the likely result of early detection of diseases such as cancer.

RLS: What do you see as the largest emerging challenge for analysis?

BLK: Analytical chemists consistently report that sample throughput is rapidly increasing. Automation and parallel processing or sample pooling (multiplexing) are necessary components of high throughput. This growth will continue as there remains a critical need to generate results with statistical power to make scientific conclusions. One unintended consequence of this is that analytical chemistry, and especially life science research, are quickly becoming an informatics science. I expect improvements in data processing and especially the human–data interface. Artificial intelligence may finally start to deliver utility that matches the attraction of the name.

RLS: Any other emerging challenges?

BLK: As has been true throughout my career, it is not sufficient for an analytical chemist to simply be focused on his/her technical expertise. One has to understand the problems that need to be solved and design effective approaches that lead to answers to those problems. This requires that the analytical chemist understand, for example, the biology involved if one is operating in the life science area. If one works for a pharmaceutical/biotechnology company, one needs to be aware that the results obtained have to be put in a regulatory context, in other words, can your prove that your results are correct?

RLS: What do you feel is your legacy?

BLK: In looking back over my 55+ year career, I am most proud of the many students and staff whom I helped mentor. Many became key contributors to separation science. It’s a wonderful experience to sit in the audience and listen to the talks of my former students. I also am proud of founding and growing the Barnett Institute at Northeastern University, which today is internationally recognized for its research and training in the field of bioanalytical chemistry.

RLS: Any final words?

BLK: The times are very exciting, with rapid advances being made. One can already look back at how “primitive” our methods were a quarter of a century ago. As I have said many times at the end of my lectures, I wish I were younger!

Robert L. Stevenson, Ph.D., is Editor Emeritus, American Laboratory/Labcompare; e-mail: [email protected]