The 2013 Emerging Leader in Chromatography: Davy Guillarme

LCGC’s 2013 Emerging Leader in Chromatography award winner Davy Guillarme, a senior lecturer at the University of Geneva, University of Lausanne, in Geneva, Switzerland, recently spoke to us about how his career began and where his research is leading him now.

Where or how did your interest in analytical chemistry begin?Guillarme: My interest in analytical chemistry really began during my masters thesis that I did in collaboration with Dr. Sabine Heinisch in the laboratory of Professor Jean-Louis Rocca in Lyon (France) in 2000–2001. At that time, I evaluated the possibilities and limitations of high-temperature liquid chromatography (LC) coupled with UV and flame ionization detection (FID). This was a very exciting research topic because the interest from the scientific community on elevated temperature in high performance liquid chromatography (HPLC) (up to 150–200 °C) was still limited then. The reason why I particularly appreciated this year of internship was that I learned a lot about the theory of chromatography with my supervisor, who was always very enthusiastic. In addition, nobody in the laboratory had experience with HPLC at high temperature, so it was very exciting when we obtained new results or findings. At the end of my internship I decided to continue in the same laboratory for my PhD work on the same subject, since the results obtained during the internship were encouraging.

What was the most important thing you learned in graduate school?Guillarme: The most important thing that I learned in graduate school was that theoretical background is of prime importance to interpret or support experimental observations. In addition to theory, a significant amount of experimental work is also extremely important, as this is generally how unexpected observations can appear. By trying to interpret them, important findings can be obtained. In conclusion, to carry out good research, it is recommended to find the best balance between theory and practice and also between pure academic work and real life applications.

Since receiving your PhD in 2004, you have published more than 77 peer-reviewed papers and become a senior lecturer at the University of Geneva. How have you accomplished so much in such a short time span?Guillarme: There are several reasons that can explain this scientific production. First of all, we have a long-term collaboration with several column and instrumentation providers. Thanks to this partnership, it has been possible to have at my disposal innovative or prototype systems very early on. For example, this was the case with ultrahigh-pressure liquid chromatography (UHPLC) in 2004, modern supercritical fluid chromatography (SFC) in 2012, columns packed with core–shell particle technology, and so on. In addition, I work with talented and motivated colleagues, including Professors Jean-Luc Veuthey and Serge Rudaz, as well as numerous PhD students and post-docs. Obviously, all the published work in which I have been involved represent teamwork, and, to further extend our knowledge of chromatography, we have developed a lot of connections with research groups involved in chromatography (such as Professor Gert Desmet at the Free University of Brussels, Professor David McCalley at the University West of England, Professor Carlo Bicchi at the University of Turin, and Professor Jeno Fekete at the University of Budapest, to name a few). Last but not least, I have had the chance to work in a very pleasant environment and our theoretical evaluation of instruments and columns is systematically applied to real case studies in the fields of pharmacochemistry, phytochemistry, and forensic and clinical toxicology in direct collaboration with close colleagues, such as Professor Pierre-Alain Carrupt, Professor Jean-Luc Wolfender, and Dr. Julie Schappler, all from the University of Geneva.

What kind of research is your group currently involved in?Guillarme: Our research group focuses its activities on separation techniques. The group works on developing pharmaceutical separations by LC, SFC, and capillary electrophoresis (CE). Various types of detectors are coupled with these separation approaches including UV, evaporative light-scattering detection (ELSD), and mass spectrometry (MS). In the field of LC and SFC, for which I’m the specialist in the laboratory together with Professor Jean-Luc Veuthey, the goal is to evaluate new solid chromatographic supports and groundbreaking instruments. Thanks to these technical innovations, we are developing new strategies to gain selectivity or sensitivity compared to the original methods. For example, hydrophilic interaction liquid chromatography (HILIC) and ultrahigh performance supercritical fluid chromatography (UHPSFC) are currently compared to reversed-phase liquid chromatography for the analysis of ionizable compounds of diverse polarity. The sensitivity achieved with UHPSFC–MS and HILIC–MS is also systematically compared to that of reversed-phase LC–MS and the achieved results have been surprisingly good. On the other hand, we are also interested in the reduction of analysis time without compromising efficiency or improvement of resolution at reasonable throughput for highly complex samples. The kinetic performance of state-of-the-art LC phases is indeed a topic of high relevance for the scientific community.

Can you tell us about your contributions to UHPLC separations, specifically comparing different approaches using various sample types? Guillarme: As I mentioned, we have been involved very recently in UHPLC. Indeed, we were one of the first groups in Europe to have such a system at our disposal at the end of 2004. Since then, we have acquired several other UHPLC systems coupled with UV and MS or MS-MS detectors, as we are really convinced by this technology. In the beginning, we focused our attention on the possibility of achieving fast analysis. Our first studies dealt with the kinetic comparison of columns packed with sub-2-µm particles from different providers at ambient and higher mobile phase temperature, thanks to van Deemter curves or kinetic plots. Very early on, we stressed to users that the system itself could be a non-negligible source of band broadening in UHPLC conditions. Followingthese preliminary studies, the UHPLC strategy was compared in a systematic way to conventional HPLC, monolithic columns, and core–shell technology. Using numerous model analytes (small pharmaceutical molecules or large biomolecules), we proved that UHPLC significantly outperforms traditional HPLC. However, core–shell technology was a strong competitor and even if the achieved efficiency was still lower on a column packed with sub-3-µm core–shell particles vs. fully porous sub-2-µm particles, the back pressure was also two- to threefold less. More recently, we investigated, in detail, the possibility of high resolution separation in UHPLC (efficiency > 50,000 plates or peak capacity > 500). We saw that by coupling up to three or four 150-mm-long, 1.7-µm d_p columns in series, the performance can be drastically enhanced. We have applied this strategy to the characterization of complex plant extracts or biomolecules and to the field of metabolomics. Finally, last year I guest edited a book with my colleague Professor Jean-Luc Veuthey entitled UHPLC in Life Sciences, which summarizes current knowledge on UHPLC strategies. This could be a good starting point for people who want to learn more about this promising approach.

How did you develop the Excel calculator for transferring methods between HPLC and UHPLC? Did you anticipate that it would be so widely used by your peers?Guillarme: It is important for our laboratory to be in contact with industrial partners (mostly pharmaceutical and chemical), because we don’t want to have our research disconnected from real-life problems. Very early after the launch of the first UHPLC systems, we had a lot of requests from our partners asking how to successfully transfer methods from conventional HPLC to UHPLC. Because the geometric rules for transferring a method can be easily implemented in an Excel spreadsheet, we came up with the idea to provide UHPLC users a calculator available for free on our laboratory website at the following URL: http://www.unige.ch/sciences/pharm/fanal/lcap/telechargement-en.htm. The first version of the calculator was made available in 2006 and, at that time, such a tool was not accessible from providers. We are now on the third version and it has been downloaded more than 7000 times. Briefly, this calculator allows users to automatically determine the new conditions for isocratic and gradient modes (injected volume, flow rate, and gradient profile) when modifying column dimensions (length, internal diameter, or particle size) in HPLC and UHPLC. In addition, it also provides the expected performance under isocratic (plate count) and gradient (peak capacity) conditions for any flow rate, temperature, and column dimensions.

How did your research in reversed-phase LC and wide-pore phases get started? And what are your latest discoveries in that field?Guillarme: Because I’m working in an academic laboratory affiliated with the school of pharmaceutical sciences, the project that I develop should follow the trends of pharmaceutical industry. In recent years, the importance of therapeutic proteins and monoclonal antibodies (glycoproteins of ~150 kDa) has been growing in the pharmaceutical field and the expectations are high for this new class of compounds. However, the analytical characterization of biopharmaceuticals remains difficult and there is a need to improve the current analytical tools. The main providers of chromatography consumables and instruments are aware of this need and they have recently commercialized some wide-pore core–shell or fully porous sub-2-µm particles that are able to successfully analyze biomolecules with high molecular weight. Since 2011, we have had a very talented senior researcher in our lab, Dr. Szabolcs Fekete, who has a strong industrial expertise in biomolecules analysis and in modern LC in general. In the last two years, together we have published a series of papers dealing with the possibilities of several modern wide-pore reversed-phase LC phases for the analysis of large biomolecules. The achieved results prove that reversed-phase LC can be an interesting approach for the characterization of intact proteins and monoclonal antibodies. Indeed, our studies have shown that the kinetic performance of these materials is excellent, analysis throughput is high, and MS detection can be used to gain additional information from the sample.

What do you plan to focus on next? Is there one big problem in separations science that you really want to tackle?Guillarme: Currently, I’m involved in a detailed evaluation of reversed-phase UHPLC, HILIC, and UHPSFC coupled with UV or MS detection, for the analysis of pharmaceutical compounds. There are still a lot of things to explore with these analytical techniques and then I would like to focus on these methods before evaluating alternative separative techniques. In reversed-phase UHPLC, there are always new innovative stationary phases (such as smaller particle size, columns with higher mechanical or chemical stability, and new bonding) released on the market or available as prototypes to be tested. In HILIC, we are particularly interested in exploring the analysis of biological fluids with MS detection, since the strategy looks powerful. In UHPSFC, there are only a few applications dealing with MS detection. We wish to explore the real advantages and limitations of UHPSFC–MS compared to the well-established UHPLC–MS.

Currently, there is not one particular problem in separation science that I would like to tackle. However, we are collaborating with a lot of people or research groups who regularly have separation problems and we need to find some solutions for them. Sometimes it is extremely tricky, particularly when dealing with human biomarkers that are always very polar or appear in a dirty matrix. In addition, we have to develop tools or methods that can be easily applied in other laboratories, which can be an additional constraint.

Click here for the full article on the 2013 LCGC awards, including more information about Guillarme's career.