Highlights of HPLC 2013, Part I

The 39th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2013) was held June 16–20 in Amsterdam, The Netherlands, for the second time. Part I of this coverage reviews some of the technology and application advances discussed at HPLC 2013 as well as the overall liquid phase chromatographic trends. We also provide a summary of the awards presented. Part II will discuss the column technology highlights observed at the symposium.

The 39th International Symposium on High Performance Liquid Phase Separations and Related Techniques, which alternates between Europe and North America with occasional side meetings in Australasia, was held June 16–20, 2013, in Amsterdam, The Netherlands, for the second time and the third time in Holland. More affectionately known as HPLC 2013, the symposium is the premier scientific event for bringing together the myriad techniques related to separations in liquid and supercritical fluid media. Cochaired by Professors Peter Schoenmakers and Wim Kok of the University of Amsterdam, HPLC 2013 assembled more than 1500 participants from all over the world. This number includes vendor representatives from 55 exhibitors for the three-day instrument, software, and consumables exhibition. The number of conferees was the highest in recent years for this series, with an especially high attendance from European countries, which may indicate that their economic crisis may be abating.

The venue for HPLC 2013 was the colossal RAI building, the location of many Dutch and European exhibits and conferences. The five-day-plus event had a total of 168 oral presentations, perhaps a new record, in plenary and parallel sessions and a whopping 945 posters in sessions with 36 different themes. Lunch was served on-site as part of the registration fee, so conferees didn't have a time-consuming search of restaurants in the area and could focus on science. With an ample social event schedule including three receptions and a symposium dinner and party, 18 vendor workshops, 20 tutorial educational sessions, and four short courses (held during the previous weekend), attendees had their hands full deciding how to allocate their time. The tutorials were particularly well attended and covered current topics such as monoliths, micro- and nanofluidics, hydrophilic interaction liquid chromatography (HILIC), columns and stationary phases, various liquid chromatography–mass spectrometry (LC–MS) topics, applications (environmental, residues, lipidomics, chiral), and comprehensive LC.

This installment of "Column Watch" is part I of my HPLC 2013 coverage. Here, I present some scientific highlights of HPLC 2013 and cover the various awards and honorary sessions that took place. Because it was virtually impossible for one person to adequately cover all oral and poster papers, my coverage will somewhat reflect a personal bias, although I was able to get presentation notes from some of my colleagues, who are acknowledged at the end of this installment.

Trends in Liquid-Phase Technology and Techniques

Obviously, high performance liquid chromatography (HPLC) and ultrahigh-pressure liquid chromatography (UHPLC) were the predominant technologies in the technical sessions at the symposium. The organizers stressed four main themes throughout:

• High impact LC (applications-oriented presentations)

• "Hyper-formance" LC (fundamental, mechanistic, and column- or stationary phase–oriented presentations)

• Education (tutorials, overview talks on established and new methodologies)

• HPLC–MS (all aspects of MS including capillary electrophoresis (CE)–MS, quantitative MS, and data analysis).

Beginning with the Sunday afternoon opening plenary and plutorial talks, sessions were organized under these four "umbrella" themes. By far the highest concentration of oral, poster, and tutorial presentations was in the category of applications with food, environmental, and proteomics leading the way. Columns and stationary phases came in second, followed by mass spectrometry, which was a dominant theme for HPLC 2013. A hot topic was the increased use of LC×LC (comprehensive LC) and column switching for improved resolution and on-line sample preparation. Somewhat disappointing was the small number of sample preparation papers — there was a single "Sample Preparation" oral session with only two papers, but buried within many of the applications-oriented papers were numerous examples of the importance of sample preparation in the development of successful methods.

The three "hot" areas in column technology this year were as follows:

• An extraordinary emphasis on superficially porous packings (SPPs, also referred to as shell particles, poroshell, and fused-core packings) that rival the sub-2-μm particles in terms of column efficiency, but with substantially lower pressure drops. With Waters joining the SPP ranks with its sub-2-μm Cortecs product and more companies jumping in with their own versions, it was almost as if the sub-2-μm porous particle columns fell off the end of the earth. I guess that they are so commonplace now that column suppliers are expected to have a full line of sub-2-μm columns.

• Monoliths remained an important topic, but with a much greater amount of interest in polymeric monoliths. This year there were almost 60% more presentations on polymer-based monoliths than silica-based monoliths; last year it was the other way around (1). For the first time, organosilica hybrid monoliths constituted a category, mostly using the sol-gel process. The organic portions were mostly methacrylate-based. Interestingly, carbon- and zirconia-based monoliths are being investigated.

• Two-dimensional (2D) chromatography again generated a great deal of interest, probably partially driven by the commercial availability of easy-to-use column switching systems. Orthogonal phases may be the next big area, because the power of 2D techniques rests in having major separation differences in the two modes (or columns).

Areas of Application

Table I is a brief breakdown of the most popular application areas reported at HPLC 2013. Oral and poster presentations on life science topics again dominated the applications with the "omics" (for example, metabolomics, proteomics, and lipidomics) out in front with post-translational modifications (such as glycosylated and phosphorylated proteins), monoclonal antibodies, and the search for biomarkers as major fields of study. LC–MS and LC–MS-MS techniques are an absolute requirement for these studies; hence, there was an overwhelming number of papers with these MS technologies presented at this meeting.

Table I: Papers presented by major application area

Pharmaceutical and biopharmaceutical companies are still one of the most prolific users of HPLC and UHPLC, and, even though this industry has been hit with some difficult times, drug discovery is still a big business. Most of the other application areas were roughly in the same percentage as applications papers in previous years (1,2).

Awards and Honors at HPLC 2013

The HPLC meetings have become the venue for chromatography awards presented by various groups for best posters, best oral presentation by a young investigator, and awards from the Chromatographic Society. This year, a new award for significant contributions by industrial chromatographers was added to the mix.

Horváth Award

For the 8th year in a row, the Horváth Award sessions, named for the late Professor Csaba Horváth, one of the founders of this series and a mentor of young scientists, were featured. This award, which is supported by HPLC Inc., a nonprofit group under the guidance of the Permanent Scientific Committee, was established for young scientists in the separation sciences under the age of 35. The award is based on the best oral lecture presented in the Horváth Sessions. The winner is selected by a jury named by the Permanent Scientific Committee, and the award consists of a cash prize, invitation to present an oral lecture at HPLC 2014, and a crystal trophy. This year there were 10 nominees, all with strong research credentials.

The winner of the 2013 Horváth Award was James Grinias of the University of North Carolina in Chapel Hill, North Carolina. The title of his winning oral presentation was "Characterizing Extra-Column Effects in Ultra-High Pressure Liquid Chromatography." In his work, he investigated those features of the UHPLC systems that contribute to extracolumn peak broadening effects (such as injectors, detectors, connecting tubing, and system connections), which are especially important for modern columns such as smaller particles (<2 μm) and superficially porous supports. In this study, injector effects on a capillary UHPLC system were measured using electrochemical detection (with carbon fiber electrodes of extremely low detection volume) and analyzed using an exponentially modified Gaussian function to separate contributions from poorly swept volumes (exponential-contribution) and open-tube Taylor-Aris dispersion (Gaussian-contribution). The data suggested that effects that were previously thought to be independent may in fact have some coupling characteristics. Initial injector output profiles and changes because of band spreading in open tubes were both characterized. Two injection modes of a Valco valve were investigated: full-loop and what he called time-pinch injection. The latter proved to provide a sharper injection profile. The impact of total extracolumn effects on low-volume packed beds in both capillaries and microfluidic devices was observed and differences between electrochemical and UV–vis detection were compared. Data acquisition rates and digital filtering in the measurement of extracolumn peaks at high flow rates also were found to be very important.

Poster Sessions and Best Poster Awards

The mainstay of HPLC 2013 was the poster sessions, where more detailed applications and methodology studies were reported, often in very specific areas, and face-to-face discussions with the authors were conducted. Fortunately, many of the poster authors were kind enough to provide small reproductions of their poster papers that could be taken for later perusal. Some authors collected business cards and addresses for sending poster reprints by mail or e-mail. Compared to HPLC 2012 (1), the number of posters greatly expanded. Only about half of the poster presenters elected to have their poster evaluated for the Best Poster Contest, which made the job a bit easier for the 80 reviewers that were assigned to the Poster Committee. The posters were up for four days, which gave viewers plenty of time to find them. It appeared that there were fewer "no shows" than had been observed in previous HPLC Symposia.

The Poster Committee Chairman was Dr. Gerard Rozing, recently retired from Agilent Technologies. The bulk of the Poster Committee devoted a great deal of time and worked very hard to narrow down the huge collection of posters by the end of the third day to 30 finalists. From these 30 finalists, new reviewers were chosen to help select seven winners by the Thursday afternoon of the symposium. The selection criteria were based on three factors: inspiration (creativity, newness, uniqueness, originality), transpiration (experimental execution, completeness of work), and presentation (overall readability, visual impression, author's explanation) and winning posters were viewed by all of the final committee jurors. For a different slant, this year there was a popular vote by all conferees for three poster winners and they were added to the seven selected by the Poster Committee to make a total of 10 winners.

The Best Poster Awards, sponsored by Agilent Technologies, were announced at the closing session on Thursday afternoon. For HPLC 2013, the Best Poster Award winners (along with their affiliations) that were present at the closing session are shown in Figure 1. Their poster titles follow their names. Each prize winner received a 500 euro cash prize. Because of the large number of awards granted as well as space limitations, detailed technical coverage of each of the award winning posters cannot be provided. It suffices to say that all the winners should be proud of their accomplishments since they represent the top 1% of all posters presented at HPLC 2013.

Figure 1: Best poster award winners at HPLC 2013. In the photo from left to right: Stefan Schütte, General Manager of Agilent Liquid Phase Analysis Division, Waldbronn, Germany; Theresa Kristl of University of Salzburg, Austria, "Quantitative Proteomic Profiling with HPLC–MS-MS: Comparison of Various Labeling Strategies Using iTRAQ and TMT"; Melissa Phillips of NIST, Gaithersburg, Maryland, "LC–MS and LC–MS-MS for Determination of Water-Soluble Vitamins in Foods"; Gerard Rozing, Best Poster Award Chairman; Bert Wouters of Vrije Universiteit in Brussel, Belgium, "Design of Cyclic Olefin Copolymer-Based Microfluidic Devices Designed for Spatial Two- and Three-Dimensional Chromatography"; Daniel Wilffert of the University of Groningen in the Netherlands, "Quantitative, Antibody-Free LC–MS-MS Analysis of Recombinant Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) in Serum"; Kenichiro Todoroki of the University of Shizuoka in Japan, "Dress-Up Chiral Columns for the Enantioseparation of Amino Acids Based on Fluorous Separation"; Martin Laher of the Institute of Polymer Science, JKU Linz in Austria, "Nanoscale Characterization of Polymer Monoliths Using Atomic Force Microscopy and Confocal Raman Imaging"; Mohammed Ibrahim of the University of Alberta in Edmonton, Alberta, Canada, "HILIC-Phase Selectivity Chart for Characterization of HILIC Stationary Phases"; Wim Smits of Vrije Universiteit Brussel, Belgium, "Computational Flow Study of the Optimal Design and Operating Conditions of the Flow Split Ring Used in Parallel Segmented Flow Columns"; Martin Lopatka, University of Amsterdam, The Netherlands, who organized the public voting for three of the Best Poster positions; Peter Schoenmakers, University of Amsterdam, the symposium chairman. Not pictured: Fernando Benavente of Department of Analytical Chemistry, University of Barcelona, Spain, "Development of an Immunoaffinity Sorbent with Fab’ Antibody Fragments for the Analysis of Neuropeptides by IA-SPE-CE-MS" and Antonia García-Fernández of CEMBIO, Universidad CEU San Pablo in Boadilla del Monte, Spain, "Search for Markers of Bladder Cancer with a Metabolomic Approach." (Photograph courtesy of Ken Broeckhoven, Free University of Brussels, Belgium).

Chromatographic Society Awards

In 1978, Nobel Prize winner Professor A.J.P. Martin gave permission for his name to be associated with the "Martin Medal." The Martin Medal is awarded to scientists who have made outstanding contributions to the advancement of separation science. The Chromatographic Society celebrated its Silver Jubilee in 1982, and to commemorate the event it instituted the Jubilee Medal. This award is presented in recognition of the contributions of younger scientists.

At HPLC 2013, two Martin Medals and one Jubilee Medal were awarded to scientists in the field of liquid-phase separations. Professor Günther Bonn, head of the Institute of Analytical Chemistry and Radiochemistry in Innsbruck, Austria, was awarded a Martin Medal for his sustained and important contribution to the promotion of separation science — reflected in his strong presence in peer-reviewed scientific literature, numerous patents, recognition as a speaker at national and international separation science meetings, and membership on numerous editorial boards. The breadth of his work spanning the synthesis of novel chromatographic and enrichment supports coupled with his vast body of work across the genomic, proteomic, metabolomic, and phytomic areas was recognized by the committee in this award.

The second Martin Medal was awarded to Professor Frantisek Svec of Lawrence Berkeley National Laboratory and The Molecular Foundry in Berkeley, California, for his world-leading contribution to chromatographic science. His prolific work in the development of polymeric stationary phases and adaptation of these to multiple column and chip formats was recognized by the committee as ground-breaking research. The pivotal approaches he has pioneered for the fabrication of monolithic supports and functionalized polymer beads have been adopted widely by numerous commercial organizations and exemplifies the practical and important nature of his work.

This year's Jubilee Medal went to Dr. Fabrice Gritti of the Department of Chemistry at the University of Tennessee in Knoxville, Tennessee, for his important contribution to the development of chromatographic science. Dr. Gritti's strong presence in the peer-reviewed scientific literature and prominence as a speaker at international separation science meetings was a key aspect in his recognition. His prolific work fundamentally understanding the adsorption and the mass transfer of analytes at the liquid–solid interface and using this to characterize and design LC stationary phases was recognized by the selection committee through this award. His recent work on the understanding of mass transfer kinetics on solid-core particles was particularly commended. Remarkably, Dr. Gritti has already published more than 170 peer-reviewed papers in his short research career.

Uwe Neue Award

Uwe Neue, who passed away two-and-a-half years ago, was an industrial chemist working for Waters who gained international respect for his contributions to the field of separation science, especially for work that turned into commercial products. The Uwe Neue Award, sponsored by Waters, is directed to scientists like Uwe, who have had great careers in industry while contributing to the further development of chromatography. The inaugural winner announced at HPLC 2013 was Jack Kirkland, who exemplified the notion that major contributions can still be made by industrial scientists. While working for Dupont, Rockland Technologies, Hewlett-Packard (which later became Agilent Technologies), and most recently Advanced Materials Technology, Jack contributed heavily in gas chromatography (GC), field-flow fractionation, and HPLC column technology and has already received numerous major awards, patents, and publications for his work. The concept of superficially porous particles that Jack pioneered and reintroduced to the LC field is one of his most important contributions and is quickly becoming the preferred approach to high efficiency LC and UHPLC separations today. In his award address, Jack talked about a new commercial SPP product (3.4 μm) with a 0.2-μm shell and a 3.0-μm core) that was developed by his group for the separation of larger biomolecules. The pore size was 400 Å, which is sufficiently large for proteins up to 400 kDa. They investigated different shell thicknesses and core sizes. A thin shell gave higher efficiency, but a thicker shell gave more sample loading. The research group compromised on the above dimensions to have the optimum efficiency and sample capacity. They compared the optimized packing to a totally porous particle (TPP, 1.7 μm) and found that the efficiency of a packed column was basically the same while the pressure drop under the same experimental conditions was a third (57 bar for SPP versus 172 bar for the TPP). They investigated several reversed-phase chemistries and found that an end-capped, C₄ alkyl chain gave the best overall chromatographic performance. The column was found to be stable to 90 °C and provided good recovery for cytochrome C (100 ± 5.8%) and catalase (92 ± 18%).

Opening Session and Plenary Lectures

Typical of Dutch unconventionality, this year's opening session at HPLC 2013 was quite different from those of recent years. Firstly, to get people loosened up, everybody in the audience was given an African drum and, with a leader and helpers on stage, lessons in rhythmic drum beating were orchestrated until everybody's hands were totally numb. Then, the lectures started. Each lecture was chosen to fit one of the four themes suggested above. Under the heading of high-impact LC, the first lecture was more of a typical opening plenary lecture by Jos Beijnen of Slotervaart Hospital in Amsterdam, The Netherlands. His talk was entitled "The Fight Against Cancer" and gave an excellent overview of steps that have been taken to gradually reduce death rates. Prevention measures such as stopping smoking and early detection have helped, but improved diagnostic tools and better treatment, including chemotherapy, have improved the chances of controlling cancer development. A basic understanding of the molecular processes underlying cancer development has increased tremendously and new targets that can be exploited for drug development have been successfully used. Cancer is a DNA disease, and the patient's tumor genome is now dictating pharmacotherapy. This form of personalized medicine is already becoming widely accepted by oncologists. Mixtures of drugs appear to be effective against certain forms of cancer, but new drugs are continually needed for mutated cancers.

The second lecture of the session, a plutorial, was under the theme of education and was given by yours truly. After a brief recognition ceremony for my contributions to education via this magazine and other activities, I presented the "Top 10 HPLC Column Myths," which was recently published (3), so I won't dwell upon the subject here!

In the HPLC–MS umbrella theme, the third lecture, with a provocative title, "Forget Everything, Except Mass Spectrometry" was quite interesting and presented by Michel Nielen of Wageningen University and RIKILT in Wageningen, Netherlands. Dr. Nielen gave several examples where simple front-end systems such as flow-injection analysis or "dilute and shoot" suffices to present a sample into the mass spectrometer that permits identification and analysis by MS or MS-MS. However, one usually must know ahead of time what one is searching for. He cited examples where direct analysis in real time (DART) MS has been able to solve real problems (for example, xenobiotics on fruit, fungicides on orange peel) and the technique can identify substances (solids, liquids, and gases) without any sample preparation or separation required. In the use of triple-quadrupole MS, he did admit that the sample matrix–dependent ionization suppression phenomenon was observed, precluding quantitative and robust analysis results. In those cases, sample preparation and even separation by LC may be required. So, at the end, he modified his title to read "Forget Almost Everything, Except Mass Spectrometry!"

Under the theme of hyper-formance LC, the fourth talk before the welcome reception was presented by last year's Horváth Award winner Stefan Bruns of Philipps-Universität Marburg in Marburg, Germany. His talk, entitled "From Pore Level Reconstruction to Morphological Analysis of Eddy Dispersion," reviewed recent progress by his research group in the three-dimensional reconstruction of hard and soft matter materials by optical and electron microscopy. One of their goals was to understand the fundamentals of column packing procedures to guide optimum slurry packing processes. Using sophisticated measurement tools such as confocal laser scanning microscopy they were able to perform nondestructive imaging of silica-based stationary-phase materials and, with other techniques, to image polymer-based capillary monoliths. After a reconstruction of the materials, they were able to examine heterogeneities on different length scales in the bed structures. Slurry-packed particulate columns were investigated for the influence of specific packing parameters, such as the particle size distribution, column inner diameter, and slurry density on the bed morphology. For monolithic columns, the morphological features limiting their efficiency were identified. Recognizing and eventually understanding the morphological origins of dispersion paves the way for a rational optimization of the preparation conditions of particulate and monolithic supports.

Part II

In part II of this series, we will review the column technology highlights observed at the symposium.

Acknowledgments

I would like to acknowledge the contributions of my Agilent colleagues who supplied notes on some of the sessions: Xiaoli Wang, Wu Chen, and Norwin Von-Doehren. A very special thanks goes to Maureen Joseph, also of Agilent, who took very copious and thorough notes at many of the columns sessions. Also, I would like to thank Professors Carol Collins from the University of Campinas in Brazil and David McCalley of the University of the West of England for sharing their notes with me.

References

(1) R.E. Majors, LCGC North Am. 30(9), 804–827 (2012).

(2) R.E. Majors, LCGC North Am. 29(9), 802–816 (2011).

(3) R.E. Majors, LCGC North Am. 31(7), 522–537 (2013).

Ronald E. Majors "Column Watch" Editor Ronald E. Majors is a Senior Scientist in the Columns and Supplies Division at Agilent Technologies (Wilmington, Delaware), and is a member of LCGC's editorial advisory board. Direct correspondence about this column to lcgcedit@lcgcmag.com.

Ronald E. Majors