OR WAIT 15 SECS
This “Column Watch” installment reports on highlights from the 48th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2019), held June 16 to 20 in Milan, Italy.
The 48th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2019), chaired by Alberto Cavazzini and Massimo Morbidelli, was held from June 16 to 20 in Milan, Italy. This installment of "Column Watch" covers many of the highlights observed at the symposium. In addition, trends and perspectives on future developments in HPLC, and related techniques gleaned from the conference, are presented.
The 48th International Symposium on High Performance Liquid Phase Separations and Related Techniques, or HPLC 2019, was held in the beautiful city of Milan, Italy (Figure 1). This was the first time this important conference has convened in Italy. The conference primarily took place at the University of Milano-Bicocca and spanned June 16–20, 2019. The HPLC symposium, which continues to be the premier event bringing together leading scientists in the field of liquid chromatography and related techniques, attracted approximately 1200 delegates from numerous countries. The conference was co-chaired by Professor Alberto Cavazzini of the University of Ferrara and Professor Massimo Morbidelli of the Polytechnic Institute of Milan. The program had a strong focus on encouraging contributions from young scientists, as well as promoting the more traditional fundamentals of separations science.
The conference included 308 oral presentations and 508 posters. Of special note were a couple of specific events geared toward young scientists, namely "Separation Science Slam" and "HPLC Tube" (see further discussions below), both of which were highly entertaining and well attended. The social programs included a beautiful opening ceremony at the Milan Conservatorio and Gala Dinner at the Central Courtyard of the Univerista delgi Studi di Milano (Figure 2). In this installment of "Column Watch," observed highlights and trends from the conference are reported.
In a similar fashion to previous HPLC review articles (1,2), several colleagues in attendance at the symposium were asked for their insights regarding the most interesting topics they observed at the event. Much of what follows is a synopsis of their responses along with some personal views.
In reviewing highlights from the past several symposia, multiple areas of interest stood out, including 3D printing, advances in large-molecule separations, multidimensional separations, and hydrophilic interaction liquid chromatography (HILIC). The main 2019 symposium topics were largely similar to recent years. One notable difference, however, was the inclusion of dedicated sessions to thin-layer chromatography.
Three-dimensional printing and microfabricated structures continue to increase in interest. The 2019 program was the first year that there was a session dedicated solely to 3D printing and microfabricated structures. As an illustration of the level of interest this topic generated, many attendees were willing to wait approximately 40 minutes in humid air and hot temperatures, due to an air conditioning issue, for the session to start. Simone Dimartino led off the session, speaking about the design of 3D-printed stationary phases (3). There were also a couple of presentations demonstrating the advantages of 3D printing by Peter Schoenmaker's group, who continue to actively investigate multidimensional separations (4,5). Along with new developments in column design through 3D printing, devices can be readily designed and tested, using the technique as shown by Jackie Sosnowski in the talk entitled, "3D Printing as a Flexible Tool to Customize Liquid Introduction with Electrospray Mass Spectrometry (6)." Dimartino also conducted a well-attended short course on 3D printing, which provided an overview of how 3D printing is influencing separation science. As noted in a recent review of 3D-printed stationary phases, the manufacture of highly efficient chromatographic columns is becoming a reality as 3D printers become more affordable and accessible and their resolution, speed, and material flexibility continue to grow (7).
Large-molecule separations were again at center stage during the 2019 conference. The importance of the topic was indicated by the high number of sessions dedicated to advances in the separation of proteins and related molecules. As noted in previous symposia reviews (1,2), many different modes of chromatography are necessary to fully characterize such complex molecules. Chen, for example, presented on the development of suitable columns based on sub-2 µm particles for size-exclusion chromatography (SEC), an invaluable technique used to assess molecular size variants of protein therapeutics (8). Chen described the importance of blending adequate pore volume, mechanical strength of particles and bonding coverage when designing SEC phases in small-particle formats. The reader is referred to a recent LCGC article authored by Chen and colleagues for more details (9).
It was noted by several colleagues that alternative particle types are being used for large-molecule separations. Several talks were presented involving large-molecule separations using porous graphitic carbon (PGC) and polymeric supports. It was observed that these alternative supports are finding utility for "omics" studies that rely on resolving highly complex samples prior to analysis by mass spectrometry. This trend has been apparent for a number of years where alternative supports that once found uses for small-molecule separations are now being applied to large molecule challenges. One observer noted that there seems to be a shift of emphasis from monoclonal antibody (mAb) and antibody–drug conjugate (ADC) analyte separations, and more towards bispecific antibodies (bsAbs) and fusion proteins, which are being proposed as new formats for biotherapeutics.
One "omics" talk that was identified as being particularly interesting was presented by Michael Lämmerhofer, future co-chair of the HPLC conference, to be held in Düsseldorf, Germany, in 2021. The talk entitled, "Lipidomics: A Window of Opportunity for Clinical Analysis," compared and contrasted an number of different approaches toward performing lipidomic studies (10). A focus of the talk centered on the benefits of both HILIC–MS/MS and reversed-phase-MS/MS methods coupled to quadrupole time of flight (QTOF) mass spectrometry (MS).
Multidimensional chromatography (MDLC) continues to be a hot topic. Sessions on two-dimensional and multidimensional separations were held each day, and on several days there were more than one. Multidimensional chromatography (MDLC) is finding its way in to a number of areas of application, including pharmaceutical analysis, indicating this is more than an academic exercise. Koen Sandra presented within a pharmaceutical session where he emphasized the utility of MDLC for biopharmaceutical analysis (11). Sandra noted many different approaches to MDLC, HILIC × reversed-phase, and size-exclusion chromatography (SEC) × reversed-phase, among others, as being important for characterization of large biopharmaceutical analytes.
Frederic Lynen presented an interesting talk on yet another combination of techniques: temperature responsive chromatography combined with reversed-phase LC (12). Lynen described recent efforts to prepare and utilize temperature responsive polymer stationary phases, such as poly(N-isopropylacrylamide) coupled to reversed-phase separations. One possible advantage of temperature responsive chromatography versus other forms is that the aqueous mobile phase employed in the temperature responsive dimension is generally a weak solvent for the second dimension, thus allowing for refocusing of the bands. Further information can be found in a recent article from the Lynen group (13).
Andre de Villiers presented on the topic of incorporating ion mobility mass spectrometry (IMS) into one- and two-dimensional workflows for the analysis of complex sets of phenolic compounds in natural products (14). For the one-dimensional analysis, de Villiers combined ultrahigh performance liquid chromatography (UHPLC) with electrospray ionization (ESI), IMS, and TOF-MS for the analysis of the ornamental flower Protea. The author noted that 41 phenolic acid esters, 25 flavonoid derivatives, and 5 anthocyanins were observed. During the presentation, de Villiers went on to add a second dimension of HILIC chromatography for the analysis of a variety of phenolic classes in chestnuts. The author noted that the incorporation of IMS can provide improved mass spectra quality, complementary separation, and automation. Ion mobility continues to improve and many mobility techniques are being adopted for the successful separation of complex samples.
HILIC was discussed in a number of different sections, including one session solely dedicated to the technique. Marti Roses of the University of Barcelona presented an interesting talk attempting to utilize the Abraham model, or linear solvation energy relationships (LSER), to predict retention and help elucidate HILIC retention mechanisms (15). The model was applied to a ZIC-pHiLIC column with acetonitrile:water and methanol:water mobile phases in the range 80–95% v/v of organic solvent. The study cast some light on the most important retention contributions to HILIC, but has not as yet been applied to ionizable solutes.
David McCalley presented on the often-discussed topic of re-equilibration in HILIC (16). Although full equilibration is considerably longer in HILIC versus reversed-phase chromatography, McCalley's work demonstrates that reproducible chromatography is obtained as long as re-equilibration times are controlled, even if full equilibration is not achieved. McCalley also discussed the impact of flow rate and temperature on equilibration rates in HILIC. The results indicate steps can be taken to optimize the process even when full equilibration is required. For more details, the reader is referred to recent papers by the McCalley group (17,18).
As noted in last year's review of the HPLC meeting (2), HILIC is being applied to large-molecule analyses. Work in this area continues as demonstrated by Andrea Gargano of the University of Amsterdam in his work characterizing an enzyme used in bread production (19). Compared to reversed-phase chromatography, HILIC has found particular utility for the separation of intact protein glycoforms. The reader is referred to recent publications from Gargano and colleagues for further details (20,21).
For the first time in memory, thin-layer chromatography (TLC) was the focus of not only one, but two, full sessions. Both sessions focused on advanced TLC techniques utilizing modern, high performance TLC plates (often referred to as HPTLC). Modern plates are characterized as being relatively fast, providing excellent resolution due to the sharp bands produced and of higher throughput due to lower dispersion (compared to, for instance, traditional 20 cm × 20 cm silica gel plates). The presentations demonstrated continued interest in utilizing HPTLC for studying a wide variety of natural products, several of which were focused on studying bioactivity of components. Another area of interest lies in the coupling of HPTLC with mass spectrometry to aid in identifying active molecules in complex samples.
Most notable in all the impressions received from colleagues regarding the 2019 program was that the symposium was highly geared toward young scientists. The 20-minute oral format allowed for the inclusion of many talks during the allotted time, providing many opportunities for young investigators to present. Mariosimone Zoccali, a young scientist from the University of Messina, presented on carbon dioxide extraction and subsequent separation techniques for the analysis of a complex set of carotenoids and derivatives (22). Zoccali explained the steps of supercritical fluid extraction (SFE) in detail, and then went on to demonstrate that SFE coupled to supercritical fluid chromatography (SFC) and triple-quadrupole MS could reduce extraction time, provide faster run times, reduce the potential for sample contamination, and improve precision as compared to published methods. Zoccali went on to note that the developed methodology allowed the observance of previously never reported apocarotenoids in human blood and colostrum. Omar Ismail of the University of Ferrara gave another notable young investigator presentation on the topic of zwitterionic teicoplanin-based chiral selectors (23). The study centered on kinetic and thermodynamic comparisons of the chiral selector bonded to core shell silica supports of varying particle sizes (2.0-, 2.7-, and 3.4-µm) and pore sizes (90, 160, and 400 Å). Ismail concluded that the 2.7-µm, 160 Å support provided the greatest overall performance.
In addition to the excellent talks from the young scientists, two innovative evening programs, the "Separation Science Slam" and the "HPLC Tube" contests, provided young scientists with an alternative means of communicating their research in a fun and creative manner. The "Separation Science Slam" asked young scientists to engage in a competition to creatively inform the audience about their research projects in a manner of their choice. From inspiring videos to, let us say, "interesting" raps, the audience was indeed entertained and inspired. The "HPLC Tube" contest provided young participants the opportunity to create a 3-minute video to communicate their research. Many of the videos were highly enjoyable, and exhibited ingenuity in delivering their message. Both of the sessions were very well attended. Many young scientists were also included in the organizing committee and as session co-chairs for the conference. The consensus from discussions with colleagues on the topic is that the engagement of young scientists in the program is important, and that the co-chairs of HPLC 2019 set the bar very high in this regard.
Posters represent a significant part of the HPLC symposia. In 2019, there were 508 submitted posters, separated into 14 scientific themes and 6 poster sessions. Agilent, once again, sponsored the "Best Poster Awards" that are graded by a group of expert reviewers for novelty, quality, presentation, and impact of the work (Figure 3).
HPLC 2019 was a lively symposium that engaged researchers interested in analytical science from around the globe. Most of the "hot topics" noted in recent symposium reviews remained the same at this year's conference. Three-dimensional (3D) printing, multidimensional chromatography, large-molecule separations and specific techniques such as HILIC continue to dominate discussions. The 2019 program also included two sessions focused on thin-layer chromatography.
The most significant impact from HPLC 2019 may be the focus of engaging our young scientists. From innovative programs such as the HPLC tube to the inclusion of many young investigators in the organizational committee, the conference set a new and hopefully continuing precedence for encouraging, inspiring, and engaging our talented young scientists. Based on the success of HPLC 2019, I suspect this is not the last time we will see Italy host this important conference.
Coverage of such a large symposium is impossible without a great amount of assistance. The author would like to acknowledge the invaluable assistance of Dr. Jason Anspach, Dr. Michael Dong, Professor André de Villiers, Professor Dwight Stoll, Cory Muraco, Professor David McCalley, Professor Deirdre Cabooter, Benjamin Peters, Dr. Mariosimone Zoccali, Dr. Martina Catani, Paul Connolly, and Professor Andrea Gargano for providing notes, text, insights and fruitful discussions regarding the content of various sessions.
(1) D.S. Bell,LCGC North Amer.35(9), 650–658 (2017).
(2) D.S. Bell, D.S., C.E. Muraco, and C. Flannery, LCGC Europe31(11), 628–634 (2018).
(3) S. Dimartino, U. Simon, and Q. Jiang, "Experimental and Modelling Design of 3D Printed Stationary Phases," presented at the 48th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Milan, Italy, 2019.
(4) N. Abdulhussain, et al., "Multidimensional Separation in 3D-Printed Devices by Fused Deposition Modeling," presented at the 48th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Milan, Italy, 2019.
(5) S. Nawada, et al., "Using 3D-Printing for Multi-Dimensional Spatial Separations," presented at the 48th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Milan, Italy, 2019.
(6) P. Sosnowski and G. Hopfgartner, "3D Printing as a Flexible Tool to Customize Liquid Introduction with Electrospray Mass Spectrometry," presented at the 48th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Milan, Italy, 2019.
(7) C. Salmean and S. Dimartino, Chromatographia82(1), 443–463 (2019).
(8) W. Chen, et al., "New Sub-Two Micron BIOSEC particles for High Resolution and High Speed Separation of Monoclonal Antibodies in HPLC," presented at the 48th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Milan, Italy, 2019.
(9) T.W. Chu, LCGC North Amer.37(6), 352–367 (2019).
(10) M. Lämmerhofer, et al., "Lipidomics: A Window of Opportunity for Clinical Analysis," presented at the 48th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Milan, Italy, 2019.
(11) K. Sandra, "Advances in the Characterization of Biopharmaceuticals," presented at the 48th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Milan, Italy, 2019.
(12) Lynen, F., Recent Advances in Comprehensive Temperature Responsive Liquid Chromatography, presented at the 48th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Milan, Italy, 2019.
(13) M. Baert, S. Martens, G.Desmet, A. de Villiers, F. Du Prez, and F. Lynen, Anal. Chem. 90(8), 4961-4967 (2018).
(14) De Villiers, A., et al., "Incorporating Ion Mobility Spectrometry into One and Two-Dimensional LC-MS Workflows for Phenolic Analysis," presented at the 48th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Milan, Italy, 2019.
(15) M. Rosés, "Characterization of HILIC Retention by the Solvation Parameter Model. Comparison to Reversed and Normal Phase Retention," presented at the 48th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Milan, Italy, 2019.
(16) D.V. McCalley, "Understanding and Managing the Equilibration Time in Hydrophilic Interaction Chromatography," presented at the 48th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Milan, Italy, 2019.
(17) J.C. Heaton, N.W. Smith, and D.V. McCalley, Anal. Chim. Acta1045, 141–151 (2019).
(18) D.V. McCalley, J. Chromatogr. A1554, 61–70 (2018).
(19) A.F.G. Gargano, G. van Schaick, T. Senard, F. Guidoni, O. Schouten, M. Akeroyd, N. Abello, B.W.J. Pirok, L. Roca, R. Haselberg, M. Wuhrer, E. Domínguez-Vega and G.W. Somsen, "Sweet and Sour: Analysis of Intact Proteins by HILIC-MS," presented at the 48th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Milan, Italy, 2019.
(20) A.F.G. Gargano, J.B. Shaw, M. Zhou, C.S. Wilkins, T.L. Fillmore, R.J. Moor, G.W. Somsen, and L. Paša-ToliÄ, J. Proteome Res. 17(11), 3791–3800 (2018).
(21) G. van Schaick, B.W.J. Pirok, R. Haselberg, G.W. Somsen, and A.F.G. Gargano, J. Chromatogr. A 1598, 67–76 (2019).
(22) M. Zoccali, M., et al., "Carbon Dioxide-Based Extraction and Separation Techniques for the Analysis of Carotenoids and Their Derivatives," presented at the 48th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Milan, Italy, 2019.
(23) O. Ismail, "Exploring the Potential of Zwitterionic Teicoplanin-Based CSPs by Using Macroporous Superficially Porous Silica Particles 2.7 µm 160Å AND 3.4 µm 400Å," presented at the 48th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Milan, Italy, 2019.
David S. Bell is a director of Research and Development at Restek. He also serves on the Editorial Advisory Board for LCGC and is the Editor for "Column Watch." Over the past 20 years, he has worked directly in the chromatography industry, focusing his efforts on the design, development, and application of chromatographic stationary phases to advance gas chromatography, liquid chromatography, and related hyphenated techniques. His main objectives have been to create and promote novel separation technologies and to conduct research on molecular interactions that contribute to retention and selectivity in an array of chromatographic processes. His research results have been presented in symposia worldwide, and have resulted in numerous peer-reviewed journal and trade magazine articles. Direct correspondence to: LCGCedit@ubm.com