Highlights of HPLC 2013

September 1, 2013
Ronald E. Majors
Ronald E. Majors

Ron Majors, editor of "Column Watch" and "Sample Prep Perspectives," has been with LCGC North America for over 26 years. Currently a senior scientist with Agilent Technologies, Wilmington, Delaware, Ron is known industry-wide as one of the premier chromatography experts in the field. He is also a member of LCGC's editorial advisory board.

LCGC Europe

LCGC Europe, LCGC Europe-09-01-2013, Volume 26, Issue 9
Page Number: 505–516

A review of the trends at this year's symposium, including discussions of column technology, sample preparation, and detector usage.

The 39th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2013) was held 16–20 June in Amsterdam, the Netherlands, for the second time. This instalment of "Column Watch" covers some of the technology and application advances discussed at HPLC 2013. We review the overall liquid phase chromatographic trends, provide a summary of the awards presented, and 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 16–20 June, 2013, in Amsterdam, the Netherlands, for the second time, and for 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. Co-chaired 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 could indicate that their economic crisis may be abating.

The venue for HPLC 2013 was the colossal RAI building, location for 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.

In this instalment of "Column Watch," I will present some additional scientific highlights of HPLC 2013. This report also covers the various awards and honourary 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 instalment.

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 and 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) which 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 1 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 1: 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 Honours 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, USA. 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 fibre electrodes of extremely low detection volume) and analysed 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 were also 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 Labelling Strategies Using ITRAQ and TMT"; Melissa Phillips of NIST, Gaithersburg, Maryland, USA, "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 Brussels, Belgium, "Design of Cyclic Olefin Copolymer-Based Microfluidic Devices Designed for Spatial Two- and Three-Dimensional Chromatography"; Daniel Wilffert of the University of Groningen, the Netherlands, "Quantitative, Antibody-Free LC–MS–MS Analysis of Recombinant Tumour 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, 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, USA, 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, USA, 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 (FFF), and HPLC column technology and has already received numerous major awards, patents, and publications for his work. The concept of SPPs 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 than 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 tumour 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, the 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 must usually know ahead of time what they are 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 can 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.

New Column Technology Highlights

Many oral, poster, and tutorial sessions were devoted to stationary phases and column technology, always topics of high interest at this series of meetings.

Superficially Porous Shell Particles: From observations made at HPLC 2013, a continuing "hot topic" for the third year in a row was the application of SPPs for developing faster separations and generating more column efficiency at lower pressure drops than the smaller porous particles that they replace. Four years ago, the focus was on comparing sub-2-µm totally porous columns to porous particles in the 2–3 µm range and larger. Three years ago the discussions were focused on the superiority of superficially porous columns over sub-2-µm particle columns. Last year the focus was on the theory and mechanisms on how SPP work relative to the sub-2-µm packed columns. This year's focus seemed to be mostly on the SPPs themselves. There are still differences in the nomenclature, these particles were referred to as SPPs and fused-core, shell, porous shell, solid core, and poroshell particles. It is now generally accepted that SPPs bring the greatest advantage to users over the smallest porous particles (<2 µm) and, at HPLC 2013, barely a whisper could be heard on the latter technology that spurred the whole topic of UHPLC. The only question now: How soon will both larger and smaller (perhaps down to 1-µm diameter) SPP in various pore sizes and different selectivities be made available? Table 2 gives a brief update on the current status of commercial SPP products for large and small molecules as of HPLC 2013.

Table 2: Status of superficially porous particles as of HPLC 2013.

Users who have already purchased UHPLC equipment are less concerned about the lower pressure drops of these columns but difficult separations, long columns, and lots of theoretical plates are always in demand. Hence, pressure availability is always a concern. Currently, many of the SPP columns on the market do not have the upper pressure limits of some of the UHPLC instruments, but that should change in the near future. However, one aspect of the UHPLC systems that users should be aware of is their greatly decreased extracolumn band dispersion that was optimized for the sub-2-µm columns, especially if interested in columns with smaller internal diameters, such as 2 mm and under.

The majority of the commercial SPPs have particle diameters in the range of 2.6–2.7 µm. Besides the movement of SPPs to larger particle sizes as outlined earlier by Kirkland, Carl Sanchez and colleagues from Phenomenex presented their studies on 1.3-µm core–shell reversed-phase particles that provide efficiencies approaching 450,000 plates/m (equivalent to ~22,000 plates for a 50 mm × 2.1 mm column). Users feared that frictional heating would be a problem with such small particle columns but core–shell phases provide improved thermal conductivity compared to porous particles of the same dimension so this has not been a factor. In the worst case, for a 50 mm × 2.1 mm column, a radial temperature gradient of 1.5 °C was measured, but in most cases the temperature rise was less than one degree. In a gradient mode, these SPP columns showed a 36% improvement in peak capacity (the best measure of gradient "efficiency") over a 1.7-µm bridged ethyl hybrid column for a bovine serum albumin digest. Although these columns offer some of the highest efficiencies on the market, instruments may not have low enough extracolumn variance to make full use of their power.

The new Waters Cortecs SPP product was introduced at HPLC 2013. The particle diameter is 1.6 µm and the core is 1.1 µm, making the shell 0.25 µm. The plate count for a 50 mm × 2.1 mm column was given as 19,700, about 39% higher than the company's BEH 1.7-µm particle column. The Cortecs column has a higher external porosity (and thus a lower bed density), resulting in a lower back pressure such that the 1.6-µm shell particle column has about the same back pressure as their 1.7-µm porous particle column. It was reported that the new core–shell particle is based on conventional silica rather than the bridged ethyl hybrid chemistry. Similar to other SPP products, the outer surface is rather bumpy. The particle distribution is narrow like other SPPs. The pore size is 90 Å, making it suitable for small molecule separations. Three surface chemistries are provided: C18, C18+ (similar process to charged surface hybrid), and bare silica, with the bonded phases having trifunctional bonding. New low dispersion column hardware is available.

James Treadway of the University of North Carolina in Chapel Hill, North Carolina, USA, reported on the difficulty of translating shell particles to a capillary format. Using 30-, 50-, and 75-µm capillaries with a special carbon fibre microelectrode (no band spreading), they packed sub-2-µm experimental shell particles using an acetone-based slurry. The particles were retained using sintered particles as frits. The smallest internal diameter with a 31.7-cm length gave the best performance of 162,000 plates with a reduced plate height (h) of 1.27. For the largest internal diameter, the performance was reduced with h = 1.65 but the particles were slightly different. Other workers have found that packing smaller internal diameter columns with either fused-core or totally porous packings give less efficient columns than for larger internal diameters.

New Column Materials and Related Studies: Each year at this meeting, new column materials are presented in oral research papers, applications papers, poster papers, and at exhibitors' booths. This year was no exception. Tony Edge from Thermo Fisher Scientific presented a paper coauthored with Peter Myers of Liverpool University, Liverpool, UK, and others that outlines a totally new particle called a sphere-on-sphere (S-o-S) silica particle (4). Unlike some of the SPP preparation procedures, the S-o-S particles are manufactured using a one-step synthesis delivering a nearly monodisperse particle and core–shell morphology. The morphology of the particle has been designed to deliver the real advantages of the core–shell particles. The total diameter of the particle and the core diameter can be controlled together with the effective pore diameters on the surface of the material ranging from 3 to 10 µm with an effective pore size range up to 1.5 µm. The pressure drops observed for packed columns is quite low compared to traditional totally porous particles of similar size. Examples have been shown with bonded-phase S-o-S particles used in normal-phase and reversed-phase separations of small molecules and also proteins. Examples were shown of how these particles can be used as scaffold particles or carrier particles in which new, novel stationary phases can be trapped in the outer layer.

Jeremy Glennon and coworkers extended their work on core–shell particles by incorporating nanoparticles of gold and silica into fused-silica columns and, by using capillary electrophoresis, enhanced selectivity was shown for low-molecular-weight biomarkers. With amperometric detection they used a boron-doped diamond electrode that gave greater sensitivity. They also incorporated latex nanoparticles on core–shell and then used them for anion exchange separations on a chip. By coating a fused-silica capillary with gold nanoparticles embedded in a cationic polymer, they were able to separate catecholamine metabolites from urine and aminothiols in plasma. In a related poster paper, they modified a core–shell particle with a chelating functionality (iminodiacetic acid) and used the packing for the high performance chelation ion chromatography (HPCIC) separation of transition and heavy metals. An isocratic separation of a mixture of 14 lanthanides and yttrium required just 8.5 min with one of these core–shell chelators.

Kazuki Nakanishi and coworkers from Kyoto University and GL Science in Japan reported on recent developments in a macroporous poly(methylsilsesquioxane) (PMSQ) monolith family that has produced a novel type of siloxane-based macroporous polydimethylsiloxane (PDMS) analogues suitable for many kinds of sample preparation purposes. These so-called "marshmallow gels," very soft, low density, highly hydrophobic, and highly permeable monoliths with micrometre-range continuous pores, are synthesized by controlled copolymerization of methyltrimethoxysilane and dimethyldimethoxysilane (MTMS-DMDMS) precursors. Because of the presence of dense surface methyl groups, all the surfaces of micrometre-range skeletons are highly hydrophobic, exhibiting a similar or faster equilibration rate than those of other PDMS-based solid-phase extraction (SPE) devices. Physical properties are comparable to conventional PDMS materials; marshmallow gels exhibit no degradation under temperatures as high as 600 K, and no obvious glass transition in the temperature range as low as 150 K. Furthermore, even under liquid nitrogen (77 K) the material still exhibits rubbery elastic deformation and recovery. In the above broad temperature range, the extraction of hydrophobic substances from a polar solvent system can be dramatically accelerated by absorbing the mixture and simply squeezing it out. The marshmallow gels may find use in both in GC and LC sample preparation purposes. Not limited to the PDMS analogue, the chemical modification of the pore surface is easy by choosing the appropriate precursors. Typical processing requires only a few hours in any shape and size below 100 °C.

Chuck Lucy and colleagues from the University of Alberta talked about carbon phases for ion chromatography (IC) and HILIC. In general, IC gives nice separations of inorganic ions but longer separation times because of the large particle size of the packing materials used. Using small-particle pyrolytic graphitized carbon (Hypercarb, Thermo Fisher Scientific) as a base material, they modified the surface with aromatic carboxylic groups onto the surface via diazonium chemistry and used these modified columns for HILIC applications. These columns appear to have a unique selectivity to commercially available HILIC phases because of the interaction with the underlying carbon. The column is stable in 100 mM sodium hydroxide and provides good separations at high pH unlike silica-based HILIC phases.

Monoliths: Monolith columns have long been desirable because they exhibit high permeability and low pressure drop (a result of increased bed porosity), show reasonable separation efficiencies, require no frits to confine the packing material, are easy of fabricate, and nowadays can be made fairly reproducibly. Although this technology has been around for several years, as a routine tool it has yet to see widespread acceptance on the commercial side but improvements continue to be made for both polymer- and silica-based monoliths. Although a number of papers and two excellent tutorials discussed the use of monoliths in various applications, unfortunately, there were few fundamental studies reported on monoliths at HPLC 2013.

Emily Hilder and coworkers from the University of Tasmania in Hobart, Tasmania, Australia, gave a keynote lecture on designing polymeric monoliths for chromatography of large and small molecules. Since their introduction more than 20 years ago, polymer monoliths have been shown to provide excellent separation performance for the separation of large biomolecules. Despite incredible promise based on both the ease and flexibility of synthesis, and favourable flow properties, for many people polymer monoliths have failed to live up to the "hype". In particular, the question still remains whether their performance for separations of a wider range of molecules can compete with particle packed columns. Hilder's presentation introduced a wide range of synthesis methodologies and applications that their laboratory has explored to improve both the separation efficiency and selectivity for both small and large molecules using polymer monoliths, demonstrating that these materials can be used for high performance applications. These include grafting phases in layers, incorporating two different functionalities into a single monolith, new synthetic approaches such as the incorporation of nanoparticles into the monolithic structure, or synthesis using cryopolymerization approaches (with and without unidirectional freezing), as well as approaches to extend the operating conditions for these column types, particularly through the use of very high temperature gradients (at temperatures up to 200 °C) or rapid pulses. Separations were shown for both small (for example, inorganic ions and small organic molecules) and large (such as proteins and biopharmaceuticals) molecules. Examples were also provided in which the performance of polymeric monolithic columns was superior to that of particle packed columns.

Although it is a widely used related technique, results obtained using thin layer chromatography (TLC) are not often presented at a HPLC conference. Martin Medal winner Frantisek Svec gave a keynote lecture on monolith columns for TLC separations. In its basic use, this very simple yet powerful separation method does not require any sophisticated instrumentation. His group developed glass-supported monolithic thin layers and used them for the separation of small molecules, peptides, and proteins in both one-dimensional (1D) and 2D formats. Svec first shared with the audience several tricks that they were using to prepare well performing layers from a variety of monomers. Whereas poly(butyl methacrylate-co-ethylene dimethacrylate) and poly(glycidyl methacrylate-co-ethylene dimethacrylate) layers with optimized porous structure were prepared using ultraviolet (UV)-initiated polymerization, poly(styrene-co-divinylbenzene) and poly(4-methylstyrene-co-chloromethylstyrene-co-divinylbenzene) monolithic layers had to be prepared using thermal initiation because these monomers are not UV-transparent. UV-initiated grafting was also used to manipulate the pore surface chemistry. In one of the examples showing excellent performance, they used a moving shutter to spatially control the exposure to UV light, which initiated photografting. This process formed a diagonal gradient of hydrophobic lauryl methacrylate on the top of a hydrophilic monolith. This novel, simple concept was used for the separation of peptides that encountered the gradient during each of the two sequential developments in a 2D regime. In a different experiment, the poly(4-methylstyrene-co-chloromethylstyrene-co-divinylbenzene) layer was hypercrosslinked using Friedel-Crafts alkylation reaction to create a multiplicity of mesopores and significantly increase the surface area. This thin layer then enabled excellent separations of small molecules.

HILIC: HILIC is still one of the most popular chromatographic techniques, particularly for small polar analytes that are eluted from reversed-phase columns way too fast. One of the most interesting papers studying the mechanism of HILIC separations was presented by Ulrich Tallarek of Philipps University in Marburg, Germany. He modelled the entire HILIC operation. It is generally agreed that the mechanism of HILIC retention involves the water-rich layer at the polar stationary phase boundary. He contends that retention in HILIC shows a shallow increase with increasing acetonitrile, but then a sudden leap in retention occurs at 75–95% acetonitrile. Water and acetonitrile prefer to be in an environment of their own kind (like prefers like). He postulated that there are three distinct regions in the pores:

  • Immediate surface region: The rigid layer; here the silanols strongly adsorb water. This region is "ice-like" where the water molecules have a long residence time that may be 100 times larger than in the bulk region. The composition of water is unchanged no matter what the concentration of water is in the mobile phase. Here, the water molecules are all bound to the silanols.

  • Intermediate (interface) region: The diffuse layer; this region responds somewhat to the water concentration in the mobile phase but lags behind it. Here it quickly decays to water-water hydrogen bonds. It gradually relaxes to the bulk bonded phase.

  • Bulk region: In the bulk region, there is some hydrogen bonding between water and the acetonitrile.

  • Even at 95% acetonitrile, the water molecules are mostly in contact with other water molecules. Methanol is much more like water so it behaves quite differently in HILIC compared to acetonitrile. The ratio of water mole fraction at or near the surface and the bulk water concentration grows nonlinearly with the acetonitrile fraction in the mobile phases and ends with a "jump", thereby explaining the sudden leap in retention at higher acetonitrile concentrations. Regions 1 and 2 make up about 56% of the pore volume.

Column Hardware Design: Every once in a while a new column hardware design comes along that proves quite interesting. This year's HPLC meeting saw an innovative concept introduced by Andrew Shalliker of the University of Western Sydney in Australia. His talk was entitled "Active Flow Technology Presenting the Infinite Diameter Column in a Curtain Flow Mode of Operation." Many years ago, Professor John Knox noted that an infinite-diameter column would be the best type of column of all because the analyte injected into the centre of a column would never reach the walls where the flow velocity profile is upset by the particle–wall interface. So for many years injectors were designed to inject the sample into the centre of the column. Most likely though, the analytes did diffuse in a radial direction and did reach the wall to disrupt the nonparabolic flow that one would like. Curtain flow technology means segmenting the injection end of the column to ensure the analyte sees the middle of the packed bed. Active flow technology relies on sampling the column effluent in the very middle of the column. How is this done? Figure 2 is a special column hardware design used to sample flow near the walls as well as in the middle of the elution profile so that the parabolic flow pattern could be sampled. By segmenting the flow and selecting just the middle portion, improved efficiency was obtained by this virtual "wall-less" column.

Figure 2: Design of active flow technology column. Courtesy of Andrew Shalliker of the University Of Western Sydney in North Parramatta, Australia.

If you have 21% of the total flow on a 4.6-mm i.d. column passing through its centre, you emulate a 2.1-mm virtual column. If you have 43% of the total flow going through the centre then you emulate a 3.0-mm virtual column. These observations are the same as cutting the parabolic Poiseuille flow profile, especially evident at a high flow rate. Shalliker first showed data for a regular monolithic column with normal endfittings with 107,116 total plates with a tailing factor of 1.5. With the curtain flow technology, the same column resulted in a total plate count of 111,813 and a tailing factor of 1.14, a definite improvement especially with the latter. He also indicated that a column packed with SPPs behaved better than a column packed with totally porous particles.

Extracolumn Contributions for Modern High Efficiency Columns

Extracolumn contributions to band spreading has also become an important topic with the high efficiency SPP and totally porous columns now commonplace. Extracolumn contributions are defined as any volume outside of the column bed itself from the point of sample introduction to the exit of the detector and includes the injector, connection capillaries, endfittings including frits, and the detector cell volume. Most older instruments do not have the ability to effectively work with these modern columns and if such a column is installed into the chromatograph, it will perform poorly. When column volumes decrease, such as what is encountered when moving to short 2.1- or even 1.0-mm i.d. columns, the problem becomes magnified. Adding to this topic of extracolumn dispersion effects highlighted earlier by the Horváth Award winning lecture, was a keynote lecture by Monica Dittmann of Agilent Technologies, where she showed the practical limitations of further decreases in extracolumn effects such as the high pressure generated by small-internal-diameter capillary tubing (for example, ~50-µm i.d.). For low k' (such as <3) analytes on 2.1- and 1.0-mm i.d. columns, the extracolumn variance has a larger impact in isocratic separation than in gradient work, and for a 50 mm × 2.1 mm column with modern SPPs or sub-2-µm TPPs, the variance must be reduced to below 1 µL2 to realize the full potential of today's high efficiency columns. For gradient separations, the external contributions have a smaller influence because of the larger peak volumes and the focusing of the analytes at the head of the column. For UV detection, she showed that 250-nL flow cells may be preferred over 800-nL flow cells for fast gradients and low k' isocratic analysis. Fixed-loop injectors may not be the best for low dispersion and therefore newer autosampler needle seat designs with a low variance contribution (~1.5 µL2) may be the answer. When using electrospray ionization (ESI)-MS, peak capacities are comparable to optimized UV detection, but when low sampling rates are used in MS, the peak capacity can be lower for fast gradients in high efficiency columns.

Gert Desmet of the Free University of Brussels in Belgium presented a tutorial on system performance. He put forward an overview of the underlying theory needed to understand the influence of the instrument design (system and dwell volume, oven design and operating mode, flow rate, and pressure range of the pump) on the observed column performance. A useful part of his lecture showed the audience how to calculate the extracolumn variances and other practical parameters of the various parts of HPLC and UHPLC systems. These calculations may be of importance because of the difficulties of measuring these quantities experimentally. Some useful equations were presented. For example, to calculate the pressure (ΔP) imposed by empty capillary tubing, equation 1 was presented:

where η is the mobile phase viscosity, L the column length, F is the flow rate, and d the internal diameter.

Pressure Effects in HPLC and UHPLC

Since the advent of UHPLC and with pressures sometimes greatly exceeding the 400-bar limits of yesteryear, the impact of column pressure on retention has brought about concerns that were never expressed before. We briefly addressed the myth "pressure has no effect on retention time" in a recent article (2). This year at HPLC 2013 two notables in the field, David McCalley of the University of the West of England in Bristol, United Kingdom, and Nobuo Tanaka, now with GL Science in Japan, presented papers on pressure effects in UHPLC.

McCalley evaluated retention time changes with different pressures for five different 5-µm stationary phases, all with the same mobile phase, using a restriction capillary at the end of his system. He confirmed Martin and Guiochon's results on the effects of the compressibility of the organic portion of the mobile phase. He also showed that almost all solutes showed the same effects. As the pressure increased from 53 bar to 246 bar there were some retention time changes resulting in some peak elution reversals that he interpreted as coming from selectivity changes, although efficiency (N) was not affected. Comparing "rigid" to "flexible" molecules, there are more changes in retention time for the more rigid (planar) molecules with pressure increases. When comparing monomeric and polymeric stationary phases, different effects are seen, with the polymeric stationary phase showing more changes. Within this category, polar and ionizable compounds show larger effects than nonpolar ones, although size also has an influence.

Tanaka investigated pressure effects from 7.6 MPa to 48 MPa on the separation of monounsaturated fatty acids, using a polymeric stationary phase. Peak symmetry is better at higher pressures. A similar effect was seen with fatty acid methyl esters (FAMEs). Comparing a monomeric C18 stationary phase with a C30 stationary phase, he reported that the C30 showed reasonable separations for some FAME and some triacylglycerols while the C18 phase was not selective. On the other hand, the monomeric C18 could effect separation of the α, β, and γ tocopherols, although polymeric C18 was better. The elution order is different for the tocopherols on C18 versus C30. One interesting observation was that lower temperatures improved separation on the C30 phase by increasing retention time and that combining lower temperature with increased pressure gives even better results.

Tanaka suggested three categories of pressure effects on compounds:

  • Neutral nonpolars — small effects

  • Weak acids and bases near their pKa — changes of pKa with pressure can cause changes in retention

  • Ions, neutral hydrophilic compounds

The outcome of his lecture seemed to be that certain selective stationary phases can give interesting changes in selectivity with pressure. Increasing the pressure can increase the retention of a favoured isomer. The C18 and C30 stationary phases gave a big difference for flexible molecules. If there is a closer distance between the solute and stationary phase caused by pressure increases, dispersive interactions will increase. Nonselective stationary phases will not show these differences.

Multidimensional and Comprehensive Liquid-Phase Separations

This year multidimensional separations were a hot topic. In the 2D short course, presented the weekend before HPLC 2013, more than 50 conferees elected to come early to Amsterdam to learn about the latest technology. One of the better educational-themed presentations was that of Paola Dugo of the University of Messina in Messina, Italy, whose tutorial "Comprehensive 2D LC" pointed out some of the problems encountered including

  • Coupling of different modes is difficult.

  • Problems are encountered with immiscible solvents in the two modes.

  • Specific software is needed.

She amply pointed out that in heart-cutting chromatography equation 2 holds:

where Nc is the total peak capacity of the combined LC columns, c1 and c2, whose individual peak capacities are Nc1 and Nc2, respectively, while in comprehensive LC (usually depicted as LC×LC), equation 3 holds:

which shows the strong separation power of comprehensive chromatography.

To obtain high 2D resolution, each peak in the first dimension should be sampled at least 3–4 times to prevent the loss of 1D information. You have to think that if you have a 1D peak with three components and three different retention times, then each component will show in 2D, but to different extents.

To connect the two columns, a 10-port valve with two loops can be used. One can use two sampling loops although two trapping columns may also be used. In the former case, the loops must be large enough to hold the entire volume content from the effluent of column 1 while the complete separation of the contents of the other loop is being injected onto and separated on column 2. Generally, in the final setup, one detector is needed because all the material from 1D is transferred to 2D; thus, there is no need for a 1D detector. Generally, the 1D column is long and operated at low flow. The 2D column must provide fast analysis, usually a couple of minutes or less. Here, a narrow-bore column can be used to prevent sensitivity loss. Fast gradients are very common. The column needs to have a fast equilibration time so core–shell, monoliths, or sub-3-µm columns are frequently used. It is possible to use an array of 2D columns in parallel. The problem with that approach is that two columns are rarely identical. However, if you use two columns in the 2D, you can double the 2D analysis time because the one 2D column can be reconditioned while the first 2D column is performing the analysis. Usually the modulation time is 30 s to 2 min.

Dugo also showed some nice food applications examples in her presentation including lipid analysis (such as triacylglycerols and phospholipids) and a combination of reversed-phase (separations based on hydrophobicity) and argentation (Ag+) chromatography (separations based on degree of unsaturation).

Kelley Zhang of Genentech talked about 2D LC applications in pharmaceutical chemistry. Some of her practical applications included excipient analysis (for example, mannitol and lactose) by HILIC, salt in active pharmaceutical ingredients (APIs) using multimodal columns, a stability indicating method, quantification of coeluting impurities, separation of multiple chiral centre compounds, and 2D LC–MS switching from a non-MS-compatible mobile phase to a MS-compatible mobile phase.

Thorsten Teutenberg and coworkers from the Institut für Energie- und Umwelttechnik e. V., Duisburg in Duisburg, Germany, strongly support the use of LC-LC (selective fraction collection) or LC×LC (continuous fraction collection) to reduce dependence on the "magic" of MS, especially for environmental samples. The use of nanoLC in both dimensions helps reduce the mobile phase "incompatibility" problem. His application example was wastewater in which he encountered >60 compounds.

HPLC 2013 (Australia) and HPLC 2014 Are Next

The next major symposium in this series, the 40th Symposium also held in 2013, will be in Hobart, Tasmania, Australia, from the 18–21 November. The chairman of this meeting will be Paul Haddad, who will be retiring after HPLC 2013 Australia. For more information go to www.hplc2013-hobart.org. In May 2014, the series returns to the United States for the 41st Symposium. HPLC 2014 will be held in New Orleans, Louisiana, USA, always a favourite place to visit. The Symposium Chair will be J. Michael Ramsey of the University of North Carolina in Chapel Hill, North Carolina, USA. You can learn about this meeting at the following website: www.hplc2014.org. Bookmark both of these websites so that you can keep up on the latest happenings.

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.

"Column Watch" Editor Ronald E. Majors is a senior scientist at the Columns and Supplies Division, Agilent Technologies, Wilmington, Delaware, USA and is a member of LCGC Europe's editorial advisory board. Direct correspondence about this column should be addressed to "Column Watch", LCGC Europe, 4A Bridgegate Pavilion, Chester Business Park, Wrexham Road, Chester, CH4 9QH, UK, or e-mail the editor-in-chief, Alasdair Matheson, at amatheson@advanstar.com

References

(1) R.E. Majors, LCGC Europe 25(9), 490–511 (2012).

(2) R.E. Majors, LCGC Europe 24(9), 468–483 (2011).

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

(4) A. Ahmed, W. Abdelmagid, H. Ritchie, P. Myers, and H. Zhang, J. Chromatogr. A 1270, 194–203 (2012).

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