Highlights of HPLC 2011

HPLC 2011 was held June 19–23 in Budapest, Hungary, for the first time in its long history. Columnist Ron Majors attended the event and reviews some of the advances in technology and new applications presented. In this installment of "Column Watch" he reveals the overall trends in liquid-phase chromatography observed at the symposium, as well as the awards presented, the opening plenary session, column technology highlights, an update on comprehensive liquid chromatography (LC×LC), and detector usage.

The 36th International Symposium on High Performance Liquid Phase Separations and Related Techniques, which alternates between Europe and North America, with occasional side meetings in Japan and China, was held June 19–23, 2011, in beautiful Budapest, Hungary. More affectionately known as HPLC 2011, the symposium is a major scientific event for bringing together the myriad techniques related to separations in liquid and supercritical fluid media.

Chaired by Professor Attila Felinger of the University of Pécs (Hungary) with an Honorary Chairman László Szepesy of the Budapest University of Technology and Economics (Hungary), HPLC 2011 assembled 1332 participants from all over the world. This number included vendor representatives from more than 60 exhibitors for the three-day instrument, software, and consumables exhibition. Students represented a good proportion of the conferees, which is a good omen for the future of separation science. Based on the number of attendees and exhibitors, the worldwide economic crisis did not play heavily into the support for this important conference. The attendance was up about 10% from HPLC 2010 (1).

HPLC 2011 had 135 oral presentations in plenary and parallel sessions and 789 posters in sessions with 21 themes. With an ample social event schedule, 13 vendor workshops (some with free snacks), nine tutorial educational sessions, and six short courses (held during the previous weekend), attendees had their hands full deciding how to best allocate their time. The tutorials were particularly well attended and covered current topics such as liquid chromatography–mass spectrometry (LC–MS), multidimensional chromatography (MDC), hydrophilic interaction liquid chromatography (HILIC), solid-phase microextraction (SPME), microfluidics, chemometrics, supercritical fluid chromatography (SFC), and overpressured-layer chromatography (OPLC).

Trends in Liquid-Phase Technology and Techniques

Obviously, high performance liquid chromatography (HPLC), including its subset, ultrahigh-pressure liquid chromatography (UHPLC), was the predominant technology in the technical sessions at the symposium, but sample preparation and the use of capillary electrophoretic techniques showed strong growth. From a perusal of the poster and oral presentation abstracts, I broke down some of the major areas of coverage in this year's symposium. These tables are useful to spot trends in LC technology, applications of liquid phase separations, and detection principles that were introduced this year.

Table I provides a rough breakdown of the coverage of liquid-phase technology and techniques in the separation sciences. Compared with HPLC 2010 (1), some slight shifts in the emphasis of technology were noted. Again this year, new column technologies were predominant with nearly a third of the papers relating to column development, column studies, and column-related methods. Over a quarter of the columns' papers dealt with monoliths but this year papers on polymeric monoliths exceeded those of silica monoliths by 15%. Polymeric monoliths have now been tweaked to cover small molecules as well as large molecules. Column efficiency of the polymeric monoliths have also increased. Silica gel-based monoliths are experiencing their second generation of commercial products with better efficiency but with slightly higher pressure drops as a result of the change in the macropore–mesopore domain ratios.

Table I: HPLC 2011 papers presented by technology or technique

Three other "hot" areas in column technology this year were noted:

• There is widespread attention to HILIC for the separation of polar analytes that are not well retained by reversed-phase chromatography. More than 20% of the papers on column technology and a tutorial dedicated to HILIC were presented.

• More emphasis on the new breed of superficially porous packings (SPPs) — which are 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. A fifth of all papers on columns covered various aspects of SPPs, including a number of theoretical studies on where in the particle design and packing technology the high efficiency originates.

• The use of MDC, especially comprehensive MDC (LC×LC), where every fraction from column one is directed for further separation to a secondary column. With chromatographers encountering more complex samples, sometimes with thousands of compounds present, these multidimensional techniques are about the only way to tackle such mixtures. Many of the papers were devoted to actual applications of MDC.

The interest in new research into sub-2-µm porous packings has waned a bit this year, probably because these columns are now firmly established in the UHPLC world with nearly 30 vendors supplying these products (2).

Sample preparation technology was well represented in the poster papers. Most prominent were improved solidphase extraction (SPE) technologies with emphasis on new phase chemistries. Strong interest was noted in molecularly imprinted polymers (MIPs) and immunosorbents. The new technique of dried blood spotting (DBS) made its appearance. Tiny volumes (< 20 µL) of whole blood are spotted on filter paper and other media, allowed to evaporate and the analytes extracted from the dried blood spot for further separation and detection, usually by tandem MS techniques (3). The technique appears to be spreading to other biological fluids (for example, urine, plasma, and cerebral spinal fluid) so may be renamed as dried media (or matrix) spotting (DMS).

After a weak showing at HPLC 2010 (1), electrodriven separation techniques, such as caplllary electrophoresis (CE), capillary zone electrophoresis (CZE), micellar electokinetic chromatography (MEKC), and isoelectric focusing (IEF), showed a significant increase, and the number of papers more than doubled at HPLC 2011. A continued lack of interest in capillary electrochromatography (CEC) was noted with only eight presentations this year, the same as last year. Only a few years ago, lecture rooms on CEC topics spilled out into the hallway and the technology was forecasted to replace both HPLC and CE. However, the "killer" application for the technique was never found; any separation performed by CEC could be done by gas chromatography (GC), SFC, or LC without all the associated problems.

This year's symposium showed a small drop-off in papers devoted to theoretical aspects, retention mechanisms and the like, and a significant drop-off in papers dealing with new instrumentation and software. Perhaps the latter fall-off may have been a result of the decreased presence of American-based instrument company scientists which often happens when the HPLC series moves to Europe or Asia.

Applications

Table II is a breakdown of the most popular applications reported at HPLC 2011. The number of oral and poster presentations on proteomics, biomarkers, protein and peptide separations and identification again led the way, but fell as a percentage of the total compared with HPLC 2010 (1). I have noted previously that whenever the HPLC series is held in Europe, there is a decrease in papers devoted to the life sciences and this trend continues. There appears to be more life science research, at least in separations technology, being done in the US. The areas of major protein–peptide studies were in the separation and characterization of glycosylated, phosphorylated, and recombinant proteins, monoclonal antibodies, and various biomarkers.

Table II: Papers presented by application area

Pharmaceutical and biopharmaceutical assays in drug discovery, therapeutics, formulations, and active pharmaceutical ingredients (APIs) also had wide coverage this year. A trend noted was the increased reports on the separation of biopharmaceuticals, which reflected big pharma and startup companies' interest in nontraditional approaches in drug development. Work is progressing on understanding the retention mechanisms of peptides and proteins and research groups are attempting to predict retention times.

A strong growth area for applications was the use of HPLC, UHPLC, and other liquid-phase techniques to analyze drugs and their metabolites, endogenous compounds, and toxins in biofluids and tissues. Here the use of LC–MS-MS for trace analytes made a strong showing. The other application areas that also showed strong growth were separations of foods, beverages, and food safety; natural products and nutraceuticals; and environmental compounds. This increased attention to the safety of our food supply is no doubt a result of reports on toxic food contaminants continually making the headlines.

In this installment, I will present some additional scientific highlights of HPLC 2011. This report will also cover some of the awards and honorary sessions that took place.

It would be 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 paper.

Awards and Honors

Horváth Award: For the fifth year in a row, the Horváth Award sessions, named after the late Professor Csaba Horváth, one of the founders of this series, a mentor of young scientists, and notable member of the Hungarian chromatography "mafia," were featured. This award, 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, based on the best oral lecture presented in the Horváth Sessions, was selected by a jury named by the Permanent Scientific Committee and consists of a cash prize, invitation to present an oral at HPLC 2011, and a trophy.

This year's winner was Matthias Verstraeten, a graduate student in the laboratory of Gert Desmet, Vrijie Universiteit (Brussels, Belgium). Coauthors were Matthias Pursch and Patric Eckerle of Dow Deutschland (Rheinmunster, Germany) along with Jim Luong of Dow (Ft. Saskatchewan, Canada). Verstraeten's presentation was entitled "Novel Thermal Modulation for Multi-Dimensional Liquid Chromatography Separations using Low-Thermal-Mass LC." In his Horváth Award presentation, Verstraeten used a concept borrowed from GC×GC in making LC×LC a more efficient process. Using a thermal modulation device consisting of an aluminum low-thermal-mass LC heating sleeve containing a capillary packed with highly retentive porous graphitic carbon (PGC) placed between the primary column and the secondary column, he was able to "cold" trap analytes eluted from the first column and then by rapidly heating the capillary (1800 °C/min), elute them in a narrow band onto the secondary column.

Compared with a non-thermally modulated system, he was able to get an increase in peak response by a factor of 35. This interface device not only preconcentrates but also serves as an injector for the second dimension. This oral paper was a follow-up to their third place finish in the Best Poster Award competition at HPLC 2010 in Boston (1). Interestingly, Matthias also won the best poster award (see later) at HPLC 2011, quite a remarkable achievement in his young career.

Poster Sessions and Best Poster Awards: The mainstay of HPLC 2011 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 2010 (1), the number of posters increased from 585 to 789, an increase of 35%. The European posters are in portrait configuration so they could be positioned in very close proximity, but sometimes it was a struggle for multiple people to view them. The posters were split into two two-day sessions; by staggering the days that the posters were staffed by presenters, the sessions did not appear to be too crowded. Unfortunately, the posters were located in two buildings, one of which was quite far from the lecture halls and exhibition. Posters that were placed in the remote building didn't get as big of an audience as the ones closer to the main activities.

The Poster Committee co-chairs at HPLC 2011 were Peter Schoenmakers, a member of the Permanent Scientific Committee from the University of Amsterdam, (The Netherlands) and Gerard Rozing from Agilent Technologies (Waldbronn, Germany). The 56 reviewers of the Poster Committee devoted a great deal of time and worked very hard to narrow down the nearly 800 posters to 26 nominations by the end of the third day. Selection criteria were based on three elements: inspiration (for originality, newness of the work), perspiration (for the amount of effort put in), and presentation (general presentation, explanation by the authors). The nominated posters were finally reviewed by a number of committee jurors to select three winners and six excellent posters by the Thursday afternoon of the symposium. The Best Poster Awards, sponsored by Agilent Technologies (Wilmington, Delaware), were announced at the Closing Session.

This year's winning poster was entitled "Switching from Constant Flow Rate to Constant Pressure Elution Mode" by senior author Matthias Verstraeten (pictured in Figure 1), along with coauthors Ken Broeckhoven and Gert Desmet of the Vrijie Universiteit (Brussels, Belgium); Frederic Lynen and Pat Sandra of the Pfizer Analytical Research Centre (PARC) in Gent, Belgium; Konstantin Choikhet, Monika Dittmann, and Klaus Witt of Agilent Technologies (Waldbronn, Germany); and Klaus Landt, Research Institute for Chromatography (RIC) in Kortrijk, Belgium.

Figure 1: Group photo of the award winners: from left to right, Professor Peter Schoenmakers, University of Amsterdam (The Netherlands), co-chairman of the Poster Committee; Matthias Verstraeten, Free University of Brussels, Belgium. (combined Horvath Young Investigator Award winner and First Place Best Poster Award); Dieter Verzele of the Pfizer Analytical Research Center, Department of Organic Chemistry, Gent University, Belgium (Second Place, Best Poster Award); and Andrea Gargano of the University of Vienna (Vienna, Austria) (Third Place, Best Poster Award).

This award-winning work discussed the chromatographic effects when switching from the customary constant flow-rate gradient elution mode to constant-pressure (cP) gradient elution mode. Constant-pressure gradient elution was recently introduced to LC; during the mobile phase gradient, the pressure drop is maintained at a set pressure, which results in an increase in flow rate for a decreasing mobile phase viscosity.

When the same volumetric mobile phase program is applied, the accuracy in both modes is maintained when converting the cP-mode data to the volume-based units. The same peak area is obtained for concentration-sensitive detectors in both modes and the linearity is maintained. However, small deviations in selectivity are found as a result of pressure and viscous heating effects, which are inevitable when speeding up the analysis by applying a higher pressure and which are comparable when switching from HPLC to UHPLC. Identical repeatability of the peak area and retention time is obtained in both elution modes.

The constant-pressure elution mode is also compatible with masssensitive detection, such as MS, charged aerosol detection (CAD), or evaporative light scattering detecionr (ELSD), although the peak area will differ between both modes because of the nonlinear flow-rate dependency of the droplet size in the nebulizing interface.

Second place in the Best Poster contest went to Dieter Verzele of the Pfizer Analytical Research Center, Department of Organic Chemistry, Gent University, Belgium. His coauthors were Frederic Lynan and Pat Sandra of the same institute and Adrian Wright and Melissa HannaBrown from the Pfizer European R&D Headquarters (Sandwich, UK). The poster, entitled "Development of a Sphingomyelin Biomimetic Stationary Phase for Immobilized Artificial Membrane (IAM) Chromatography," showed a new silicabased stationary phase that more closely mimics cell membranes than current phosphatidylcholinebased IAM colums. The synthesis was straightforward, and Verzele showed some good analytical applications of IAM chromatography.

Third place went to Andrea Gargano of the University of Vienna (Vienna, Austria). The title of the poster was "Selectivity of Mixed-Mode Chromatography for Structural Isomers of Phosphorylated Carbohydrate Metabolites," and it was coauthored by Helmut Hinterwirth, Michael Lämmerhofer, and Woflgang Lindner of the same institution. In this poster, the authors developed an HPLC–MS-MS platform for the quantitative profiling of many intra- and extra-cellular metabolites in fermentation broths that allowed them to measure individual isomers of phosphorylated carbohydrates. The key to the separation of these difficult compounds was the development of a mixed-mode reversed phase–weak anion exchange phase that allowed them to completely resolve a complex mixture of six hexose phosphate structural isomers.

Opening Session and More Awards

The opening ceremony plenary lectures, presented on the first evening of the symposium, are supposed to be cutting-edge, thought-provoking lectures to inspire attendees to think outside the box. After the opening ceremony with welcoming remarks from Attilla Felinger, there was a very interesting and entertaining sound and lights presentation by a "sand master," who uses thin sand and his bare hands to create art forms on an overhead projector depicting various caricatures of humans, animals, and scenes — all to the setting of a musical background.

After the opening theatrics, various prizes were announced, including the Martin Gold Medal, which was awarded to Peter Schoenmakers. Schoenmakers is widely recognized for his contributions to the separation and analysis of polymeric materials in GC, chemometrics, column technology, and, recently, in investigations on the use and optimization of LC×LC for complex mixtures.

He is also active in molecular-topology fractionation (MTP), matrix-assisted laser desorption–ionization (MALDI) MS, and chip separations. The prize was presented by The Chromatographic Society's Paul Ferguson. The Chromatographic Society is based in the United Kingdom with international connections and was created for the promotion and development of separation science. In 1978, Professor A.J.P. Martin gave permission for his name to be associated with the Martin Gold Medal. The Martin Medal is awarded to scientists who have made outstanding contributions to the advancement of separation science.

Other awards were also presented at the Opening Ceremony. Professor Gyula Vigh of Texas A&M University (College Station, Texas) was awarded the Halász Medal. Professor Halász, also a Hungarian pioneer in chromatography, was an early worker in GC and HPLC and, along with Csaba Horváth, developed some of the earliest column packings, which opened up HPLC in the late 1960s. Professor Vigh is best known for his work in the area of analytical and preparative electrophoretic separations such as isoelectric focusing (IEF) in its various forms such as parallel isoelectric trapping (IET) and pH-based IET. His IEF–IET work was mostly applied to proteins, and he has also developed chiral-based reagents for the separation of enantiomers by electrophoretic means.

The Csaba Horváth Memorial Award was presented jointly by the Connecticut Separation Science Council and the Hungarian Academy of Science to Professor Gunther Bonn of the University of Innsbruck, Austria. He was recognized for his work in the development of stationary phases, biopolymer analysis, and the understanding of pharmacological effects of both synthetic drugs and natural drugs in which chromatographic and electrophoretic separations play a key role. He has also been active in the development of electrochemical methods of analysis.

Plenary Lectures

The opening session on Sunday had two plenary lectures. The first by Professor Georges Guiochon of the University of Tennessee (Knoxville, Tennessee) and Oak Ridge National Laboratory (Oak Ridge, Tennessee) was entitled "Recent Progress in Column Technology Begets Progress in our Understanding of Column Efficiency." Along with coauthor Fabrice Gritti, Professor Guiochon has been fascinated by the new column-packing technologies that have appeared in the marketplace in the last few years.

Packings of sub-2-µm totally porous particles (TPP) and the 2.7µm and 1.7-µm superficially porous particles have been able to generate efficiencies that outperform the classical HPLC particles by a factor of five.

His interest is to understand why these particles, especially the SPPs, are able to perform so efficiently. The core–shell particles are of particular interest. In recent studies that the two scientists have performed, they found that the eddy diffusion (the A term of van Deemter equation, which is difficult to measure) and axial diffusion (B term, easy to measure by the peak parking method) both are smaller than porous particles of equivalent size and have determined reduced plate height values for SPP of 1.2–1.5. The C terms (also easy to measure) are somewhat equivalent. They have established that beds packed with SPP are radially more homogeneous than those packed with sub-2-µm particles and have a higher permeability. They have observed that the SPPs also have a higher optimum velocity and can therefore provide faster separations. Also the radial distribution of the mobile phase velocity from the center of the packed bed to the column wall has a velocity differential smaller for the SPPs (0.7% for Halo, 1.6% for Kinetex), larger for the TPPs (4–5%) and even larger for current silica-based monoliths (6–8%). This observation cannot be explained by the narrow particle size distribution (RSD 5–6%) of the SPP compared to the sub-2-µm particles (RSD 20%).

The good radial distribution observed for the SPPs could be explained by the surface roughness of the packings. The particles do not slip against each other easily or settle and rearrange during use. The researchers found that heat conductivity of the shell particles is much greater than fully porous particles.

This observation is important because frictional heat is generated when the packed columns with small particles are subjected to high pressure and fast flow: materials with heat conductivity will direct the heat away from the chromatographic bed. Guiochon suggested that the cores of the shell particles be made of a different material chosen for its good heat conductance properties. For example, alumina has 30 times the heat conductance of silica. He also suggested that when making such column measurements, the data should always be corrected for instrumental contributions to band broadening. As particles get even smaller and more efficient, instruments must be optimized to keep up with the gains in column efficiency.

The second lecture by a local scientist, Professor András Perczel of Laboratory of Structural Chemistry and Biology, Eötvös University (Budapest, Hungary) was entitled "Dynamical Structure Activity Relationship of Peptides and Proteins." His presentation involved no separation chemistry, but talked about the complex interactions that take place between macromolecules such as peptides, carbohydrates, proteins, and so forth and how they affect signaling, energy transfer, or cell division. Most of the time workers talk about structureactivity relationships, but he has proposed that the dynamics of the systems be taken into account and thus an enhanced model of dynamics-structure-activity relationships should be considered. He gave several examples where the dynamics are constantly changing, including aggregation, folding and partner-binding properties of proteins and other dynamic situations such as side-chain rotation for which time scales are of the order of milli- and microseconds; even some protein motions in the nanosecond and picosecond can take place.

New Column Technology

As seen in Table I, the development and study of columns and stationary phases still dominates new technologies. Both oral sessions and poster sessions were devoted to column technology, particle technology retention mechanisms, fundamental aspects of separation, stationary phases, high-throughput applications, UHPLC, and the like. If one combines all papers relating to column technology, about a third of the presentations at HPLC 2011 covered this topic. Despite all the advances made in column technology to date, investigations on further developments in academia as well as the commercial side are still taking place.

Superficially Porous Shell Particles From what I observed last year at HPLC 2010 (1), the SPPs received a great deal of attention and this trend continued at HPLC 2011. At the Budapest meeting, three more companies joined the four companies already marketing such columns. There was a continued focus on SPPs by both the theoreticians and the practitioners.

In a nutshell, these columns provide the efficiency of sub-2-µm particles but at around half the operating pressure. I counted a total of 41 papers (oral and poster) devoted to these columns. The academics are focusing on understanding why SPPs perform better than sub-2-µm fully porous particles. The practitioners are using the particles for high-speed separations and as the second column in a comprehensive LC×LC setup where separation times have to be in the order of seconds.

Although users who have already purchased UHPLC equipment are less concerned about the lower pressure drops of these columns, for difficult separations long columns and lots of theoretical plates are always in demand. Hence, pressure availability is always of concern. Currently, 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 future. Of more concern is the band dispersion observable with some commercial UHPLC instruments, with which these columns sometimes yield peaks less than a microliter wide; they cannot tolerate much tubing and system dead volume.

The Monday morning session on new particle technology consisted of four lectures on various aspects of SPPs. Fabrice Gritti of Guiochon's laboratory elaborated on some of their studies comparing SPPs with TPPs. He also presented some practical guidelines on using the SPP columns. He discussed sources of band broadening that can affect efficiency measurements: endfittings, connecting fittings (minimize number), tubing internal diameter (smallest possible, 0.12-mm i.d. best compromise), injection volume (keep below 4 µL), detector flow cell volume (smallest possible for required sensitivity), needle seats (use smallest internal diameter capillary). Gritti also studied the effect of surface roughness of SPP on column efficiency. He packed 2.5µm i.d. Luna TPP particles (smooth surface) and the same coated with an 0.25-µm silica shell (rough surface) into 100 mm × 3.0 mm columns. The interstitial porosities of the columns were 0.36 for the Luna and 0.41 for the Luna with the shell.

He concluded that surface roughness led to less densely packed beds as a result of high shear stress between the SPPs during packing consolidation. Also noted was the observation that the shell Luna yielded lower eddy dispersion for the unretained peak uracil. To conclude his lecture, Fabrice speculated that 1-µm SPPs are possible but the column lengths would have to be 15 mm or lower and internal diameters would have to be of the order of 0.5 mm to prevent frictional heating effects. He also suggested that high temperatures of 150–200 °C would be required. He estimated that pressures of 3800 bar would be needed to drive solvent through these tiny particles.

In the same session, Joseph Stankovich, representing a group from the Irish Separation Science Cluster (Cork, Ireland), studied the effect of particle size distribution (PSD) on column packing of C18 SPP. They used 1.16-µm, 1.8-µm, and 2.6-µm unclassified core–shell particles and mixed them in different proportions to achieve different PSDs. The 1.16-µm particles represented the fines and the 2.6-µm SPPs represented the large particles. Six mixtures were made with increasing amounts of large particles but the average particle diameter was kept at 1.8 µm. Using inverse size-exclusion chromatography (SEC), the interstitial porosity decreases from 0.405 to 0.37 with increasing amounts of large particles and interestingly the column permeability went up. The h_min was fairly poor for all six batches and decreased from the set with the least fines to the set with mostly larger particles. The batch with the most 2.6µm particles showed a minimum reduced plate height (h_min ) of 3.8, about three times higher than the best observations of others but this PSD gave the best peak shape, just the opposite of what one would expect. He concluded that sub-2-µm core–shell particles with narrow PSD are difficult to pack (in agreement with others) and their packing pressure of 800 bar was insufficient to provide the best packed bed efficiency. He suggested that 1500 bar would be a better packing pressure.

In his lecture on core–shell columns, Tivadar Farkas of Phenomenex (Torrance, California), also cautioned users about sources of band broadening in modern UHPLC instruments. He suggested the dilution of the injection sample with water concentrates the sample at the head of the column and eliminates precolumn dispersion.

He also found reduced frictional heating with SPP compared with TPP. For 150 mm × 3.0 mm SPP columns, even with 30-W/m heat generated, there was no effect on the separation. He speculated that for a 1.1-µm SPP, pressures would rise more than 2.3 times current columns and plate heights would decrease by 20% with frictional heating limiting the column length and internal diameter that could be used. System contributions to band broadening would have to improve considerably for such columns to be feasible.

Barry Boyes and coworkers of Advanced Materials Technology (Wilmington, Delaware) presented a paper on the investigation of new hydrophilic bonded phases on an SPP particle suitable for high performance HILIC. Using 90-Å SPP silicas as base materials, they bonded various phases with –OH functionality via a linking group. As the number of –OH groups increased (from one to five), there was an increment in retention of their polar test compounds in HILIC mode with an acidic mobile phase. Most of the phases showed less retention than bare silica, but the pentanol phase (five –OH groups) showed retention exceeding silica, with the exception of strong bases. For example, k values were higher on this phase for catecholamines and for organic acids, as well as phenylalanine and tryptophan (zwitterionic amino acids), whereas they were lower for benzylamine and amitriptyline; as examples of basic compounds. Interestingly, retention on the pentanol phase was largely independent of ionic strength, supportive of a significant reduction in acidic silanol interactions, and favorable for LC–MS applications of HILIC.

A number of practical comparison papers appeared again showing the superior performance of the SPP when compared with the totally porous sub2µm particles, especially when the pressure drop is taken into account. In addition, numerous applications of the SPPs appeared at HPLC 2011 ranging from separations of pharmaceuticals (basic drugs, α-tocopherol, and various others), biopharmaceuticals (insulin), environmental samples (hydrocarbons, polynuclear aromatic hydrocarbons, bisphenol A, and pesticides), life science (metabolomic studies, peptides, and enzymes), natural products (silybin diastereomers and carotenoids), food (milk phospholipids and polyphenols) and organic chemicals (polystyrene and perfluorinated chemicals) were noted.

Applications of SPP for SFC and as the secondary dimension in LC×LC were also discussed. It was commented that users experienced less plugging with SPP columns compared with sub-2-µm columns, presumably because of the larger porosity frits used on the inlet side of the columns. The sub-2-µm columns traditionally use 0.5-µm porosity frits while the SPP use 2-µm porosity frits, the same frits used on 5-µm columns widely used in HPLC.

Monoliths: Monolith columns have been desirable for a long time because they exhibit high permeability and low pressure drop (as a result of increased bed porosity), show good separation efficiency, have the absence of frits to confine the packing material, are easy to fabricate, and 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, which have shown further improvements in performance.

This year, polymeric monoliths appear to be gaining ground on the silica-based monoliths. Once confined to the separation of macromolecules, by careful control of the mesopore–macropore ratio, laboratories have been able to demonstrate column performance for small molecules approaching the silica monoliths.

Ivo Nischang of the University of Linz (Leonding, Austria) reviewed porous polymer monoliths for small molecule separations. Most laboratories are using methacrylic and styrenic precursors but their approach was to use copolymerization of mono- and divinyl precursors with a micro- and macro-porogenic diluent. The group can control the polymerization to make it slower by using low temperatures and a low amount of radical initiator. Stopping polymerization in the early stages results in a monolith that separates small molecules more effectively. Longer polymerization times change the porous structure and one achieves poorer separation of small molecules. The total porosity goes down and the small molecule efficiency decreases with increased polymerization times. The mesoporous pore space is enabled by the microporogenic diluents in the polymerization precursor mixture.

Merck scientists Karin Cabrera and coworkers have also developed a second generation of silica monoliths with improved separation efficiencies as well as improved peak shapes, especially for basic drugs. To obtain better efficiency, the macropore size had to be increased, which, in turn, increases the pressure drop (which is still quite low compared with particle columns). The best monolith gave an H_min of about 5 µm, roughly equivalent to 200,000 plates/m. She contended that the second-generation monolith is more structurally homogeneous and should be able to compete with particle-packed columns. The columns show flat van Deemter curves that do tend to curve up somewhat at higher velocities.

Kazuki Nakanishi of Kyoto University in Japan, has combined monolith technology with core–shell technology to improve the overall performance of silica-based monoliths. The so-called "fused-core monoliths" are composed of fully sintered macroporous skeletons with their surfaces homogeneously coated with submicron additional silica layers. These monolithic gels should perform better than conventional monolithic columns as a result of the reduced diffusion distance, but they should not suffer from higher back pressure.

They investigated two different approaches to generate these fusedcore monoliths. Using the concept of pillar stationary phases, they were also able to coat the pillars with nanoparticles of silica forming a shell-like structure. The pillar microfluidic phase showed a surface area of 66 m² /g after sintering. Another approach to make core–shell monoliths involves the use of mesoporous silica nanoparticles that are synthesized by reacting tetraethyl orthosilicate with a template made of micellar rods. Nakanishi and coworkers used Pluoronic P123, a block copolymer, instead of the more common SBA15 silica as a template. The resulting monolith material was strengthened by sintering to 900 °C. This monolith had a porosity of 90% and X-ray diffraction showed that the mesopores were highly aligned. This sintered monolith had a very high mechanical stability and showed 170,000 plates/m efficiency. In addition, the surface area was over 300 m² /g.

Thus, in the monolith world, the technology is advancing and the promise of silica and polymeric monoliths competitive to particle-packed columns may be right around the corner.

HILIC: With the growing interest in HILIC (see Table I), phases are being developed that will retain polar compounds in relatively high percentages of acetonitrile in water mixtures, a favorite mobile phase combination. David McCalley of the University in the West of England (Bristol, UK) presented a standing-room-only tutorial on HILIC. His coverage was very balanced and he showed many examples of successful separations from his laboratory and from the work of others. Just as in reversed-phase chromatography, buffers are needed as retention is higher when the solute is fully ionized (low pH for bases and high pH for acids). The buffers are needed to stabilize retention. Some favorite buffers are ammonium acetate and ammonium formate in the 5–20 mM range; both are suitable for LC–MS. It was not recommended to use formic or acetic acid alone. The choice of injection solvent is equally important: Don't inject in pure water like one might in reversed-phase chromatography — water is a strong solvent in HILIC!

Column Types: McCalley defined five different types of columns that are used in HILIC:

• Bare Silica: Charge on the surface depends on mobile phase composition; silanols are more acidic than in reversed-phase chromatography; for basic compounds getting retention is sometimes a problem; advantage: no phase to bleed.

• Neutral Bonded Ligands: Most popular are amide and diol ligands.

• Charged Ligand: Positively charged amino phase or negatively charged poly-2-sulfoethylaspartamide ligands.

• Zwitterionic Phase: Quaternary amine (positive charge) and sulfonic group (negative charge) on same bonded chain.

• Mixed Reversed-Phase/HILIC Phase: Long alkyl chain with diol groups.

Advantages: The advantages of HILIC include

• The order of elution is usually the opposite of reversed-phase chromatography.

• Low column back pressure as a result of high organic content.

• Flatter van Deemter curves means methods can run be run at higher flow rates.

• Overloading of bases is less serious than in reversed-phase chromatography.

• Fast separation on long columns and columns can be stacked in series.

Disdvantages: Some disadvantages of HILIC include

• Little retention of nonpolar compounds

• The high acetonitrile content of the mobile phase is not environmentally friendly.

• The retention mechanism is not well understood.

• Poor solubility of solutes in mobile phase

• Limited choice of buffers that are soluble in high organic

• Longer equilibration times

• Difficult to manipulate separation

In his lecture on novel HILIC phases, Professor Wolfgang Lindner of the University of Vienna (Vienna, Austria) suggested that mixed-mode interactions can be used to explain HILIC retention. Using phases containing ion exchange–reversed phase functionality, analyte ionization or phase ionization can cause additional electrostatic attraction or repulsion which explains the impact of pH on the elution of certain compounds.

In his lecture, he introduced "chocolate" phases based on the Maillard reaction of reducing carbohydrates with an aminopropyl-modified silica. Another novel HILIC phase was based on dimedone derivatives immobilized on to thiolated silica. Tohru Ikegami of the Kyoto Institute of Technology (Kyoto, Japan) presented a new test method for the chromatographic characterization of HILIC stationary phases. Using radar plots and his test method to characterize a large number of HILIC phases, he was able to group phases by their hydrophilicity, which could then be useful for selecting the appropriate HILIC column for the separation at hand.

MDC and Comprehensive Liquid-Phase Separations: As samples become more complex, even with the best and most efficient columns available, the peak capacity of a single column is no longer sufficient to resolve all the peaks to baseline. Peak capacities of 1000 can be achieved with a single column in a very long analysis time but to handle samples with thousands and tens of thousands of components, multiple columns with different separation mechanisms are needed. The total peak capacity of a multidimensional system is equivalent to the peak capacity of the initial column times the peak capacity of the second column if the two chromatographic modes are orthogonal.

MDC has been around for many years but now has been generating a high level of interest. Comprehensive LC, termed LC×LC, is a technique that attracted the most interest at HPLC 2011. In this approach, every single fraction from the first dimension is directed to a second dimension to be further separated and identified. There can be off-line and on-line approaches to address this coupling. In the on-line LC×LC approach, switching is accomplished without shutting down the flow of the first-dimension column by storing the effluent from column one in a loop or packed bed and switching that storage device onto the second column after it has finished its analysis. The implication here is that the second dimension must be extremely fast in the time scale of the storage of entire effluent from column one.

Some of the highlights noted at HPLC 2011 were the better design of orthogonal separation systems to achieve maximum resolving power. New instruments are being developed for routine 2D analysis. New interface developments, such as the one cited above for the Horváth Young Investigator Award winner, will help to provide better transfer of the effluent from the first dimension to the second dimension without suffering injection band broadening. One particular observation was the large number of applications papers using MDC and LC×LC techniques, both off-line (easier to carry out) or on-line.

Detection Techniques

By a perusal of the abstracts, I tabulated the detection principles (Table III) that were used on a relative basis in the various presentations at HPLC 2011. Not every abstract indicated the detector that was used, so only those that provided this information was counted. The category assignments were based on the main emphasis of a particular scientific paper as well as separation and detection techniques used. Again, MS clearly dominates the detection category. If one adds up the use of MS in chromatography and electrophoretic techniques, 58% of the papers presented at HPLC 2011 (up from 54% last year at HPLC 2010 [1]) used this detection technique. Despite the higher cost, the remarkable selectivity and sensitivity of the tandem MS techniques are favored by chromatographers and MS practitioners alike. They are quickly taking over as the workhorse detectors in many laboratories.

Table III: Types of detection techniques used at HPLC 2010

After MS, as might be expected, UV detection, especially diode-array detectors (DAD) was the second favored detection technique, mostly in application papers. Fluorescence detection and ELSD–CAD showed a slight dip in relative use, while electrochemical detection increased slightly, but all of these other detection techniques are used relatively infrequently.

HPLC 2011 (China) and 2012 (USA)

The next major symposium in this series, the 37th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2011 Dalian) will be held in Dalian, China, October 8–11, 2011. The chairmen of this upcoming event will be Professor Yukui Zhang of the Dalian Institute of Chemical Physics, Chinese Academy of Science (Dalian, China) and Professor Peter Schoenmakers. Co-chairmen will be Professor Guowang Xu, Dalian Institute of Chemical Physics, Chinese Academy of Science and Professor Koji Otsuka, Kyoto University (Kyoto, Japan). For more information consult the official website: www.hplc2011.dicp.ac.cn

The next time the symposium will be held in the United States will be in 2012 in Anaheim, California. The dates will be June 16–21, 2012 and the chairman will be Professor Frank Svec of University of California (Berkeley, California) in conjunction with CASSS. A website, www.hplc2012.org, is already available; bookmark this website so that you can keep up on the latest happenings.

Acknowledgments

I would like to acknowledge the contributions of my Agilent colleagues Maureen Joseph, Xiaoli Wang, and Bill Barber of Wilmington, Delaware, who supplied their notes on some of the sessions.

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

Ronald E. Majors

References

(1) R.E. Majors, LCGC North America 28(9), 764–781 (2010).

(2) R.E. Majors, LCGC North America 29(3), 218–235 (2011).

(3) R.E. Majors, LCGC North America 29(1), 14–27 (2011).