Highlights of HPLC 2008: Part I

August 1, 2008
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 North America

LCGC North America, LCGC North America-08-01-2008, Volume 26, Issue 8
Page Number: 676–691

Columns | <b>Column: Column Watch</b>

HPLC 2008 was held in Baltimore in May. Columnist Ron Majors covers the highlights of this major symposium, held in the U.S. every two years.

The 32nd International Symposium on High Performance Liquid Phase Separations and Related Techniques, which alternates between Europe and North America, was held, for the second time in Baltimore, Maryland, May 10–16, 2008. More affectionately known as HPLC 2008, 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 Georges Guiochon of the University of Tennessee (Knoxville) and Oak Ridge National Laboratory (Oak Ridge, Tennessee) and Stephen Jacobson of Indiana University (Bloomington, Indiana), HPLC 2008 assembled just under 1200 scientists from a total of 42 countries. This number includes vendor representatives from over 67 exhibitors for the three-day instrument, software, and consumables exhibition.

The five-day plus event had a total of 138 oral presentations, many given during simultaneous sessions, over 460 posters in sessions with 30 themes. With an ample social event schedule, 11 vendor workshops (some with free lunch), six tutorial educational sessions, two discussion sessions and 10 short courses, the latter held during the previous weekend, attendees had their hands full deciding how to allocate their time. The tutorials and discussions were particularly well attended with provocative titles such as "Is Silica Walking into the Sunset?," "Comprehensive 2D LC: The Good, the Bad and the Ugly," and "Packed Columns vs. Monoliths: Where Are We Now and Where Are We Going?"

Obviously, high performance liquid chromatography (HPLC) was the predominant technology in the technical sessions at the symposium. 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 the technology and applications of liquid phase researchers in the world. Many of the topics that are currently "hot" in the separations sciences were introduced in this series.

Table I provides a rough breakdown of the coverage of liquid-phase technology and techniques in the separation sciences. Compared with HPLC 2007 (1,2), some definite shifts in technology emphasis were noted. Although column technology always leads the pack, this year, about a third of the columns' papers dealt with monoliths, with more emphasis this time around on polymeric-based monoliths which have less intellectual property protection compared to silica-based monoliths. Three other areas were "hot" in column technology this year: the continued interest in sub-2-μm porous packings with ultrahigh-pressure liquid chromatography (UHPLC) being the latest buzzword; the increasing interest in hydrophilic interaction liquid chromatography (HILIC) for the separation of polar analytes and; the new breed of superficially porous packings (also referred to as pellicular, porous layer beads, and fused core packings) that are said to rival the sub-2-μm particles in terms of column efficiency but with substantially lower pressure drops. Discussion sessions weighed on the virtues and tradeoffs of monoliths vs sub2-μm particles, vs. 2–3 μm particles vs. superficially porous particles. A well-covered topic was the use of 2D and comprehensive LC×LC, which had four oral sessions, a discussion session, and a tutorial devoted to it. With chromatographers encountering more complex samples, sometimes with thousands of compounds present, these multidimensional techniques are about the only way to tackle such mixtures.

Figure 1: (a) Graduate student Ken Broeckhoven (left)of the Free University of Brussels after receiving his first place Best Poster Award from Poster Chair Ron Majors (right). (b) Winner of the Second Best Poster, Pelin Yang of Dow Chemical (Midland, Michigan). (c) Winner of Third Best Poster, Petr Cesla of the University of Pardubice, Czech Republic.

Sample preparation topics, always in the top three categories of technology, that were prominent included solid-phase extraction (SPE) and solid-phase microextraction (SPME), selective phases such as molecularly imprinted polymers (MIPs), immunoextractions, and restricted access media (RAM) and various forms of liquid–solid extractions (for example, matrix-solid-phase dispersion, accelerated solvent extraction and pressurized fluid extraction, and superheated water as a solvent) getting attention. Other areas that had great interest were oral and poster papers devoted to theoretical topics and retention mechanisms and electrophoretic techniques such as capillary electrophoresis (CE), capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), and isoelectric focusing (IEF). After last year's poor representation, microfluidics and chip-LC showed a stronger presence, presumably because the meeting moved back to the U.S. where more of this science is being pursued. The biggest drop-off compared to last year was for capillary electrochromatography (CEC), which had only six presentations that mentioned the technique. Only a few years ago, lecture rooms on CEC topics spilled out into the hallway. The "killer" application was never found.

Table II is a breakdown of the most popular application areas. The order of the popularity for the first eight application topics of Table II is exactly the same as last year (1) with proteomics and the separation of proteins and peptides the leading the way in terms of absolute numbers and growth of presentations.

Table I: HPLC 2008 papers presented by technology or technique

In this installment of "Column Watch," I will present some of the scientific highlights of HPLC 2008. This report also will emphasize the various awards and honorary sessions taking place, the scope of the plenary lectures, column and phase developments; temperature studies, multidimensional and comprehensive chromatography, detection highlights and a few significant applications trends. Because it was virtually impossible for one person to cover all of the oral presentations and poster papers adequately, my coverage will somewhat reflect a personal bias, although I was able to get meeting summaries from some of my colleagues who are acknowledged at the end of this column.

Awards and Honors at HPLC 2008

Horvath Award: For the third year in a row, the Horvath Award sessions, named for the late Professor Csaba Horvath, one of the founders of this series and a mentor of young scientists, were featured. This award was supported by HPLC Inc., a nonprofit group under the guidance of the Permanent Scientific Committee. The award was established for young scientists in the separation sciences under the age of 35. The award, based upon the best oral lecture presented in the Horvath Sessions, was selected by a jury named by the Permanent Scientific Committee and consists of a travel grant to and free registration for HPLC 2009 as well as a trophy. This year there were 10 nominees and half were female scientists. This year's winner was Jude Abia from the University of Tennessee and the Oak Ridge National Laboratory in Oak Ridge. His co-authors were G. Guiochon and K. Mriziq from the same institutions. The title of his award winning presentation was "Radial Homogeneity of Silica-Based Wide-bore Monolithic Column." The researchers investigated the elution profiles of a flat injected band recorded at the exit of a silica-based monolithic column using an on-column amperometric detector. This detector configuration allowed the recording of elution profiles at different spacial positions throughout the column cross section. They observed lower efficiency along the wall than in the center of the column due to radial heterogeneity of the monolith. These results may explain the large value of the A term of the van Deemter or Knox equations for such columns.

Table II: Papers presented by application area

Poster Sessions and Best Poster Awards: The mainstay of HPLC 2008 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 collected business cards and addresses for sending poster reprints by mail or e-mail. Compared with HPLC 2007 (1), the number of posters was substantially reduced. The posters were split into two 2-day sessions; by staggering the days that the posters were staffed by presenters, the sessions did not appear to be crowded. From walking around the poster areas, there appeared to be quite a number of "no shows," often from third world countries who apparently did not get funding to attend or merely wanted their abstracts to appear in the program.

For the last time in 10 successive years, I once again had the privilege of leading the Poster Committee, which chooses the top three posters of the Symposium. For the 40 members of the Poster Committee who devoted a great deal of time and worked very hard to narrow down the collection of posters to 17 by the end of the fourth day and then helped to select the three winners by Friday morning of the symposium, I want to want to thank them again for their contribution to HPLC 2008. Their names and affiliations are provided in Table IV.

Figure 2: Ron Majors receiving the Martin Gold Medal from The Chromatographic Society's President John Lough.

The Best Poster Awards, sponsored by Agilent Technologies (Wilmington, Delaware), were announced at the Closing Session. After narrowing the field down to the top 17 posters, the three winners selected as the topic vote getters received Amazon.com gift checks. This year's winning poster was entitled "Future of HPLC and UPLC: Are Higher Pressures and Smaller Particle Opportune?" by authors Ken Broeckhoven, K. Choikhet, and G. Desmet, Free University of Brussels (Brussels, Belgium), and G. Rozing of Agilent Technologies (Waldbronn, Germany).

Using computational flow dynamics and experimental studies, the authors determined flow and temperature profiles inside the packed bed and the column wall for the most common thermal boundary condition. They showed that for ultrahigh pressures, the heat transport in columns must be taken into account when studying the effect of viscous heating for, even at currently available operating pressures (~1000 bar), a measurable band broadening can occur. The authors went on to conclude that, due to the viscous heating, further increases in operating pressure beyond 1000 bar will quickly result in a deterioration of performance and resolution, especially for retained components. The prediction of this study was that further reduction in particle size will require higher pressures that, at the optimum flow velocity, will reduce the anticipated gain in performance due to these viscous heating effects. Figure 1a shows a photograph of graduate student Ken Broeckhoven of the Free University of Brussels receiving his prize at the Closing Session of HPLC 2008. Of particular note is that this Belgium research group has finished in the top three poster awards in four out of the last five years.

Figure 3: David McCalley receiving the Silver Jubilee Medal from The Chromatographic Society's President John Lough.

Winners of the second best poster entitled "Application of Fused-Core Particle Technology in the Separation of Natural Products and Impurities" were authors Peilin Yang, G. Litwinski, M. Pursch, H. Cortes, T. McCabe, K. Kuppannan, and C. Crouse from Dow Chemical (Midland, Michigan; Indianapolis, Indiana; and Rheinmuenster, Germany).

Their poster compared the isocratic separation of 15 natural products standards on totally porous 3-μm and 1.7 μm columns and on fused-core, superficially porous columns (2.7 μm). The plates per meter for the respective columns for the same peak were 70,000 (3 μm), 220,000 (1.7 μm) and 150,000 (2.7 μm) while the pressure drops were 2600, 6000, and 3100 psi and separation times were 33, 10, and 12 min, respectively. Some batch-to-batch variations were noted on the columns but the authors concluded that fused-core particle technology allowed high-speed separations to be performed on conventional instruments without high backpressure and comparable performance to the sub-2-μm columns. Figure 1b provides a photo of first author Pelin Yang who received her award for the second Best Poster prize.

Winners of the third best poster, entitled "Comparison of Detection Techniques for LC×LC: Separation of Natural Compounds in Beverages," were Petr Cesla, T. Hajek, and P. Jandera, University of Pardubice (Pardubice, Czech Republic). The authors developed an almost orthogonal 2D separation of 27 phenolic and flavone natural antioxidants in wine, beer, and plant extracts using combinations of polyethyleneglycol- or phenyl-silica columns in the first dimension and reversed-phase columns in the second dimension. A system of parallel gradients with matching profiles in both dimensions improved the orthogonality. Three detection techniques (multichannel coulometric electrochemical detection, diode array, and atmospheric pressure chemical ionization [APCI]-MS [offline]) allowed the identification of sample components. Software for handling the 2D chomatograms was developed including an algorithm correcting for shifts in retention in the second dimension induced by the parallel 2D gradient.

Table III: Poster committee members 2008 (in alphabetical order)

Figure 1c shows graduate student Petr Cesla who received his third Best Poster award.

Pfizer Innovation in Pharmaceutical Analysis Awards: At HPLC 2008, the Pfizer Analytical Research Center (Parc) presented three awards for the best poster contributions in pharmaceutical analysis. The winners were: First prize: "On-line Coupling of Monolithic Enzymatic Microreactor, Liquid Chromatography, and Mass Spectrometry for Analysis of Therapeutic Monoclonal Antibodies," by J. Krenkova and F. Svec, Lawrence Berkeley National Laboratory (Berkeley, California); second prize: "Development of Immobilized GPR17 Receptor Liquid Stationary Phases for On-line Screening of Potential Drug Candidates," by C. Temporini, E. Calleri, S. Ceruti, S. Ferrario, C. Lambertucci, R. Volpini, M.P. Abbracchio, G. Caccialanza, G, Gristali, and G. Massolini, University of Pavia (Pavia, Italy); third prize: "Protein Two-Dimensional Separation on Spatially Multiplexed Polymer Microchip Platform," by S. Yang, J. Liu, and D. DeVoe, University of Maryland (College Park, Maryland).

The Chromatographic Society Awards: The Chromatographic Society is based in the United Kingdom with international connections and was created for the promotion of 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. At HPLC 2008, the 2007 Medal was presented to Ronald E. Majors, Senior Scientist at Agilent Technologies (Wilmington, Delaware) for his contributions to separation science in the areas of sample preparation and surface chemistry and his distinguished career on the manufacturer–vendor side of separation science.

In addition, The Chromatographic Society awarded its Jubilee Silver Medal to Professor David McCalley of the University of the West of England, Bristol, United Kingdom for his widely cited fundamental studies on the mechanism of reversed-phase separations including overloading effects, effect of pH and its measurement in aqueous–organic solutions and kinetic effects for "difficult" compounds. The Jubilee Medal is awarded to up-and-coming separation scientists who have made major use of separation science or to scientists who have made meritorious contributions to a particular area of separation science. Figures 2 and 3 show Majors and McCalley after receiving their medals presented by the Chromatographic Society's president, Professor John Lough of the University of Sunderland (Sunderland, UK).

Plenary Lectures

Opening Ceremony plenary lectures, presented on the first morning of the symposium, are supposed to be leading edge, thought-provoking presentations that are to inspire attendees to think beyond the box. This year two notables gave plenary lectures that were oriented towards cell signaling. The first lecturer was Donald F. Hunt of the University of Virginia (Charlottesville, Virginia), a mass spectroscopist, who discussed cell signaling using innovative mass spectrometry technology. He focused on the application of the electron transfer dissociation (ETD) technique for intact proteins. Professor Fred McLafferty of Cornell University (Ithaca, New York) developed this technique. In ETD, positively charged proteins in the gas phase (by electrospray ionization [ESI]) are reacted with fluoranthene radical anions to form random fragments on the amide bond of the protein backbone. In a second reaction, the multiple charged fragment ions are deprotonated through an ion–ion reaction with the carboxylate anion of benzoic acid. This results in singly, doubly, and triply charged peptide species of 15–60 amino acids at the N- and C-termini of the protein. Together with the measured mass of the intact protein, it is possible to identify unknown proteins, posttranslational modifications, and splice variants without the need for proteolytic digest before analysis. In his plenary lecture, Professor Hunt provided some nice examples. For example, the technique was successfully applied for the study of the 70S ribosome from E. coli where he was able to identify 48 out of 52 proteins, the class I and II MHC-antigen pathway in malignant melanoma, and the complex phosphor proteome pattern of focal adhesion proteins.

In the second plenary lecture, Clay Fuqua of Indiana University (Bloomington, Indiana) did not talk much about HPLC but nevertheless his presentation was quite interesting from a scientific viewpoint. Fuqua illustrated the different possibilities of bacterial communication (intra- and interspecies). Bacterial communication pathways might be an interesting target of drug targets in infectious diseases. Bacteria excrete substances (for example, toxin release) that can trigger certain responses of the bacterial population. It is not sufficient that only one bacterium releases a trigger for a certain response but, by excretion from several or many bacteria, a critical concentration must be reached to cause such a combined response. This process is called "quorum sensing." In Gram-negative bacteria, these signals are often acylated homoserin lactones while Gram-positive species mostly employ oligopeptides. Examples were demonstrated for chemiluminescence, pathogen-host interaction and formation of biofilms. As detection methods, gas chromatography (GC)–MS and LC–MS-MS were used to illustrate the signaling pathway. Different labeling strategies were used to demonstrate populations with and without a caused signal response.

New Column Technology Highlights

As seen in Table I and similar to the last seven years of HPLC symposia coverage (1–9), the development and study of columns and stationary phases still dominates new technologies. At least nine oral sessions and an equivalent number of poster sessions were devoted to column technology, retention mechanisms, high-throughput applications, UHPLC, and the like. If one combines all papers pertaining to column technology, about a third of the presentations at HPLC 2008 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.

From what I observed at HPLC 2008, the current hot topic in HPLC column technology is still the discussion of the alternative approaches for developing faster separations and generating more column efficiency at lower pressure drop and which approach will be favored in the long run. Discussion sessions, tutorials, and general sessions were devoted to these topics. In my lecture in the advances of column technology session, I reviewed these approaches:

Packed columns with small porous particles (sub-2 μm)

Packed columns with small porous particles (2–3 μm)

Monoliths (silica and polymeric)

Superficially porous packings (~2.7 μm), which are also referred to as fused core–, pellicular, or porous layer bead-particles.

In general, all these approaches work in balancing column efficiency, phase ratios, and column permeability. At least 14 companies now produce columns with sub-2-μm packings in the 1.5–2.0 μm range (10). Short versions (50 mm and less) have reasonable pressure drops while displaying excellent separation speed while longer columns, up to 150 mm, generate sufficiently high back pressure to require higher pressure pumps and systems for the most effective use.

This high-pressure requirement has led at least seven companies to provide more moderate pressure columns packed with particles in the 2.1–2.9 μm range (10). These columns provide better efficiency than the 3.0–3.5 μm columns but lower pressure drop than the sub-2-μm columns. Both "schools" advocate the use of higher operating temperatures that tend to reduce mobile phase viscosity, thereby lowering column back pressure, improving efficiency, and decreasing retention. Superficially porous packings generated lots of attention at HPLC 2008. These columns appear to have the efficiency of the sub-2-μm particles but the pressure drop of 2.7-μm particles.

Monoliths: Monolith columns have been desirable since they exhibit high permeability and low pressure drop (due to 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 but with continued academic advances reported at HPLC 2008, if commercialized, users should take more advantage of these columns. Reports were devoted to improving both polymer- and silica-based monoliths. Overall observations indicate that silica-based monoliths seem to work best for small molecules while the polymeric monoliths are best for macromolecules. Both theoretical and practical studies have been applied to understanding how to increase phase ratios and homogeneity of the co-continuous structures of silica skeletons.

In the silica monolith approach, at least in the academic setting, improvements have been made in the homogeneity of the continuous beds and improvements in efficiency by adjusting the relative sizes of the macro- and mesopores, which can be independently varied using the sol-gel synthesis process, has led to better overall chromatographic columns. By varying this ratio, monolith permeability can be affected, sometimes in a detrimental way taking away one of the advantages of monoliths over packed beds. Nobuo Tanaka and coworkers of Kyoto Institute of Technology (Kyoto, Japan) further described million plate monolithic capillary columns having highly efficient mesoporous skeletons with high permeability due to macropores. These long columns (possible up to 2 m) have fair reproducibility but when a 1-m column was cut into 200-mm short columns, some showed better efficiency than others indicating tha the monolith within the column was not uniform. In the future, monoliths with diverse chemistries (for example, ion exchange, HILIC, and size exclusion) should become very useful for 2D separations, especially in the second dimension where fast chromatography is a necessity. So far, these improvements have not been adopted commercially.

Karen Cabrera of Merck KGaA (Darmstadt, Germany) described the recent developments in the company's commercial silica monoliths, especially the new 50 mm × 2 mm columns that have reduced radial inhomogeneities and show faster separations (11). Higher temperatures were shown to increase efficiency while reducing asymmetry.

R. Skudas of the Johannes Gutenberg University (Mainz, Germany) discussed the importance of macropore structures in silica monolith, as the plate height is pressure dependent. He described four procedures that were used to evaluate macropore and mesopore contents: hydrodynamic chromatography, liquid permeation, mercury intrusion, and scanning electron microscopy. All give reasonable values for larger monolith columns but mercury intrusion is not possible for the capillary monoliths. In addition, neither mercury intrusion nor hydrodynamic chromatography give skeletal information. The latter technique also requires too many assumptions (such as cylindrical pores). Comparisons with applications indicate that larger macropore content leads to lower efficiencies but increased porosities (that is, faster analyses).

Jose Rivera from the University of Buffalo (Buffalo, New York) group of L. Colon described recent results on preparing monolithic columns containing other metal oxides. Depending upon the metal, these types of monoliths should have better resistance to pH variations compared to pure silica monoliths. However, the hybrid monoliths that they prepared did not show improved pH resistance. Pure metal oxide monoliths based upon hafnium or zirconium prepared using N-methylformamide as porogen in the presence of polyethyleneoxide were better. Pore size depended upon the amount of N-methylformamide and on the temperature of subsequent sol-gel curing. X-ray diffraction was used to confirm the monolithic structure.

Polymeric monoliths appear to have the best potential for use in microchannels and chips since they are more easily fabricated than attempting to efficiently pack particles into very narrow channels. Frank Svec, an expert on polymeric monoliths, studied the geometrical shape of channels and found that porosities of cylindrical, square and retangular shapes were virtually the same. Using thermally initiated polymerization for either methacrylate or polystyrene–divinylbenzene (PS-DVB)–based monolithic chips, it could be demonstrated that the corners of the channels are completely filled and that the monoliths adhere to the wall without any special treatment, thereby negating wall effects. Svec went on to fabricate 200 μm × 200 μm polyimide chips and used them to separate two different peptide samples using a 2-min gradient. In the same monolith session, Hanfa Zhou and coworkers from the Dalian Institute of Chemical Physics (Dalian, China) used a biphasic monolith (strong cation exchange and reversed phase) within a single 100-μm i.d. capillary column for the on-line multidimensional separation of tryptic digests of yeast proteins. The column contained an integrated ESI tip. Using this microLC–MS-MS setup and a 14-step multidimensional separation, the group positively identified over 2000 distinct proteins and over 8500 unique peptides. Dr. Zhou found that there was 12–19% better column efficiencies for the monoliths when compared to packed columns and speculated that monoliths will become more important in proteomics in the future.

By changing the monolith "recipe," in the Closing Session, Christian Huber from Saarland University (Saarland, Germany) described small molecule applications of PS-DVB monoliths modified with C18 groups. The pore size of the polymeric phase was decreased by changing the amount of micro- or macroporogen during the synthesis. By increasing the amount of porogen, columns became more permeable, which enabled the implementation of longer monolithic columns with moderate back pressure. Plate counts up to 100,000 plates/m were obtained in isocratic separations of dansylated amino acids. Most of these polymeric columns are made in-situ so that making them column-by-column is somewhat of an inconvenience.

G. Bonn and coworkers of the University of Innsbruck (Austria) also discussed ways to prepare organic monoliths for the separation of biopolymers as well as low molecular weight compounds. Using such chemistries as polyacrylates, styrene–vinylaryl copolymers, and organosilane–vinylaryl copolymers by varying the polymerization parameters such as the monomer-to-porogen ratio, microporogen nature and content, and polymerization temperature and time, the porous structure of the monoliths could be controlled. In his paper, he also gave a fast overview of using monoliths for sample preparation cleanups. The monolithic material is placed in a pipette tip and several sample preparation procedures were successfully performed, such as phophopeptide enrichment using a monolith with TiO2 and ZrO2 embedded in polydivinylbenzene (DVB); serum desalting using a monolith-diamond phase; and monolithic enzyme beds.

Butyl methacrylate and lauryl methacrylate monolithic columns (each 250 mm × 75 μm) and a conventional Zorbax C18 SB (150 mm × 75 μm) were compared for an 11-peptide mixture in the gradient separation mode by Peter Pruim and coworkers from the University of Amsterdam (The Netherlands). As a measure of gradient performance, peak capacity was selected. For long gradients, the conventional packed column was superior, especially compared to butyl methacrylate monolith. The poorer performance of the monolith was attributed to peak broadening of highly retained peptides. Performance of the lauryl methacrylate monolith approached that of the Zorbax column. In general, the author noted that for monolith columns, long, shallow gradients should be avoided since highly retained peptides are eluted in broad peaks, which results in a lower peak capacity.

In his tutorial on particle packed columns versus monoliths, K. Unger of the Johannes Gutenberg Universitat (Mainz, Germany) spent most of his time discussing the various attributes of packed columns of the types cited previously. But in the discussion session on the same topic a couple days later, the subject of monoliths was discussed more thoroughly when the lack of the widespread use of silica monoliths was brought up as an issue. Surveys have shown that less than 1% of chromatographers are routinely using monolithic columns. Part of the answer could be that, although polymeric monoliths are available from several suppliers, silica-based monoliths are available from a single source due to patent exclusivity. Thus, the lack of a second bonafide supplier of silica monoliths may preclude their acceptance. Some pharmaceutical companies wish to have a second source before they validate a regulated method; lack of this source may discourage them from using a single source column. The higher cost of monoliths was brought up as a detriment for routine application. Other reasons may be pegged to the requirements of regulatory agencies for revalidation of methodology when one of the parameters (for example, column type or packing) changes. The high flow rate requirement for earlier monoliths to provide shorter retention times discouraged some potential users, especially the mass spectroscopists. The newer 2-mm i.d. silica monolith columns may negate this objection. Reproducibility of monolith columns was another factor that was brought up; nowadays, these columns also have seen an improvement in column-to-column reproducibility, although it may take some time for users to see that improvement since they do not use them that often. Unger brought up the point that it took chromatographers 30 years to perfect packed columns with stable C18 bonded phases with reduced particle diameters and higher temperature stability so monoliths may still be in their "infancy." Frank Svec mentioned that in his opinion, monoliths are the only way to obtain a repeatable preparation process for capillary columns, which he considers the future of HPLC and for its environmentally friendly aspects.

Superficially Porous Particles: Superficially porous particles (SPP) (~2.7 μm), a subset of the 2–3-μm particles in terms of pressure, have a solid inner core and a porous outer core. The outer core is sufficiently thin to allow rapid mass transfer into and out of the stationary phase, and since the inner core is solid fused silica, analytes cannot penetrate any further. This diffusion path length is shorter than the porous particles of approximately the same diameter (decreased C-term in van Deemter) and roughly equivalent to that of the sub-2-μm particles. There is also a smaller A-term since the particle size distribution is extremely narrow compared with porous particles, an observation made later in presentations by J.J. Kirkland of Advanced Materials Technology (Wilmington, Delaware) and M. Dittmann of Agilent Technologies, Waldbronn, Germany, who also reported that there may even be contribution in the lowering of axial diffusion.

Proponents for SPP here say that you get the lower pressure drop of the larger particle and the efficiency of the smaller sub-two particle. With a shell thickness of 0.5 μm, the available stationary phase is roughly about 75% that of a comparable totally porous particle so the reduced amount of available stationary phase is not as great as superficially porous structure might suggest.

A number of papers showed results using these newer superficially porous particles. Majors in his columns talk introduced Poroshell 120 for the rapid separation of small molecules. In the same session, Kirkland showed applications of the Halo column for peptides and intact proteins using a capillary column format. The thin porous layer of these particles is especially important for the separation of larger biomolecules where slow mass transfer causes loss of resolution with fast separations on totally porous particles. As an example, he provided data for a six-protein digest on a 90-Å C18 Halo 150 mm × 0.2 mm column. He was able to demonstrate a total peak capacity of 400 in the gradient separation mode. He also discussed intact protein separation of a C8 column and stated that carryover and clogging are much less frequent than with totally porous particle capillary columns.

The transfer of methods and speedup of methods on the SPP columns was the subject of an oral presentation by H.K. Brandes and coworkers at Sigma-Aldrich/Supelco (Bellefonte, Pennsylvania). Some of the differences between conventional porous particle columns and these "fused core" particles that should be taken into account are the 25% reduction in stationary phase volume due to the presence of the solid core and expected differences in retention and selectivity. Currently, a limited number of stationary phases are available but that is sure to change in the future as these products become more widely accepted.

The SPP (and monoliths) are useful when a rapid separation is needed at high flow rates and low operating pressure. An ideal application is the second dimension of a typical LC×LC experiment. Also, for conventional HPLC systems limited to 400 bar pressure, the low pressure drops noted for the SPP and the high efficiency rivaling the sub-2-μm columns are ideal for having to avoid updating instrumentation to UHPLC capability.

Sub-2-μm Columns: Under the technology category (Table I), the sub-2-μm columns were intertwined with papers on UHPLC. Other terms used in this area were ultra-pressure LC, fast LC, ultrafast LC, rapid resolution LC, high speed LC, ultra speed LC, and high throughput LC. No wonder people are confused with the nomenclature in this sub-2-μm world. In reality, the sub-2-μm particles are the normal progression of the decrease in packed column particle sizes that has occurred over the years from 10 μm in the 1970s to 5 μm in the 1980s and 1990s to the 3–3.5 μm particles in the 1990s and early 2000s. There is nothing magic about the use of these particles except that to take full advantage of their speed, especially when they are packed into very short (less than 30 mm) and small internal diameter (less than 2.0 mm) columns, one must pay more attention to such experimental parameters as extracolumn effects (for example, detector cell volume, injection volume, and connecting tubing internal diameter), time constants, and gradient delay volumes (dwell volume). Possible frictional heating with the sub-2-μm particles was brought up several times during lectures and discussion. This impact of frictional heating on the separation performance and reproducibility needs a thorough unbiased study as this may limit further reductions in particle size.

These features were further elucidated during a lecture in the high-speed HPLC session. Prof. Unger suggested that there might be a minimum particle size on the horizon. Today's sub-2-μm particles in short columns have resulted in higher plate numbers, better resolution, reduced analysis time, increased detectivity due to narrower, taller peaks, and sometimes better robustness and reproducibility. The flipside is the increased pressure and the buildup of higher internal (frictional) temperature gradients. However, this latter observation may be turned to an advantage if properly recognized and is less of a problem with smaller internal diameter columns where the heat is dissipated. Unforeseen problems may arise from the effects of higher pressures on the vital mobile phase–stationary phase equilibria, on the solubility of analytes, and even on the miscibility of the components of the mobile phase. A further problem with changing particle size is the "transferability" of already developed (validated) methods as, even with manufacturers' descriptions indicating "the same phase," this is often not the case. Unger recommends that users should buy columns from companies that control the entire column process from making the silica to the final tested packed column. In Unger's opinion, the race for smaller and smaller particles should end. Particles smaller than the current sub-2-μm particles will not lead to significant improvements in plate number, but will significantly increase back pressure and its concomitant problems. He ended his lecture with a prediction that, in the future, more emphasis will be given to mechanically stable monoliths with a wider range of selectivities.

Other Stationary Phases and Retention Mechanisms: Researchers now seem to be developing bonded phase chemistries that are useful outside of the regular modes of C8 and C18 reversed phases. P.G. Stevenson of the Centre for Complementary Medicine (Sydney, Australia) described work with several silica-based phenyl phases each having different alkyl chains to bond the phenyl group to the support. From computer predictions of minimum energies, the "pictures" obtained indicated that, when the alkyl chain has an even number of carbons (n = 2, 4), the phenyl is essentially horizontal with respect to the silica surface while with odd n (n = 1, 3), the ring is perpendicular to the surface. This prediction would suggest that n = 3 would have more "height," possibly leading to better retention.

Experimentally, he and his coworkers found that the longer alkyl chains (n = 3, 4) produced shorter retention times for large aromatics (for example, PAHs) due to hindered access while n = 2 (ethyl) gave more selectivity. He concluded that multiple mechanisms must be involved.

One of the more unique supports presented at HPLC 2008 was diamond particles, which were first introduced at Pittcon 2008. M.R. Linford of Brigham Young University (Provo, Utah) functionalized the diamonds to produce amino, C18, perfluoro, phenyl, and sulfonic acid stationary phases for HPLC and SPE. Two general strategies were employed: hydrogen-terminated diamond reacted with a mixture of radical initiator and monomers to produce bonded polymer films (~3–6 nm thick) and self-limiting polymer adsorption to make phases, which are also in nanometer thicknesses. An example of the latter approach, poly(allylamine) adsorbs in this manner to produce an amino phase, which upon crosslinking with a diepoxide creates an extremely stable phase. Stability tests on his phases indicate, in general, that they are stable for many hours in 2.5 M hydrochloric acid or 2.5 M sodium hydroxide.

In a tutorial entitled "Is Silica Walking into the Sunset?," Professor Patrick Sandra of Ghent University (Belgium) first compared the number of phases in GC, which used to number in the hundreds, to the fact that 90% of the work can be done on one support (fused silica) with only four phases and that with these four phases, the selectivity and efficiency are nearly independent of the column manufacturer. Contrast that to HPLC, where different modes, different column formats, and different supports (inorganic and organic) and particle formats add up to thousands of configurations. Columns from one manufacturer of the same phase cannot, for the most part, be interchanged with columns of another vendor. During this tutorial, Dr. Sandra challenged the audience to think outside the box and consider other ways to increase resolution through the α term (for example, selectivity tuning via 2D LC, supercritical fluid chromatography, use of temperature to affect selectivity), which provides more clout than working on the efficiency term only (~ N1/2). The use of HILIC was encouraged where the silica backbone is deactivated with water and even unstable compounds can give excellent peak shape. He furthermore suggested that chromatographers also consider the use of polymeric columns that give good van Deemter curves and reduced plate heights of 2.3–2.4 at high temperature. At high temperatures (120 °C), the amount of organic solvent (for example, acetonitrile) in a reversed-phase application can be significantly reduced. Most polymers display no catalytic activity that sometimes occurs with silica with possible sample degradation. With polymers there is usually no bleeding of stationary phase and they can work at a very wide pH range and high temperatures.

HILIC: Probably the biggest interest in these sub-modes has been in HILIC. HILIC is a separation technique for highly polar analytes that gets around some of the problems associated with reversed-phase chromatography such as low retention or phase collapse (dewetting). HILIC uses a polar stationary phase such as bare silica gel or polar bonded phase (for example, diol) and requires a high percentage of a nonpolar mobile phase, similar to the requirements for normal-phase chromatography. However, unlike normal phase, which uses nonpolar solvents like hexane and methylene chloride and tries to exclude water from the mobile phase, HILIC requires some water in the mobile phase to maintain a stagnant enriched water layer on the packing surface into which analytes may selectively partition. In addition, water-miscible organic solvents are used. Under HILIC, polar analytes are well retained and are eluted in order of increasing hydrophilicity. The high organic solvent content of the mobile phase is especially attractive for LC–MS since ionization efficiency is improved and suppression effects reduced.

David McCalley continued his work on HILIC, this time studying the properties of SPP for charged analytes (mostly basic) that may involve partitioning, adsorption, ionic and reversed-phase (hydrophobic) interactions. A special characteristic of HILIC is that hydrophilic bases have good retention, somewhat opposite of reversed-phase LC. The HILIC columns show greater loadability than reversed-phase columns, especially for ionizable compounds. Dr. McCalley studied the loading of the SPP column Halo and found that, even though there is a smaller volume of stationary phase in the superficial surface, the capacity still rivals totally porous reversed-phase columns for ionized solutes. He also investigated SPP at high flow rates and high temperature where earlier work reported poor performance; his generated van Deemter curves showed normal behavior and he was able to observe a reduced plate height of 1.5. In another set of experiments, he estimated the extent of the water layer in the pores of the silica-based SPP HILIC column. Using pychometry, he determined the maximum column void volume and then compared it with the injection of a small hydrophobic solute (benzene) as void volume marker into acetonitrile–water phases of high organic content. Benzene does not partition into the water layer and is, thus, partially excluded from the pores of the phase up to a water content of 30%; after this concentration, the mechanism reverts to hydrophobic interaction. He was able to estimate that about 4–13% of the pore volume of a silica phase is occupied by the water-rich layer in the range of 70–95% acetonitrile.

HPLC 2009 is Next

The next major symposium in this series, the 33rd International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2009), moves back to Europe and will be held for the first time in Dresden, Germany, June 28–July 2, 2009. The chairman of this upcoming event will be Prof. Christian Huber of the Saarland University (Saarland, Germany). For more information consult the official website http://www.hplc2009.com.


I would like to acknowledge the contributions of my Agilent colleagues: summaries from Martin Vollmer and Gerard Rozing (Waldbronn, Germany), K.M Robotti from Agilent Labs (Santa Clara, California), and detailed notes from Maureen Joseph (Wilmington, Delaware). I also would like to give a special thanks to Professor Carol Collins of State University of Campinas (Sao Paulo, Brazil) for her excellent summary of many of the columns' talks that I was unable to attend. Also, thanks to Olcay Sagirli of Istanbul University (Istanbul, Turkey) for supplying some of the photos that were reproduced in this article. Finally, last but not least, I want to again thank the Poster Committee members who worked very hard reviewing (and sometimes rereviewing) the posters at HPLC 2008.

Ronald E. Majors

Ronald E. Majors

"Column Watch" Editor Ronald E. Majors is business development manager, Consumables and Accessories Business Unit, Agilent Technologies, Wilmington, Delaware, and is a member of LCGC's editorial advisory board. Direct correspondence about this column to "Sample Prep Perspectives," LCGC, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail lcgcedit@lcgcmag.com.


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