Highlights of HPLC 2010

The 35th 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 in Boston, Massachusetts for the third time, June 19–24, 2010. More affectionately known as HPLC 2010, the symposium is the premier scientific event for bringing together the myriad techniques related to separations in liquid and supercritical fluid media. Chaired by Dr. Steve Cohen of Waters (Milford, Massachusetts), HPLC 2010 assembled 1232 scientists from a total of 40 countries. This number included 305 vendor representatives from over 59 exhibitors for the three-day instrument, software, and consumables exhibition. Students constituted nearly a fifth of the conferees, which speaks highly of the next generation of separation scientists. Based upon the number of attendees and exhibitors, the worldwide economic crisis did not play heavily into the support for this important conference; the attendance was flat from HPLC 2009 (1).

The five-day-plus event had a total of 114 orals in plenary and parallel sessions and 585 posters in sessions with 29 themes. With an ample social event schedule, 12 vendor workshops (some with free snacks), 9 tutorial educational 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 were particularly well attended and covered current topics such as capillary electrophoresis–mass spectrometry (CE–MS) and liquid chromatography (LC)–MS, multidimensional LC, hydrophilic interaction liquid chromatography (HILIC), solid-phase microextraction (SPME), regulatory issues, microfluidics, quality by design, and the development of commercial liquid chromatography.

Trends in Liquid Phase Technology and Techniques

Obviously, high performance liquid chromatography (HPLC) was the predominant technology in the technical sessions at the symposium but sample preparation, use of electrophoretic techniques, mostly in a capillary format, and an increase in supercritical fluid chromatography (SFC) topics were strongly evident. 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, applications of liquid phase and detection were introduced in this series.

Table I: HPLC 2010 papers presented by technology or technique

Table I provides a rough breakdown of the coverage of liquid-phase technology and techniques in the separation sciences. Compared to HPLC 2009 (1), some slight shifts in technology emphasis were noted. Again this year, new developments in column technology led the pack. About a quarter of the columns' papers dealt with monoliths. Although not yet considered a commercial success, research interest in this technology is still running high. Silica gel–based monoliths are seeing their second generation of commercial products with better efficiency but slightly higher pressure drops due to the change in the macropore/mesopore domain ratios. Presentations on polymer-based monoliths outnumbered silica-based monoliths 2:1. A continuation of new developments in polymeric monoliths devoted to the separation of small molecules has shown improvements in column efficiency; originally the small molecule domain was for the silica-based monoliths only. Three other "hot" areas in column technology this year were

• More emphasis on the new breed of superficially porous packings (also referred to as shell particles, poroshell, and fused-core packings) that rival the sub-2-μm particles in terms of column efficiency but with substantially lower pressure drops.

• The widespread attention to the technique of HILIC for the separation of polar analytes that are not well retained by reversed-phase chromatography.

• A very hot but rather specialized topic was the use multidimensional chromatography, especially comprehensive LC×LC, which had over 50% more papers than at HPLC 2009 (1). With chromatographers encountering more complex samples, sometimes with thousands of compounds present, these multidimensional techniques are about the only way to tackle such mixtures

The interest in new research into sub-2-μm porous packings has waned a bit this year, probably because these columns now are firmly established in the ultrahigh-pressure liquid chromatography (UHPLC) world with over 25 vendors supplying these products (2) and nine instrument vendors providing UHPLC instruments to handle them.

Sample preparation technology was well represented in the poster papers. Most prominent were improved solid-phase extraction (SPE) technologies with new phase chemistries: monoliths, thermally responsive polymers, molecular-imprinted polymers (MIPs), and mixed-mode phases providing new selectivities. A novel E-SPE technique was noted, in which an electrical potential was imposed across the cartridge. By variation of the applied voltage, retention of charged analyte and matrix compounds could be adjusted allowing a new variable in SPE method development.

After years of a strong showing, electrodriven separation techniques (for example, CE, capillary zone electrophoresis [CZE], micellar electrokinetic chromatography [MEKC], and isoelectric focusing [IEF]) showed a significant dropoff with about half the number of papers presented this year compared to 2009 (1). A continued lack of interest in capillary electrochromatography (CEC) was noted with only eight presentations at HPLC 2010. 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 nearly a doubling of the number of presentations dealing with theory, modeling, and separation mechanisms. Researchers appear to be reinvestigating some of the fundamentals of liquid phase separations. Topics of interest in this area included the addition of gradient theory to the Kinetic Plot Model, the mechanism of turbulent flow chromatography, many papers on the mechanism of HILIC, computer modeling of the packing of columns, modeling of convective diffusion mass transport in a random walk particle tracking approach, modeling of the retention of biomolecules applied to posttranslational modifications, and insights into chiral recognition mechanisms in SFC.

Areas of Application

Table II is a breakdown of the most popular application areas reported at HPLC 2010. The number of oral and poster presentations on proteomics, biomarkers, protein, and peptide separations and identification again led the way. The number of papers increased by 35% over HPLC 2009 (1) perhaps indicating the strength of research in the life sciences in the U.S. The areas of major applications were glycosylated proteins and monoclonal antibodies. Pharmaceutical and biopharmaceutical assays in drug discovery, therapeutics, formulations and active pharmaceutical ingredients (APIs) had wide coverage this year. Another application area with a strong showing was for liquid-phase techniques in foods, beverages, and food safety. This increased attention to the safety of our food supply is no doubt a result of reports on toxic food contaminants continually making the news in the past year. The fourth major area was the use of HPLC in the analysis of drugs, toxins, and endogenous compounds in biofluids and tissues in pharmaceutical and clinical testing.

Table II: Papers presented by application area

One particular area of focus was the growing interest in glycobiology. On Monday, one of the plenary lectures was delivered by Pauline M. Rudd of the Dublin-Oxford Glycobiology Laboratory, NIBRT, Dublin, Ireland. Her title was "Linking Glycome and Genome: Robotic HPLC Based Platform Establishes the Variability, Heritability, and Environmental Determinants of Human Plasma N-Glycome." Glycosylation plays an essential role in the functions of the proteins that contain this common posttranslational modification and it is a parameter that determines the efficacy, pharmacokinetics, and safety of most biological drugs. These include erythropoietin, the pituitary hormones and monoclonal antibodies, of which 31 are currently licensed by the FDA and 200 more are in the pipeline. Glycosylation processing pathways are complex and not under tight genetic control; therefore, monitoring and controlling glycosylation in bioprocessing plants is a significant challenge. Rapid, detailed, quantitative glycan analysis is required at all stages of bioprocessing,from quality by design, to process analytical technology, to drug licensing and in-vivo tracking. Dr. Rudd has found HPLC–MS-MS with the modes HILIC, reversed phase, and weak cation exchange to be indispensable techniques for the separation of glycans. The glycans are cleaved from the glycoproteins by specific enzymes. In Europe, the regulatory bodies are more rigorous than the U.S. FDA and require the determination of the different proportions of glycans in glycosylated drugs. In fact, different lots of monoclonal antibodies can be monitored via glyco(protein) functions.

In this installment of "Column Watch," I will present some additional scientific highlights of HPLC 2010. This report also will cover the various awards and honorary sessions that took place. Because it was virtually impossible for one person to cover all oral and poster papers adequately, my coverage will reflect a personal bias to a certain degree, 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 at HPLC 2010

Horvath Award: For the fifth 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, 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 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 cash prize, invitation to present an oral at HPLC 2011, and a trophy. This year's winner was Jesse O. Omamogho (pictured in Figure 1a) along with coauthors E. Stack and J.D. Glennon of the Irish Separation Science Cluster at the University College, Cork, Ireland. The title of the award winning presentation was "Structural Variation of Solid Core and Thickness of Porous Layer of Core-Shell Particles on Chromatographic Performance." This timely research explored the influence of structural variation of solid core and shell diameter on the chromatographic properties and performance of core-shell silica packing materials. Three types of core-shell silica particles that varied in solid-core/shell diameter (1.0/0.35; 1.2/0.25; 1.5/0.1 μm) and having identical external particle diameter of 1.7 μm were synthesized. The surface areas of the three shell packing varied (205, 130, and 80 m²/g) proportionally to the porous shell thickness, providing identical pore size of 10 nm for each core-shell packing material. The measured total column porosities (0.43, 0.39, and 0.29, respectively) were in the same order. The particle size distributions were very narrow with a d_90/10 ~1.2 for all three packings. The particles were derivatized with C₁₈ silane, packed into 50 mm × 2.1 mm columns and tested chromatographically using a well-retained analyte, naphtho[2,3-a]pyrene. Van Deemter and various versions of kinetic plots were constructed. From the van Deemter plots, the reduced plate heights for the columns were determined to be 1.9, 2.2, and 2.5 (with decreasing shell thickness) comparing favorably with commercial shell core products. Compared to totally porous particles, he reported that the C-terms of the van Deemter curves were substantially lower indicating better mass transfer characteristics.

Figure 1: Photos of (a) the Horvath Award winner Jesse O. Omamogho (Irish Separation Science Cluster, University College, Cork, Ireland) and the Top Three Winners of the Best Poster Awards at HPLC 2010: (b) First Place: Iva Urbanova from the E.0. Lawrence Berkeley National Laboratory; (c) Second Place: Michael Fogwill, University of Calgary, along with Agilent Technologies presenter Elaine Ricicki; (d) Third Place: Matthias Verstraeten, Free University of Brussels, Belgium.

Poster Sessions and Best Poster Awards: The mainstay of HPLC 2010 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 2009 (1), the number of posters was about the same. 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 crowded. About 10% of the posters indicated in the abstract booklets were "no shows," often from third-world countries who apparently didn't get funding to attend or merely wanted their abstracts to appear in the program. Unfortunately, this behavior is difficult to stop but creates gaps in the poster session as well as extra expense for the sponsors because they must pay for unused poster boards anyway.

The Poster Committee co-chairs at HPLC 2010 were Prof. Peter Schoenmakers, a member of the Permanent Scientific Committee from the University of Amsterdam, The Netherlands and Dr. Gerard Rozing, Agilent Technologies, Waldbronn, Germany. The 52 members of the Poster Committee devoted a great deal of time and worked very hard to narrow down the huge collection of posters by the end of the third day and then helped to select the three winners by the Thursday afternoon of the symposium. The selection criteria were based upon three factors: inspiration, transpiration, and presentation, and winning posters were viewed by a large number of committee jurors.

The Best Poster Awards, sponsored by Agilent Technologies (Wilmington, Delaware), were announced at the Closing Session. After narrowing the field down to nine posters, the three winners selected as the top vote getters received cash prizes and a certificate commemorating their achievement. This year's winning poster was entitled "Monolithic Polymer Layers for Separation of Peptides and Oligonucleotides Using Pressurized Planar Electrophoresis and Electrochromatography" by authors Iva Urbanova (pictured in Figure 1b), Scott D. Woodward, David Nurok, and Frantisek Svec from the E.0. Lawrence Berkeley National Laboratory, Berkeley, California, and Indiana University/Purdue University, Indianapolis, Indiana.

In this award-winning work, specifically designed superhydrophobic porous polymer monolithic thin layer plates were constructed using photopolymerization. Separations of peptides and oligonucleotides in the electrophoretic mode were performed using the hydrophobic poly(butyl methacrylate-co-ethylene dimethacrylate) layer with an optimized pore structure that did not contain any ionizable functionalities. Rapid separations were obtained in 2 min. In contrast, the separation in the pressurized planar mode was carried out using a negatively charged thin layer prepared via specifically designed process comprising cografting of 2-acrylamido-2-methylpropanesulfonic acid and 2-hydroxyethyl methacrylate on top of the hydrophobic monolithic thin layer. The pressurization overcame accumulation of mobile phase on the plate surface and drying of the layer due to Joule heat, thus affording significantly better separations. In the electrochromatography mode, separations of peptides was made in 1 min. Adjustments of the chemistry of the monolithic layer and the use of higher applied voltages improved the separations and further increased speed.

The second place winner of the Best Poster contest went to Michael Fogwill (pictured in Figure 1c) and his professor Kevin B. Thurbide of the University of Calgary (Calgary, Canada) and was entitled "Supercritical Fluid Chromatography Employing Water as a Stationary Phase." Using a water-coated long capillary as the stationary phase and supercritical carbon dioxide as the mobile phase, the authors were able to effect separations with very good sample capacity, peak symmetry and retention time reproducibility (~1% RSD). Since altering temperature and pressure can change CO₂ density and polarity of the stationary phase, by optimizing such parameters as column temperature, CO₂ pressure, and CO₂ flow rate, rapid separations of alcohols, caffeine in an energy beverage and E10 gasoline were demonstrated in isocratic and gradient programmed modes as well as isothermal and temperature programming modes. The method is free of organic chemical components, is environmentally friendly and allows the use of the universal flame ionization detector.

The third place winner of the Best Poster award went to an international group consisting of Dow people from Germany (M. Pursch and P. Eckerle).and Canada (J. Luong) and an academic group from the Free University of Brussels, Belgium (G. Desmet and Matthias Verstraeten, who presented the poster and whose picture is shown in Figure 1d). The title of their poster was "Low Thermal Mass Liquid Chromatography: Modeling the LTMLC Setup." LTMLC is a novel technique that applies fast temperature programming to capillary LC separations. The technology is based on resistive heating and is similar in concept to low-thermal mass gas chromatography. Temperature program rates are as high as 1800 °C/min. The goal of this work was to see whether the heat transfer in the current LTMLC setup could be improved. By a combination of theoretical studies on such things as the temperature gradient inside of a cylinder when heating the wall and contribution to radial temperature gradients to band broadening along with experiment and numerical methods, they were able to come up with a recommendation of a final design of maximizing the inner diameter as a function of the heating rates. Some important practical conclusions were that the mobile phase must be preheated to minimize band broadening and thermal paste should be used to enhance the heat conduction.

Opening Session and Plenary Lectures

Opening Ceremony plenary lectures, presented on the first evening of the symposium, are supposed to be leading edge, thought-provoking presentations that are to inspire attendees to think beyond the box. After the Opening Ceremony involving welcoming remarks by Dr. Cohen, various prizes were announced including the Martin Gold Medal awarded to Prof. Peter Carr of University of Minnesota. Prof. Carr is widely recognized for his contributions to the separations theory, development of new column materials, and, recently, the investigations on the use and optimization of multidimensional chromatography (comprehensive LC×LC). This prize was presented by The Chromatographic Society's John Lough of the University of Sunderland, Sunderland, UK. 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, Prof. 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.

In his Plenary Lecture, Professor Carr first gave a brief introduction to 2D LC, where he defined the optimum peak capacity as the maximum number of equally resolved peaks that can fit in the separation space. Next, he outlined the three conditions necessary to obtain the maximum peak capacity for a 2D separation:

• The separation modes in the 1⁰ and 2⁰ dimensions must be orthogonal (totally different types of chromatographic interactions).

• Peaks must cover the entire separation space.

• The separation in the first dimension must be maintained in the 2⁰ dimension.

An early paper by Murphy, Schure, and Foley (3) pointed out that one must take at least four samples per base width (8σ) of a peak at a resolution of one. If one undersamples, then information will be lost.

One conclusion from his studies was that it does not make sense to struggle to get a high peak capacity in the 1⁰ dimension but one should strive to make the 2⁰ dimension really fast. Following are some of the suggested experimental approaches to increase the speed (cycle time) of this dimension:

• Use high temperatures (>100 °C).

• Use high mobile phase velocity.

• Use short analytical columns (with a short dead time) with small particles (<2 μm), monoliths or superficially porous particles.

• Use rapid gradient elution via dual gradient LC.

The cycle time of the 2⁰ dimension sets the overall speed of analysis because the total time of analysis is equal to the cycle time of the 2⁰ dimension run times the number of 2⁰ run.

Although not dealing with liquid-phase separations, the second plenary lecturer Prof. George Whitesides of Harvard University gave a very thought provoking lecture on diagnostics in the third world where money and resources are virtually unavailable. So the challenge he presented was how to provide low cost or no cost testing to places where there were no doctors, no infrastructure and not even understanding of disease. He discussed paper-based diagnostic systems where colorimetric immunoassays (for example, enzyme-linked immunosorbent assay [ELISA]) might be used in-situ. Something like a cell phone might be used to read the test sheet and either transmit test data or have the phone make the test itself. One could envision three-dimensional patterned paper where perhaps electrochemistry could be used as the driving force. The power would be supplied by button or flashlight batteries. Electrochemistry and paper colorimetry could be used on the same platform. Perhaps on the paper evaporative concentrators could be used to focus analytes. He even envisioned mechanical eggbeaters used as centrifuges. Overall, it was an interesting presentation that could cause analytical chemists to think outside the box in coming up with new approaches for low cost, simple chemical and biological analysis.

New Column Technology Highlights

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, retention mechanisms, high-throughput applications, HPLC/UHPLC, and the like. If one combines all papers pertaining to column technology, about a third of the presentations at HPLC 2010 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 at HPLC 2010, one of the current "hot topics" in HPLC column technology was 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. Last year's focus was on comparing sub-2-μm totally porous columns to the superficially porous columns. This year's focus seemed to be more focused on the superficially porous particles (SPP) themselves because they provide the efficiency of sub-2-μm particles but at around half the operating pressure. I counted a total of 31 papers devoted to these columns. There is some confusion about the nomenclature and during HPLC 2010, these particles were referred to as SPP, fused-core, shell, shell fused core, porous shell, porous shell core, solid-core shell, and poroshell particles.

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 near future.

Besides the Horvath Award winning lecture discussed earlier, at HPLC 2010, a number of papers on SPP were presented. Fabrice Gritti and Georges Guiochon of the University of Tennessee investigated mass transfer in columns packed with SPP core shell particles. They fully characterized the packing materials by using scanning electron micrographs, low-temperature nitrogen adsorption, Coulter counter, pycnometry, van Deemter, peak parking method, total pore blocking method and local electrochemical detection to look at all possible particle parameters. With respect to the van Deemter coefficients, they concluded that for small molecules, a smaller A term (eddy diffusion term was 40% less than totally porous particles) and a smaller B term (axial diffusion term was 25% less) have the biggest contribution to improving the efficiency of the SPP. They concluded that the C term (mass transfer) was much smaller and had little or no contribution. In his talk, Guiochon reported that heat effects are less important for SPP particles compared to totally porous particles because of their high heat conductivity. He supported the idea of G. Desmet (Free University of Brussels, Belgium) that was presented in another lecture to use segmented columns to increase heat dissipation since this is a problem with the tiny totally porous particles.

In addition, Gritti and Guiochon have done considerable work on minimizing extra column effects. Peaks from these SPP columns are quite narrow and connecting tubing, injection volumes and detector flow cell volumes have to be optimized. In his lecture on core shell columns, Tivadar Farkas of Phenomenex (Torrance, California) suggested the dilution of the injection sample with water thereby concentrating the sample at the head of the column and eliminating precolumn dispersion. This procedure, which he termed "performance optimized injection sequence," can be easily accomplished with modern autosamplers that can first draw up water from a reservoir and then sample from a vial. Their studies found that a 3:1 ratio of water to sample was best to accomplish the trace enrichment.

Stephanie Schuster and colleagues of Advanced Materials Technology presented a paper titled "Fused-Core Particles: Varying Shell Thickness and Pore Size." First, a comparison was done on a standard 90-Å pore size (two shell thicknesses of 0.3 and 0.5 μm) SPP to a 160-Å pore size (0.3-μm shell thickness) for a small molecule. For a small molecule, the thinner shell gave higher efficiency but less retention. The experiment was run for biomolecules with molecular weights ranging from 555 Da to 14.7 kDa. For the 90-Å pore size particle, the larger biomolecules gave poor performance because they were excluded from the smaller pores. Repeating the same experiment on a 160-Å pore SPP, better performance was noted.

Further tests were conducted on the wider pore SPP (160-Å) a column more suitable for peptide and small protein separations. Using a 1.7-μm solid core, a porous shell of 0.5 μm or 0.3 μm was added making a particle size of 2.7 and 2.3 μm, respectively. For higher molecular weight compounds, the thinner shell gave better efficiency but for sample loading a thicker shell was a bit better. For the 0.3-μm thick shell, the reduced plate height was 27% lower compared to the 0.5-μm shell. As far as peak capacity, both SPP packings gave a similar figure. For the thicker shell, there was a slightly longer retention for the same test compound.

Some novel core-shell phases were developed by a group from Brigham Young University led by Matthew Linford. One approach used 1.7-μm zirconia cores with a 0.5-μm porous nanodiamond shell. After functionalizing with C18, the columns were used to separate aromatic analytes. The disadvantage of this approach was the high cost of the raw materials so the authors switched to a graphite core which was also coated with nanodiamonds but the peak shape was undesirable. However, pH stability was quite remarkable and further work on improving the phase chemistry could result in useful packing.

A number of comparison papers appeared again showing the superior performance of the SPP when compared to the totally porous sub-2-μm particles, especially when the pressure drop is taken into account. In addition, numerous applications of the SPP are beginning to appear and at HPLC 2010, separations of pharmaceuticals (antibiotics, pain management drugs, anti-inflammatory drugs and various others), aflatoxins, drugs of abuse, genotoxins, small proteins and peptides, melamine/cyanuric acid and food and beverage additives were noted. Also, applications of SPP for SFC and as the secondary dimension in LC×LC are showing up.

Monoliths: Monolith columns have been desirable since they exhibit high permeability/low pressure drop (due to increased bed porosity), show good separation efficiency, have the absence of frits to confine the packing material, are easy of 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. In his Keynote Lecture in the Closing Session, Frantisek Svec of Lawrence Berkeley National Laboratory, Berkeley, California gave an excellent overview of the porous polymer monoliths. Last year reports at HPLC 2009 showed that polymeric materials can be synthesized that have better performance for small molecules. Svec and his colleagues, among others, have been working on new polymerization techniques for second-generation polymeric monoliths. By generating large throughpores with massive surface areas in the mesopores, small molecules can now be separated on polymeric monoliths. Monolithic poly(styrene-co-vinylbenzyl chloride-co-divinylbenzene) columns were prepared and subsequently modified using hypercrosslinking reaction to afford monoliths containing an array of small pores. These monolithic columns exhibit a large surface area of up to 500 m²/g that is more than one order of magnitude larger than that found for the nonmodified precursor columns. The presence of mesopores in these hypercrosslinked monolithic columns enables very good separations of small molecules in isocratic mode using aqueous-acetonitrile as the mobile phase. The column efficiency for uracil was found as high as 73,000 plates/m. However, there was still a small amount of tailing. The addition of tetrahydrofuran gave much better peak shapes. His group has been able to increase the surface area by the addition of carbon nanotubes chemically cut into short lengths and embedded into the structure of the monolith. With a high hydrophobicity and large surface area, these modified monoliths have shown further improvements in performance. Their latest research involves the functionalization of GMA monoliths with C₆₀ (fullerene) that has increased the surface area to 800 m²/g and increased efficiency to over 100,000 plates/m. In a new area, the Svec group has been able to tailor monoliths with new selectivities by incorporating gold and hydroxyapatite nanoparticles into them.

On the silica monolith front, the second generation monoliths are already underway. Nobuo Tanaka of the Kyoto Istitute of Technology has already demonstrated improvement in the permeability of monolithic silica capillary columns by controlling the feed ratio of alkoxysilanes. Long monolithic silica capillary columns (up to 450 cm) prepared from a mixture of TMOS and methyltrimethoxysilane (MTMS) and modified with C₁₈ moieties were able to generate 2,000,000 theoretical plates for aromatic hydrocarbons with four connected columns of 15 m at a pressure of 50 MPa. Single columns of 80–450 cm long can generate approximately 100,000–500,000 theoretical plates with a t₀ of 10–60 min under a pressure below 18 MPa at 30 °C. Generation of 10,000–1,000,000 theoretical plates is feasible by HPLC using the rod-type and capillary-type monolithic silica columns.

Tanaka also reported on new formats that are being constructed. Monolithic silica rods were prepared from teramethoxysilane, modified with octadecyl-silyl (C₁₈) moieties, and the columns were fabricated by encasing the silica rod in a stainless-steel protective column with two polymer layers between the silica and the stainless-steel tubing (2-mm thick, 8-mm o.d.). The advantage of the stainless steel construction is that the columns can withstand more pressure compared to the PEEK encapsulated commercial columns. The columns (MonoClad, GL Sciences, Tokyo, Japan) are silica rods with a 3-mm diameter, 3–25 cm length, surface area of 200 m² /g, through-pore size of 2 μm, and mesopore size of 18 nm) that provide a plate height of about 7 μm at optimum (at approximately 25 mm/s) linear velocity. A 25-cm silica rod column can generate 35,000–38,000 theoretical plates for aromatic hydrocarbons with t₀ of 1.8 min at 0.8 mL/min and pressure drop of 7.5 MPa in 80% acetonitrile at 40 °C. High-speed separation is also possible with a 5–10 cm column generating 7000–14,000 theoretical plates with t₀ of about 10 s. Short MonoClad columns are also suitable as a second-dimension column of 2D-HPLC.

Kazuki Nakanishi and Japanese coworkers from Kyoto University, GL Science, and the Kyoto Institute of Technology discussed an approach to better-performing semimicro columns by improving the structural homogeneity by increasing the volume fraction of silica skeletons in the bicontinuous macropore structure. The adoption of a different polymer additive, poly(acrylic acid), than conventional poly(ethylene glycol), was a key to successful preparation of macroporous silica monolith with improved structural homogeneity especially in the wall-region of the rod-shaped monolith. With a reduced domain size, in exchange with increased column pressure by a factor of two, the efficiency increased by approximately 50 % (N increased from 90,000 to 140,000/m). Together with a novel cladding technique, it is now possible to manufacture narrow bore columns with 1–3 mm internal diameters and 50–250 mm lengths with a pressure 50–33% that of 3-μm particulate columns. Another approach is based upon the high-porosity (90%) silica monolith having average macropore size of 1 μm comprising very thin (~200-nm) twig-like silica skeletons embedded with long-range-ordered cylindrical mesopores (~10 nm). Because of its high macroporosity, the van Deemter curves exhibit a considerable contribution of longitudinal diffusion (larger B-term). The minimum plate height value, however, is more than an order larger than theoretically possible limit based on the skeleton thickness and macropore diameter. This column is expected to give much lower plate height under electro-driven conditions, where the homogeneity in flow profile becomes much higher.

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 such as triptylines. For this purpose they have reduced the domain size from 3.3 μm down to 2.0 μm with a corresponding macropore size of about 1.2 μm. The resulting columns show N/m values of about 150,000–160,000, which is double those of the first generation. A typical mobile phase consisting of 60:40 (v/v) acetonitrile–water creates a column back pressure on these columns of only 50–55 bar (725–800 psi) at a flow rate of 2 mL/min. Thus, operation of these columns with conventional low-pressure HPLC systems is possible.

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.

New Technologies: Each HPLC meeting, new column designs are presented and this year was no exception. A most interesting paper by Mary Wirth and coworkers of Purdue University dealt with capillary columns packed with silica colloidal crystals. Her on-going work demonstrated a quantum leap in column efficiency compared to incremental gains in plates normally reported. Her particles were of submicrometer dimensions in the range of 150–500 nm. Although the particles were nonporous, because they are extremely small and densely packed, the surface areas are quite adequate to demonstrate their retentivity. Packing the small particles still represented a challenge. The first mode of separation was CEC and a microscope was used to follow the separation. Using proteins as test solutes, they found that the plate heights for 300-nm particles were limited by molecular diffusion but the protein peaks looked good and plate heights of 10 nm were measured for CEC. Next, they tried pressurized flow and used very short capillaries (30 mm). Injections were made electrokinetically, then hydraulic pressure applied. Under pressurized flow conditions, they achieved plate heights of 50 nm and achieved 300,000 plates for the column. However, next they switched to the larger 500-nm particles but still were able to achieve 150,000 plates and a 100-nm plate height. The pressure across their column was 6500 psi and the t₀ was determined to be 3 s. For these tiny columns, she determined that for the van Deemter parameters the A and C terms are zero while the B term is very low.

In another paper related to nanodimensions, Prof. Susan Olesik of Ohio State University developed nanoscopic polymeric and carbon fibers using the technique of electrospinning. The fibers are generated by forcing polymer solutions through a small, electrically charged orifice. The properties of the nanofibers are dependent on the polymer solution properties and the concentration of the polymer. Her first attempt was to electrospin polyacrylonitrile fibers in a mat configuration for application to thin-layer chromatography (TLC). The mat thickness was approximately 25 μm and the TLC efficiency was extremely high; thicker mats could generate even greater efficiency. As the fibers get smaller in size, the mats are slower running. Further work with carbon nanofibers has generated even more efficiency: 2-cm lengths have given up to 60,000 plates. Work on carbon nanofibers processed at higher temperatures has shown the potential of even higher efficiency. With more precise and smaller spotting devices, it is anticipated that TLC efficiencies could be even greater.

Further approaches to new column packings are under development. One area of continued research involves organic–inorganic hybrid packings. These packings are noted for their wider pH range than silica particles alone but provide efficiencies in the same range. New hybrids were discussed that apparently used a multilayered approach that provided good efficiency and pH performance. Hybrid polymeric HPLC chiral stationary phases (CSP) were reported to have been synthesized by a grafting-from photoinduced radical polymerization of enantiopure N,N'-diacryloyl derivatives of chiral diammines (trans-(1,2)-diaminocyclohexane and (1,2)-diphenylethylenediamine), initiated by the system consisting of trichloroacetyl groups on mesoporous silica particles and manganese decacarbonyl under UV light irradiation.

Waters scientists recently have expanded the scope of the ethylene-bridged hybrid (BEH) technology by re-engineering the hybrid particle to create porous particles with almost a twofold increase in pore volume compared with the original BEH particle. These have been found to be especially useful for size-exclusion separations. In addition, the group has developed charged surface hybrid (CSH) particles where a small percentage of positive charge is introduced to the surface before bonding the stationary phase. This process was developed to improve peak shape for bases at low pH and to overcome the slow equilibrium when the materials are exposed to alternating high- and low-pH mobile phases, particularly those useful for LC–MS. The columns also offer additional selectivity choices.

Dionex (Sunnyvale, California) scientists have introduced nanopolymer silica hybrid (NSH) technology. Their new material consists of high-purity porous spherical silica particles coated with nanopolymer beads via an electrostatically driven self-assembly process. This process results in a distinctive spatial separation of the anion exchange and cation-exchange regions, and it allows both retention mechanisms as well as hydrophobic interactions with the backbone to function simultaneously and be controlled independently, making a trimodal packing material.

Multidimensional and Comprehensive Liquid-Phase Separations

As pointed out by Prof. Carr in his Plenary Lecture, 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 in order 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.

Multidimensional chromatography 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 2010. 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 online 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 once 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.

Prof. Paola Dugo of the University of Messina, Italy updated her group's applications work on approaches to obtain high peak capacity on real samples using an automated LC×LC system. Earlier using combinations of normal-phase and reversed-phase columns in the first and second dimensions, they were able to resolve complex mixtures of naturally occurring substances. The first-dimension column was usually a long column with a small internal diameter that offered high resolving power while the second-dimension column provided a very fast separation using a superficially porous particle. An example shown this year was the analysis of triacylglycerols in corn oil using LC×LC–MS-MS. The first-dimension column was a silver ion impregnated stationary phase which separated by the number of double bonds while the second-dimension column was a reversed-phase column that separated by carbon number. Another example was in the area of proteomics but in this case both modes were reversed-phase LC only the mobile phases were at different pH values for the primary and secondary dimensions. The sample shown was a tryptic digest of human serum albumin. Four microbore C18 columns were used in the first dimension while in the second dimension a single Ascentis Express (Supelco, Bellefonte, Pennsylvania) was used but at a high 4-mL/min flow rate. The cycle time was 0.6 min and MS was used to identify some of the eluted peptides.

Prof. Pavel Jandera of the University of Pardubice, Czech Republic, investigated various combinations of gradients in the first and second dimension. Just like in one-dimensional chromatography, gradient elution provides significant improvement in peak capacity in comprehensive two-dimensional LC×LC, However, second-dimension gradients are limited to a short time period. For fast columns investigated in the second dimension (for example, sub-2-μm, monolith, porous shell), he also had to apply a fast gradient. Three types of second-dimension gradients were compared: steep gradients with an equal mobile phase composition change in each second-dimension run; "continuously shifting" shallow gradients in subsequent second-dimension runs; and a new type of steep second-dimension gradients with different partial mobile phase composition changes in several subsequent segments into which the two-dimensional time is divided. The type and the profile of the gradient affects significantly the quality of separation in the second dimension, namely the bandwidths, the peak capacity, the separation time and the range of lipophilicity of sample compounds that can be separated in the second-dimension reversed-phase time period. The third type of the gradient combines some advantageous features of the first and the second type. An approach was developed for calibration, prediction and optimization of gradient conditions for fast two-dimensional separations of samples with broad lipophilicity distribution, avoiding undesirable wrap-around of noneluted compounds between the subsequent second dimension fractions. The researchers applied this approach to comprehensive two-dimensional separations of natural antioxidants with a polar column in the first dimension and a porous-shell fused-core C18 column in the second dimension.

Because there were a large number of papers dealing with multidimensional LC separations, I tallied the types of phases that were used in the first and second dimensions and came up with Table III, which shows the modes being coupled. In some cases, the mobile phases are readily compatible while in other cases special considerations must be given to coupling incompatible liquids. Of course, when mobile phases are incompatible, off-line techniques with solvent exchange are possible.

Table III: Two-dimensional (LC×LC) techniques reported at HPLC 2010

Detection Techniques

By a perusal of the abstract, I tabulated the detection principles (Table IV) that were used on a relative basis in the various presentations at HPLC 2010. Not every abstract indicated the detector that was used so only those that provided this information was counted. The category assignments were based upon 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, 54% of the papers presented at HPLC 2010 used this detection technique. Compared to last year (1), there was about a 25% growth in the MS-MS category, while LC–MS dropped by a third. Despite the higher cost, the remarkable selectivity and sensitivity of the tandem techniques are favored by chromatographers and mass spectroscopists alike.

Table IV: 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 examples. Fluorescence and evaporative light scattering detection showed a slight growth while use of electrochemical detection dropped a bit but all of these other detection techniques are used infrequently.

HPLC 2011 (Europe) and 2012 (U.S.) Are Next

The next major symposium in this series, the 36th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2011), Budapest, Hungary, June 19–23, 2011. The chairman of this upcoming event will be Prof. Attila Felinger, University of Pécs, Pécs, Hungary. For more information consult the official website: http://www.hplc2011.com. 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 Prof. Frank Svec of University of California, Berkeley 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.

Acknowledgment

I would like to acknowledge the contributions of my Agilent R&D colleagues of Wilmington, Delaware, who supplied notes on some of the sessions. I would also like to thank Dr. Xiaodong Liu of Dionex Corp. for his summary of some of the highlights of HPLC 2010. I also would like to commend the CASSS organization for running a well organized meeting that met everyone's expectations. I also appreciate the statistical information used in my introduction that was supplied by Stephanie Flores, Executive Director of CASSS.

Ronald E. Majors Ronald E. Majors "Sample Prep Perspectives" 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 to "Sample Prep Perspectives," LCGC, Woodbridge Corporate Plaza, 485 Route 1 South, Bldg F, First Floor, Iselin, NJ 08830, e-mail lcgcedit@lcgcmag.com

References

(1) R.E. Majors, LCGC North America 27(9), 796–815 (2009).

(2) R.E. Majors, 2010 Pittsburgh Conference, Orlando, FL, paper number 2890-4, March 4, 2010.

(3) R.E. Murphy, M.R. Schure, and J.P. Foley, Anal. Chem. 70, 1585–1594 (1998).