The technology and market for liquid chromatography columns continue to mature, with applications-specific columns on the rise. The chromatography columns target specific assays in the clinical diagnostics, foods, and environmental segments. Columns are supported with methods that help the nonchromatographer get results and largely avoid optimization of runs for specific instruments. This is easier for isocratic separations. Methods involving gradient elution are potentially more difficult since differences in delay volume can change gradient profiles. However, several firms provide simple computer programs and even cell phone applications to guide the transition.
An applications example is YMC-Omega from YMC (www.ymcamerica.com) for assay of polyunsaturated fatty acid (PUFA) esters. The column has a reversed-phase liquid chromatography (RPLC) surface chemistry on 10-μm silica. Ligand density is controlled to provide good resolution for all isomers, including omega-3 and 6 fatty acids. The analytical column is used to monitor fatty acids in foods and dietary supplements. Corresponding preparative columns enable large-scale purification of fatty acids with YMC’s process LC.
The GlycanPac AXH-1 is a new mixed-mode phase designed for separation of anionic glycans. The conventional GlycanPac from Thermo Fisher Scientific (www.thermoscientific.com) separated by hydrophilic interaction liquid chromatography (HILIC) mode, which works well for neutrals, but Thermo’s column wizard Chris Pohl reasoned that adding anion exchange selectivity could expand the separation space to include glycoanions such as sialic acids. One can adjust the mobile phase to provide a class separation (monosialic, disialic, trisialic, etc.), or, with a slower gradient and longer run time, the individual class members are resolved. Beta evaluators are very impressed.
For neutral glycans, Thermo added a new particle size to the Accucore HILIC. This column seems to have a particular sweet spot for chromatography of neutral, native glycans for LC/MS. If better detection sensitivity is required, the column also works very well with fluorescently tagged glycans.
Thermo’s column chemists added two new HPLC columns to the Acclaim line, which is based on spherical silica. The Acclaim Q1 was developed for LC/MS of diquat and paraquat herbicides in environmental studies. The Acclaim SEC (size exclusion chromatography) column is optimized for water-soluble polymers in the size range of 100 Dalton to 1 megaDalton.
QuEChERS method for dicyandiamide
Recent concern about dicyandiamide (DCD) in dairy products led Bonna-Agela Technologies, Inc. (www.agela.com) to develop a QuEChERS method for assay with LC/ UV or LC/MS. Both methods use the company’s Unisol 5-μm HPLC column (4.6 × 250 mm). The UV method uses rapid gradient elution and detection at 220 nm. The quantitation limit was about 0.5 μg/g. With LC/MS on a 2-mm-i.d. Unisol Amide and multiple reaction monitoring (MRM), the limit of quantitation was 2 ng DCD/g/sample. Since the toxicity profile of DCD has not been finalized, required control limits have not been set.
Columns for general LC
Previously, I summarized the features and dimensions of LC columns in charts supported by text. However, in the interest of conserving space, I’ve avoided discussing column dimensions since most vendors provide a wide range covering capillary to preparative, at least at the lab scale. Without exception, you can get the details online.
On the core/shell stage, Thermo introduced Accucore 4-μm, and Phenomenex (www.phenomenex.com) presented Kinetex® 5-μm particles. Both replace the totally porous 3–5 μm column packings with the core/shells. The columns must be compatible with legacy HPLCs, primarily flow, pressure, and detector dispersion.
At the 5-μm end, replacing a 5-μm column packed with 4- or 5-μm core/shell packings should be easy and entail only minor tuning of the mobile phase composition. One should expect to see moderate improvements in resolution and peak height. In the case of Accucore 4-μm, some may be concerned about replacing a 5-μm particle with a 4-μm. Would this require revalidation? Not likely, since the USP allows a 20% change in particle diameter as a minor (permitted) adjustment.
However, replacing a 3-μm totally porous particle with a 4- or 5-μm core/shell does not meet the 20% criteria. But if the method is not subject to strict regulatory validation, one can make the switch and probably see a reduction in working pressure with no loss in resolution or peak height. Columns packed with all of these packings should be compatible with legacy HPLC instruments. Indeed, one may be able to increase flow rate modestly to gain a corresponding reduction in run time. Plus, the larger core/shell particles may have advantages in directly scaling up analytical separations obtained on 1.3-, 1.7-, or 2.6-μm diameter core/shells to lab-scale prep.
For prep, Phenomenex packs Kinetex 5-μm phases in its Axia™ column hardware. Axia columns have developed a following with chromatographers separating drug candidates, intermediates, and natural products.
Supelco (www.sigmaaldrich.com) also settled on 5-μm diameter for its extension of the Ascentis® Express 5-μm core/shell phases. Surface chemistries include C18, C8, phenyl–hexyl, HILIC, Pentafluorophenyl (PFP or F5), ES-Cyano, and OH5.
Thermo added to the Accucore line with the XL series, which features a 4-μm dp. Taking advantage of Accucore’s thin skin design, the XL series offers efficiency comparable to 3-μm-diam porous silica but with a lower pressure drop. In contrast to 5-μm porous particles, the efficiency is higher, as are the peak heights. The XL series includes C18 and C8 surface chemistries.
Five surface chemistries were announced to round out Thermo’s original Accucore core/shell (2.6-μm-diam, 80-Å pore) product line: 1) The C30 phase provides shape selectivity for nonpolar analytes; 2) the PFP (pentafluorophenyl) phase shows strong selectivity for halocarbons; 3) for HILIC, an amide surface chemistry was developed; 4) for aromatics, Phenyl-X shows higher retention; and 5) for RPLC with highly aqueous mobile phases, the “AQ” was developed. For larger molecules, Thermo introduced the Accucore 150-C18, 150-C4, and 150-Amide-HILIC; “150” designates a 150-Å pore.
Hybrid core/shell packings
Traditional core/shell column packings have porous shells of silica that are usually further modified with various surface chemistries including C18, C8, and PFP. Imtakt USA (www.imtaktusa.com) announced a novel hybrid core/shell with a dense silica core and porous polymer on the surface. The particles are 1.5 μm in diameter, which gives slightly better efficiency and resolution compared to conventional sub-2s (1.7–1.9 μm).
HILIC is emerging as the mode of choice for assay of saccharides. Amino phases are ideal candidates due to unique selectivity of the amine–carbonyl interaction. The Asahipak NH2P series from Shodex™ (www.shodex.net) are an excellent choice since the polyvinyl alcohol particle is immune to base attack, which can provide a high background signal in the evaporative light scattering detector (ELSD) with silica particles. The amine is pentaethylenehexamine. The composite is stable over the pH range 2–13.
This year, Shodex introduced the NH2-40P 3E, which features a 4-μm particle size to improve column efficiency. These come in a 3-mm-i.d. column diameter, which is compatible with most HPLC instruments. Columns packed with 4-μm particles yield about 30% greater plate count than 5-μm particles.
COSMOSIL HILIC is a new column from Nacalai USA, Inc. (www.nacalaiusa.com). Compared to the legacy HILIC phase on 5-μm particles, the new 2.5-μm packing gives baseline separation in one-sixth of the time. The surface chemistry is triazole bonded to 2.5-μm silica particles with 120-Å pore. The particles are large enough that the column pressure does not require ultrahigh-performance liquid chromatography (UHPLC).
Silica offers the highest efficiency of any of the column packing particles. Its major weak point is susceptibility to hydrolytic attack at a pH less than 3 or higher than 7. For decades, chromatographers have tried various end-capping strategies to extend the operating window to lower and higher pH. ACE® SuperC18™ is the latest effort by Advanced Chromatography Technologies, Ltd. (www.ace-hplc.com) to shield the silica surface from attack. The company calls it “Encapsulated Bonding Technology,” or EBT™. Animation showed that the silica surface is encapsulated and thus protected from acids and bases. Further, it showed an absence of silanols. Lifetime tests at pH 1.8 and 10.7 revealed no significant loss in performance after 30 days and 2000 injections. A chromatographer can therefore use one column to exploit the full selectivity of the C18 stationary phase at a range of pH values. ACE SuperC18 is available in 2-, 3-, 5-, and 10-μm dp, 90-Å pore.
C18 on PS/DVB beads
Many may recall that Hamilton Company (www.hamiltoncompany.com) was one of the first vendors of HPLC column packings. These were based on polystyrene/divinylbenzene (PS/DVB) copolymer. The beads were nearly indestructible by acid or base, but lacked the efficiency and hence resolution offered by silica particles. Usually the low efficiency was attributed to slow mass transfer and mixed mode, particularly π–π interactions.
However, our understanding of good chromatographic particle design and surface chemistry has improved greatly in the last 45 years. Hamilton figured it was time for a fresh start; after all, polymeric column particles should still be more stable to low or high pH and heating up to 100°C, especially compared to silica particles. Ligand bleed is not detectable. There are no silanols that require end-capping. Column efficiency, measured in reduced plate height, is about 4—not outstanding, but a great improvement over conventional PS/DVB columns, which were much larger. The particle size is 5 μm. The short column (50 mm) gives sub-10-min separations for complex mixtures.
ACE EXCEL™ 2-μm Ultra Robust UHPLC columns from Advanced Chromatography Technologies are designed for high durability and long life. A series of surface chemistries are available, even within RPLC. To meet the needs of high-throughput screening, Imtakt introduced Unison UK-C18 high-throughput columns, which are short (75 mm) and hence fast. The column packing is C18 bonded to 3-μm-diam silica. Imtakt’s proprietary polymeric end-capping provides long life, even at low pH.
For glycopeptides, Imtakt introduced three new mixed-mode C18 phases under the Scherzo brand name for chromatography of sialylglycopeptides: Scherzo SS C18 has strong anion and cation exchange groups bonded to 3-μm silica, and Scherzo SW C18 has a lower capacity of the strong ionophores. Scherzo SM offers a mixture of weak anion and cation groups. Packed columns are available in a wide range of lengths and diameters.
“A diamond is forever” comes to mind as the chromatographers at Diamond Analytics (www.diamond-analytics.com) explained the careful, elegant synthesis of diamond core/shell particles as the basis for a C18 stationary phase. Starting with a microdiamond core, the porous shell is created layer-by-layer by immersion of the core in solutions of polyallylamine followed by nanodiamond. When the proper shell thickness is reached, the structure is cross-linked with a C18 diepoxide. This provides a robust solid particle with excellent mass transfer performance. Chromatographic efficiency of a column packed with 4-μm particles is 100,000 N/m for a reduced plate height of 2.5. The column can be run indefinitely at pH 11.3 at 120 °C, and repeatedly regenerated. Applications include separation of herbicides in less than 2 min, and tricyclic antidepressants at pH 12.
Graphite is another of the many allotropes of carbon. United Science (www.uniscicorp.com) is entering the HPLC column market with novel columns with graphitized carbon black deposited type B silica. Since other graphite phases strongly adsorb certain analytes, the surface coverage is partial. Low coverage promotes elution. The silica provides good mechanical strength. I wonder if we will soon have other bonded allotropes such as buckyballs and nanotubes for unique selectivity.
Waters (www.waters.com) extended the product range of the novel ACQUITY UPLC® SEC columns for proteins by adding a 450-Å pore particle that extends the exclusion limit to 1.5 megaDalton. The diol surface chemistry reduces nonspecific adsorption. As a result, the BEH 450 can be used with biopolymers such as IgG (~150 kDa) and thyroglobulin (660 kDa). Previously, the BEH 200 SEC and BEH 125 SEC column packings used a 1.7-μm particle, but the BEH 450 SEC is on a 2.5-μm particle. These columns support the ACQUITY® Advanced Polymer Chromatography™ System (APC™) described in the LC instrument review (http://www.americanlaboratory.com/913-Technical-Articles/136940-Pittcon-2013-Developments-in-Liquid-Chromatography/).
Kromasil®: 2.5 μm and 25 years
For 25 years, Kromasil from Akzo Nobel (www.akzonobel.com) has earned respect as an excellent series of column packings for bioprocessing. The initial concept was to focus on column packings for process scale that were generally larger than 20 μm in diameter. However, at least two factors interrupted this plan. Biochemists would like to avoid developing a new method for bioprocessing. It is preferable to scale from lab-scale analytical to bioprocessing since the impurity profiles should be identical or nearly so. Scaling in particle size is well understood. Variations in surface chemistry are less so. Thus, scaling up from the discovery lab to production scale is an anticipated route for a successful therapeutic. Kromasil 2.5 has C18, C8, and C4 phases to cover a range of polarities.
Product line extensions from Thermo
In response to the success of UHPLC, Thermo also introduced Bio columns encased in PEEK-lined stainless steel tubes. The column names carry a suffix of “RS,” i.e., MABPac-RS. The columns are rated to 10,000 psi. They are particularly recommended for compatibility with the Dionex Bio-RS instrument introduced last year.
Syncronis HPLC columns were first available with only 1.7- or 5.0-μm-diam silica particles. This was a large gap for chromatographers. This year, Thermo split the difference by introducing a 3-μm particle size for the Syncronis line. The pore diameter is 100 Å for all sizes.
A productive year at YMC
YMC introduced several new columns for a range of applications. YMC-BioPro Q and S 3-μm nonporous columns supplement the 5-μm columns introduced previously for ion exchange chromatography. The particles are a novel polymer with very low nonspecific adsorption. For high-speed SEC, YMC introduced BioPro Diol on a 3-μm porous particle. Available pore sizes range from 60 to 300 Å, which covers peptides to midsize proteins.
For lab-scale prep, YMC presented the LCForte semipreparative HPLC instrument for use with columns with 10–30 mm i.d. This is supported by YMC-BioPro 6-μm nonporous and 10-μm porous Q and S ion exchange columns and bulk packing for capture and polishing of biotherapeutics. Previously, the size of the column packing was either 30 or 75 μm. Users wanted more efficiency.
YMC continues to extend the range of the popular Triart series by offering new 1.9-μm particle size and phenyl and pentafluorophenyl surface chemistries. The new Triart TS-5 is a series of columns specifically optimized, packed, and QC’d for supercritical fluid chromatography (SFC). For chiral, YMC introduced four carbohydrate-coated stationary phases on 10- or 20-μm-diam particles for LC and SFC.
Sepax Technologies (www.sepax-tech.com) focused on high-throughput bioseparations with introduction of Zenix™ SEC-80 and Proteomix® ion exchangers. The Zenix series starts with 3-μm 80-Å pore silica spheres. However, the surface chemistry is novel. The surface chemistry of Zenix SEC is a neutral brush-type hydrophilic polymer called a “stand-up” monolayer. In contrast, the Zenix-C phase uses the same particle but the surface chemistry is polymerized in a “lay-down” configuration. The operating range is 100–50,000 Da for both phases. The calibration curves for both phases are similar but different. Some proteins show better resolution on one phase than the other. The question is: Which one is best for a particular application? The answer: You probably need to try both. Proteomix columns are now available on 1.7-μm spherical silica.
In 2011 and 2012 I found so many instruments and columns for SFC that it justified a separate article focused on SFC. This year, activity in instruments and columns was much lower.
As SFC develops into a major factor in chromatography, we are finding idiosyncrasies along the way: 1) SFC columns are more difficult to pack, especially when the particles are smaller than 5 μm. 2) Pyridine and other nitrogen heterocyclics are the preferred phases due to efficiency and useful selectivity. For example, ES Industries (www.esind.com) has developed a line of columns for SFC packed with 2-μm particles for analytical and 3-μm particles for prep. The prep columns have an i.d. of 10–30 mm. Phases include imidazole, pyridylamide, ethylpyridine, aminophenyl, nitro, pentafluorophenyl, and diethylamine propyl.
Only in the very early days of LC did instrumentation lead advances in column technology. Over the last 40 years, column technology has set the pace. I expect that I will be reporting on even more advances, perhaps even a breakthrough, at Pittcon 2014 in Chicago. I hope to meet you there.
Robert L. Stevenson, Ph.D., is a Consultant and Editor of Separation Science for American Laboratory/Labcompare; e-mail: firstname.lastname@example.org.