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Liquid chromatography is easier and more efficient with every new rendition of tools
In 1900, Russian-Italian botanist Mikhail Semyonovich Tsvet invented liquid chromatography. A variety of historical and cultural obstacles, plus some technical ones, kept the technique from being used for a decade. But then, spread it did. On December 29, 2017, a search for “liquid chromatography” or “LC” on PubMed turned up 213,798 articles.
That amount of work is reflected in the number of tools that scientists can consider. Here, experts from some top manufacturers talk about some of the newest products.
Souped-up C18
When asked about the newest LC column for biologics characterization from Agilent Technologies (Santa Clara, CA), Suma Ramagiri, product marketing manager for biocolumns, says, “One of our new LC columns is the AdvanceBio Peptide Plus column, a reversed-phase C18 column with a charged surface chemistry designed for optimum separation of peptides with formic acid-containing mobile phases for LC/MS.”
In describing the specifications of this column, Ramagiri points out that “its unique surface chemistry results in exceptionally sharp peaks under formic acid mobile phase conditions, as well as different selectivity and higher loading capacity versus traditional C18 columns.” She adds, “Another key feature is the 2.7-micrometer Poroshell particle that this chemistry is built on, which allows the column to achieve 80 to 90% of the efficiency of a sub-2-micron column, but only about half the backpressure.”
Agilent’s experts designed this column for peptide-mapping applications with LC/MS. As Ramagiri notes, “It particularly shines with detection of especially long or hydrophobic peptides, high sample loads, and resolution of post-translational modifications, such as deamidations.”
Representation of the polymerically bonded C18 phase found in the Kinetex PAH Core-Shell Particle. (Image courtesy of Phenomenex.)Other manufacturers introduced unique application-specific C18 columns, such as the Kinetex PAH Core-Shell HPLC/UHPLC Column from Phenomenex (Torrance, CA). This is a 3.5-micrometer polymerically bonded C18 designed for food and environmental testing of polycyclic aromatic hydrocarbons (PAHs). “With its unique selectivity combined with core-shell particle technology, Kinetex PAH provides high efficiency and increased sensitivity for polycyclic aromatic hydrocarbons at low HPLC backpressure,” says Simon Lomas, strategic marketing manager for new products at Phenomenex. “This core-shell phase is also an excellent option for the separation of closely related compounds, such as isomers.”
GL Sciences in Japan makes the MonoCap C18 HighResolution 2000, a monolithic silica capillary column that is 2 meters long. The company notes that this column was “designed for identifying an extremely high number of peptides/proteins for proteome research via LC-MS/MS.”
Polar improvements
To separate polar compounds, Agilent developed its InfinityLab Poroshell 120 HILIC-Z column. Jason Link, marketing manager for small molecule, GPC, and preparative HPLC columns at Agilent, says that the column uses “a new zwitterionic chemistry that separates polar compounds using hydrophilic interaction chromatography,” or HILIC. He adds, “This column is based on the Agilent 2.7-µm Poroshell particle platform, enabling fast analyses with low backpressure, with the HILIC-Z being a proprietary zwitterionic HILIC chemistry available only from Agilent.”
Polar compounds must be separated in many labs, including environmental, food, and pharmaceutical ones. Link says that, “many [labs] have had to settle for slower workflows with older HPLC technologies when they are performing amino-acid analysis, detecting polar pesticides, or analyzing organic acids.” He adds, “InfinityLab Poroshell 120 HILIC-Z can separate these challenging compounds quickly and without derivatization, improving throughput and reducing operating costs in the laboratory.”
New column family for bioseparations
Waters developed a new family of columns for biopharmaceutical analysis. (Image courtesy of Waters.)Waters (Milford, MA) just launched the first member of a new family of columns for biopharmaceutical analysis. The BioResolve RP mAb, Polyphenyl 450-Å 2.7-µm column is intended for reversed-phase analysis of monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs). “BioResolve represents our next-generation column technologies that are purposefully engineered with our customers’ challenges and solutions in mind,” says Priya Jayaraman, principal product marketing manager at Waters. “They are a must-try if you are looking to tackle complex analytical problems in various biopharma applications.”
The BioResolve column brand will deliver more than just performance-driven columns. “Bioseparations are complex, but being successful does not have to be complex,” Jayaraman explains. “To ensure that our customers start and remain successful, our BioResolve brand of columns will provide application-focused standards, methods, and exclusive support to its users so that scientists can consistently and reliably achieve state-of-the-art separations.”
BioResolve RP mAb, Polyphenyl columns were engineered to solve some of the complex analytical problems encountered in the area of reversed-phase LC and LC/MS analyses of intact and subunits for mAbs and ADCs in trifluoroacetic acid or formic acid. According to Jayaraman, Waters’ optimized solid-core particle design establishes a new industry standard for separation efficiency (i.e., peak capacity), sample recovery, and low injection-to-injection carryover for this defined application, even when operated in high-throughput applications. The use of an innovative, patent-pending polyphenyl ligand technology, Jayaraman notes, delivers unique separation selectivity of minor from major component peaks for high-resolution methods.
“At Waters, we take quality very seriously, as getting consistent and reliable data is the underlying goal of any analytical method that ultimately affects patient safety,” says Jayaraman. “The material used in our BioResolve RP mAb columns must pass more than 300 defined measurements in addition to a well-defined gradient separation criteria tested using a reduced, IdeS-digested, NIST mAb standard developed by Waters.” She adds, “This level of attention to quality helps ensure batch-to-batch and column-to-column consistency when developing and validating methods.”
As Jayaraman points out, the 2.7-µm BioResolve RP mAb, Polyphenyl columns can deliver near-equivalent performance on HPLC, UHPLC, and UPLC instrumentation without the penalty of high backpressures. This means that the same 2.7-µm particle size can be used across all three platforms, which minimizes concerns about transferring methods from development to manufacturing/QC. The available internal diameters will be 2.1 or 4.6 millimeters and lengths of 50, 100, or 150 millimeters.
Around the market
The LC segment is broad and complex. In the past 100 years and more, the choices available to researchers kept changing and continue to do so.
Restek (Bellefonte, PA) Raptor LC columns rely on superficially porous particles, and “bond rugged 1.8-, 2.7-, and 5-μm superficially porous particles with Restek’s unique Ultra Selective Liquid Chromatography (USLC) phases to offer chromatographers the best of both worlds,” notes the company.
The LC market supplies a wide range of columns, and many manufacturers also make a wide variety themselves. Berlin-based Knauer, for example, produces many different kinds of LC columns. “In the field of analytical HPLC columns, we can offer a solution for nearly any analytical separation task,” its website states. In addition, the company “offers about 6000 columns for analytical HPLC and UHPLC applications as well as for purification tasks in the preparative scale.”
Another company in Germany, Bischoff Chromatography, also makes a range of columns for HPLC. “It is almost always possible to find the right separation column for your own separation problem,” the company asserts, adding, “We have our own filling materials—ProntoSIL, HiPAK, PolyEncap—as well as filling materials from other well-known manufacturers.”
Going back to the beginning of the 1900s, Tsvet could hardly have predicted what LC would become. The technology started along a bit of a bumpy research road, but now it drives an important market that gives scientists the tools to separate more compounds in samples that even Tsvet couldn’t imagine. His work changed the world of analytical science.
Mike May is a freelance writer and editor living in Texas. He can be reached at [email protected]