High-performance liquid chromatography (HPLC) is a widely used technique to separate molecules from heterogeneous solutions for further characterization. HPLC is often used prior to mass spectrometry in the identification of peptides, proteins and other molecules. Ultra-high performance liquid chromatography (UHPLC) can provide better resolution, increased sensitivity and faster separation times compared with traditional HPLC, but it can also be less reproducible. This article will focus on recent advances in HPLC technology and its expansion into separation of larger biomolecules.

HPLC and UHPLC systems

"One of the key attributes of HPLC is its robust reliability, which researchers may prefer for established methods over an increase in resolution or reduction in run time—especially if the HPLC protocol is well defined."

“Confidence that the instrumentation will perform what they want the system to do is paramount,” says Evert-Jan Sneekes, strategic marketing manager for HPLC at Thermo Fisher Scientific.

Although there isn't a clear dividing line between HPLC and UHPLC, one distinction is the diameter of particles (the resins used) within the columns. Typically, particles used in HPLC are larger than 2 µm, whereas sub-2-µm particles are used for UHPLC. But the distinction has blurred thanks to recent advances in particle technologies and tool providers’ recognition that researchers want the best separation possible—no matter what it is called or traditionally used for. Tool providers are assisting researchers by providing an array of columns with varying particle sizes to address different assay formats. For example, Waters’ new CORTECS C8 and Phenyl columns for small-molecule separations have solid-core particle sizes of 2.7 µm for HPLC and 1.6 µm for UHPLC. Likewise, Waters’ new XBridge columns for size-exclusion chromatography (SEC) separations of proteins and other biomolecules come with either 3.5-µm particles for HPLC or 1.7-µm particles for UHPLC.

Waters’ ACQUITY Arc System is designed for researchers who want better and faster HPLC separations but can’t switch to UHPLC. “They might have regulatory constraints, for example, that prohibit them from upgrading to a UHPLC,” says William Foley, senior director of separations marketing at Waters. “The Arc Multi-flow Path Technology lets them run an HPLC method, and then if they so wish, experiment with a lower-dispersion flow path and a smaller particle column, to explore the advantages of UHPLC in terms of sensitivity, resolution and speed of analysis.” The new Vanquish platform from Thermo Fisher Scientific is designed to perform both HPLC and UHPLC with ease of use in mind.

Refining particle technologies

Both HPLC and UHPLC continue to benefit from advances in particle technology. Fused-core, solid-core and core-shell particles—consisting of a solid core fused to a porous outer layer—improve efficiency by reducing the diffusion path length of molecules traveling through the column and also allow better separations at lower flow rates and pressures.

Particles likely will continue to evolve, says Åke Danielsson, research director at GE Healthcare’s Life Sciences business, potentially leading to UHPLC-type separations using larger particles that today would be used for HPLC (e.g., greater than 2 µm). “Then it will be possible to achieve ultra-high resolution separations using equipment that does not require very high pressure tolerance, for instance,” he says.

Column selectivity is also improving. This is particularly valuable in characterizing biopharmaceuticals, for instance, says Sneekes. More than one type of HPLC column chemistry can be used (individually or in mixed-mode columns) for analyzing peptide sequences, aggregates, charge variants, intact structures and glycan profiles. Phenomenex offers new selectivities for reverse-phase LC with its expanded Kinetex® core-shell HPLC/UHPLC column line. Conventional pentafluorophenyl (PFP) stationary phases—an alternative to traditional C18 phases in reverse-phase HPLC—struggle with batch-to-batch reproducibility, according to Jeff Layne, senior marketing manager at Phenomenex. The new Kinetex F5 phase, constructed using proprietary methods, retains PFP selectivity and improved reproducibility. Additionally, the Kinetex EVO C18 core-shell phase is designed for stability at both low and high pH values (pH 1 to 12), “[with] a modified organo-silica surface chemistry that improves the separation and peak shape of basic analytes,” says Layne.

Advances in HPLC detectors

Advances in HPLC detectors are also emerging. Jasco’s new 4000 series of HPLC/UHPLC products encompasses a range of HPLC detectors, including dual-wavelength UV, photdiode array (PDA), refractive index, circular dichroism, optical rotation and fluorescence detectors. Jasco also offers a new single-quadrupole mass spectrometer (MS)-based detector for mass confirmation. According to Tom DePhillipo, regional chromatography specialist at Jasco, researchers are increasingly interested in simpler, more compact, less expensive MS detectors rather than a high-performance MS system. “Many laboratories who previously could not afford MS detection would settle for less sensitive universal types of detection, such as evaporating light scattering detection (ELSD), charged aerosol detection (CAD) or refractive index,” says DePhillipo. “While these types of detection serve their purpose in the analytical and preparative realms, they cannot provide the sensitivity that MS provides.”

Similarly, Waters offers the ACQUITY QDa mass detector for verifying the mass of analytes. “It’s great for a quality-control lab or for situations where a standalone mass spectrometry system might be more than what’s called for,” says Foley. Waters also plans to release a new version of its conductivity detector (used in both HPLC and UHPLC) with updated electronics.

Separating larger biomolecules

HPLC originally was developed to separate small molecules; proteins can prove more challenging. Larger biomolecules diffuse at a slower rate than small molecules—and also don’t separate as efficiently with reduced particle size (i.e., sub-2 µm). “Smaller particle size does not give considerably higher efficiency [with protein separations],” says Layne. “This is the reason behind offering Aeris® WIDEPORE media in only the 3.6-µm particle morphology.”

Separation of proteins by SEC, on the other hand, can benefit from sub-2 µm particles, because slower flow rates tend to keep back pressures low enough for use on most HPLC systems. Phenomenex recently released Yarra® 1.8-µm SEC-X150 columns for SEC separations of proteins up to 450 kDa. In spring 2016, Phenomenex will release Yarra 1.8-µm SEC-X300 columns for molecules up to 700 kDa, such as high-molecular-weight protein aggregates and large biosimilars. In addition, GE Healthcare’s new SEC columns use different agarose-based resins to separate proteins in different size ranges: Superdex 200 Increase resin for 10-kD to 600-kD proteins; Superose 6 Increase resin for 5-kD to 5,000-kD proteins; and Superdex 75 Increase resin for 3-kD to 70-kD proteins (expected to be released in spring 2016).

Also designed for biomolecules, MilliporeSigma’s BIOShell™ column particles have a 400A pore size that is larger than in conventional columns. “We designed it this way because biotherapeutics are getting larger and more complicated,” says Wayne Way, market segment manager at MilliporeSigma. “The larger pore size allows for better chromatography since large biomolecules don’t adsorb onto the column, which helps provide a better peak shape for drug products.” BIOShell™ also offers more specialized columns for working with glycoproteins or with biotherapeutics such as antibody-drug conjugates.

Sneekes expects that future advances will bring HPLC and mass spectrometry closer together. “Chromatography is about more than separating—the analytical question is only answered after the analytes are detected,” he says. And with improvements in HPLC and UHPLC every year, better separations are making it easier to detect those analytes.