Expanding separation technologies

The adoption of UHPLC, with the advancements made in both UHPLC instrumentation and UHPLC column technology, has changed scientists’ expectations of chromatographic performance.

In many realms of science and applied technology, the first step in a process takes things apart. Doing that can involve breaking down information or physically separating things. Many scientists use liquid chromatography (LC) to separate solutions into components that can be analyzed with other technologies, often mass spectrometry (MS). From LC, the technology advanced to high-performance LC (HPLC) and then to ultra-HPLC (UHPLC or UPLC). Despite being the newest form of LC, UHPLC platforms come in many forms. Over 10 years ago, the first UHPLC system with the respective column technology was commercialized. The use of these sub-2-micrometer particle columns and UHPLC systems with reduced dead volumes, and increased backpressure capabilities, enabled chromatographers to significantly speed up an HPLC assay

As much as any other metric in modern analytical equipment, speed plays a fundamental role. Although big data is a big deal these days – maybe the most popular buzz phrase in science for the past few years – scientists and manufacturers have searched for ways to speed up technologies since the start of science, and LC is no exception. Despite all of the improvements in LC speed, in several cases the throughput gains are sometimes not enough, even with UHPLC technology instead of HPLC technology. Sometimes, there was just no easy way to speed up an application. On top of the speed still being less than desired, changing an existing HPLC application to a UHPLC application requires some work in method development and validation.

Along with the instrument, there have been significant innovations in particle technology used to drive separation performance. Silica-based particles have been widely used over the years because of their wide applicability for a broad range of separation conditions. Many traditional silica-based columns that are used for HPLC have particles with open pore structure and high pore volume, which makes them mechanically unstable when operated at UHPLC pressures. Ethylene-bridged hybrid particles, which combine attributes of both silica particles and polymeric particles, were the first to provide increased mechanical stability, while maintaining separation efficiency. Hybrid particles also have better chemical stability over silica-based particles, giving them the ability to be used over a broader mobile-phase pH range. Innovations in silica-based particle synthesis allowed manufacturers to produce higher-strength silica particles that can tolerate UHPLC operating pressures. With the development of high-strength silica, chromatographers are able to transfer methods developed on HPLC silica particles to UHPLC. Superficially porous silica particles were the next innovation to increase separation efficiency. Columns packed with these particles further reduce the eddy dispersion and also reduce longitudinal diffusion. Understanding these particle technology developments has allowed manufacturers to provide innovative columns applicable for other areas outside of routine small-molecule analysis, such as size-exclusion chromatography and supercritical fluid chromatography.

Evolution of UHPLC for size-exclusion chromatography
Size-exclusion chromatography (SEC) and gel-permeation chromatography (GPC) differ only in application areas. They are both size-based separation modes with SEC commonly associated with biomolecule separations and GPC referring to separations for synthetic or natural polymers. Significant improvements in UHPLC technology using smaller particle columns coupled with low-dispersion fluidic paths increased resolving power with a subsequent reduction in analysis time for both techniques. As innovation in biomolecular research continues, scientists are required to fully characterize and identify biomolecular species present in the sample that may impact the efficacy or safety of the drug product. In the polymer industry, the physicochemical properties of the polymer, such as chemical resistance or tensile strength, are correlated to molecular data to tailor the material characteristics required for the end-use product. For these examples, size-exclusion chromatography is the preferred technique.

Similar to small-molecule separations, any extra-column band spreading, resulting from a poorly optimized UHPLC system, can undermine the gains achieved from the highly efficient separation. For SEC, the separation takes place within the pore volume contained within the column; it is even more important to minimize the contribution of extra column-band broadening. For this reason, isocratic systems with extremely low-system dispersion are preferred.

The lack of modern column chemistries optimized for high-performance GPC have limited the potential for developing new, more efficient UHPLC GPC separations. To fully realize the efficiency benefits of smaller particle columns, the mobile phase needs to be pumped through the chromatographic bed at a higher linear velocity. Using this approach for traditional 7.8 mm i.d. SEC columns will waste a considerable amount of mobile phase for very little chromatographic benefit. Additionally, achieving maximum resolution for SEC separations requires stationary phases with large pore volumes. This is challenging for small particles because increasing pore volume significantly decreases the structural integrity of the particle. Poor particle strength is the main disadvantage of polymer-based resins, where the relatively non-rigid structure can compress or collapse under high-pressure operating conditions. The most recent developments for GPC stationary phases use fully porous hybrid particles, which maintain the required mechanical rigidity while providing the improvements in separation efficiency.

Evolutions in SFC
Another recent advancement for UHPLC separations is the exploration and use of alternative solvents and mobile phases to achieve analyte selectivity and retention. Supercritical fluid chromatography (SFC) is one such area that uses alternative solvents, such as liquid carbon dioxide along with co-solvents like methanol, as the mobile phase. The pressure required to maintain carbon dioxide in the critical fluid region is typically between 100 and 400 bar. Carbon dioxide is a highly compressible fluid, which places greater demand on the fluidics and pumping algorithms compared to systems designed for pumping non-compressible fluids commonly used for LC. Any small deviation in system pressure will change the solvent density and subsequent solvent strength, thus impacting analyte-retention reproducibility. Historically, older HPLC instrument technology could not consistently deliver compressed carbon dioxide; so, method reliability and repeatability suffered.

Most LC instrumentation is designed to transfer incompressible fluids and, therefore, struggle to maintain compositional accuracy and flow precision under SFC operational conditions. Improvements in UHPLC system engineering changed the understanding of pump design and pump-control algorithms to compensate for compressible fluids to allow for reproducible retention times and extremely low baseline noise. Additionally, gradient SFC separations are now commonplace, compared to methods that were primarily limited by older technology and isocratic conditions.

Backpressure regulation within the system is the most important aspect to achieve reliable SFC separations. Poor pressure regulation greatly affects mobile-phase density and the resulting chromatographic reproducibility. Traditional SFC instrumentation was plagued by poor pressure-monitoring capability and slow-to-react feedback loops. By using both active and static pressure control, the user can accurately control both pressure and flow rate to achieve highly repeatable results under all separation conditions. In this case, the static control maintains a set minimum pressure while the enhanced active control fine tunes the set point required by the user.

The higher diffusion rates of SFC separations, combined with smaller particles, results in faster mass transfer of the analyte between the stationary phase and the mobile phase. This results in highly efficient separations, using high mobile phase linear velocities. For example, chiral separations that were developed for normal-phase conditions can now take advantage of SFC to significantly reduce sample run times and solvent cost.

Way forward
Combining columns packed with small particles with a low-dispersion UHPLC system is the key to UHPLC technology. Small-particle technology has been around for many years; however, the particle alone cannot provide the expected increases in chromatographic performance. It is the holistic approach to system design that considers the instrument, column hardware, and particle development that provides true UHPLC performance.

The evolution of UHPLC technology over the past 14 years has enabled scientists from different application areas to advance their research. Adapting UHPLC concepts that originated with small molecule theory are now being applied to additional chromatographic techniques such as SEC and SFC. SEC and SFC are not designed to replace conventional UHPLC techniques but rather complement it by providing different separation modes. Independent of the application, UHPLC has produced significant advancements to reduce analysis time, produce sharper peaks for increased sensitivity, and enhance resolution for better characterization and quantification of sample constituents.