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HPLC going places

Chromatography may have been around for decades, but by no means has the technique and its applications stopped evolving. The method will continue to be adapted to meet novel challenging applications in 2021.

Since its inception, high-performance liquid chromatography (HPLC) has undergone technological enhancements in all aspects, whether in detectors, better accuracy, and precision or new column chemistries. Since the millennium, most of the advances have been centered on performing faster separations through the advent of UHPLC (ultra-high-performance liquid chromatography), complemented by smaller column particles and diameters. That is not to say that other enhancements have not been made in the areas of robustness, reliability, and detection sensitivity. All of these developments have given rise to increases in productivity.

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 owing to 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 increased 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.

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 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.

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 sixteen 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 technique 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.

Moving forward
Chromatography may have been around for decades, but by no means has the technique and its applications stopped evolving. The method will continue to be adapted to meet novel challenging applications in 2021. The rise in environmental awareness will likely exert influence on how chromatography is performed so that green-chemistry requirements can be satisfied.

Let us look at some of the trends in analytical chemistry that are driving ongoing research and are poised to have a major impact for years to come.

Miniaturization. Thanks to rapid developments in instrumentation, microelectromechanical systems (MEMS) are becoming increasingly diminutive in size – making it possible to decrease demands in working space, water, reagents and power, as well as a number of other high-cost variables.

In particular, the rise of mobile laboratories and portable benchtop spectrometric devices has enabled more varied analytical applications of the technology, giving analytical chemists greater scope for out-of-laboratory work. In the past, most spectrometers were cumbersome instruments that were large enough to fill a room. Today, a Raman spectrometer can fit inside a chemist’s pocket and can be integrated with smartphones for monitoring.

With greater miniaturization, analytical chemists will be able to use spectrometric devices in ways that were not thought possible a decade ago.

Automated analysis. In recent years, the automation of analytical instruments (such as autosamplers, databases, data treatment, and control of instruments using microprocessors) has accelerated the advancement of analytical chemistry techniques.

Automation has long been employed to solve a number of industrial and laboratory problems. In the chemical and petroleum industry, these automatic analytical systems are typically based on gas chromatography, a technique used for separating and analyzing compounds that can be vaporized without decomposition.

In metallurgical plants, similar systems such as fast sampling tools, pneumatic mail for transporting samples to a laboratory, and atomic emission spectrometers are already in use.

In the laboratory, automatic systems for analysis often include continuous flow (particularly in the agriculture and pharmaceutical industries) or flow-injection analysis.

Improvements in software and computational technology have also led to the generation of ever-expanding data sets that need to be sorted, stored, and analyzed. As such, analytical chemists working with these complex data sets need to be well versed in statistics and chemometrics, as well as being specialists in sampling, instrumentation, and interpretation.

The use of laboratory robots is also on the rise, with some fears that the advent of robotics may kill this traditionally hands-on discipline. However, the vast amounts of data generated by modern techniques and instrumentation actually represent an opportunity for analytical chemists because their cognitive skills are needed to render the data useful.

More cost- and time-effective research. The direct knock-on effect of the developments explored above is a cheaper and faster end-to-end research process.

More advanced separation techniques and the increasing use of computer simulation modelling removes the need for additional experiments, helping to save time and money. Specific method development and optimization software programs like DryLab, for example, can even enable researchers to predict chromatograms using a small number of data points.

As analytical instruments and protocols become more advanced, it will also become possible to perform complex analytical tasks with higher sensitivity and greater accuracy.

Ultra-performance liquid chromatography (UPLC), for example, offers a more accurate analysis than traditional HPLC owing to its higher throughput and enhanced ability for separation.

For this reason, the use of UPLC and other hyphenated techniques have become increasingly commonplace in certain industries, particularly the pharmaceutical sector.

Currently, multidimensional gas techniques like GCxGC and LCxLC are widely used in a range of industries, while developments are taking place in column technology and mass spectrometry. Another emerging area is tandem mass spectrometry (MS/MS), a technique in which two or more mass spectrometers are coupled up to better analyze chemical samples.

Green analytical chemistry. As is the case across all areas of scientific research, sustainability is becoming a key theme in analytical chemistry.

Green analytical chemistry is an emergent field that focuses on minimizing the consumption of dangerous substances, as well as maximizing safety, both for operators and the environment.

The miniaturization of equipment and procedures also forms a key part of sustainable analytical chemistry strategies.

HPLC is certainly going places, and the importance of the discipline cannot be understated. By making key breakthroughs, HPLC enables the development of various other fields that deploy its tools and equipment. As technology continues to progress exponentially, one can expect to see a spate of new discoveries in the coming years.

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