Separation Science: How Far We’ve Come, How Far We Have to Go

The National Academies of Sciences, Engineering and Medicine published last month a report on separation science entitled “A Research Agenda for Transforming Separation Science,” the Academies’ first dedicated report on the topic since 1987. The report defines separations as “the division of a chemical mixture (a mixture of molecules or colloidal particles) into its constituent or distinct elements.” Separations are most commonly utilized for the enrichment or separation of a component from a chemical mixture. A chemical mixture is “a mixture of molecules or colloidal particles, in which the separation of those components is based on inter-molecular interactions [such as electrostatic interactions, dispersion or hydrogen bonding] or chemical reactions and not mechanical mechanisms, such as filtration.”

The report addresses problems in separation science, which in turn can affect advancements in fields as diverse as energy production, drug manufacturing and public health, among many others. As the report makes clear, “Without separations, access to such necessities as chemicals, medicines, clean water, safe food and energy sources would not be possible.”

The report outlines how to improve separation science research and how future technology and software developments can advance this field. It also makes a series of recommendations for research priorities and government action. In particularly, two themes are highlighted (see below): 1) the need for separation systems that are more selective, increase capacity and occur at a higher rate, and 2) insight into the effect of time on separation processes. The report also stresses the need for separation standards and reference materials, and the application of new software tools to study separations.


Source: National Academies of Sciences, Engineering and Medicine


In line with Instrument Business Outlook’s (IBO’s) editorial focus on analytical testing, this blog will concentrate on the report’s conclusions as they relate to analytical separations of chemical and biological substances, including for bioprocessing.

Unfortunately, separation science is hurt by a lack of understanding of the basic mechanisms and dynamics of separations. As the report lays out, “Overall, fundamental knowledge on how matter interacts in complex environments is insufficient for the design and manufacture of highly selective separation systems that have high capacity and throughput.”

Nonetheless, separation science for chemical and biological analysis has progressed in terms of both scientific understanding and commercial solutions, according to the report. These successes in improving separations are evident in lab and process-scale chromatography and sample preparation. Yet these developments also reveal current shortcomings.

The report delves into the progress made in materials science and development that have advanced separation science and increased selectivity, including the introduction of new types of separation materials, such as polymeric and mix-matrix membranes, solid absorbents and ionic liquids. For example, refinements in the design and production of silica particles have improved high-performance liquid chromatography (HPLC); specifically, the development of sub-2 µm particles, sub-µm-shell superficially porous particles, and silica with wider pores for laboratory- and process-scale HPLC. For biological samples, new coatings have been used to create magnetic nanoparticles and multimodal ligands.

In the area of sample preparation, molecular-recognition techniques have contributed to improved selectivity in liquid-liquid extraction (LLE) of solvents and development of new configurations of liquid-membrane separations. Advances are also evident in terms of separations from dilute solutions. The report points to the use of preconcentrations for trace analysis as an example, including the commercialization of solid phase microextraction techniques (SPME). However, the report contends that greater progress lies ahead, advising, “SPME is not perfect: the coatings used in SPME can be stripped from the supporting fiber, and the coatings will swell in some solvents.”

The report also describes the advances made possible by the study of the interface between two bulk phases, which has also been informed by materials design, such as surface modification. In this instance, the report cites HPLC, describing protein-resistant membranes used for bioseparations as well as utilization of carbon materials in LC media.

As such, the development of new materials and insight into surface-sample interaction have also encompassed breakthroughs in separation throughput and capacity. Describing the importance of each to more efficient separations, the report provides LC examples, reflecting, “In batch operations, such as chromatography, the capacity of the column for the analytes might be the main determinant of throughput and therefore a more useful measure of throughput. Nonetheless, the overall cycle processing time (the time it takes to process all the species) is the focus, so both throughput and capacity are important.”

Meeting the needs for higher flow rates are new membrane materials that address issues such as fouling. In this case, the report points to the use of membranes for LC-based purification of monoclonal antibodies. Also regarding throughput, the report presents ultra HPLC (UHPLC) as an example of the progress made in increasing throughput and efficiency. Further innovations, such as superficially porous packing materials for LC, continue to build upon these advances. “When the method is combined with superficially porous packing materials, further improvement in separation speed and efficiency is observed, typically by a factor of 8 to 10 compared with 5-μm particles (Hayes et al. 2014),” states the report.

Another sign of progress in separation science is the greater use of multidimensional separation. The report identifies the use of LC x LC and GC x GC (gas chromatography) for separation of complex mixtures. The benefits of multidimensional separations span both chemical and biological analyses, including applications in proteomics and metabolomics. But the report advises, once again, that more R&D is needed, commenting, “[T]here is considerable room for further advances in column selectivity and instrumentation for comprehensive chromatography media.”

Other achievements in separation science are developments impacting process equipment for separations, energy-efficient separations and sustainability. Compared with standard HPLC, UHPLC’s smaller volumes is one example of sustainability through systems design. In fact, the report asserts, “If HPLC instruments were replaced by UHPLC separation technology, the same high-quality separations could be achieved in a similar time frame with reductions in solvent use and waste generation of more than 60%.”

Because materials development and modification are key factors in advancing separation science, a portion of the report is specifically devoted to materials synthesis and the need for an interdisciplinary approach that has so far been lacking in separations R&D in general. “The design and realization of materials with targeted applications have historically relied largely on trial and error. Driven by advances in theory and computation and new characterization tools, materials synthesis is rapidly transforming into a more science-based approach,” explains the report.

Advances here would encompass systems engineering, control of field gradients, and greater application of advanced instrumentation to characterize materials, such as exploring material composition. The report specifies HPLC as an example of the current limitations, recognizing, “The speed of liquid chromatography, especially size-exclusion chromatography, depends on the molecular-scale pore structures formed by the aggregated nanoparticles, but these structures have been beyond the limits of direct observation and characterization.” In addition, the report emphasizes that data science will be critical to materials development in the form of simulations, modeling, and extensive data analysis.

The report further points to the requirement of understanding the impact of operating conditions on separations. In particular, this understanding could be applied to the separation of complex mixtures. The report cites trace analysis using preconcentration and extraction in the form of LLE or SPME with LC/MS as progress in this area, stating, “Target compounds can now be measured in μg/L down to ng/L.” But the report cautions, “Nevertheless, such detection limits cannot be reached for all compounds of interest, and often the limits are still not low enough. In addition, achieving those limits in the case of many nontarget compounds remains challenging.”

A method to increase selective separation of components is to target a wider dynamic range. The report describes the detection of proteins in human blood plasma, such as for biomarker identification, as a particular challenge. Unfortunately, according to the report, “No current multistep process that involves preconcentration, separation and detection can analyze proteins across such a large dynamic range. Various methods, such as prefractionation of high-concentration proteins, have been attempted, but some of the lower-concentration proteins are also removed with these methods (Rassi and Puangpila, 2017; Wu et al., 2016).” This applies to specific indicators of disease states. “If a particular biomarker is identified, a method of selectively preconcentrating and detecting it is possible. However, nontarget analysis is still difficult or impossible.”  More work to understand the fundamental nature of materials and separation is encouraged through study of physical forces, temporal changes and chemical reactions.

In detailing areas for further research, the report provides insight into the challenges in explaining the complexity of sample and separation interface, particularly molecular interactions. “A fundamental understanding of inter-molecular forces at interfaces is critical for improving the performance of capillary electrophoresis, chromatography, membrane filtration, adsorption, ion exchange, affinity multimodal systems and others.” Research also address needs to address understanding the effects of  operating conditions, and alterations such as degradation.

The report also devotes attention to the need for standards and methods, which would improve data reliability and reproducibility. Specific concerns are data validation and computer modeling of systems. Describing techniques for the separation and analysis of monoclonal antibodies in drug production, and referring to the increasing use of mass spectrometry as well as its drawbacks, the report points to the National Institute of Standards and Technology’s (NIST) development of monoclonal antibody reference material to assist proteomic analysis during biopharma development as a success case. However, the report underlines the need for membrane standards. Further progress in standardization can expect to be made via data science applications in the areas of theory and modeling.

Report recommendations focus on education opportunities, collaboration among chemists and chemical engineers, and federal involvement in championing cross-disciplinary projects. As the report puts it, “Through the implementation of the research agenda, separation science will have the opportunity to address many questions that sit at the forefront of chemistry and chemical engineering. The advances will require improvements in separation-science education, collaborations, community building, and use of experimental and computational resources.”

For further information, including market data, about lab and process analytical purification, see SDi’s “2019 Filtration and Purification Report.” For information on the HPLC market, see “HPLC 2018 Market Analysis and Perspectives.”