Suspect Screening of Lupin-Produced Phytotoxins in Environmental Samples

The Future of Biomolecule Analysis Using HPLC/UHPLC

Comprehensive Small Molecule Analysis Using Trapped Ion Mobility Spectrometry 

A Look at How Green Chemistry Applies to GC

Green Chemistry: What Is It (and What Is It Not)? 

Analysis of PFAS in Locally Acquired Food Containers

Events

HPLC 2023 Programme Highlights

John McLean previews his plenary lecture at HPLC 2023, where he will describe emerging analytical strategies using liquid chromatography–ion mobility–mass spectrometry (LC–IM–MS) for untargeted molecular phenomics in systems, synthetic, and chemical biology.

One of the most successful big science projects in modern history is the Human Genome Project. Among the many motivations for sequencing the human genome was to better understand what made us human and healthy. The frontiers of our knowledge rapidly expanded and opened new research avenues that continue to grow—such as major advances in genetic engineering and synthetic biology. This knowledge expansion facilitates many big, comprehensive, systems-wide biology projects to better understand the intricate molecular interplay of life—beyond the central dogma of molecular biology—and is largely built upon omics strategies (1).

Omics fields strive to understand the relationships among molecules from a global perspective. This often requires massive molecular cataloging experiments and data organization efforts to interpret these relationships. In many cases, the answers that are sought are tantamount to asking questions such as “who says what, where, when, and to whom?” These efforts initially centered on one or several classes of molecules, which gave rise to individual fields such as genomics, transcriptomics, proteomics, lipidomics, glycomics, and metabolomics, among an astounding array of omics areas of study today. In each of these efforts, experiments focus on cataloging the broad-scale changes in a molecular inventory.

In phenomics, these experiments seek the comprehensive molecular characterization of a phenotype in both space—for example at a cell, tissue, and organismal level—and time—such as healthy vs. disease, lifespan, or in response to exposures, treatments, and lifestyle choices (2). This places enormous demands on measurement technologies, including minimal sample preparation, fast measurements, high concentration dynamic range, low limits of detection, and high selectivity. In general, the omics fields utilize three primary technologies that embody these traits—most typically spectroscopically‑based and array technologies (for example, genomics and transcriptomics), nuclear magnetic resonance (NMR, for example, metabolomics and metabonomics) and mass spectrometry (MS, for example, proteomics, lipidomics, glycomics, metabolomics). The data density resulting from such experiments is enormous and requires computational approaches to organize the millions of potential species present in vanishingly small spatial coordinates (3,4). The interplay between phenomic datasets and bioinformatics forms the nexus of translating phenomics data into actionable information and understanding.

Since the first descriptions of combining MS data with computational approaches for proteomics (5), significant efforts have centred on the ability to expand the analytical capacities of mass spectrometry and computational approaches in omics research. On the data generation side of the equation, chief among these are developments to increase peak capacity for complex biological samples. These enhancements in selectivity are driven by two parallel advances. One is through integrating additional separation dimensions with MS detection to leverage the multiplicative nature of peak capacity (6). The second is through enhancements in resolving power and resolution in each of the individual separation dimensions, including both chromatography and mass spectrometry, to increase peak capacity in each individual dimension. Residing at the intersection of these two is the emergence of gas-phase electrophoretic molecular separations based on ion mobility (IM).

Separation selectivity in time‑dispersive IM is achieved through the electromigration of ions through a neutral gas, the latter providing an attenuating force that is proportional to the number of collisions between the ion and the neutral gas species (7). This in turn is proportional to the molecular ion surface area (measured in units of Å

 (CCS) (8). Importantly, since separations are performed post-ionization in the gas-phase following ionization, IM is readily interfaced between liquid-phase separations such as chromatography and the mass spectrometer without any increase in total analysis time. In the first proteomic experiments combining liquid chromatography (LC)–IM–MS, this feature was referred to as 

, since the LC dimension provided separations in minutes, the IM dimension provided separations in milliseconds, and the MS dimension provided separations in microseconds (9). In this way, there is no tradeoff of increased peak capacity for sample throughput. A second critical aspect of IM for omics studies are the prevailing structures that different classes of biomolecules preferentially adopt. Based on the monomeric units from which different biopolymers are comprised, different classes of molecules occupy specific regions of conformation space, or the correlation of CCS with the mass of the molecule (10). This results from the prevailing gas-phase folding forces of each class of molecule. For example, lipids adopt less dense structures than peptides or carbohydrates. These predictable and highly reproducible correlations of CCS vs. mass have opened new descriptors for performing omics measurements of multiple classes of molecules simultaneously. When combined with retention time, accurate mass, and fragmentation information, CCS provides another measurement to support high accuracy molecular annotation. Like the advances in peak capacity afforded by high resolving power MS, significant recent enhancements in IM resolving power have been a critical area of advancement in IM technologies.

This plenary lecture at HPLC 2023 will describe recent advances in IM–MS-integrated omics measurement strategies in the analyses of complex biological samples of interest in systems, synthetic, and chemical biology. New advances in artificial intelligence (AI) and machine learning based on developments in internet commerce and astronomy will also be described, to approach biological questions from an unbiased and untargeted perspective and to quickly mine these massive datasets. These techniques will be highlighted through selected examples, ranging from the creation of microfluidic human organs-on-chip, to replace animal testing in drug development workflows, to probing the outcomes of fast genetic editing experiments using CRISPR in the optimization of synthetic biology for fine and commodity chemical production. While enormous challenges in phenomics remain, there is immense promise in terms of diagnostics and predictive capabilities for health and medicine, to help better understand what makes us human.

Sherrod, S. D.; McLean, J. A. Systems-Wide High Dimensional Data Acquisition and Informatics Using Structural Mass Spectrometry Strategies. 

 (1), 77–83. DOI: 10.1373/clinchem.2015.238261

Holmes, E.; Wilson, I. D.; Nicholson, J. K. Metabolic Phenotyping in Health and Disease. 

 (5), 714–717. DOI: 10.1016/j.cell.2008.08.026

May, J. C.; Goodwin, C. R.; McLean, J. A. Ion Mobility-Mass Spectrometry Strategies for Untargeted Systems, Synthetic, and Chemical Biology. 

, 117–121. DOI: 10.1016/j.copbio.2014.10.012

May, J. C.; Gant-Branum, R. L.; McLean, J. A. Targeting the Untargeted in Molecular Phenomics with Structurally-Selective Ion Mobility-Mass Spectrometry. 

, 192–197. DOI: 10.1016/j.copbio.2016.04.013

Eng, J. K.; McCormack, A. L.; Yates, J. R. An Approach to Correlate Tandem Mass Spectral Data of Peptides with Amino Acid Sequences in a Protein Database. 

 (11), 976–989. DOI: 10.1016/1044-0305(94)80016-2

Giddings, J. C. Two-Dimensional Separations: Concept and Promise. 

 (12), 1258A–2170A. DOI: 10.1021/ac00276a003

May, J. C.; McLean, J. A. Ion Mobility-Mass Spectrometry: Time-Dispersive Instrumentation. 

May, J. C.; Morris, C. B.; McLean, J. A. Ion Mobility Collision Cross Section Compendium. 

 (2), 1032–1044. DOI: 10.1021/acs.analchem.6b04905

Valentine, S. J.; Kulchania, M.; Barnes, C. A. S.; Clemmer, D. E. Multidimensional Separations of Complex Peptide Mixtures: A Combined High-Performance Liquid Chromatography/Ion Mobility/Time-of-Flight Mass Spectrometry Approach. 

 (1–3), 97–109. DOI: 10.1016/S1387-3806(01)00511-5

McLean, J. A. The Mass-Mobility Correlation Redux: The Conformational Landscape of Anhydrous Biomolecules. 

 (10), 1775–1781. DOI: 10.1016/j.jasms.2009.06.016

 is Stevenson Professor and Chair of the Department of Chemistry and Associate Provost at Vanderbilt University in the USA. He also serves as Director of the Center for Innovative Technology, which facilitates phenomics research for large-scale consortia projects in health and medicine. He studied at the University of Michigan and George Washington University, and subsequently performed postdoctoral research at Forschungszentrum Jülich in Germany and Texas A&M University. He is a fellow of the National Academy of Inventors and the American Association for the Advancement of Science. He has published over 200 manuscripts in the areas of technology development in ion mobility, mass spectrometry, and omics research.

John McLean previews his plenary lecture at HPLC 2023, where he will describe emerging analytical strategies using liquid chromatography–ion mobility–mass spectrometry (LC–IM–MS) for untargeted molecular phenomics in systems, synthetic, and chemical biology. 

Molecular Phenomics in Systems, Synthetic, and Chemical Biology

Measuring chemical exposure is extremely challenging due to the range and number of anthropogenic molecules encountered in our daily lives, as well as their complex transformations throughout the body. To broadly characterize how chemical exposures influence human health, a combination of genomic, transcriptomic, proteomic, endogenous metabolomic, and xenobiotic measurements must be performed. However, while genomic, transcriptomic, and proteomic analyses have rapidly progressed over the last two decades, advancements in instrumentation and computations for nontargeted xenobiotic and endogenous small molecule measurements are still greatly needed.

Upon completion of The Human Genome Project, it was determined that greater than 90% of human diseases are not solely due to a person’s genetics but a combination of genetic factors and environmental influences (1–4). Linking environmental exposures and biological effects is however extremely challenging. Humans are exposed daily to a broad range of xenobiotic chemicals in drinking water, foods, household items, and everyday consumer products, such as plastic bottles, containers, clothes, and cosmetics. While many xenobiotics do not cause harm when contacted or ingested, some disrupt normal bodily processes and change how hormones, organs, and the immune system function, either instantly or later in life. Identifying xenobiotics with adverse effects and evaluating how their chemical exposure influences human health is therefore of utmost importance, but it requires advanced instrumental and computational analysis capabilities.

While genetic factors have been readily assessed to date using rapid genome sequencing technologies, measuring environmental factors is much more challenging and requires the implementation of direct and indirect molecular measurements (5,6). In direct measurements, specific xenobiotic chemicals of interest are analyzed in environmental and biological samples such as water, plants, biofluids, and human tissues. These measurements are extremely complex, as thousands of xenobiotics exist and can also be transformed in the environment and body. Furthermore, many xenobiotics and their transformation products are often excreted before biological responses ever occur, causing difficulties in linking specific exposures to phenotypic outcomes. Indirect analyses are therefore utilized to evaluate molecular changes occurring due to chemical exposure. In indirect measurements, one or more complementary omic techniques such as transcriptomics, proteomics, metabolomics, or lipidomics are often performed on each sample. However, these multi-omic measurements must be completed in a relatively short period of time to quickly assess exposure effects and potentially provide treatments. Novel developments in both analytical and computational methods are therefore imperative.

Traditionally, both direct and indirect measurements have focused on a subset of target compounds largely selected from toxicology studies that have highlighted hazardous characteristics or known functional disruptions. In targeted approaches, the extraction, analysis, and detection of the known chemicals of interest is optimized to enable the highest sensitivity for the limited set of molecules. However, targeted direct analyses create a huge knowledge gap in understanding exposure, since thousands of new chemicals are introduced each year but not included in targeted studies until deemed to be of concern. Furthermore, the additional transformation products that occur due to degradation and metabolism of the xenobiotics are often not monitored by targeted assays, even though some can be more toxic than the parent molecules. Nontargeted analyses have therefore become a focus of recent exposure assessments (7). However, these evaluations are challenged by the great number and structural diversity of xenobiotics and the endogenous small molecules they affect. The chemical makeup of these molecules is unlike the biopolymers in genomics, transcriptomics, and proteomics studies, which have a limited number of building blocks, that is, nucleotides and amino acids), with some synthetic xenobiotics pushing the boundaries of chemistry itself. For example, xenobiotics such as per- and polyfluoroalkyl substances (PFAS) have numerous fluorine atoms within each species, and some pesticides have various types of halogens within the same molecule. Broad coverage of nontargeted measurements therefore requires different extraction methods, chromatography techniques, ionization sources on mass spectrometers (electrospray ionization vs. atmospheric pressure chemical ionization), and analysis polarities (6,8–12). This multiplies the number of instrumental and data analyses performed on each sample and brings forth infinite possibilities for molecular studies. Budgets are therefore used to prioritize and limit the number of nontargeted analyses, even making them somewhat targeted and biased to certain molecules. Thus, to extend the number of nontargeted studies possible, the development of higher throughput approaches capable of assessing many samples following distinct extractions, ionization sources, polarities, and separations is essential.

The identification of unknown features is another important challenge for nontargeted analyses (13,14). While thousands of features are often detected in small molecule nontargeted analyses of complex matrices, normally only a few hundred can be identified confidently (15). Furthermore, the fragmentation of small molecules is not as intuitive or informative as peptides, which always break at known locations based on the fragmentation method used. Molecular standards are therefore often utilized for the identification of unknown features in these studies. Unfortunately, this is also a limitation, as only a fraction of the small molecules that exist have a commercially available standard. Two alternate approaches often used to identify unknowns are nuclear magnetic resonance (NMR), which allows accurate structural identification (16), and the application of 

 tools that provide plausible chemical class or structural matches to the experimentally measured characteristics (17,18). While NMR has proven very useful for unknown structure identification, it also has challenges, such as the need for high concentrations of the molecule of interest and its low throughput, ultimately limiting the number of novel molecules identified per year (19). 

 tools are much faster, but small testing sets and errors between the theoretical numbers and experimental analyses (for example, chromatography retention time differences) often only allow plausible candidate matches until validated with a standard (17). Therefore, in my keynote presentation at HPLC 2023, I will address the challenges of xenobiotic and endogenous small molecule measurements and demonstrate how multidimensional analyses combining liquid chromatography, ion mobility spectrometry, collision induced dissociation, and mass spectrometry (LC–IMS–CID–MS) and computational developments enhance direct xenobiotic analyses and indirect multi-omic evaluations. I will also showcase applications where these developments are leading to a better understanding of molecular responses occurring due to chemical exposures.

Vermeulen, R.; Schymanski, E. L.; Barabási, A. -L.; Miller, G. W. The Exposome and Health: Where Chemistry Meets Biology. 

 (6476), 392–396. DOI: 10.1126/science.aay3164

Wild, C. P. Complementing the Genome with an “Exposome”: The Outstanding Challenge of Environmental Exposure Measurement in Molecular Epidemiology. 

 (8), 1847–50. DOI: 10.1158/1055-9965.EPI-05-0456

Wild, C. P. The Exposome: From Concept to Utility. 

Miller, G. W; Jones, D. P. The Nature of Nurture: Refining the Definition of the Exposome. 

Thomas, D. Gene--Environment-Wide Association Studies: Emerging Approaches. 

Walker, D. I.; Valvi, D.; Rothman, N.; et al. The Metabolome: A Key Measure for Exposome Research in Epidemiology. 

, 93–103. DOI: 10.1007/s40471-019-00187-4

Exposure Science in the 21st Century: A Vision and a Strategy

Athersuch, T. Metabolome Analyses in Exposome Studies: Profiling Methods for a Vast Chemical Space. 

, 177–86. DOI: 10.1016/j.abb.2015.10.007

Ruddigkeit, L.; Awale, M.; Reymond, J. L. Expanding the Fragrance Chemical Space for Virtual Screening. 

O’Hagan, S.; Kell, D. B. Understanding the Foundations of the Structural Similarities Between Marketed Drugs and Endogenous Human Metabolites. 

Wishart, D. S. Advances in Metabolite Identification. 

 (15), 1769–82. DOI: 10.4155/bio.11.155

Peironcely, J. E.; Reijmers, T.; Coulier, L.; Bender, A.; Hankemeier, T. Understanding and Classifying Metabolite Space and Metabolite-Likeness. 

 (12), e28966. DOI: 10.1371/journal.pone.0028966

Schymanski, E. L.; Jeon, J.; Gulde, R.; et al. Identifying Small Molecules via High Resolution Mass Spectrometry: Communicating Confidence. 

Schrimpe-Rutledge, A. C.; Codreanu, S. G.; Sherrod, S. D.; McLean, J. A. Untargeted Metabolomics Strategies–Challenges and Emerging Directions. 

 (12), 1897–1905. DOI: 10.1007/s13361-016-1469-y

Monge, M. E.; Dodds, J. N.; Baker, E. S.; Edison, A. S.; Fernández, F. M. Challenges in Identifying the Dark Molecules of Life. 

 (1), 177–199. DOI: 10.1146/annurev-anchem-061318-114959

Garcia-Perez, I.; Posma, J. M.; Serrano-Contreras, J. I.; et al. Identifying Unknown Metabolites Using NMR-Based Metabolic Profiling Techniques. 

 (8), 2538–2567. DOI: 10.1038/s41596-020-0343-3

Plante, P. L.; Francovic-Fontaine, É.; May, J. C.; et al. Predicting Ion Mobility Collision Cross-Sections Using a Deep Neural Network: DeepCCS. 

 (8), 5191–5199. DOI: 10.1021/acs.analchem.8b05821

Foster, M.; Rainey, M.; Watson, C.; et al. Uncovering PFAS and Other Xenobiotics in the Dark Metabolome Using Ion Mobility Spectrometry, Mass Defect Analysis, and Machine Learning. 

 (12), 9133–9143. DOI: 10.1021/acs.est.2c00201

Socha, O.; Osifová, Z.; Dračínský, M. NMR-Challenge.com: An Interactive Website with Exercises in Solving Structures from NMR Spectra. 

 (2), 962–968. DOI:10.1021/acs.jchemed.2c01067

 is an associate professor at the University of North Carolina in Chapel Hill, North Carolina, USA. To date, she has published over 150 peer-reviewed papers utilizing different analytical chemistry techniques to study both environmental and biological systems. Over the last four years, Erin also helped grow the Females in Mass Spectrometry group, where she served as the Events Committee Chair from 2019–2022 and hosted 35 online and two in-person events. She is currently serving as the Vice President of Education for the International Lipidomics Society, a mentor for Females in Mass Spectrometry, and as an associate editor for the 

Journal of the American Society for Mass Spectrometry

. She has received seven US patents, two R&D 100 Awards, and was a recipient of the 2016 ACS Rising Star Award for Top Midcareer Women Chemists, 2022 ASMS Biemann Medal, and 2022 IMSF Curt Brunnée Award. Currently, her research group utilizes advanced separations and novel software capabilities to examine how chemical exposure affects human health.

Measuring chemical exposure is extremely challenging due to the range and number of anthropogenic molecules encountered in our daily lives, as well as their complex transformations throughout the body. To broadly characterize how chemical exposures influence human health, a combination of genomic, transcriptomic, proteomic, endogenous metabolomic, and xenobiotic measurements must be performed. However, while genomic, transcriptomic, and proteomic analyses have rapidly progressed over the last two decades, advancements in instrumentation and computations for nontargeted xenobiotic and endogenous small molecule measurements are still greatly needed. 

Assessing How Chemical Exposures Affect Human Health 

Gas chromatography (GC) has received a major boost with the introduction of a novel stationary phase composed of a metal organic framework (MOF) and an ionic liquid (IL) pseudophase. This new development, led by Daniel W. Armstrong at the University of Arlington, was published in the journal 

, and has unlocked distinct separation properties and expanded the capabilities of GC (1). By investigating the behavior of four different MOFs in 18 ILs, researchers were able to identify the optimal combination for achieving remarkable separation performance.

A metrology laboratory specialist takes a compressed gas cylinder for testing and calibration with a chromatograph. A man connects a hose for gas analysis. Gas analyzer and chromatography. | Image Credit: © NEZNAEV

The team successfully created a hybrid stationary phase by uniformly dispersing colloidal ZIF-8 (a type of MOF) in an imidazolium-based dicationic IL. This pioneering configuration enabled the first-ever separation of permanent gases through hybrid gas-liquid chromatography. Furthermore, the newly developed stationary phase demonstrated exceptional separation proficiency for various organic compounds, including alkanes, ketones, alcohols, ethers, and Rohrschneider-McReynolds compounds.

To gain deeper insights into the behavior of solutes within this innovative pseudophase system, the researchers derived equations for a three-phase model. These equations facilitated the calculation of distribution constants, providing valuable information on solute partitioning between the MOF, IL, and gas phases. Notably, the study emphasized the substantial impact of the MOF on solute partitioning to the stationary phase, highlighting the pivotal role of MOFs in the separation process.

This groundbreaking research opens new possibilities for gas chromatography, enhancing its efficiency and expanding its analytical capabilities. The combination of MOFs and ILs as a stationary phase represents a significant advancement in the field, with potential applications ranging from environmental analysis to pharmaceutical development. The improved separation performance and versatility offered by this novel system hold promise for researchers and scientists across various disciplines. As the scientific community awaits future developments, this breakthrough sets the stage for a new era of gas chromatography with enhanced precision and efficiency.

(1) Patel, A.; Aslani, S.; Firooz, S. K.; Armstrong, D. W. A Metal Organic Framework + Ionic Liquid Pseudophase System as a Gas Chromatography Stationary Phase. 

https://doi.org/10.1007/s10337-023-04258-z

Articles

Metal organic frameworks (MOFs) and ionic liquids (ILs) combine to form a powerful gas chromatography stationary phase, enabling improved separation properties and expanded application possibilities. This innovative system offers enhanced separation capabilities and paves the way for advancements in gas chromatography analysis.

Revolutionizing Gas Chromatography: Metal Organic Framework + Ionic Liquid System Offers Unprecedented Separation Performance

Exploring ways to improve the efficiency of high-throughput analysis in small-molecule drug discovery, researchers at Biocon Bristol-Myers Squibb R&D Center (BBRC) in Bangalore, India, have harnessed the potential of superficially porous particle (SPP) column technology in combination with traditional high performance liquid chromatography (HPLC) systems. The study, published in 

, was led by Khemraj Bairwa and Amrita Roy, and aimed to reduce runtime without compromising analysis quality (1).

Close up scientist changing a column in HPLC system. Analytical chemistry laboratory. Preparation before analysis. | Image Credit: © vladim_ka - stock.adobe.com

SPP columns have a solid core surrounded by a thin, porous layer. This design provides advantages such as improved efficiency, enhanced separation capabilities, and reduced backpressure. The thin porous layer increases the surface area available for analyte interactions, allowing for faster mass transfer and shorter analysis times. The reduced particle size and narrow particle size distribution of SPP columns contribute to improved peak shapes and resolution. Overall, SPP columns offer a promising solution for achieving higher throughput and increased efficiency in HPLC analysis.

SPP columns have gained popularity due to their superior performance compared to fully porous particle (FPP) columns. However, their full potential had not been effectively explored in traditional HPLC methods. By optimizing the experimental parameters such as flow rate, gradient time, and photodiode array (PDA) detector acquisition rate, the researchers demonstrated the effectiveness of SPP column technology.

Two SPP columns, namely Kinetex EVO C18 and Accucore C18, were employed in the study. The optimized method exhibited remarkable improvements compared to the regular method. The SPP column achieved enhanced peak capacity, peak symmetry, theoretical plate numbers, and peak resolution, all within a significantly reduced runtime of just 7 minutes.

The results showcased a 50% reduction in runtime and approximately 31% net organic solvent savings, demonstrating an eco-friendlier approach to method development. The combination of SPP column technology and optimized parameters provided a greener and more efficient solution for high-throughput HPLC analysis in small-molecule drug discovery.

The study highlights the potential of SPP column technology to enhance the performance of HPLC methods, offering researchers in the pharmaceutical industry an efficient and sustainable approach for drug discovery. The utilization of SPP columns not only improves analysis efficiency but also contributes to a more environmentally conscious laboratory practice by reducing solvent consumption. The findings of this study serve as a foundation for future advancements in chromatographic techniques, ultimately benefiting the pharmaceutical industry and its pursuit of innovative therapies.

(1) Tellakula, N.; Guna, M.; Das, M.; Bane, V.; Chauthe, S. K.; Mathur, A.; Bagadi, M.; Bairwa, K. Roy, A. Leveraging Superficially Porous Particle Technology to Develop High-Throughput HPLC Methods for Small-Molecule Drug Discovery. 

https://doi.org/10.1007/s10337-023-04259-y

Superficially porous particle (SPP) column technology is leveraged to optimize high-throughput HPLC methods for small-molecule drug discovery, resulting in improved efficiency and reduced runtime.

Enhancing High-Throughput HPLC Methods for Small-Molecule Drug Discovery Using Superficially Porous Particle Technology

Researchers at Beijing Technology and Business University have made significant progress in the field of countercurrent chromatography by developing a general separation strategy. The study, published in the 

, focused on the separation of sanshools from 

 oleoresin, a plant known for its rich amide compounds (1).

Formamide (methanamide) solvent molecule | Image Credit: © molekuul.be - stock.adobe.com

Countercurrent chromatography is a liquid-liquid chromatographic technique that utilizes the partitioning of solutes between two immiscible liquid phases for separation. Unlike traditional chromatographic methods, countercurrent chromatography does not require a solid stationary phase. Instead, it employs two liquid phases, typically an organic solvent and an aqueous solution, which flow in opposite directions. This counterflow creates a stationary phase through the equilibrium distribution of solutes between the two phases. The technique offers advantages such as high resolution, low sample loss, and the ability to separate compounds with similar chemical properties. Countercurrent chromatography has found applications in various fields, including pharmaceuticals, natural product research, and biochemistry, for the purification and isolation of complex mixtures.

Sanshools are a series of amide compounds extracted from 

, which possess various potential pharmacological properties. However, due to their similar structures, polarities, and dissociation constants, achieving their complete separation through countercurrent chromatography presented a considerable challenge. To overcome this hurdle, the researchers proposed a solvent-system-selection strategy to identify an appropriate solvent system for efficient separation.

The team successfully established a separation procedure that incorporated multi-elution modes selection. Through careful experimentation, they determined that a solvent system consisting of 

-hexane, ethyl acetate, methanol, and water in a ratio of 19:1:1:5.67 yielded the best results. Using the recycling elution mode, the researchers obtained high-purity sanshool compounds from the crude extract. Specifically, hydroxy-ε-sanshool (8.4 mg; purity: 90.64%), hydroxy-α-sanshool (326.4 mg; purity: 98.96%), and hydroxy-β-sanshool (71.8 mg; purity: 98.26%) were successfully separated from a 600 mg sanshool crude extract.

The developed solvent-system-selection strategy and separation procedure provide valuable guidance for countercurrent chromatography users, particularly those dealing with compounds that possess highly similar chemical properties. This breakthrough not only opens doors for the separation and purification of sanshool compounds from 

 oleoresin but also offers a general approach that can be applied to other challenging separations in the field of chromatography.

Countercurrent chromatography, with its ability to achieve high-resolution separation and purification, continues to be a promising technique in the field of analytical chemistry. The findings of this study contribute to the advancement of countercurrent chromatography methodologies and have the potential to benefit various industries, including pharmaceuticals, natural product research, and biochemistry, by facilitating the isolation and characterization of valuable compounds from complex mixtures.

(1) Han, T.; Ling, T.; Fu, Z.; Cao, X. Development of a general separation strategy by countercurrent chromatography using sanshools from Zanthoxylum bungeanum oleoresin as a case study. 

An analytical team has successfully developed a novel strategy for the separation of sanshools from Zanthoxylum bungeanum oleoresin using countercurrent chromatography, providing valuable insights for the separation of compounds with similar chemical properties.

Researchers Develop Innovative Strategy for Countercurrent Chromatography Separation of Sanshools From Zanthoxylum bungeanum Oleoresin

Liquid chromatography (LC) is the single largest analytical field in terms of people involved and money spent. LC is crucial for almost all public and private sectors and the technique has seen tremendous technological advancements. Nevertheless, separations are often performed under suboptimal conditions and technological capabilities remain unused. Because expert knowledge and method development time are increasingly scarce, methods are often inefficient. Exploiting the full technological capabilities of liquid-phase separation technology requires deep knowledge and great time investments. Method optimization strategies that can simultaneously optimize the large number of parameters involved are therefore of great interest to chromatographers. This review examines different workflows that have been designed and used to facilitate and/or automate method development. In particular, focus is paid to the implementation of computer-aided workflows for the optimization of kinetic and thermodynamic parameters in LC, as well as on the possibilities to conduct this in a closed-loop fashion. Finally, the opportunities to use machine learning to achieve these goals is addressed.

Decades of research and development have allowed liquid chromatography (LC) to become the reliable and robust technique that we know today. This has paved the way for the development of increasingly advanced two-dimensional LC (2D-LC) methods with increased peak capacities, better use of mass spectrometry (MS), and new opportunities such as in-line reactions (1). For complex samples a 2D-LC method can be much faster than a one-dimensional (1D)-LC counterpart (2,3).

The development of 2D-LC has largely been driven by the growing interest of society and industry in understanding the increasingly complex and diverse samples, each with their own challenges associated.

The need to implement more powerful separation technologies is growing faster than the number of qualified users of multidimensional chromatography. The leading cause hampering further proliferation is the considerable amount of resources required for method development. This task is ever more daunting as method parameters are numerous and often interdependent, increasing the experience required to use all capabilities effectively.

This is illustrated in Figure 1, where the technological complexity (that is, the cost) is sketched against the information density (the benefit). There is a disproportionate increase in resources required to obtain further information. State-of-the-art separation technology such as 2D-LC can only make an impact if it can be efficiently employed.

To facilitate this, researchers worldwide have been designing strategies with software to simplify method development (4–6) These and other method development and optimization strategies capitalize on theoretical understanding to simplify the method development process, as well as maximize its potential similar to the motto “work smarter, not just harder” (7). This review will map the method development process and identify categories of parameters that can be targeted. Different method optimization approaches that simplify or even automate sections of this workflow will then be examined. Finally, the opportunities to use machine learning to achieve in this context will be addressed.

Objectives of Chromatographic Method Development

Chromatographic method development is affected by the aim of the method. The optimization approaches for LC method development can be roughly classified as targeted and untargeted (8). Targeted approaches focus on specific analytes, whereas untargeted methods attempt to characterize the entire sample. This article will focus on untargeted methods, as targeted methods tend to have dedicated goals that cannot easily be generalized.

Generally, an analyst will start method development by using any information available on the sample chemistry, for example, Giddings’ sample dimensions (9), literature, and other resources to select an initial column (often a general-purpose column available in the laboratory) and generic method parameters (a generic linear gradient).

After the initial instrument parameters are established, the method can be evaluated based on its ability to provide the required critical information. When the method does not provide this information, method parameters can be adjusted to improve the method. This process is generally termed 

. In chromatographic literature, the term 

 often represents divergent intentions based on the objective and the stage of the method development process. Indeed, “optimization” can refer to sample preparation (10), kinetic parameters that affect efficiency (11), the selection of a suitable stationary-phase chemistry (12), a reduction in analysis time (8), and so on. The common denominator in each case is that method parameters are adjusted with the aim of obtaining the desired information more effectively. Typically, this is evaluated through metrics that quantify the quality of separation, also known as 

To maximize the ability of a method to characterize a sample, untargeted approaches are often driven by such quality descriptors. For example, in the case where an analyst encounters a chromatogram saturated with peaks, maximization of the peak capacity (that is, the number of peaks that can be separated by the method) can be employed as a quality descriptor.

Untargeted optimization suffers from the vast number of adjustable parameters and the large interdependence between these. It is inherently difficult to predict whether a change in parameters will result in an improved method. Improving one metric (analysis time) may result in worsening another metric (resolution). Computer-aided development of a chromatographic method thus requires a balanced approach.

Resolution to Adjust a Chromatographic Method:

 One useful quality descriptor that quantifies the degree of separation, yet also instructs on possibilities to improve it, is the resolution (equation 1):

Here, the separation between analytes 1 and 2 is expressed with 

 and σ representing the retention time and peak standard deviation. When assuming that both analytes experience a similar column efficiency (that is, 

), we can re-write equation 1 as follows (13):

Now, we have resolution quantified as the product of three contributions: efficiency (

), selectivity (α), and average retention factor (

). Equation 2 demonstrates that the only means to adjust the separation is to change something within either of these three parameters.

At this point, it is important to acknowledge the significance of sample preparation and the crucial effect this has on the method development workflow. Sample preparation tends to be highly sample dependent, and it has been extensively treated elsewhere (14,15). It is outside the scope of this review.

Equation 2 can be schematically expressed in the context of method development workflows as depicted in Figure 2, which represents a typical case often faced by chromatographers. After having used their experience to select an appropriate column and initial method parameters, chromatographers are recommended to first adjust the method conditions by altering thermodynamic parameters that affect retention and selectivity. For example, they may change the gradient program or—if necessary—adjust the selectivity dramatically by selecting a different column or mobile-phase chemistry. Finally, they may opt to improve the efficiency or analysis time of the method by regarding the kinetic parameters. Different approaches that have been developed to facilitate method development through automated workflows or chemometrics will be reviewed in the following sections.

Improving the efficiency (that is, the theoretical plate number) of the separation provides a clear metric relevant to method optimization as it increases the peak capacity for the separation and decreases the likelihood of coelution. For 1D-LC it is possible to predict how different parameters will affect the efficiency. Another method is the use of kinetic plots (16). However, the square-root dependence of 

 implies a rapidly diminishing return on investment.

In comprehensive two-dimensional liquid chromatography (LC×LC) kinetic optimization schemes can be applied to substantially improve the method. This is attributed to the additional parameters, including the second-dimension (

D) column dimensions, and phenomena such as under-sampling (of the 

D sample-dilution (17). Vivo-Truyols et al. proposed the use of sample-independent Pareto optimization (PO) for LC×LC (18). In this chemometric-driven approach, multiple method parameters (column dimension, flow rates, gradient slope) were varied and the effects thereof were evaluated using (theoretically related) quality descriptors such as peak capacity, analysis time, and dilution factor. The authors demonstrated that optimizing the kinetic parameters can be highly rewarding and that this approach is suited to deal with trade-offs between different objectives. The PO method provided optimal values for a large number of parameters, including 

D particle sizes and column diameters, while taking into account pressure restrictions.

This work was extended by the group of de Villiers (11,19,20), who applied the PO strategy for the chromatographic separation of a phenolic red-wine extract (19), implemented a predictive kinetic-optimization tool for a hydrophilic interaction liquid chromatography×reversed-phase liquid chromatography (HILIC×reversed-phase LC) separation of procyanidins (11), and used the same tool to compare reversed-phase LC×HILIC and HILIC×reversed-phase LC for phenolic analysis (20). In all cases, optimization of kinetic parameters provided higher peak capacities and produced valuable knowledge on the influence of under-sampling, dilution factors, and band broadening.

Selectivity in liquid chromatography is influenced by the thermodynamic parameters related to analyte interactions with the mobile and stationary phases. Changes in mobile phase composition, gradient program, stationary phase chemistry, pH, or temperature can significantly affect the distribution of peaks in a chromatogram. While modifying the mobile phase, pH, or temperature is relatively straightforward in theory, in practice this may not suffice to meet the objectives of the separation. Changing the stationary phase in principle requires a re-optimization of the mobile phase, making it more expensive in terms of effort (apart from the column cost), but generally provides broader changes in selectivity.

Various strategies and workflows have been described to select appropriate chemistries for both the mobile and stationary phases in LC. One such strategy involves optimizing the parameters through a Design-of-Experiments (DoE) workflow, in which sets of parameters (for example, mobile phase, temperature, pH) are varied and an extensive screening is performed, often on multiple columns, to determine the optimum approach. Similar approaches are commonly used in the industry (21). They may be efficient in terms of manpower required, but can be quite inefficient in terms of numbers of measurements, time, solvents and instruments required.

For column selection, stationary phase characterization methods aim to simplify the end user’s choice of stationary phases based on characterizing the physicochemical interactions with small-molecule probes. For an extensive review the reader is referred elsewhere (22). The best-documented example is the hydrophobic subtraction model (HSM) developed by Snyder, Dolan, and co-workers, which may be used to select columns with different selectivity (23).

Recently, workflows have emerged related to selectivity screening for 2D-LC method development. For example, Zhang et al. developed a multiple heart-cut 2D-LC setup for pharmaceutical purity testing that enabled automated screening of the 

D selectivity (24). In other work, Wang et al. (25) used an automated column screening framework for analyzing chiral and achiral impurities, with online multiple heart-cutting 2D-LC; in a similar study, Zhang and co-workers described fast chiral and achiral profiling of compounds with multiple chiral centres by an LC–multicolumn-LC approach (26). Together with automated data acquisition, these workflows constituted a step forwards, but interpretation and processing of the data are still dependent on manual input and user experience.

In an ideal scenario, a method development workflow must be capable of integrating all previously discussed parameters and optimizing these in an automated and interpretive manner. In this context, the word 

 implies that the chromatograms are interpreted as a result of the elution of the individual analytes. In the workflow the peaks should be tracked and simple models may be established for each analyte—all of this in an unsupervised and automated manner. Based on the analyte models, many chromatograms can be simulated and the objective function can be optimized. Such a method development workflow has the potential to minimize manual input and the expertise required for developing complex methods.

Bos et al. developed the AutoLC approach, in which the mobile phase gradient was optimized in a closed-loop fashion (27). Based on an earlier theoretical LC×LC proof-of-principle study (28), and the pioneering work of Dolan and co-workers (29), Bos et al. designed an algorithm that could accommodate different chemometric optimization strategies. Two examples were presented: one using empirical retention modelling, and one using the machine-learning Bayesian optimization technique. In both cases, the algorithm developed multi-segment gradient programs to optimize the retention and selectivity of separations of a monoclonal antibody digest by LC–MS.

The examples above demonstrate the possibility of including computer-assisted optimization strategies for kinetic and thermodynamic parameters in automated workflows. However, despite the significant progress made, three major challenges remain that need to be addressed to achieve a closed-loop, interpretive algorithm for developing LC methods in a completely unsupervised and automated manner.

The first and arguably most important challenge is to define quality descriptors that can assess the quality of a separation. The success or failure of the optimization process critically depends on the objective function. The latter depends on quality descriptors that, in turn, may depend on chemical and physical parameters. The objective function serves as the primary driving force for every algorithm. Single-number descriptors, known as 

 (CRFs), have been developed to quantify the critical information that the method is intended to provide. Various CRFs have been proposed in the past (30). Although many CRFs exist for 1D-LC, few are available for 2D-LC (31–36). Alvarez-Segura and co-workers recently included peak prominence in their CRF (33,34), Huygens et al. (35) introduced a CRF for 2D-LC based on peak purity, and Boelrijk et al. (36) presented a CRF based on connective components in graph theory to assess both separation quality and time. It has also been demonstrated that the selection of inappropriate quality descriptors can lead to suboptimal separations (27). Therefore, it is crucial to create CRFs that can effectively weigh quality descriptors, while the program provides quantitative information regarding various practical aspects of analytical queries, such as method time and sensitivity.

The second challenge is related to the data processing. The quality of the output of an algorithm relies strongly on the input data and the data processing. Any error in background correction, peak detection, or peak tracking can lead to cascading errors in later iterations (27). While MS provides information to reduce the errors in 1D-LC, data processing remains a significant challenge for LC×LC. This is especially the case for peak tracking, where an algorithm must establish the location of each analyte across multiple signals and account for incorrect clustering of coeluting peaks (37,38). In addition, it is difficult to quantitatively compare the performance of the large number of data processing approaches available (27,39).

The third challenge concerns the computation costs associated with the large number of simulations and the number of parameters that need to be optimized. Improving the performance of the algorithm either requires more measurements or more simulated data that resemble the experiments more closely. The former increases the amount of time and solvent needed, while the latter requires more computation power. Machine learning techniques may be employed to reduce the computation time required.

Interest in machine learning methods is rapidly gaining momentum in the field of chromatography (7). The increasing computation power available, combined with the demand for advanced simulation tools, has led to the exploration of machine learning approaches for accelerated method development (40,41). Machine learning has the potential to enhance various stages of the chromatographic-analysis workflow, including baseline and noise correction (42), retention-time or retention-index prediction (43–45), peak detection (46,47), peak annotation (48), and classification (49). This section will focus on the utilization of machine learning to optimize the gradient program, which frequently relies on several of the aforementioned stages.

One type of machine learning is artificial neural networks (ANNs). The application of neural networks in chromatography has been explored in the past (50–53). In a recent study, Kensert et al. (42) investigated the potential use of reinforcement learning for selecting optimal isocratic scouting runs for retention modelling. The neural network was trained on simulated data using a Q-Learning algorithm and evaluated based on the accuracy of the retention predictions for 57 small-molecule analytes 

 experiments. While the work is promising, the algorithm from this study has only been tested on isocratic data for a specific reversed-phase LC setup with a limited number of simplistic molecules. A downside of the use of artificial neural networks is that they require a large amount of data for training. This can be mitigated with simulated data, but this carries a risk of the model not capturing the actual chromatographic space with sufficient accuracy (46).

Several authors have explored the potential of evolutionary algorithms to optimize gradient profiles (35,54). In particular, Hao et al. (54) used a genetic algorithm to optimize multi-linear gradient profiles for the separation of lignin-degradation products. They validated their simulations by comparison with experimental measurements and found good agreement. Huygens et al. (35) investigated evolutionary strategies for optimizing 

 experiments of a LC×LC separation of 100 experiments. Their genetic algorithm required less than 100 experiments to reach a better CRF than a grid search of 625 experiments. This suggests that evolutionary algorithms have the potential to guide search-based method development, accelerate computational model-based method development, and deal with many parameters simultaneously. However, the authors simplified the experimental conditions by assuming perfect orthogonality, equal concentrations, and perfect Gaussian peaks. This raises concerns over the risk of oversimplification and the underutilization of the full range of variations in chromatographic conditions, as can also occur with neural networks.

One of the biggest limitations of applying machine learning is the required amount of data. An emerging optimization method that is less dependent on the amount of input data is Bayesian optimization, a type of machine learning tool that can optimize expensive-to-evaluate functions (expensive in terms of computation cost), even when there is a low amount of data available. Due to the compatibility with black box functions, only the score itself needs to be ascertainable, while the underlying mechanics or processes do not necessarily need to be understood or known. Boelrijk et al. investigated the potential use of Bayesian approaches for LC×LC 

 (36); they developed an unsupervised closed-loop algorithm to predict 1D-LC gradient profiles for a complex dyestuff mixture (55) and compared Bayesian optimization to retention modelling for automated method development (27). These studies demonstrated the versatility of Bayesian approaches and highlighted their applicability in method development workflows, with the added benefit of being less reliant on retention models and peak tracking. However, a potential limiting factor for LC×LC is the maximum number of parameters that can be optimized simultaneously. Due to the multivariate nature of the method, the number of necessary measurements for each additional parameter increases exponentially. The authors emphasized that the efficiency of Bayesian optimization is heavily dependent on the accurate definition of the objective function.

Machine learning approaches show tremendous potential to optimize the gradient program, yet the most advanced applications often have limitations that render them unsuitable for widespread use. This is partly due to the fact that these models can only handle scenarios that were present in the training data. Insufficient data can stem from a lack of appropriate standards or from insufficient resources to perform all the necessary measurements. One way to alleviate this issue is by employing simulated data, but this approach only works effectively if the simulated data closely resemble real-world situations, which is frequently not the case. With the rapid advancements in the field of machine learning, the issues currently encountered are expected to be addressed in the near future.

Driven by the ever-increasing need for more information, separation technology is advancing faster than our ability to use it to its full potential. Despite the enormous size and importance of the field, separations are often performed under suboptimal conditions and technical capabilities remain unused. For advancements in LC, such as 2D-LC, to make a significant impact, it is imperative that our community develops tools to fully exploit the current potential.

To this end, the chromatographic community must continue to deepen its understanding of fundamental concepts. This review has underlined the huge benefits and impressive advancements by scientists who have developed tools to streamline method development, focusing on kinetic or thermodynamic parameters—or both. Many of these advances are expressed as improved method development workflows that capture the experience and knowledge of the chromatographer.

However, the chromatographic community cannot do it alone. Indeed, we need the help of computers and information sciences. Automated LC method development workflows are rapidly advancing through the implementation of chemometric and machine learning algorithms. Nevertheless, despite the potential and promise of such algorithms to expedite method development, the challenges are still daunting and require a combined effort by the chromatographic and chemometric communities. Better ways must be found to mathematically express the chromatographic end goals, improve data analysis (peak detection), and invent smart algorithms that can interpret chromatographic data and combine these with fundamental kinetic (kinetic plots) and thermodynamic (retention models) concepts to create better methods. Meanwhile there is also the continuous need for chromatographers to deepen the understanding of physicochemical interactions of analytes inside our columns. The use of machine learning has recently been demonstrated by several groups to show great promise (35,55).

From the above-mentioned strategies it is clear that optimization workflows, especially for selectivity, remain heavily dependent on prior knowledge of the sample dimensionality or a trial-and-error approach. New machine learning algorithms should be able to optimize more parameters without needing unreasonable amounts of data. Other approaches may entail optimizing subsets of parameters at a time to lower the dimensionality of the problem or combining retention modelling with machine learning; in that way aspects that can be well described do not need to be captured in the machine learning models. The practical challenges are mostly on the required number of measurements or computations. Every optimization process is quicker when starting with a good setup. By scanning various columns at the start, a good estimate can be made of which columns show potential and are candidates for orthogonal separation in 2D-LC.

Computer-aided method development for LC has been under development for some four decades and it has failed to become common practice. However, with the rapid advances in LC, the gap between contemporary LC practice and potential has grown to a point where computer-aided method development is becoming a must. This need, in combination with the enormous growth in computation power and the dramatic advances in artificial intelligence, suggests that the time has come for computers to outperform analysts in LC method development.

This publication is part of the project Unleashing the Potential of Separation Technology to Achieve Innovation in Research and Society (UPSTAIRS) (with project number 19173) of the research programme TTW-VENI, which is financed by the Dutch Research Council (NWO).

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This review examines different workflows that have been designed and used to facilitate and/or automate method development in liquid chromatography (LC).

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