Our Exclusive Interview with Monica Cahilly

Ensuring Data Integrity in the Cloud

Tips for Liquid Chromatography Coupled with Charged Aerosol Detection

Not (Only) Reversed-Phase Liquid Chromatography–Mass Spectrometry

Evaluating the Goodness of Instrument Calibration 

Looking at Improved Aroma Profiling of Foods 

SCM, the symposium series on the Separation and Characterization of natural and synthetic Macromolecules, is a leading platform for discussion on the latest developments in large‑molecule analysis. Since 2003 live conferences have been held at the beginning of every second year, mainly in Amsterdam, The Netherlands, but in 2013 in Dresden, Germany. SCM is organized by a large group of researchers from the Centre for Analytical Sciences Amsterdam (CASA) that is formed by the two Amsterdam universities: the University of Amsterdam and the Vrije Universiteit.

SCM has developed into an enthusiastic community, with lively meetings and exciting science. While 2021 will be different from any previous—and hopefully future—year, the idea of the organizers is to cherish the traits that have made SCM an anchor point for scientists in the field. SCM‑X will focus the attention of an extended community on the great science that keeps being performed.

On the proposed days for SCM (27–29 January 2021), two‑hour virtual sessions will be organized in late afternoon (European time) for the SCM community to promote the work highlighted on the SCM-X website and, importantly, to stimulate interaction between members of the community. Researchers from anywhere in the world may participate at no charge, provided they register through the website and are willing to adapt to the time schedule, which, unfortunately, is challenging for those living in Asia or Australia.

SCM‑X provides great opportunities for researchers to advertise their science within and beyond the community. All scientific papers with publication year 2019, 2020, or 2021 that are within the scope of the SCM meeting can be highlighted at SCM‑X. MSc and BSc theses may qualify if they can be downloaded from the website of the responsible educational institute.

By presenting a 3‑min pitch on their paper or thesis, scientists may amplify interest in their work and trigger other researchers to read their publication, which is made accessible from the conference website. Young scientists may also draw attention to themselves and, in doing so, enhance their chances on the job market.

Past and future sponsors of the SCM conferences may contribute in two different ways. They may advertise their scientific papers as part of the regular scientific programme and they may also submit a pitch highlighting their latest technical accomplishments with respect to instruments, software, or consumables, with a reference to their website.

The virtual sessions on January 27, 28 and 29 will feature presentations and discussions on new developments in the field and ample attention for the featured papers, culminating in live pitches and an awards presentation on Friday January 29. Among the confirmed speakers is André Striegel from NIST (Gaithersburg, Maryland, USA), who is arguably the greatest expert on size-exclusion chromatography (SEC) today and who will discuss recent developments in the area. Ewoud van Tricht from Janssen Vaccines in Leiden (The Netherlands) will address the hottest topic of the moment, namely the role of separation and characterization in the development and production of vaccines.

Papers can be proposed for incorporation in SCM‑X using the form on the conference website. If the paper is accepted, the presenting author is asked to make the accompanying pitch available within 30 days. Papers can be submitted until the end of 2021. However, to feature in the discussions on 27–29 January and to qualify for the awards, pitches should be available before January 6th, 2021.

Peter Schoenmakers comments: “If you have attended an SCM conference before, you will sure want to see what is happening at SCM-X. If you have not, there is a very low threshold for you to get to know the conference”.

For more information and registration, visit the SCM‑X webpage at
Website: 

Articles

The International Symposium on the Separation and Characterization of Natural and Synthetic Macromolecules (SCM-X) will take place virtually, from 27 to 29 January 2021, with the live event potentially taking place in 2023. This preview offers a flavour of what visitors can look forward to from SCM-X’s “Zoomposium”.

SCM-X 2021 Preview

	You're invited to publicize your 2020-2021 new product introductions in 

. Here's how to get your products included.

 limited to new products introduced at Pittcon! For our 2021 review, you may submit any product launched between May 2020 and March 2021.

	We have four forms, covering different types of products. Participate in as many categories as you wish. Limit 5 products per category.
	
	Links to the surveys appear below. Please download the forms you need, fill them out, and send them to 

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Click on the links below to download the forms:

HPLC and Mass Spectrometry Systems, Modules, and Data Software

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	Download the forms online and email all materials to: John Chasse, Managing Editor at 

John has a new email address, reflecting our current corporate ownership. Emails to his old UBM address will not go through!

If you are unable to complete the forms yourself, please forward them to the appropriate person. If your company distributes products for other companies, please send this announcement to the manufacturer.

Include examples of appropriate chromatograms or other technical data.

Information on every product you submit will be held in strict confidence prior to publication.

Careful consideration will be given to all surveys received. Submitted information will be held in confidence until publication. Final decisions regarding what details are included for publication is based on editorial judgment, reader interest, and available space.

LCGC 2021 New Product Review–Call for Submissions

In March 2020, the United States was in the early stages of the COVID-19 pandemic. We shut the entire country down and ground the economy to a halt in an effort to slow the spread of the virus. Think back to March, and how much uncertainty we were living under.

Nine months later, the FDA approved two COVID-19 vaccines under emergency authorization. Before New Year’s Day, millions of Americans had received the vaccine, including front-line physicians and health care providers and nursing home patients, our most vulnerable citizens.

Nine months. Take a moment to let that sink in.

The mainstream media has crafted a narrative around the COVID-19 pandemic that’s almost entirely negative. For the purpose of ratings, they have described the U.S. response to the pandemic as blundering from one mistake to the next. This narrative is false.

There is another way — a more accurate and underappreciated way — to tell the story of the last nine months. It is a story of heroism, innovation, and precise science, performed under unbelievable pressure.

Let’s not mince words: The United States and the world needs to appreciate the role of the pharmaceutical industry — the researchers, physicians and business leaders — who are rescuing the world from COVID-19. It’s the medical breakthrough of our lifetime.

Instead of dwelling on why many in the media are ignoring this, let’s review some facts.

Since the discovery of COVID-19, here is what scientists have accomplished: They identified a novel virus, unlocked and sequenced its genetic code, created new therapies to save lives, and developed multiple safe and effective vaccines using messenger RNA technology, a technology hopefully applicable to future vaccine development. Margaret Liu, MD, a member of the MJH Life Sciences COVID Coalition, called it a breakthrough for mRNA vaccines.

The United States has two vaccines approved for emergency use, one from Pfizer/BioNTech and another from Moderna, and the AstraZeneca/Oxford vaccine has been approved for emergency use in the UK. In addition, there are 64 vaccines undergoing clinical trials at the moment, including 20 in phase 3 trials. In the United States and around the world, the pharmaceutical industry has answered the call and invested heavily in this effort.

This was the fastest vaccine development program in history, and it’s not even close. David Pride, MD, a microbiologist at the University of California San Diego, estimates that vaccines typically take 10 to 15 years to develop. Until the COVID-19 pandemic, the fastest development timeline was four years, for the mumps vaccine.

Many government systems moved quickly to lessen the burden of onerous regulations and provide funding so that vaccines could be developed quickly but with still rigorous standards. Perhaps it should be a lesson to all of us that regulation/innovation can be calibrated more effectively during “normal” times as industry races to develop new therapies for our world’s other pandemics — cancer, diabetes, heart diseases, and more.

The next step of the process — distribution of the vaccine — will be as challenging as the development phase, if not more so. But again, the pharmaceutical industry is rising to the occasion. Factories around the world are working in overdrive to produce hundreds of millions of vaccine doses.

Already, less than a month after the Pfizer vaccine was approved, more than 15.4 million doses of vaccine have been distributed across the country, and more than 4.6 million people have received their first dose, according to CDC data. Many patients are already receiving their second dose.

While 15.4 million doses are impressive, some expected 20 million doses. But even that is moving the goal line a bit, as six months ago many observers didn’t think we’d get a vaccine until 2021.

Members of our COVID Coalition told us that the holidays slowed the rollout considerably. Nancy Messonnier, M.D., a physician with the National Center for Immunization and Respiratory Diseases at the CDC, expects a rapid jump in administered vaccines during these first few days of 2021.

Every day, more people will be vaccinated. After health care workers and our most vulnerable citizens, other frontline workers will be next. Teachers will be vaccinated so our children can return to school. And soon, all Americans will be able to go to their doctor or walk into a CVS or Walgreens and receive the vaccine.

Remember, we did all this in nine months, with the help, dedication and expertise of our pharmaceutical industry heroes. Next time you turn on the TV and see negativity, turn it off and imagine instead where we will be nine months from now.

Mike Hennessy Sr is the founder and chairman of MJH Life Sciences.

The COVID-19 vaccine, and the speed at which it was developed, is the medical breakthrough of our lifetimes.

How Pharmaceutical Innovation is Saving the World

Kevin A. Schug is a Full Professor and Shimadzu Distinguished Professor of Analytical Chemistry in the Department of Chemistry & Biochemistry at The University of Texas (UT) at Arlington. He joined the faculty at UT Arlington in 2005 after completing a Ph.D. in Chemistry at Virginia Tech under the direction of Prof. Harold M. McNair and a post-doctoral fellowship at the University of Vienna under Prof. Wolfgang Lindner. Research in the Schug group spans fundamental and applied areas of separation science and mass spectrometry. Schug was named the LCGC Emerging Leader in Chromatography in 2009, and most recently has been named the 2012 American Chemical Society Division of Analytical Chemistry Young Investigator in Separation Science awardee.

 and the American Chemical Society Analytical Division Subdivision on Chromatography and Separations Chemistry.

In April 2020, on the heels of the pandemic shutdown, the price of crude oil fell to a negative value for the first time ever. The pandemic sent ripples through an oil and gas industry, which had more ebbed than waned in the United States over the past 10–15 years, primarily due to major successes in extracting oil and gas from low permeability unconventional shale resources, using technologies like hydraulic fracturing and horizontal drilling. But the 2020 shutdown hit many oil and gas companies hard. In some cases, major players in some shale plays have essentially withdrawn all of their development interests. Other companies have been left with insufficient means to service the debts they had accrued during the better times. Going forward, there is a generally positive sentiment about the importance of oil and gas extraction to our collective energy economy, but the downturn has created significant introspection across the industry.

While companies lick their wounds and decide their next moves, an important concept called environmental and social corporate governance (ESG) has come greater into focus. The hydrocarbon extraction and processing industry as a whole has certainly come under greater scrutiny for its potential environmental implications (especially, greenhouse gas emissions) in the past several years. The truth of the matter is that some companies are more progressive and responsible with their actions towards documenting, limiting, and mitigating deleterious environmental and social impacts than others. Even if the fine details about how it is regulated have not been fully formulated, ESG promises to involve some overall metric to rank the environmental and social responsibility efforts and actions of companies. Favorable metrics will be necessary to attract investment dollars in the industry going forward.

It is worth mentioning that, while ESG is discussed very prominently in the hydrocarbon processing industry (HPI), it is a concept that is permeating corporate America, as a whole. A closer look would likely find that virtually all major corporations now have some executive sustainability officer who is focused on reducing waste and improving environmental and social sustainability.

While our research has primarily been involved with assessing the potential environmental impacts of upstream oil and gas production from shale, inevitably we also became involved in the midstream logistics of handling the product, and more importantly the wastewater byproducts. In most shale plays, the volume of water byproduct (so called, produced water) far exceeds the volume of hydrocarbons extracted from an oil and gas well. We have worked with small and large companies to assess the effectiveness of different produced water treatment technologies—the goal being to recycle or reuse the treated water, rather than to simply dispose of it underground, where it has been shown to cause unwanted seismic activity. The success of these technologies will ultimately be driven by their cost, throughput, performance, and robustness.

Our analytical chemistry research has been largely driven by industrial support. We have had the opportunity to discuss the problems with industry leaders in upstream and midstream HPI, and to help develop and improve solutions, through our ability to speciate complex mixtures.

That said, going forward, downstream HPI is likely a bigger opportunity for industrial–academic partnerships. Downstream HPI deals with turning raw products into those that can used by the consumer, including fuels, plastics, and other feedstocks. A prominent way a company can improve their ESG is to reduce reliance on virgin feedstocks and instead, to recycle used products. For example, an abundance of used plastic products can be broken back down into their constituent monomers and used anew.

This concept screams for more development in chemical measurements and analysis. As researchers develop new catalysts, which can be used to selectively decompose plastics, those catalysts need to be characterized (for example, pore structure, porosity, chemical make-up). The next level of complexity comes from the wide range of different plastics desired to be processed. Plastics are essentially reduced into a range of pyrolysis oils. Imagine the varying heteroatom content present both in the plastic feedstock and the finished pyrolysis oil. Different heteroatoms can have particularly deleterious effects on catalyst performance. Depending on the desired use of the recycled feedstock, not only the heteroatom content, but also the olefin and aromatic content of the product oils are desired to be determined.

The downstream HPI market, especially that associated with plastics recycling and driven heavily by a desire for improved ESG, is expected to double in the next 30 years. In many cases, the standard chromatography–mass spectrometry research tools available from manufacturers will not have sufficient capabilities for adequate characterization of recycled hydrocarbon feedstocks. This is a major opportunity for academia to re-engage in the HPI, through the development of novel hardware and software solutions. Gas chromatography literally got its start in HPI.Instead, now think multi-dimensional separation and detection systems implemented for routine process monitoring. It is no small challenge, but it is one modern analytical chemists are ready to address. On the flip side, HPI companies are looking for an edge and will certainly have significant budgets associated with establishing themselves as a responsible leader in the industry through ESG. 

The time should be right for the developing of more industry–academic partnerships in HPI, and the driving force is to achieve overall greater sustainability. This feels like it could be a “win” all around for everyone and the environment.

Kevin A. Schug is a Full Professor and Shimadzu Distinguished Professor of Analytical Chemistry in the Department of Chemistry & Biochemistry at The University of Texas (UT) at Arlington. He joined the faculty at UT Arlington in 2005 after completing a Ph.D. in Chemistry at Virginia Tech under the direction of Prof. Harold M. McNair and a post-doctoral fellowship at the University of Vienna under Prof. Wolfgang Lindner. Research in the Schug group spans fundamental and applied areas of separation science and mass spectrometry. Schug was named the 

Emerging Leader in Chromatography in 2009 and the 2012 American Chemical Society Division of Analytical Chemistry Young Investigator in Separation Science. He is a fellow of both the U.T. Arlington and U.T. System-Wide Academies of Distinguished Teachers.

 and the American Chemical Society Analytical Division Subdivision on Chromatography and Separations Chemistry (ACS AD SCSC). The goals of the subdivision include

promoting chromatography and separations chemistry

organizing and sponsoring symposia on topics of interest to separations chemists

developing activities to promote the growth of separations science

increasing the professional status and the contacts between separations scientists

For more information about the subdivision, or to get involved, please visit 

https://acsanalytical.org/subdivisions/separations/

In April 2020, on the heels of the pandemic shutdown, the price of crude oil fell to a negative value for the first time ever. The shutdown hit many oil and gas companies hard. But while companies lick their wounds and decide their next moves, an important concept called environmental and social corporate governance (ESG) has come greater into focus.

The LCGC Blog: Environmental and Social Governance (ESG) in the Hydrocarbon Processing Industry (HPI) is an Opportunity for Industrial–Academic Partnerships

Agilent Technologies Inc. has honored Professor Anurag Rathore, a scientist at the Indian Institute of Technology (IIT) Delhi, with the Agilent Thought Leader Award, for his contributions to the field of biopharmaceutical research and his work with advanced methods for molecular characterization of biosimilars.

“I feel humbled to be chosen for an Agilent Thought Leader Award,” said Rathore. “As one of the many researchers at IIT Delhi working in the new COVID-19 normal, this is an encouraging and welcome event. The award will propel me to work harder as we strive to bring the technologies and products we have been working on to market.”

The award Rathore received includes new Agilent instrumentation equipment, including a liquid chromatography–QTOF-mass spectrometry (LC-QTOF-MS) instrument, an automated liquid handling platform, and a two-dimensional LC (2D-LC) system. Rathore also received a grant that will be used to promote academic-based research on critical areas that are impacting the biopharmaceutical industry.

Agilent is invested in developing workflows to streamline the analysis of critical charge and glycosylation heterogeneity of monoclonal antibodies. Rathore’s laboratory is focusing on the best practices toward molecular characterization of monoclonal antibody–based biosimilars. The goal of Rathore’s laboratory work is to create best practices that facilitate commercialization of biotech drugs.

Rathore, in addition to his work at IIT, also serves as a regular columnist for 

, where he is the editor of the “Focus on Biopharmaceutical Analysis” column.

The Agilent Thought Leader Award program promotes fundamental scientific advances by contributing financial support, products, and expertise to the research of influential thought leaders in the life sciences, diagnostics, and chemical analysis space. Further information is available on the Agilent Thought Leader Award 

Professor Anurag Rathore, a scientist at the Indian Institute of Technology (IIT) in Delhi, is the first researcher based in India to receive this award.

LCGC Columnist Anurag Rathore Receives Agilent Thought Leader Award

Articles for this special issue should address issues in food and beverage analysis using chromatographic or mass spectrometry techniques, or a combination of those. Suggested topics include food and beverage safety, pesticide residue analysis, analysis of particular nutrients, flavor analysis, food and beverage quality, authentication of origin, speciation analysis of metal contaminants, or other relevant topics.

Submissions from equipment manufacturers will be considered but must be devoid of promotional content. Discussions of new technology should not focus on a specific manufacturer’s instrument.

Manuscripts should be approximately 2500­­–3500 words long, plus up to four figures and tables combined. Manuscripts should follow a standard experimental article format, including an abstract of 150–200 words. Figures and tables, along with their captions, should appear at the end of the manuscript, and figures also must be sent as high-resolution separate files. References should be called out using numbers in parentheses and listed at the end of the manuscript in numerical order.

Due date for abstract submission: January 18, 2021

Due date for completed articles: February 25, 2021

Send all proposals and completed articles to Editor Laura Bush, at 

 has been the gold standard relied upon by chromatographers for unbiased, nuts-and-bolts technical information with a practical focus. 

’s columns and peer-reviewed articles continue to bring readers practical technical advice from respected experts in liquid and gas chromatography, including hyphenated techniques; capillary electrophoresis; supercritical fluid chromatography; and more. 

 is indexed in Science Citation Index; Web of Science: Science Citation Index Expanded; EBSCOhost; Scopus; and Journal Citation Reports.

LCGC is seeking contributed manuscripts for an April 2021 supplement on food and beverage analysis. 

Call for Papers: April 2021 Supplement on Food and Beverage Analysis

Announces the 2021 Winners of the Lifetime Achievement and Emerging Leader in Chromatography Awards

, the leading resource for separation scientists, is proud to announce that Paul Haddad and Erik L. Regalado are the winners of the 14th annual 

Lifetime Achievement and Emerging Leader in Chromatography Awards, respectively. Haddad and Regalado will be honored in a symposium as part of the technical program at the Pittcon 2021 virtual conference in March 2021.

The Lifetime Achievement in Chromatography Award honors an outstanding professional for a lifetime of contributions to the advancement of chromatographic techniques and applications.

Haddad, the 2021 winner, is an Emeritus Distinguished Professor at the University of Tasmania, Australia. He is best known for his research on the theory, mechanism, and applications of ion chromatography, and computerized approaches to method development.

His work has focused on analytical separation science in liquid phases, with particular emphasis on the separation and quantification of ionic species. A large body of his research has centered on the separation mechanisms and methods of detection of high performance liquid chromatography (HPLC), ion chromatography (IC), capillary electrophoresis (CE), and capillary electrochromatography (CEC), with a view to improving fundamental understanding and to apply this understanding to the development of new chromatographic and electrophoretic methods. This work has involved making detailed measurements of the retention or migration of analytes in the desired system to derive mathematical models that describe these observations, and to use the mathematical models to devise strategies for computer-assisted optimization of separations for particular applications. Other major themes of his work include the investigation and manipulation of separation selectivity, studying potentiometric detection using reactive metallic electrodes, and the separation of complexed metal ions and sample handling methods.

Haddad served as the foundational director of the Australian Centre for Research on Separation Science (ACROSS), which he established in 2001. ACROSS is recognized worldwide as a leading center of research excellence in separation science. Over the past 19 years the center has been responsible for the publication of more than 1150 refereed papers, graduating 120 PhD students, and presenting more than 1600 oral papers or posters at international scientific meetings. In 2006 he established the Pfizer Analytical Research Centre (PARC), a multimillion dollar research center funded by Pfizer.

Haddad is the author of 585 publications, encompassing 13 books, monographs, or special issues of journals, 33 book chapters, 533 journal articles, and 8 patents, for a total of >14,000 literature citations and an 

index of 59 (including books). He is the author of the acclaimed reference text, 

“Ion Chromatography: Principles and Applications,”

 which has been cited more than 700 times. He has given more than 800 talks, including 140 plenary and keynote lectures.

. He is a current member of the editorial boards of 

Haddad was named a University of Tasmania Distinguished Professor and an Australian Research Council Federation Fellow. He was also named a fellow of the Australian Academy of Science, the Australian Academy of Technological Sciences and Engineering, the Royal Australian Chemical Institute, the Royal Society of Chemistry, and the Federation of Asian Chemical Societies.

Key international awards he has received include the American Chemical Society (ACS) Award in Chromatography in 2013, the Marcel Golay Award in 2011, the CASSS Award in 2018, the Desty Memorial Lectureship in 2011, the AJP Martin Gold Medal of the Chromatographic Society in 2002, and the Royal Society of Chemistry Analytical Separation Methods Award and Medal in 2003. He was the first Australian to receive each of these international awards. Key national awards include an Australian Museum Eureka Prize in 2019, the Royal Australian Chemical Institute (RACI) Applied Research Medal in 2010; the RACI H.G. Smith Medal in 2007, the RACI Leighton Memorial Medal in 2020, the Federation of Asian Chemical Societies (FACS) Foundation Lectureship Award in 2001, and the Royal Society of Chemistry Australasian Lectureship Award in 2001.

The Emerging Leader in Chromatography Award recognizes the achievements and aspirations of a talented young separation scientist who has made strides early in his or her career toward the advancement of chromatographic techniques and applications.

Regalado, the 2021 winner, received his PhD in chemistry in 2011 at the University of Havana, in Cuba, in collaboration with the University of Nice Sophia Antipolis in France. He is currently a Principal Scientist in the Analytical Research & Development department of Merck Research Laboratories, where he leads the Method Screening and Purifications group. His research focuses on analytical and preparative enabling technologies that accelerate development of new pharmaceuticals, including automated method screening, multidimensional chromatography, high-throughput analysis, and ultrafast and computer-assisted separations.

Regalado recently introduced the concept of automated multicolumn two-dimensional liquid chromatography (2D-LC) screening for easy selection of stationary and mobile-phase conditions in both dimensions. He has also introduced the concept of chaotropic effects in sub- and supercritical fluid chromatography (SFC). This new strategy has enabled Regalado’s group at Merck to deliver highly pure cyclic peptides at the kilogram scale, something unprecedented in purification laboratories. In addition, the concept has extended the reach of SFC to molecules that were previously considered out of the scope of the technique. Other major themes of his work include advanced reaction monitoring of pharmaceutical processes and the deployment of new approaches combining SFC with photodiode array detection and mass spectrometry as a framework for impurity fate and purge studies.

Regalado also has become a leader among both academic and industry researchers in the development of universal chromatographic methods for use in the research and development of new drug substances—an approach that is transforming analysis in many pharmaceutical laboratories. He has also been involved in advancing the development of ultrafast chiral chromatography, as well as pioneering its introduction to high-throughput enantiopurity analysis and comprehensive 2D-LC workflows. Very recently, Regalado’s team introduced multifactorial peak crossover (MPC) in analytical and preparative chromatography, a new software-based technique that enables convenient switching of the elution order of target analytes to gain large chromatographic productivity increases for preparative purifications, as well as substantial improvements of the signal-to-noise ratio for trace analysis.

In total, he has 90 publications and 2 patents, for a total of 2260 literature citations and an 

index of 28. He has given more than 50 oral and poster presentations at scientific conferences.

Regalado has received numerous awards, including, among others, the ACS Young Investigator Award; the Merck Green & Sustainable Science Symposium Award; a Merck Research Laboratories Innovation Award; the postdoctoral HPLC Travel Grant; a MRL Postdoc Publication Award; the Cuban National Academy of Sciences Award (twice); Cuba’s Environmental Agency Award (twice); the European Molecular Biology Organization (EMBO) Fellowship; the Phytochemical Society of Europe Award; the University of Havana Award; two Boehringer Ingelheim Funds Fellowships; a United Nations Educational, Scientific and Cultural Organization (UNESCO) Research Grant; and a UNESCO Fellowship.

 or for information on how to nominate a candidate for the 

LCGC Announces the 2021 Winners of the Lifetime Achievement and Emerging Leader in Chromatography Awards

Selected highlights of innovative chromatography products.

Instrumental Innovations 2020

Q. What trends do you see emerging in sample preparation?

 Sample preparation should be a simple and transferable stage of the analytical process. Simplicity of sample preparation based on cost-effective materials should be the driving force behind research in sample preparation. This would also help to increase the interest in the university research sector from the production sector.

 From the perspective of a product manager for automated sample preparation, I see that a number of established, effective, and rugged sample preparation techniques are still performed manually by many laboratories worldwide—even though they could be automated with commercially available instruments. Automation is definitively still on the rise, contract laboratories and the chemical industry want to automate as many manual workflows as reasonably possible and this applies to all types of chemical and biochemical analysis, not just LC– and GC–MS applications. The best example are the large pipetting robots that handle our Corona PCR tests and facilitate such incredible throughput, helping us to get through the current pandemic. This means that instrument manufacturers need to invest more in the development of innovative hardware and especially software solutions. I see the trend of coupling diverse instruments from different manufacturers under one communication protocol. This could give the analytical world a boost similar to what we experienced during the “golden years” after GC–MS and LC–MS had first emerged.

Coupling or hyphenating instruments requires new soft- and hardware interfaces. Establishing generic communication/interface standards such as the standardization in laboratory automation (SiLA 2) or open platform communications unified architecture (OPC UA) and the derived Laboratory Agnostic Device Standard (LADS) could be a valuable first step. Our efforts should result in the availability of a toolbox of techniques that “just” need to be connected to gain significant new benefits for the user. Mobile robots delivering samples from the refrigerator to a pipetting robot and finally to an analysis system are not just a dream any more. The future of analytical chemistry will increasingly be the domain of software/firmware programmers—like most areas in our future life. Accordingly, laboratory staff may need a higher level of software training.

Sample preparation in the proteomics field is one of the most critical and under-focused areas as a result of the meticulous research that is required before upgrading conventional procedures. While the research may be time-consuming, the impact to the results are substantial, such as speed, reproducibility, sustainability, and cost-effectiveness. Some examples of these advances are trypsin spin columns for quick peptide mapping, rapid glycan analysis tools for digestions, labelling, and clean‑up, and specialized spin cartridges/columns for complex post-translation modification analysis. As the goal of sample preparation is to balance time savings and the integrity of results, developing high-throughput workflows while minimizing protein modification helps to achieve this.

 I think sample preparation is going “micro”. Microextraction has been around for 30 years, so it is definitely not new. A lot of microextraction is published in the scientific journals, but implementation into routine laboratories is slow. I also think the next generation of analytical chemists will be much more concerned about the use of hazardous chemicals, and they will prefer microextraction for environmental reasons. This generation are also born with smartphones in their hands, and will use them as much as they can for chemical measurements. Of course, this requires sample preparation, not with separation funnels, but with microextraction integrated into devices compatible with smartphones!

Q. In your opinion, what is the future of sample preparation?

 We should emphasize that although sample preparation is very often identified as a bottleneck, sometimes it is an unavoidable step. Bearing this in mind, reduced sample manipulation, either by automation or the design of selective extractant phases that could isolate the target analyte or family of analytes is essential. Additionally, the integration of these extraction units with the instrumental techniques, and specifically high-resolution mass spectrometry is a clear trend in sample preparation.

Generally, I see a bright future for sample preparation. Questions of cost for each sample, miniaturization, and sustainability are gaining importance. In my opinion, microscale sample preparation techniques that can be combined and automated will have a promising future.

A good example is the analysis of microplastics in different environmental samples. Currently, the analysis is performed predominantly manually using spectroscopic methods or even counting particles under a microscope after sample pretreatment. These approaches are extremely tedious and result in a throughput of only a couple of samples a day without proper mass conversion factor for calculating concentrations. The combination of pyrolizing a complex soil or filter sample in a thermogravimetric oven, collecting the evolved breakdown products, and analyzing them for characteristic compounds by thermal desorption GC–MS offers a quick screening tool (TED-GC–MS) for microplastics in complex samples.

Moreover, microsampling and analysis techniques for blood are gaining importance. In this context dried blood spot (DBS) sampling and volumetric absorptive microsampling (VAMS) play an important role because they enable safe and simple self-sampling in any location on the world. This translates to more freqent analysis of important blood parameters and ultimately better patient care, a more effective fight against doping and drug abuse, and more meaningful pharmaceutical study results, respectively. One current example is the detection of SARS-CoV-2 IgG antibodies by standard enzyme-linked immunosorbent assay (ELISA) tests using dried blood spots sampled by the patients themselves at home.

 Currently, with the technology that exists on the market, proteomic scientists can easily analyze protein modifications with moderate time and minimal errors. In the future, with improvements to the current workflow, it would make it possible to save even more time, including resources and human errors, while maximizing automation to increase output. Innovations in sample preparation automation equipment will lead to increases in productivity so that all laboratories can process samples in a fast-paced laboratory environment for quicker and more reliable results.

 As I mentioned before, future sample preparation will be based on microextraction, tailor-made for smartphones and other hand-held devices. Microextraction will also be implemented in biomedical research to assist soft extraction. In this way we can do extractions in living biological systems, without disturbing biochemical equilibria.

Q. What is the LC or LC–MS application area that you see growing the fastest?

 Untargeted analysis is one of the fastest growing application areas in any chromatographic mode (either LC or GC) when coupled with high-resolution mass spectrometric detectors. The non-directed analysis makes the technique more versatile and capable of being adapted to the analysis of samples where the analytes should not be specifically limited to a previous selection made by the analyst.

 In my view the broad field of “omics-applications”, such as proteomics, lipidomics, metabolomics, and so on, drives the application of LC–MS the fastest. The hope is that these applications will lead to better diagnostic tools, more effective cures for diseases, and better nutrition for the world’s population, three major issues for mankind today.

 In biological therapeutics, the majority of drug development occurs in relation to different types of immunoglobulin G (IgG). The research revolves around identifying modifications and subclasses of IgG such as charged species and oligomers or fragments. One of the latest trends for LC is to analyze both of these parameters using 2D-LC to facilitate and take advantage of the two different modes. For LC–MS, the native MS workflow is growing in use because it produces data without altering the protein properties. Native MS also offers fewer intermediate steps between LC and MS or MS/MS, providing better results with no additional time.

Q. What one recent development in the area of sample preparation would you say is the most important?

 It is not easy to choose one, but in my field of expertise, I would say that the use of nanostructured materials and new extractant phases such as supramolecular solvents (SUPRAS) or metal‑organic frameworks (MOFs) have clearly contributed to increase the efficiency of sample preparation and to reduce the sample volume/chemicals, making sample preparation more sustainable.

 In my view there has been no single ground-breaking new development. Nevertheless, combining different existing techniques will catalyze significant advances in the area of sample preparation and analysis overall. Reinventing the wheel for individual techniques is not as likely to make a difference as just combining established techniques.

One of the most important recent developments in sample preparation is rapid glycan clean-up. Traditionally this workflow would take over two days of preparation and subsequent analysis. In order to get the results, the protocol required a daily glycan digestion followed by a tedious protein removal, 4-hour-long fluorophore tagging, and eventually led to clean-up by solid phase extraction prior to the LC–fluorescence detection (FLR) or LC–MS analysis. With improvements to this method, the workflow has been optimized to only a 30 min sample preparation procedure while still leading to accurate results.

 From my perspective, which is from a pharmaceutical and biomedical angle, I think recent research on in-vivo SPME and LPME/EME are very important, but I am probably a little biased.

Q. What obstacles do you think stand in the way of sample preparation development?

 One issue that needs to be solved is the difficulty associated with the validation of analytical processes that implement new tools at the sample preparation level. The time required to modify official or reference methods always makes the advances proposed at the R&D step obsolete, even if eventually they are approved.

 Analytical chemistry and sample preparation in particular only get limited attention in chemical research. This is illustrated by the relatively small number of professorships for analytical chemistry and the low impact factors of analytical chemistry journals. I think this is one key factor that slows down new developments.

Manufacturers of sample preparation tools and systems have invented numerous new techniques over the past years. Nevertheless, many users take a lot of convincing and stick to tried and trusted products and concepts, which in many cases are methods that have been validated and proven over years.

From the manufacturers’ point of view, regulations also slow down innovation. This is especially seen in the field of clinical diagnostics. Instruments and tests need to be developed and manufactured according to in-vitro diagnostic (IVD) legislation. After having passed such a complex and costly process successfully, the manufacturer of an in-vitro diagnostic test has no interest in changing anything in the workflow or instrumentation because this would result in the need for a recertification. The outcome is most clearly seen in clinical laboratories worldwide where LC–MS techniques have arrived with a delay of 10–15 years compared to other application areas, such as food safety analysis. The in-vitro diagnostic legislation is a barrier against using calibration-based techniques such as LC–MS and GC–MS, which are solely calibrated by the end user and not by the instrument manufacturer. Assuming that the instrument and the method are generally ”fit for purpose”, the quality of results is much more dependent on the quality of the work performed in the laboratory than on the instrument being manufactured according to IVD guidelines. Case in point: Personally, I would rather have my hormone levels determined by a validated non-IVD LC–MS test in an accredited clinical laboratory than by an IVD immunoassay in the same laboratory. Whatever the IVD regulations require, in my view a mass spectrum says more than an immunoassay. In my perspective, IVD legislation is the main impediment to the implementation of state‑of-the-art techniques in the clinical laboratory and also slows down the development of innovative techniques, especially in small, innovative companies.

 What we commonly see as a hurdle for sample preparation is not only a knowledge barrier around complete solutions but also having to work with analytes that are constantly changing, such as biologics in drugs development that are diversified in advanced segments of discovery biology. It is difficult to have a deep and extensive understanding for all of the biological workflows for diverse analytes that also have different clean-up and analysis requirements.

 Analytical methods used in routine analysis are validated, and this is a big advantage in the way that we then know the data are accurate. Validation takes time and costs money, and therefore routine laboratories prefer to use their existing methods, including the sample preparation methods. Although a lot of new sample preparation methods are published in the scientific literature, they are implemented into routine analysis to a very small extent.

Q. What was the biggest accomplishment or news in 2019/2020 for sample preparation?

 After several years of evaluation of novel extractant phases and (micro)extraction formats, we have been able to catch the interest of the scientific community with sample preparation. This can be visualized through the consolidation of dedicated international conferences on this topic. Also, the proposal of thematic networks that join scientists working on sample preparation for sharing knowledge and providing visibility to the topic. The EuChemS Sample Preparation Task Force and the Spanish Network on Innovation in Miniaturized Sample Treatment exemplify this.

 Analytical chemists are increasingly focused on the sustainability of their laboratory operations, their techniques, and their methods. Guidelines for rating analysis methods with regard to environmental friendliness and sustainability have already been established over the past couple of years. Becoming aware of this was my personal highlight of the last year. For example, I listened to a presentation from a member of the “Green Analytical Project”. Analytical chemists have started to feel responsible for the future in terms of reducing the environmental impact of their activities, partly driven by young activists that make their voices heard all over the globe. That has impressed me and I propose that we, as analytical chemists, should strive for greener and more sustainable analysis techniques. For example, the liquid–liquid extraction (LLE) with 50 mL of toxic dichloromethane is still widely used, but completely outdated and often unnecessary. We as a community should fix that, as a helping hand to future generations.

From analytical to large molecule sample preparation, some of the biggest accomplishments have come from coupling multi-functional sample preparation products with automation. The advantages of using multi-well products that can clean-up, extract, concentrate, and remove matrix effects in parallel, while also using semi-automation or automation to speed up the method is a big achievement. Even using something as simple as a high‑throughput magnetic separation device for immunocapture using magnetic beads has massive implications for improving on traditional ELISA methods.

It is very difficult to select a single accomplishment. I think the accumulated sample preparation efforts are the biggest accomplishment, to establish this as a scientific discipline and attract the attention of analytical chemists.

 is a Professor of Analytical Chemistry at the University of Córdoba, Spain.

 is Product Manager, Automated Sample Preparation at Gerstel GmbH & Co. KG.

 is the Global Product Manager of Biologics-Sample Preparation at Phenomenex.

 is a Professor of Pharmaceutical Analytical Chemistry at the University of Oslo, Norway.

A panel discussion on the latest advances and future developments in sample preparation.

Trends and Developments in Sample Preparation

I’ve recently been involved in troubleshooting some issues with a Pharmacopeial method which have highlighted some important general issues in the practical implementation of high performance liquid chromatography (HPLC) and ultrahigh-pressure liquid chromatography (UHPLC) methods.

The method is used to determine Related Substances for Hydroxychloroquine and is briefly summarized below.

 Dilute sulphuric acid (approximately pH 2.8) : Methanol 50:50

Water containing potassium dihydrogen phosphate, sodium heptane sulphonate adjusted to pH 7.0 with triethylamine

 25 mg of test substance diluted to 50 mL with sample diluent

 Test Solution 1 diluted 1:10 (v/v) with sample diluent

 Various dilutions of Hydroxychloroquine sulphate with known impurities B and C

 End-capped ethylene-bridged octadecylsilyl silica gel for chromatography (hybrid material) R (1.7 μm)

 4 µL of test solution (1) and reference solutions.

For reference, Figure 1 shows the structures and some physicochemical information for hydroxychloroquine and impurities B and C for which limits are specifically set within the method.

It should be noted that in total there are seven named impurities for this related substance determination, but only those which have individual limits are shown for illustrative purposes.

The issues highlighted by my correspondent included:

Non-reproducible quantitative response (peak areas) for specific analytes

Carry-over of the active pharmaceutical ingredient and inability to obtain a “blank” chromatogram despite flushing the system with various reagents

Noisy and erratic baselines, especially after filtration of the mobile phase.

You may be asking yourself why this very specific set of problems with a very particular Pharmacopeial method warrant Incognito column inches. Simply put, this method, and the problems which have been encountered, highlight some very useful considerations when undertaking liquid chromatographic analysis and the skill of “critical evaluation” of methods, that is, the evaluation of a method to help understand the possible and actual problems encountered. So, whether you work in the pharmaceutical or any other industry, the following guidance should be generally applicable.

Let’s start by looking at the mobile phase and its constituents.

For me, with over 30 years experience in practising chromatography, the constituents of the mobile phase are recognisable but certainly not what one could call “modern”. However, for some less experienced chromatographers, the mobile phase additives, and the fact that the pH is titrated to a set value using one of the reagents will be, frankly, alien.

Figure 2 shows the structures of the additives used in the mobile phase and it’s worth spending a little time on why these reagents may be used.

Potassium dihydrogen phosphate is a traditional buffer which is used to resist small changes in pH when the sample in its diluent (approximately pH 2.8) mixes with the eluent within the instrument tubing and primarily at the head of the analytical column. As we wish to maintain a pH of 7.0 to enable reproducible separation, and because the pKa of the phosphate buffer is 7.21, this obeys the general rule that the buffer will be most effective when the desired solution pH is within 1 pH unit of its native pKa. This should lead to an improvement in the robustness of retention time and peak shape. We will further discuss this particular reagent later.

Sodium heptane sulphonate is a so‑called ion pairing reagent, which has two modes of action. In the bulk mobile phase, the anionic reagent will form an ionic equilibrium with basic analyte functional moieties to form a neutral complex, which will be more highly retained on the highly hydrophobic stationary phase surface. As can be seen from the analyte pKa values in Figure 1, at an eluent pH of 7.0, several of the analytes will have protonated functional groups and therefore will be capable of ion pair formation. The second mode of action of the ion pair reagent is to strongly partition into the stationary phase via the highly hydrophobic moiety of the pairing reagent which will effectively add anionic groups (SO3-) to the stationary phase surface, with which any unpaired cationic analyte species may undergo electrostatic attraction, and therefore improve retention.

Apart from its use to obtain the correct mobile phase pH, triethylamine is used as a silanol group masking reagent. Figure 3 can give us further insight into the mechanism and requirement for this reagent.

Species 5 in Figure 3 represents an acidic silanol group which has not been effectively treated with an end capping reagent (such as that shown in species 8) which will be anionic under the mobile phase conditions of our method. If left untreated, these silanol anions will form electrostatic attractions with cationic analyte molecules and unwanted secondary interactions can lead to issues with peak broadening and tailing as well as extended column equilibration times and possible issues with peak area reproducibility. Therefore, ionogenic masking reagents such as triethylamine were traditionally added to mobile phases to effectively “end-cap” anionic surface sites, as the triethylamine, which is fully protonated below pH 7.2, is electrostatically attracted to the ionised surface silanol residues.

So, given the analytes of interest and the eluent pH, this mobile phase system would seem to be very well designed to overcome any issues with analyte reproducibility of analyte retention and peak shape. So far so good.

Let’s now look at the column being used.

The so-called bridged ethylene hydride columns were introduced by Waters in around 2004 in order to fulfil several requirements, including increased ruggedness under UHPLC conditions and increased resistance to extremes of eluent pH (especially high pH), using organic, and inorganic hybrid substrates.

However, we must go back even further to highlight some of the issues with the method under consideration. The lone (acidic) silanol groups which are masked using the triethylamine additive, were particularly prevalent when manufacturers used Type A silica as stationary phase substrate materials. This silica contained metal ions which are known to “activate” surface silanol groups towards interaction with basic analytes, and was not treated to avoid the presence of lone silanol groups. The later development of Type B silica effectively reduced silanol activity by removing target metal ions from the silica matrix and used surface treatments to reduce the number of lone silanols and promote the amount of inter-hydrated or vicinal silanol groups (species 2 in Figure 3). These developments were very effective at reducing the interactions between basic analytes and surface silanol species, effectively negating the requirement for masking reagents such as triethylamine. The bridged ethylene phases (species 1 in Figure 3) are known to further reduce the number of inherent silanol species capable of interaction with basic analytes in their ionised state.

Given the fact that, due to these new stationary phase materials, the use of silanol masking reagents have not been required for over 20 years now, one must therefore question why triethylamine is included in this method? Further, triethylamine is relatively volatile, and will be lost from the mobile phase to the atmosphere on standing, which may give us a clue as to the reason behind the drift in analyte retention over time as the inherent pH of the mobile phase will change as the triethylamine concentration lowers. In my own experience, it is also possible for the triethylamine to “settle” within the eluent, again leading to drifting or changing analyte retention. Further problems with this reagent also exist. As it inherently alters the stationary phase surface, one has to be mindful when equilibrating the HPLC column as extended flushing of the phase is often necessary to obtain reproducible retention times for the first injections of a sequence. One also must question the possible interaction between the anionic ion pairing reagent and the triethylamine cation. The possibility of this interaction, forming a neutral complex, may render the masking reagent ineffective and one needs to wonder what robustness issues this may create.

Let’s now consider the role of the ion pairing reagent in this separation. Modern stationary phases such as those used in this method, are capable of withstanding high pH mobile phase environments and as such we must also call into question the use of this type of buffer in the method. Given the pKa values of the analytes shown and their logP(D) values, it is conceivable that, by adjusting the mobile phase pH to pH 10.5 or 11, the degree of ionisation of some of the analyte basic moieties will be reduced and therefore retention may be gained, even using the more hydrophobic C18 stationary phase. Further, there are many stationary phases available which contain more polar functional chemistry or are designed to work with very little or no organic modifier within the mobile phase at the beginning of the mobile phase gradient. The use of ion paring reagents such as sodium heptane sulphonate has not been necessary for many years, since the introduction of higher quality silica, combined with more polar (so-called Polar Embedded) and more pH-resistant stationary phases can be used to gain sufficient reversed-phase retention, even for more polar analytes. Less experienced or younger chromatographers may be puzzled by the use of this reagent under these circumstances.

Lastly, let’s consider the use of the buffer reagent with the current column and method conditions. The column contains 1.7 μm particles which are designed for use with UHPLC systems, given the higher inherent back‑pressure created when using such small diameter particles. Intrinsically, these systems have very small internal volumes and very narrow internal diameter tubing and connectors, to reduce the effects of extra column (bed) band broadening on the separation. The use of a solid, involatile buffer with UHPLC systems is not recommended as any precipitation of the buffer through the use of highly organic eluents or via evaporation of the mobile phase on standing, will lead to significant problems with system blockages and over pressure situations. Further, the column end frits used with UHPLC columns containing 1.7 μm particles typically have a porosity of around 0.2 μm which renders them even more susceptible to blockages with solid particles. It is possible to filter the eluent solution to remove any particulate materials initially present in the eluent system, however one must again be very mindful that the triethylamine additive is relatively volatile and therefore this may be partially lost if filtration is achieved using vacuum apparatus. The target eluent pH of 7.0 is also relatively problematic, given that the pKa of at least one of the ionogenic analyte functional groups in each of the analytes is close to 7.0, we might expect a relatively large change in the ionic character of the analyte (and therefore the analyte LogD value and its polarity), unless the pH of the mobile phase is very accurately controlled each time it is made. Anyone studying robustness in undergraduate chromatography studies will be warned to stay away from the analyte pKa values to optimize the method reproducibility, due to the potential for large variations in analyte retention with relatively small changes in eluent pH. At the very least, it would surely have been better to quote a volume or weight of triethylamine required to achieve the desired pH, which could be controlled much more reproducibly.

From this brief review it would very much appear that the current method has been “stitched together” using a very traditional mobile phase with a modern stationary phase which should not require any of the now “exotic” mobile phase additives in order to achieve a suitable separation. Without the requisite experience in critical evaluation of chromatography methods, this combination may be baffling and the resulting problems very difficult to interpret or overcome.

However, this is not the end of the story. There are other idiosyncrasies of the method which require some further thought, and which highlight some more general principles in chromatography.

On studying the solubility of hydroxychloroquine and impurities B and C, I can see no evidence of limited solubility in aqueous media, although at this point, I should declare that I’ve not undertaken any further study on the solubility of the remaining five impurities. It seems strange that the pH of the sample diluent solution is adjusted to approximately 2.8, presumably to improve the aqueous solubility by protonating the analyte molecules, but then to dissolve in 50:50 aqueous organic seems very strange. Given that the mobile phase starting composition is just 10% organic, the peak shape of the analytes will be poorer than it needs to be as the diluent composition will give a more highly eluting local environment as the sample plug enters the chromatographic bed, typically leading to peak tailing and/or splitting. There may be explainable mitigation in the solubility of the other impurities, however the sample diluent composition does seem rather counter intuitive.

The issues of equilibration have not been highlighted in the method details and, in modern times, this could be a cause of much consternation with practical application of this method. As explained above, the initial equilibration of a new column will be much longer than perhaps is usual, as there are at least two reagents which need to interact with the stationary phase to a steady state. Granted, the internal volume of the column may be relatively low (approximately 120 μL), it may take many tens if not hundreds of column volumes of mobile phase to properly equilibrate this column. The requirement for extended re-equilibration between sample injections may also be extended (see next paragraph). Further, as we are extensively modifying the stationary phase surface with the luent additives, it is good practice to dedicate the column to use only with this specific mobile phase, to avoid the requirement for extended column flushing and re‑equilibration.

It should be further noted that the detection is at a fixed wavelength of 2254 nm and this can be problematical when the ionic strength of the mobile phase changes to such a large extent during the analysis, as is the case here, where the aqueous component ranges from 90% to 15% during the course of the analysis. Without the judicious set‑up of the detector, for example by employing a reference wavelength, such changes in ionic strength might well give rise to retention time shifts, peak shape changes and unstable baselines, unless sufficient time is given to allow re-equilibration of the column between sample injections. It may be far more judicious to use a ternary system in which the ionic strength is maintained using a third eluent system which contains the buffer and additives at a higher concentration (that is, all reagents at 10× concentration and added at a constant 10% of mobile phase composition during the analysis).

I haven’t begun to address the carry‑over issues mentioned earlier, as these may well be system- rather than method-related.

In summary, this particular method appears to be sub-optimal and could be very difficult to implement under practical circumstances by less experienced users. I should also say that I’m aware that there is a new hydroxychloroquine related substances method in draft which is due for introduction in June of 2021.

Incognito leads us through the complex method used to determine related substances for hydroxychloroquine, exploring the practical implementation and critical evaluation of a less than optimum ultrahigh-pressure liquid chromatography (UHPLC) pharmacopeial method.

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