Advances in HPLC

Analytically Speaking Ep.4

Demystifying Sample Preparation for Headspace Analysis Using Direct Injection Mass Spectrometry

Ester Formation in Acidified Mobile-Phase Solvents

Chemistry in a Bottle

A Hydrophilic Strong Anion-Exchange Hybrid Monolith for Capillary Liquid Chromatography

Trends and Developments in Sample Preparation

Agilent Technologies (Santa Clara, California) is launching a partnership with Delaware State University (DSU) (Dover, Delaware), a prominent historically Black university, to increase the share of underrepresented students entering science, technology, engineering, and math (STEM) fields. This goal is a fundamental mission of the company’s philanthropic work through the Agilent Foundation.

Agilent will donate $1 million along with new laboratory instrumentation to help DSU expand educational opportunities and advance research in applied chemistry, biological sciences, food science, molecular, and cellular neuroscience, and related disciplines. The company’s support will also contribute to building research capacity of a consortium of historically Black colleges and universities (HBCUs) in the mid-Atlantic region, led by Delaware State.

“DSU has a tremendous pool of talented STEM students,” Agilent President and CEO Mike McMullen said in a statement. “This partnership will help us provide direct support to these students and encourage more scholars at HBCUs across the mid-Atlantic to consider opportunities at Agilent and within the broader life sciences sector.”

Agilent’s donation arrives as DSU breaks ground on a new 24,000-square-foot building for its College of Agriculture Science and Technology (CAST), which includes space for new laboratories.

“Many believe that this is a renaissance moment for HBCUs. It’s not,” DSU President Dr. Tony Allen said in a statement, “We’ve been doing the work for 175 years—building the most useful pipeline for African Americans to the American middle class and breaking new ground in every field of human endeavor.” He praised Agilent for understanding the work being done and connecting with DSU’s system, which he said produces 25% of all the Black STEM graduates in the United States. 

Agilent’s donation will fully fund 21 graduate and undergraduate students pursuing STEM degrees. The company is also providing analytical laboratory instrumentation, supplies, services, and other infrastructure to equip CAST’s new laboratory space. Additionally, Agilent will provide mentorship opportunities for DSU students with Agilent scientists, engineers, and researchers and will help fund DSU’s summer internship program for STEM students.

Articles

Agilent Technologies (Santa Clara, California) is launching a partnership with Delaware State University (DSU) (Dover, Delaware), a prominent historically Black university, to increase the share of underrepresented students entering science, technology, engineering, and math (STEM) fields.

Partnership between Agilent and Delaware State University Promotes Diversity in Science, Technology, Engineering, and Math (STEM) Fields

Do you remember your first time? Perhaps it was a tie-dye party. Maybe you experienced marker pigments dividing across a paper towel dipped in liquid. Was it a few spots crawling up a plate in organic chemistry class? Possibly it was pushing the plunger on a syringe and seeing what was once invisible turn into beautiful, Gaussian artistry walking across a chart recorder or computer monitor. Whenever it was, if you’re reading this journal, then you’ve most likely applied chromatography and remember the first separation that sparked your love for the field. That magical moment you felt was more than a waxing poetic memory. In fact, it was probably the time you were most apt to learn chromatography…or… apt to learn anything!

I recently read a wonderful book called “Beginners:The Joy and Transformative Power of Lifelong Learning” by Tom Vanderbilt (2021 Penguin Random House). At face value, it has absolutely nothing to do with chromatography. The author went on what some may view as a self-indulgent experiment to learn a variety of new skills as a middle-aged adult. How in the world can a book about some journalist trying to learn to surf, play chess, juggle, and a few other adventures have to do with becoming a better separation scientist? Interestingly, throughout his journey, the author made frequent stops with top researchers in the field of learning to understand what was changing within himself as each skill was attempted. What was most fascinating was that regardless of the task, the pattern was similar and borne out by the science. We learn best when we approach something new. What’s more, if we can revert to a childlike innocence, our ability to gain competency (not to be confused from mastery) is rapidly accelerated. Our steepest learning curves come when we deviate from skills we dismiss as already acquired. For example, learning to sing to a level for public performance or drawing beyond doodles are so hampered by already-ingrained mindsets that progressing those skills is more difficult than learning something entirely new. Consider how much harder it is to learn a second language in adulthood, when your brain has already been taught how to converse, 

 as a child that has never had to diagram an English sentence.

Along his journey, Vanderbilt visits the Infant Action Center at New York University’s Center for Neural Science. When learning about learning, it makes sense to watch babies as they grasp the basics. As any parent knows, infants figuring out how to walk tend to fall. A lot. For months. In fact, those that fall more frequently tend to ultimately conquer walking 

 than those that shy away from testing gravity. They also get upright mobility quicker by trying lots of variations, random walking if you will, many of which attempts don’t work. It’s the process, however, that leads to ultimate success. Babies are lucky in that they have inherent advantages to take these falls, with built-in cushioning and flexible bones. Adults are more rigid – both physically and mentally. As we age, we tend to become more defensive and protective of our bodies. We know we’re more breakable as a lifetime of experiential cuts and bruises (possibly trips to the hospital) will attest to. We also more carefully protect our perceived view of ourselves. “I can’t run this new supercritical fluid chromatography (SFC) instrument because it’s different software, there are settings I don’t understand, and I’ll look dumb to my co-workers.” Yet this example, assuming you are versed in another area of separations, is a perfect opportunity, as it’s likely within the zone of proximal development. This is an optimal learning space between what you can do unaided and what you can do with a little help. It may feel like the edge of impossible at first, but it’s closer than you realize.

Ideally, we would all have the time, money, and support to take in-depth courses with personalized coaching on whatever skill we wished to advance. In the real world, there are likely some low-resource opportunities in our everyday lives to advance our separation skills. When trying to elevate something that has become routine, the key to advancement is in variable learning. A runner that takes the same path at the same pace will quickly plateau in their training. If they add some sprint intervals or hill climbs or anything new, they’ll achieve more progress. A chromatographer that runs the same gradient on that 5 µm C18 column every day could also benefit from changing things up. If your environment frowns on these kinds of deviations, other avenues exist. If you’re a gas chromatography (GC)-minded scientist, take a chance and read that article a few pages over on liquid chromatography (LC) (and 

 for the liquid phase folks). Curious about what’s going on in that mass spectrometer at your column outlet? Hop on 

 and wander through the available content (there’s tons for free). Lucky enough to be at a conference (hopefully we’ll see many of you at Pittcon 2023 for the ACS SCSC annual meeting!)? Try attending a session outside your comfort zone. Alternatively, take a lunch break and watch a webinar outside your daily sphere, such as at 

www.ACS.org/content/acs/en/acs-webinars.html

. A pharmaceutical scientist may find the latest advances in forensic technology as a breakthrough innovation in their own field. The key is to not be a robot running the same routine. Tangents aren’t just for peak integrations.

A key lesson from the book is that it didn’t really matter what the specific new skill was. Learning one new talent helped subsequent attempts at other new competencies. Scientifically, you can read about neuroplasticity in the book. I prefer to think of it as becoming more comfortable with falling and regaining my childlike mindset. Take a stroll through your local library’s shelves and pick up a 101 book on whatever stokes your passion, whether it be coding or cooking. Download a new language course and listen on the commute to work. Sign up for that community class on plumbing, which will also be directly applicable to LC and GC! Go ahead—indulge yourself in that new skill, whether it be juggling, forging, or multidimensional separations. Learn to learn again!

Jonathan Shackman is an Associate Scientific Director in the Chemical Process Development department at Bristol Myers Squibb (BMS) and is based in New Jersey, USA. He earned his two B.S. degrees at the University of Arizona and his Ph.D. in Chemistry from the University of Michigan under the direction of Prof. Robert T. Kennedy. Prior to joining BMS, he held a National Research Council position at the National Institute of Standards and Technology (NIST) and was a professor of chemistry at Temple University in Philadelphia, PA. To date he has authored more than 30 manuscripts and two book chapters. He has presented more than 30 oral or poster presentations and holds one patent in the field of separation science. Jonathan has proudly served on the executive board of the ACS Subdivision on Chromatography and Separations Chemistry (SCSC) for three terms.


What’s the fastest way to acquire a new chromatographic skill? The answer lies with those who are just beginning to learn.

The LCGC Blog: Remember the First Time

A new $215 million precision chemical manufacturing facility built by Waters Corporation recently achieved Leadership in Energy and Environmental Design (LEED) certification by the U.S. Green Building Council. The 140,000 square-foot facility, located in Taunton, Massachusetts, features sustainable design elements that significantly reduce energy consumption estimated to decrease water use by 20% and cut industrial waste volume by 50%.

The Waters Taunton site is the first and only LEED-certified chemical manufacturing facility in Massachusetts and is among a small number of LEED-certified industrial manufacturing projects in the United States. The site nearly triples Waters’s manufacturing footprint. It has created 25 new jobs and provides future growth capacity.

The facility incorporates features that include state-of-the-art, on-site industrial waste containment and treatment technology designed to reduce overall building emissions by an estimated factor of six over the company’s legacy manufacturing facility; wastewater recycling technology that reclaims and filters wastewater for use in property irrigation, climate control, and restroom facilities; and a LEED design optimized for energy efficiency and indoor air quality that incorporates building materials that promote sustainability.

A new $215 million precision chemical manufacturing facility built by Waters Corporation recently achieved Leadership in Energy and Environmental Design (LEED) certification by the U.S. Green Building Council. 

Waters Achieves LEED Certification for Chemical Manufacturing Facility in Massachusetts

Peter Schoenmakers of the University of Amsterdam is the 2022 recipient of the American Chemical Society (ACS) Award in Chromatography. The award was presented to him at the ACS fall meeting in Chicago on August 23.

Schoenmakers retired in June from his post as a professor of chemistry at the University of Amsterdam, where he was also the director of the van ‘t Hoff Institute of Molecular Science (HIMS) and a founder and the education director of a public-private-partnership organization on analytical chemistry called “Comprehensive Analytical Science and Technology (COAST).”

Schoenmakers has made significant contributions to the field of chromatography, from his early publications on the theory of gradient elution in reversed-phase chromatography and its optimization, to his work to advance the analysis of polymers, and to his pioneering and ongoing work in developing two- and three-dimensional liquid chromatography (LC) methods.

Schoenmakers received his PhD under Professor Leo de Galan in Delft and Professor Barry Karger in Boston, Massachusetts. He then he worked for Philips in Eindhoven (The Netherlands) and for Shell in Amsterdam and in Houston, Texas. While at Shell, he became a part-time professor in Polymer Analysis at the University of Amsterdam in 1998, and a full-time professor in 2002.

Schoenmakers has received multiple awards throughout his career. Recent international awards include the American Chemical Society (ACS) Award in Chromatography (2022); the Dal Nogare Award (2019), the Fritz-Pregl Medal (2018), the CASSS Award (2015), the Csaba Horváth Memorial Award (2015), the John H. Knox Medal of the RSC (2014), the Martin medal of the Chromatographic Society (2011), and the EAS Award for Excellence in Separation Science (2010). In 2016 he received a European Research Council (ERC) Advanced grant of 2.5 million Euros for the “Separation Technology for A Million Peaks” (STAMP) project.

In June, Schoenmakers was named a “Knight of the Order of the Netherlands Lion.” This royal commendation is one of the oldest and highest civil honors in the Netherlands. King Willem I established the Order in 1815. Candidates who have made an exceptional contribution to society, especially in science, art, sports, and literature, are eligible for this honor.

and is a member of the Editorial Advisory Board of

Peter Schoenmakers of the University of Amsterdam is the 2022 recipient of the American Chemical Society (ACS) Award in Chromatography. 

Peter Schoenmakers Wins ACS Award in Chromatography

Civica, Inc., a nonprofit generic drug company, will invest $27.8 million to establish a new laboratory testing facility at Meadowville Technology Park in Chesterfield County, Virginia. The company will construct a 55,000-square-foot facility to support its Petersburg, Virginia pharmaceutical manufacturing operation through quality testing and development of new products. The project will create 51 new jobs.

The construction of the laboratory will be supported by an award from the U.S. Economic Development Administration to the Alliance for Building Better Medicine, a coalition that aims to expand essential medicines manufacturing in the Richmond-Petersburg region. Civica will provide matching capital.

Civica is a nonprofit generic drug company launched in 2018 to address the problem of chronic generic drug shortages and high drug prices. In January 2021, Civica announced that it will establish a 140,000-square-foot state-of-the-art facility to manufacture sterile injectable drugs in the City of Petersburg. The company’s new laboratory testing facility in Chesterfield County is expected to be operational soon after the Petersburg plant reaches commercial scale in 2024.

Civica, Inc., a nonprofit generic drug company, will invest $27.8 million to establish a new laboratory testing facility at Meadowville Technology Park in Chesterfield County, Virginia. 

Civica Invests $27.8 Million for a New Testing Facility

A snapshot of key trends and developments in sample preparation according to selected panellists from the chromatography sector.

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

We see multiple and diverse trends in sample preparation. On the one hand, next‑generation mass spectrometry (MS) detectors allow for the analysis of low concentrated samples without prior enrichment, but on the other hand, regulatory agencies continuously ask for lower detection and quantification limits, putting more pressure on the laboratory scientists for results. Due to these trends, we see more solutions, such as protein precipitation and phospholipid removal, being developed for a quick removal of matrix components to achieve a level of cleanliness in the sample with good recovery and relatively simple procedure. Other trends we are seeing are more sophisticated sample preparation like multi‑layer solid-phase extraction (SPE), automation of the extractions, and integration of the sample preparation to the analytical workflow in the case of online SPE or two-dimensional liquid chromatography (2D-LC) in combination with MS detection.

I am frequently in contact with customers from industry and academia working to meet their needs. All of this feeds into our perspective on requirements and future developments. I am also specifically involved in automating sample prep processes.

In the last sample preparation technology forum (1), I “complained” that users stick to tried and trusted products and concepts, mainly relying on manual sample preparation. This seems to have changed since then. Companies are investing in automation for sample preparation, starting with simple yet effective things like preparing standards based on liquid handling, agitation, as well as vial de-capping and capping steps. The idea of automating sample preparation has clearly arrived in laboratories throughout the world in all application areas and markets. For users it is beneficial to rely on an automation platform on which they can start with basic workflows and extend functionalities over time as new challenges arise.

In that context, downscaling is often needed to enable automation. Standard operating procedures (SOPs) were until recently frequently written around manual sample processing and therefore employed large amounts of sample, solvents, and reagents. Automation and downscaling help to cut cost for chemicals, including for their safe disposal. In addition, automation reduces the exposure of laboratory personnel to hazardous substances. A greener and more cost-efficient chemical analysis is the result. A recently developed method for the determination of perfluoroalkyl and polyfluoroalkyl substances (PFAS) in water also profits analytically from smaller sample vessels because these can be efficiently flushed with limited amounts of solvent and the rinse effluent added to the SPE step, eliminating loss by adsorption on surfaces.

As more high-throughput laboratories tend to employ automation for sample cleanup, miniaturization of the starting sample and corresponding solvent volumes has become possible. Coupling various instruments used in sample preparation has gained popularity because it adds convenience to the overall user experience. A good example would be processing samples on well plates using a liquid handler combined with a manifold. Products that allow routine complex workflows to be simplified without sacrificing analyte recoveries are also attracting analysts from all industries.

Filtration columns and tips with certain chemistries allow for a reduction in the number of steps while removing matrix components to an acceptable level.

Also, with an increase in the legalization of marijuana for recreational and medical use, demand for extracting and separating tetrahydrocannabinol (THC) isomers from challenging matrices has been on the rise. To better understand the impact of PFAS toxicity on the environment and human health, more emphasis than ever is being put on analyzing PFAS in various water and food samples.

In my opinion, more and more analysts engage towards a greener and more sustainable future for sample preparation. “Going green” in sample preparation is not a trend; it is a necessity. Current global challenges and stressors highlight the urgency at which sustainability issues must be addressed. Going in this direction, the application of environmentally benign sample preparation methods should be a priority for all researchers, practitioners, and routine analysts for the benefit of the environment, human health, and society. Green sample preparation is sample preparation. It is not a new subdiscipline of sample preparation but a guiding principle towards sustainability.

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

With a focus on sustainability across the globe, we expect further miniaturization and reduced environmental impact (solvent consumption) as some of the main drivers for new developments in sample preparation solutions. In addition, we believe that in the clinical diagnostics space, there will be a greater number of LC–MS/MS methods replacing classical immunoassays. Based on this assumption, we expect a larger need for easily automatable sample preparation solutions for some of these assays.

 Sample preparation will remain important for the foreseeable future. For many matrices, sample preparation is indispensable. A colleague of mine likes to point out that “an LC–MS system has no door through which the lettuce can be introduced for pesticide analysis”. That is why we need sample prep! For other applications, sample preparation makes sense as well because we need to ensure that we arrive at accurate results using rugged analysis methods while continuing to have long maintenance intervals and high throughput.

Two years ago, I described the seamless integration of different manufacturers’ instruments into one software control platform as a trend. In this matter, having been overly optimistic, I have to correct myself. We are at the early stages of such a development, in which “all” laboratory equipment will be integrated into one control software connected to a laboratory information management system (LIMS) using generic interface standards. This setup will eventually allow us to generate comprehensive automated workflows with robots that fetch samples from the refrigerator before transferring them to a sample preparation platform and finally on to the analysis system. Alternatively, workflows with human intervention, such as a manually executed weighing step, are possible. The analysis results are finally transferred to the LIMS. Today, customers are requesting interaction and synchronization between instrument control software and LIMS. For example, vials can simply be placed in the autosampler tray, the barcode scanned, the sample ID established, and the correct analysis method and sequence table set up and executed automatically.

Advances in automation appear to guide the future of sample preparation. SPE products that are compatible with various components integrated into an all‑in‑one workstation will significantly maximize productivity. Liquid handlers ensure consistent and accurate pipetting of extremely small volumes, overcoming critical errors that would be introduced when using a manual approach. Apart from saving time, the robots minimize the risk of exposure to chemicals on a daily basis and drastically reduce solvent consumption, contributing to a cost-effective method set-up. Treating all the samples the same way without many variations leads to reliable results and excellent reproducibility. The ability to modify the extraction procedures stored on the software is another favourable feature of automation.

Sample preparation will continue to grow in importance, with sustainability issues guiding future trends in speed, automation, energy, materials, chemicals, and size. Meanwhile, cooperation and coordination with national and international standardization bodies will be established with the aim of replacing hazardous official standard sample preparation methods with green high‑performers. Sample preparation will most likely become increasingly interdisciplinary to promote problem‑driven research and address the world’s increasingly complex and interconnected problems. Tearing down disciplinary walls will also facilitate knowledge transfer from other “distant” disciplines and increase our understanding of the fundamentals underlying sample preparation. We should also expect breakthroughs in low‑cost, robust, and networked sensors for remote all-in-one sampling, sample preparation, and analysis. This foreseen rise in remote analysis could also lead to the much‑discussed fusion with big data and artificial intelligence (AI), creating miraculous benefits.

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

There is a tendency to avoid sample preparation by using more sensitive detectors. This is driven by the fact that in many cases sample preparation is still a manual process and requires a thorough method development and method validation. Even with advances to the detectors, sample preparation is a vital step that needs to be performed by experienced laboratory scientists to ensure reliability and to provide accurate results.

Many companies develop and manufacture innovative sample preparation equipment. Nevertheless, the majority of the “big” gas chromatography (GC)–MS and LC–MS instrument manufacturers have less focus on sample preparation, preferring to market (high-end) mass spectrometers and to omit sample preparation wherever possible, using a “dilute-and-shoot” or direct injection approach. This strategy may lead to less robust analysis methods and an increased need for instrument maintenance, leading to instrument downtime. A closer collaboration between instrument manufacturers and sample preparation equipment manufacturers could benefit all parties because customers today are asking more and more frequently for comprehensive solutions from the sample as received to the final analysis result.

Highly sensitive modern analytical instruments leave no room for residual contamination that would have usually gone under the radar. It becomes tremendously vital for the companies to closely monitor the manufacturing process and for the laboratories to ensure a contamination-free environment with no carryover issues. While some matrices are exceptionally difficult to work with, more realistic expectations need to be held, especially when it comes to achieving high recoveries and minimal matrix effects for a large panel comprised of analytes with varying chemistries.

The biggest obstacle to sample preparation is the weak connection between academia and the private sector. Research in sample preparation is blooming but perhaps left unnoticed by outsiders. We have not been effective enough at projecting these advances and have acquired the image of a slow‑paced discipline developing laborious and time‑consuming procedures. The lack of a coherent strategy to promote the criticality and benefits of modern sample preparation technologies has resulted in many official standard methods still relying on old-fashioned and hazardous analytical procedures. Newer, more powerful, and greener sample preparation methods are ignored in favour of more traditional and standard approaches, which may not fully address the analytical challenge at hand. Private and industrial laboratories see new and green sample preparation alternatives as an extra burden, rather than an opportunity to adopt powerful techniques that have a shared value for both the productivity and society. Strengthening the interface between academia and the private sector is an obstacle that has to be overcome, and this is a major task of the EuChemS-DAC Study Group and Network Force, which I have the honour of leading.

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

The biopharmaceutical market has consistently had strong growth for the past couple of years, especially with the global distribution of the COVID-19 vaccine. Biopharma laboratories need to be able to consistently clean up their biological samples without losing their sample while also maintaining high recovery. By using, for example, a magnetic bead as a reproducible sample preparation option, this addresses both those needs, but can add a lengthy amount of time to the workflow.

I do not see one single most important development in particular in techniques or applications. The quest for greener analysis and greener sample preparation techniques seems to be gathering momentum. The good news is that there is progress in that area in terms of underlying concepts as well. In that context I would like to recommend the article “The Ten Principles of Green Sample Preparation” in 

 by Angela I. López‑Lorente et al. (2), in which 10 fundamental principles are described that the sample preparation of the future should stick to. The authors plan to develop metrics for assessing “greenness” of sample preparation steps. Further, the IUPAC project “Greenness of official standard sample preparation methods” is guaranteed to become recommended reading (3).

The recently developed QuEChERSER Mega-Method is an upgrade over the regular QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure, as it covers analytes within a broader polarity range. This new approach seems to be a significant development and eliminates the need for using separate methods to analyze pesticides, veterinary drugs, environmental contaminants, and mycotoxins in food.

Early sample preparation methods were a major source of the total negative impact of analytical methodologies on the environment, mainly due to the large energy demands and quantities of hazardous substances used and generated. For this reason, the first of the 12 Green Analytical Chemistry (GAC) principles suggested applying direct analytical techniques to avoid sample preparation. The first principle of GAC was commonly misinterpreted and created the false impression that omitting the sample preparation step was a green approach, fully neglecting the “green” technological advances in the field. The “exclusion” of sample preparation from GAC also created a gap, as removing this step was practically impossible for complex samples and when high sensitivity is needed.

I think that the most important recent development was the formulation of the 10 principles of green sample preparation and the introduction of AGREEprep, the first metric tool focusing on sample preparation. Defining sample preparation within the context of green chemistry and GAC was a great necessity in the area of sample preparation. We now have the approach formulated in hand and a metric to evaluate the environmental impact of methods. Currently, decisions about methods are based on method performance and price. Thanks to these developments, they can now be complemented by a sustainability factor, which is a measure of how green the method is.

Q. What was the biggest accomplishment or news in 2021/2022 for sample preparation?

The development of products that are able to solve multiple problems in one extraction for scientists—combining two different sorbents or products to help create a faster solution.

The sample preparation community is increasingly organized; for example, the “Sample Preparation Study Group and Network” was installed in the Division of Analytical Chemistry of the European Chemical Society in 2020. This group held European Sample Preparation e-Conferences in 2021 and 2022, and this year the conference was even accompanied by the 1st Green and Sustainable Analytical Chemistry e-Conference. Moreover, 

, a dedicated open-access scientific journal, was established in 2021. This shows that the community is active and is receiving more and more attention in the analytical chemistry world. These are the big accomplishments of the last two years.

EPA 1633 draft method for the analysis of 40 PFAS in non-drinking water matrices was a piece of important news this year. The sample cleanup is accomplished using weak anion exchange (WAX) and graphitized carbon black (GCB) in powder or cartridge format.

In my opinion, 2021–2022 was a landmark year for sample preparation, as this was the launch year of Elsevier’s open access journal 

. SAMPRE was the first academic journal dedicated to sample preparation. The launch of this new journal is of particular importance to our scientific community, as it recognizes the extraordinary technological and scientific growth in the area. For many years, senior members of our community were proposing the creation of this journal but major publishers could not identify the niche to fill. At that time the growth was not sufficiently large and new papers could be absorbed into existing journals. The launch of SAMPRE formalized sample preparation as a discipline and illuminated the dynamics, importance, and impact of the field.

https://iupac.org/project/2021-015-2-500/

Dirk Hansen is Regional Market Development Manager, Europe at Phenomenex.

Oliver Lerch is Head of Automated Sample Preparation at Gerstel GmbH & Co. KG in Mülheim, Germany.

Ritesh Pandya is Technical Specialist at UCT.

Elia Psillakis is a professor in the School of Chemical and Environmental Engineering, Technical University of Crete.

A broad range of trace-level volatile organic compounds (VOCs) was detected in drinking water using headspace–trap sampling. Analysis of a 72-component standard mix using gas chromatography–mass spectrometry (GC–MS) with selected ion monitoring (SIM) acquisition provided mean detection limits as low as 2 ppt, with excellent mean linearities (R2 0.999), recoveries (97.8%), and repeatabilities (4.7% relative standard deviation [RSD])—a performance that is comfortably lower than required by all major regulations. In addition, tap water was found to contain a range of VOCs at low-to-medium levels (2–200 ppt), in addition to ppb-level chlorinated compounds.

Rachael Szafnauer received an M.Sci in forensic science from the University of South Wales, UK, where her final‑year project focused on fingerprinting emerging psychoactive substances using advanced techniques such as GC×GC–TOF MS, in collaboration with Markes International. She later took up the role of thermal desorption product specialist at Markes, providing technical and application support to the commercial team, before taking on her current role at the start of this year, specialising in the development of applications using extraction and enrichment techniques.


Elinor Hughes obtained her B.Sc. in chemistry and Ph.D. in organic chemistry at Bangor University, UK. After working for a chemical manufacturing company for three years, she moved to the Royal Society of Chemistry where she worked in journals publishing for six years and on Chemistry World magazine for four years. This was followed by five years as a freelance copyeditor and science writer. Her current role is technical copywriter at Markes International.

Chemical contamination of rivers, reservoirs, and groundwater used as sources of drinking water originates from intentional and accidental discharges from industry, agriculture, and urban pollution. This contamination requires extensive treatment, but such processes can also result in the formation of contaminants such as the trihalomethanes that are formed by the reaction of the common oxidant chlorine with organic matter. Such contamination is naturally of concern, and acceptable levels for the volatile organic compound (VOC) content of drinking water are specified by a variety of regulatory bodies, including the US Environmental Protection Agency (EPA), Chinese EPA, European Environment Agency (EEA), Canadian Drinking Water Quality Guidelines (DWQG), and the World Health Organization (WHO).

Headspace analysis is a well-established and robust method for the determination of VOCs in water, but in certain aspects it offers limited flexibility. In particular, the injection of larger headspace sample volumes to improve sensitivity can cause undesirable chromatographic effects such as broad or split peaks, and options such as multiple injections are usually not possible. There are also limited options for water management, meaning that analyses can suffer from reduced analyte response and repeatability, as well as negative impacts on column and detector lifetime.

In this study, we demonstrate how the use of a backflushed, cryogen-free focusing trap packed with multiple sorbent beds, in conjunction with gas chromatography–mass spectrometry (GC–MS), can overcome such issues for the headspace analysis of residual VOCs in drinking water. In particular, we show how selected ion monitoring (SIM) acquisition allows quantitation of target analytes at low-ppt levels, while avoiding issues relating to interference from water.

A set of calibration standards at seven levels from 50 ppt (50 ng/L) to 20 ppb (20 µg/L) was prepared by volumetric dilution of a 2000-ppm stock solution using high performance liquid chromatography (HPLC)-grade methanol. The mix contained 72 components, including one internal standard (fluorobenzene) and two surrogate standards (4-bromofluorobenzene and 1,2-dichlorobenzene-d4). The volatility of the compounds in the standard mix ranged from dichlorodifluoromethane (b.p. –29.8 °C) to 1,2,3-trichlorobenzene and naphthalene (b.p. 218 °C) and included six compounds that are gases at ambient temperature. The methanol solutions were spiked into HPLC‑grade water (10 mL) contained in a standard 20-mL crimped‑top vial prior to headspace–trap GC–MS analysis. Calibration curves and performance results were calculated based on the on-column concentration as detected by the MS system.

 Tap water (10 mL) from a source in South Wales, UK, was added to a 20-mL headspace vial containing sodium sulfate (2.5 g; 25% w/v). The sample was spiked with the internal standard (2.0 ppb; 2.0 μg/L) and capped.

Instrument: Centri (Markes International); headspace–trap: headspace sample: 1 mL; incubation: 80 °C (10 min); injection: 200 °C (2 min).

Preconcentration: Focusing trap: ‘TO-15/TO‑17 Air toxics’ (part no. U-T15ATA-2S, Markes International); purge flow: 50 mL/min for 1 min; trap low: 20 °C; trap high: 280 °C (0.5 min); split ratio: 5:1.

GC: Column type: 30 m × 250 μm, 1.4-μm MEGA-624 (Mega); column flow: 2 mL/min (constant flow); purge flow: 3 mL/min; oven program: 35 °C (3 min), then 10 °C/min to 100 °C, then 30 °C/min to 220 °C (1 min). Quadrupole MS: Transfer line: 180 °C; ion source: 300 °C; mass range: 

 35–300; SIM groups: multiple; tune type: E-tune.

 Figure 1 shows the overlaid SIM responses for all VOC compounds of the standard mix at 100 ppt on-column. For accurate quantitation of the early‑eluting gaseous compounds at these low concentrations, peak shape is important. The inset in Figure 1 shows the SIM responses for the first three peaks in the standard mix at 20 ppt on-column. These are difficult compounds to analyze at low-ppt concentrations, but in this study the peak shape was highly symmetrical.

 Excellent linearity was obtained for all target compounds, with a mean R2 value for the seven-point calibrations from 10–4000 ppt being 0.999, and with the lowest value being 0.988 for 1,2-dichlorobenzene. The linearity was also calculated as the relative standard deviation (RSD) of the relative response factor for each analyte against the internal standard, using four replicate injections per level across the calibration range. The results indicate that very high confidence in quantitation is possible down to low-ppt levels.

The use of R2 as a measure of how well a set of data fits a calibration curve is well‑established. However, there is a growing desire within certain organizations and regulatory bodies to use a separate metric, the relative standard error (RSE) (1,2). The key difference is that whereas R2 tends to under‑represent deviations at low concentrations, RSE is equally sensitive to deviations across the concentration range. Acceptable values for RSE are method‑dependent or based on RSD values, but in general, values below 20% are indicative of a good fit.

Figure 2 shows the R2 and RSE values for the six most volatile compounds in the standard mix (all of which are gases under ambient conditions). RSE values range from 4.2% (chloromethane, #2) to 18.5% (trichlorofluoromethane, #6), confirming a good curve fit across the concentration range.

 Recoveries were calculated based on 10 replicate injections of the standard mix at 100 ppt on-column, using raw peak area values. The mean recovery was 98%, with a mean RSD of 4.6%, and with RSDs less than 10% for the most volatile compounds, indicating very high stability in overall system performance.

Method detection limits (MDLs) were calculated using injections of the standard mix at 10 ppt on-column. The mean MDL was 1.9 ppt, with values ranging from 0.6 ppt (for bromochloromethane, #19) to 10.7 ppt (1,2-dichlorobenzene, #66).

Also determined were the practical quantitation limits (PQLs). The PQL is the minimum measurable concentration of an analyte for which there can be a high degree of confidence that the analyte is present at or above that concentration. The PQL is typically set at 6× to 10× the MDL, and in this study, we used 6× MDL. This gave a mean PQL of 11.4 ppt, with the most volatile compounds all having values <20 ppt.

Table 1 compares the limit levels for several key VOCs specified by several agencies for water quality (including the relatively stringent EEA Directive 98/83/EC2) (3) with the PQL values obtained in this study. This shows that the current work provides detection levels significantly lower than required by all major regulations.

Performance for Internal Standard and Surrogate Standards: 

The stabilities, repeatabilities, and recoveries of the internal standard and the two surrogate standards were determined by running 12 replicate injections at 20 ppt on-column and plotting the raw peak area against run number. This provided a practical way of assessing the stability of the system, making it straightforward to identify when re‑calibration was required.

Mean recoveries (and RSDs) for fluorobenzene (#27), 4-bromofluorobenzene (#50), and 1,2-dichlorobenzene-d4 (#65) were 103.0% (RSD 7.0%), 100.4% (RSD 6.5%), and 99.7% (RSD 8.0%), respectively. Figure 3 shows the results graphically and indicates high levels of recovery and stability at this low concentration (20 ppt).

 A tap water sample, spiked with the internal standard mix at 2 ppb on-column, was analyzed for residual VOCs and the concentrations are shown in Figure 4. In addition to three chlorinated VOCs present at low ppb levels, a number of ppt-level VOCs were found, of which benzene, ethylbenzene, and the xylenes are regulated and were present well below stipulated levels.

This study has shown that static headspace sampling combined with preconcentration on a multi-mode platform, in conjunction with GC–MS in SIM mode, allows the detection of low-ppt VOCs in water. Limits of detection were significantly below limit levels specified in a variety of regulations, and the system stability, precision, and accuracy were all excellent, which allowed quantitative analysis across a broad concentration range.

The use of a backflushed, cryogen-free focusing trap packed with multiple sorbent beds provided good retention and release of analytes, ranging from the very volatile gases through to the higher-boiling compounds. The availability of a trap also allowed re-collection of split samples for repeat analysis, with key benefits in this case being streamlined method validation and detection using different methods.

To further enhance sensitivity, the platform could be operated in splitless injection mode, and set to allow multiple samples from the same vial to be preconcentrated onto the same focusing trap, prior to desorption.

Calculating RSE, NELAC Institute, 2017: http://nelac-institute.org/docs/comm/emmec/Calculating RSE.pdf

Basic RSE calculator, NELAC Institute, 2017: http://nelac-institute.org/docs/comm/emmec/Basic RSE calculatorv4.xlsx

Council Directive 98/83/EC of 3 November 1998 on the quality of water intended for human consumption, European Commission, 1998: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A31998L0083

 received an M.Sci. in forensic science from the University of South Wales, UK, where her final‑year project focused on fingerprinting emerging psychoactive substances using advanced techniques such as GC×GC–TOF-MS, in collaboration with Markes International. She later took up the role of thermal desorption product specialist at Markes, providing technical and application support to the commercial team, before taking on her current role, specializing in the development of applications using extraction and enrichment techniques.

 obtained her B.Sc. in chemistry and Ph.D. in organic chemistry at Bangor University, UK. After working for a chemical manufacturing company for three years, she moved to the Royal Society of Chemistry where she worked in journals publishing for six years and on 

 magazine for four years. This was followed by five years as a freelance copyeditor and science writer. Her current role is technical copywriter at Markes International.

A broad range of trace-level volatile organic compounds was detected in drinking water using headspace–trap sampling.

Analysis of Volatile Organic Pollutants in Water Using Headspace–Trap GC–MS: Maximizing Performance for ppt‑Level VOCs

This month we interview Simona Felletti, a postdoctoral researcher in analytical chemistry in the Department of Chemical, Pharmaceutical, and Agricultural Sciences at the University of Ferrara, Italy, about her work in the mass transfer phenomena and thermodynamic properties of packed columns under both liquid chromatography (LC) and supercritical fluid chromatographic (SFC) conditions, and the hazardous potential that uncharacterized minor cannabinoids pose.

Q. When did you first encounter chromatography and what attracted you to the subject?

 It was back in 2013 when I was a bachelor student at the University of Ferrara in Italy. I have always loved TV series based on forensic science, where the culprit is caught thanks to chromatographic analysis. I like the concept of “find what you do not see”, and with chromatography you can find out what is hidden in your sample. At the beginning, what attracted me the most was the application of chromatography to environmental analysis, but I later discovered other interesting aspects of chromatography, such as the theoretical aspects behind adsorption, thanks to my mentor, Alberto Cavazzini.

Q. Can you tell us more about your Ph.D. thesis?

My Ph.D. research project focused on the investigation and understanding of rather complex phenomena occurring in packed beds through a combination of experimental and theoretical approaches of linear and nonlinear liquid chromatography (LC). This allows the information needed regarding the kinetics and the thermodynamics of the chromatographic separation process to be obtained. From a theoretical viewpoint, kinetic factors (diffusion, eddy dispersion, mass transfer resistance, finite rate of adsorption/desorption process) directly affect the efficiency of a chromatographic separation, while thermodynamic factors (adsorption equilibria) have an impact on retention and selectivity.

The knowledge of all these contributions is pivotal to developing selective, ultra-fast, and highly efficient separation methods, and for the design of novel stationary phases.

Q. What chromatographic techniques have you worked with?

I currently work with different scales of chromatography, going from analytical to preparative applications. For analytical, highly efficient, and fast separations, I work with supercritical fluid chromatography (SFC) and ultrahigh-performance liquid chromatography (UHPLC). In preparative-scale chromatography, I work with traditional single-column batch LC and also with multi-column, continuous techniques for the purification, isolation, and enrichment of compounds, such as multi‑column countercurrent solvent gradient purification (MCSGP), simulated moving bed (SMB), and N-Rich techniques. In other words, my working range goes from sub-2-µm to 20‑µm porous particles, or from 0.1 to 10 mL/min. 

Q. Your primary research focus is on the study of mass transfer phenomena and thermodynamic properties of packed columns under both LC and SFC conditions—what specifically attracted you to this area of research?

I am a curious person and I like discovering and knowing the reasons behind something. This also happens with my research, where fundamental studies contribute to the (almost) complete understanding of the theoretical reasons at the basis of the separation process.

Indeed, the knowledge of mass transfer and adsorption phenomena is important, not only for the comprehension of retention and molecular recognition mechanisms but also for practical reasons, because it is the basis of the modelling of separation under different experimental conditions—including overloading and gradient. The possibility of predicting both the retention and peak shape of a target compound and how they can be modulated by modifying the experimental conditions (mobile phase composition, flow rate) allows the chromatographic method to be optimized to maximize speed, resolution, and selectivity.

For example, regarding chiral chromatography, these studies contribute to shedding light on some fundamental questions, such as the relationship between chiral discrimination (enantioselectivity) and adsorption-desorption kinetics, and its dependence on some experimental parameters, including the loading of chiral selector, the particle geometry, and the pore size.

Much work has been done in this field (1,2), but there is much more to be discovered, and we are trying to make our contribution.

Q. You have recently published a paper investigating the effect of the polarity of the stationary and mobile phases on the retention of cannabinoids in normal-phase LC (3). What advantages does it offer over reversed-phase LC?

Reversed-phase LC is currently the preferred and most employed chromatographic mode for the separation of cannabinoids. However, particularly for preparative purposes where large amounts of compounds are processed, water‑based eluents show some limitations, namely scarce solubility of cannabinoids, large back pressure, and energy-consuming solvent removal. In this field, normal-phase LC could offer several advantages over reversed-phase LC. Indeed, the use of apolar solvents facilitates sample preparation (leger solubility of hemp extracts), makes solvent evaporation from purified fractions easier, and allows for faster runs (the lower back pressure permits the use of higher flow rates). Moreover, thanks to the different retention mechanism (adsorption vs. partition), different selectivities may be obtained with respect to reversed-phase LC.

However, conversely to reversed-phase LC, no fundamental studies about the employment of normal-phase elution mode for the separation of cannabinoids were available in the literature. In this work, we investigated from a fundamental viewpoint the effect of polarity and the type of interaction mechanisms of four different adsorbents on the retention of selected cannabinoids using a variety of mobile phase compositions under normal phase conditions.

The results of this work may be useful for the setting up of purification procedures, or in cases where orthogonal methods to reversed-phase LC are needed, to help in the selection of the proper stationary phase and experimental conditions for cannabinoids analysis and isolation.

Q. Are there any challenges involved with using this method?

A critical point when using normal phase for the separation of cannabinoids is the resolution of the early eluting compounds. However, peaks can be resolved (or partially resolved) by modulating either the percentage or the type of the polar modifier in the mobile phase.

Another thing to keep in mind is the dissolution of apolar compounds, such as waxes, that occurs when sample extraction is performed with heptane or hexane. The removal of these compounds is pivotal, especially in preparative scale where large amounts of sample are processed, in order not to damage the chromatographic column.

Last but not least, the use of proper instruments compatible with apolar solvents
is required.

Q. No fundamental studies about the employment of normal-phase LC or chiral chromatography for the separation of cannabinoids have been performed. Is this something you plan to explore further in the future?

 L. has been known for millennia, the analysis of cannabis products and extracts is a relatively new topic, having only taken place in recent years after the legalization and depenalization of cannabis in many states. As a consequence, scientists have focused their attention on the development of experimental procedures for the extraction and the analytical separation of cannabinoids. Nevertheless, since new cannabinoids are continuously discovered and isolated, new analytical methods with different and enhanced selectivity with respect to the traditional C18 columns are required. Therefore, our research is focused on the development of alternative analytical methods for cannabinoids separation, and we have started with some theoretical studies devoted to the understanding of the interaction mechanisms between cannabinoids and different stationary phases in normal-phase LC and chiral reversed‑phase LC. We are planning to perform complete kinetic and thermodynamic studies on different stationary phases (both achiral and chiral), including SFC elution mode.

Q. There is concern about the hazardous potential of uncharacterized minor cannabinoids, including chiral ones. The development of LC methods for the enantioseparation of cannabinoids is therefore very topical. Another of your recent papers discusses the retention behaviour of natural cannabinoids on differently substituted polysaccharide‑based chiral stationary phases under reversed-phase LC conditions (4). Please could you discuss your results and how it could help in the future characterization of cannabis samples.

 Up to date, there is still no analytical method that permits the complete characterization of cannabis samples in terms of purity, amount, and enantiomeric excess.

Theoretically, in chiral analysis, the number of cannabinoids to be resolved is (almost) double with respect to achiral analysis, since both (+) and (–) enantiomers could be present. This makes the development of effective separation methods even more challenging. In this context, our work is intended to start the investigation of the most suitable chiral stationary phase (CSP) for the chiral characterization of hemp samples. In this work, a screening of stationary and mobile phases was performed to understand the effect of substituents (either electron‑donating or electron-withdrawing) present in the polysaccharide-based CSPs on the retention and selectivity of cannabinoids.

The choice of the correct stationary phase chemistry may help in the comprehensive characterization of the sample, which is useful in regulatory environments for the detection of impurities, and in forensic toxicology. It also assists in the development of preparative methods for the isolation of single enantiomers. These methods will permit investigatation of their biological activity to produce analytical‑grade standards.

For example, if the quantification of cannabichromene (CBC), sold as a racemic mixture, is performed with an achiral method, results may be misleading (being the sum of both +and – enantiomers). The same happens with other cannabinoids, like tetrahydrocannabinol (Δ9-THC), whose (+)-enantiomer has been found with an enantiomeric excess of 0.27% in medicinal marijuana.

Q. What projects are you currently working on?

 On the analytical side, we are currently exploring retention behaviour and performing kinetic studies of several biopharmaceuticals and other molecules of industrial interest on different column chemistries (traditional C18, doped phases, polar phases, polysaccharide‑based, Pirkle‑type), particle types (totally porous, superficially porous), and elution modes (normal‑phase LC, reversed‑phase LC, SFC). We are also focused on the replacement of toxic solvents commonly used in LC, such as acetonitrile, with greener and harmless ones (for example, dimethyl carbonate and dimethyl isosorbide). The aim is to understand whether green solvents can be used as an alternative to traditional ones to perform cost-effective, efficient separations of the molecules
under investigation.

On the preparative side, by using data collected in the analytical scale, and also through the use of simulation and modelling codes, we are developing purification methods for both peptides and cannabinoids, using single-column and multi-column techniques (MCSGP and SMB), in collaboration with several companies. We are also comparing the purification performance of traditional and green solvents.

Completing all these studies is an ambitious goal, but the obtained data will help chromatographers in the selection of the proper combination of stationary and mobile phases depending on their purpose.

Q. You recently came first in the HTC Tube Contest at the HTC-17 conference in Ghent, Belgium. How was it presenting your research in a three-minute video?

I like new challenges and I try to push myself beyond my comfort zone because I am a very creative person. It was really stimulating to present my research in an unconventional way through a short and funny video. At the HTC conference, I was very excited and I can say that the audience appreciated my video and had a good time. As I really enjoyed this experience, I decided to create another video and to participate to the HPLC Tube Contest in San Diego (California, USA) (HPLC 2022). I also managed to thrill the audience on this occasion, winning the first prize!

I would like to thank my mentor, Alberto Cavazzini, for giving me the opportunity to join his group at the University of Ferrara. During these six years, I have had the chance to spend different periods in qualified research centres and to meet esteemed researchers all around the world. I also want to thank my research group and all the people involved in our projects for the support and for believing in me. I would never be where I am today without them.

S. Felleti, C. De Luca, G. Lievore, et al., 

O.H. Ismail, M. Antonelli, A. Ciogli, et al., 

C. De Luca, A. Buratti, Y. Krauke, et al., 

C. De Luca, A. Buratti, W. Umstead, et al., 

is a postdoctoral researcher in analytical chemistry in the Department of Chemical, Pharmaceutical, and Agricultural Sciences at the University of Ferrara, Italy, in the group of Alberto Cavazzini. Her research focuses on the study of mass transfer phenomena and the thermodynamic properties of packed columns under both liquid and supercritical fluid chromatographic conditions (LC, SFC), as well as the development of methods for the purification of bioactive molecules, such as peptides and cannabinoids using both single-column and multi-column continuous techniques. She is author or co-author of 25 articles in peer-reviewed journals, and she has presented her work in more than 20 national and international meetings. In 2021, she was the recipient of the Ervin Kováts Award for Young Scientists from the Hungarian Society for Separation Sciences. She achieved second place at the Separation Science Slam Competition at the HPLC 2019 symposium (Milan, Italy) and, more recently, first place at both the HTC Tube Contest at the HTC-17 symposium (Ghent, Belgium, in 2022) and at the HPLC Tube Contest at the HPLC 2022 symposium (San Diego, California, USA).

This month we interview Simona Felletti, a postdoctoral researcher in analytical chemistry in the Department of Chemical, Pharmaceutical and Agricultural Sciences at the University of Ferrara, Italy, about her work in the mass transfer phenomena and thermodynamic properties of packed columns under both LC and SFC conditions, and the hazardous potential that uncharacterized minor cannabinoids pose.

Rising Stars of Separation Science: Simona Felletti

The final part of a review of the recent Chromatographic Society (ChromSoc) spring symposium meeting focusing on small molecule analysis

Christina Jayne Vanhinsbergh is a researcher at the University of Sheffield, UK. Her Ph.D. research focused on the development of multidimensional chromatographic methods for the analysis of therapeutic oligonucleotides. Currently, she is researching DNA alkylation from environmental compounds using LC–MS methodologies. Christina is the membership officer for the Chromatographic Society, UK.

The final part of a review of the recent Chromatographic Society (ChromSoc) spring symposium meeting focusing on small molecule analysis.

Session 4: Challenges in Small Molecule Analysis: Polar and Chiral Compounds: 

John Lough from the University of Sunderland was the keynote speaker for the first session of day 2. His talk on “The Role of Chiral Screening in the Analysis of Small Molecules; Asymptotic Growth?” was an in-depth evaluation of chiral screening over the past two decades. By asymptotic, John meant a steady growth in throughput over time. John evaluated the current state of the art in chiral stationary phases and how columns and instrumentation have changed over the years. John discussed supercritical fluid chromatography (SFC) and how it became implemented in chiral screening strategies. He demonstrated the plethora of columns available for screening and the different methods he has utilized within his research. He added that even simple screens can be appropriate methods, as he demonstrated with his work screening anti-cancer compounds. John highlighted some future challenges for growth in chiral separations, such as the analysis of multi-chiral drugs, improvements in SFC technology, the development of tiered screens, and increasing throughput.

The next presentation was “The Dark Art of Chiral SFC” by Kiera Bailey from Reach Separations. Kiera explained that supercritical fluid chromatography harnesses supercritical fluids for separations as they have the low viscosity of a gas and the solubilizing power of a liquid. The high diffusivity value of CO2 leads to increased column efficiencies, which can then be used for chiral separations. Extensive screening approaches use 88 chromatographic conditions to find the optimal separation and, often, SFC is used over high performance liquid chromatography (HPLC) due to its higher throughput. Kiera presented some case studies of chiral and achiral separations and introduced the sustainability aspect of SFC: lower waste generation, usage of safer solvents, and renewable feedstocks—all of which will be potential analytical drivers in tomorrow’s world.

The final presentation of the session was from Luisa Pereira from Unilever, “Porous Graphitic Carbon—a Unique Stationary Phase for Challenging Separations”. Luisa presented an in-depth overview of the chemistry and retention mechanisms associated with this type of stationary phase. Porous graphitic carbon (PGC) is very pH-stable and highly retains polar analytes by inducing strong dipole–dipole interactions. She presented some case studies of separations, such as the separation of purines and pyrimidines using reversed-phase conditions, and commented on the complementarity between porous graphitic carbon separations and hydrophilic interaction chromatography (HILIC). Luisa also discussed high-temperature liquid chromatography using PGC stationary phase and just water for the mobile phase. High temperature separations demonstrate high efficiency, faster analysis times, and higher resolutions.

Session 5: Challenges in Small Molecule Analysis: Environmental Analysis:

 The keynote speaker for session five was Helena Rapp-Wright from Imperial College London, who presented a talk on “Analyzing Small Molecules in the Environment: Challenges and Opportunities”. She changed the title of the presentation to fit better to the theme and presented an engaging talk on characterizing contaminants of emerging concern (CEC). These CECs have the potential to be bioactive, entering the food chain and inducing antimicrobial resistance and other toxic effects. Helena’s work was to sample water routes around specific locations and analyze CECs using direct injection LC tandem mass spectrometry (MS/MS) and a 5-mm biphenyl stationary phase. Helena explained the method optimization strategy that facilitated the analysis of over 100 CECs in a 4-min run, allowing the analysis of 261 injections per day for high-throughput analysis. The case examples that she presented covered the analysis of pesticides and other toxic CECs, as well as illicit drugs within water effluent streams. Helena discussed how matrix effects can impede the sensitivity integrity of analytical data, and therefore her group has developed three-dimensional (3D)-printed passive sampling systems that capture CECs over a longer period and across a range of seasons to concentrate low-level analytes and increase sensitivity. She concluded that the increase in ability to characterize CDCs will be complemented by implantation of machine learning to large data sets in the future, and that biomonitoring remains challenging.

Following Helena, Simon Hird from Waters presented “Bringing Reliable Determination of Anionic Polar Pesticides in Food to the Routine Laboratory”. Simon introduced his talk by discussing food analysis and the challenges involved in extracting pesticides in food samples, such as getting the extraction technique correct to facilitate detection. The quick polar pesticides method (QuPPe) can extract multiple pesticides; however, it can be affected by LC–MS/MS matrix effects due to there being no sample clean-up step. LC–MS/MS analysis of pesticides found in food was performed using a HILIC/weak anion exchange (WAX) mixed-mode column originally designated for SFC, and great results were obtained from the phase chemistry. Simon presented a tool to investigate matrix effects and enable acquisition or multiple reaction monitoring (MRM) and full-scan data simultaneously. He concluded that flexibility is key for method optimization and that there remains the need to investigate improved sample clean-up options.

The final presentation was “Exploiting Flat Plate Heating to Double VOC Analysis Throughput, Whilst Improving GC Peak Shape for the Most Volatile Components”, by Bryan White from Agilent. Bryan quickly polled the audience to investigate how many of them use or have used gas chromatography (GC), and went on to argue how GC is not as challenging to approach as many might think. Cartridge‑type GC columns make installation much easier and, couple that with more user-friendly software, can help reduce operational errors. He discussed volatile organic compounds (VOC) analysis using flat plate heating technology, which can rapidly heat and cool to facilitate shorter analysis times. Faster temperature ramps can lead to band broadening, so shorter columns can overcome this and smaller column diameters can overcome
resolution limits.

Session five was followed by the Chromatographic Society annual general meeting where the society activities and expenditures throughout 2021 were described by Paul Ferguson, Tony Edge, and Greg Jonas.

Session 6: The Future of Small Molecule Analysis: 

Peter Myers from the University of Liverpool was the keynote speaker for the final session. His presentation “A World Without Chromatography” highlighted a vision to move away from the need to separate molecules. Peter talked about historical events, such as witnessing the birth of new detector types, and how people would attend symposia to witness technological breakthroughs. He argued that we haven’t moved on much from the observation of peaks in recent years and there is still so much we have the opportunity to do with technology. Peter introduced the idea of transitioning to pattern recognition and sensor technology for the future. He used many examples to highlight this concept, one of which was the “electronic nose”, which senses odours and identifies the pattern that is created. This technology is currently limited by a requirement for 1000s of sensors to match what we have naturally in the nose. He discussed the need to couple powerful computing with high-resolution sensors to understand complex patterns in the future, as well as alternative technologies for sensors, such as electrical pore wetting and de-wetting or nanowires. His key message was that if we can identify a sample pattern, then we would not need to separate the mixture using chromatography.

The final presentation was a closing discussion from Tony Edge, from Avantor, discussing “Chromatography of the Future”. Tony argued that chromatography is not dead and that we are actually on the cusp of something very exciting in the future. He discussed technological advancements, such as the development of sub-micrometre particles and the challenges they introduce to the field, such as increases in back pressure. We need faster chromatography and greater selectivity for complex samples through alternative approaches to separations. Tony identified the need for in situ analysis and instantaneous results, which may be facilitated by advancements in chromatography. He discussed future opportunities in modelling approaches to separations and improved stationary phase chemistries with defined pore structures to improve selectivity of complex mixtures. Printed or etched columns respective to our separation needs may be a product of the future, which might also further in situ analysis approaches. Tony also discussed high temperature analysis, which makes water behave more like an organic solvent and improves sustainability. He concluded with the idea that chromatography is a world full of opportunity!

To view the on-demand event, please click here:

Part 2 of a review of the recent Chromatographic Society (ChromSoc) spring symposium meeting focusing on small molecule analysis.

ChromSoc Meeting Report: Challenges in Small Molecule Analysis in the Pharmaceutical Industry: Part 2

Sample preparation for headspace analysis of volatile organic compounds (VOCs) by gas chromatography (GC) is well understood. Newer direct‑injection mass spectrometry (DIMS) techniques have found application in headspace analysis, but with some necessary modifications due to (i) the removal of the chromatographic column, and (ii) the soft chemical ionization that is used in these techniques. These factors place important constraints on the total load of VOCs—whether of solvent(s), analytes, or other VOCs in the matrix. This article summarizes the basic differences between DIMS and GC approaches, and describes the strategies by which headspace-DIMS can be successfully adopted.

Headspace analysis is widely used with gas chromatography (GC) for the analysis of volatile organic compounds (VOCs) in solution or solid matrices (1). In addition to the obvious requirements—that the sample be placed in a sample vial and headspace be developed—the GC technique itself can impose additional sample preparation requirements, including dry purging, derivatization of polar compounds, and headspace enrichment. These factors, coupled with the time it takes to separate the compounds in the column, can have a marked effect on sample throughput.

In recent years, direct sample analysis techniques have become available that provide high specificity without the need to utilize chromatographic separation. The most effective of these are based on direct‑injection mass spectrometry (DIMS), which use soft chemical ionization coupled with mass spectrometry (MS) to provide specific analysis. Where it is applicable to the matrix and analytes, DIMS methods can simplify sample preparation and increase sample throughput, providing lower cost per sample.

Using selected ion flow tube mass spectrometry (SIFT-MS; see [2]) as representative of DIMS techniques developed for gas-phase VOC analysis, this article describes the fundamental differences between DIMS and GC, and considers briefly how these impact on sample preparation requirements for headspace analysis using DIMS.

DIMS and Gas Chromatography: Key Differences

Elimination of the chromatographic column in DIMS techniques is the most obvious difference compared with GC and is addressed first. The second and more subtle point is that suitable ionization must be utilized to enable DIMS to be applied. Here, the principles are illustrated using the soft chemical ionization applied in SIFT-MS.

DIMS analysis is chromatography‑free. In DIMS techniques, the sample is continuously delivered to the ionization region of the instrument, so there is no means by which a solvent front or matrix VOCs can be switched out. This must be addressed through use of compatible (low reactivity) solvents and ionization agents—so-called 

 in SIFT-MS. This is addressed in the next section.

In GC, rapid headspace injection, whether from a pressurized sample loop, syringe, cryotrap, and so on, is used to provide the focused peak shape required for good chromatographic separation. This approach is ill-suited to DIMS techniques, where the best result is obtained when headspace is introduced into the DIMS instrument continuously at a steady flow rate for simultaneous ionization and detection of the analytes. To date, this has been most effectively achieved by integrating syringe‑injection autosamplers with SIFT-MS instruments, providing high repeatability even when no internal standard is utilized (2)—in contrast to the potentially poor repeatability when large headspace volumes are injected into a GC inlet liner. Figure 1 shows a headspace injection into a SIFT‑MS instrument, where the instrument is continuously analyzing sample during the slow, steady sample introduction. The figure shows the concentration generated as a function of time, with the reported value taken from the mean over the injection. Note that solid-phase microextraction (SPME) and similar headspace preconcentration approaches are not so well suited to DIMS due to small capacity and rapid sample desorption.

DIMS techniques with very soft ionization are most suitable. In DIMS instruments, removal of the chromatographic column shifts responsibility for achieving specific analysis entirely to the combined ionization and detection system. Since electron ionization (EI) is too harsh to provide specific analysis without chromatography, soft chemical ionization is typically utilized because minimal fragmentation occurs for most VOCs. In SIFT‑MS instruments, three positively charged reagent ions (H3O+, NO+, O2+) and up to five negatively charged reagent ions (OH-, O-, O2-, NO2, NO3-) are available. These reagent ions provide highly sensitive analysis of most VOCs, and they have very efficient ionization mechanisms, resulting in non-discriminatory sample analysis—even for underivatized volatile fatty acids. Analysis is achieved directly from air because the ions are “blind” to the bulk components, namely oxygen, nitrogen, argon, and carbon dioxide in positive ion mode, and they react only very slowly with water.

Specific analysis is obtained by the use of reagent ions that usually ionize a given VOC via different mechanisms—the relevance of a given mechanism being dependent on the functional group (3) and molecular weight of the VOC—and the fact that, uniquely in SIFT‑MS, these ions are switchable in real time (Figure 1[b]).

Matrix Compatibility and Solvent Selection

Continuous flow of sample through the DIMS instrument with no temporal pre‑separation of components creates some special challenges for transfer of methods from chromatographic techniques. The overarching principle that needs to be followed for quantitative analysis is that the bulk of the headspace must be non‑reactive—at least with one reagent ion. Although it varies based on composition and the relative sensitivities of each reagent ion, for commercial SIFT‑MS instruments a typical upper limit to the total headspace concentration for all reactive compounds is 100 parts‑per‑million by volume (ppmV). This headspace could be dominated by solvents and/or matrix compounds.

Solvents—whether used for extraction, derivatization, preconcentration, or in the creation of calibration standards—need to be selected with care in SIFT-MS. In contrast to GC, water is the preferred solvent for SIFT-MS because its reagent ion reactions are very slow (4). For organic solvents, at least one reagent ion must have significantly lower sensitivity to the solvent and/or the headspace partitioning of the solvent needs to be poor. Typically, an intermediate dilution step (in water) is also required (2). Work is currently underway to quantify the maximum proportion of organic solvent in water that headspace-SIFT-MS can accommodate under the standard headspace conditions described in Perkins and Langford (2021) (4). For dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), methanol (using NO+ only), and triacetin, from 5
to 10% solvent in aqueous solution appears workable.

Assessment of the sample matrix takes a similar approach. If the headspace composition is not known—whether for solutions or the solid phase, such as polymers—untargeted analysis assists identification of matrix species and assessment of the impact that these will have on quantitative SIFT-MS analysis. If total concentrations are likely to exceed the SIFT‑MS instrument’s dynamic range, then dilution, for example, through slower headspace injection or dilution in aqueous, can be used. Clearly, limits of quantitation will be reduced proportionately.

Headspace Enrichment, Preconcentration, and Drying

SIFT-MS quantifies VOCs to sub part‑per‑billion concentrations (by volume, ppbV) direct from headspace without preconcentration. Given the robustness of SIFT-MS analysis to water, analytes that partition readily to headspace from aqueous solution can usually be analyzed to low ppb concentrations in water without requiring dry purging, enrichment, or preconcentration. Hence the use of dynamic headspace analysis, purge-and-trap, or “workaround” approaches, such as dispersive liquid–liquid microextraction, can usually be avoided by implementing DIMS. Figure 2 shows linearity data obtained for benzene, toluene, ethylbenzene, and the xylenes (BTEX) measured directly from unmodified aqueous solution without the use of an internal standard (2). Relative standard deviations (RSDs) less than 10% were obtained over six replicates in the 1–9 μg/L concentration range in solution.

The differences between DIMS and GC approaches are presented.

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