Confidence in Your Cannabis: Exploring New Horizons

What Are My Options to Improve My Separation? 

A Look at Immersive Sorptive Extraction, GC×GC–TOF-MS, and Chemometrics

Comparing the Flavour Profiles of Soft Drink Brands 

Rising Stars of Separation Science

The Rise of Automation—Evolve or Die?

High Performance Liquid Chromatography Analysis of Cinnamon from Different Origin

Tony Taylor is Group Technical Director of Crawford Scientific Group and CHROMacademy. His background is in pharmaceutical R&D and polymer chemistry, but he has spent the past 20 years in training and consulting, working with Crawford Scientific Group clients to ensure they attain the very best analytical science possible. He has trained and consulted with thousands of analytical chemists globally and is passionate about professional development in separation science, developing CHROMacademy as a means to provide high-quality online education to analytical chemists. His current research interests include HPLC column selectivity codification, advanced automated sample preparation, and LCâMS and GCâMS for materials characterization, especially in the field of extractables and leachables analysis.

On December 1 2022, the newly harmonized General Chapter <621> (Chromatography) of the United States Pharmacopeia (USP) became official. The British Pharmacopeia (BP), European Pharmacopeia (EP), and Japanese Pharmacopeia (JP) followed suit in January 2023.

Most chromatographers that use pharmacopeial methods will be aware of this chapter, and the various updates to the “System Suitability” subsection titled, “Adjustment of Chromatographic Conditions,” which have been released since 2009. Initially introduced to allow adjustments to chromatography conditions to meet system suitability requirements, this section has evolved to allow more widespread changes. These allowable changes can also be used to modernize monograph methods which use older HPLC columns to shorten analysis time, improve method performance, and reduce solvent usage. This is highly attractive not only to meet analytical throughput requirements, but to reduce the environmental impact of laboratory methods, which is becoming increasingly important.

I’ve previously written on various updates to this chapter (1,2) and given illustrative examples of how allowable changes may be implemented, but in this case, I want to focus on a significant change in the allowable changes to the column dimension and particle size changes which are now allowed with gradient chromatography methods, introduced in the latest update to the chapter in December 2022.

Before we get to this discussion, I want to draw attention to some important considerations for any chromatographer adjusting Pharmacopeial methods.

The Adjustment of Chromatographic Conditions section outlines various allowable changes to Monograph methods and clearly states that changes outside of these ranges require full method revalidation. It is important to highlight that the section also states:

Multiple adjustments can have a cumulative effect on the performance of the system and are to be properly evaluated by the users.

If adjustments are made to a pharmacopeial procedure, additional verification tests may be required.

Compliance with the system suitability criteria is required to verify that conditions for satisfactory performance of the test or assay are achieved.

Adjustment of conditions with gradient elution HPLC is more critical than with isocratic HPLC, since it may shift some peaks to a different step of the gradient, potentially causing partial or complete coelution of adjacent peaks or peak inversion, and, thus leading to the incorrect assignment of peaks and to the masking of peaks or a shift such that elution occurs beyond the prescribed elution time.

These caveats are very important, especially where we are making several changes to a monograph method to take advantage of more modern HPLC technologies such as smaller particles, superficially porous particles (SPPs) and the attendant reduced column dimensions. In fairness, there will be few chromatographers working with Pharmacopeial methods who would not undertake verification of the adjusted method, even if full revalidation is not mandated. Parameters which may be affected, and therefore need verification within the adjusted method, will depend on the nature of changes made, the purpose of the procedure (that is, assay or stability indicating method), and the ability of the system suitability requirements to verify that affected method characteristics remain valid. For assistance in deciding which criteria may be affected, one might refer to General Chapter <1226> Verification of Compendial Procedures. As stated in the caveats above, this is most important where adjustments to gradient methods are made because of the complexity of the retention mechanism. It is also important to remember that for gradient HPLC methods, further system suitability requirements are specified within the chapter, but more on that later. Lastly, it’s also important to remember that the allowable changes are all relative to the original monograph and not the version of the method in your own standard operating procedures (SOPs). Your in-house methods may have already had changes made against the original monograph and any further changes to these methods may take your overall adjustments beyond those of the allowable changes.

Table I below shows a summary of the allowable changes to monograph methods as defined within USP General Chapter <621>.

Table I: A summary of the allowable changes to monograph methods as defined within USP General Chapter <621>.

The most significant change in the latest update to General Chapter <621> is the possibility to alter the L (column length)/d

 (particle diameter) ratio for gradient elution separations. As such, it’s worthwhile demonstrating how one might go about calculating the changes for a gradient elution HPLC monograph method, and the determination of organic impurities in Olanzapine formulations is a convenient example to use.

The monograph method for olanzapine impurity determination includes the following details:

Buffer: 68 mMol Sodium Dodecyl Sulphate (pH 2.5)

Mobile phase gradient: (see Table II below)

Table II: The mobile-phase gradient for solution A and solution B.

Column: 4.6 mm x 25 cm; 5-mm packing L7 (Octyl Silane (C8) chemically bonded to porous silica)

Resolution: NLT 3.0 between olanzapine related compound A and olanzapine

Tailing factor: NMT 1.5 for the olanzapine peak

Relative standard deviation (RSD): NMT 2.0% from four replicate injections for the olanzapine peak

Typically, the first decision to make is the new column dimensions that one might like to use and to investigate if the target column dimensions are available with the stationary phase you are using.

 ratio of the monograph method column is 50,000. So we need column length to particle size ratio columns which lie in the range 37,500 to 75,000.

Some widely available column dimensions which fall within the allowable range include:

Let’s assume we wish to significantly speed up this method and we select column c) 75 mm x 2.1 mm x 1.9 mm (also assuming that we are using an HPLC system which can handle higher back pressures and is optimized for low dwell volume).

We can then adjust the flow rate to give an equivalent linear velocity for the new column dimensions:

Each segment of the gradient timetable is then adjusted as such:

Given the reduction in column volume, it is unsurprising that the gradient times are very much reduced compared to the original monograph method. However, as I mentioned above, one needs to ensure that the system on which the adjusted method is run can repeatably achieve a 100% change in eluent composition in 1.2 min (that is, only very low dead volume, dwell volume systems will be capable of this).

The reduction in column volume will also necessitate a reduction in injection volume, which is calculated as follows:

A side-by-side comparison of the adjusted parameters is shown below in Table III.

Table III: A side-by-side comparison of the adjusted parameters.

In practice, the new conditions would be used for the separation and a view taken on how feasible the method is, typically by assessing several repeat injections against the system suitability criteria. If the criteria can be satisfied, and especially where the method is changed to the extent we suggest above, method verification should be performed on the critical criteria of the method. Typically, this might include specificity (especially in the case of adjustments to gradient HPLC methods), selectivity, precision, intermediate precision, and sensitivity (limit of detection), but these are merely suggestions, and one would need to identify the critical criteria for the method under adjustment and the conditions that are altered. Specifically for gradient elution HPLC methods, General Chapter <621> also requires:

Where the column dimensions are unchanged that the principle peaks elute within ±15% of their respective retention time in the original method

Any changes to the composition of the mobile phase and/or gradient timetable ensure that the first peaks are sufficiently retained, and the last peaks are eluted

My last point is around the allowable changes to the stationary phase used for the adjusted method in which changes are allowed, provided that the new phase is within the same USP classification (L1, L7, etc.). Most readers will fully appreciate that the selectivity of various phases within these classifications can vary enormously. At the last count, I noted over 200 phases, which purported to fall within the L1 classification! To maximize chances of success, one would be advised to stick to the same manufacturers stationary phase, as undoubtedly the transfer between the different column dimensions and/or particle size and morphology is more likely to be scalable; however, that being said, bonding methods and surface coverage should be checked to ensure that they fall within the allowable changes as outlined within General Chapter <621> (that is, that the surface modification, chromatographic support, and extent of surface modification should be similar). The chapter explicitly states that the change from fully porous to SPPs is allowed, provided that the previous criteria can be met; however, one would need to be mindful regarding the extent and type of surface modification when changing particle morphology as the manufacturer may adjust these parameters between the fully porous and SPPs to optimize column performance.

Of course, if one did wish to change column manufacturer, provided one could satisfy the requirements above, then this is possible. However, to save a great deal of time and heartache, you may wish to refer to one of the several stationary phase characterization tools that exist to help justify this change, and to choose columns which are similar in terms of their primary selectivity characteristics.Such tools are described in the literature (3,4). Working with your column vendor can also help greatly when considering such changes.

It should go without saying that the effort required to verify equivalence of the adjusted method should be very carefully considered when changing between column manufacturers.The potential for retention and selectivity differences is higher and therefore the likelihood of a simple scaling of method parameters as described above, less likely to succeed out of the box.

In summary, the new General Chapter <621> does reflect the great work of the Pharmacopoeias in helping chromatographers to use more modern technology for their regulated methods, speeding up analysis, and reducing waste. The latest revision allows us to adjust column dimensions for gradient separations, which is a significant advance. Although these freedoms are welcomed, one must be very mindful of the requirements or limits of the allowable changes and the verification work that underpins our demonstration of equivalent performance of the adjusted method. Although in theory these changes avoid the necessity for full revalidation of the analytical method, it is often that when making extensive changes to the method, the verification work required will approach that of a full revalidation. The end user must assess the benefits of change versus the work required to prove method performance equivalence.

https://www.crawfordscientific.com/chromatography-blog/post/usp-chapter-621-changes

https://www.chromatographyonline.com/view/lcgc-blog-shape-things-come-possible-changes-usp-chapter

https://apps.usp.org/app/USPNF/columns.html

 is the Chief Scientific Officer of Arch Sciences Group and the Technical Director of CHROMacademy. His background is in pharmaceutical R&D and polymer chemistry, but he has spent the past 20 years in training and consulting, working with Crawford Scientific Group clients to ensure they attain the very best analytical science possible. He has trained and consulted with thousands of analytical chemists globally and is passionate about professional development in separation science, developing CHROMacademy to provide high-quality online education to analytical chemists. His current research interests include HPLC column selectivity codification, advanced automated sample preparation, and LC–MS and GC–MS for materials characterization, especially in the field of extractables and leachables analysis.

Articles

The new General Chapter <621> does reflect the great work of the Pharmacopoeias in helping chromatographers to use more modern technology for their regulated methods, speeding up analysis, and reducing waste. The latest revision allows us to adjust column dimensions for gradient separations, which is a significant advance. Although these freedoms are welcomed, one must be very mindful of the requirements or limits of the allowable changes and the verification work that underpins our demonstration of equivalent performance of the adjusted method.

Allowable Changes to Chromatography Methods for HPLC – More Freedom for Chromatographers Granted by the Pharmacopoeias?

Katerina (Kate) Mastovska has been named the Deputy Executive Director and Chief Science Officer of AOAC International, a nonprofit organization that serves to ensure the safety of foods and other products that impact public health. She will join the organization on January 30.

Mastovska has been an active member of AOAC International since 2004 and received the association’s highest scientific honor, the Harvey W. Wiley Award, in 2021. She has extensive experience in research chemistry—working for the University of Chemistry & Technology in Prague, the U.S. Department of Agriculture, and founding her own independent consulting business, Excellcon International. In 2009, she joined Covance Laboratories, where her responsibilities grew rapidly. Following the acquisition of Covance Food Solutions by Eurofins Scientific, Mastovska continued to serve in roles of increasing importance, most recently as the Chief Scientific Officer of the Eurofins US Food Division.

AOAC International, founded in 1884, is an independent, third party, not-for-profit 501(c)(3) association that develops voluntary consensus standards. The 

 database is used by food scientists around the world to facilitate public health and safety and to promote trade.

In addition, two current AOAC staff members, each with almost 20 years of experience at AOAC, Dawn L. Frazier and Deborah McKenzie, have been promoted.

Frazier has been promoted to the position of Deputy Executive Director of Engagement. Previously she served as the Senior Director of Membership, Marketing, and Communications. Her responsibilities include leading and implementing the organization’s engagement strategy, which includes developing and maintaining relationships with key stakeholders and partners, as well as overseeing outreach and communication efforts — all to achieve the organization’s strategic plan.

McKenzie has been promoted to the new role of Deputy Assistant Executive Director and Chief Standards Officer. McKenzie was previously the Senior Director of Standards and Official Methods. Her responsibilities include overseeing implementation and execution of voluntary consensus standards processes and the official methods. She and her team coordinate and administer associated activities with standards development and method approval programs.

Katerina Mastovska Named AOAC International Chief Scientific Officer

interviewed Varoon Singh from the National Dope Testing Laboratory in India, about his work in sample preparation using chromatography with mass spectrometry (MS), and the development of novel materials for analytical applications.

Videos

The Column interviewed Varoon Singh from the National Dope Testing Laboratory in India, about his work in sample preparation using chromatography with mass spectrometry (MS), and the development of novel materials for analytical applications.

Rising Stars of Separation Science: Varoon Singh

Chris Burgess, PhD, is an analytical scientist at Burgess Analytical Consultancy Limited, ‘Rose Rae,’ The Lendings, Startforth, Barnard Castle, Co Durham, DL12 9AB, UK; Tel: +44 1833 637 446; chris@burgessconsultancy.com; www.burgessconsultancy.co

The scope of data integrity is shown by the 

 (seen in the figure below), which sits within the overall pharmaceutical quality system of a regulated laboratory. Analysis takes place at Level 3 in this model, but to ensure both data quality and integrity it is imperative that the elements of the Foundation and Levels 1 and 2 are in place. The analogy of this model is building a house.

: A house starts with the foundations; otherwise the building falls down. The data integrity model also starts with a Foundation comprising management leadership, quality culture, and ethos, using trained and competent staff. To ensure that the Foundation works, it is essential to keep up to date with current regulatory trends—monitoring both new regulatory guidance documents and trends in non-compliance.

 At Level 1 is the right analytical instrument and computerized system for the job. Here, a regulated laboratory should be aware of the trends in qualification of analytical instruments, such as the proposed changes in 

Analytical Instrument and System Qualification (AISQ

Also at Level 1 is computerized system validation (CSV), which is often thought of as being a regulatory burden rather than a business benefit. FDA is proposing changing CSV to computer system assurance (CSA), but is this new approach really needed? Is there the flexibility already available in current regulations and industry guidance? And is the conservative nature of the pharmaceutical industry as much of a barrier as the regulations?

 Level 2 is the right analytical procedure for the right job. 

 chapter <1220> on analytical life cycle management presents a different way of validating analytical procedures compared with ICH Q2(R1). In 

 <1220> method development is the critical component of the life cycle and a scientifically sound approach to the design of the analytical procedure begins with the defining the analytical target profile (ATP).

It is critical that the Foundation and Levels 1 and 2 are in place and effective, otherwise any analysis work performed at Level 3 will have data integrity vulnerabilities.

For more description of the data integrity model, please see the following articles:

McDowall, R. D. Understanding the Layers of a Laboratory Data Integrity Model. 

https://www.spectroscopyonline.com/view/understanding-layers-laboratory-data-integrity

McDowall, R. D. Data Integrity Focus, Part 1: Understanding the Scope of Data Integrity. 

https://www.chromatographyonline.com/view/data-integrity-focus-part-1-understanding-scope-data-integrity

The scope of data integrity is shown by the data integrity model, which sits within the overall pharmaceutical quality system of a regulated laboratory. 

The Data Integrity Model

Chiral separation is challenging and requires thorough method optimization. Selectivity plays a critical role in chiral separation. In this study, various ways of achieving resolution by playing with selectivity are presented. This resolution improvement can be realized from the stationary phase—the type of polysaccharide, nature of the chiral selector, position of the functional group in the chiral selector—the mobile phase composition, and from temperature. To optimize such challenging separations, it is important to explore all of these parameters through a chiral screening process across various chiral column chemistries.

Chirality is the potential of a molecule to occur in two asymmetric forms (enantiomers) that are non-superimposable mirror images of each other without changing the atomic composition, atom­-atom connections, or bond orders. Chirality is a property of the whole molecule, but the cause of chirality is the chirality centre (asymmetric centre) within the molecule. It is important for an active chiral ingredient to be in one chiral form for a few reasons—the other chiral isomer can be inactive in the biological system or can have adverse effects. A good example of the adverse effects of a chiral isomer is described in the case of thalidomide (1–7), where pregnant women treated with this drug gave birth to children with birth defects due to the (S)‑enantiomer being teratogenic. To separate chiral isomers and quantify the amount of each isomer in a given mixture, a column that can recognize small spatial differences between one chiral isomer and the other is required. Many column phases have been developed to attempt to separate enantiomers and stereoisomers. This includes ligand exchange, protein-based, chiral crown ether‑based, cyclodextrin‑based, pirkle‑type, polysaccharide‑based, macrocyclic glycopeptide‑based, and cinchona alkaloid‑based phases. The most popular column currently used for this purpose is a polysaccharide-based chiral stationary phase (CSP). There are several advantages to using a polysaccharide-based CSP. Polysaccharide CSPs have a wide chiral recognition and selectivity, multiple chiral selectors, can be easily manipulated to reverse elution order, high pressure stability, high loading capacity, high efficiency, and—most importantly—scalability from analytical to preparative separation. Polysaccharide-based CSPs are also amenable to various separation modes such as reversed phase, normal phase, polar organic, and supercritical fluid. For the scope of this study, only chiral separation from polysaccharide stationary phases will be discussed.

Resolution improvement in chromatography can be brought about by three different factors: efficiency term, selectivity term, and retention term. Out of these three, selectivity is the most influential term to bring drastic improvement in resolution. Selectivity for chiral separation can be realized from three main sources: the stationary phase, the mobile phase, and temperature. Within the stationary phase, selectivity differences arise from the type of polysaccharide and type of selector. Amylose or cellulose are the common polysaccharides used in chiral stationary phases and bring a difference in selectivity. The nature and the position of the functional group can have a huge impact on the selectivity and eventual resolution of chiral isomers. This will be discussed in detail in the following sections.

Selectivity can come from the polysaccharide chosen for the stationary phase. Cellulose produces a tight, layered structure, whereas amylose has a more helical structure to help separate enantiomeric analytes (Figure 1). For example, tebuconazol was run on both a cellulose- and amylose-based chiral column with the same chiral selector. Figure 2 shows that enantiomers of tebuconazol can be separated depending on the polysaccharide. On the cellulose-based column, there was only a single peak that eluted. However, the helical structure of amylose allowed for the separation of the enantiomeric pair of tebuconazol and resulted in two peaks.

The chiral selector that is attached to the polysaccharide base can also affect the selectivity of a column. The chiral selector directs analytes towards the sugar moiety, thereby allowing for more specific selectivity depending on the analyte. As an example, fenarimol was analyzed using four cellulose stationary phases, each with a different chiral selector. Figure 3 shows how, depending on which chiral selector is used, the separation and retention times can be very different.

The enantiomers of diniconazole have very different separation or no separation at all depending on which stationary phase and chiral selector are used. Finally, the position of the side groups can also affect the selectivity of analytes. When analyzing myclobutanil, a simple position change of the methyl group allowed for the enantiomers to be separated completely (Figure 4).

The content and composition of the mobile phase can also affect the selectivity of a chiral separation. The addition of an additive or adjusting the concentration of an additive can not only affect the separation of enantiomers but can also change the elution order. This can be seen with Fmoc-N-Isoleucine separation. As the concentration of formic acid (FA) increased in the mobile phase, the two enantiomers eluted together to form one peak, and then changed elution order, with the L- form eluting first with 0.5% FA in the mobile phase. This same shift was seen when FA was maintained and the ratio of the other two components of the mobile phase were changed. As the percentage composition of hexane increased, the selectivity change was clearly visible from the flip in elution order of enantiomers of Fmoc‑n-Isoleucine (Figure 5).

Selectivity can be enhanced by the addition of a mobile phase additive. For example, an analyte with three chiral centres (eight isomers) was analyzed. Under the current conditions with a two-component mobile phase, one of the critical pairs was not separated. However, with the addition of a third component to the mobile phase—the addition of 6% dichloromethane to the water and acetonitrile mixture—brought the critical pair resolution. The use of a chlorinated solvent such as chloroform or dichloromethane are allowed only on specialized phases (Figure 6).

Temperature change can bring a drastic change in selectivity in chiral separation. Fmoc-N‑Isoleucine was further explored for the effect of temperature. The stationary phase and mobile phase were maintained, but the temperature was increased. At 5 °C the enantiomers were fully resolved, with the D-form eluting first. As the temperature was increased, the enantiomers were coeluted, and then finally a reversal of elution order was seen at 50 °C. The peak shape and peak efficiency were higher at higher temperatures (8).

Finally, the combination of the mobile phase and the chiral stationary phase can affect selectivity of enantiomers. The same mobile phase was used with different stationary phases to analyze Fmoc-N-Isoleucine, giving different peak shapes, separation, and elution order. It is advantageous to screen multiple phases for a higher chance of success and to obtain the desired order of elution.

When screening a specific chiral isomer by normal, reverse, and polar organic mode, the initial screening in normal phase with one specific polysaccharide column does not resolve the enantiomers; however, with reversed‑phase mode, the compounds were separated on the same column. Chiral separation is a three‑dimensional separation, unlike traditional reversed-phase separation. In reversed-phase chromatography using a C18 column, highly probable predictions can be made by looking at the structure of molecules, their hydrophobicity difference, and log 

 (octanol-water partition coefficient). However, with chiral separation, one should screen analytes through multiple phases and multiple modes to get the optimal resolution (9). Once the screening results are explored and the best separation is identified, further fine-tuning can then be done to optimize the method for the ideal separation (8).

Chiral separation is three-dimensional, and the resolution of a given separation can be improved by optimizing the selectivity. Selectivity in chiral separation can come from the mobile phase, the stationary phase, and the temperature of the analysis. To obtain high resolution and the right peak order that meets the goal of the analysis, it is important to consider optimizing all these aspects of selectivity before settling on the separation. Chiral screening is a prudent approach to explore the selectivity aspects of chiral separation.

Burley, D. M.; Lenz, W. Thalidomide and Congenital Abnormalities. 

, 271–272. DOI: 10.1016/S0140-6736(62)91217-5

Lenz, W. A Short History of Thalidomide Embryopathy. 

, 203–215. DOI: 10.1002/tera.1420380303

Stephens, T. D. Proposed Mechanisms of Action in Thalidomide Embryopathy. 

, 229–239. DOI: 10.1002/tera.1420380307

Bartlett, J. B.; Dredge, K.; Dalgleish, A. G. The Evolution of Thalidomide and its IMiD Derivatives as Anticancer Agents. 

Wu, J. J.; Huang, D. B.; Pang, K. R.; Hsu, S.; Tyring, S. K. Thalidomide: Dermatological Indications, Mechanisms of Action and Side-Effects. 

, 254–273. DOI: 10.1111/j.1365-2133.2005.06747.x

Melchert, M.; List, A. The Thalidomide Saga. 

, 1489–1499. DOI: 10.1016/j.biocel.2007.01.022

Chankvetadze, B.; Farkas, T.; Peng, L.; et al. Enantiomer Elution Order Reversal of Fluorenylmethoxycarbonyl-Isoleucine in High-Performance Liquid Chromatography by Changing the Mobile Phase Temperature and Composition. 

, 6554–6560. DOI: 10.1016/j.chroma.2011.06.068

Chiral HPLC/SFC Method Development Poster

. https://www.phenomenex.com/ViewDocument?id=chiral+hplc_sfc+method+development+poster&fsr=1 (accessed 2022-12-19).

 has been in the chromatography industry for over 20 years and has hands-on and troubleshooting experience in chromatography. He has earned a master’s degree and doctoral degree in analytical chemistry from Seton Hall University, New Jersey, USA, with specialization in microextractions, multidimensional gas chromatography, and tandem mass spec techniques. He has developed and validated several regulatory compliant methods in the pharmaceutical, food, and environmental industries. He joined Phenomenex in August 2014 and serves as Global Product Manager-Gas Chromatography at Phenomenex, USA.

 earned a bachelor’s degree in biochemistry and genetics from Texas A&M University (Texas, USA) and a doctoral degree in translational biology and molecular medicine from Baylor College of Medicine (Texas, USA). He joined Phenomenex in September of 2020 and he serves as Technical Marketing Writer at Phenomenex, USA.

In this study, various ways of achieving resolution by playing with selectivity are presented. 

Playing with Selectivity for Optimal Chiral Separation

Immersive sorptive extraction coupled with comprehensive two-dimensional gas chromatography and time-of-flight mass spectrometry (GC×GC–TOF-MS) was used to compare flavour profiles from popular brand soft drinks with those of imitation products.

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.

Unlike counterfeit goods, replica and imitation products are legal because they do not use the branded product’s trademark. In the food and beverage industry, imitation products attempt to mimic the taste experience of popular branded products.

However, flavour profiles are extremely complex and consist of a broad range of chemical classes, the combination of which ultimately determines the consumer’s preference for a particular brand. It is important to be able to confidently identify these volatiles during product development, as well as in quality and authenticity studies.

Traditional sample preparation methods, such as headspace and solid-phase microextraction (SPME), are widely used but are often limited in terms of sensitivity. Additionally, sampling is generally restricted to the headspace volatiles, meaning that there is a bias towards nonpolar analytes. This article demonstrates the use of high‑capacity sorptive extraction with novel trap-based focusing to provide enhanced sensitivity and improved chromatographic performance, as well as the ability to perform both headspace and immersive sampling for compatibility with a wider range of polar and semi-volatile analytes. This improved performance, coupled with improved separation by comprehensive two-dimensional gas chromatography (GC×GC) and detection by time-of-flight mass spectrometry (TOF-MS), gains greater insight into sample composition.

However, sampling, separation, and detection is just the beginning—the resulting datasets must then be reduced to discover significant differences and ultimately allow meaningful conclusions to be reached. This article will also demonstrate the use of chemometrics to help transform complex datasets into usable results. The approach used ensures that trace peaks are not ignored and enables automated workflows to be adopted,. This workflow will be presented for the comparison of brand and imitation cola‑flavoured soft drinks.

Five store-bought cola-flavoured soft drinks were purchased from different suppliers. Sampling and analysis were performed in triplicate for each soft drink.

The following experimental conditions and instrumentation were used. For immersive sampling, HiSorb high-capacity sorptive extraction probes were used (PDMS/CWR/DVB, Markes International). Full automation was performed on the Centri extraction and enrichment platform (Markes International). GC×GC: Insight flow modulator (SepSolve Analytical); modulation period (PM) = 2.5 s. TOF-MS: BenchTOF2 mass spectrometer (SepSolve Analytical); mass range: 

= 40–350; ionization energy: tandem ionization at 70 eV and 14 eV. Software: ChromSpace software (SepSolve Analytical) for full instrument control and processing was used, with chemometric comparisons by ChromCompare+ (SepSolve Analytical)

Coupling high-capacity sorptive extraction with GC×GC–TOF-MS ensured that a wide range of chemical classes were efficiently separated and confidently identified.

The additional chemical detail provided on the composition of soft drinks is clearly evident in Figure 1. Four compounds are highlighted that would have coeluted in a conventional one-dimensional (1D) analysis but have been separated in the second dimension and identified confidently using the spectral quality and mass accuracy of TOF-MS (Figure 2).

In this study, five “cola” soft drinks from different manufacturers were analyzed using this workflow. To determine the differences in composition between each brand of soft drink, a nontargeted, tile‑based workflow was then applied in the software to compare all of the raw data, and the most significant differences between the sample classes were identified automatically.

The resulting principal component analysis (PCA) score plot (Figure 3) shows distinct clustering of the different brands. Interestingly, brands B and E are both “diet” soft drinks from different manufacturers and cluster separately from the “zero sugar” brands.

Benzyl benzoate was found to be a key differentiator of brand D (Figure 4, left) and likely contributed a balsamic, fruity flavour. Additionally, trans-cinnamaldehyde (Figure 4, right) was found to differentiate the “diet” and “zero sugar” classes.

Volcano plots in the software were also used to directly compare the imitation colas against popular brand products—an example of which is shown in Figure 5.

Figure 5 also shows the reference-quality spectra of the MS analysis. This helped to ensure confident identification of potential markers of brand and/or imitation cola products. It should also be noted that tandem ionization data were acquired in this study at 70 eV and 14 eV. The tandem data were used to confirm positive hits, improving the discovery of subtle, trace differences by reducing the frequency of false positives (2).

This study has demonstrated an end‑to‑end nontarget workflow for aroma profiling of food and beverages comprising fully automated immersive sampling of wide‑ranging aroma-active volatiles using high-capacity sorptive extraction. Enhanced separation by GC×GC using consumable-free flow modulation obtained comprehensive aroma profiles. Sensitive detection and confident identification of analytes were achieved using TOF‑MS. Chemometrics offered automated workflows for alignment and comparison of complex chromatograms.

The Good Scents Company Information System (search facility)

. http://www.thegoodscentscompany.com/search2.html (accessed 2022-06-20).

SepSolve Analytical White Paper 041: Improving discovery workflows using Tandem Ionisation data

. https://www.sepsolve.com/white-papers/overview/technical-note-improving-discovery-workflows-using-tandem-ionisation-data.aspx (accessed 2022-12-19).

 received an M.Chem. in chemistry from the University of St Andrews, UK, followed by an M.Sc. in forensic science at the University of Strathclyde, UK. Her Ph.D. in environmental forensics, also at the University of Strathclyde, focused on the chemical fingerprinting of environmental contamination using advanced techniques such as GC×GC−TOF-MS. In her current role at SepSolve Analytical, she specializes in the application of GC×GC and TOF-MS to challenging applications.

 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.

 spent nine years working within an environmental analytical laboratory, where he was responsible for method testing, development, and implementation. In his current role at SepSolve Analytical, James supports customers through the development, demonstration, and handover of analytical methods across the company’s portfolio of instruments and software.

Comparing the Flavour Profiles of Soft Drink Brands Using Immersive Sorptive Extraction, GC×GC–TOF-MS, and Chemometrics

This month we interview Martina Catani from the University of Ferrara, Italy, about her work on the separation of enantiomers by high performance liquid chromatography/ultrahigh-pressure liquid chromatography (HPLC/UHPLC) and the study of the kinetic and thermodynamic properties of chiral stationary phases, and the separation and purification using LC analysis (single-column and continuous multi-column) of biomolecules of high therapeutic interest.

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

The first time I encountered chromatography was during my bachelor’s degree in chemistry. Even though at that time I was only studying it from a theoretical point of view, I became fascinated by the great versatility of this technique and its capability for understanding the composition of unknown complex mixtures. However, the moment at which I said, “OK, this is what I would like to do for the rest of my life” was during my master’s thesis. My work was focused on the design of enzymatic packed‑bed microreactors for the enantioselective formation of C–C bonds in aqueous environment. I had the opportunity to functionalize silica particles with the enzyme, to pack the columns with this adsorbent, and to fully characterize the performance of the microreactor not only in terms of catalytic conversion but also from a chromatographic point of view. In addition, since the scope of the work included the evaluation of the enantiomeric excess of the products, I also performed several enantioseparations by using a chiral column in gas chromatography (GC) and I started developing a passion for separating enantiomers (a topic that is still dominating a great part of my work).

During that period, I started seriously thinking about the idea of pursuing a Ph.D. in analytical chemistry—especially based on chromatography.

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

 I started my Ph.D. in 2014 at the University of Ferrara under the supervision of Alberto Cavazzini. My Ph.D. project was mainly focused on the investigation of theoretical aspects of liquid chromatography (LC), including mass transfer characteristics and thermodynamic properties of different kinds of porous media commonly used for ultrafast, high efficiency separations. I considered not only achiral adsorbents (such as C

 or perfluorinated stationary phases) but also (and especially, I would say) chiral ones.

In this context, I performed innovative studies to unravel the complex mass transfer phenomena dominating kinetics of chiral chromatography and I also investigated the thermodynamics of adsorption through nonlinear chromatographic means (namely, 

). In particular, these investigations were devoted to the understanding of the impact of kinetic aspects (adsorption–desorption kinetics and eddy dispersion), particle geometry (fully porous vs. core–shell), and physicochemical characteristics (specific loading of chiral selector, pore size) on the performance of these columns.

These studies not only opened up new perspectives in this field by reviving the debate about the best particle format for ultrafast separation, but they have also been the basis for the design, characterization, and operation of high‑efficient columns for ultrafast chiral chromatography. The paper (written in collaboration with the group of Francesco Gasparrini of the University of Rome “La Sapienza”) for the realization of ultrafast separations where the enantiomers of some compounds of pharmaceutical interest were separated in less than one second with extremely high efficiency should be noted (1).

Q. What chromatographic techniques have you worked with?

I have mainly worked with high performance liquid chromatography/ultrahigh-pressure liquid chromatography (HPLC/UHPLC) coupled with different detection techniques such as UV–vis diode array, mass spectrometry, fluorescence, or refractive index detection. I have applied different chromatographic modes such as normal phase, reversed phase (including separations with perfluorinated and mixed-mode adsorbents), hydrophilic interaction liquid chromatography (HILIC), ion-exchange, affinity, and chiral chromatography.

I have also worked with preparative liquid chromatography—both single‑column and multi-column countercurrent. More recently, I have also worked with supercritical fluid chromatography (SFC) coupled with UV–vis diode array detection, and, as I mentioned earlier, I had the chance to work with GC coupled with flame ionization detection (FID) during my master’s thesis.

Q. Your research focus currently lies in the separation of enantiomers by HPLC/UHPLC and the study of the kinetic and thermodynamic properties of chiral stationary phases—what specifically attracted you to this area of research?

As I mentioned earlier, I have always been fascinated by the power of chromatography to separate enantiomers. In particular, I am stimulated by the fact that, experimentally speaking, the separation of enantiomers could be a very easy operation (once the optimal experimental conditions are found), but if we think about the mechanism that leads to their separation, this is anything but easy, and many aspects related to enantiorecognition are not yet fully understood. This is why I am very interested in widening the knowledge of mass transfer phenomena and the thermodynamic mechanisms affecting chiral chromatography.

Q. You are also active in the field of separation and purification using LC analysis (single-column and continuous multi-column) of biomolecules of high therapeutic interest, such as polypeptides, proteins, and oligonucleotides. You recently published a paper on the enrichment and recovery of oligonucleotide impurities by N-Rich twin-column continuous chromatography (2). What is continuous chromatography? What challenges did you face during this analysis?

 Yes, this is another research field that we began a few years ago, after a research period at ETH Zurich in the group of Massimo Morbidelli.

Continuous chromatography is considered to be the most suitable solution for process intensification in the (bio)pharmaceutical industry, and to partially overcome intrinsic limitations of single-column (batch) preparative chromatography—the most widely used approach for the industrial purification of (bio)pharmaceuticals. When only one column is used, the loading of the feed is a discontinuous operation. In a continuous (or semi-continuous) process, the feed is continuously (or following a time cycle) loaded into the purification unit, which is usually made by (at least) two identical columns. Multi-column processes typically exploit the concept of countercurrent chromatography, where the columns are connected by a series of valves that allow simulation of the movement of the stationary phase in the opposite direction to the mobile phase. Such countercurrent movement increases the interphase mass-transfer rates, therefore making the process more efficient. In addition, the overlapping regions, eluting from one column and containing both the target product and impurities, are recycled into the other column with increased yield of the purification process and less solvent consumption.

This kind of approach can also be applied to the enrichment and isolation of species present in very low amounts by applying the so-called 

, which is particularly beneficial in the isolation of critical impurities or minor compounds, as in the paper mentioned.

The most difficult part in setting up multi‑column continuous purification is the choice of characteristic times defining the recycling and collection windows, which requires much experimental work to find the optimal conditions.

Q. Another of your recent papers presents the use of multi-column countercurrent chromatography for the purification of either target peptides/oligonucleotides or their impurities (3). What benefits does this method offer over existing ones?

 As mentioned before, single-column chromatography has several intrinsic limitations. In particular, when several product-related impurities are present in the mixture, they can coelute with the target product both in the front and in the end of the peak, generating critical peak overlapping. This situation, called 

, leads to a yield-purity trade-off, which means that to achieve high purity, overlapping portions of the chromatogram need to be discarded, thus the collection window is narrowed at the cost of yield. Vice versa, higher yield can be obtained by enlarging the collection window (therefore also collecting impurities), but purity unavoidably decreases. The employment of multi-column countercurrent techniques allows this limitation to be partially overcome by automating the recycling of the overlapping portion of the chromatogram into the system. As a consequence, higher yield can be obtained at the same purity constraint, solvent consumption decreases, and the purification process can be completely automated.

Q. What are your next steps with this research?

We are working on an ambitious project aimed at finding green solutions to increase the sustainability of liquid chromatography, particularly in preparative conditions (4).

Q. What advice would you give to anyone wanting to enter this field?

 Well, the very first step to becoming a researcher is to get a Ph.D. degree. One should be aware that this track can be characterized by many ups and downs, but my advice is to never give up and keep on rocking, especially during the “downs”. I have personally grown a lot during my Ph.D. degree, not only from the scientific aspect but also from a more personal perspective. Connected to this, I also encourage people to spend some time abroad since it helps a lot in the development of skills that are fundamental for researchers and helps to create a network with other scientists. Indeed, my final advice is that, in my opinion, it is of fundamental importance to remember that science is not an individual activity and teamwork can make science work better.

Q. Is there any other recent research or work you have been doing lately that you think is important and want to highlight?

 Yes, I would like to mention that our group is also active in the separation of natural cannabinoids through HPLC and SFC. Specifically, we have recently published some interesting data regarding chirality of cannabinoids, about which almost nothing is known (5). Indeed, many cannabinoids are chiral but they are preferentially produced as (-)-L-trans form by the plant. For this reason, common separation methods are based on the use of achiral columns, irrespective of cannabinoids stereochemistry, and there are only a few papers in the literature where chiral cannabinoids were separated—mostly dating back to the beginning of the 1990s. However, in recent years, due to the increasing popularity of cannabis products, there has been a growing interest towards the characterization of cannabinoids in terms of enantiomeric purity. For instance, cannabichromene (CBC) is one of the rare examples of natural racemic (or scalemic) mixtures, as also demonstrated by our work, where both CBC enantiomers from a real sample have been separated in LC with two polysaccharide-based chiral stationary phases.

Ismail, O. H.; Pasti, L.; Ciogli, A.; et al. Pirkle-Type Chiral Stationary Phase on Core–Shell and Fully Porous Particles: Are Superficially Porous Particles Always the Better Choice Toward Ultrafast High‑Performance Enantioseparations? 

, 96–104. DOI: 10.1016/j.chroma.2016.09.001

Lievore, G.; Weldon, R.; Catani, M.; Cavazzini, A.; Müller-Späth, T. Enrichment and Recovery of Oligonucleotide Impurities by N-Rich Twin-Column Continuous Chromatography. 

, 123439. DOI: 10.1016/j.jchromb.2022.123439

De Luca, C.; Felletti, S.; Bozza, D.; et al. Process Intensification for the Purification of Peptidomimetics: The Case of Icatibant through Multicolumn Countercurrent Solvent Gradient Purification (MCSGP). 

, 6826−6834. DOI: 10.1021/acs.iecr.1c00520

Ferrazzano, L.; Catani, M.; Cavazzini, A.; et al. Sustainability in Peptide Chemistry: Current Synthesis and Purification Technologies and Future Challenges. 

De Luca, C.; Buratti, A.; Umstead, W.; et al. Investigation of Retention Behavior of Natural Cannabinoids on Differently Substituted Polysaccharide-Based Chiral Stationary Phases Under Reversed-Phase Liquid Chromatographic Conditions. 

, 463076. DOI: 10.1016/j.chroma.2022.463076

 is an assistant professor of analytical chemistry in the Department of Chemical, Pharmaceutical, and Agricultural Sciences at the University of Ferrara (Italy), and she received her Ph.D. in chemical sciences in 2018 from the same university. During her career, she has spent research periods at VUB Brussels (Belgium) in the group of Gert Desmet, and at the University of Pécs (Hungary) under the supervision of Attila Felinger. In 2019 she was a postdoctoral research fellow at ETH Zurich (Switzerland) in the group of Massimo Morbidelli. Martina works in the field of LC for both analytical and preparative purposes. Her main research activities are focused on the purification of polypeptides, oligonucleotides, and proteins by means of single-column and continuous countercurrent multi-column preparative LC; separation of natural cannabinoids; and investigation of kinetic and thermodynamic phenomena in chiral and achiral HPLC and SFC. She is co-author of almost 60 papers in peer‑reviewed journals and has presented her research at more than 20 national and international scientific meetings. She has been the recipient of many awards, including the “Csaba Horváth Young Scientist Award” conferred at HPLC2018 Washington (USA) and the “2021 Young Researcher Award” conferred by the Analytical Chemistry Division of the Italian Chemical Society.

Rising Stars of Separation Science:  Martina Catani

Agilent Technologies Inc. has announced that it has become the first “Angel” level sponsor of My Green Lab, a nonprofit organization devoted to building a global culture of sustainability in science.

Agilent Technologies Inc. (Santa Clara, California, USA) has announced that it has become the first “Angel” level sponsor of My Green Lab, a nonprofit organization devoted to building a global culture of sustainability in science.

Agilent is also sponsor of the My Green Lab Certification programme, and is working hard to ensure its global on-site customer demonstration laboratories are Green Lab Certified. The company has already achieved the highest level certification for the Waldbronn, Germany, Cheadle, UK, and Santa Clara, USA, sites.

“Sustainability and environmental, social, and governance (ESG) standards are central to our mission to advance the quality of life, and our sponsorship of My Green Lab helps us to drive this forward,” said Darlene Solomon, senior vice president, and chief technology officer at Agilent. “Being an ‘Angel’ level sponsor will further cultivate our knowledge of creating sustainable green labs, which we can share with our customers globally and support them on their sustainability journey.”

Recognized by the United Nations Race to Zero campaign as a measure of progress for pharmaceutical and medical technology companies toward a zero-carbon future, the certification indicates that a laboratory has achieved strong sustainability practices in energy use, water consumption, recycling, and waste production.

Agilent Achieves “Angel” Level Sponsorship of My Green Lab

Thermo Fisher Scientific is opening a site in Hangzhou, China, as part of its global effort to help companies provide therapies to patients faster.

Thermo Fisher Scientific (Waltham, Massachusetts, USA) is opening a site in Hangzhou, China, as part of its global effort to help companies provide therapies to patients faster. The 80,000-square‑metre current Good Manufacturing Practices (cGMP) facility will offer integrated clinical and commercial drug substance and drug product capabilities, including process development, cell line development, biologics drug substance manufacturing, and sterile fill-finish.

“Hangzhou is a strategic addition to our global pharma services network and the latest example of our commitment to meeting customer demands globally,” said Mike Shafer, senior vice president and president, pharma services. “We continue to invest in capabilities to support the manufacturing needs of our customers, enabling them to serve more patients throughout the world.”

“Thermo Fisher has been in China for 40 years, serving this market through its bioproduction, clinical research and pharma services businesses,” said Hann Pang, president, Thermo Fisher China. “Committed to our ‘in China, for China’ localization strategy, we are enhancing our workflow capabilities to fully support pharmaceutical and biotechnology companies in China and worldwide in helping more local innovations go global.”

Thermo Fisher Scientific is opening a site in Hangzhou, China, as part of its global effort to help companies provide therapies to patients faster. 

Thermo Opens New Site in China

Determination of molar mass distributions and the molar mass averages derived therefrom are the main objectives of gel permeation chromatography/size-exclusion chromatography (GPC/SEC) analysis. But what is the meaning of these averages and how are they influenced by the setting of baselines and integration limits? This instalment of Tips and Tricks in GPC/SEC will try to provide a better understanding.

Gel permeation chromatography/size‑exclusion chromatography (GPC/SEC) has become the main technique used to determine molar mass distributions and molar mass averages. In GPC/SEC, macromolecules are separated according to their size, which for most polymers correlates to molar mass. At the appropriate conditions the high molar mass species elute first, while smaller molecules elute later from the column. The use of a calibration curve allows the chromatogram to be translated into a molar mass distribution from which average molar masses, such as the number (

), can be obtained. These average molar masses can then be used for quality control or to release products for further use. For example, pharmacopoeias often request molar mass averages to lie within defined limits before the materials can be used for pharmaceutical applications.

But what is the meaning of these molar mass averages? Most of us know how to calculate these averages, but what do these values tell us?

 are the weight concentration, number, and weight fraction of molecules with molar mass 

. The sums must be evaluated over all species present in the sample. Other versions of the above formulas that use integrals do exist. The difference between the integrating versions and the summations above result from the assumption of the existence of discrete molar masses (summation) or a continuum of molar masses (integrations) present in the sample. The latter assumption is often applied in theoretical calculations.