 spoke to Giorgia Purcaro from the University of Liège in Belgium, about her work investigating mineral oil hydrocarbons (MOH) using GCxGC and the practical advantages that a novel LC–GCxGC–TOF-MS/FID method offers the analyst.

Interview by Alasdair Matheson, Editor-in-Chief, LCGC Europe

Q. Mineral oil hydrocarbon analysis is a hot topic of investigation at the moment. Why is it so important to analyze this group of compounds? What are the main challenges when analyzing them?

 The presence of mineral oil hydrocarbons (MOH), which are divided into two main groups—mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH)—has been known since the 1990s. However, attention to this type of contamination only began in 2008, when a significantly high amount of MOH was found in sunflower oil originating from Ukraine, followed shortly after by a concerning amount found from the migration of recycled cardboard into dry food (pasta) (1,2). Since then, several scandals have been reported in the press, particularly by Foodwatch International (3), thus raising concerns about this very complex class of contaminants. This complexity makes the topic challenging, both from the toxicological evaluation—for which it would be necessary to characterize the toxicity of each compound and their interactions (synergism or antagonism)—and the analytical determination. Indeed, a reliable and detailed characterization is needed to perform the risk assessment and assure consumer safety. The analytical methods for MOSH/MOAH analysis in food are particularly challenging because these contaminants are closely related to the lipid fraction with which they share many similar chemical structures. Therefore, the sample preparation and purification is rather extensive, and many interferences may still remain in the final determination, requiring expert evaluation for interpretation of the chromatograms and achieving accurate results. Moreover, the routine method applied, that is, liquid chromatography–gas chromatography–flame ionization detection (LC–GC–FID), only provides information on the total MOSH/MOAH in a specific volatility range but no further details on the chemical structure (the number of aromatic rings or the degree of alkylation). Comprehensive two-dimensional gas chromatography (GC×GC) is the key analytical technique that can provide this additional information, and it has started to gain attention in recent years. In fact, the enhanced chromatographic separation and the formation of clear chemical patterns in the two-dimensional (2D) space offer a detailed characterization according to the sub-classes composing either MOSH and MOAH, which have different relevance from a toxicological standpoint. Moreover, the 2D separation and visualization offer great support in the interpretation of the chromatograms, separating the bleeding and chromatographically removing some of the interferences from the compounds of interest.

Q. You have developed a liquid chromatography–comprehensive multidimensional gas chromatography time-of-flight mass spectrometer/flame ionization detector (LC–GC× GC–TOF-MS/FID) method for mineral oil analysis that was validated using interlaboratory comparison data (4). Can you tell us more about this?

I have been working with GC×GC since my Ph.D., and I am a big supporter of the technique; I think that it deserves a more central place in routine analysis thanks to its enhanced peak capacity and simplified data interpretation. In the field of MOH, it has shown its potential since 2009, when it was used to clarify the contamination found in sunflower oil (5,6). Since then, it has often been used as a confirmation tool, but it had never moved beyond being used for routine quantification—despite some attempts (7–10). Therefore, we decided to take a step forwards and propose a unified LC–GC×GC–TOF-MS/FID platform to perform both routine and confirmatory interpretation in a single analysis. Nevertheless, we had to solve an urgent problem related to the specific integration required to quantify MOSH and MOAH by developing a dedicated 2D software (11). To prove it reliable and convincing for use on a routine level, we had to exit our normal research environment, participating instead in two interlaboratory comparisons (ILCs) to establish the reliable performance of the proposed platform and the robust and accurate quantification (4).

Q. What were the advantages of the method you devised compared to previous techniques and why was interlaboratory comparison data important?

 Besides the aforementioned advantages related to the better characterization of the unresolved complex mixture (generally called “hump”) generated by the MOSH and MOAH thanks to the GC×GC enhanced separation for answering toxicological questions and related data occurrence, the use of a completely unified platform allowed for a higher degree of automation, thus avoiding cross-contamination and losses and simplifying the sample introduction. In fact, not all of the GC inlet can assure an injection without any discrimination in the C10–C50 volatility range, as required by the JRC guidance (12). Therefore, if a programmable temperature vaporization (PTV) injector is fine for just confirmatory information, to assure correct quantification without discrimination, an on-column injector exploiting the partially concurrent eluent evaporation techniques is needed. As this is a rather dedicated introduction system, hyphenating the GC×GC device to the LC system simplifies the overall methodology.

We believed that participating in the ILCs would be a strong proof of the reliability of the proposed system. We therefore joined two ILCs organized by the JRC and the German Society for Fat Science (DGF) and the French Technical Institute for the Study and Research of Fats (ITERG) for the determination of MOAH in infant formula and edible oil, respectively. The ILCs lasted approximately two years, during which we provided all the data to the organizer following the standard operating procedure (SOP) request; in parallel we performed the LC–GC×GC–FID/MS analysis to compare the results (4).

In a preliminary trial on pure mineral oil (13), the JRC set the required performance of the LC–GC–FID method (that is, relative standard deviation [RSD] lower than 5% and recovery between 92 and 100%). We showed that using the LC–GC×GC platform offered superior results in terms of recovery and repeatability. Repeatability and recovery data for the LC–GC×GC–FID analysis were 2.5% of RSD and 99.1% of recovery at the highest point. These results indicated no losses and no discrimination during the transfer of the fraction from the LC into the GC system, and no modulation problems causing poor release of the high boiling compounds or poor trapping of the lighter one.

Also, the analysis of the real-world samples (oils and infant formulas)—for which the impact of sample manipulation is much more important—showed results completely aligned with the outcomes of the ILCs, thus supporting the reliability of the quantitative analysis performed in GC×GC.

The results clearly showed that the proposed platform could successfully perform quantitative analysis, and provide the confirmatory information that may be required in particular cases. Moreover, the routine analysis by LC–GC×GC–FID—implying the reliable quantification of the 2D data—can provide additional information related to the MOAH with 1–2 rings and the MOAH with more than 3 rings (11,14–16), thus filling a critical gap for the risk assessment.

Finally, the 2D separation allowed the chromatographic removal of some interfering peaks, thus providing a more straightforward interpretation, more robust results, accurate quantification, and a deeper characterization of the contamination (14).

Q. Are you planning to explore this research further?

We will certainly continue this important research because more detailed data are needed to be able to perform a complete risk assessment of the MOSH and, in particular, the MOAH fractions; this has also been requested by the recent EFSA opinion, available for public consultation since the middle of March (17).

It is indeed my opinion that now that all the necessary tools and proof of reliability are available, GC×GC should be applied at a routine level to provide the necessary level of information.

We will therefore continue to generate data to convince of the validity of the proposed approach. We are also starting a project that combines the detailed characterization provided by this advanced chromatographic technique with 

(1) Hochegger, A.; et al. Mineral Oil Risk Assessment: Knowledge Gaps and Roadmap. Outcome of a Multi-Stakeholders Workshop. 

(2) Sdrigotti, N.; Collard, M.; Purcaro, G. Evolution of Hyphenated Techniques for Mineral Oil Analysis in Food. 

(3) FoodWatch, Toxic mineral oil found in food products. 

https://www.foodwatch.org/en/news/2021/toxic-mineral-oil-found-in-food-products/

(4) Bauwens, G.; Barp, L.; Purcaro, G. Validation of the Liquid Chromatography Comprehensive Multidimensional Gas Chromatography-Time-of-Flight Mass Spectrometer/Flame Ionization Detector Platform for Mineral Oil Analysis Exploiting Interlaboratory Comparison Data. 

(5) Biedermann, M.; Fiselier, K.; Grob, K. Aromatic Hydrocarbons of Mineral Oil Origin in Foods: Method for Determining the Total Concentration and First Results. 

(6) Biedermann, M.; Grob, K. Comprehensive Two Dimensional GC after HPLC Preseparation for the Characterization of Aromatic Hydrocarbons of Mineral Oil Origin in Contaminated Sunflower Oil. 

(7) Biedermann, M.; et al. Mineral Oil in Human Tissues, Part II: Characterization of the Accumulated Hydrocarbons by Comprehensive Two-Dimensional Gas Chromatography. 

(8) McCombie, G.; et al. Compliance Work for Polyolefins in Food Contact: Results of an Official Control Campaign. 

(9) Purcaro, G.; et al. Detailed Elucidation of Hydrocarbon Contamination in Food Products by Using Solid-Phase Extraction and Comprehensive Gas Chromatography with Dual Detection. 

(10) Zoccali, M.; Tranchida, P. Q.; Mondello, L. OnLine Liquid Chromatography-Comprehensive Two Dimensional Gas Chromatography with Dual Detection for the Analysis of Mineral Oil and Synthetic Hydrocarbons in Cosmetic Lip Care Products. 

(11) Bauwens, G.; Pantó, S.; Purcaro, G. Mineral Oil Saturated and Aromatic Hydrocarbons Quantification: Mono- and Two-Dimensional Approaches. 

Guidance on Sampling, Analysis and Data Reporting for the Monitoring of Mineral Oil Hydrocarbons in Food and Food Contact Materials,

 Bratinova, S.; Hoekstra, E., Eds.; Publications Office of the European Union, Luxembourg, 2019. DOI: 

(13) Goncalves, C.; et al. Determination of MOSH/ MOAH in Shell SN500 Mineral Oil; JRC IF 2021- 03 - The 3rd Interlaboratory Comparison. 

(14) Bauwens, G.; Conchione, C.; Sdrigotti, N.; Moret, S.; Purcaro, G. Quantification and Characterization of Mineral Oil in Fish Feed by Liquid Chromatography-Gas Chromatography-Flame Ionization Detector and Liquid Chromatography-Comprehensive Multidimensional Gas Chromatography-Time-of-Flight Mass Spectrometer/Flame Ionization Detector. 

(15) Bauwens, G.; et al. Investigation of the Effect of Refining on the Presence of Targeted Mineral Oil Aromatic Hydrocarbons in Coconut Oil. 

(16) Biedermann, M.; Eicher, A.; Altherr, T.; McCombie, G. Quantification of Mineral Oil Aromatic Hydrocarbons by Number of Aromatic Rings via Comprehensive Two-Dimensional Gas Chromatography: First Results in Food. 

https://connect.efsa.europa.eu/RM/s/publicconsultation2/a0l09000006qqHf/pc0400

Articles

Food Analysis

LCGC Europe spoke to Giorgia Purcaro from the University of Liège in Belgium, about her work investigating mineral oil hydrocarbons (MOH) using GCxGC and the practical advantages that a novel LC–GCxGC–TOF-MS/FID method offers the analyst.

Modern Mineral Oil Hydrocarbon Analysis

"GC Connections" presents the column's annual review of new developments made in the field of gas chromatography (GC), made available in 2022–2023.

I am pleased to present our annual review of new products in gas chromatography (GC), introduced between spring 2022 and spring 2023. With the COVID-19 pandemic mostly behind us, Pittcon reconvened in person for the first time since 2020, in Philadelphia, Pennsylvania, USA, from 20–22 March 2023. In general, trade shows have been trending smaller over the past two decades, and Pittcon is no exception. While the smaller show has a more intimate feel, one can easily see the widening of the aisles, the greater spacing of the booths, and reduced efforts from major vendors. This reduced effort is unfortunate; I hope that major vendors will reconsider attending and marketing at these expositions as a means for “waving the flag”, not only for themselves, but for the analytical science industry as a whole.

Pittcon 2024 will be held from 24–28 February 2024, in San Diego, California, USA. Now that we are out of the pandemic, I challenge everyone to attend a professional conference over the next year. Also consider participating in a local or regional chromatography discussion group or local section of the American Chemical Society (ACS) meeting. In the remainder of 2023 and early 2024, I will be attending at least four meetings, including the American Chemical Society in San Francisco in August 2023, the Eastern Analytical Symposium in Princeton, New Jersey in November 2023, and Pittcon in San Diego. I have used the rise of the virtual conferences to attend additional meetings that I would not have attended otherwise due to the travel costs. Two years of global pandemic were extremely difficult for conference organizers, and for the many vendors, contractors, and economies that conferences support. They still need your help, even more than last year. Conference and meeting attendance is still the best way to improve skills, learn about new techniques, train, and educate yourself.

In the specific space of gas chromatography, I highly recommend the International GC×GC Symposium. The 20th edition of the symposium is scheduled for Canmore, Alberta, Canada, on 28 May–1 June 2023. This meeting will include many of the best minds in GC×GC and GC, and is of special interest to anyone who has heard about GC×GC and wants to learn more about it. The list of speakers is an international all-star list of top chromatographers. For a no-cost virtual experience, look out for the 2023 ChromTalks, in May, sponsored by LCGC and ChromAcademy, its online training platform.

GC continues to have a strong presence on the conference circuit and in the literature. Topics such as cannabis analysis, biofuels, food and beverages, forensics, pharmaceuticals, and general applications of both GC and GC×GC are seen in a robust publication space. GC itself is trending towards being less the driver of research than its application to scientific problems. The most effective online search for GC today probably begins with phrases such as “GC of ____”, with your analyte or sample of interest filling in the blank.

The information presented in this article is based on vendors’ responses to questionnaires and additional information from press releases, websites, and product literature, not on actual use or experience of the author. Every effort has been made to collect accurate information but because of the preliminary nature of some of the material, 

 cannot be responsible for errors or omissions. This column cannot be considered a complete record of all new GC products introduced in the past year because not all vendors responded to the questionnaire, nor is all the submitted information necessarily included here, because of the limited available space and the editors’ judgment as to its suitability. Table 1 provides a listing of the vendors highlighted in this review, some of whom are lesser known in the areas they are listed, who showcased new products this year, or both. For a more complete picture of the GC-space today, I encourage you to also review the three previous years’ reviews (1–3).

In new instruments, trends seen over the past few years towards smaller, more automated systems, systems that are more specialized, and spectrometric detectors continue this year. Table 2 shows a list of new complete instruments introduced over the past year. All these systems are designed with optimizing and simplifying workflows, operation, and maintenance. One concern is that systems are becoming so automated and convenient to use that training and education may be viewed as less necessary, a seemingly reasonable view until something goes wrong. They represent a broad range, from a portable instrument about the size of a small briefcase, to a benchtop instrument, to a range of three options for GC–mass spectrometry (MS) and GC–MS/MS.

Stationary phases continue to be updated by most of the vendors, with nearly all making incremental improvements or showing new applications in their online application note libraries. The two new stationary phases that appear on this year’s survey were capillary column phases tailored to specific applications. These are highlighted in Table 3. This incremental trend in capillary column stationary phase development for specific specialized problems continues. The 2021 review included two phases for fatty acid methyl esters and dioxins. Users are encouraged to contact and keep up with your column vendor; new phases and improvements are happening continuously.

In 2022–2023, developments in new accessories, sampling devices, and detectors continue to trend towards simplifying analysis and making chromatographers’ work easier. We saw new advances in mass detection, with new ion sources focused on making soft ionization techniques more accessible, a new vacuum ultraviolet detector, and a new sulfur chemiluminescence detector. On the sampling side, new systems for purge and trap and gas mixing were presented. For more information on sample preparation, see the specific sample preparation article by Doug Raynie in next month’s issue of 

 (4). As with the GC reviews, it is worthwhile to review the past two years for a complete picture of sample preparation products (5,6). Table 4 provides a listing of products released in the past year that illustrate this trend. Many of the accessories are instrumental improvements in detectors and sampling devices.

Software and online resources continue to evolve along with the new technology. Two significantly updated systems for MS and comprehensive two-dimensional GC (GC×GC)–MS analysis were released, along with updates to a free online method development platform. Along with the GC platform, a complimentary liquid chromatography (LC) method development program is now available. Table 5 highlights these developments in software and online resources.

Our emergence from the pandemic provides more opportunities to continue education and training. Besides numerous short courses associated with Pittcon and other major conferences, many additional training and educational resources are available for 2023. The pandemic has resulted in much growth in online training and seminar offerings by vendors and training organizations, so check with your vendor. Opportunities for in-person short courses, seminars, and training are moving again and are offered by most major instrument vendors and through many conferences and organizations, including the American Chemical Society, Eastern Analytical Symposium, local and regional chromatography discussion groups, and, of course, Pittcon. LCGC’s online training system, ChromAcademy, is continuously adding content and is again hosting ChromTalks, a virtual symposium in May 2023.

As gas chromatography has passed its 70th birthday, development and innovation are ongoing and include all areas: sampling and supplies, new instruments, columns, detectors, data systems, and educational opportunities. I look forward to more innovation and advancements in the coming years, and I hope to see many of you at EAS 2023 in my home state of New Jersey, or at Pittcon 2024 in San Diego.

(1) Snow, N. H. New Gas Chromatography Products for 2021–2022. 

 (4), 136–141. DOI: 10.56530/lcgc.na.la4187p6

(2) Snow, N. H. New Gas Chromatography Products for 2020–2021. 

(3) Snow, N. H. New Gas Chromatography Products for 2019–2020. LCGC Europe 2020, 33 (5), 248–256.

(4) Raynie, D. E. New Sample Preparation Products and Accessories. 

(5) Raynie, D. E. New Sample Preparation Products and Accessories 2021–2022. 

(6) Raynie, D. E. New Sample Preparation Products and Accessories. 

 is the founding endowed professor in the Department of Chemistry and Biochemistry at Seton Hall University, USA, and an adjunct professor of medical science. During his 30 years as a chromatographer, he has published more than 70 refereed articles and book chapters and has given more than 200 presentations and short courses. He is interested in the fundamentals and applications of separation science, especially gas chromatography, sampling, and sample preparation for chemical analysis. Direct correspondence to: 

GC Connections

"GC Connections" presents the column's annual review of new developments made in the field of gas chromatography, made available in 2022–2023.

From Pittcon 2023 and Beyond: The Gas Chromatography Products to Watch This Year

The recent legalization of cannabis in many countries and its increased use in scientific and medical research as a result of its therapeutic properties have focused attention on the need for large quantities of highly purified cannabinoids. This is particularly important for the production of (new) analytical-grade standards, used as reference materials, and for the execution of clinical trials to test the biological activity of the single compounds that could potentially be used as new drugs or active pharmaceutical ingredients (APIs). To date, various purification strategies, usually based on several steps, have been proposed with different outcomes. However, all these methods have both advantages and disadvantages. This review will report on the challenges and future perspectives in the purification and isolation of major and minor cannabinoids from cannabis extracts.

 L. is one of the most discussed crops in the world. It has been used in food, industrial, agricultural, and medical applications for a long time (1–6). However, its psychoactive effects have led to social and political controversies that have resulted in its classification as an illicit drug. For example, in the US, substances are classified into five schedules under the Controlled Substances Act (CSA), based on their therapeutic potential and the risk of dependency, addiction, and/or abuse. Therefore, cannabis has been categorized as a hallucinogen, with no accepted medical use in the most restricted class of drugs (schedule I) (3,7). Nevertheless, in recent years, cannabis and its related products are increasingly being approved for both medicinal and recreational use. Indeed, their potential therapeutic uses in different diseases, extending from epilepsy to multiple sclerosis and Parkinson’s disease, have emerged.

 L. are the result of phytocannabinoids, which represent the most abundant class of bioactive compounds contained in the plant. They encompass a large family of chiral and achiral secondary plant metabolites, directly produced by the plant in an acidic form, with more than 150 compounds discovered so far. Chemical structures of the main cannabinoids are presented in Figure 1. Despite this structural variability, the vast majority (> 90%) of scientific investigations have focused only on the achiral analysis of two cannabinoids, thanks to their natural abundance in the plant: (–)-Δ

-THC is known for psychotropic effects, while CBD is not psychoactive. Moreover, despite the large number of discovered cannabinoids, only roughly 20% are available as analytical standards, with some of them available only as racemic or scalemic mixtures, and not as single enantiomers (8).

The absence of well-characterized reference standards imposes limitations on the identification and quantification capability of analytical methods. Indeed, to date, the three official methods for cannabis inflorescence analysis with achiral liquid chromatography (LC) (monographs of cannabis flos of the German, Dutch, and American Herbal Pharmacopoeias) offer the quantification of fewer than 10 cannabinoids, regardless of the stereochemistry of the analytes (9–11). Consequently, the lack of a universally accepted standardized analytical method for the chemo- and enantio-selective analysis of phytocannabinoids and the limited number of cannabinoid reference standard materials make it difficult to obtain a comprehensive characterization of the sample in terms of purity, identification, and concentration of the single species. In addition to the main compounds, other minor non-psychoactive cannabinoids, such as cannabigerol (CBG) and cannabichromene (CBC), have shown interesting qualities, and recently discovered cannabinoids are being reported (8,12). These findings create new perspectives on the possible medical use of cannabinoids. However, specific studies to test the therapeutic or toxic health effects on humans (and animals) of minor cannabinoids, including single enantiomers, have not yet been held. This can be explained by the difficulty involved in isolating sufficient purified quantities of these compounds because of their relatively small concentrations in cannabis plants and extracts—hence the need for accurate and high-throughput purification methods able to isolate sufficient quantities of pure cannabinoids for the production of standards for use in medicinal, analytical, and regulatory applications (8,13).

This review will report on the current challenges and future perspectives of the purification of cannabinoids from cannabis extracts.

Challenges of Current Purification Methods

The growing interest in cannabis and its beneficial effects have led to an increase in the demand for highly efficient and high-throughput methods for the separation of cannabinoids. Particular emphasis is also given to the environmental impact of the whole process. The development of fast, cost-effective, environmentally friendly, and selective methods applied to cannabis is still a challenging task; a sample can contain more than 500 phytochemicals in a highly complex sample matrix (14).

Various methods, based on different techniques, have been described and used for the purification, isolation, and extraction of cannabinoids from different types of samples (8). Among these, one can find both chromatographic and non-chromatographic methods. Chromatography-based methods can be subdivided into two main categories: solid–liquid and liquid–liquid techniques. Solid–liquid techniques include single-column high performance liquid chromatography (batch HPLC), multicolumn countercurrent chromatography (MCCC), and medium-pressure (flash) chromatography; liquid–liquid techniques consist of countercurrent chromatography (CCC) or centrifugal partition chromatography (CPC). Other non-chromatographic-based methods make use of subsequent extraction and recrystallization steps with solvents of different polarity.

However, since cannabis is a botanical matrix, a combination of different methods is usually employed for sample treatment and purification. Indeed, current purification procedures are based on various steps that can be summarized in: (i) decarboxylation, (ii) extraction, and (iii) purification. Other intermediate steps can be included in the procedure, such as precipitation, dissolution, and crystallization.

In the following section the most important applications of large-scale purification methods of cannabinoids based on column chromatography are discussed. Special emphasis is given to the advantages and disadvantages of the different approaches.

The first step in processing cannabis botanical samples is the extraction of cannabinoids from the matrix. If neutral cannabinoids are the target, a decarboxylation step is included before or after the extraction to convert acidic species to neutral ones. Since cannabinoids are highly lipophilic molecules with a very limited solubility in water (less than 1 μg/mL), they need to be extracted with lower polarity solvents. As an example, CBD has a solubility > 30 mg/mL in ethanol and > 100 mg/mL in hexane (15,16).

Extraction is usually performed in subsequent steps using organic solvents with different polarities or, more recently, with supercritical carbon dioxide (SC-CO

) (13,17–21). The most used solvents are methanol, ethanol, dimethyl sulfoxide (DMSO), methyl tert-butyl ether (MTBE), hexane, heptane, and iso-octane, to name but a few. These solvents represent a clear hindrance to the greening of the entire cannabis purification process, as a result of their toxicity to humans and the environment (22). In order to overcome this issue, supercritical fluid extraction (SFE) is gaining popularity as a greener alternative to organic solvents extraction (OSE). SFE makes use of low toxicity solvents, such as SC-CO

, and produces solvent-free extracts (13).

However, the sole use of OSE or SFE procedures for the isolation of cannabinoids is not sufficient to achieve the desired product purity, which is usually > 90%. Indeed, extraction methods have a limited capacity to discriminate between different cannabinoids because of the very similar chemical structures (Figure 1). This is the reason why extractions are often followed by one or more chromatographic steps, mostly based on flash chromatography (17–19,21,23,24).

Preparative single-column LC is by far the most commonly used technique for the purification of different samples, including cannabinoids (8,25). Its success is down to the possibility of using columns packed with porous particles of different sizes, geometries, and functionalization and operated under different elution conditions.

Concerning cannabinoids purification, most examples make use of flash or solid-phase extraction (SPE) column chromatography, either under normal-phase or reversed-phase conditions. The most used stationary phases are silica or alumina for normal-phase operation and C18 for reversed phase. Solvents used in normal-phase applications include pentane, hexane, heptane, iso-octane, petroleum ether, organic ethers, alcohols, esters, or ketones. Solvents for reversed-phase chromatography include water or an acidic buffer, alkanol (methanol, ethanol, and iso-propanol), or acetonitrile (20,21). Many applications include activated charcoal filtration as an optional treatment or as a pre-purification step prior to other chromatographic procedures (17,19).

The introduction of column chromatographic step(s) improves final product outcomes in terms of purity and throughput. However, the use of very large particles with a broad size distribution (20–35 μm or more) as adsorbents, typical of flash and SPE chromatography, is detrimental to the quality of the separation. Indeed, it is widely known that chromatographic efficiency and resolution strongly depend on particle size and its distribution, being inversely proportional (26,27). This causes broad peaks that can overlap, negatively affecting product purity. To alleviate this problem, packing particles with a smaller diameter (between 5 and 15 μm), typically used for HPLC applications, can be effectively used for cannabinoids purification. As an example, Figure 2 compares chromatograms obtained under the same experimental conditions with a HPLC preparative column (100 × 21.2 mm, 5-μm C18) and a flash column (C18, 20–35 μm, 20 g/column) for the separation of a CBD-rich hemp extract (28). The flash column offers very poor resolution, as a result of poor chromatographic efficiency. From a preparative viewpoint, this will result in a collected sample having lower purity and higher dilution when compared to that from the HPLC column separation. Indeed, undesired compounds not fully separated from the target substance will lead to peak contamination and the large peak broadening will force the collection of a wider time window to recover the entire peak.

 Reversed-phase HPLC, with octadecyl silica (C18) as the stationary phase and water–organic mobile phases, still remains the preferred elution mode for the quantification, separation, and isolation of cannabinoids (4,8,29–31). However, despite the great success and the greenness of water-rich mobile phases, they have a clear disadvantage concerning the preparative aspects of cannabis purification. Indeed, as already mentioned, cannabinoids have a very limited solubility in water and are highly lipid-soluble substances (15,16). Therefore, sample extraction and/or dilution need to be performed with organic solvents (ethanol or hexane). This does not present an issue if analytical-scale chromatography is concerned, since injection volume is typically in the range 1–100 μL (that is, < 4% of the total column volume, CV). Otherwise, when dealing with preparative-scale separations, larger injection volumes are often used (> 1 CV). In this case, the diluent has a great influence on sample retention, with high organic contents leading to unretained analytes. This translates into the impossibility of loading volumes as high as in other preparative applications, forcing a decrease in the injection volume to a few microlitres to produce acceptable cannabinoids retention. Consequently, sample preparation and sample injection represent a typical bottleneck of reversed-phase chromatography applied to cannabis purification.

Different preparative methods have been described and proposed for cannabinoids purification. These methods make use of C18 columns and mixtures of water and alcohol as the mobile phase.

In Table 1 (28,32–36), column and injection volumes are reported and compared. These preparative methods use volume percentage related to the ratio of injection and column volumes that are typical of analytical chromatography. This lessens the typical advantages of preparative chromatography when compared to analytical chromatography, that is, high throughput, high productivity, and small solvent consumption. As a matter of fact, current reversed-phase HPLC preparative methods can produce only very small amounts of pure cannabinoids (in the milligram scale) by using hundreds of millilitres of solvents.

Another drawback of reversed-phase elution mode is, despite its high efficiency, the poor selectivity—that is, the difficulty of achieving baseline separation between critical pairs of cannabinoids (CBD/CBG, Δ

-THC, and cannabicyclol [CBL]/CBC). This aspect is magnified in nonlinear chromatography, when peaks deviate from the Gaussian profile because of overloading conditions (37). This translates into a product contaminated by other species, depending on the initial composition and purity of the sample. This indicates that performance and outcomes of reversed-phase LC methods are highly dependent on the sample composition.

Nevertheless, preparative reversed-phase HPLC can be used to isolate several cannabinoids (32), where the purification of CBD, cannabinol (CBN), Δ

-THC, CBL, and CBC from a CBD-rich oil is described. A preparative chromatogram showing the collected fractions is presented in Figure 3(a). The results of the analysis of the five highlighted fractions indicate that the purity of CBD was increased from 72 to 93% (fraction 1), CBN from 0.6 to 33% (fraction 2), CBL from 0.5 to 12% (fraction 3), THC from 2.2 to 80% (fraction 4), and CBC from 5.7 to 97% (fraction 5). As an example, the analytical chromatogram of fraction 1 can be seen in Figure 3(b). As already mentioned, the selectivity between CBG and CBD peaks is a critical factor with reversed-phase methods, as shown in Figure 3(b), where CBG cannot be depleted from CBD.

To date, no examples of HPLC columns operated under normal-phase conditions for this purpose are reported. Alternatively, normal-phase conditions have been effectively applied in flash chromatography for cannabinoids purification, using stationary phases based on bare silica, alumina, or cross-linked dextran-based resin (20,24). Regarding HPLC, some recently published studies have demonstrated the feasibility of other types of adsorbents, namely CN, NH

, and diol stationary phases, for fast cannabinoids isolation (38,39) in normal phase.

It has been proven that normal-phase elution mode—as a result of a different retention mechanism with respect to the reversed-phase mode—provides better resolution and selectivity for some compounds. An example is shown in Figure 4, where the very high selectivity obtained for the pair CBD/CBG (almost coeluting under reversed-phase conditions) is shown.

Moreover, the use of apolar organic solvents boosts sample solubility, facilitating sample preparation and increasing column loading. This aspect of the normal-phase mode is of pivotal importance because it leads to smaller solvent consumption, higher throughput, and higher productivity compared to its reversed-phase counterpart. Indeed, with this separation mode, it is possible to extract cannabis samples with an apolar solvent and directly inject the extract into the chromatographic column with high injection volumes (>> 1 CV), without the risk of unretained compounds. Another important aspect to be considered is that the use of low-viscosity solvents makes solvent removal from purified fractions easier, as a result of their volatility, and allows for higher flow rates (faster runs), which is less problematic in terms of pump backpressure.

Concerning the toxicity and adverse effects of apolar organic solvents, the use of heptane, rather than hexane, MTBE, or dichloromethane (DCM), is recommended. Indeed, heptane is classified as a “problematic” solvent, like acetonitrile—the most commonly used mobile phase modifier in reversed-phase chromatography (22).

Supercritical fluid chromatography (SFC) is rapidly gaining in popularity thanks to its interesting characteristics. This technique makes use of mixtures of high-pressurized CO

 mixed with a co-solvent (usually an alcohol). SC-CO

 is a relatively nonpolar solvent, and it has lower viscosity and higher diffusion coefficients compared to liquid mobile phases, permitting an increase in the flow rate without detrimental effects on efficiency and backpressure. Moreover, SC-CO

 is an environmentally friendly solvent that rapidly evaporates, allowing easy sample concentration. For all the reasons mentioned above, SFC has become a reliable and green alternative to normal-phase LC. Indeed, in recent years, it has been largely employed for the enantioseparation of cannabinoids using chiral chromatography on an analytical scale (8,40).

Some works have hypothesized the feasibility of SFC for the preparative isolation of cannabinoids by using achiral columns packed with 5 μm particles (41,42). Indeed, preparative SFC can offer several advantages when compared to classical preparative LC, such as lower organic solvent consumption, higher productivity, rapid solvent removal, and lower environmental impact. However, very few applications of preparative SFC for the isolation of cannabinoids are available.

A 2005 patent (43) describes a two-step purification of chemically synthesized (–)-Δ

-THC by preparative SFC, consisting of a chiral and an achiral separation. The chiral method uses a polysaccharide-based stationary phase supported on macroporous silica gel (20 μm), while the achiral method involves a silica gel or derivatized silica gel separation (10 μm). Subsequent feed injections of 0.85 mL were performed and resulted in the isolation of (–)-

-THC with more than 99% enantiomeric purity.

To date, preparative SFC is not yet a routinely used technique for the green purification of cannabinoids, probably due to its expense and complex constructional setup. However, its broader use in the preparative industrial field may be seen in the near future.

Chirality of cannabinoids has been known for decades, with the first report about optical activity of CBD published in 1940 (44). However, this topic has been only marginally considered until recently, when the complete characterization of cannabis extracts and products started to become a topic of medical interest. Indeed, it was discovered that the natural (–)-

 isomer of THC has psychotropic effects, while the corresponding (+)-enantiomer does not. As a consequence, the analysis of the composition of cannabinoids must be carefully evaluated not only in terms of content (the so-called 

) but also considering their enantiomeric purity and stereostability (8,45).

This has focused attention on possible side effects (or even positive properties) of isomers present in natural or chemically synthesized cannabis samples that cannot be detected with classical achiral chromatography. For example, the presence of racemic CBC in cannabis extracts has been observed (46,47), but no information on the biological activity and medicinal properties of the single enantiomers has been reported yet. This draws attention to the need for preparative chiral methods to isolate sufficient amounts of pure single enantiomers to perform clinical trials.

Nevertheless, the employment of chiral preparative chromatography for cannabinoids isolation is still a largely unexplored area, with very few examples of application.

As previously mentioned, a preparative SFC chiral method, combined with an achiral method, was able to produce +99% pure (−)-

-THC (43). Flash chiral chromatography has also been used for the purification of optically pure (−)-

-THC (> 99%), from the racemic mixture, on an amylose-based chiral stationary phase (48).

Evidence showing that both reversed-phase and normal-phase chiral HPLC can be used for enantio-purification based on polysaccharide stationary phases is reported in some recently published studies (49–51).

The isolation of pure cannabinoids from real matrices can be highly demanding, as many commonly used methods do not provide sufficient selectivity and resolution. This results in peak overlap that causes a very narrow region of the chromatogram where the target compound can be collected with acceptable purity. This intrinsic limitation of single column chromatography is called 

. However, it can be overcome by multicolumn countercurrent continuous (or semicontinuous) techniques (8,25).

Among these techniques, simulated moving bed (SMB) has long been used for the purification of cannabinoids in both laboratory- and pilot-scale procedures. SMB can be effectively applied in binary or pseudo-binary separations, as it is able to divide the feed mixture into two fractions. This also represents the biggest limitation of this process because it can only be applied to relatively simple mixtures. Nevertheless, SMB allows for higher productivity with respect to the batch process, thanks to a continuous sample loading and a reduction of solvent consumption. Moreover, it can provide 100% recovery in case of peak overlap (52).

In some patents (53,54) SMB technology has been applied to the purification of Δ

-THC obtained by enantiopure synthesis. The raw feed product had a Δ

-THC content of 65–75%, and 20–30% of Δ

-THC as the main impurity. The purification process was conducted using C18 columns with particle size of 10 to 100 μm under reversed-phase conditions, using a mobile phase mixture of methanol (62%), tetrahydofuran (17%), and water (21%), leading to > 99% THC purity. Two patents (55,56) describe a method for the purification, separation, and enrichment of CBD that includes a sequence of purification steps and SMB process using modified activated carbon (45 to 1700 μm), modified hydrophobic adsorbent (styrene-divinylbenzene) resin, or a poly-(methyl-methacrylate) (PMMA) resin (25 μm and 300 μm) under reversed-phase conditions.

SMB chromatography can be used for continuous THC remediation from a sample, using only one chromatographic step, and with clear advantages from an industrial and commercial viewpoint.

In the very near future, HPLC columns may be coupled to the SMB technique and operated under normal-phase elution conditions. This combination could be highly advantageous for cannabinoids purification, for all the reasons mentioned, allowing for continuous streams of highly concentrated feed and purified compounds.

Another recently developed multicolumn technique is multicolumn countercurrent solvent gradient purification (MCSGP). This technique permits gradient elution, ternary separations, and the internal recycling of overlapping areas, with clear advantages for productivity and solvent consumption when compared to single-column purification. Twin-column MCSGP has been applied to CBD purification under reversed-phase conditions using C18 columns (15 μm). The sample had an initial purity of > 85% and the final product was > 99.5% pure. However, the product also contained small quantities of cannabidivarin (CBDV), cannabidiolic acid (CBDA), and CBG (34). As has already been seen for reversed-phase chromatography, the outcome of the purification process depends on the composition of the feed mixture, making the process not universally applicable.

To date, no universally applicable preparative method has been developed for cannabinoids purification. As a consequence, purification methods need to be chosen depending on the complexity of the sample (initial target purity and concentration, number of other cannabinoids).

Various methods based on column chromatography are currently available with this aim at different degrees of purification. Flash chromatography has been the method of choice to produce pure cannabinoids or as a pre-purification step, but preparative HPLC is becoming established as a more efficient option. Preparative reversed-phase HPLC, using either achiral or chiral separations, is only successful on a very small scale because of the limited solubility of cannabinoids in water-based solvents. Valuable alternatives are normal-phase HPLC and SFC.

The future of preparative chromatography applied to cannabinoids purification will be devoted to the development of efficient, green, cost-effective, and fast methods able to produce highly pure compounds. In this regard, the coupling of HPLC columns operated in normal-phase elution mode to continuous multicolumn techniques, such as SMB and MCSGP on the one hand, and the use of SFC on the other, means there will be plenty of room for improvement in the field, contributing to increased research productivity.

(1) Crowther, S. M.; Reynolds, L. A.; Tansey, E. M. 

The Medicalization of Cannabis Wellcome Witnesses to Twentieth Century Medicine

, Vol. 40; Wellcome Trust Centre for the History of Medicine, 2010.

(2) Potter, D. Cannabis Horticulture. In 

Pertwee, R., Ed.; Oxford University Press, 2014.

(3) Mead, A. Legal and Regulatory Issues Governing Cannabis and Cannabis-Derived Products in the United States. 

(4) Capriotti, A. L.; Cannazza, G.; Catani, M.; et al. Recent Applications of Mass Spectrometry for the Characterization of Cannabis and Hemp Phytocannabinoids: From Targeted to Untargeted Analysis. 

(5) Cerino, P.; Buonerba, C.; Cannazza, G.; et al. A Review of Hemp as Food and Nutritional Supplement. 

(6) Sirangelo, T. M.; Ludlow, R. A.; Spadafora, N. D. Multi-Omics Approaches to Study Molecular Mechanisms in 

The Controlled Substances Act: Regulatory Requirements

(8) Felletti, S.; De Luca, C.; Buratti, A.; et al. Potency Testing of Cannabinoids by Liquid and Supercritical Fluid Chromatography: Where We Are, What We Need. 

(9) Deutsches Arzneibuch 2018 (DAB), Allgemeiner Teil. Monographien Cannabisblüten-Cannabis flos; Deutscher Apotheker Verlag, 2018.

(10) Dutch Office for Medicinal Cannabis, CIBG Ministerie van Volksgezondheid, Welzijn en Sport; Analytical Monograph Cannabis flos, Version 7.1, 2014.

https://american-safe-access.s3.amazonaws.com/documents/AHP_Cannabis_Monograph_Preview.pdf

(12) Citti, C.; Russo, F.; Sgrò, S.; et al. Pitfalls in the Analysis of Phytocannabinoids in Cannabis Inflorescence. 

(13) Gallo-Molina, A. C.; Castro-Vargas, H. I.; GarzónMéndez, W. F.; et al. Extraction, Isolation and Purification of Tetrahydrocannabinol from the 

 L. Plant Using Supercritical Fluid Extraction and Solid Phase Extraction. 

(15) Wright, S.; Wilkhu, J.-D. ORAL CANNABINOID FORMULATIONS. WO 2018/150182 Al, 2018.

(16) Technobis, Optimizing the CBD Crystallization Process. Technobis Crystallization Systems Application Note, 2023. 

https://www.crystallizationsystems.com/publications/appnoteoptimizi zg-the-cbd-crystallization-process/

(17) Goodwin, N. J.; Archer, N. J.; Murray, C.; Greenwood, A. K.; McHattie, D. PRODUCTION OF Δ9 TETRAHYDROCANNABINOL. US 7 592 468 B2, 2009.

(18) Bhatarah, P.; McHattie, D.; Greenwood, A. K. Production of Delta 9 Tetrahydrocannabinol. WO 2009/133376, 2009.

(19) Flockhart, I.; Wheatley, G. W.; Dring, S.; Archer, L. Method of Preparing Cannabidiol from Plant Material. US 2006/0167283 A1, 2006.

(20) Roura, X. N. Methods of Purifying Cannabinoids, Compositions and Kits Thereof. US 2016/0214920 A1, 2016.

(21) Elsohly, M. A.; Ross, S. A. Method of Preparing Delta-9-Tetrahydrocannabinol. US 6 365 416 B1, 2002.

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

(23) Flockhart, I.; Wheatley, G. W.; Dring, S.; Archer, L. Methods of Purifying Cannabinoids from Plant Material. WO 2004/026857, 2004.

(24) Wohlfarth, A.; Mahler, H.; Auwärter, V. Rapid Isolation Procedure for Δ9-tetrahydrocannabinolic Acid A (THCA) from Cannabis Sativa Using Two Flash Chromatography Systems. 

(25) De Luca, C.; Felletti, S.; Lievore, G.; et al. Modern Trends in Downstream Processing of Biotherapeutics Through Continuous Chromatography: The Potential of Multicolumn Countercurrent Solvent Gradient Purification. 

(26) Gritti, F.; Bell, D. S.; Guiochon, G. Particle Size Distribution and Column Efficiency. An Ongoing Debate Revived with 1.9μm Titan-C18 Particles. 

(27) Ismail, O. H.; Catani, M.; Pasti, L.; et al. Experimental Evidence of the Kinetic Performance Achievable with Columns Packed with New 1.9μm Fully 130 LCGC Europe April 2023 FELLETTI ET AL. Porous Particles of Narrow Particle Size Distribution. 

(28) Preston, J.; Orlowicz, S. THC Remediation from CBD Extract to Demonstrate Running Flash Chromatography on a Prep HPLC Instrument. Phenomenex Application Note, 2020.

(29) Raharjo, T. J.; Verpoorte, R. Methods for the Analysis of Cannabinoids in Biological Materials: A Review. 

(30) Nahar, L.; Onder, A.; Sarker, S. D. A. A Review on the Recent Advances in HPLC, UHPLC and UPLC Analyses of Naturally Occurring Cannabinoids (2010-2019). 

(31) Citti, C.; Braghiroli, D.; Vandelli, M. A.; Cannazza, G. Pharmaceutical and Biomedical Analysis of Cannabinoids: A Critical Review. 

(32) Krauke, Y.; Monks, K. Purify CBD and other Cannabinoids by Preparative HPLC. Knauer Wissenschaftliche Geräte GmbH, VPH0073 Application Note, 2020.

(33) Krauke, Y.; Monks, K. Purification of Cannabidiol from CBD Oil by Preparative HPLC. Knauer Wissenschaftliche Geräte GmbH, VPH0074 Application Note, 2020.

(34) ChromaCon, Highly Pure Cannabidiol (CBD) by Twin-Column Chromatography (MCSGP). ChromaCon Application Note, P2.V2, 2019. 

https://www.chromacon.com/resources/public/lava3/media/kcfinder/files/CBD_MCSGP_application_note.pdf

(35) Rieck, F. Purification of Cannabinoids from a Cannabis Sativa Extract. Agilent Technologies Inc. Application Note 5994-1773EN, DE.1299305556, 2020. 

https://www.agilent.com/cs/library/applications/applicationcannabinoids-purification-1290-infinity-iiautoscale-prep-lc-msd-5994-1773en-agilent.pdf

(36) Aubin, A. Purification of Cannabidiol from Hemp Oil Using the Prep150 LC System. Waters Corporation Application Note, 2015. 

https://www.waters.com/content/dam/waters/en/appnotes/2015/720005287/720005287-en.pdf

(37) Guiochon, G.; Felinger, A.; Shirazi, D. G.; Katty, A. M. 

Fundamentals of Preparative and Nonlinear Chromatography,

(38) De Luca, C.; Buratti, A.; Krauke, Y.; et al. Investigating the Effect of Polarity of Stationary and Mobile Phases on Retention of Cannabinoids in Normal Phase Liquid Chromatography. 

(39) Felletti, S.; Krauke, Y.; Stephan, S.; Monks, K. Determination of Retention Behavior of Cannabis Extract by Column Screening. Knauer Wissenschaftliche Geräte GmbH, VFD0189 Application Note, 2020.

(40) Felletti, S.; Ismail, O. H.; De Luca, C.; et al. Recent Achievements and Future Challenges in Supercritical Fluid Chromatography for the Enantioselective Separation of Chiral Pharmaceuticals. 

(41) Przybyciel, M.; Kohler, D. A. The SFC Isolation and Purification of Cannabinoids using Application Specific Stationary Phases Under Optimised Conditions. 

(42) Franklin, E. G.; Wilcox, M. Purification of CBD Extracts using SFC and a Celeris 4-Ethyl Pyridine Column. Regis Technologies Application Note, 2019. 

https://www.registech.com/download/purification-of-cbd-extracts-using-sfc-and-a-celeris-4-ethyl-pyridine-column/

(43) Geiser, F. O.; Keenan, J. J.; Rossi, R.; Sanchez, A.; Whelan, J. M. Process for Purifying (–)-δ9-TransTetrahydrocannabinol. WO 2005/061480 Al, 2005.

(44) Adams, R.; Hunt, M.; Clark, J. H. Structure of Cannabidiol, a Product Isolated from the Marihuana Extract of Minnesota Wild Hemp. I. 

Peer Reviewed

The current challenges and future perspectives of the purification of cannabinoids from cannabis extracts are presented in this review article.

Purification and Isolation of Cannabinoids: Current Challenges and Perspectives

Many high performance liquid chromatography (HPLC) users are confronted with questions about how to improve the performance of an existing or recently developed method. These days, we have many technological options to consider (for example, using higher pressures or temperatures, or both), but how do we choose one (or a few) to try? A systematic approach to method improvement can save time and resources by using methods that are likely to be better, and more robust, than those developed using a trial-and-error approach.

During a recent visit to a local laboratory here in Minnesota, USA, I was asked a question that I initially thought would be easy to answer, but then it caused me to pause and think a lot. And the more I thought about it, a series of “LC Troubleshooting” articles unfolded in my mind, and this will be a frequent discussion topic here in 2023. The essence of the question was (paraphrasing) as follows: “I’ve got this validated method that isn’t great, but it gets the job done. The method is approximately 20 years old, and I’ve heard and read that high performance liquid chromatography (HPLC) technology has changed a lot in 20 years, so it seems to me it must be possible to improve this separation a lot. What are my options for doing that?” Of course, as someone deeply committed to all things chromatography, I immediately felt the urge to say, “Why yes, a lot has changed, and you can improve that separation. But… you have a lot of options, and… it’s a little complicated.” Nevertheless, I am certain there are many HPLC users that have this exact question on their minds, and feel unsure about where to start. In a series of “LC Troubleshooting” articles, we are going to confront this question directly and make it more manageable to think about how to proceed. Not all of the options we will discuss will be available to all users, but it is valuable to understand what the full array of options looks like, so that you can feel confident in making an informed decision about how to proceed.

Some Analytical Challenges Need To Be Treated Differently

HPLC users routinely face a staggering variety of analytical challenges ranging from relatively fast (tens of seconds) separations of just a few analytes to long separations (several hours) of complex mixtures that contain thousands of analytes. Figure 1 shows a comparison of chromatograms obtained for simple and complex mixtures using contemporary technologies. In the case of the simple mixture, there is a lot of empty space in the chromatogram, and one means of decreasing analysis time would be to reduce the amount of wasted, empty space. On the other hand, the entire chromatogram for the complex sample is full of peaks. In this case, improvement may mean decreasing the peak widths on average so that fewer analytes are coeluted, but it is very unlikely that all of the analytes in the mixture can be fully resolved, even if the analysis time is extended beyond one hour. These disparate challenges necessarily require different chromatographic solutions, and improving on old or poor separations in current use for these applications require different lines of thinking.

When dealing with relatively simple mixtures, we generally have more paths to improve the separation, including changing mobile-phase chemistry, the stationary-phase chemistry, or both, and adjusting the retention and optimizing efficiency (that is, finding the most time-efficient way to achieve the chromatographic efficiency needed to meet the chromatographic objectives). On the other hand, when dealing with complex samples, our options are more limited. A big difference is that changing the mobile-phase chemistry, the stationary-phase chemistry, or both will move peaks around, but there will still be coelution, so a simple reordering of the peaks may not be all that helpful—and usually mass spectrometry (MS) is critically helpful for resolving compounds that are not separated chromatographically. In this case, the most effective way of improving the separation would be to improve the efficiency as a means of improving the peak capacity—roughly, the number of peaks that can be fit side-by-side into a separation space. We will discuss the concept of peak capacity in more detail in a future instalment focused on improving separations of complex mixtures.

Relative to the situation 20 years ago, HPLC users currently have many options to consider as possible means to improve upon the performance of old methods. Some of the most prominent options currently are changes in particle morphology and size, operating pressure, column temperature, and an increasingly diverse array of stationary-phase chemistries (for example, consider the growing number of “mixed-mode” phases that contain both charged and lipophilic functional groups).

We can roughly divide these options into two categories: those that affect peak width (kinetic adjustments), and those that affect peak spacing, or relative retention (thermodynamic adjustments). Here, I provide a full list of options simply to make the point that there is a lot to consider. We won’t discuss any one of these in detail now, but we will discuss each of them in detail in future instalments in this series.

Kinetic adjustments—reducing peak width relative to analysis time, which include:

changing the particle size (usually decreasing it, but not always);

moving to a higher pressure range (allows increase in flow rate, increase in column length, decrease in particle size, or both);

moving to a higher temperature range (allows increase in flow rate, increase in column length, decrease in particle size, or both); and

changing the particle morphology (for example, moving from totally porous particles to superficially porous ones).

Thermodynamic adjustments—improving peak spacing (that is, reducing the fraction of the time axis that is empty), which can include:

finding a stationary-phase chemistry that provides more even distribution of peaks across the time window;

adjusting the mobile-phase chemistry in a way that moves towards more even distribution of peaks; and

adjusting the column temperature can also be considered here.

Resolution is a Key Metric of Separation Performance in This Context

There are many ways to measure separation performance, including analysis time, efficiency (that is, plate number), selectivity, retention, and resolution. Among these, resolution (

A systematic approach to method improvement can save time and resources by using methods that are likely to be better, and more robust, than those developed using a trial-and-error approach.

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