We saw in our last column how capacity factors can be used to calculate separation factors, which are a measure of the quality of the separation between two analyte peaks. However, we found that because the separation factor ignores peak widths it can sometimes give misleading results. The solution to this problem is to calculate peak 

, which takes both peak positions and widths into account giving a more accurate measurement of separation quality.

As in all previous columns on chromatographic theory, for more details I refer you to these references (1,2).

Recall (3) that the separation factor for a pair of chromatographic peaks, which we will label as a and b, is given by 

We also learned last time (3) that we can derive that the separation factor equals the ratio of the capacity factors for two peaks as such, seen in 

where kb is the capacity factor for peak a and ka is the capacity factor for peak b and k

 shows a chromatogram with two well separated analyte peaks marked with their capacity factors.

Using Equation 2 we can calculate the separation factor for peaks a and b using 

Note that analyte peaks a and b are well separated with a separation factor of 3. This is why the separation factor can be used as a measure of separation quality.

The problem with the separation factor is that it only uses elution time—the position of the top of the chromatographic peak—but ignores the fact that peaks have a width as well. We saw last time (3) that when peaks are unusually broad this does not work, as illustrated in 

The separation factor for analyte peaks a and b for the chromatogram in Figure 2 is 3, the same as for the chromatogram in Figure 1. The problem is that the peaks in Figure 2 are poorly separated, whereas in Figure 1 they are well separated, and yet both chromatograms have the same separation factor. In Figure 2, the separation factor is not a good measure of separation quality because of large peak widths. What metric then can we use that will work for all chromatograms and all peak widths? To be able to do this we need a method of measuring chromatographic peak widths.

Now that chromatographic systems are computerized, peak width measurements can be made easily by many different algorithms. When reporting peak widths, it is important to state how they were measured because different algorithms may produce different results. An old fashioned but useful way of measuring chromatographic peak widths is triangulation. This technique is illustrated in 

This method involves drawing tangent lines on either side of a peak as seen in Figure 3. These lines are then extended so they intersect with the baseline. The distance between the two points of intersection is the triangulated peak width. Note in Figure 3 that the width of peak a, w

, is 2 min, and that the width of peak b, w

The peaks in Figure 3 are well separated, making the calculation of the triangulated peak width easy. This is not the case with broad peaks as seen in Figure 2. In this case, we have two poorly separated peaks that are quite overlapped. How do we measure peak width in this case? We first draw a baseline underneath the overlapped peaks. Tangent lines are then drawn as best we can, along the sides of the portions of each peak visible, and then the tangent lines are extrapolated down to the baseline. The peak width, as above is the distance between these two intersection points. Note in Figure 2, that the width of peak a is 5 min and of peak b is 6 min. Note that these peak widths have a ~ in front of them, and there is a question mark underneath the chromatogram.

The problem with the peak width calculations made on the chromatogram in Figure 2 is that when we extrapolate the tangent lines there is a region under the chromatogram where the tangent lines for the two peaks cross, denoted by the question mark, and it is not obvious where the right edge of peak a and left edge of peak b fall. The triangulation method assumes chromatographic peaks are symmetrical and baseline separated. The purpose of the question mark is to show that this assumption may not be true.

 of two peaks in a chromatographic separation uses both peak position and width to measure how well peaks are separated from each other. Its form is seen in 

Note in the numerator of Equation 5, that the elution times for two chromatographic peaks are subtracted from each other. The stipulation that tb be greater than ta exists so that the numerator, and hence R, have a positive value. Note that the denominator in Equation 5 contains the width of peaks a and b. Finally, we have a measure of separation quality that includes width. Remember that the widths of peaks a and b are added together and divided by 2; that is, the denominator is the average of the two peak widths. Since the average of the peak widths is in the denominator, as this average goes up R goes down. That is, broader peaks give a lower resolution value than narrower peaks.

We saw above that the separation factor fails as a measure of separation quality for broad peaks, and the separation factors for peaks a and b in Figures 1 and 2 were the same, even though these separations are clearly of different quality. How then does our new metric, the resolution, handle this situation? For the well separated peaks in Figure 3 the resolution is seen in 

For the poorly separated peaks in Figure 2 the resolution is seen in 

Ha! We finally have it, the resolution, a metric that properly measures separation quality in the presence of broad peaks by using peak width in its calculation. Note that the well separated peaks in Figure 3 have an R value well above 1, while the poorly separated peaks in Figure 2 have an R value below 1. A resolution of 1 means two peaks are 3% overlapped, and an R of 1.5 or greater means two peaks are baseline resolved, as is the case in Figure 3. Remember that chromatography is used primarily as a quantitative technique, and that peak heights and areas are used to determine concentrations of analytes in samples. For this quantitation to be at its most accurate, baseline resolution of all peaks in a chromatogram is necessary. Thus, when developing methods our goal is to make sure that R for all pairs of peaks is at or above 1.5. The question then becomes what experimental variables can be adjusted, in what direction, to achieve R values of 1.5 or greater? You will have to wait for future columns for the answer.

This column has been devoted to including peak width in our separation quality calculations. But why are some chromatographic peaks narrow and others wide? In other words, what determines chromatographic peak width? You will have to read the next column to find out.

We reviewed the concept of separation factors, and found them wanting because they ignore peak widths, leading to situations where well separated and poorly separated pairs of peaks have the same value of α. The solution to this problem necessitates developing a method for measuring peak width. A tried-and-true method that was illustrated was triangulation. We then introduced the concept of chromatographic resolution for a pair of peaks, and since this quantity includes peak widths, we found it accurately measures peak separation even in the presence of broad peaks.

Skoog, D.A., West, D.M., and Holler, E.J., Analytical Chemistry: An Introduction,

Robinson, J.W., Undergraduate Instrumental Analysis, 

Smith, B., Chromatographic Theory, Part IV: Measuring Separation Quality with Capacity and Separation Factors, 

PhD, is Founder, CEO, and Chief Technical Officer of Big Sur Scientific. He is the inventor of the BSS series of patented mid-infrared based cannabis analyzers. Dr. Smith has done pioneering research and published numerous peer-reviewed papers on the application of mid-infrared spectroscopy to cannabis analysis, and sits on the editorial board of 

. He has worked as a laboratory director for a cannabis extractor, as an analytical chemist for Waters Associates and PerkinElmer, and as an analytical instrument salesperson. He has more than 30 years of experience in chemical analysis and has written three books on the subject. Dr. Smith earned his PhD on physical chemistry from Dartmouth College. Direct correspondence to: 

Smith, B., Chromatographic Theory, Part V: Chromatographic Resolution, 

Articles

Cannabis Analysis 

A review of the concept of separation factors and a discussion on developing a method for measuring peak width including triangulation followed by the introduction of the concept of chromatographic resolution for a pair of peaks.

Chromatographic Theory, Part V: Chromatographic Resolution

In our previous article, we touched on the prospective advantages of utilizing clustered regularly interspaced short palindromic repeats (CRISPR) in the production of high-quality cannabis cultivars. This biotechnology has the potential to boost resilience to pests and microbial agents commonly found in cannabis cultivation through the introduction and the extraction of specific genetic traits. This is especially advantageous considering the idiosyncratic challenges that are inherent in cannabis cultivation. These include, but are not limited to: susceptibility to a variety of pathogens, low production of rare or minor cannabinoids, uncontrollable or undesirable ratios of tetrahydrocannabinol (THC) and cannabidiol (CBD), and instability of cannabis genetics. Collectively, these challenges pose significant obstacles to achieving optimal yield and quality. This article provides insights into the opportunities involving the genetic modification of cannabis. With its precision and versatility, CRISPR technology can enable targeted genome editing to enhance pathogen resistance, optimize cannabinoid and terpene profiles, and improve overall plant quality. By harnessing the potential of CRISPR technology, the new frontier of medicine can continue to expand throughout the agricultural sector, meeting the growing demand for high-quality cannabis products, and paving the way towards a more sustainable future.

 with an established history of medicinal, recreational, and industrial applications. The legalization and widespread acceptance of cannabis and hemp as effective treatments for chronic conditions are driving the expansion of this previously stigmatized plant. As a result, the global demand for cannabis is soaring, leading to the emergence of a substantial enterprise on par with other major crops. In this article, we discuss the challenges associated with cannabis cultivation and propose an innovative solution to overcome these obstacles that can hinder the production of consistent products.

In recent years, there has been significant progress in genome editing technology, which can be utilized for inducing genetic and phytochemical variability in plants such as cannabis. In contrast to traditional plant breeding methods, revolutionary genome editing techniques are distinguished by their higher efficiency and specificity. Through the application of these advanced genetic engineering techniques, it is possible to significantly improve the overall efficiency of cannabis cultivation by modulating specific genes that are involved in conferring resistance to pests and viruses. Additionally, genetic modifications can potentially increase the production of rare or minor cannabinoids, or fine tune the expression of specific tetrahydrocannibinol (THC) and cannabidiol (CBD) ratios.

Despite being a relatively new field, cannabis biotechnology is rapidly expanding due to the accessibility to large-scale genetic engineering techniques. In particular, the clustered regularly interspaced short palindromic repeats (CRISPR or Cas) system has garnered significant notoriety for its ability to perform with high efficiency, specific gene knock-ins (adding of a specific gene), knock-outs (removal of a specific gene), and genetic modifications (altering a specific genetic sequence to attenuate an undesirable trait or accentuate a favorable trait). Initially, this tool was discovered in the microbial “immune system” that prokaryotes—bacteria and archaea—use to prevent infection by viruses called phages. Viruses are small infectious agents that can invade cells and replicate, causing harm to the cell. Comparative genomic analyses between bacteria and archaea suggests that CRISPR and Cas proteins (the cas gene products) work together to provide prokaryotic cells with an acquired immunity system that defends against invading viruses and plasmids, analogous to the eukaryotic ribonucleic acid (RNA) interference (RNAi) system. Interspersed between the short deoxyribonucleic acid (DNA) repeats of bacterial CRISPRs are similarly short variable sequences called spacers, which have been found to be highly homologous to virus or foreign plasmid DNA sequences. These spacers are derived from DNA of viruses that have previously attacked the host bacterium and hence serve as a ‘genetic memory’ of previous infections (1). If another infection by the same virus should occur, the CRISPR defense system will act as “molecular scissors,” cutting out any viral DNA sequence matching the spacer sequence to protect the bacterium from viral attacks. If a previously unseen virus invades, a new spacer is created and added to the chain of spacers and repeats. This technology enables precise genome editing by introducing modifications to the expressed traits of any organism, including the cannabis plant. As a result, high quality unique cultivars with tailor-made levels of phytochemical content and resilience to environmental stressors (pests, bacterial, fungi, and viruses) can be created rapidly, compared to the painstaking and laborious processes required through traditional ‘Mandelian’ breeding techniques.

CRISPR Applications in the Cannabis Industry

Cannabis, much like any other plant, is highly susceptible to pests and pathogens, which can wreak havoc on both indoor and outdoor cultivation environments. Modern cannabis growers are constantly seeking innovative ways to protect their cultivars from harm posed by these pathogens. These pathogens can cause substantial post-harvest losses especially in larger commercial cultivation facilities that present larger degrees of risk. Consequently, growers in a number of domestic and international markets are unable meet the industry demands in production. There are various types of pathogens such as, viruses, bacteria, fungi, nematodes, and insects that are capable of deteriorating the quality of cannabis at any point in the cultivation cycle. Once plants are exposed to these infestations, it can hinder their growth and commercial viability. Even more concerning is the fact that these tiny pathogens can be introduced and spread easily through various means, including wind, rain, soil, insects, contaminated seeds, irrigation, and human contact.

One example of a pathogen that could be controlled through CRISPR-Cas9 intervention is the hop latent viroid (HpLVd). Cannabis is taxonomically related to hops and is a host for the seemingly ubiquitous HpLVd that is the causative agent of “dudding disease” or “cannabis virus.” It consists of a small single-stranded RNA molecule that is transmitted by infected seeds or contaminated plant material. The HpLVd infects the phloem tissue of cannabis plants, which is responsible for transporting nutrients and sugars throughout the plant. Cannabis plants infected with HpLVD can transmit the virus via vegetative propagation of contaminated clones and seeds, which often go unnoticed because the virus can remain latent for prolonged periods of time. The asymptomatic plants make early detection and overall control of the virus extremely challenging. The symptoms of observable indications of HpLVD infections can include stunted growth, yellow misshapen leaves, and brittle stems (2). Additionally, cannabis plants infected with HpLVD exhibit reduced flower mass and reduction of trichome production, which are the highly valuable resinous glandular hairs found on the plant surface that are vital for the production of different classes of phytochemicals (for example, cannabinoids and terpenes). This results in a catastrophic decline in both yield and quality, including as much as a 50% reduction in cannabinoid and secondary metabolite production (3). Unfortunately, HpLVd has a tremendous impact on both financial expenditures and the overall cultivation environment. Growing less efficacious plants requires managing and utilizing larger square footage to produce the same overall phytochemical content, thus contributing to a more resource-intensive enterprise.

Through CRISPR-Cas9 technology, specific genes within the HpLVd genome can be targeted and cleaved, thus preventing the replication, and spread of the virus. Various methods can be used to introduce this system into cannabis plants, such as 

-mediated transformation. This method uses a bacterial pathogen, 

, to introduce genes of interest into the host and editing for CRISPR-Cas9 systems. There is a stable transformation within the nucleus, where DNA is integrated into the cannabis genome and is capable of being passed on to future generations. Transient transformation, on the other hand, permits the DNA to transiently remain within the nucleus and not fully integrate into the plant’s genome, while simultaneously being transcribed to produce desirable traits (4). By utilizing the CRISPR approach, cannabis strains resistant to HpLVd could be developed. With that being said, it is imperative that we understand the genetic makeup of HpLVD, as well as the specific genes and sequences responsible for the virus’ virulence. Prior to introducing modified cannabis cultivars into agricultural practices, it is pivotal to ensure that it does not pose any unintended consequences, such as off target mutations or decreased viability.

The potential applications of CRISPR-Cas technology in cannabis cultivation go beyond the control of plant pathogens. There is a current effort by scientists to utilize CRISPR technology to develop novel cannabis strains with desired traits, such as the elevated expression of rare or minor cannabinoids, secondary metabolites, and the precise regulation of THC and CBD ratios (see 

). To accomplish this, the plant’s genome is precisely edited, and the genes responsible for producing these compounds are introduced and enhanced. For example, utilizing CRISPR-Cas9 to target and attenuate downstream enzymes such as the primary cannabinoid synthases (tetrahydrocannabinolic acid [THCA], cannabidiolic acid [CBDA], and cannabichromene acid [CBCA]) could result in cellular resources being reallocated towards the production of other cannabinoids of interest. This could result in the creation of novel strains that exhibit greater expression of rare or minor cannabinoids and ancillary compounds such as tetrahydrocannabivarin (THCV). Why is this important? Well for one, THCV up-regulates energy metabolism, making it valuable pharmaceutical compound for weight loss, obesity, and Type II diabetes. However, the expression of THCV in cannabis (Group I THC-dominant in particular; generally non-existent in Groups II and III cannabis cultivars) is limited to less than 2% weight/weight (w/w). In addition to the aforementioned example (attenuating the expression of the THCAs, CBDAs, and CBCAs synthases), CRISPR could also potentially amplify the expression of the tetrahydrocannabivarin acid (THCVA) synthase enzyme, which is responsible for the conversion of its progenitor molecule, cannabigerovarinic acid (CBGVA), into THCVA.

Another example of low-hanging fruit for CRISPR utilization is in the large-scale production of CBCA and cannabichromene (CBC). As with the THCVA synthase, CRISPR can target the CBCA synthase enzyme to potentially exploit high CBGA genetics to enhance the production of CBCA and CBC. Due to the extremely low levels of CBCA and CBC in commercial cannabis (~1% w/w), these isolates are extremely expensive and require a tremendous amount of flower material to be processed to produce commercially-relevant amounts of these important neuroprotectants. As such, wholesale pricing for a kilogram of CBC in its purest form (95-99% purity) is approximately $10,000 (6). In addition to labor, resources, and profit, these prices account for all of the expenses associated with producing this isolate. However, CRISPR could potentially create high-CBC producing cannabis strains that can be harvested at a lower cost and eliminate time consuming extraction methods. This would result in affordable and accessible isolates for targeted treatment options for both recreational and medicinal users alike.

The cannabis market is brimming with a plethora of curated strains that have been meticulously crafted to provide cannabis enthusiasts with a one-of-a-kind experience. Based on the plant morphology and foliar arrangement, products were traditionally classified as either 

. However, due to widespread crossbreeding and mislabeling of these sub-species, these terms are deceiving and fail to reflect the cannabinoid profile (which is, after all, the primary interest of most consumers). This issue has given rise to a relatively new classification scheme in which the relative levels of THC and CBD expression determine the sub-type of a specific cannabis strain. For example, Group I cannabis cultivars are THC-dominant (>20:1 THC–CBD); Group II consists of ratioed amounts of THC and CBD, (that is, ~1:1, 1:2, or 2:1 THC– CBD); and Group III cannabis (hemp) cultivars are exclusively CBD-dominant (that is, >20:1 CBD–THC).

One scenario where these classifications are paramount to operational success is in the hemp industry. In accordance with the hemp guidelines, a plant that contains more than 0.3% (w/w) of THC cannot be categorized as hemp and must be classified as cannabis. As such, if an organization is designed to cultivate hemp and is doing so with a hemp license—and not a cannabis license—then a lack of the compliance with the 0.3% total THC requirement could result in a partial or complete destruction of their crop, ultimately having catastrophic economic implications. As such, growers in the hemp industry are constantly struggling to find compliant hemp genetics that perform as advertised, which has been one of the major contributing factors to the current struggles endured by those in the American hemp industry. CRISPR has the potential to be the solution to the uncontrollable ratios of THC and CBD. Utilizing CRISPR to target and modify the genes encoding for the CBDA and THCA synthase enzymes, it is possible to increase or decrease their activity within the plant thus modulating the ratios of CBD to THC, respectively. In particular, modulation of the gene encoding CBDA synthase appears to be the best angle of attack compared to trying to dampen THC expression, for the simple fact that THCA is integral to plant viability by allowing cannabis plants to metabolize certain wavelengths of light (7).

Though CRISPR technology is still in its nascent stages, it has the immense potential to revolutionize the cannabis industry. In comparison with traditional breeding techniques, CRISPR's simplicity and versatility makes it a highly efficient tool for enhancing the overall sustainability in cannabis cultivation. This tool can dissect and regulate the cannabinoid biosynthetic pathway that is essential to the improvement of quality and diversity in cannabis. CRISPR has the ability to knock in or knock out the expression of transcription factors or key enzyme genes in the synthesis of effective components or down-regulate them in the competitive pathway to increase the content of active components. Utilizing CRISPR, novel cannabis strains with enhanced resistance to pests and diseases, can be developed, which can result in reduced reliance on harmful chemical pesticides and herbicides. Additionally, CRISPR technology can increase both biological and economic yields by creating cultivars with desirable phytochemical compounds. Overall, with the continued rapid expansion of the cannabis industry, CRISPR is poised to become an indispensable tool for breeders who strive for competitive advantages in a crowded market that is inherently driven by innovation.

Guo, M.; Chen, H., Dong, S.; et al., CRISPR-Cas gene editing technology and its application prospect in medicinal plants. 

Warren, J.G.; Mercado, J.; Grace, D., Occurrence of hop latent viroid causing disease in Cannabis sativa in California, 

Adkar-Purushothama, C.R.; Sano, T.; Perreault, J.P., Hop Latent Viroid: A Hidden Threat to the Cannabis Industry, 

Li, L.; Yu, S.; Chen, J.; Cheng, C.; Sun, J.; Xu, Y.; Deng, C.; Dai, Z.; Yang, Z.; Chen, X.; Tang, Q.; Su, J.; Zhang, X., Releasing the Full Potential of Cannabis through Biotechnology, 

Jacques, A. and Hildenbrand, Z., Hemp Regulations and the Restrictions on Innovation, 

https://www.gvbbiopharma.com/product/cbc-isolate/

Hildenbrand, Z.L.; Grosella, A.; Manes, R.J.; Liden, T.; Jacques, A.; Spurlock, M.; West, C.; and Schug, K.A., Variable Red Light Exposure Affects Phytochemical Content in Group III Cannabis Cultivars, 

 is a research Professor at the University of Texas at El Paso, the principal founder of Inform Environmental, a partner of Medusa Analytical, and is a director of the Curtis Mathes Corporation (OTC:CMCZ). Direct correspondence to: 

 is an MS Chemistry student at The University of Texas at El Paso.

Mendoza, H., Hildenbrand, Z., Maximizing Cultivation Efficiency Through the Utilization of Genetic Technology,

The Cultivation Classroom

This article provides insights into the opportunities involving the genetic modification of cannabis. 

Maximizing Cultivation Efficiency Through the Utilization of Genetic Technology

There has been a movement underway now gaining traction within cannabis extractors to make cannabis edibles more functional in their effect and therefore broader in their appeal. Call them designer cannabis products, if you will, or just call them next generation cannabis products with a wellness purpose.

Shaping that reality are edible makers who have found better ways to extract various minor cannabinoid compounds to make a more terpene-heavy product favored by consumers, that comes with a controllable level of psychoactive components (or none at all), which consumers believe they can trust. These edibles are targeted as go-to sleep aids, anxiety aids, anti-inflammation assistance, or other comfort and wellness aids that are not about just getting as high as possible—which was the goal of the first generation of cannabis edibles back in 2014.

Extractions support a clearer market direction now, in both the recreational and medical side. But there’s more to its rise in popularity. The increasing number of countries decriminalizing cannabis has become one of the key trends in the cannabis-infused edible products market during 2021–2025 (1). The global edibles market is expected to reach $25 billion by 2025, with 75% of that growth coming from North America.

This installment of “Tech Innovations” discusses new ideas in extraction that are beginning to find out more about the plant and its capabilities. Some topics covered include extractions that help adjust bioavailability; cleaner extractions that are safer for the consumers; and how cannabis extraction methods are embracing some of the old-school extraction technologies used for decades, even as the cannabis industry begins to take the lead in the technological evolution of extraction.

“We’re starting to see evolution of all these traditional techniques, like CO

, ethanol, hydrocarbon,” said Jessie Kater, senior vice president, innovation, research, and development at Curaleaf (2). “And as good as they’ve gotten, there are pros and cons to each of those techniques. There is no universally best technique. You have to choose which one meets your business needs, or your product needs, the best.”

Vince Harkiewicz, CEO and co-founder of Grownetics, a farm and grow automated system developer, is seeing a push-back against the huge wave of isolates, he mentioned, as he watched the development of the industry in Colorado (3). “It was fascinating to see the innovation go towards how to do fractional distillation and isolate out all these terpenes, all these secondary metabolites,” he said.

The push-back came when it was determined that if you isolate all those terpenes out there is actually very little evidence and very little research that’s been done showing that isolated compounds have the same effect as the whole plant, or as even more complex compounds from the whole plant. “So, there’s this huge shift towards live rosin, fresh frozen products, to get that whole plant experience to the consumer. And that’s where the connoisseur has also gone,” Harkiewicz said.

Curaleaf, headquartered in New York, makes edibles with nanoparticles. “People describe a much more social, less anxious experience with these products,” explained Kater. “So, people will be able to go to the bar and socialize with friends with a nano gummy product.”

Curaleaf recently released its JAMS (4) edible product line, made using nano and liposomal technology. JAMS includes a food grade nano emulsification technology that’s completely all-natural, Kater said. “It’s taking those fat-soluble cannabinoids, surrounding them with kind of a more water soluble outer shell. So, you get more of the cannabinoid intact and able to be delivered to the target receptor sites where we want them to have their efficacy, ” said Kater.

With nanotechnology, Kater explained that they’re seeing initial onset as fast as 10 to 15 min, and peak onset around the 30 min mark. “It’s a powderized formulation,” he said. “So, we call it a pre-liposomal, meaning the cannabinoids are not liposomal in the product itself. But when it hits the aqueous saliva in your mouth, it assembles into liposomes.”

The product, offered in wide range of dosage options and promoted for cannabis consumers just starting out with edibles, features an improved bioavailability. “Typically, you wouldn’t see technologies like this being deployed—this self-assembling-when-it-hits-saliva,” he added.

Curaleaf primarily and historically has done CO

 extraction, along with some ethanol and hydrocarbon extraction, Kater stated. “CO

 has really kind of been our national workhorse. I feel good about CO

 from a safety perspective because of the fact that CO

 is a big component of the air we breathe. We use food grade CO

 (5), where when you release it from pressure, it dissipates so there’s no residual CO

 often isn’t thought of as a solvent because it’s a gas, he said. “But CO

One new innovative extraction device of note is the Towers Highrise press (6), a scalable, solventless rosin press that has just been released. “It’s substantially different to anything else,” Harkiewicz mentioned. “This is the sort of innovation we’re seeing in the extraction solvents side, which is these mechanical kind of systems that haven’t existed yet.” The unit costs about $60,000, he added.

Duncan Mackie, the Director of Pharmacology & Experimental Therapeutics at MedPharm Holdings (7), commented that MedPharm’s extraction methods are done using cryo-ethanol. “That’s a real big one,” Mackie said. “Throughput-wise, it’s allowing us to extract large amounts of cannabis now, doing everything at -40. We’re keeping everything really cold so that we’re not pulling through fats and waxes.”

 to get full high terpene-rich mixtures at MedPharm. “We’re investigating terpenes, minor cannabinoids, flavonoids, and just about any kind of compound that we can isolate from that plant, or extract from the plant,” he said.

MedPharm is looking at these compounds, and their activity at the cannabinoid 1 and cannabinoid 2 receptors, as well as orphan receptors. “So, there’s GPR55 (a novel cannabinoid receptor [8]) and a couple other orphan receptors that we’re starting to see really cool pharmacology,” Duncan said. “It’s a really exciting time to be in cannabis as a scientist with my background. It’s a rarity that you get to work in an area that should have been explored 100 years ago.”

Antiflammatory Medicines Are the New Calling for Cannabis

Cannabis has developed great compounds for anti-inflammatory treatments. One study (9) found that future approaches for improved usage of cannabis “demand the development, transformation and formulation of full-spectrum cannabis extracts into active plant ingredients to achieve higher effectivity,” and that cannabis components can be combined with other pharmaceutical drugs to reduce inflammation.

Mackie said that MedPharm is looking at the inflammatory condition in the context of neurodegeneration in Alzheimer’s, Parkinson’s, Huntington’s, traumatic brain injuries, and concussions, such as chronic traumatic encephalopathy (CTE) with the National Football League (NFL). CTE has been found in 99% of NFL players, according to a research at Boston University (10). “It doesn’t have to be tetrahydrocannabinol (THC). It doesn’t have to have that euphoric side effect,” Mackie said. “We’ve selected for that compound over all these years of selective breeding. But these minor kind of compounds, we don’t know much about them.”

There’s tetrahydrocannabivarin (THCV) anecdotally used as an appetite suppressant as well as cannabinol (CBN), anecdotally discussed as well, with a little more research getting behind it, looking at sleep disorders (11). “You’re starting to see people take interest in these minor cannabinoids,” Mackie said. “I’ve been interested from the beginning. It was like that’s where the medical value is going to come from.”

The body’s way of healing itself is through inflammation. “When the inflammation goes awry in the brain, you have to figure out a way to temper it down,” said Mackie.

COVID-19 is one newer example of inflammation. According to the National Institutes of Health, (NIH) prolonged inflammation after COVID-19 infections (12) have been shown to cause permanent damage to lungs and kidneys. “There might even be a place for cannabinoids for treatment there,” Mackie added. “The plant itself is quite amazing. And the medical value is through the roof.”

To isolate the minor cannabinoids, Mackie uses high performance liquid chromatography (HPLC) (13) in their laboratory, where they can take a mixture of 120 cannabinoids at very different percentages and isolate them at 99% pure, which is pharmaceutical grade purity. “We have the ability to actually isolate super high purity minor cannabinoids, and can work to address questions such as what does this combination in this ratio do to a neuron, or to a microglia (the immune cells of the brain), or to a muscle cell in a cell culture system?

“It’s actually getting that mechanistic work and that’s the basis of how we need to be going about it from a scientific standpoint, in the drug discovery lab,” said Mackie, adding that it’s a complex plant that is going to have poly-pharmacology. “You’re going to have to have components that are going to go together because they almost need to go together. Because that’s how the plant did it.”

Kater has been doing a lot of live rosin work, which is different from live resin. “When I think about live resin, I think about fresh frozen being extracted with a hydrocarbon solvent, which is a derivative of petrochemical things like propane and butane,” he said. “When done at cold temperatures, and with a fresh frozen material, you get a really terpene rich extract, which consumers really like.”

The trade-off is a petrochemical derivative is used to do that extraction as your solvent-based extraction. Live rosin, on the other hand, is truly solventless, Kater stated. “In this case, we’re taking fresh frozen material, and just using a physical process.”

With this process, they are freezing the trichomes in an ice water bath, and then just using physical means to agitate and break those THC and terpene-containing trichomes off the plant. Through a heating process, they convert THC and terpene-containing trichomes off the plant and convert them into an oil or a concentrate. “It’s a little bit more labor intensive,” he said. “It’s a little bit less of a yield than a solvent extraction. But you’re really getting the purest expression of just natural cannabis trichomes containing the cannabinoids and terpenes with no interference of any solvents or rigorous chemical processing at all.”

He thinks using this process is the closest that an extractor can get into concentrated vapable experience to the actual flower itself. “It may be even preferred to some extent because you’re getting rid of some of the plant components, like carbohydrates, that really aren’t made for inhalation,” said Kater. “I think a big part of the growth we’re seeing in the rosin categories is because people appreciate that full plant flavor taste.”

Kater explained that the company recently filed a provisional patent on an extraction process that uses nothing but water. “It’s different from the rosin process because we’re using water as a solvent. It’s a unique process, because you don’t typically think of cannabinoids as water soluble, because they’re not. But we’ve we found an interesting way to go about making them water soluble and being able to use water as an extraction solvent. So that’s an innovative process that we’re developing. There are others,” he added.

Kater noted that they’ve gone from the days where potency was the only thing that mattered, to where consumers want a broader spectrum of cannabinoids. “Then you get into terpenes, a wide variety of terpenes,” he said. “We want them intact as they are in the plant, not altered, not degraded. That brings out that full flavor. Consumers are really starting to realize that the terpene profile has a massive influence on the type of experience they have with a particular strain, from sedated to silly to a happier experience. That’s really starting to drive the market, particularly on the vape and concentrate side of the market.”

Harkiewicz mentioned that all the innovation right now is happening around the molecular chemistry, and through solventless extraction methods, and the different pressing recipes such as squishing hash through filter bags with a low temperature press. “That is still so young, in such its early days,” he said. “It’s been around for a while, but there hasn’t been a lot of innovation that’s reached the market yet in that space, because we’re dealing with so many new markets still coming online, figuring this all out for the first time.”

He believes that the extraction discussion is really about the craft side of things, because there are not tools to do this sort of extraction at large scale yet. “So, it’s really interesting to see all this innovation happening in the craft market,” Harkiewicz expressed. “A lot of our clients are much larger and are wanting to dip their toes in and wanting to get into solventless. But then not understand that, okay, there are not tools built for that scale in the solventless realm yet.”

If you want a designer cannabis experience, there are still challenges in getting that right. “I think it’s because, first of all, the effect isn’t consistent because depending on your genetic disposition, it is or is not going to affect you in the same way,” he said. “That’s another uniquely challenging side of designer effects.”

There’s so much mystery about the plant on the therapeutic side, Kater said, about what cannabis strain and what cannabinoids and what terpenes are there, and why they should be used for therapeutic application. “That can all be unraveled through understanding the biochemistry of how these cannabinoids and terpenes are interacting with the receptors, and the downstream cascade of signaling that takes place, as well as the pharmacokinetics of these products.”

Extraction to process plants is not new. The technology can be traced back thousands of years (14). In fact, CO

, ethanol, and hydrocarbon extraction have been used in industries long before there was legal cannabis in the US. The cannabis industry and its industry-specific innovation has attracted some of the old school extractors working in soaps, perfumes, and even beer. For example, the focus on terpene production has also been picked up by hops farmers, Harkiewicz said. “That’s all they care about as well, like massive terpene production, especially with the trend for all these very, very fragrant IPAs,” he observed. “So, there are companies who are doing hop extracts, which seems like they took a note from the cannabis industry providing crop extracts to the brewers now as well.”

Mackie commented that he is starting to see people really latch on to the well-known analytical chemistry methodologies and physical chemistry methodologies. “The knowledge just wasn’t there before. And there was more a lack of the expertise in the field.”

Now he’s coming across a lot more people with more advanced degrees in the cannabis space, who have the scientific knowledge, and have been trained. “And now we’re finally applying (that scientific process) to this plant which is really exciting,” he said.

“Stoners are pretty innovative,” Mackie said. “You give them the tools, they generally run with it. Extraction-wise, we’ve gotten really advanced over the last decade. Now it’s the biomass. It’s really trying to find those unique cultivars that give you the cannabinoid variants at a high enough concentration that we can extract them and then actually do the pharmacology on them.”

Technavio. Cannabis-infused Edible Products Market Size to Grow by USD 25.27 Billion: Find Future Trends, Analysis, and Insights: Technavio 

https://www.prnewswire.com/news-releases/cannabis-infused-edible-products-market-size-to-grow-by-usd-25-27-billion--find-future-trends-analysis-and-insights--technavio-301561762.html

https://ir.curaleaf.com/management-team?item=68

Curaleaf Holdings Inc. Curaleaf Expands Brand Portfolio with Launch of JAMS™ Cannabis-infused Edibles 

https://www.prnewswire.com/news-releases/curaleaf-expands-brand-portfolio-with-launch-of-jams-cannabis-infused-edibles-301781463.html

https://www.airproducts.com/gases/carbon-dioxide?utm_feeditemid=&utm_device=c&utm_term=food+grade+co2&utm_source=google&utm_medium=ppc&utm_campaign=North-CO2&hsa_cam=12689956503&hsa_grp=127986210680&hsa_mt=p&hsa_src=g&hsa_ad=537149132299&hsa_acc=1445747373&hsa_net=adwords&hsa_kw=food+grade+co2&hsa_tgt=kwd-300898220303&hsa_ver=3&gclid=CjwKCAjwrJ-hBhB7EiwAuyBVXUyPvWr1F9MgZZcdzbefWpasrRVO31DFIxVnKxK_iGtP1Wizvhx-mhoC-AUQAvD_BwE

Duncan Mackie, Ph.d. - Director of Pharmacology ... - Linkedin 

https://www.linkedin.com/in/duncan-mackie-ph-d-3bb1892b

Ryberg, E.; Larsson, N.; Sjögren, S.; Hjorth, S.; Hermansson, N.O.; Leonova, J.; Elebring, T.; Nilsson, K.; Drmota, T.; Greasley, P.J. The Orphan Receptor GPR55 is A Novel Cannabinoid Receptor 

https://pubmed.ncbi.nlm.nih.gov/17876302/

Anil, S. M.; Peeri, H.; Koltai, H. Medical Cannabis Activity Against Inflammation: Active Compounds and Modes of Action 

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9124761/

https://www.sleepfoundation.org/sleep-aids/cbn-for-sleep

Long Covid Symptoms Linked to Inflammation 

https://www.nih.gov/news-events/nih-research-matters/long-covid-symptoms-linked-inflammation

https://www.fs.usda.gov/wildflowers/ethnobotany/medicinal/index.shtml

 has written for many cannabis publications, and organized or moderated sessions at national and international cannabis trade shows. He was voted the 2018 Journalist of the Year by Americans for Safe Access, the world’s largest medical cannabis advocacy organization.

Hodes, D., Innovations in Extraction Targeting Specific Wellness Goals, 

Tech Innovations

Searching for purer, minor cannabinoids is the goal of new extraction research as extractors dig deeper into the diversity of the plant.

Innovations in Extraction Targeting Specific Wellness Goals

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, 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 

. 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 steps 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, 

 compares chromatograms obtained under the same experimental conditions with an HPLC preparative column (100 x 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 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 microliters 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.

 (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 milliliters 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 

. 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 

. As already mentioned, the selectivity between CBG and CBD peaks is a critical factor with reversed-phase methods, as shown in Figure 3b, 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 

, 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 Δ8-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.

This article was originally published by our sister publication, 

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.

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

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

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