In our ongoing quest to understand how to optimize methods for chromatographic separations, we will see how to calculate retention times and introduce the concept of the capacity factor. The capacity factor normalizes retention time so that these quantities can be compared to each other even if separations are performed under different conditions. The capacity factor will help us predict how well analytes will be separated from each other, and we will find that this depends upon an important concept from the last column, the fraction of time the analyte spends on the stationary phase, F

Following up on our discussion of quantitating the separation of mixtures from the last column, here we will discuss further how to calculate the expected retention time of an analyte, and then define what are called capacity factors. The capacity factor is a way of normalizing retention times so we can compare chromatograms that are measured, for example, at different flow rates.

Recall that in the last column (1), we derived an equation that allows us to calculate the elution time for an analyte, as seen in 

 is the fraction of time spent adsorbed on the stationary phase.

 goes up, that is, the higher the fraction of time an analyte spends adsorbed and immobile on the stationary phase, (1-F

 goes up. If you imagine our different types of analyte molecules are in a horse race as in the last column (1), Equation 1 says, for example, that a horse that spends more time standing around grazing, as horses are wont to do, and not moving will get to the finish line later than a horse that spends all its time running.

Equation 1 is, in my opinion, the most important equation in chromatography because it relates the quantity we care about as measured by a chromatograph, the elution time of an analyte, to a property of the analyte­—the fraction of time it spends on the stationary phase. We will be seeing Equation 1 a lot going forward because it will provide us with the theoretical foundation for understanding how different chromatography separation methods work. As I have mentioned (1), and will probably mention again, the tricky part in achieving a separation is getting F

 to be different for all the analytes in a mixture you wish to separate.

 shows a chromatogram from last time (1). We assumed the sample was tetrahydrocannabinol (THC) dissolved in ethanol, and that the ethanol molecules had no affinity for the stationary phase, did not adsorb, and hence had a F

 value of 0. We also established last time (1) that the t

 for ethanol is 2 min. The elution time of ethanol then using Equation 1 is:

which shows that for unadsorbed molecules the elution time is the void time, which here is 2 min.

 were an easy calculation because we were not dealing with an analyte. Last column we assumed (1) that our THC molecule spent ½ of its time on the stationary phase, or in other words its F

 is 0.5. How then would we calculate the retention time of this analyte?

The reason THC has an elution time of 4 min in Figure 1 is that 2 x 2 min = 4 min. Note that the elution time for THC is twice that of ethanol because it spent half its time stationary whereas the ethanol spent no time stationary.

The beauty of Equation 1 is that it allows us to calculate the retention time of an analyte as long as we know F

. The practical problem here is that we rarely know F

 exactly because it depends upon several factors including the chemical natures of the analyte, mobile phase, and stationary phase. Thus, our lack of knowledge of the exact value of F

 means we must experiment with chromatographic conditions to optimize separations. However, Equation 1 is still true and can help guide our experiments. Given knowledge of the chemical structures of the analytes to be separated, one can deduce based on Equation 1 what types of experiments to perform to obtain different values of F

 for the analytes and hence achieve a separation. More on that in future columns.

As with all things chromatography, I refer you to publications (2,3) and references therein for a more detailed discussion of what is presented here.

So far, we have only considered a single chromatogram measured under controlled conditions. A problem with chromatography is that variations in the experimental variables affect retention time. For example, when we first discussed Figure 1 (1), we stated that the flow rate was 1 mL/min which gave t0 = 2 min. Now, if we slow down the flow rate for this separation to 0.5 mL/min, an unadsorbed analyte molecule will take twice as long to elute as before and give a t

 of 4 min. Note that in the calculation for the retention time of THC in 

says that the retention time for THC equals 2t

. By slowing down our flow rate to 0.5 mL/min and increasing t

 to 4 min the elution time for THC is now 8 min, compared to 4 min at a flow rate of 1 mL/min. These results mean that separations performed at different flow rates have different retention times, which ultimately means retention times in chromatograms run at different flow rates cannot be compared to each other.

A practical problem with chromatography, particularly with the pumps used in liquid chromatography, is that a given pump may not give the same exact flow rate over time. Worse yet, pumps on different instruments set to the same flow rate may give measurably different flow rates. This means that elution times for the same sample can vary from instrument to instrument, which is a real problem when comparing data across instruments and developing methods.

Fortunately, there is a solution to this problem. We calculate for each peak in a chromatogram what is called its 

. The capacity factor gives us a way of normalizing retention times so we can compare them across separations on different chromatographs. For example, capacity factors allow us to compare retention times when separations are performed at differing flow rates. The equation for calculating a capacity factor for a given chromatographic peak is seen in 

Let’s calculate the capacity factors for the two peaks in Figure 1. For ethanol t

) = 0 hence k = 0. This shows that for all void time peaks the capacity factor is 0. From Figure 1 t

 shows the chromatogram from Figure 1 with its peaks labeled with their capacity factors.

We said above that if we slowed our flow rate down to 0.5 mL/min t

 for our separation becomes 4 min and the t

 for THC becomes 8 min. We have established that the capacity factor for all peaks at t

 is 0. What is the capacity factor for THC at this new flow rate?

Note that despite the change in flow rate the capacity factor for THC is still 1 as seen in both 

. The reason for this is that in equation 8 the numerator is in units of time, the denominator is in units of time, and when we calculate k the two units cancel, meaning the capacity factor is a unitless quantity that does not depend upon flow rate. This means that by using chromatograms labeled with their capacity factors we can compare retention times to each other even if they are measured at different flow rates.

 shows a chromatogram with a void volume peak and two analyte peaks. In this chromatogram t

 is 2 min and the analyte elution time for peak A is 4 min and the elution time for peak B is 8 min. What are the capacity factors for the peaks in this separation? Well, the first two peaks are the same as in Figure 1, so their capacity factors are still 0 and 1, respectively. The capacity factor for peak B, k

 because there is no overlap between them and there is a flat baseline between them. Note that they also have quite different capacity factors, which indicates that the relative capacity factors of two peaks might be a measure of the quality of a separation. More on that next time.

We presented the equation from last time for how to calculate elution time, emphasizing the importance of the fraction of time, F

, an analyte spends adsorbed and immobile on the stationary phase. We then calculated some elution times based on assumed values of F

. We found that elution times for the same separation measured at different flow rates are different, and that the capacity factor, k, is a flow rate independent quantity that normalizes retention time and allows us to compare retention times measured under different conditions. Finally, we saw that for baseline separated peaks their capacity factors are different.

Smith, B.C., Chromatography Theory, Part II: How Separations Occur Illustrated and Quantitated, 

Skoog, D.A.; West, D.M.; and Holler, E.J.; 

Robinson, J.W.; Undergraduate Instrumental Analysis, 

, 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., Chromatography Theory, Part III: Calculating Elution Times and Capacity Factors, 

Articles

Cannabis Analysis 

Part III of this series will delve into understanding how to optimize methods for chromatographic separations, Smith will also introduce the concepts of capacity factor and selectivity.


Chromatography Theory, Part III: Calculating Elution Times and Capacity Factors

Cannabis is known for its distinct aroma. This aroma is due to the terpene profile during the flowering stage of the growth cycle. The aroma is more complex and amplified after the product has been dried and cured. The terpene profile of the plant is vastly different when it is freshly harvested as opposed to dried and cured. Manufacturers of cannabis concentrate and vape products have brought a new terpene profile to market by extracting cannabis that has been freshly harvested and frozen. In this article, we explore the difference in chemical profile between fresh and dry cannabis flower and why manufacturers may choose to extract frozen or dry cannabis.

Terpenes and terpenoids are aromatic compounds in the essential oils of plants, including cannabis. While many cannabis plants have a characteristic aroma, there is a wide array of cannabis phenotypes that produce a complex variety of terpene profiles. These unique aromatic profiles produce cannabis that smells like skunks, pineapples, blueberries, and many other familiar things. The cannabis plant can produce hundreds of terpenes based on genetics and environmental factors.

Cannabis plants produce glandular trichomes, which are bulbous plant appendages. Trichomes are found in only 30% of all plants. Terpenes and terpenoids are produced in the glandular trichomes of the cannabis plant. These glandular trichomes produce and house many metabolites of the cannabis plant, which include cannabinoids, terpenoids, and flavonoids. Three types of trichomes have been identified on the cannabis plant: sessile, bulbous, and stalked (

These three trichomes have different structural characteristics. However, they are also located in different parts of the plant and house different compounds. Stalked trichomes have a large head and house high concentrations of cannabinoids and monoterpenes. They are mainly found on the flowers of the female plants. Sessile trichomes are glandular with a short stalk and are mainly found on the outer leaves of the flower, known as sugar leaves, and produce both cannabinoids and sesquiterpenes. However, sessile trichomes produce much lower concentrations of cannabinoids and much higher concentrations of sesquiterpenes. Bulbous trichomes are the smallest of the three trichomes. They are more often located on the stalk and stems of the plant, help the plant maintain moisture, and protect the plant from the sun. When cannabis is harvested, the flowers are selected to obtain the highest concentrations of cannabinoids and terpenes.

The terpene profile of any given cannabis plant is dependent on many variables including genetics, environment, and grow method. However, when the plant has been freshly harvested the concentration of monoterpenes is higher than after a drying and curing process (2,3). The most common monoterpenes found in cannabis are beta-myrcene, limonene, alpha-pinene, linalool, and terpinolene. The most common sesquiterpenes found in cannabis are caryophyllene and humulene. The stalked trichomes exist in much higher concentrations than sessile trichomes, which leads to the conclusion that in cannabis there will be higher concentrations of monoterpenes. Terpenes and terpenoids are valuable components of the cannabis chemical profile. They not only give the plant its aroma, but also affect the physiological effects of cannabis.

Historically, the main method of consumption for cannabis flower has been smoking. The flower is dried and cured prior to consumption to lower the water content of the plant, increase the concentration of the desired compounds, and make it possible to smoke. In this drying process, depending on the storage conditions, the terpene profile will change. When done properly, the drying process will increase the total concentration of terpenes and develop a richer profile. When done improperly, all terpenes can be lost, and cannabinoids can degrade (2).

The curing process involves drying the product in a controlled environment and cycling oxygen in the curing environment. During this curing process, the concentration and composition of terpenes can change as a result of several factors, including:

Evaporation: As the flower dries, some terpenes may evaporate and dissipate, leading to a reduction in their overall concentration.

Decomposition:Over time, some terpenes may undergo chemical reactions or degradation, altering their molecular structure, and affecting their aroma and flavor.

Conversion: Some terpenes can be converted into other compounds through natural processes such as oxidation or enzymatic reactions.

Development: As the flower continues to age, new terpenes may be produced or existing terpenes may increase in concentration, leading to changes in the overall terpene profile.

The composition of terpenes in the cured flower will be vastly different from that of the fresh flower.

Cannabis flower has become a commodity in most mature adult-use markets. This is because of an excess in production of cannabis biomass. With this much biomass available, extraction operators and cultivators are looking for ways to preserve and store large quantities of biomass as well as convert it into a wider variety of products that are stable.

Most extracts have come from cured cannabis biomass; however, a shift has occurred. Many extractors, especially those using hydrocarbon solvents or solventless processes, have begun using freshly harvested cannabis in extractions. As a result, these extracts offer a completely different terpene profile from extracts produced with cured biomass. The cannabinoid profile of freshly harvested biomass will also be different from that of cured cannabis. Enzymes in the cannabis plant continue to convert cannabinoids and terpenoids in the plant, even after harvest.

This category of extracts has been named “live resin” and has taken the market by storm. To preserve the freshly harvested cannabis, the product must be frozen, or the product is at risk of developing mold. Instead of dedicating time and space to the drying and curing process, cultivation and extraction companies can simply freeze the biomass. This has created a streamlined storage process for large quantities of biomass. The downside is that freezers are not an efficient use of space and do require constant electricity. Alternatively, some companies are utilizing freeze drying technology to dry the cannabis while mitigating terpene loss.

Based on the growth of this segment of the market, it is clear that consumers are finding live resin to be unique and enjoyable. The main difference is the terpene profile, as live resin products will have more monoterpenes than extracts produced from cured cannabis. In the midst of the exponential growth of this product segment, cannabis companies have taken advantage of the lack of standardized taxonomy in the industry (4). In an effort to create a foundational standard for manufacturing terms in cannabis, the National Cannabis Industry Association (NCIA) produced a Glossary of Commercial Manufacturing Terms (5). In this glossary, the definition is (5): “Live Resin (n.): A cannabis extract product produced from plant material that was harvested and stored such as to preserve the chemical profile of the living plant, typically by freezing or freeze drying the material shortly
after harvesting.”

There are countless companies using the term “live resin” on products that are not produced from fresh frozen cannabis. Many products labeled “live resin” are simply distillate infused with cannabis derived terpenes from fresh or cured plant material, leaving out the entire cannabinoid profile of the freshly harvested biomass. This exposes a serious flaw in the cannabis industry as consumers can purchase products that are perceived to be one thing but are not. It will be interesting to watch this segment and uncover why consumers are truly drawn to these products.

Sommano, S.R.; Chittasupho, C.; Ruksiriwanich, W.; Jantrawut, P.; The Cannabis Terpenes. Molecules. December 8, 2020; 25 (24). DOI: 10.3390/molecules25245792. PMID: 33302574; PMCID: PMC7763918.

Bueno, J.; Leuer, E.; Kearney, M.; The preservation and augmentation of volatile terpenes in cannabis inflorescence. J Cannabis Res, 2020, 2, 27. DOI: 

https://doi.org/10.1186/s42238-020-00035-z

Wietstock, C. Researchers Reveal How Curing Cannabis Affects Terpene Levels 

https://www.ganjapreneur.com/researchers-reveal-how-curing-cannabis-affects-terpene-levels/#:~:text=After%20one%20week%20of%20drying,and%20Beta%2DCaryophyllene%20were%20increased

Luebke, D. What are live resin vapes? why are live resin vapes popular? 

https://www.heylocannabis.com/post/what-are-live-resin-vapes-and-why-are-they-popular

The National Cannabis Industry Association Cannabis Manufacturing Committee. Glossary of Commercial Manufacturing Terms 

https://drive.google.com/file/d/1rogqYBmQpe3VlqBchfsJ0iysjfbSq5bn/view

 is the founder, CEO, and Chief Extractor of Heylo. With a background in chemistry and clinical research, Lo was inspired to explore cannabis as a medicine and to enter the emerging industry. She joined Eden Labs, a leading CO

 extraction equipment manufacturer to support and expand a Research and Development department. There she managed the development of their latest and greatest CO

 extraction system. In 2017, after working with Eden Labs and another cannabis processor, Lo launched Heylo with a mission to help people get more out of life with cannabis.

Friesen, L., The Transformation of Cannabis Terpenes from Harvest to Shelf, 

Extraction Science

In this article, we explore the difference in chemical profile between fresh and dry cannabis flower and why manufacturers may choose to extract frozen or dry cannabis.


The Transformation of Cannabis Terpenes from Harvest to Shelf

Cannabis, much like any other plant, is susceptible to changes incurred by the surrounding environment. When it comes to cultivation, the peripheral environment is unquestionably critical, with factors such as soil, water, nutrients, and lighting quality and quantity, collectively having a profound impact on the terminal yield and phytochemical profile (cannabinoids, terpenes, and flavonoids) of a resulting crop. Additionally, the sensitivity of individual cultivars to unpredictable environment stressors (for example, pests and microbes) can also alter their phytochemical expression in ways that we don’t fully understand yet. As such, there is a tremendous impetus for the cannabis industry to continue studying the interplay between phytochemical content and environmental factors, which will undoubtedly require us to wade into the relatively unchartered waters of cannabis epigenetics.

In the realm of biochemical research, epigenetics is an aspect of gene regulation that has garnered considerable interest over the past decade. In essence, epigenetics is the study of how environmental factors can alter genetic material, thus influencing the expression of genetic traits. Contrary to genetic mutations, whereby individual changes are made in the nucleotide sequences of deoxyribonucleic acid (DNA), epigenomic modifications do not directly affect these building blocks, and therefore do not result in permanent changes to an organism’s genetic code. Epigenetic modifications occur through DNA methylation, a process involving the transfer of a methyl group (CH3-) onto DNA nucleotides, which can also be mediated (methyl groups removed) by enzymes called DNA demethylases. Collectively, these epigenetic modifications affect how DNA strands interact with histone proteins that are involved in DNA storage and regulation, thus ultimately serving as molecular ON/OFF switches modulating gene expression.

Arguably the most interesting aspect of epigenetic modifications to one’s genetic code is the discovery that these changes can be passed down to subsequent generations. For example, recent studies have now shown that smoking tobacco can have a distinguishable effect on the individual who is smoking (nothing new there), in addition to altering gene expression in their gametes (sperm or egg cells), and gene expression in the gametes of their offspring. This is to say that the genotype/phenotype of a grandchild can be heavily influenced by the environmental conditions experienced by their parents and even grandparents.

The study of epigenetics likely hasn’t started or hit its full stride in the cannabis industry; primarily due to the federal regulations on cannabis-related research. However, we can learn from the seminal work that has been done in other plants. For example, there is growing evidence that epigenetic regulators play critical roles in fruit development and ripening in flowering plants, and that these molecular actuators might work together with hormones and transcription factors. One example of this is in the way that climacteric fruits (such as tomatoes and bananas) ripen through respiration and ethylene production. Epigenetic modifications have been found to play a role in this process. A distinct example of this is the tomato variant Colorless Non-Ripe (CNR), which exhibits an inhibition of fruit softening and the absence of carotenoid and flavor compound accumulation. Studies have shown that the CNR variant arises from the hypermethylation of a 286 bp region that is located 2.4 kb upstream of a SQUAMOSA promoter binding protein-like transcription factor (SBP box/SPL). In result, this leads to a spontaneous mutation known as CNR. The CNR mutant is characterized by a non-ripening phenotype where the fruits turn white and then yellow but remain firm during the ripening period. This study supports the notion that natural methylation polymorphisms can influence the fruit phenotype of tomato plants under specified environmental conditions, demonstrating the importance of epigenetic variation in tomato plants (1).

As it turns out, the ripening of orange fruits is also attributable to hypermethylation. This involved the active expression of several hundreds of genes including genes involved in abscisic acid, a prominent plant hormone responsible for a myriad of physiological processes (2). These findings demonstrate that epigenetic modifications play an integral role in orange fruit ripening during early stages of plant development.

Additionally, natural variation in vitamin E concentrations found in tomato fruits has also been associated with changes in DNA methylation at the promoter of the enzyme 2-methyl-6-phytylquinol methyltransferase (1). As such, it is clear that epigenetic processes play critical roles in a number of physiological, metabolic, and molecular changes in fruit-bearing plants, the extent of which remains to be determined. However, it is plausible that the interplay between DNA methylation and demethylation has a discernible role in the relative expression of pigmentation, soluble sugars, and aromatic volatile compounds. It is technical findings such as these that could yield “fruitful” results in the cannabis industry.

In cannabis (Groups I, II, and III), understanding the genetics and what phytochemical contents are influenced by the environment is crucial to the expression of various phenotypes in a genetic cultivar. As such, the use of epigenetic analyses in the cannabis industry could help us differentiate chemical characteristics that are predominantly genetic-based from those that may be attributable to the cultivation environment. Ultimately, this would allow us to “dial in” the cultivation variables for a certain genetic so that it yields a precise phytochemical profile for the treatment of specific ailments.

While the production of phytochemical content and yields in cannabis depends on a number of environmental factors (soil, water, nutrients, and so forth) lighting is unequivocally the most influential variable. During photosynthesis, light can provide energy to the plant, but also act as a sensory signal that affects growth and development, physiological functions, and biochemical pathways. Various studies have begun exploiting the spectral flexibility of light emitting diodes (LEDs) to examine the effect of different spectral combinations on cannabis production and phytochemical diversity under in controlled environment agriculture (3-4).

With that being said, experimenting with different lighting spectra and intensities seems like the most fertile ground for epigenetic studies in the cannabis industry to better understand the interplay between horticultural lighting and cannabinoid, terpene, and flavonoid expression, particularly for any downstream clones or offspring (seeds) (see Figure 1).

For example, we may discover that how you treat your seeds and mother plants can have a noticeable effect on multiple subsequent generations. Hypothetically speaking, if you “pop” seeds under a T4 bulb and then switch them over to a full-spectrum LED to be “mothered” for clones, it is possible that the lack of full spectrum photons (400–700 nm) from the T4 could have downstream implications for the subsequent clones or even any pollen or seeds that those clones may produce. We already know that cannabis can experience “spectral shock” if the spectral changes between two points in the cultivation cycle are too drastic. This is primarily an issue going from the sparce spectrum of a T4 bulb in cloning to a full spectrum light in veg. You’ll notice that for the first 7-10 days of this switch, the plants will appear as though they are struggling to thrive, or potentially “burning up” completely. As such, it seems completely plausible that if these effects are noticeable in the current generation then this change in environmental stimuli could have significant downstream implications. While this phenomenon is poorly understood, propagating clones from tissue culture may be one way to address the accumulation of poor treatment or “epigenetic baggage.”

Across the past decade there has been an explosion of new knowledge with respect to how to improve cannabis production. This is largely the result of new research designed to further elucidate the interplay between phytochemical expression and the quality of the surrounding environment. However, how do environmental factors, such as lighting, affect the chemical composition of downstream products? It is still unclear how epigenetic inheritance works in cannabis and how epigenetics contributes to the phenomenon of nature versus nurture. Although this research is still in its infancy, it has been established that DNA methylation/demethylation is another layer through which the genome’s language is interpreted and, ultimately, demonstrated in an organism’s phenotype. As such, it is going to be extremely exciting to see how all of this unfolds in the cannabis industry for years to come. The hope is that a better understanding of the cannabis epigenome will provide cultivation experts with another opportunity to produce highly efficacious products that can be tailor-made for the effective treatment of specific ailments.

Gallusci, P., Hodgman, C., Teyssier, E., and Seymour, G. B., DNA Methylation and Chromatin Regulation during Fleshy Fruit Development and Ripening, 

Huang, H., Liu, R., Niu, Q., Tang, K., Zhang, B., Zhang, H., Chen, K., Zhu, J. K., and Lang, Z., Global increase in DNA methylation during orange fruit development and ripening, 

Proceedings of the National Academy of Sciences of the United States of America

Hildenbrand, Z.L., Liden, T., Jacques, A., Spurlock, M., West, C., Westerfield, R.E., and Schug, K.A., Differentiation of Group III cannabis into distinct sub-classes. 

Hildenbrand, Z.L., Grosella, A., Manes, R.J., Spurlock, M., Jacques, A., West, C., Love, O.R., Westerfield, R.E., Pecore, M., Liden, T., Gao, S. 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., Epigenetics: Decoding the Effects of Environmental Factors in Cannabis Cultivation,

The Cultivation Classroom

A look at what epigenetics means, how other plant industries have used or studied them, and how that can be applied to cannabis to improve yields, plant genetics, and solve plant illness.


Epigenetics: Decoding the Effects of Environmental Factors in Cannabis Cultivation

As the recreational cannabis market matures, and the medical market relies on precise dosage, there are some surprising new ideas in processing and sustainability being explored. This installment will discuss new methods of cannabis oil production, and manufacturers' reaction to the rise of the demand for cannabis artisan extracted products. We’ll also take a look at how new gene technologies are allowing growers to enhance the THC content in their plants even to the point of a specific THC target level. Finally, we’ll talk about safety as an ongoing issue in cannabis manufacturing.

When the cannabis industry first began selling recreational adult products in Denver, Colorado in 2014, there was a rush to get the highest-level tetrahydrocannabinol (THC) products. In fact, according to an article in 

 (1), in some states the legalization of cannabis for adult and medical use has increased the availability and sales of cannabis products, such as extracts, that have a THC content greater than 70%.

The cannabis industry has resisted proposals to cap THC content by arguing that people who use high potency cannabis extracts reduce their THC dosage to achieve the same desired psychoactive effects. They may, for example, reduce the amount smoked when using high THC products. They may also inhale smaller puffs or do so less often when using higher potency products. The 

 (1) article concluded that there is an “urgent need for larger and better controlled experimental and observational studies of the extent to which cannabis users can and do titrate their THC doses when using more potent cannabis products, such as cannabis extracts and high potency cannabis flower. This research is needed to inform policymakers on how to reduce harms from the use of high potency cannabis products. It may indicate the need for caps on the potency of cannabis products or higher taxes on more potent cannabis products to discourage their heavy use. It is also needed to inform the labeling of THC doses in legal cannabis products that may include standardized THC doses analogous to standard units of alcohol.”

Indeed, medical cannabis regulators in states such as Vermont, Texas, and Florida—among others—have discussed or implemented THC caps for medical cannabis (2-4). This installment discusses manufacturers reaction to the rise of the demand for new types of cannabis artisan products with varying strengths of THC, and other “designer” cannabis that the consumer wants. We’ll also look at how new gene technologies are allowing growers to enhance the THC content in their plants—even to the point of a specific THC target level. Finally, we’ll talk about safety as an ongoing issue in cannabis manufacturing, with a note from the Sustainable Cannabis Coalition (SCC) and a quick mention of new fire safety standards being developed by the National Fire Protection Association (NFPA) for oil extraction facilities.

One of the new ways to help determine THC levels in a cannabis plant is through genetic engineering. It’s being used now to study and try to control the THC levels of hemp, which has a tendency to “go hot” after being planted, meaning that the 0.3% THC required for it to be considered legal hemp (5) actually rises, and becomes more concentrated, due to various soil and climate conditions.

When that happens, it becomes an illegal cannabis plant. A hemp farmer could have his whole crop burned if even part of it was discovered to have a greater percentage of THC than is allowed by law.

Samuel Proctor, CEO of GT Research, which is a plant genetics research organization made up of geneticists, computer scientists, and bioinformatic specialists, said their focus has been on building the best platform or computational analysis of the genome and the epigenome of a plant organism, and leveraging that understanding in terms of bio-engineering.

“Understanding the genome is fundamental, although there's plenty of progress being made,” he said. “For cannabis, there is far less understanding of its genome than compared to crop things like corn or soy—crops that have been industrialized and heavily studied and modified by industry for decades. So, we are very much building for the present and future.”

He said that GTR is agnostic in the sense that they don’t try and make the plant do this or that. “We deploy our technology on behalf of partners and customers,” he explained. “So, we really just let them say what they need, and then use our expertise and capabilities to see if we can deliver that.”

“It was clear to us from the beginning that the there was a commercial need for a hemp line where one could be assured of the level of Delta 9 output, and that's what GTR has been working on for almost two years,” Proctor said. “We've achieved a number of milestones in terms of demonstrating our ability to use the technology. It is patented. And we can share that patent. It's publicly available if you want to take a look at it. We're very optimistic that this cultivar will be in the market sometime this calendar year. We're deploying the technology in partnership with producers, so those producers will bring to market this cultivar, which essentially will have been created using our technology.” He added he doesn’t know what the resulting cultivar will be called.

Could GTR then essentially design a certain type of cannabis plant for a different niche market? “The way we think about it is a series of sub or niche markets in terms of the plants,” Proctor said. “One market certainly is the legal cannabis US retail consumer market. The demand point is clearly related to Delta 9 output. But there is a whole set of markets for particular, rare cannabinoids. We're all about focusing on the place where there's the best overlap of the Venn diagram (7) in terms of commercial need, and the ability to deploy our technology to achieve those needs.”

The way their process generally works is by starting with what someone wants to achieve and working backwards, Proctor explained. “We started with the problem, which is: What causes the levels of Delta 9 to fluctuate, and why is it never zero? The answer is, as we discovered when we looked, that there is a multiplicity of copies of the THC gene,” he said. “So any dilution of that has to deal with the fact that there are multiplicity of copies. They turn themselves off and on. Our core competency lies in understanding what the genetic and epigenetic mechanisms are underlying the phenotypic outcome. And then identifying and using the right computational and bio-engineering tools to deal with whatever mechanisms are identified.”

OK, so GTR has found a way to measure and manipulate THC. But is there anything else that can be done with this technology? “We think the sky's the limit,” Proctor said. “It's an amazing plant, with just a whole universe of compounds and effects which we are still at the beginning of understanding. So, it's clear that minor cannabinoids are in demand, and that modulation or enhancement of them could be an attractive outcome. But there's plenty of cannabinoids that we haven't yet really characterized and don't really understand what they do.

“We're generally of the view that, over time, we will go in the direction that other agricultural commodities have. Science will continue to develop its understanding of the genome. Through a number of means, both in terms of modifying the plant and through synthetic biology, we will create a host of products that key off of the plant and what it produces,” said Proctor.

Some of those products could be the higher end, and sometimes higher THC, cannabis artisan products—a growing niche market today (8).

Proctor said that the computational tools that are available to analyze the genome of any living thing continue to advance, and are very powerful. “We think that everyone should be doing whole genome sequencing of their cultivars to gain a core genomic understanding of their plants,” he said. “That has nothing to do with genetic modified organisms (GMOs [9]). That's just computing.”

GTR offers a general overview of a cultivar for $3000 that includes whole genome sequencing. The sequencing technology will continue to improve. It also depends on what someone wants to look at. “At a base case, doing whole genome sequencing and analyzing key pathways, that's important,” Proctor said. “But a particular grower might say, ‘Is this cultivar favorable or unfavorable for increasing THC yields,’ just as an example. So, those are the types of questions that the computational platform is necessary to answer, and we definitely have the ability to deliver answers to those questions to growers in a very cost-effective manner.”

As GTR and other companies are exploring ways of controlling THC in the laboratory, the product development side of the industry keeps churning out new ways of consuming cannabis products, some that are beginning to look more like pharmaceuticals with controlled dosage, which helps broaden their appeal.

“Smoking is not exactly an accepted way of consuming cannabis across all of consumers,” said Robert Sedlock, vice president of Natoli Scientific (10), a tablet compression tooling and development company. “Because you have older people that don't want to smoke. So, with tablets, it's a consistent dosage form. Tablets have been around for over a century and it's an accepted process in the medicine world and in the dietary supplements world. So, moving the cannabis industry into a dosage form like tablets is definitely the future in my mind because now you've got an exact dosage form.”

Natoli is based in a 14,000-square-foot facility in Telford, Pennsylvania with four PhD scientists, mechanical engineers, and technicians. The company provides services to the pharma, dietary supplements, cannabis industry, and food industry.

 (11) identified tablets as being the largest group of delivery systems for the pharmaceutical and dietary supplement industries. This technology offers an opportunity to the cannabis industry to allow orally administering of a consistent and defined amount of cannabidiol (CBD) or THC.

Sedlock doesn’t have a Schedule 1 Drug Enforcement Administration (DEA) license to work with Delta-9 THC product directly (his partner grows both hemp and Delta-9 cannabis), so he works with CBD distillate.

“You as a customer might have a THC product, but your CBD distillate is the same consistency, the same kind of viscosity as your THC,” he said. “So, I’ll develop (a tablet operation) using CBD and then I’ll send it to them and they’ll approve it. Then you tell them ‘OK when you guys manufacture, don’t use CBD, use your THC.’ It’s the same thing.”

Natoli follows pharmaceutical practices and regulations in developing their tablets. “So, they're proven safe and compliant procedures,” Sedlock said. “It’s the wild, wild west right now in terms of cannabis regulations in various states, so eventually the FDA [Food and Drug Administration] is going to come in and make some rules. You better hope that you're following the procedures of the pharma process.”

Most sustainability issues deal with water and electricity, and what to do with the waste from processed cannabis, in a broad process that includes managing safety issues as well.

But there is new light being shed on what to do with the actual product packaging, which is generally made of hard plastic, and difficult to open, because industry regulators appear to be erring on the side of overdoing it with their recommendations for packaging safety.

For example, due to regulated safety requirements, the type of packaging used in cannabis often weighs four to 30 times their contents. A single gram of cannabis has been found to be packaged in 70 g of plastic packaging (12).

Trulieve, and their program, TruRecycle, based in Florida (13), has reportedly recycled more than 3500 lbs of cannabis packaging waste since October 2021. Their program allows patients to return select container types to any Trulieve dispensary location in the state, so long as they are clean, free of labels, and clear of residue. Patients return packages in-store and they remain in zero waste boxes that are shipped with a UPS carbon neutral shipping partner to their recycling partner.

Shawn Cooney (14), founder and CEO of Cloud Farming (15), and a spokesperson for the Sustainable Cannabis Coalition (SCC), sees the problems with packaging in the industry. “Yeah, you got that one wired,” he said, referring to the problem of hard plastic packaging of recreational cannabis products. “Part of it is legislation, and the regulators saying you have to do it (package) a certain way, whether you agree with them or not. Everyone has to sort of go along with that.”

One company Cooney talked to is getting a lot of plastic coming back now, but they've had to ask their customers to do that. “They still don't have a really great way of disposing it. So, they put it in to some kind of chopping system to do something with it. That’s not the best way to get rid of it,” he said.

“There are no industry facts and figures about getting rid of or recycling plastic packaging,” Cooney said. But there is work under way.

The SCC is working with the American Society of Testing and Measurement (ASTM) and the ASTM’s cannabis group, Committee D37 (16). So far, that committee has published more than 40 standards and has 100-plus currently in development (16). There are 16 subcommittees (17) under the D37 committee, addressing standards issues of sustainability, processing and handling, laboratory, quality management, and others.

There's also a packaging group and a labeling group. Cooney and his wife, Jennifer, are the chair and vice chair of the cannabis sustainability group at the ASTM. “We've got 110 folks just focused on sustainability, working on what to do with the cannabis waste stream,” he said. “We are starting a whole standards thing. We will have some standards that legislators can share with regulators.”

“What the ASTM is trying to come up with is a consensus on industry standards that can convince legislators and regulators who can look at that and say, ‘Okay, this is what the experts have agreed on, maybe we should start moving in that direction.’ As opposed to looking at each other and pointing fingers, or relying on anecdotal stories,” Cooney said. “That's what we're really pushing for, is to start the standards process on that from a sustainability side.”

He also shared that the SCC is focusing on greenhouse gas emissions in the cannabis production, or what's called Scope 3 emissions, which is really never talked about. Scope 3 emissions are the result of activities from assets not owned or controlled by the reporting organization, but that the organization indirectly affects in its value chain, according to the Environmental Protection Agency (EPA) (18). “And that's the hardest to account for,” Cooney said. “But generally, it's basically 70% of the company's greenhouse gas emissions.”

Measuring greenhouse gases is on the short list of what cannabis producers and packagers need to understand better for an overall safer operation.

Other safety standards are still being explored by The National Fire Protection Association (NFPA), which is developing new cannabis fire protection standards, hoping to expand on the NFPA1 fire code, which addresses fire protection and energy efficiency concerns within growing and processing facilities.

NFPA1 (19) only addresses concerns, it doesn’t set in stone specific regulations. New standards will address the protection of those facilities and set education, inspecting, testing, and maintenance requirements in part to reduce excess energy consumption. (Extraction operations are already semi-well regulated, according to U.S. Chemical Storage [20], because of well-established rules regarding hazardous flammable chemicals such as butane, hexane, and ethanol; and hazardous gases, such as carbon dioxide.)

Controlling the entire production and packaging process better is top of the agenda for any cannabis production operation. That means from the
micro-level—the genetics of the plant—to the macro level—the machinery and environment inside the production facility. Everything must work in sync to get the most efficiency—both out of the plant and the production process using the plant.

In terms of energy and sustainability, there are some hidden electricity greenhouse gas generators that not everyone takes into consideration beyond just the energy costs of growing the plant. “If a cannabis producer is running a kitchen, he’s got a lot of heavy equipment. You've got chillers, compressors, ovens, vacuum pumps, and on and on and on,” said Cooney. “So, they're in a real similar position to a grow operation. They may not be quite as big, but the energy intensity for that piece of the business is really high.”

Cooney added that SCC is pushing the industry to all get on the same page when it comes to the broader picture of sustainability. “Every cultivator has its own sustainability problem—indoor, greenhouse, or outdoors. They've got something to talk about. And it’s time to start measuring and fixing it,” he said.

https://www.frontiersin.org/articles/10.3389/fpsyt.2021.630602/full#B1

https://ccb.vermont.gov/response-15-thc-cap-recommendation

https://texasoriginal.com/2021/05/19/the-texas-medical-marijuana-thc-cap-explained/

https://norml.org/florida-oppose-arbitrary-thc-caps/

https://www.cdc.gov/marijuana/featured-topics/CBD.html

https://www.lucidchart.com/pages/tutorial/venn-diagram

https://www.farmhouse.delivery/collections/cannabis-goods

https://www.nature.com/scitable/topicpage/genetically-modified-organisms-gmos-transgenic-crops-and-732/

https://natoli.com/services/natoli-scientific/

https://www.cannabissciencetech.com/view/cannabis-and-hemp-tableting-challenges

https://www.cbc.ca/news/canada/new-brunswick/cannabis-packaging-excess-1.4870682

https://www.trulieve.com/discover/blog/trulieves-cannabis-packaging-recycling-program-trurecycle

https://www.sustainablecannabiscoalition.com/spokespersons

https://www.astmcannabis.org/cannabis-standards-and-publications/

https://www.astm.org/get-involved/technical-committees/committee-d37/subcommittee-d37

https://www.epa.gov/climateleadership/scope-3-inventory-guidance

https://www.nfpa.org/codes-and-standards/all-codes-and-standards/list-of-codes-and-standards/detail?code=1

https://www.uschemicalstorage.com/safe-hazmat-storage-compliance-in-the-growing-cannabis-extraction-industry/

 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., Targeting Consistent THC Levels in Sustainable Manufacturing and Processing, 

Tech Innovations

This installment discusses new methods of cannabis production and manufacturers reactions to the rise of the demand for cannabis artisan extracted products.

Targeting Consistent THC Levels in Sustainable Manufacturing and Processing

The cannabis industry has experienced significant growth momentum in the last decade as several states and countries outside the US have legalized cannabis for medical and recreational use. Alongside it, the nature of the products in the market has diversified and consumers now have a wide spectrum of products to choose from. This has prompted a shift from flower, which has been the mainstay of the unregulated market, towards more sophisticated products created from extracts. Given the high value of cannabis extraction as a niche, starting a cannabis extraction facility can be a potentially lucrative venture. However, the regulatory environment around cannabis is rather nuanced, with fluid laws that change from one state to another. This tutorial article outlines the key considerations that must be put in place when setting up a cannabis extraction facility.

The cannabis extracts market size has grown in leaps and bounds in the last couple of years. From a current estimated value of about $2.7 billion, the value of the global extract market size will probably hit $3.5 billion by 2030 if the Compound Annual Growth Rate (CAGR) of about 20.6% is maintained during the period (1).

Cannabis extraction is an integral part of the cannabis value chain. Once cannabis has been cultivated and harvested, the phytochemicals are then extracted and used to create different products. Extraction laboratories can either exist independently or they can be a part of a vertically integrated cannabis operation.

Cannabis extraction is one of the ways through which laboratories can venture into the cannabis industry. Setting up an extraction laboratory for business requires a huge investment in terms of time, resources, and expertise (2). The extraction of cannabinoids and terpenes from cannabis is a fine balance between science and art, calling for high precision in the scientific technique while at the same time, employing the finest art in curating a high-quality extract. In addition, there are rules and regulations that cannabis extraction laboratories must comply with. Cannabis is classified under Schedule I of the Controlled Substances Act (CSA), technically implying that it has a high risk of abuse and has no known medical use in the US (3). This also means that cannabis is an illegal compound under federal law, even though more than two thirds of the states have legalized medical cannabis. Cannabis is thus a highly regulated industry and so is cannabis extraction.

When setting up a cannabis extraction facility, several factors must be taken into consideration. Like any other business, several key questions need to be asked: “where” to set it up means the location, the “why” refers to the business objectives, the “how” illustrates the business model, the “who” is the human resource, and the “which” refers to the kind of equipment or extraction methods that will be used. In addition to this, there will be compliance requirements that the laboratory must follow. This includes obtaining a license to operate an extraction facility and setting up the facility in compliance with industry standards. Much as the considerations are broad, we have identified 10 key areas that must be considered before you set up an extraction facility.

An extraction laboratory is a business like any other and will therefore require a business plan to direct all its activities. Your business plan should be specific and have quantifiable objectives. It is important to involve different experts when crafting your business plan to ensure that it is authentic, efficient, and feasible. Start by first defining your vision and mission. Is profitability the sole purpose of your business and, in that case, how soon do you expect to achieve this? What do you want to offer the market and does the market need it in the first place? Who is your ideal customer and what are their unique needs and preferences? Having a clear roadmap before you begin the journey will provide you with the much-needed focus, direction, and resilience should the going get rough. On the flip side, you might even discover that you do not want to venture into extraction after you lay all the facts bare on the table.

Unlike other businesses, you cannot just set up a cannabis extraction facility in any vacant area of your choice. States and municipalities dictate where cannabis businesses can be located. First, you will need to find a state that has legalized cannabis and has friendly cannabis laws. Most states require that cannabis businesses are located at a minimum distance away from public facilities such as daycares, elementary schools, parks, libraries, playgrounds, and residential areas. This is usually about 1000 ft away. Some states will also require a minimum distance away from other cannabis businesses to prevent oversaturation in one area.

Other than the laws, you should also consider a location that best supports the implementation of your business plan. Where are your target customers located? Where are you likely to face the least competition? What about accessibility to raw materials? You must answer these questions and make decisions judiciously to maximize profitability.

Without a license, you cannot run a cannabis extraction facility legally in the US. However, you should only apply for a license after you have drafted your business plan and found a suitable location. If you are venturing into the cannabis industry for the first time, it will be advisable to engage an expert, such as a cannabis attorney, to help you with the license application process (4). Ensure that you have all the documents that are required for the application and that they are valid and accurate. Most states and municipalities will also require you to pay a non-refundable license application fee. Some states have programs for equity applicants, and you can make use of such if your situation applies. Familiarize yourself with the zoning laws in your area to ensure that your license application is not declined based on your location.

Once you have succeeded at getting a license, it is time to design your extraction facility. You will need to hire personnel with the best expertise in the niche to help you come up with a facility that fully optimizes your workflows while ensuring the highest levels of safety (5-7). There should be a consistent flow from the time the raw materials get into the laboratory to the point where they are transported out of the laboratory. This should support the highest level of safety and efficiency. Extraction laboratories should also comply with the current good manufacturing practices (cGMP) (8).

cGMP has about 10 principles that dictate how manufacturing facilities should handle people, raw materials, processes, and equipment. This preempts the need for standard operating procedures that outline the minimum quality threshold for extraction processes, staff training, and equipment maintenance. cGMP standards ensure that all extracts meet internationally acceptable safety standards.

Extraction laboratories handle volatile compounds that can easily start a fire. Consequently, they need to adhere to safety measures to prevent fire outbreaks and minimize injury, loss of property, and death as a result of a fire outbreak. Consequently, laboratories are required to have fire extinguishers, alarms, smoke detectors, sprinkler heads and pipes, and fire safety messages strategically placed in the facility. The staff should also be adequately trained on fire safety and should countersign every training, including fire drills. There needs to be a fire policy detailing what should be done should a fire outbreak happen.

National Fire Protection Association Local Fire Codes

The National Fire Protection Association (NFPA) is an organization that promotes fire safety in workplaces and other public spaces. It has created more than 3000 fire codes that apply to different scenarios and situations (9). Here are a few that apply to extraction facilities:

This code provides general fire safety measures to be followed in case of a fire outbreak.

 This code provides guidance on how flammable materials should be handled and stored.

: This code provides guidance on how liquefied petroleum gas (LPG) should be handled.

 This code, also called the National Electrical Code (NEC), provides guidance on how electrical installations should be done.

The cannabis extraction industry is relatively new and at the same time very specialized. It requires a high level of expertise that may not be easily accessible. You will need legal expertise to help you navigate the licensing and other compliance processes, skilled engineers and architects to design the facility, and master extractors and chemists to handle the extraction process. You can use sources such as LinkedIn to get referrals or you may also opt to use a hiring agency that has expertise in the cannabis space. Once you have put together a team, you will need to ensure that they are adequately trained and have all the resources that they need to execute their job.


While your business plan should have already specified the extraction equipment that you will need for your laboratory, your team of extraction experts should validate your choice. You will need to be aware of the target lead times and how long the installation process will take. The equipment that you select will determine the nature and quality of your extracts, it will also determine the quantity of the output. Ensure that your equipment is in tandem with the general objectives of your extraction facility and that it also fits your budget. Lastly, your extraction equipment must meet regulatory requirements. Once your equipment has been installed, the municipality will send a state-licensed engineer to verify that the required standards for safety and functionality have been met.

Cannabis extraction produces a lot of waste that will need to be effectively managed. Because of the federal status of cannabis, wastes from the plant and any unused materials and products must be treated in accordance with federal laws (10). Some municipalities will require that such wastes be destroyed on-site while others will allow third parties to do that at designated sites. On-site disposal methods include burning, shredding, and burying, but the waste must first be destroyed to a point where it becomes both unusable and unrecognizable. You will need to familiarize yourself with the cannabis waste disposal laws in your municipality before settling on a waste disposal method. Cost and convenience should also be factored in since you will be constantly churning out waste.

After setting up your facility, hiring the best team, and getting your equipment in place, the next thing you should be thinking about is how to manage your supply chain. Extraction, being a mid-stream process, has several inflows and outflows. Supply chain management involves considering the demand for products and services and matching them up with the raw materials and resources that are coming in. This ensures that at any given time, all the resources in your extraction facility are working at optimal levels and that there is no wastage or redundancy. If your laboratory resources and processes are not optimized, it will be difficult to achieve profitability despite having sufficient sales (11). It is important to hire experts to handle your lab’s supply chain.

10. Leveraging a Laboratory Software for CBD and THC Laboratories for Automation

One final step, automation of laboratory workflows. Extraction laboratories handle a lot of materials and data at any given time. Managing information and workflows can be quite hectic for the lab staff and hence increase the chances of human error. With a laboratory software for cannabidiol (CBD) and tetrahydrocannabinol (THC) laboratories, known as a laboratory information management system (LIMS), extraction laboratories can automate their processes and streamline workflows for maximum productivity (12). A cloud-based LIMS plays a key role in ensuring the success of an extraction laboratory since it can be accessed from anywhere and at any time (13).

With these 10 considerations taken into account, you should be ready to launch your extraction facility. Depending on your locality, the “Authority Having Jurisdiction” (AHJ) may need to conduct a final inspection at your facility. This is done to ensure that everything within your laboratory is safe and meets regulatory requirements. Once you launch your facility, stick to your business plan to avoid operational inconvenience and to maximize profitability.

Cannabis Extract Market Size, Share & Trends Analysis Report By Product Type (Oil, Tinctures), By Extract Type (Full Spectrum Extracts, Cannabis Isolates), By Sources (Hemp. Marijuana), By End Use, By Region, And Segment Forecasts, 2022 - 2030 

This tutorial article outlines the key considerations that must be put in place when setting up a cannabis extraction facility.

March 2023

Chromatography Theory, Part III: Calculating Elution Times and Capacity Factors

The Transformation of Cannabis Terpenes from Harvest to Shelf

Epigenetics: Decoding the Effects of Environmental Factors in Cannabis Cultivation

Targeting Consistent THC Levels in Sustainable Manufacturing and Processing

Cannabis Extraction Facility Setup 101

Keeping it Clean: Recapturing Terpenes for Added Value and Cleaning Wastewater Streams