Throughout our discussion of chromatographic theory (1-7) we have emphasized the importance of F

, the fraction of time an analyte spends stationary, as a key determiner of elution time and separation quality. Does the Master Resolution Equation make sense in light of this? Yes, but there is a lot of math involved. Here, we will explore how the different parameters in the Master Resolution Equation relate to F

, and what that means for chromatographic experiments.

In previous columns (1-7), I have discussed and reviewed at length the concepts of elution time, capacity factor, separation factor, and plate number. The equation for elution time is shown in 

 is the elution time, to is the void time, and F

 is the fraction of time an analyte spends adsorbed on the stationary phase.

I introduced the Master Resolution Equation last time (1), which pulls together most of what we have learned about chromatography in one place. This is given as 

where R is resolution, N is the plate number, α is the separation factor, and k is the capacity factor.

To review very briefly, the parameters in Equation 2 are defined as follow.

Recall (4) that the separation factor is calculated for two peaks with elution times t

 = elution time for a chromatographic peak, and w is the chromatographic peak width measured with the triangulation method.

 one or more elution times are present. In other words, each of the quantities in the Master Resolution Equation depends on elution time, and according to equation 1 elution time depends upon F

. This means all the quantities in the Master Resolution Equation depend upon F

For example, let’s take the first term in the Master Resolution Equation, (√N/4). If we rearrange Equation 5 to solve for t

, and then substitute into equation 1 we obtain 

where all these terms have been defined above.

Remember from the Master Resolution Equation (Equation 2) that N is proportional to R, and anything that increases N will increase chromatographic resolution. Peak width is in the denominator in Equation 6, which makes sense since as peaks get narrower N, and hence R, will get better. Note the presence of (1-F

) in the denominator in Equation 6. As Fs goes up, (1-F

) gets smaller, which means N, and hence R, get bigger.

This makes sense since the greater the fraction of time an analyte spends adsorbed the less chance it has to diffuse and broaden a peak and hence decrease resolution.

From Equation 2, we know that as k goes up R goes up. Equation 3 shows us the dependence of capacity factor on elution time t

If we substitute Equation 1 into Equation 3 and rearrange we obtain 

. The ~ in Equation 7b means the two quantities are proportional. Note in Equation 7b that (1-F

) is in the denominator again as in Equation 6. Again, this means that as F

 goes up, k goes up. The capacity factor k depends upon the elution time, t

. The elution time is determined, in part, by the affinity of an analyte for the stationary phase. There is a maxim in chemistry when thinking about solutions that “likes dissolve likes.” Similarly in chromatography, “likes adsorb likes.” Consider the structures of ethanol and toluene seen in 

. A polar ethanol molecule with an O-H group is much more likely to adsorb onto a silica stationary phase containing Si-OH groups than a non-polar toluene molecule. In this case, F

for ethanol will be greater than for toluene, toluene will elute before ethanol, and k for ethanol will be greater than that for toluene. This term “affinity” of the analyte for the stationary phase can be thought of as its “stickiness.” Sticky analytes will bind more strongly to the stationary phase and thus spend a greater fraction of their time adsorbed than less sticky ones.

For the separation factor α, if we substitute Equation 1 into Equation 4 we derive 

 is the fraction of time analyte a spends adsorbed on the stationary phase, and F

 is the fraction of time analyte b spends adsorbed on the stationary phase.

Note that Equation 8 involves the ratio F

, the fractions of time analytes a and b spend adsorbed on the stationary phase.

From Equation 2, note that as α goes up R goes up. Does this make sense? To maximize α and hence R, in Equation 8 we want the numerator to be as large as possible, and the denominator to be as small as possible. To maximize the numerator, we want F

) is large; that is, we want analyte a to spend as small a fraction of its time stationary as possible and hence elute as early as possible. Whereas to minimize the denominator, we want F

) is small, which means we want analyte b to spend as large a fraction of time as possible on the stationary phase so it elutes as late as possible. This all makes sense because the larger the difference between F

 the greater the difference in their speeds through the column and the greater the difference in their elution times. This will maximize R.

Real World Use of the Master Resolution Equation

Now that we have discussed the Master Resolution Equation and its components in detail, how do we make use of this knowledge to optimize chromatographic separations?

Let’s begin with the first term involving plate number, N. Recall (5,6) that N is a measure of the number of times an analyte molecule adsorbs as it works its way down a chromatographic column. So, how do we increase the number of sites a molecule can adsorb on? One way is to increase the column length; the longer the column the more adsorption sites there are and hence the more chances an analyte molecule has to adsorb. Column length is a variable used often in gas chromatography (GC) to optimize separations. For example, there are GC columns available in lengths from 15 to 60 m (8). In high performance liquid chromatography (HPLC) columns of different length are also available, for example from 50 to 300 mm (11). So, when developing a chromatographic method it makes sense to have columns of different length available as it is easy enough to swap out chromatographic columns to change column length and hence N. However, a disadvantage of increasing column length is that it increases analysis time; the longer the column, the more time it will take an analyte molecule to travel its length for a given flow rate. Another problem with increasing column length is increased backpressure, which can put a strain on HPLC pumps.

A second way of increasing N is to reduce the particle size of the packing material. Small particles have a greater surface area than large particles, which means they have more adsorption sites available compared to large particles. Increasing the number of adsorption sites gives molecules more places to adsorb, increasing N. Thus, both GC and HPLC columns can be purchased with different particle sizes varying from a few microns (µm) to 100 µm or more (8,10). Like increasing column length, a disadvantage of decreasing particle size is increasing backpressure.

The second term in the Master Resolution Equation involves the separation factor, α. Recall from the discussion above that α is calculated for a pair of chromatographic peaks. One of the most convenient variables we can change to alter the chemistry of a separation is the polarity of the mobile and stationary phases. In HPLC (10), what is known as “normal phase” chromatography involves a polar stationary phase and non-polar solvent. Whereas in “reversed phase” chromatography (10) a non-polar stationary phase and polar solvent are used.

Let’s imagine that we want to separate a mixture of toluene and ethanol. Again, their respective chemical structures are seen in Figure 1. As noted above, because of their chemical structures ethanol is more polar than toluene.

For a normal phase HPLC separation of these analytes we could use a polar silica stationary phase containing Si-OH groups and a non-polar mobile phase such as hexane. In this case, the non-polar toluene will have a low affinity for the polar stationary phase, F

 for it will be small, and it will elute early. On the other hand the polar ethanol will have a high affinity for the polar stationary phase, it's F

Reversed-phase chromatography is sometimes performed with silica particles that have been covered with a hydrocarbon coating such as “C18” (CH

) (10,11), providing non-polar adsorption sites, and a polar mobile phase such as water. In this case, polar ethanol will have a low affinity for the stationary phase, its F

 will be small, and it will elute first. On the other hand the non-polar toluene will have a low affinity for the mobile phase, a high affinity for the stationary phase, its F

 will be large, and toluene will elute later.

In summary, changing the nature of the column packing or mobile phase impacts the separation factor and hence can be used to optimize a separation. Note in the above example, that the elution order and elution times for the same two analytes differed greatly depending upon the experimental conditions. That is, elution time is not just a function of the chemical structure of the analyte but also the nature of the stationary and mobile phases as well. This means that elution times are not unique for a given molecule and should not be used for chemical identification, more on that later.

To summarize, to optimize a separation we can experiment with the variables in the Master Resolution Equation as such:

The plate number, N, can be increased by increasing column length or reducing stationary phase particle size.

The separation factor, α, can be optimized by changing the type of mobile phase or stationary phase. For example, the polarity of the two phases can be changed to effect a separation.

The capacity factor, k, can be optimized, for example, by changing the composition of the mobile phase.

Equation 2, the Master Resolution Equation, tells us that the quality of a chromatographic separation depends upon N, a, and k. Equations 6-8 show us that all these parameters are related to F

, the fraction of time an analyte molecule spends stationary. This is because F

 determines the speed at which a molecule moves through a chromatographic column, and at the end of the day this is what determines elution time. Thus, all of the experimental parameters we have discussed here that can be varied are all done simply to get F

 to be different enough for the analytes in a mixture that their retention times are different enough to achieve baseline resolution. This is your mission as a chromatographer, if you choose to accept it.

In future columns I will use these concepts to help us understand different types of chromatography, including a detailed analysis of my favorite cannabis potency liquid chromatography method (11).

We reviewed concepts previously developed for capacity factor, separation factor, and plate number. I then presented the Master Resolution equation, that shows that to optimize a chromatographic separation we want N, α, and k to be maximized. We saw that all of these parameters depend upon F

 in some form. We then discussed how to vary a number of chromatographic experimental variables to maximize R.

Smith, B., Chromatographic Theory, Part VII: The Master Chromatographic Resolution Equation, 

Smith, B., Chromatographic Theory, Part VI: Peak Widths, Band Spreading, and Plate Number, 

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

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

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

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

Smith, B., Principles of Chromatography, Part I: Theory, 

Carlin, M., and Dean, J., Forensic Applications of Gas Chromatography, 

Smith, B., Quantitation of Cannabinoids in Dried Ground Hemp by Mid-Infrared Spectroscopy, 

Bayne, S., and Carlin, M., Forensic Applications of High Performance Liquid Chromatography, 

Giese, M., Lewis, M., Giese, L., and Smith, K., 

, 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 VIII: Pulling It All Together, 

Articles

Cannabis Analysis 

Here, we explore how the different parameters in the Master Resolution Equation relate to Fs, and what that means for chromatographic experiments.

Chromatography Theory, Part VIII:  Pulling It All Together

Cannabinoids and terpenoids, the biologically active compounds found in cannabis and hemp, have gained significant attention for their diverse physiological effects. However, the complex nature of cannabinoid extracts pose challenges in their isolation and, for some products, repeatability. Chromatography, a widely utilized separation technique, offers promising solutions to overcome these challenges. This article explores chromatographic separation techniques for the efficient purification of cannabinoids along with the benefits and drawbacks of these separation methods.

Chromatography is a versatile and widely used separation technique employed to separate and analyze mixtures of chemicals or substances into their individual components. It’s based on the principle that different components in a mixture have different affinities for a stationary phase and a mobile phase. By exploiting these differences, chromatography allows for the separation of compounds based on their various properties, such as size, charge, polarity, and interaction with specific chemical groups.

The basic components of a chromatographic system include:

: This is a solid or liquid phase that remains fixed in place while the mobile phase moves through it. The choice of stationary phase depends on the specific separation requirements.

: This is a fluid (liquid or gas) that carries the sample mixture through the stationary phase. The mobile phase’s composition and flow rate can be adjusted to influence the separation process.

: The mixture to be separated is introduced into the chromatographic system. As the mobile phase moves through the stationary phase, the components of the mixture interact differently with the two phases, leading to separation.

: This is used to identify and quantify the separated components as they elute (come out) of the column or stationary phase. Different detectors are used depending on the properties of the compounds being analyzed, such as ultraviolet-visible (UV-vis) spectroscopy, mass spectrometry, or refractive index detection.

There are various types of chromatography techniques, each with its own principles and applications. These include:

: In this technique, the stationary phase is a solid, and the mobile phase is a liquid. High performance liquid chromatography (HPLC) is a common form of liquid chromatography used for a wide range of applications including quantification of cannabinoids.

: In this technique, the stationary phase is a liquid or a solid, and the mobile phase is a gas. GC is often used for the separation and analysis of compounds like terpenes or pesticides.

: In TLC, the stationary phase is a thin layer of a solid adsorbent (like silica gel) on a flat surface, and the mobile phase is a liquid. It’s often used for qualitative analysis.

: In this technique, the stationary phase is packed into a column, and the mobile phase is allowed to flow through it. Preparative column chromatography is used for large-scale purification of compounds.

The cannabis plant can have significant variability in its chemical profile when grown from seed. When grown from a clone, the chemical profile will remain relatively similar, but will still have some variability in the exact quantity of tetrahydrocannabinol (THC), other cannabinoids, and terpenoids. More variability can be introduced when the plant material then undergoes extraction. Column chromatography can be used as a large-scale method for purification and separation of the plant extract chemical profile. With a method such as this, cannabinoids, terpenoids, and other physiologically beneficial compounds can be concentrated and isolated. Purified compounds afford manufacturers the opportunity to formulate a wide array of chemical profiles and products in a reliably reproducible manner.

Column chromatography is a separation technique used to separate and purify components from a mixture based on their different interactions with a stationary phase and a mobile phase. It’s one of the most fundamental and widely used methods in chemical and biochemical laboratories for purification purposes. The technique involves packing a column with a stationary phase material and passing a mobile phase (solvent) through it, which carries the mixture to be separated. As the mixture travels through the column, the components interact differently with the stationary and mobile phases, leading to their separation. The efficiency of the separation depends on factors like the choice of stationary phase, mobile phase, column length, and the properties of the mixture being separated.

Here’s how column chromatography works:

: A glass, plastic, or stainless steel column is packed with a solid stationary phase material. Common stationary phase materials include silica gel, alumina, and specialized resins. The stationary phase is chosen based on the properties of the mixture components and their interactions with the stationary phase.

: The mixture to be separated is carefully applied to the top of the column. This is often done using a solvent that’s compatible with the stationary phase and the mixture.

: A mobile phase (solvent or solvent mixture) is gradually added to the top of the column. As the mobile phase passes through the column, it carries the mixture compounds down the column. The components in the mixture interact with the stationary phase to varying degrees.

: Components that have strong interactions with the stationary phase will move more slowly down the column, while those with weaker interactions will move faster. This differential movement results in the separation of the mixture’s components.

: Fractions of the eluent (the liquid coming out of the column) are collected in tubes or vials. Each fraction typically contains one or more separated components.

: The collected fractions can be analyzed using various techniques, such as TLC, GC, or HPLC, to determine the purity
and composition of the separated components.

One of the commonly used stationary phases in large scale chromatography is silica gel due toits versatility and compatibility with various compounds. Silica gel is a porous material made from silicon dioxide (SiO

) and has a high surface area, which provides ample binding sites for compounds to interact with.

For cannabinoid purification, silica gel is often selected as the stationary phase because cannabinoids, including THC and cannabidiol (CBD), interact with it based on their polarity. The less polar compounds tend to move faster through the column, while more polar compounds, such as some impurities or isomers, might have stronger interactions and move more slowly.

Here are some reasons why silica gel is often chosen as the stationary phase for cannabinoid purification:

: Cannabinoids can have different degrees of polarity. Silica gel’s polar surface can effectively differentiate and separate these compounds based on their polarities.

: Silica gel is readily available and cost-effective, making it suitable for large-scale purification processes.

: It is compatible with a variety of solvents commonly used in chromatography, which allows for flexibility in method development.

: Silica gel has the capacity to separate cannabinoids from various impurities and closely related compounds.

: Silica gel can be used in both laboratory and industrial-scale chromatography setups, making it a practical choice for large-scale cannabinoid purification.

While silica gel is a common stationary phase for cannabinoid purification, it’s important to note that the choice of stationary phase might vary depending on the specific requirements of the purification process, the target compounds, and the impurities present in the initial mixture. In some cases, specialized stationary phases or mixed-phase columns might be used to achieve optimal separation and purification results.

The mobile phase is one of the most important factors when seeking to achieve the best separation of compounds. In column chromatography, a mobile phase consisting of a mixture of organic solvents is commonly used when seeking to separate and purify cannabinoids. The choice of mobile phase composition depends on the specific chromatographic technique, the stationary phase, and the desired separation characteristics. For cannabinoid purification, especially in preparative column chromatography, a mixture of organic solvents is typically used to achieve efficient separation and elution of cannabinoids.

A common mobile phase mixture for cannabinoid purification in column chromatography could include:

: Hexane is a nonpolar solvent that helps elute less polar compounds. It can be used as a component of the mobile phase to
facilitate the separation of certain cannabinoids.

: Ethyl acetate is a moderately polar solvent that is often used in mobile phase mixtures for cannabinoid purification. It can help elute a range of cannabinoids with varying polarities.

: These polar solvents are commonly used in chromatography as they can effectively elute polar compounds. They are usually used in combination with less polar solvents to create a gradient elution.

The specific ratios of these solvents in the mobile phase can be adjusted to achieve the desired separation. A gradient elution is often used in column chromatography for cannabinoid purification, where the proportion of more polar solvent (such as methanol or acetonitrile) is gradually increased over time. This helps to separate cannabinoids with different polarities effectively.

The compounds are collected after passing through the column and undergoing separation. If done correctly, the fractions that are collected will each contain single and pure compounds. Analysis is crucial during this phase to ensure that the fractions are pure and determine when the next fraction of compounds should be collected. Following collection and analysis, the fractions can undergo solvent recovery, resulting in purified target compounds.

While the target compounds are often purified fractions of cannabinoids for product formulation, column chromatography can also be used to remediate undesirable compounds from an extract. Undesirable compounds can include pesticides, cannabinoid isomers, or to produce THC-free CBD extracts.

Column chromatography has many advantages including versatility, scalability, cost of purification, and repeatability. However, the drawbacks include the startup cost, difficulty of method development, and navigating the complex matrix of compounds that cannabis extracts can contain. Implementing refinement and distillation of cannabis extracts has the potential to increase the efficacy of chromatography. Its versatility and scalability is attractive to manufacturers seeking to create products that are repeatable and unique in their cannabis formulae. As the industry expands, this method will likely become a more commonly applied method of purification. Minor cannabinoids are gaining attention from consumers and cannabinoid separation can support manufacturers with formulating unique ratios of minor cannabinoids. The future is bright for large-scale chromatography in the cannabis industry, however, the market demand for uniquely formulated products is too small to encourage most manufacturers to invest in purification methods, such as chromatography.

 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., Advancements in Chromatographic Separation Techniques for Efficient Cannabinoid Analysis and Purification, 

Extraction Science

This article explores chromatographic separation techniques for the efficient purification of cannabinoids along with the benefits and drawbacks of these separation methods.

Advancements in Chromatographic Separation Techniques for Efficient Cannabinoid Analysis and Purification

Microbiological organisms play an important role in many biological processes for health and harm. In cannabis, as with many other consumer products, the presence of microbiological organisms can not only potentially cause deleterious health issues but also cause economic losses for the cannabis producers. Understanding the conditions in which microorganisms thrive, grow, and multiply is important in both controlling microbial growth and also optimizing for microbial testing.

Contamination of food, water, and consumer products is a huge concern around the world causing hundreds of thousands of illnesses and deaths. The major contributor to these exposures is microbiological organisms. One only must turn on the television or pull up the US Food and Drug Administration (FDA) website to see another product being alerted or recalled because of microbial contamination. The first step in producing safe cannabis products is understanding the conditions which allow microbial viability, encourage growth, and promote propagation. Both at the point of product and in the testing laboratory, it is important to understand the factors which persevere the accurate biota to allow for accurate testing.

Testing laboratories tasked with protecting the consumer product supply, including cannabis, use many different methods to detect and in some cases quantify the presence of microorganisms including cell culture, chromogenic media, enzyme-linked immunosorbent assays (ELISA), and polymerase chain reaction (PCR). Some of the most used methods of detection and propagation of microbiological organisms include cell culture and chromogenic media.

The first order of survival for organisms is to find nutrition to grow, survive, and propagate. Organisms can be classified by how they obtain nutrients. 

 are organisms that have an internal process to convert the energy in light into nutrients (that is, algae with chlorophyll). 

 obtain nutrients from chemicals produced outside of the organism (most of the typical microorganisms of interest to cannabis). Under chemotrophs, organisms can either be 

 (obtaining nutrients from organic compounds) or 

 (nutrients obtained from inorganic compounds).The method the organisms obtain organic or inorganic chemicals can either be from decaying or dead organisms or tissues (

The major elements that can impact microorganism growth are carbon, oxygen, hydrogen, nitrogen, phosphorus, and sulfur. Minor elements that are important biological factors, such as vitamins and coenzymes, include copper, iron, potassium, magnesium, calcium, molybdenum, manganese, and zinc.Carbon is one of the most important of the major elements utilized by chemotrophs. The source of the carbon can either be elemental sources of inorganic carbon (autotrophic) such as carbon dioxide or organic carbon sources (heterotrophic) (see 

). Most organisms including many bacteria, fungi, and animals are chemoorganoheterotrophs.

Another important element for microorganisms is oxygen.Bacteria that thrive in oxygenated environments are 

and ones that are most negatively impacted by the presence of oxygen are called 

. Aerobic microorganisms can use molecular oxygen to produce more energy than anaerobic organisms, but they are exposed to reactive forms of oxygen which can damage cells. Aerobic organisms often require antioxidants or enzymes like peroxidases, catalase, or mutases to survive in oxygen environments.

Many microorganisms fall somewhere between the two extremes of anerobic and aerobic into five categories: obligate aerobes, facultative anaerobes, aerotolerant anaerobes, microaerophiles, and obligate anaerobes (see 

Obligate aerobes require oxygen for growth and prefer high oxygen environments. These organisms produce enzymes to counteract oxidative effects which allow them to effectively use oxygen to produce nutrients. In tube media, these microorganisms congregate at the top of the media with higher oxygen concentrations. For the cannabis industry, molds are most often obligate aerobes.

Facultative anaerobes can exist in high and low oxygen environments, but require oxygen for increased growth.They produce enzymes to combat oxidation. In growth media, these microorganisms grow best at the top area of higher oxygen concentrations, but can be found distributed throughout the media tube.Microorganisms of concern to the cannabis industry, such as yeasts, 

Microaerophils prefer low oxygen to anaerobic environments and grow with small amounts of oxygen. These microorganisms do not produce enzymes to combat oxidation and will be found in media tubes near the center of the tube with low oxygen.

Aerotolerant anaerobes tolerate a wide range of oxygen levels. Growth is not affected by oxygen levels, and they have limited enzymes for combatting oxidation. In a growth media tube, they can be found evenly distributed throughout the media.

Obligate anaerobes survive in low to no oxygen environments and have almost no tolerance to oxygen. Growth occurs in anaerobic conditions, and they lack the ability to combat oxidation. Oxygen can create toxic effects to these anaerobes because of a lack of enzymes against oxidation.

Nutrients are only one component in the growth of microorganisms. Temperature is a critical factor in many organisms’ growth. Most living organisms exist in the "Goldilocks range" of approximately 0 °C to 40 °C. As temperatures drop below the low end of the range, enzymatic reactions slow or stop and eventually cell death occurs. In the opposite direction, as temperatures rise above the higher range, proteins denature, and cell death occurs for most organisms. Microorganisms, bacteria in particular, have been known to exist in a multitude of extreme conditions including extreme temperatures (see Table II). Microorganisms of concern in the cannabis industry have moderate temperature ranges (mesophilic) within the same range of most organisms. Mesophiles include the most targeted microorganisms in cannabis testing such as 

Each microorganism has its range of temperature limits that, once exceeded for a given time period, inactivate or kill that organism. Food safety guidelines are created based on exposing products to various (usually high) temperatures for a set period of time to eliminate potential pathogens (see 

These inactivation temperatures can become important during product or sample processing if products are exposed to high temperatures for extended periods of time. In cases of product processing, heating consumable items can eliminate living microorganisms, but the improper handling of product samples heading to an analytical laboratory can falsely reduce the microbiota being tested.Sample processing in the laboratory, which may produce heat for a period of time, can also reduce or damage the target microorganisms that will affect the analytical results for some testing methods that require living cells.

Other factors that can affect the growth of microorganisms can include pH. The pH is a measure of the acidity or alkalinity of a solution. Most bacteria and other microorganisms prefer an ideal environment around a neutral pH ranging from 5–8 (neutrophiles). There are some organisms or strains that can survive in acidic environments under 5.5 pH (acidophiles) or in alkaline conditions above 8.5 pH (alkalophiles). Cannabis microbiological targets can cover a wide range of pH—from molds, which run the pH spectrum, to 

 which prefers a narrow range of pH from 4.4 to 8.5. This wide range of pH means some target organisms can survive many cannabis production processes (see 

The availability and activity of water is an important factor in the growth and metabolism of organisms. Water is available either as bound water (bound to molecules), which is not available to microorganisms, or free water (unbound to molecules in food). 

) of a product is the ratio between the vapor pressure of the product itself and the vapor pressure of DI water under ideal conditions. Water activity is a measurement of free water available to microorganisms. A water activity value of 0.9 means that the vapor pressure is 90% of the free water. Water activity increases with increasing temperatures.

Water content, in contrast, is the amount of water by mass of a product or material while water activity is a measurement of water available for microbial growth. Water content is part of the composition of a product whereas water activity is a quality control aspect to sustain shelf life of a product. Reduction of water activity lowers the availability of water to the organisms, which can disrupt metabolic processes and slow growth. Many of the organisms important to cannabis have water activity limits from 0.7 to 0.9 (see 

). Ideally cannabis flower has a water activity of about 0.6; with potential microorganism growth occurring above 0.65. Keeping the water activity of products lower than the water activity levels of the common microorganisms inhibits growth.

When favorable conditions of nutrients, pH, moisture, and temperature occur microbiological organisms can profligate quickly either intentionally or unintentionally. Generation time is the amount of time required for a microbial population to double. The mean generation time (g) is equal to the time (t) in minutes divided by the generation number (n) (see 

), while the generation number (n) is ratio of the log of the number of cells at the beginning of the culture (N

For organisms of interest to cannabis testing, the generation time can range under optimal temperature (20–40 °C) and media conditions from less than 30 min for some 

). In native settings, depending on the conditions the generation time or doubling time can be either accelerated or delayed by the availability of nutrients, water, temperature, and so forth.

The ability to understand the optimal conditions and factors for microbial growth of concern in cannabis aids both the laboratories and industry control and testing for those organisms. Changes in conditions of cannabis products during all phases of the product life can adversely affect the microbiota, which will be tested in the laboratory. Knowing the conditions that restrict growth, increase propagation, or kill the organisms can help the industry to optimize methods to preserve samples for testing. In the laboratory, the knowledge of the correct conditions to store and process samples so as not to significantly change the microorganism populations becomes key to accurate testing.

In the next column, we will continue the discussion of detection and quantitation of the diverse types of cannabis microbiological targets starting with microbiological cultures and various types of chemogenic and plating techniques and media.

Atkins, P., Measuring Microbiology, Part I: A Look at the Microbiological Contaminants in Cannabis, 

Carter, B.P., The What, How, and Why of Water Activity in Cannabis, 

Boyar, K., Cannabis Microbial Testing - Methodologies & Considerations. 10.1201/9780429274893-6 (2021).

is a Senior Applications Scientist with Spex, an Antylia Scientific company and has been a member of many cannabis advisory committees and working groups for cannabis including NACRW, AOAC, and ASTM.

Atkins, P., Measuring Microbiology, Part II: Understanding the Conditions of Microbiological Growth and Viability, 

Navigating the Lab

A look at why understanding the conditions in which microorganisms thrive, grow, and multiply is important in both controlling microbial growth and optimizing for microbial testing.

Measuring Microbiology, Part II:  Understanding the Conditions of Microbiological Growth and Viability

Cannabis Science Conference (CSC) Fall took place in Providence, Rhode Island on September 20-22nd. The conference featured several exciting tracks: analytical science, cultivation, medical cannabis, and psychedelics. Here we share some interview highlights with Dr. Peter Grinspoon, Jini Glaros, Dr. Miyabe Shields, and Adam Jacques to learn how they are each helping move the cannabis industry forward.

Dr. Peter Grinspoon is a primary care doctor at Massachusetts General Hospital, an Instructor in Medicine at Harvard Medical School, and a 25-year cannabis specialist. He was a keynote speaker at Cannabis Science Conference Fall on September 22, 2023. Here, Dr. Grinspoon shares his background and interest in medical cannabis as well some policies he’d like to see changed in the future.

Q: Can you tell us a little bit about yourself and how you became interested in the cannabis industry?

: Well, it goes back a long time and there are several reasons that sparked my interest. The first thing is that my brother Danny, in the early 1970s, was fighting an unsuccessful battle with childhood leukemia. My parents saw him suffering so much from the chemotherapy that they illegally procured him cannabis in the early 1970s, right when Richard Nixon was just embarking on his war on cannabis. It was just amazing to see my brother when he didn’t use cannabis, he’d just be lying in his bed with a towel over his head barfing. But when he used cannabis, he was able to eat, hold down food, maintain his weight, strum on his Fender Stratocaster (which he loved to do), and, most importantly to us, play with his little brothers. So, I saw at a very early age how impactful medical cannabis can be and that really stuck with me for my entire medical education and my entire medical career.

The other thing I should mention is that my father, Dr. Lester Grinspoon, was a legendary psychiatrist at Harvard Medical School, and he wrote a pioneering book in 1971 called 

, which was actually reviewed on the front page of 

 in glowing terms. At that time, when only 12% of Americans supported full legalization of cannabis, my dad called for the legalization of cannabis, saying, “Sure, there are some harms associated with it. Teens, pregnancy, certain people shouldn’t use it. But the harms of criminalization are much worse than the harms of actually using cannabis.” So, growing up, I had all kinds of cannabis advocates and activists and proponents in my living room, sort of smoking and talking, and these were people that were actually changing the world. I started to associate cannabis not only with healing because of my brother Danny, but with sort of an intellectual lubricant because all these super motivated people were using it in my home. So, I’ve been interested in it my entire medical career.

Q: Can you explain the top two biggest misconceptions about cannabis that you encounter frequently?

: Sure. Well, number one, medical marijuana doesn’t mean smoking. No doctor recommends smoking. I mean, if you’re dying of cancer or you have chemotherapy and you need to take something really quickly so you don’t throw up, maybe there’s certain exceptions. But I had a friend huffily say to me, “How can burning smoke be medicine?” So, I explained that you could use a tincture under your tongue, one of these old fashioned medicinal tinctures. You can take a small dose of an edible very carefully. You can actually use a skin patch, an inhaler, a suppository, or a topical. There are many different delivery methods. So, one misconception is that to use medical cannabis is to be smoking cannabis. There are many different delivery options.

Another misconception is that there’s no such thing as medical marijuana and people are just trying to get high. In fact, the doses of medical marijuana are often much lower than the doses of adult use or recreational marijuana. Also, using cannabidiol (CBD) and other medicinal cannabinoids besides tetrahydrocannabinol (THC) often people get comfortable with no high effect or a minimum of the high that people associate it with. So, it doesn’t mean you’re stoned all the time. It just means you’re getting effective symptom relief. So those are a couple of misconceptions.

Jini Glaros is the Chief Scientific Officer (CSO) at Modern Canna Labs in Lakeland, Florida. She received her bachelor’s degree with honors in Biochemistry from the University of West Florida. In May of 2022, Glaros earned a Master’s degree in Medical Cannabis Science and Therapeutics from the University of Maryland, Baltimore. Glaros gave a presentation at CSC Fall in an analytical-focused general session titled “Sample Preparation, Compound Error, and Potency Inflation.” Here, Glaros shares how she got her start in cannabis testing and more.

Q: Can you tell us about your background and how you got involved with cannabis testing?

: Of course. So, if you had asked me 10 years ago if I would be doing cannabis testing, like probably a lot of people in the industry, I would have said, ’No, there’s no way.’ My original plan was I wanted to get a medical degree and be a doctor. I worked in a lab during my undergraduate education and fell in love with it. I ended up taking a gap year after graduation, which was when I worked at the National Institute on Aging in Baltimore, Maryland. During that time, I realized that it was lab life for me; I wanted to be in the lab. There was no doubt about it. So, I thought maybe I’ll pursue my MD and PhD. I’ll still get to be in a lab and still get to be a doctor—kind of the best of both worlds. Of course, sometimes life happens and that didn’t work out, but everything happens for a reason. I ended up working at the University of Florida for a while in one of their labs, before moving back to my hometown of Lakeland, Florida, which is where the Modern Canna Labs’ headquarters is located and where I’m working currently. This opportunity really just fell into my lap, and I couldn’t be happier about my role and thrilled that I was given such an opportunity. I got in the industry when things were very fresh and new in Florida. I started working for Modern Canna back in 2017, so I’ve been with them for quite some time now, and really have been able to watch the company grow.

Q: Can you share what your talk at CSC Fall focused on?

: Potency is a highly scrutinized analysis, whether that be from cultivators, producers, or even consumers, right? You hear all the time that consumers want the most “fire” weed, or they want cannabis with the highest THC. It’s great that they want high THC flower, but that’s not the only thing that matters. When it comes to high THC flower, by the industry pushing for higher and higher potencies, it’s led some laboratories to maliciously manipulate their data. However, the reality is that, in most cases, I really don’t think that’s the intention of all laboratories. I think, ultimately, that some of these labs just don’t have the tools and the techniques to know when they’re doing something that could be leading to potency inflation or could be leading to results that are skewed in one way or another. So, the goal of my talk was to uncover some of those things that lead to compound error, which may in turn lead to potency inflation. We’re dove into sampling bias, sample weighing, sample extraction, sample dilutions—the whole nine yards—and how each step of that process impacts the final result. I also discussed how laboratories can improve their processes and what cultivators, producers, and regulators can look for when they’re looking to a lab to produce accurate data.

Miyabe Shields, PhD, is a queer and neurodivergent stoner scientist and avid psychonaut with more than 13 years of experience in academic and industrial research. Dr. Shields has dedicated their life to understanding the inner workings of the neurodivergent brain on drugs. Now, a beginner farmer, they aim to address health equity through natural medicines. Dr. Shields organized an exciting panel discussion in the psychedelics track at CSC Fall titled “Barriers to and Learnings from Psychedelics Research.” Here, Dr. Shields shares their background in cannabis and psychedelics research and more.

Q: Can you tell us about your background and how you got involved with cannabis and psychedelics?

: Yes, absolutely. My background is in pharmaceutical sciences. I have my PhD in drug discovery, and I studied the endocannabinoid system. The reason why I originally went into drug discovery in the endocannabinoid system and in pharmaceuticals is because cannabis is the medicine of my life. But, previously, I had so much guilt and shame around using cannabis as medicine that I was really interested in understanding the molecular mechanisms of how cannabis works, and if it was possible (ideally) to create a synthetic version that would be able to be given to me in a pill. Halfway along the track of doing my PhD, I had a more rounded understanding of why certain medicines are stigmatized over others. That really affected the progression of where I saw my research going and where my career went. So, after I finished my PhD, that’s why I transitioned into doing research in the cannabis industry. I co-founded a company that researched heat transformation of cannabinoids.

In terms of my interest and research into psychedelics, that was more recently introduced by my wife, Lane, and there’s always been a synergy there between the serotonin system and the endocannabinoid system that has just fascinated me. I always liken it to our marriage because Lane has a lot of serotonergic things—migraine disorders are serotonergic. I see it as a balance and a mixture of a balance and a synergy. That’s sort of how I got interested in this space, and it’s how I’ve been singularly obsessed with being in the space for most of my life. I started using cannabis when I was 15, so it’s been a long journey for me. Cannabis is a lifelong medicine and something that I’m very certain I want to dedicate my life to and be able to give back to the community. I also want to add some information and add some interesting context and perspective, I think, that can sometimes get lost even though we’re doing our best, but research can be difficult. It can be a difficult vacuum.

Q: Can you share a summary of your panel presentation at CSC Fall?

: Sure. So the team that I did the panel with was myself and my two colleagues, Dr. Riley Kirk, who also has her PhD in pharmaceutical sciences, but studied secondary metabolites, which are the molecules that are in natural products like plants and fungi that cause the effects, and Marné Garretson, who is a real-world evidence data scientist and is focused on using real world data versussay, clinical settings. Our panel “Barriers to and Learnings from Psychedelic Research” was about how we have come together as a multidisciplinary team and as a group—our team also has medical doctors and other professionals—that are in the industry. All of us have come across some challenges in the space and challenges within ourselves too, so we discussed how we are going to frame this research, interpret the research, conduct the research, publish the research, and make everything accessible while at the same time highlighting the need for more research and the benefits of it. So, I think it was a really great discussion, and a back and forth that we have, every time that we meet about the intersection of all of our different areas and also how our studies are a representation of that intersection.

Adam Jacques is a world-renowned expert with more than 20 years of experience cultivating unique strains of cannabis. Having developed more than 300 high-CBD strains of cannabis, Adam has established himself as the foremost expert on CBD genetics and was recognized as the most influential person in the Northwest and Canadian cannabis industry in 2016 by CannAwards. Adam participated as a CSC Fall speaker on an exciting fireside chat in the Cultivation Track General Session titled “Industry Answers with Adam Jacques.” Here, Adam shares his background in cannabis cultivation, his passion for learning, and more.

Q: Can you tell us about your background and how you got involved with cannabis cultivation?

: I started, kind of, my love affair with cannabis as a teenager. When I was a teenager, it was a much different time, right? So, like 1994 is probably when I used cannabis for the first time. I’m a neurodiverse individual, and back then, there wasn’t a lot of knowledge surrounding that, you were either a kid that was a troublemaker or you weren’t. I found, when I used cannabis, it really dialed in my brain and allowed me to focus. I was using cannabis for medical purposes without understanding that I was using it for medical purposes, and that went on through college. I did quite well.

What really started it all was that college was expensive and I was poor, so I started growing cannabis for myself at home. It was very difficult to get any new varieties of cannabis because it wasn’t necessarily legal, and they just weren’t around. So, I taught myself that you can breed cannabis. When I was around 23 or 24, I started breeding my own cannabis strains, looking to make them in a specific way or to make a strain in a way that I needed for me, personally, medically. I would say that’s kind of where I started. It started from a place of trying to treat my own illness, slowly over time. My educational background was not in biology or horticulture. This is all self-taught. After about 30 years, I’m okay at it now. But it started from a point of necessity. There wasn’t really anybody serving medical patients. There was no real good information out there, everything was hearsay and pseudoscience and things of that nature. So, it was difficult to find any real help or answers. After I was working with myself for a while, I found a group here in Eugene, Oregon that was kind of clandestine, but they were doing work with medical patients, specifically cancer patients at that time. I got involved with them, donating my time, knowledge, and cannabis and genetics to them, and that kind of started my road down this medical cannabis track that I’ve been on.

I enjoy learning a lot. I would say that the majority of my free time is spent teaching myself things. It was a lot harder back in the day. There was not as much information as there is now, like online and things like that, but libraries existed, and they had a lot of good books on plants. I like to think that I kind of grew up with the industry. I came in at a very interesting time in the industry. Marijuana had just become medically legal in Oregon, which opened up a huge opportunity for me where I wasn’t worried about getting arrested for growing plants in my house. I kind of just hit all the timing perfectly.

There were some people with DEA licenses in Kentucky growing cannabis, but there weren’t really any universities doing major studies on cannabis or looking at molecules. The most important thing to me with cannabis was that I know that it works, right? I knew that it worked, I knew it was working, I knew it was doing what I needed, but I didn’t know why. And that drove me crazy. So, I spent, and I have been spending the past 20-30 years trying to answer that why. Why does cannabis affect people in the way that it does? Why does it affect everybody differently? What are the important constituents? If you do, let’s say a delta-9-THC isolate, you’re going to get a really generic effect. But it is pretty normal between everyone. Now you start adding in different cannabinoids, terpenes, flavonoids, things of that nature, and it seems like every addition creates a dynamic to the medicine that we’re using that changes the effect or what we use it for. So, trying to understand the whole picture of cannabis instead of breaking it down to THC is the drug and everything else is a waste product, which I think is terrible. Whole plant medicine has been kind of my focus. I’ve had a lot of help through the university systems, at this point, specifically working with Dr. Zac Hildenbrand, who I presented with at the conference. I met him in 2015 at a science conference and we instantly connected— we’re like brothers now. But we’ve been working with the University of Texas system, looking at a lot of different things and flavonoids and terpenes, and what we would call lesser cannabinoids. It’s been great—very interesting journey I’ve been on.

Q: What interested you in working with CBD genetics, and do you work with other cannabinoids and terpenes?

: I started working with CBD genetics because of a gentleman named Frank, who is a close personal friend of mine. Frank had cancer, and he was getting some oil made for him that was just high THC and he could not focus during the day. It was just this constant usage of THC that was making his life uncomfortable. In 2014, I started doing some research. This was pre-Stanley Brothers, so CBD genetics really weren’t being talked about. I started doing some research and found some old research from a group in Brazil in the 1970s that was looking at CBD as a treatment for epileptic seizures. I found out that hemp was the highest carrier of CBD. So, by finding hemp genetics and breeding those with higher percentage things then I was able to create—I won’t go into the science of how breeding works because we will be here forever—but I found that I could create genetics that were rich in both THC and CBD. While doing that, I also found out how CBD and THC work with the CB connectors and how you actually are going to get less of a psychological effect if you mix the two. THC fits perfectly in that receptor, CBD gums it up a little bit. We were having this effect where I was able to push more and more THC into a system as long as I was tempering that with CBD. I created a genetic then called “Frank’s Gift,” which was a 3 to 1, test it out at like 30% total cannabinoids, it was huge at the time, and we used that with him.

We started working on extraction methodologies at that point. This was right at the infancy of CO

 extraction. We got one of the Waters machines. I was working with Frank and trying to get him less high by developing these genetics. He started using them and we used them thoroughly and he went into complete remission from cancer. I was like, “Wow, this seems great.” Frank was a fairly well-off individual and he said, “I want to fund anything that you want to do to help people like you helped me.” At that point, I kind of had a ticket to create and learn, so then I just started going down this rabbit hole of CBD breeding. But it was another case of kind of trying to figure out something through necessity that ended up helping lots of people in the long term.

Watch the full interviews with these thought leaders at: 

https://www.cannabissciencetech.com/videos/editorial-videos

In this new interview series, we sit down with several speakers from 2023 Cannabis Science Conference events to learn about their expertise and backgrounds in the cannabis industry. Join us to learn more about Dr. Peter Grinspoon, Jini Glaros, Dr. Miyabe Shields, and Adam Jacques.

September 2023

Chromatography Theory, Part VIII: Pulling It All Together

Advancements in Chromatographic Separation Techniques for Efficient Cannabinoid Analysis and Purification

Measuring Microbiology, Part II: Understanding the Conditions of Microbiological Growth and Viability

Industry Thought Leaders: Meet the People Driving the Cannabis Industry Forward

The Crucial Role of GxP in the Cannabis Industry: Ensuring Quality, Safety, and Efficacy