Advancements in Chromatographic Separation Techniques for Efficient Cannabinoid Analysis and Purification

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:

1. Stationary phase: 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.
2. Mobile phase: 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.
3. Sample mixture: 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.
4. Detection system: 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:

1. Liquid chromatography (LC): 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.
2. Gas chromatography (GC): 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.

1. Thin-layer chromatography (TLC): 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.
2. Column chromatography: 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:

1. Column setup: 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.
2. Sample loading: 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.
3. Elution: 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.
4. Separation: 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.
5. Collection: 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.
6. Analysis: 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 (SiO₂) 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:

1. Polarity variation: Cannabinoids can have different degrees of polarity. Silica gel’s polar surface can effectively differentiate and separate these compounds based on their polarities.
2. Wide availability: Silica gel is readily available and cost-effective, making it suitable for large-scale purification processes.
3. Compatibility: It is compatible with a variety of solvents commonly used in chromatography, which allows for flexibility in method development.
4. Effective separation: Silica gel has the capacity to separate cannabinoids from various impurities and closely related compounds.
5. Scalability: 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:

1. Hexane: 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.
2. Ethyl acetate: 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.
3. Methanol or acetonitrile: 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.

About the Columnist

Lo Friesen 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 CO₂ extraction equipment manufacturer to support and expand a Research and Development department. There she managed the development of their latest and greatest CO₂ 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.

How to Cite This Article

Friesen, L., Advancements in Chromatographic Separation Techniques for Efficient Cannabinoid Analysis and Purification, Cannabis Science and Technology, 2023, 6(7), 12-15.