Strain-Specific Isolation of Terpenes Utilizing Microwave-Assisted Extraction

Photo credit: AVID_STOCK.ADOBE.COM

Figure 1 (click to enlarge): An Ethos X microwave system with a 5 L vessel and glass fragrance extraction kit manufactured by Milestone SRL.

Table I (click to enlarge): 5 L vessel extraction method

Table II (click to enlarge): Potency and terpene profile of fresh frozen OGKB 2.0 pre-extraction

Table III (click to enlarge): Isolated terpene profile for OGKB 2.0 fresh frozen material

Figure 2 (click to enlarge): GC–MS chromatogram for fresh frozen OGKB 2.0 terpenes.

Table IV (click to enlarge): Random sample of fresh frozen flower tested post-microwave extraction

Figure 3 (click to enlarge): Isolated terpenes from cured OGKB 2.0 flower.

Table V (click to enlarge): Potency comparison pre- versus post-microwave assisted extraction

Table VI (click to enlarge): Comparison between the potency of hydrocarbon extraction of microwaved material further processed into THC distillate

Figure 4 (click to enlarge): THC vape pen diluted with cannabis-derived terpenes isolated using microwave assisted extraction.

Figure 5 (click to enlarge): High terpene full spectrum extract.

As the cannabis industry matures, terpene isolation from cannabis plant material has become a primary issue. So far, there have only been two practical applications: steam distillation, which is time consuming and smaller in scale, or CO₂ extraction, which requires expensive equipment and an experienced operator. Solvent-free microwave-assisted extraction solves these problems with a cost-effective, efficient solution. The process requires nothing other than water, takes roughly 1 h per run, and yields tetrahydrocannabinol (THC) free, strain-specific terpenes from a variety of starting materials. Throughout this article, I discuss the capabilities of microwave-assisted extraction and how the cannabis industry can utilize it.

Percy Spencer invented the microwave in 1945 (1) and since that time it has revolutionized the way food has been cooked and prepared. However, microwaves have numerous uses outside of the kitchen. By the mid-1970s, chemists began using them for a wide variety of applications in inorganic and organic sample preparation (2) with materials ranging from soils and food to electronic boards (3). This instrumentation has been an asset to the scientific community over the last 40 years and will continue to be. In this article, we are going to discuss the capabilities of this technology in the isolation of strain-specific terpenes from the cannabis plant.

Common Terpene Isolation Methodology

Harvesting terpenes from cannabis is generally completed in two ways. One utilizes steam distillation in a Clevenger apparatus or similar set-up to facilitate terpene removal. The other process utilizes CO₂ as the extraction solvent, collecting the terpenes as an initial fraction within the closed loop system. While both processes work adequately, they each have drawbacks. Steam distillation requires a significant amount of heat under a long dwell time to achieve a full extraction. The length of time under heat can have an effect on product quality. It is also difficult to scale in comparison to CO₂ and microwave-assisted extraction techniques. While subcritical CO₂ is an efficient way to remove terpenes from plant material, the technique lacks the ability to efficiently process wet or fresh frozen material. This is because of the interaction between water and carbon dioxide forming carbonic acid (CO₂ + H₂O⟷H₂CO₃) (4), which can cause an acidic pH change in the final product affecting the overall flavor. The implementation of a commercial supercritical CO₂ extraction system is also a large initial capital investment that requires an experienced operator to achieve optimal results.

Microwave Theory

Microwave-assisted extraction solves these problems with the introduction of an affordable and efficient system that requires minimal experience to operate. Utilizing a set of magnetrons, the system channels microwaves throughout the sample cavity and uses them as a form of energy to excite polar molecules. In the cannabis application, that polar molecule is water. As the microwaves pass through the sample matrix, every change in the electric field of the wavelength causes a dipole rotation of the water molecules. This rotation generates friction, causing a tremendous amount of heat very rapidly (5).

This heat eventually causes the water to change from its liquid phase to steam. The increase in heat and pressure caused from this steam releases the terpenes from the plant material (6). As the steam begins to exit the microwave cavity into the distillation head, it carries these newly released terpenes with it. The microwave process is more efficient than standard steam distillation because instead of using convection heat, the microwaves heat evenly throughout the sample matrix.

Experimental Design

The development work was done using an Ethos X microwave system with a 5 L vessel and glass fragrance extraction kit manufactured by Milestone SRL (Figure 1). (See upper right for Figure 1, click to enlarge.) The only additional materials needed are water and cannabis biomass. The process works on trim or flower, either freshly harvested or fresh frozen or in cured form. The first portion of the work discussed is done using fresh frozen flower material, followed by applying the same process on cured flower material. The strain used in this development work is OGKB 2.0 and the strain choice was because of inventory constraints at the time of development. The intent was to compare the same strain in both fresh frozen form as well as finished shelf-grade material, and OGKB 2.0 was the only strain we had in both forms at the time. Freshly harvested material, without freezing, is not an option because of Nevada state regulations, as it must be frozen within 2 h of harvest and tested by a third party analytical laboratory before being transferred to a production license. The microwave extraction method used was the same for both varieties of material and can be seen in Table I. (See upper right for Table I, click to enlarge.)

Process and Results

Since fresh frozen cannabis material generally has a water content upwards of 70%, no additional water needs to be added in the process. To begin, the frozen cannabis was removed from a -25 °C freezer and allowed to stand at ambient temperature to thaw for 1 h. Once the plant material had warmed, it was dropped evenly into the material vessel. Packing the material with any type of pressure is not recommended because it creates a less efficient distillation. Once the vessel was filled, it was placed into the microwave cavity of the microwave system and the extraction process was started.

Since the plant was tested as frozen material after harvest, there is a reference point for potency and terpene content that can be seen in Table II. (See upper right for Table II, click to enlarge.) This test panel is done on an “as is” basis, so water content has not been factored out. This accounts for the potency and terpene content being at lower-than-average values.

Once the run completed, terpenes were collected from the burette of the microwave system. Since they are not miscible with water, they sit as an oil layer above the water layer. Water contamination is unavoidable during collection. To help mitigate this problem, the water and terpene collection was placed in a griffin beaker and allowed to fully separate at room temperature for 5 min. Once full separation was visually observed, the beaker was placed in a freezer for 1 h. During that time, the water freezes into a solid layer of ice while the terpenes remain in liquid form above the frozen water. The terpene layer can then be decanted into a collection vessel for a pure terpene fraction. This water removal can also be accomplished using a separatory funnel or by running the solution through a bed of sodium sulfate if state regulations allow.

In this batch of fresh frozen OGKB 2.0, the total terpene yield by weight was 0.51%, compared to the analytical result of 0.39% total terpenes seen previously in Table II. These pure terpenes were then sent to a third party analytical laboratory for potency and terpene profiling. The harvested terpenes tested as nondetectable for all cannabinoids. As seen in Table III, the total terpene content was found to be 59.32% total by weight. (See upper right for Table III, click to enlarge.) There are two main reasons that this number does not get near the 100% purity levels expected in the sample. The first is analytical variability found in all testing laboratories. However, that is a small factor when compared to the missing 40% we are looking for. The main issue is that most laboratories only have access to analytical standards containing the 23 major terpenes found in cannabis (7). Since there are hundreds of terpenes found in the plants system, the industry is lacking the analytical standards needed to accurately quantitate the large array of compounds found in cannabis. As seen in the gas chromatography–mass spectrometry (GC–MS) chromatogram in Figure 2, there are numerous peaks circled in red that do not have an associated analytical standard attached to them. (See upper right for Figure 2, click to enlarge.) Without these standards, the compounds cannot be accurately quantified and reported. As the industry matures and develops, more standards will be created and more compounds will be quantifiable. However, for now we are limited to accurately quantifying the standard mix of 23 terpenes, which puts some constraints on us as an industry.

Once the terpene extraction was completed, a random sample of flower was taken from the material vessel and sent out for third party analytical testing for both potency and terpene content. As seen in Table IV, only a small amount of terpenes were found on the plant material. (See upper right for Table IV, click to enlarge.)

The extraction process was then completed on cured OGKB 2.0 for a direct comparison between fresh frozen and cured materials of the same strain. The only change between fresh frozen and cured material is that cured material requires a hydration step to reintroduce water into the plant material before processing. This step is heavily dependent on the moisture content of the plant prior to rehydration; however, the best results have been seen starting with 2 g of water to every 1 g of plant material. If the product becomes over-hydrated, the microwave may have issues reaching optimal distillation temperatures during the run. However, water content can be removed and optimized while the instrument is running by allowing water to periodically drain from the burette. If the material is not hydrated enough, it can burn and release some very unpleasant compounds into the terpenes during distillation, ruining the end product.

The terpene yield by weight seen on cured OGKB 2.0 plant material was 1.59%, again beating the tested analytical value of 1.46% seen on the cured flower. The isolated terpenes were then sent out to a third party analytical laboratory for terpene and potency profiling. The results were similar to the fresh frozen sample sent out, yielding nondetect on cannabinoid levels and a total terpene content of 68.35%. This material produced clear terpenes that can be seen in Figure 3, as most material does; however, varying shades of yellows and oranges have been seen in our laboratory throughout different varieties of cannabis. (See upper right for Figure 3, click to enlarge.)

What About the Cannabinoids?

While valuable, terpenes are only part of the equation in the economics of cannabis. The plant also has a value in the cannabinoids it produces. These molecules are still intact in the plant material after having the terpenes extracted. As seen in Table V, the total potential tetrahydrocannabinol (THC) in the post extraction fresh frozen plant material closely matches the values pre-extraction. (See upper right for Table V, click to enlarge.) Both of the samples tested were chosen at random. One considerable difference is that tetrahydrocannabinolic acid (THCA) is almost fully decarboxylated post-microwave assisted extraction. The plant material is varying shades of brown, a product of the Maillard reaction, and has an extremely high moisture content. We first attempted to simply freeze the material immediately after it was removed from the microwave then extract it using butane, similar to the production of live resin. However, the yields were found to be well below expected values. It was thought that the excess moisture in conjunction with freezing the material was a factor in the yield loss, so a forced air oven was then used to completely dry the microwaved material prior to solvent extraction. This change in process provided the mathematical yields expected after solvent extraction. A sample of the resulting crude oil was sent out for potency testing, further purified into THC distillate, and then sent out for potency testing again. The results can be seen in Table VI. (See upper right for Table VI, click to enlarge.) This process was done to ensure that there were no unexpected side effects in THC production on post-microwave assisted extraction, such as isomerization or potency degradation. No negative side effects were seen throughout processing.

We Have Terpenes, What Now?

The terpenes isolated can be used in numerous applications. They have a place in aromatherapy and topicals, but our facility uses them almost exclusively in the production of vape carts. We have found that by using cannabis-derived terpenes as the diluting agent for high potency distillate we have been able to produce a vape cartridge completely derived from cannabis that provides an unparalleled flavor profile, an example of which can be seen in Figure 4. (See upper right for Figure 4, click to enlarge.) These terpenes can also be added in higher amounts to other concentrate products (such as shatter or live resin) to produce what is known to the industry as a high terpene full spectrum extract (HTFSE, Figure 5). (See upper right for Figure 5, click to enlarge.) This type of extract sacrifices some cannabinoid potency to achieve an extremely high terpene profile, creating a unique effect and flavor.

Conclusion

Cannabis extraction methods are numerous and ever evolving, with innovations in equipment and technique almost daily. One of those innovations is microwave-assisted extraction. I hope that this article helped shed light on this technique and the possibilities it provides in terpene isolation within the cannabis industry. With new states coming online and opportunities to further research the properties and effects of the cannabis plant continue to open up, the need to efficiently isolate these compounds is only going to increase.

Acknowledgments

I would like to thank Chris Wren and the team at Medizin Las Vegas for cultivating some of the best cannabis in the world; Levon Shilling and Ryan Boyle from Milestone Srl for their continued support; and DB Labs for their thorough and accurate sample analysis.

References:

1. E. Conover and A.G. Levine, American Physical Society 24(9), 2 (2015).
2. J.A. Nobrega and C. Pirola, Microwave Assisted Digestion 1st ed., Milestone Srl (2018).
3. Titan MPS Applications Notebook, “Great Results Begin With Good Preparation,” Perkin Elmer, www.perkinelmer.com/lab-solutions/resources/docs/APP_Titan-MPS-Applications-Notebook_010773B_01.pdf
4. A. Morias, et. al., Chemical Reviews 115(1), 6 (2015).
5. D. Spector, www.businessinsider.com/how-do-microwaves-work-2014-6.
6. www.doterra.com/US/en/brochures-magazines-living-winter-2013-2014-distilling-essential-oils.
7. www.restek.com/chromatogram/view/GC_GN1200/terpenes.

Stephen Markle is the Vice President of Production for Planet 13 in Las Vegas, Nevada, and is responsible for all concentrate and infused product manufacturing. Markle has over seven years of experience as an analytical chemist in the nutraceutical and cannabis industries. Direct correspondence to: stephenm@medizinvl.com

How to Cite This Article

S. Markle, Cannabis Science and Technology 2(4), 50-57, 76 (2019).