44 February / March 2018

A Systematic Approach to Developing Extraction Conditions Utilising Supercritical Dioxide

by Eric Kawka, Cattis Consulting, Montpeiler, VT 05602, [email protected]

Cannabis Sativa produce and accumulate terpene-rich within the secretory cells of glandular trichromes [1]. and are important components of Cannabis resin as they contribute to the unique attributes of different Cannabis strains. are responsible for the ’s aroma and flavour.

They possess specific medical properties Discussing terpene extraction is problematic pharmacological properties of cannabis and may act synergistically with for two reasons. strains; this relationship is commonly referred , enhancing the therapeutic to as the ‘’ [3]. There are 1. during post-processing, thermally labile benefits of the plant. The extraction of several promising applications based on the terpenes undergo degradation reactions terpenes and cannabinoids from Cannabis combined use of cannabinoids and terpenes, is a function of their solubility in different 2. isolation of terpenes from these such as pairing (CBD) with the organic solvents [2]. Solvents like methanol, is difficult due to similar boiling points monoterpenes , and ethanol, butane, and hexane are commonly Among the various extraction techniques, to treat acne [4] or adding , used in Cannabis extractions. However, we explore the use of supercritical fluids (SC) linalool and to 1:1 CBD/THC aside from safety considerations, extracts as a for the targeted extraction of extracts to treat sleeping disorders [4]. produced with such solvents are considered terpenes from cannabinoids in Cannabis. The smallest and most volatile terpenes are ‘one pot extractions’; no selectivity between Data suggests that the interaction between monoterpenes which are biosynthesised by cannabinoids and terpenes can be achieved. cannabinoids and terpenes affects the the head-to-tail addition of two

Figure 1: (A) Five carbon isoprene unit (B) head to tail linkage of two isoprene units to form the Myrcene (C) terpene-rich resin within the secretory cells of glandular trichromes (D) classification of terpenes based on the number of isoprene units. 45

Figure 2: Products of the thermal degradation of the monoterpene limonene when heated at 120°C for 24 hours. Degradation of Limonene was monitored by GC-FID. 50% of Limonene degraded in 24 Hours into the compounds depicted above [5]. units (Figure 1). An isoprene unit is a 5-carbon in Cannabis oil formulation, these organic Experiment 1 molecule upon which terpenes are built. solvents will contaminate the final product. In order to develop terpene extraction Combining three isoprene units forms a class Supercritical fluid extraction (SFE) is an conditions it is critical to understand the of compounds called sesquiterpenes which are effective method for the separation of relative solubility of terpenes in supercritical less volatile than monoterpenes. The largest monoterpenes from sesquiterpenes, their carbon dioxide. Solubility data exists for and least volatile terpenes are biosynthesised alcohol derivatives, and cannabinoids. The cannabinoids in supercritical carbon dioxide by the joining of four or more isoprene units. most popular supercritical solvent used is [[2], but limited literature is found on the Terpenes are both volatile and thermolabile carbon dioxide (CO ). CO is inexpensive and 2 2 solubility of terpenes. In Experiment 1, we compounds. They easily convert into each is a generally recognised as safe solvent. CO 2 increase our understanding of the elution other by oxidation, isomerisation, cyclisation reaches supercritical state at conditions of 31°C order of monoterpenes and their separation or dehydrogenation reactions (Figure 2). The and 74 bar and returns to a gaseous state at from sesquiterpenes at different supercritical environment in which Cannabis is stored ambient conditions. This allows for simple carbon dioxide conditions. and the methods used in processing will solute recovery and results in a solvent-free To develop and optimise conditions for effect the chemical composition of these extract [6]. By modifying the pressure and terpene extraction using supercritical isoprene-containing compounds. For example, temperature of the CO system, the dissolution 2 carbon dioxide, we used the Investigator distillation of crude Cannabis oil raises two properties of the solvent can be adjusted, Supercritical Fluid Chromatography issues. First, due to the high temperature enabling a selective extraction of cannabinoids (SFC) instrument (Waters, Milford, USA). required for distillation, delicate monoterpenes and terpenes. Here we describe a systematic Typically, the Investigator SFC system is readily thermally degrade. Second, during approach to developing terpene extraction used for analytical and semi-preparative distillation organic solvents co-elute with the conditions utilising supercritical carbon dioxide purification work [7]. However, with a few terpene fraction. If this terpene fraction is used

Figure 3: Investigator SFC System/experimental design. 46 February / March 2018

Table 1: Relative solubility of α-Pinene, β-Myrcene, d-Limonene, Linalool, β-Caryophyllene. The modified SFC Investigator system was equilibrated and SFE

parameters maintained at 50 bar, 30°C with a flow rate of 1ml/min for 30 minutes after which the density of CO2 was changed by increasing the pressure to 100 bar. Fractions were coloured coded to indicated time of elution.

minor modifications, this SFC unit can be From a starting mixture of 400 µL of terpines ratio is above 1, meaning there was a higher transformed into a bench top preparative a total of twenty-four 15 µL fractions were concentration of beta-caryophyllene than

SFE system. collected in 42 minutes employing pure CO2 linalool. This suggests that the solubility as mobile phase. The elution of terpenes of these terpenes at the given supercritical First, a 20 mL SFC column was obtained and began at 10 minutes. Fractions 1 through 11 carbon dioxide conditions is as follows: the column packing was removed, effectively were collected between 10 and 14 minutes -pinene, -myrcene, d-limonene, linalool, transforming it into an extraction vessel for α β after which the elution of terpenes stopped. -caryophylene. the bench top SFE experiments. β At minute 30, the system was equilibrated Next, a three-way valve was installed before to 100 bar, maintaining the initial column the photodiode array detector and 1/16 temperature and flow settings. Terpene Experiment 2 inch tubing was plumbed directly into the elution was once again observed at minute Next, the conditions from Experiment 1 backpressure regulator, bypassing the 36. Fractions 12 – 24 were collected between were used to extract 5 grams of Cannabis detector. The Fraction Collection Module minute 36 and 42. Each fraction was inflorescence (the mature flower of a female was disconnected and fractions, already analysed using GC-FID and the peak height plant). A similar procedure was followed: enriched with the target compounds, were recorded. Linalool and beta-caryophyllene this time a 5 mL SFC column was obtained collected as material exited the automatic were observed in each fraction. Linalool and the packing removed. Five grams backpressure regulator (ABPR) into was chosen as the normalisation peak as it of cannabis inflorescence were ground, scintillation vials. produced the highest signal. To standardise packed into the 5mL SFC column, and The SFC column was pre-packed with the data, the peak height of each terpene installed into the Investigator’s oven. The diatomaceous earth. Equal molar amounts was divided by the peak height of linalool. Investigator system was equilibrated to 50 of α-pinene, β-myrcene, d-limonene, In Table 1, the terpene fractions with the bar and the oven set to 30°C. The flow rate linalool, β-caryophyllene were sonicated highest terpene:linalool ratio are highlighted was maintained at 7 mL/min. No fractions for 10 minutes. 400 µL of this solution was in bold. By comparing these ratios, we can were observed in the first 60 minutes, at injected onto 0.5 g of diatomaceous earth determine the elution of terpenes at given which time the system was equilibrated to and loaded into the head of the 20 mL supercritical carbon dioxide conditions. 100 bar, maintaining temperature and flow pre-packed SFC column. The column was Alpha-pinene was found at its highest rate. Three fractions were collected, with installed into the Investigator’s oven, and the concentration in fraction 2, followed by the elution starting at 80 minutes. These system was equilibrated to 50 bar with the beta-myrcene and d-limonene. These fractions were analysed using GC-FID and oven temperature set to 30°C. A flow rate monoterpenes all eluted at 50 bar and compared with the terpene quantification of of 1 mL/min was maintained throughout the 30°C. A ratio turning point for beta- the starting material. extraction process. caryophyllene occurred in fraction 12; the 47

Results were not satisfactory for two reasons:

1. In this first attempt we were unable to recover our volatile terpenes

2. The length of extraction was prohibitively long

If these conditions were scaled to a 5 L system (vs our 5 mL system), the terpene extraction would take over three hours,

requiring a significant amount of CO2. This experiment was repeated multiple times, modifying both the instrumentation and supercritical carbon dioxide conditions until both monoterpenes and sesquiterpenes were recovered in an appropriate length of time. The next section will examine the modifications and conditions required to scale up to a 5 litre SFE system.

Supercritical Extraction Equipment and Terpene Extraction Procedure The scale up terpene extraction was conducted using a 5 L Bio-Botanical Figure 4: GC-FID analysis of the three terpene fractions obtained from the 5 g Cannabis extraction Extraction System (Waters, Milford, USA) pressure was maintained by the ABPR. An independently heated and each with manual modified to include a 4th terpene collection electrical jacket controlled the temperature pressure control. The high-pressure pump vessel (CS4). The system had a maximum of the extraction vessel. The system had delivered a maximum mass flow of 200 g/ operating pressure of 600 bar. Extraction three collection vessels (CS1-CS3), each min. The solvent flowed through a heat

Figure 5: Schematic drawing of 5 litre Bio-botanical Extractor (Waters, Milford, USA) modified with an extra 4-terpene cyclone collection vessel. (CS = cyclone separator) 48 February / March 2018

Table 2: Optimised Supercritical CO2 extraction conditions for targeting terpenes found in terpene rich Hemp. Both terpene fractions were analysed using GC-FID, shown in Figure 3. In stage 3 of Stage 1 Stage 2 Stage 3 the extraction, the pressure parameter Extraction Temperature (°C) 50 55 55 was increases to 300 bar for extraction. Total extraction time for Extraction Pressure (bar) 70 150 300 monoterpenes was 45 minutes, extraction Flow Rate (g/min) 100 150 200 time for cannabinoids was 5.5 hours, and Extraction time (min) 45 45 45 the total efficiency was 92% of the available CS1 target compounds were extracted. N/A 103 / 50 103 / 50 Pressure (bar) / Temperature (°C)

CS2 N/A 65 / 45 65 / 45 Pressure (bar) / Temperature (°C) Conclusion CS3 N/A 51 / 35 51 / 35 In order to develop terpene extraction Pressure (bar) / Temperature (°C) condition utilising supercritical carbon SC4 Ambient / -78 N/A N/A dioxide, it is essential to understand the Pressure (bar) / Temperature (°C) relative solubility of terpenes in supercritical

Extraction Time Terpenes 45 min carbon dioxide. It was determined that Extraction Time Cannabinoids 5.5 hours monoterpenes have high solubility at 70 bar Extraction Efficiency 92% and 50°C while sesquiterpenes have low to mild solubility at these conditions. With

exchanger to bring the liquid CO2 to a addition of CS4, volatile monoterpenes the addition of a terpene specific collector supercritical state before it entered into the evacuate with supercritical carbon (CS4), supercritical CO2 is an effective

extractor vessel. The supercritical stream dioxidefrom SC3 into the CO2 recycler. solvent for the extraction of terpenes from dissolved the target components from the With the addition of CS4, it is possible to Cannabis. The ability of supercritical carbon botanical material and directed them from create conditions that allow for the recovery dioxide to return to a gaseous state once the extraction vessel to the appropriate of volatile monoterpenes. However, even exposed to ambient conditions allows for cyclone separators. The optimised with the addition of CS4, at ambient simple terpene recovery and results in no conditions are summarised in Table 2. temperatures volatile monoterpenes detectable residual solvents. Monoterpene will evacuate with the gaseous CO . To Cannabinoids and terpenes have varying 2 fractions were obtained from the terpene remedy this, CS4 was cooled to -78°C solubility in supercritical carbon dioxide. specific collector with high purity and no with a solvent mixture of and dry Furthermore, compounds within the classes detectable cannabinoids. In order to recover ice. Once chilled, the extraction vessel of cannabinoids and terpenes have varying highly volatile monoterpenes, CS4 needed was pressurised to 70 bar and extraction solubility in supercritical carbon dioxide. The to be chilled to -78°C. Plant waxes and occurred at 50°C. Monoterpenes have high extraction of terpene rich hemp is divided cannabinoids co-elute with sesquiterpenes, solubility in supercritical carbon dioxide into three stages, with the density profile of which are collected from CS3. Due to the at these conditions, while sesquiterpenes supercritical carbon dioxide being increased more robust nature of sesquiterpenes, these have mild to low solubility. In Stage 2, both at each stage in order to target different compounds can undergo post processing temperature and pressure were increased compounds. methods such as winterization with minimal to target less volatile sesquiterpenes. to no degradation. The ability to separate To extract monoterpenes, the flow of During this stage, the flow of supercritical monoterpenes from sesquiterpenes from supercritical carbon dioxide was first carbon dioxide was directed into CS1 and cannabinoids allows for post processing directed into CS4, bypassing cyclone continued through CS3. After 45 minutes, of cannabinoids without the danger of separators one through three. Without the sesquiterpenes were collected from CS3. terpene degradation.

Figure 6: Left chromatogram: Monoterpene fraction obtained at extraction conditions of 70 bar and 50ºC. Right chromatogram: Sesquiterpenes obtained at extraction conditions of 150 bar and 55ºC. 49

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