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
Fractions were coloured coded to indicated time of elution. was changed by increasing the pressure to 100 bar.
minor modifications, this SFC unit can be transformed into a bench top preparative SFE system.
First, a 20 mL SFC column was obtained and the column packing was removed, effectively transforming it into an extraction vessel for the bench top SFE experiments.
Next, a three-way valve was installed before the photodiode array detector and 1/16 inch tubing was plumbed directly into the backpressure regulator, bypassing the detector. The Fraction Collection Module was disconnected and fractions, already enriched with the target compounds, were collected as material exited the automatic backpressure regulator (ABPR) into scintillation vials.
The SFC column was pre-packed with diatomaceous earth. Equal molar amounts of α-pinene, β-myrcene, d-limonene, linalool, β-caryophyllene were sonicated for 10 minutes. 400 µL of this solution was injected onto 0.5 g of diatomaceous earth and loaded into the head of the 20 mL pre-packed SFC column. The column was installed into the Investigator’s oven, and the system was equilibrated to 50 bar with the oven temperature set to 30°C. A flow rate of 1 mL/min was maintained throughout the extraction process.
From a starting mixture of 400 µL of terpines a total of twenty-four 15 µL fractions were
collected in 42 minutes employing pure CO2 as mobile phase. The elution of terpenes began at 10 minutes. Fractions 1 through 11 were collected between 10 and 14 minutes after which the elution of terpenes stopped. At minute 30, the system was equilibrated to 100 bar, maintaining the initial column temperature and flow settings. Terpene elution was once again observed at minute 36. Fractions 12 – 24 were collected between minute 36 and 42. Each fraction was analysed using GC-FID and the peak height recorded. Linalool and beta-caryophyllene were observed in each fraction. Linalool was chosen as the normalisation peak as it produced the highest signal. To standardise the data, the peak height of each terpene was divided by the peak height of linalool.
In Table 1, the terpene fractions with the highest terpene:linalool ratio are highlighted in bold. By comparing these ratios, we can determine the elution of terpenes at given supercritical carbon dioxide conditions. Alpha-pinene was found at its highest concentration in fraction 2, followed by beta-myrcene and d-limonene. These monoterpenes all eluted at 50 bar and 30°C. A ratio turning point for beta- caryophyllene occurred in fraction 12; the
ratio is above 1, meaning there was a higher concentration of beta-caryophyllene than linalool. This suggests that the solubility of these terpenes at the given supercritical carbon dioxide conditions is as follows: α-pinene, β-myrcene, d-limonene, linalool, β-caryophylene.
Experiment 2
Next, the conditions from Experiment 1 were used to extract 5 grams of Cannabis inflorescence (the mature flower of a female plant). A similar procedure was followed: this time a 5 mL SFC column was obtained and the packing removed. Five grams of cannabis inflorescence were ground, packed into the 5mL SFC column, and installed into the Investigator’s oven. The Investigator system was equilibrated to 50 bar and the oven set to 30°C. The flow rate was maintained at 7 mL/min. No fractions were observed in the first 60 minutes, at which time the system was equilibrated to 100 bar, maintaining temperature and flow rate. Three fractions were collected, with the elution starting at 80 minutes. These fractions were analysed using GC-FID and compared with the terpene quantification of the starting material.
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