Extraction and decarboxylation of cannabinoids: Extraction of cannabinoids was carried out from dried and ground floral plant material (high THCV strain, 3:1 - THCVA: THCA, PTC8505V from Phylos Bioscience, Portland, OR) using a Cup-30 cold ethanol extractor (Delta Separations, Cotati, CA). The extraction solvent was 200 proof food grade ethanol (Rocky Mountain Reagents, Golden, CO). The ethanol was evaporated using a Falling Film Evaporator (Delta Separations) and the resulting extract was treated at 110 °C under vacuum for 8 h for the decarboxylation of cannabinoids and elimination of residual ethanol using a 50 L glass reactor under vacuum system from Keda Instruments (Zhengzhou, China). Raw floral plant on average was 15.3% THCVA (Tetrahydrocannabivarinic Acid) and 9.0% THCA (Tetrahydrocannabinolic acid). Average extraction yield was 12.7% by mass with average extract composition having 42.8% THCV and 34.0% THC.

Distillation of cannabinoids: Cannabinoid distillate was obtained using a 6-inch stainless-steel wiped-film evaporator system from Pope Scientific (Saukville, Wisconsin). Working parameters for distillation were 120–165 °C heating mantle temperature (HMT), 80–90 °C internal condenser temperature (ICT) at a pressure of 180 mTorr.

Purification of cannabinoids: THCV was purified using a Selekt Biotage flash chromatography system equipped with a Biotage Sfär C18 400 g column (CV, dead volume, = 582 mL) and a UV detector (230 nm) and using an ethanol and water (65:35) mix as mobile phase.

Heptadecane 99% pure, liquid at room temperature (CAS# 629-73-7), ethyl linoleate ≥ 99%, liquid at room temperature (CAS# 544-35-4), cannabinoid mixture standard (Cerilliant^®, 1 mg/mL in methanol each of (−)-trans-Δ9-THC, cannabidiol, and cannabinol in methanol) product number C-220 and GC grade methanol (CAS# 67-56-1) were obtained from Sigma-Aldrich. THCV and THC were obtained from Schwazze Biosciences. Three cannabinoid mixtures, mixture 1 (85.75% cannabinoid, 16.75% THC, 0.00% CBD, 0.91% CBG, 0.53% CBC, 0.00% CBN and 67.46%), mixture 2 (90.49% cannabinoid, 44.16% THC, 0.00% CBD, 1.90% CBG, 0.55% CBC, 0.00% CBN and 43.91% THCV) and mixture 3 (89.79% cannabinoid, 23.72% THC, 0.00% CBD, 1.01% CBG, 0.75% CBC, 0.00% CBN and 64.31% THCV) were also obtained from Schwazze Biosciences. Cannabinoids and cannabinoid mixtures were a viscous liquid at room temperature.

Extraction, distillation and purification of cannabinoids performed at Schwazze Biosciences were monitored with high pressure liquid chromatography (HPLC). THCV and THC levels in cannabis extract and distillate were monitored using an Agilent 1100 HPLC system equipped with a Phenomenex Kinetex 2.6 mm C18 100 A (150 × 4.6 mm) column (Torrance, CA) and an isocratic mobile phase of a mix acetonitrile/water with 0.1% phosphoric acid (75:25), and a flow of 1.0 ml/min (12 min total run); the retention times of THCV and THC were 4.2 and 7.0 min, respectively.

Confirmation of THC and THCV cannabinoid purity at Colorado College was performed by gas chromatography-mass spectroscopy (GC-MS) analysis. Samples were prepared for GC-MS analysis by dissolving cannabinoids in GC-MS grade methanol. Cannabinoid standard and samples were analyzed on an Agilent 7890 A GC system coupled to an Agilent 5975 C inert XL MSD with an Agilent 19,091 S-433, HP-5MS, 5% phenyl methyl silox. 30 m x 250 μm x 0.25 μm column. A 3 µL injection with a 50:1 split was utilized. The Injector temperature was set to 250 °C. The column temperature program started at 40 °C with a one-minute hold and then a ramp to 220 °C at 30 °C/min, ramp to 260 °C at 10 °C/min, hold for 5 min, and a final ramp to 300 °C at 30 °C/min.

Thermogravimetric analysis (TGA) of phytochemical (heptadecane and ethyl linoleate) and cannabinoid boiling points were performed on a Waters TA Discovery TGA 5500 instrument and Differential Scanning Calorimetry (DSC) analysis of phytochemical and cannabinoid boiling points and heats of vaporization (ΔH_vap) were performed on a Waters TA Discovery DSC 250 instrument. Between three and fifteen mg of sample, mixed with 2–3 mg of alumina diluent (when used) were prepared in Tzero pans with an internal volume of 20 µL (TA instruments part number 901683.901). Heptadecane and ethyl linoleate are non-viscous liquids at room temperature and were transferred to Tzero pans with a small volume pipette. For viscous cannabinoid samples, the samples were loaded into small volume glass syringes. These syringes were heated to 50 °C in an oven and sample was transferred from the heated syringe into Tzero pans warmed on top of a hot plate to 50 °C. The heating allowed for clean transfer of sample. The Tzero pans were hermetically sealed with Tzero hermetic pinhole lids with a 75 μm diameter laser drilled pinhole (TA instruments part number 901685.901). The instrument (TGA or DSC) temperature ramp was set between 5 °C/min and 10 °C/min for each analysis.

For TGA experiments boiling point was determined by using the instrument software to find the onset temperature of the mass lost feature on TGA graph, see Fig. 2A. For DSC experiments the onset temperature of the enthalpy of vaporization peak gives the boiling point, see Fig. 2B (Goodrum and Siesel 1996) The heat of vaporization (ΔH_vap) is determined from the total energy in joules in the integration of the vaporization peak divided by the total mass of the original sample to give ΔH_vap in J/g. This number is corrected for the mass of sample lost in reaching the boiling point as determined from TGA experiments run using the same parameters as the DSC experiments (Rojas-Aguilar et al. 2005) and then converted to ΔH_vap in units of KJ/mol. On average heptadecane experienced 25% mass loss, ethyl linoleate experienced 26% mass loss, THCV experienced 25% mass loss, and THC experienced 22% mass loss in reaching their respective boiling points.

The two phytochemicals heptadecane and ethyl linoleate were selected as test compounds for boiling point determination of pure compounds and mixtures by TGA and DSC analysis. Very-long-chain (VLC) alkanes and long-chain (LC) alkanes are commonly produced by plants through biosynthetic pathways (Wen et al. 2025). Heptadecane (C₁₇H₃₆) is one such LC-alkane and is a common phytochemical found in plant extracts. Heptadecane has a normal boiling point between 288.50 and 301.85 °C (Brown and Stein 2025) with a ΔH_vap = 86.5 KJ/mol (Acree Jr. and Chickos 2025). Ethyl linoleate (C₂₀H₃₆O₂) is a common phytochemical known as an essential fatty acid. It is found in high concentrations in garlic and is one of phytochemicals that gives garlic anti-inflammatory properties (Park et al. 2014). Ethyl linoleate has an estimated normal boiling point of 355.6 °C (Wallek et al. 2018) with a ΔH_vap = 72.6 KJ/mol (Acree Jr. and Chickos 2025).

The atmospheric pressure during TGA and DSC analysis of heptadecane and ethyl linoleate ranged between 610.0 and 615.0 mm Hg. See Fig. 2 for representative TGA and DSC data. The average atmospheric pressure was recorded during each run using a mercury barometer. Within a reasonable range of atmospheric pressures, the heat of vaporization is constant. Normal boiling points (at 1 atm) were calculated by two methods for comparison. The first using data at barometric pressure recorded during the time of the experiment, the average experimentally determined ΔH_vap and the Clausius-Clapeyron equation (see Eq. 1).

Where p₁ and p₂ are vapor pressure (atm) at temperatures T₁ and T₂ (Kelvin). ΔH_vap is the heat of vaporization (KJ/mole) and R is the gas constant (0.00831447 KJ K^-1  mol^-1). Boiling occurs when vapor pressure equals atmospheric pressure.

The second method used data at barometric pressure recorded during the time of the experiment and the Sydney-Young equation (Young 2010).

Θ is the correction in centigrade or kelvin to add to the observed boiling point to obtain the boiling point at 760 mm Hg, p is the pressure in mm Hg of the observed boiling point and t is the observed boiling point. See Table 1 for summarized data using both analysis methods.

The experimental determined boiling points of heptadecane and ethyl linoleate give a reasonable estimate of the expected boiling points based on literature values with best data coming from use of the Sydney-Young equation. For both test compounds the experimentally measured heats of vaporization are lower than expected by 55% for heptadecane and by 41% for ethyl linoleate.

If it can be assumed that when two volatile compounds form an ideal solution, then Raoult’s law (see Eq. 2) can be used to determine the vapor pressure of two component mixture).

The boiling point of the mixture occurs when p_T equals atmospheric pressure. Equations 1 and 3 are utilized in a system of equations to predict the boiling point of a two-component mixture. The less ideal the solution the greater the deviation between predicted and measured boiling point for the mixture. The boiling point of three mixtures of heptadecane and ethyl linoleate were prepared were measured by TGA (see Table 2). The predicted boiling points as determined using Eqs. 1 and 2 with DSC data from Table 1 were compared to the measured boiling point. This system of equations accurately predicted the boiling points of the mixtures within ± 2.5%.

The agreement between experimental TGA mixture boiling point measurements using normal boiling point values of the pure compounds determined by TGA and DSC experiments confirms these methods for determining the boiling points of cannabinoids. The validation experiments also indicate that these methods will require further optimization in order to accurately determine heats of vaporization. The heat of vaporization calculated by these experiments are systematically about 50% lower than expected. Likely the amount of material lost to vaporization before reaching the boiling point has been underestimated. Each different mass weighed into the Tzero pans will have a different surface area to volume ratio. Experimental data will be required for each substance to appropriately factor in how the surface area to volume ratio impacts the specific amount of material lost while the DSC instrument is heating up to the boiling point. Control experiments are also needed to determine the appropriate correction factor needed to account for the aluminum mass difference between the sample Tzero pan and the reference Tzero pan. Given the results of the method verification experiments the THCV and THC boiling points are determined using the Sydney-Young equation.