-

1. Extraction, isolation and purification of tetrahydrocannabinol from Cannabis sativa L.
plant using supercritical fluid extraction and solid phase extraction
Ada C. Gallo-Molinaa,b,1, Henry I. Castro-Vargasa,c,1, William Garzónb, Ariel Martínezd,
Zully Riveraa, Jerry W. Kinge, Fabian Parada-Alfonsoa*,
a Chemistry Department, Universidad Nacional de Colombia, Av. Carrera 30 # 45-03. Bogotá
D.C., Colombia.
b Fiscalía General de la Nación, Diagonal 22B # 52-01. Bogotá D.C., Colombia.
c Faculty of Engineering, Universidad Libre, Carrera 70 No 53-40. Bogotá D.C., Colombia
d Pharmacy Department, Universidad Nacional de Colombia, Av. Carrera 30 # 45-03. Bogotá
D.C., Colombia.
e CFS, 1965 E Spinel Link#7, Fayetteville, AR 72701, USA.
1 A. Gallo-Molina and H. Castro-Vargas contributed equally to this work.
* Corresponding author.
E-mail address: fparadaa@unal.edu.co (F. Parada-Alfonso)

2. Abstract.
The aim of this study were to obtain tetrahydrocannabinol (THC) - enriched extracts from
Cannabis sativa L. plant material by supercritical fluid extraction (SFE), and to purify the THC
extract using a single solid phase extraction (SPE) step. SFE was utilized at different
temperatures (40°C-80°C), pressures (15-33 MPa) and ethanol co-solvent (EtOH) (0-5%) by a
central composite design. The effect of extraction parameters on response variables (extraction
yield and THC content in raw extract) were evaluated and the most suitable conditions for THC
extraction were found. THC quantification was performed by gas chromatography with flame
ionization detection (GC-FID). The highest extraction yield was 26.36%, obtained at 33 MPa,
80°C and 5% EtOH. On other hand, the highest THC content in raw extracts were 37.85% and
36.18%, respectively, obtained at 60°C, 2% EtOH, and 33 and 15 MPa, respectively. One extract
with high THC content was selected and submitted for furthert purification-isolation by SPE.
From SPE, two fractions with high THC purity were obtained, and combined and analyzed by
GC-FID, reverse phase high performance liquid chromatography and nuclear magnetic
resonance, in order to verify the THC purity. One final fraction having 90.1% of THC purity was
obtained using a single SPE step. A sequential SFE-SPE process thus allowed one to obtain high
purity THC from the Cannabis sativa L. plant, which suitable for a quality control material or
analytical standard.
Keywords: Marijuana, cannabinoids, green extraction, quality control material.

3. 1. Introduction
Cannabis (Cannabis sativa L.) is a herbaceous plant which originated thousands of years ago
probable in Central Asia, the Northwestern Himalayas and China [1]. It has since found its way
many regions of the world, eventually spreading to the American continent. The history of
cannabis use goes back as far as 12,000 years, which places this plant among the oldest human
cultivated crops. Cannabis belongs to Cannabaceae family [2], their taxonomiic classification has
been debated for decades, however, current nomenclature of cultivated plants is used to
differentiation cannabis plant groups. For example, relative to cannabis’ use this may be
classified as (−)-trans-Δ⁹ -tetrahydrocannabinol-drug group, fiber-hemp group, or seed-oil group
(1).
Cannabis has been used with medical, therapeutically and spiritual purposes across the world
since ancient times. The plant use and crop were criminalized in the USA in 1937, when the plant
was classified as a “Schedule I substance” (highest dangerous with potential for addiction), i.e.,
in the Controlled Substances Act (USA) in the 1970. However, currently there is a trend toward
legalization for both medicinal and recreational cannabis uses. The last World Drug Report 2017
from the United Nations’ Office on Drugs and Crime (UNDOC) established that cannabis has
been the most widely used drug in the world, with an annual prevalence of 3.8% in the adult
population. The cultivation of cannabis plant was reported in 135 countries between 2010 to 2015
[3]. A market analysis of plant-based drugs, showed in the 2017 UNDOC report, indicated that
the main South America countries that were producers of the cannabis plant were Colombia and
Paraguay [4].
Cannabis is also known for their potential uses in the treatment of different diseases such as
glaucoma, depression, neuropathic pain, multiple sclerosis and Alzheimer's disease [5-10]. In
addition, this plant and its products are recognized for alleviation of symptoms in cancer (e.g.,
weight gain, nausea and vomiting induced by chemotherapy) and in the therapy of HIV/AIDS
patients [11,12]. The Organization of American States (OAS) established evidence for the
therapeutic use of cannabis. Actually, more than 450 chemical compounds (e.g., terpenes,
phenols, fatty acids, amino acids, hydrocarbons, sugars, and others) have been identified in
cannabis plants [1,13], among these are principally the cannabinoids and terpenoids group. More

4. than 104 cannabinoids have been identified in different cannabis plant strains [14], some notable
examples are (−)-trans-Δ-9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN),
cannabigerol (CBG) and cannabichromene (CBC), THC and CBD are the most prevalent
cannabinoids. These specific (THC and CBD) have been associated with the therapeutic and
medicinal properties of cannabis plant and associated products, and THC for its psychotropic
effects [14].
Recently years substantial changes to the cannabis policy outlook has been observed; for
example, in many countries possession and marketing of cannabis is illegal, however, possession
in small quantities has been decriminalized. For 2018 some countries with the laxest cannabis
laws are: Australia, Canada, Chile, Colombia, Costa Rica, The Czech Republic, India, Israel,
Jamaica, México, The Netherlands, Portugal, South Africa, Spain, Uruguay, and some United
States regions. In addition, in many countries cannabis legalization requests are in progress, some
governments considerate legalization as a way to eliminate the illegal traffic and associated
crime, added to it could even be a source of taxes. In Colombia cannabis laws has change during
the last three years, Colombian Government (Ministry of Justice) had issued 19 licenses related to
the cultivation of no psychoactive cannabis and 14 licenses for the production of psychoactive
cannabis [15]. In this context, psychoactive cannabis is defined for the local law as plant material
with THC concentration above to 1%. Since 2016, Colombian medical specialist have been
allowed to prescribe high-THC concentration cannabis preparations for any medical condition,
actually is expected that these preparations to become widely available. Noteworthy that
Colombian laws say that medical cannabis can only be sold and exported in extract form.
The current global changes relative to cultivation, processing, trade and use of cannabis in
scientific research related to its medicinal and therapeutic properties have increased the need for
high purity cannabinoids standards. A THC standard of high purity is required for forensic
purposes (e.g., as certified reference material). For example, for forensic analyses the occurence
of THC is of great importance in order to define when a cannabis plant material or product has a
therapeutic or recreational use, according to the international or local laws (e.g., Colombian laws,
a THC concentration above to 1%). In Bogotá D.C. (Colombia) it is estimated that ten thousand

5. cannabis samples (plant material, extracts and other products) are annually-analyzed. Pure THC
is also required in the development of pharmacological investigations; since there a potential
market for THC as a pain killer in palliative treatments, i.e. as a substitute for other stronger and
addictive substances such as morphine [16,17].
Some industrial processes have been development with order to obtain high purity THC from
vegetal samples. A U.S. patent (US 2005/0171361) describes a process using organic solvents
extraction (OSE) and reversed phase liquid chromatography (RPLC) for THC purification. This
process includes four successive extraction steps using heptane, isopropyl ether, water and
methyl tertiary butyl ether (MTBE), in addition, the RPLC purification step [18]. Another patent
(WO 2009/133376), include four OSE steps using n-heptane and MTBE and one RPLC
purification step [19]. These processes have some disadvantages; multiple isolation steps are
required, the use of toxic and polluting organic solvents, and difficulty in scaling-up the process.
Supercritical fluid extraction (SFE) is a good alternative technique for THC extraction-separation
being an extraction method using a low toxicity solvent such as carbon dioxide in its supercritical
state (SC CO2). This yields extracts that ate solvent-free and extracts that are GRAS (Generally
Recognized as Safe) co-solvents. SC CO2 has been used for cannabinoid extraction (e.g. CBD
and CBN) [20,21]. However, the low polarity of SC CO2, requires that small amounts of organic
solvents (e.g. GRAS solvents such as ethanol or ethyl acetate) be used in SFE in order to improve
the yields and in some cases the selectivity of the extraction. For example, ethanol (EtOH) can be
used to aim increase extraction yield for some cannabinoids. Rovetto and Aieta explored the THC
extraction using SC CO2 by adding EtOH (CO2/EtOH), and they observed that by using EtOH,
extracts with highest THC content were obtained compared to neat SC CO2 [22].
The aim of the present study was to develop and optimized a SFE process to obtain THC extracts
from cannabis plant. In addition, a solid phase extraction (SPE) was explored as isolation-
purification technique to obtain a high purity THC standard. CO2/EtOH was used as solvent, a
central compose design (CCD) and response surface methodology (RSM) employed to study the
effects of extraction parameters (temperature, pressure and EtOH percentage) on extraction yields
and THC content in the extracts. THC from enriched extract was isolated and purified using a

6. single SPE step. Finally, SFE and SPE allowed one to obtain high purity THC from Cannabis
sativa L. plant material, suitable for a quality control material (QCMs) [23] for use in research,
analysis and forensic science.
2. Materials and methods
2.1 Chemicals
High purity carbon dioxide was supplied by Linde de Colombia. Ethanol (96%) analytical grade
was obtained from Sigma-Aldrich Chemical Co. (St. Louis-MO, USA). Cannabinoid standard
having a purity higher than 99% were purchased for Lipomed AG, Tetracosane and deuterated-
dimethylsulfoxide (99.8%) was provided by Sigma-Aldrich Co (Milan, Italy). SPE cartridges
Supeclean ENVI were purchased from Supelco (Bellefonte-PA, USA).
2.2 Cannabis sativa L. sample and preparation
Samples of cannabis with fully ripe inflorescences (determined visually when more than 75% of
stigmas turn brown and shriveled) were selected. These were harvested in Southwest of
Colombia (Corinto, Cauca). A representative amount of vegetal material was sampled and
approximately 300 g selected for further use. The selected material was dried at room
temperature (between 15 to 18 °C) until the central stem of the floral cluster broke [24], then the
dried sample was milled and sieved to a size of less than 0.5 mm. Finally, the vegetal material
was stored at 4 °C in the absence of light until the SFE.
2.3 Supercritical fluid extraction
SFE was performed using a dynamic laboratory scale supercritical fluid extraction apparatus
(Figure 1). The extraction unit included a 10 cm3 extraction cell whose temperature was
controlled using an electrical jacket and regulator. The extraction pressure and flow were
maintained constant using a backpressure regulator. Ethanol as co-solvent was provided using a
liquid pump, this was mixed with the main CO2 stream before at extraction cell. The extraction
parameters, pressure, temperature and co-solvent percentage, were varied between 15 to 33 MPa,
40 to 80 ° C, and 0 to 5% EtOH, respectively, according to experimental design shown in the
Table 1. All extractions were development keeping constant the extraction time, the cannabis

7. sample amount, and supercritical solvent flow (4 h, 8 g and 0.55 kg/h, respectively) based on
previous study [16]. Ethanol was removed under vacuum and extracts were weighted using an
analytical balance to estimate the extraction yields, these were presented as weight percentage on
a dry basis (% wt d.b.). All extracts were analyzed by gas chromatography with flame ionization
detection (GC-FID) in order to quantify their THC content.
2.4 Experimental design and statistical analysis
The effect of SFE parameters (pressure, temperature and percentage of co-solvent) on response
variables (extraction yield and THC content in the raw extract) were determined using response
surface methodology (RSM) and central composite design (CCD, with α = 1). The levels of
independent variables were established based on previous reports on cannabinoids extraction
[20,22,25]. The experimental design consisted of 19 experimental runs performed in randomized
order; the central point was replicate five times (extracts number 15 to 19). The full experiment
design is shown in the Table 1. The experimental data were fitted to second-order polynomial
model which expressed the response variables as a function of independent variables according to
equation (1) as:
2 2 2
0 1 1 2 2 3 3 11 1 12 1 2 13 1 3 22 2 23 2 3 33 3Y X X X X X X X X X X X Xb b b b b b b b b b= + + + + + + + + + (1)
where Y represents the response variable, b0 is a constant, b1, b2 and b3 are the linear coefficients,
b11, b22 and b33 are the quadratic coefficients, and b12, b13 and b23 are the interactions coefficients
for the independent variables; X1 (EtOH percentage), X2 (pressure) and X3 (temperature). The fit
of the model was evaluated by one-way analysis of variance (ANOVA) with α< 0.05. The
coefficient of determination (R2) and the absolute average deviation (AAD) was used to establish
the correlation between the predicted and observed data. Evaluation of R2, R2 adjusted and AAD
values were used to check the accuracy of the model. R2 and R2 – Such R’s must be close to
100% and the AAD must be as small as possible. The acceptable values of R2 and AAD values
mean that the model equation defines the true behavior of the system and it can be used for
interpolation in the experimental domain [26]. The processing of the data was done with the R-

8. Project software version 3.4.2 (Free Software Foundation’s GNU project, Boston, Massachusetts,
US).
2.5 THC analysis and quantification
The THC analyses and quantification were carry out using a Shimadzu GC-2010 chromatograph
equipped with Shimadzu auto-sampler AOC-20i and an analytical capillary Rtx® GC column (30
m x 0.25 mm x 0.25 μm, Restek, Bellefonte, Pennsylvania, US). The injector and detector
temperatures were maintained at 290 °C and 300 °C, respectively. One microliter of cannabis
extracts dissolved in ethanol (at 12.5 mg/mL) were injected on split mode at ratio 1:20. The oven
conditions started at 200 °C kept for 2 min, then this was increased until 260 °C at 10 °C/min and
maintained during 10 min (3). The data were analyzed using the Lab Solutions lite software
version 5.52 of Shimadzu corporation. The THC quantification was carry on using a standard
curve (10 to 500 mg/L) to which was added tetracosane as an internal standard (100 mg/L) [27].
The results are expressed as weight percent of THC respect to dry extract (% wt d.b.).
2.6 THC isolation and purification process.
The SFE and GC-FID results were used to identify and select an extract with good extraction
yield, the highest THC content and contamination (lower number of compounds extracted). This
extract was submitted to isolation and purification process by SPE to obtain high purity THC.
The selected extract was analyzed by reverse phase high performance liquid chromatography
(RP-HPLC), in addition, to the GC-FID analysis. RP-HPLC was also used to follow of SPE
process and to identify the fractions with highest THC purity. Finally, one fraction with the
higher THC content was selected and analyzed by nuclear magnetic resonance (NMR) in order to
confirm the THC purity.
2.6.1 THC isolation and purification by SPE
THC isolation and purification were development using a solid phase extraction column packed
with octadecyl-modified silica gel (5 g, 40-60 m) [28]. The extract (0.5 g) was dissolved in
solvent A (trifluoroacetic acid 0.05% in water) and injected into the SPE column, then the
compounds were eluted using a linear gradient of solvent B (trifluoroacetic acid 0.05% in

9. acetonitrile) from 0% to 100% at constant flow rate of 1 mL/min. Thirty-one fractions were
obtained each one of 12 mL. All fractions were analyzed by thin layer chromatography and RP-
HPLC in order to determinate the THC presence and to select the THC enriched fractions. The
high THC content fractions were joined (obtaining the final fraction), the solvent was removed
under vacuum and the free-solvent fraction was lyophilized, then the global yield of process SFE-
SPE was determined. The final fraction was analyzed by RP-HPLC and NMR to determinate the
purity of THC obtained.
2.6.2 Analysis by RP-HPLC
RP-HPLC analysis were performed with an Agilent Series 1260 chromatograph using a
monolithic Chromolith C18 column (50 x 4.6 mm, Merck, Darmstadt, Germany). The elution was
development using a linear gradient of solvent A (acetonitrile) in solvent B (water) from 5% to
100% in 17 minutes. Samples of 5 L at 55.6 mg/L were analyzed. The mobile phase flow was
2.0 mL/min and the compounds detected at 210 nm wavelength. THC was identified using a
standard compound based on its retention time.
2.6.3 NMR analysis
A one-dimension NMR experiment from final fraction was performed; proton (1H NMR) and
carbon-13 (13C NMR). A Bruker AVANCE 400 MHz NMR instrument equipped with an inverse
probe head with z-gradient was used. A sample of 10 mg of dry final fraction was dissolved in
deuterated dimethylsulfoxide (DMSO-d6) and NMR spectra was obtained at 23 °C. Topspin 2.1
software was used for spectra acquisition and processing (Bruker Corporation).
3. Results and discussion
The extraction results are present in Table 1; the extracts numbers correspond to each extraction
carried out under the conditions specified. The results obtained for the extraction yield are
presented as weight percentage of dry cannabis plant material, while THC content are expressed
as weight percent of THC in the dry extract.

10. 3.1 Extraction yield
Table 1 shows that the higher extraction yield was 26.36%, corresponding to the extract number 6
obtained at 33 MPa, 80°C and 5% of EtOH. At these conditions supercritical solvent has a good
solvent power due to the high extraction pressure, and in addition, the presence of co-solvent
improves the SC CO2 solvation power [22,29]. Even though higher temperatures can reduces the
SC CO2 density, at higher extraction pressures this effect lacks importance compared to the
increases in the pressure vapor of solutes; hence high temperatures can enhance the extraction
yield [30,31]. Other extract with high yield was number 4 (23.36%), obtained at 15 MPa, 40°C
and 5% of EtOH, using the lowest levels of extraction pressure and temperature. Extracts number
4 and 6 showed the highest extraction yields, however these extracts were obtained using
different levels of pressure and temperature (low versus high levels). This result can be due to
variation of solvent selectivity with the pressure and temperature changes, for example, Table 1
shows that at high levels (33 MPa and 80°C) the THC content in extract is the half compared to
lower levels of pressure and temperature (15 MPa and 40°C). SFE has been previously used on
cannabis using neat SC CO2 to recover different cannabinoids, i.e., Rovetto and Aieta [22]
reported a maximum extraction yield of 18.5% obtained at 34 MPa and 55°C, whereas Da Porto
et al. [32] achieved a maximum yield of 21.5% at 30 MPa and 40°C. These yields are lower
compared with extracts number 4 and 6, suggesting that the addition of ethanol as co-solvent
improved the yield.
The resultant experimental data were analyzed by response surface methodology (RSM)
according to the parameters described in Section 2.4. Table 2 present the ANOVA results used to
evaluate the fit of the second order model to extraction yield data. ANOVA shows that just one
extraction parameter had a significative effect on extraction yields, the linear coefficient of
%EtOH (p-value = 0.027). An adjustment to the second order model was made using the Akaike
information criterion in order to obtain an equation that best describes the effect of the extraction
parameters on the extraction yield. Since the only factor that influence significantly on extraction
yield is the co-solvent, the adjusted model has the linear and quadratic coefficient for ethanol
effect. Equation (2) present the resulting second order model equation for the extraction yield
from C. sativa L. plant by SFE (with only significant coefficients):

11. Yield (%) = 8.19 + 4.91EtOH - 0.42EtOH2 Eq. (2)
Table 2 shows the coefficient of determination R2, the R2 adjusted and the absolute average
deviation (AAD), these statistical indicators (R2= 83.6, R2 adjusted= 81.5 y AAD= 9.1) indicate
that the new second order model represents adequately the experimental data. RSM analysis
shown that the ethanol addition as co-solvent in SFE from cannabis plant is the principal factor
significantly influencing the extraction yield. This effect of co-solvent was evident at 24 MPa and
60ºC, at these conditions the extraction yield obtained using neat SC CO2 was 13.06% (extract
number 14), however, addition of 2 and 5% of ethanol increased the yields, reaching 16.77%
(average value extracts number 15 to 19) and 18.27% (extract number 13), respectively. As cited
before, these results may be explained by an increase of higher polarity compounds extracted
from vegetal matrix (e.g., phenols and sugars). This effect has been reported previously by
Rovetto and Aieta [22] who observed that ethanol use as a co-solvent improved the overall
extraction yields of extracts from cannabis plant.
3.2 THC content
THC content in raw extracts was determinate by GC-FID; Table 1 shows the obtained results.
The highest THC content, 37.85%, corresponding to extract number 9 obtained at 33 MPa, 60°C
and 2% EtOH. Other extracts with high THC content were numbers 1, 2, 4 and 11 with THC
contents above 30% (between 31.08 to 36.18%). In contrast, the lower THC content was 15.52%
corresponding to extract 6 obtained at 33 MPa, 80°C and 5% EtOH. These results suggest that
co-solvent level between 2-5% favors the extraction of THC from the cannabis plant, however an
increase of ethanol percentage to a high level can reduce the THC recovery. This effect is evident
when comparing extracts number 9 and 6 (considering no temperature effect). Previously, Omar
et al. [33] and Rovetto and Aieta [22] reported that addition of ethanol as co-solvent in SFE
enhance the cannabinoids extraction efficiency including THC, however, larger co-solvent
concentration could reduce the solvent selectivity [34].
The Omar et al. [33] study explored the cannabinoids (CBD, CBN and THC) extraction from

12. thirteen cannabis samples of different types, growing areas and seasons, using SFE with
CO2/EtOH. They studied various extraction conditions (10-25 MPa, 35-55°C and 0-40% Ethanol)
and observed THC contents between 0.45 to 32.4% in dry raw extracts. On other hand, Rovetto
and Aieta [22] work studied the THC and Δ-9-tetrahydrocannabinolic acid (THCA) extraction
from four different C. sativa L. strains by SFE with neat SC CO2 and CO2/EtOH. They using a
novel extraction procedure based multistage pressure increments and pulses of co-solvent, their
results showed THC contents in extracts ranging between 64.2 to 76.2%. Compared with the
present work, THC contents in extracts observed by Omar et al. are lower, however, THC
contents reported by Rovetto and Aieta are higher. Noteworthy is the fact that the cannabis raw
material used in the present work and the material used for Omar et al. and Rovetto and Aieta
studies had different THC content, therefore the THC content in extracts can be very different,
despite using the same extraction method, solvent and similar conditions. To evaluate the
efficiency of extraction method for THC recovery from vegetal material one should consider the
THC content in raw material. The ratio between the THC content in the raw extracts and total
THC in raw material (%THCextract / %THCTotal) may be used in order to evaluate the THC
extraction efficiency. For example, in the present work the highest THCextract/THCTotal ratio was
5.38 (37.85% THCextract / 7.04% THCTotal), while for Rovetto and Aieta it was 4.59 (76.2%
THCextract / 16.6% THCTotal). This result is very important considering that the cannabis raw
material used in Rovetto and Aieta study had a high THC content.
The data of THC content were used to determine the coefficients for the second order model
(Table 2). ANOVA shows that the extraction parameters has a significant effect with the linear
and quadratic coefficient of pressure and the linear coefficient of %EtOH. The second order
model was adjusted using the Akaike information criterion; Equation (3) representing the
resulting second order model equation adjusted for the THC content in raw extract from C. sativa
L. plant. The R2, the R2 adjusted and the ADD (Table 2) indicates that the adjusted to model is
acceptable.
THC content (%) = 72.2 + 3.46EtOH - 3.69P - 0.93EtOH2 + 0.070P2 Eq. (3)

13. Figure 2 shows the response surface graph of second order model for THC content. This plot
allows one to visualize the effect of extraction parameters (pressure and %EtOH) on the THC
content in the raw extracts. The positive linear effect of % EtOH was evident at all pressures at
the interval between 1 to 2% EtOH; for example, at 15 MPa the THC content increased
approximately from 32 to 35% when the co-solvent was enhanced from 0 to 2%. By contrast, the
negative quadratic effect of co-solvent was evident on the interval between 2 at 5% of EtOH (for
all pressures) - this effect was more pronounced at 15 MPa. Figure 2 shows that the highest THC
contents was obtained at low pressures (15 MPa), however, the increases of this parameter
between 15 to 24 MPa reduces the cannabinoid concentration in extracts. The effect of pressure
observed in the present work agree with the results reported by Rovetto and Aieta [22], they
observed that at constant temperature the increase of pressure from 17 to 24 MPa decreased the
THC content in extracts from 76.23 to 64.17%, however, the change from 24 at 34 MPa
enhanced the THC content.
The results shown above suggest that SFE with CO2/EtOH is an adequate extraction technique
for THC recovery from C. sativa L. plant. The ethanol addition as co-solvent is an important
extraction parameter to improve both extraction yields and THC content in the extracts, the
extraction pressure affected the THC content, whereas temperature has no effect on the response
variables. Two-THC enriched extracts were obtained (number 9 and 11) using middle values of
ethanol and temperature (2% and 60 °C), however, their extraction pressure levels were different
(33 and 15 MPa, respectively). Finally, extracts number 9 and 11 were considered for the
isolation and purification of THC using single step- SPE.
3.3 THC isolation and purification
The results shown at the previous section indicated that the extracts 9 and 11 were candidates for
further purification, as analyzed by GC-FID and RP-HPLC. Figure 3 shows their respective
chromatographic profiles. According to the chromatographic profiles, extract number 11 is less
contaminated than that extract number 9, hence extract number 11 was submitted for further
purification. Noteworthy that extract number 11 was obtained using low pressure level - this is a
very important parameter considering the technical implications for extraction process scale up.

14. 50 mg extract of extract number 11was injected into the column and its components were eluted
using a linear water-acetonitrile gradient. Thirty-one fractions were collected, each one was
analyzed by TLC and RP-HPLC for the presence of THC presence and purity. The
chromatographic analysis allowed identification of the highest THC content fractions, i.e., two
fractions (number 26 and 27) were selected for further characterization. Figure 4 shows the
chromatographic profiles obtained for fractions number 26 and 27. The fractions selected were
combined, the organic solvent was removed under vacuum, with water removed by
lyophilization. Finally, the dried final fraction (FF) was obtained weighing 12.7 mg. This was
subjected to NMR analysis to verify its purity with respect to THC content.
The FF was analyzed by NMR: 1H-NMR and 13C-NMR experiments. Table 3 presents the NMR
signals observed for FF and THC signals reported in the literature, Figure 5b shows the 1H-NMR
spectra obtained from FF. The 1H-NMR signals observed for FF shown high similarity with those
reported for THC by Peschel and Politi [35]: noteworthy is that the maximum differences
observed on the δ values were 0.02 ppm. Some characteristics 1H-NMR signals for THC were
observed in FF spectra (see Figure 6): the H-9 and H-10 protons of angular methyl groups at 0.98
ppm and 1.33 ppm, respectively; the H-6 proton at 1.62 ppm; the olefinic H-2 proton at 6.37
ppm; the aromatic H-5’ proton at 6.15 ppm; the H-5a and H-5b protons at 1.25 and 1.86 ppm,
respectively; and the proton on hydroxyl group which provide a distinguishable signal at 9.21
ppm. The ∆-8-THC and THCA are cannabinoids with similar structures compared to THC, the
analysis of 1H-NMR signals observed for FF permitted elimination of the presence of these
compounds in the FF. The carboxyl group on C3’ in THCA changes the stereochemistry of H-1’’
protons, therefore its δ values will be found as separate signals between 2.7 to 2.8 ppm. In ∆-8-
THC, the H-4 proton would be moved because the OH group had no deshielding effect on this
proton. On the other hand, the 13C-NMR signals observed for FF were comparable to those
assignments previously for THC by Young et al. [36].
Figure 5 shows the 1H-NMR spectra of extract number 11 and FF. Comparing these spectra it is
possible to corroborate that the extract number 11 was composed mainly of THC. Other

15. cannabinoids such as CBD or CBN could potentially be present in extract, these probably
reduced the 1H-NMR spectra signals resolution (e.g., between 0.25 to 2.25 ppm), due to
overlapping of similar NMR signals. Figure 5 analysis indicates that SFE with CO2/EtOH
provided a highly THC enriched extract, which followed by single-SPE step was purified of other
cannabinoids. RP-HPLC and NMR results shown that FF correspond to high purity THC.
Finally, the THC concentration was quantified by GC-FID and 90.1% of purity was observed.
In general, the results showed that SFE coupled SPE allows to obtain high purity THC from C.
sativa L. plant. A global balance of extraction, isolation and purification process suggest that
from 100 g of cannabis material used in this work is possible recovery approximately 5.1 and 1.2
g of THC in raw extract and FF, respectively. By the SFE-SPE process it is possible to obtain a
high purity THC with a global yield above 1%. Using a cannabis starting material with high THC
content could increase the THC yield.
Conclusions
A sequential SFE-SPE process was explored to obtain THC with high purity from Cannabis
Sativa L plan material. The effect of SFE parameters on extraction yield and THC content in the
raw extract were analyzed; ethanol as co-solvent showed the main effect on both response
variables, while pressure shown influence on the THC content. A better extraction yield was
obtained at higher levels of pressure, temperature and ethanol percentage, however, these
extraction conditions provided a lower THC content. The highest THC recoveries was reached
using low ethanol percentages (2%), additional increase in pressure from 15 to 33 MPa improved
THC extraction, however, this reduced the extraction selectivity. SFE provided two extracts with
highest THC content, number 9 and 11 with 37.85 and 36.18% of THC, respectively, extract
number 11 was selected to purification-isolation step due to less complexity. One single SPE step
allowed one to obtain THC at 90.1% purity from extract number 11, was checked by
chromatographic and spectroscopic analysis. According with our results, from 100 g of Cannabis
Sativa L plan material by sequential SFE-SPE process it is possible to obtain 36.18 g of THC-
enriched extract and 9.14 g of high purity THC, which is a promising candidate to be an “in-
house standard”, or to continue the purification until have a reference standard. This demonstrates

16. the usefulness of conjugating SFE with SPE, for the first to obtain a raw clean extract. The
foregoing allows us to have a possible standard material type in-house THC, obtained at low cost
and with fully affordable resources.
Acknowledgements
The authors acknowledge to Universidad Nacional de Colombia (División de Investigación, Sede
Bogotá) for the financial support and to Fiscalía General de la Nación for its valuable support in
carrying out this work.
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19. Figures Captions
Fig 1. Schematic diagram of SFE apparatus. (1) CO2 cylinder; (2) CO2 pump; (3) and (6)
micrometering valve; (4) co-solvent reservoir; (5) co-solvent pump; (7) preheating spiral tube; (8)
10 cm3 extraction cell; (9) electronic temperature control; (10) collection vial; (11) Flowmeter;
(12) backpressure regulator valve; (13) manometer.
Fig. 2. Surface response plot for the effect of pressure and co-solvent percentage on THC content
in Cannabis Sativa L. extracts
Fig. 3. RP-HPLC and CG-FID profiles for extracts number 9 and 11. The signals at 12.7 and 12.2
minutes corresponds to THC in RP-HPLC and CG-FID profiles, respectively.
Fig. 4. RP-HPLC profiles for fractions 26 (a) and 27 (b). The signal at 12.7 corresponds to THC.
Fig. 5. 1H NMR (400 MHz in DMSO-d6) spectra for the extract number 11 (a) and the final
fraction (b).
Fig. 6. Tetrahydrocannabinol (THC) structure. The numbering presented is based
monoterpenenoid nomenclature.