Accepted Manuscript

Title: Extraction, isolation and purification of

tetrahydrocannabinol from the Cannabis sativa L. plant using
supercritical fluid extraction and solid phase extraction

Authors: Ada C. Gallo-Molina, Henry I. Castro-Vargas,

William F. Garzón-Méndez, Jorge A. Martı́nez Ramı́rez, Zully
J. Rivera Monroy, Jerry W. King, Fabian Parada-Alfonso

PII: S0896-8446(19)30054-3
DOI: https://doi.org/10.1016/j.supflu.2019.01.020
Reference: SUPFLU 4458

To appear in: J. of Supercritical Fluids

Received date: 27 January 2019

Accepted date: 28 January 2019

Please cite this article as: Gallo-Molina AC, Castro-Vargas HI, Garzón-Méndez WF,
Martı́nez Ramı́rez JA, Rivera Monroy ZJ, King JW, Parada-Alfonso F, Extraction,
isolation and purification of tetrahydrocannabinol from the Cannabis sativa L.
plant using supercritical fluid extraction and solid phase extraction, The Journal of
Supercritical Fluids (2019), https://doi.org/10.1016/j.supflu.2019.01.020

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Extraction, isolation and purification of tetrahydrocannabinol from the Cannabis
sativa L. plant using supercritical fluid extraction and solid phase extraction

Ada C. Gallo-Molinaa,b, Henry I. Castro-Vargasa,c, William F. Garzón-Méndezb, Jorge

A. Martínez Ramírezd, Zully J. Rivera Monroya, Jerry W. Kinge, Fabian Parada-
Alfonsoa*,

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a
Departamento de Química, Universidad Nacional de Colombia, Av. Carrera 30 # 45-03.

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Bogotá D.C., Colombia.
b
Fiscalía General de la Nación, Diagonal 22B # 52-01. Bogotá D.C., Colombia.

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c
Facultad de Ingeniería, Universidad Libre, Carrera 70 No 53-40. Bogotá D.C., Colombia
d
Departamento de Farmacia, Universidad Nacional de Colombia, Av. Carrera 30 # 45-03.

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Bogotá D.C., Colombia.
e
CFS, 1965 E Spinel Link#7, Fayetteville, AR 72701, USA.
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* Corresponding author.
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E-mail address: fparadaa@unal.edu.co (F. Parada-Alfonso)

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Graphical abstract
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Highlights

 A sequential SFE-SPE process thus allowed one to obtain high purity THC from the
Cannabis sativa L. plant material.
 The effect of extraction parameters on response variables were evaluated and the
most suitable conditions for THC extraction were found.
 An extract with 36.18% of THC was obtained using CO2 at 15 MPa, 60 °C and 2%
of ethanol.

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 The THC obtained which suitable as a quality control material or analytical
standard.

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Abstract.
The tetrahydrocannabinol (THC) was obtained from Cannabis sativa L. plant material using
supercritical fluid extraction (SFE) and solid phase extraction (SPE). SFE was utilized at
different pressures (15-33 MPa), temperatures (40-80°C) and ethanol (EtOH) as co-solvent
(0-5%). The effect of extraction parameters on response variables (yield and THC content in
raw extract) was evaluated. Keeping both temperature and co-solvent concentration constant

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at 60°C and 2% respectively, the highest THC content in raw extracts reaches 37.85% at 33
MPa and 36.18% at 15 MPa. One extract with high THC content was selected and submitted

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for purification-isolation by SPE. One final fraction with a 90.1% of THC was obtained using
a single SPE step. This fraction was analyzed by GC, RP-HPLC and NMR to verify the THC

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purity. A sequential SFE-SPE process allows to obtain high purity THC from the cannabis
plant, which is suitable as quality control material or analytical standard

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Keywords: Marijuana; cannabinoids; green extraction; quality control material.
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 1. Introduction
Cannabis (Cannabis sativa L.) is a herbaceous plant which originated probably in Central
Asia, the Northwestern Himalayas and China thousands of years ago [1]. It has since found
its way to many regions of the world, including the Americas. 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 the Cannabaceae family [2]. Although its taxonomic

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classification has been debated for decades, current nomenclature of cultivated plants is used
to differentiate cannabis plant groups. For example, relative to its use cannabis may be

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classified as (−)-trans-Δ-9-tetrahydrocannabinol-drug group, fiber-hemp group, or seed-oil
group (1).

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Cannabis has been used for medical, therapeutically and spiritual purposes across the world

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since ancient times. However, its use and cultivation were criminalized in the USA in 1937

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when the plant was classified as a “Schedule I substance” (highly dangerous with potential
for addiction), i.e. in the Controlled Substances Act (USA) of 1970. Currently, however,
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there is a trend towards legalizing cannabis for medicinal and recreational uses. The 2017
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World Drug Report 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
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prevalence of 3.8% in the adult population. Cultivation of the cannabis plant was reported in
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135 countries between 2010 and 2015 [3]. A market analysis of plant-based drugs, as
contained in the 2017 UNDOC report, placed Colombia and Paraguay as the main cannabis
producers in South America [4].
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Cannabis is also known for its potential uses in the treatment of different diseases such as
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glaucoma, depression, neuropathic pain, multiple sclerosis and Alzheimer's disease [5-10].
In addition, this plant and its products are recognized for the alleviation of the side-effects of
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cancer treatment (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) have
developed protocols 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 which the cannabinoids and
terpenoids are the main groups. More 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 and have been associated with
the therapeutic and medicinal properties of the cannabis plant and associated products, but
THC is mainly recognized for its psychotropic effects [14].

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In recent years government policy towards cannabis has experienced significant changes. For

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example, in many countries possession and marketing of cannabis are illegal; however,
possession of small quantities has been decriminalized. In 2018 the countries with the most

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relaxed cannabis laws included Australia, Canada, Chile, Colombia, Costa Rica, The Czech
Republic, India, Israel, Jamaica, México, The Netherlands, Portugal, South Africa, Spain,

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Uruguay, and some states in the USA. In addition, in many other countries requests for

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legalizing cannabis are in progress; indeed some governments consider legalization as a way
to eliminate illegal trafficking and its associated crimes, and also as a source of revenue
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through taxation. In Colombia cannabis laws have changed during the last three years. The
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Colombian government through the Ministry of Justice has issued 19 licenses for the
cultivation of non-psychoactive cannabis and 14 licenses for the production of psychoactive
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cannabis [15]. In this context, psychoactive cannabis, as defined by local legislation, is plant
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material with a THC concentration above 1%. Since 2016 Colombian health specialists have
been allowed to prescribe cannabis preparations with a high THC concentration for any
medical condition; indeed these preparations are expected to become more widely available.
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It is worth noting that Colombian legislation allows medical cannabis to be sold domestically
and exported in extract form only.
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Current global changes with regards to cultivation, processing, trade and the use of cannabis
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in scientific research for 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 (i.e. as a certified reference material). For example, for forensic analysis the
occurrence of THC is of great importance in order to determine if a cannabis plant material
or product has a therapeutic or recreational use under international or local laws (e.g. a THC
concentration above 1% under Colombian law). In Bogotá D.C. (Colombia) it is estimated
that ten thousand cannabis samples (plant material, extracts and other products) are analyzed
annually. Pure THC is also required in the development of pharmacological investigations as
there is a potential market for THC as a pain killer in palliative treatments so as to substitute
for other stronger and addictive substances such as morphine [16,17].

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Some industrial processes have been developed to obtain high purity THC from vegetal
samples. A U.S. patent (US 2005/0171361) describes a process using organic solvents

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extraction (OSE) and reversed phase liquid chromatography (RPLC) for THC purification.
This process includes four successive extraction steps using heptane, isopropyl ether, water

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and methyl tertiary butyl ether (MTBE) in addition to the RPLC purification step [18].
Another patent (WO 2009/133376), includes four OSE steps using n-heptane and MTBE and

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one RPLC purification step [19]. These processes have some disadvantages such as the

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requirement of multiple isolation steps, the use of toxic and polluting organic solvents, and
the difficulty in scaling-up the process.
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Supercritical fluid extraction (SFE) is a good alternative technique for THC extraction-
separation, since it uses a solvent with low toxicity such as supercritical carbon dioxide (SC
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CO2). The resulting extracts are solvent-free. SC CO2 has been used for cannabinoid
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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 to improve the yields and in some cases the selectivity of the extraction. For
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example, ethanol (EtOH) can be used to increase extraction yield for some cannabinoids.
Rovetto and Aieta explored the THC extraction using SC CO2 with EtOH (CO2/EtOH), and
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observed that extracts with higher THC content could be obtained rather than using just neat
SC CO2 [22].
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The aim of the present study was to develop and optimize a SFE process to obtain THC
extracts from the cannabis plant. In addition, a solid phase extraction (SPE) was explored as
an isolation-purification technique to obtain a high purity THC standard. CO2/EtOH is used
as solvent. A central compose design (CCD) and response surface methodology (RSM) are
employed to study the effects of the extraction parameters (temperature, pressure and EtOH
concentration) on the extraction yields and the THC content in the extracts. THC from
enriched extracts was isolated and purified using a single SPE step. Finally, SFE and SPE
allow high purity THC to be obtained from the Cannabis sativa L. plant material and be used
as a quality control material (QCMs) [23] in research analysis and forensics.

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2. Materials and methods
2.1 Chemicals

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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

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standard with a purity higher than 99% was purchased from Lipomed AG. Tetracosane and
deuterated-dimethylsulfoxide (99.8%) were supplied by Sigma-Aldrich Co (Milan, Italy).

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SPE cartridges Supeclean ENVI were purchased from Supelco (Bellefonte-PA, USA).

2.2 Cannabis sativa L. sample and preparation

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Samples of cannabis with fully ripe inflorescences (determined visually when more than 75%
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of the stigmas turned brown and shriveled) were selected. These were harvested in the
southwest of Colombia (Corinto, Cauca). A representative amount of vegetal material was
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sampled and approximately 300 g selected for further use. The selected material was dried
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up 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.
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2.3 Supercritical fluid extraction

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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
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controlled using an electrical jacket and regulator. The extraction pressure and flow were
maintained constant using a backpressure regulator. All extractions kept constant the
extraction time, the cannabis sample amount, and the supercritical solvent flow (4 h, 8 g and
0.55 kg/h, respectively) as given by a previous study [16]. Ethanol as a co-solvent was
supplied by a liquid pump and mixed with the main CO2 stream before at the extraction cell.
The extraction parameters (pressure, temperature and co-solvent concentrations) were varied
between 15 to 33 MPa, 40 to 80 ° C, and 0 to 5% EtOH, respectively, according to the
experimental design shown in Table 1. The ethanol percentages were added to the CO2 flow
at a constant rate during the whole experiment. The CO2 to feed ratio used in all the
experiments was 275, while the ethanol to feed ratio was 5.5 for 2% EtOH and 13.7 for 5%
EtOH. After extraction, ethanol was removed under vacuum and the extracts were weighed

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using an analytical balance to estimate the extraction yields which were expressed as weight
percentage on a dry basis (% wt d.b.). All extracts were analyzed by gas chromatography

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with flame ionization detection (GC-FID) to quantify their THC content.

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2.4 Experimental design and statistical analysis
The effect of the SFE parameters (pressure, temperature and co-solvent concentration) on

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response variables (extraction yield and THC content in the raw extract) was determined

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using response surface methodology (RSM) and central composite design (CCD, with α =
1). The levels of independent variables were established based on previous studies on
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cannabinoids extraction [20,22,25]. The experimental design consisted of 19 experimental
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runs performed at random. The central point was repeated five times (extract numbers 15 to
19). The full experiment design is shown in Table 1.
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The experiment data were fitted to a second-order polynomial model which expressed the
response variables as a function of independent variables according to equation (1):
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Y 0 1 X1 2 X2 3 X3 11 X12 12 X1 X 2 13 X1 X 3 22 X 22 23 X 2 X3 33 X 32

(1)
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where Y represents the response variable, b0 is a constant, b1, b2 and b3 are the linear
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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) were used
to establish the correlation between the predicted and observed data. The values of the R2, R2
adjusted and AAD were used to check the accuracy of the model such that R2 and R2 adjusted
should be close to 100% and AAD must be as small as possible. The acceptable values of R2
and AAD mean that the model equation defines the true behavior of the system and it can be

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used for interpolation in the experimental domain [26]. The processing of the data was done
with the R-Project software version 3.4.2 (Free Software Foundation’s GNU project, Boston,

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Massachusetts, US).

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2.5 THC analysis and quantification
The THC analysis and quantification were carry out using a Shimadzu GC-2010

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chromatograph equipped with Shimadzu auto-sampler AOC-20i and an analytical capillary

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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
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microliter of cannabis extracts dissolved in ethanol (at 12.5 mg/mL) were injected on split
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mode at ratio 1:20. The oven conditions were started at 200 °C kept for 2 min, then increased
until 260 °C at 10 °C/min and maintained during 10 min [3]. The data were analyzed using
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the Lab Solutions lite software version 5.52 of Shimadzu Corporation. The THC
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quantification was carry out using a standard curve (10 to 500 mg/L) to which tetracosane
was added as an internal standard (100 mg/L) [27]. The results were expressed as weight
percentages of THC with respect to dry extract and as weight percentages of THC with
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respect to dry cannabis sample (% wt d.b.).

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2.6 Total THC content in the Cannabis sativa L. raw material

The THC present in the raw material was extracted using the Soxhlet method. The extractions
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were performed under reduced pressure to avoid the thermal degradation of the target
compound and the artefacts formation. The extractions were carried out in a six-hour period,
using a solvent polarity gradient - first hexane, then ethyl acetate, and finally ethanol. Briefly,
4.0 g of raw material was extracted with hexane, obtaining the non-polarity fraction.
Subsequently, the residual cake was extracted with ethyl acetate, producing the middle-
polarity fraction. Finally, the next residual cake was extracted with ethanol, thus getting the
polar fraction. All fractions were concentrated under vacuum and analyzed by GC-FID. The
total THC content in the raw material was given by the sum total of each fraction THC
content.

2.7 THC isolation and purification process.

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The SFE and GC-FID results were used to identify and select an extract with good extraction
yield, the highest THC content and lowest contamination (with the lowest number of

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compounds extracted). This extract was submitted to an isolation and purification process by
SPE to obtain high purity THC. The selected extract was analyzed by reverse phase high

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performance liquid chromatography (RP-HPLC), in addition to the GC-FID analysis. RP-
HPLC was also used as a follow-up of the SPE process to identify the fractions with the

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highest THC purity. Finally, one fraction with the highest THC content was selected and

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analyzed by nuclear magnetic resonance (NMR) to confirm the THC purity.
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2.7.1 THC isolation and purification by SPE
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The isolation and purification of THC were developed using a solid phase extraction column
packed with octadecyl-modified silica gel (5 g, 40-60 m) [28]. The extract (0.5 g) was
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dissolved in solvent A (trifluoroacetic acid 0.05% in water) and injected into the SPE column,
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then the compounds were eluted using a linear gradient of solvent B (trifluoroacetic acid
0.05% in acetonitrile) from 0% to 100% at a constant flow rate of 1 mL/min. Thirty-one
fractions of 12 mL each were obtained. All fractions were analyzed by thin layer
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chromatography and RP-HPLC to determine the presence of THC and to select the THC
enriched fractions. The high THC content fractions were joined – thus obtaining the final
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fraction – and the solvent removed under vacuum. The resulting free-solvent fraction was
lyophilized, and the global yield of the SFE-SPE process was determined. The final fraction
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was analyzed by RP-HPLC and NMR to assess the purity of THC thus obtained.

2.7.2 Analysis by RP-HPLC

RP-HPLC analysis was performed with an Agilent Series 1260 chromatograph, using a
monolithic Chromolith C18 column (50 x 4.6 mm, Merck, Darmstadt, Germany). The elution
was developed 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 were detected at 210 nm wavelength. THC was
identified using a standard compound based on its retention time.

2.7.3 NMR analysis

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A one-dimension NMR experiment for the final fraction was performed: proton (1H NMR)
and carbon-13 (13C NMR). A Bruker AVANCE 400 MHz NMR instrument equipped with

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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

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°C. Topspin 2.1 software was used for spectra acquisition and processing (Bruker
Corporation).

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3. Results and discussion N
The extraction results are shown in Table 1. The extract numbers correspond to each
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extraction carried out under the conditions specified. The results obtained for the extraction
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yield are expressed as a weight percentage of dry cannabis plant material, and the THC
content expressed as a weight percentage of THC in the dry extract and as weight percentages
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of THC in dry cannabis sample.

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3.1 Extraction yield

Table 1 shows that the highest extraction yield was 26.36%, corresponding to the extract
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number 6 obtained at 33 MPa, 80°C and 5% EtOH. At these conditions the supercritical
solvent has a good solvent power because of the high extraction pressure. Also, the presence
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of co-solvent improves the SC CO2 solvation power [22,29]. Even though higher
temperatures can reduce the SC CO2 density, at higher extraction pressures this effect lacks
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importance compared to the increases in the pressure vapor of solutes; hence high
temperatures can enhance the extraction yield [30,31]. Another extract with high yield was
number 4 (23.36%), obtained at 15 MPa, 40°C and 5% EtOH, using the lowest levels of
extraction pressure and temperature. Extract numbers 4 and 6 showed the highest extraction
yields; however these extracts were obtained using different levels of pressure and
temperature (low versus high levels). It is worth mentioning that a similar results were
previously reported by Da Porto et al. [32] for hemp cannabis oil extraction. These results
can be due to variation of solvent selectivity with the pressure and temperature changes. For
example, volatile compounds such as terpenes can be more efficiently extracted at high
temperatures, whereas cannabinoids extraction is facilitated at low temperatures. Previously,
Omar et al. [33] observed that for SFE with CO2/EtOH the extraction of sesquiterpenes from

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the cannabis plant was favoured at high temperatures, whereas cannabinoids extraction was
better at low temperatures. These results agree partially with the present study, high THC

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content were observed in the extracts obtained using low temperature levels (e.g. extract
numbers 1 and 4). On the other hand, SFE has previously been used on cannabis using neat

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SC CO2 to recover different cannabinoids, e.g. 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

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a maximum yield of 21.5% at 30 MPa and 40°C. These yields are lower compared with

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extract numbers 4 and 6, suggesting that the addition of ethanol as co-solvent improves the
yield.
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The resulting experiment data were analyzed by response surface methodology (RSM)
according to the parameters described in Section 2.4. Table 2 shows the ANOVA results used
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to evaluate the fit of the second order model on the extraction yield data. ANOVA shows that
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just one extraction parameter has a significant effect on extraction yields, the linear
coefficient of %EtOH (p-value = 0.027). An adjustment to the second order model is
introduced using the Akaike information criterion to obtain an equation that best describes
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the effect of the extraction parameters on the extraction yield. Since the only factor that
influence significantly the extraction yield is the co-solvent, the adjusted model has the linear
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and quadratic coefficient for the ethanol effect. Equation (2) shows the resulting second order
model equation for the extraction yield from the Cannabis sativa L. plant by SFE (with only
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significant coefficients):

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). The 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 shows that the ethanol addition as co-solvent in SFE is the principal factor which
significantly influences the extraction yield. This co-solvent effect was evident at 24 MPa
and 60ºC. At this pressure and temperature the extraction yield using neat SC CO2 was

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13.06% (extract number 14). However, the addition of ethanol at 2 and 5% increased yields,
reaching 16.77% (the average value of extract numbers 15 to 19) and 18.27% (extract number

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13), respectively. These results may be explained by an increase of higher polarity
compounds extracted from the vegetal matrix (e.g. phenols and sugars). This effect has

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previously been reported by Rovetto and Aieta [22], they observed that ethanol, used as a co-
solvent, improved the overall extraction yields.

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3.2 THC content N
THC content in raw extracts was determinate by GC-FID. Table 1 shows the experiment
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results. The highest THC content, 37.85% (extract number 9) was obtained at 33 MPa, 60°C
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and 2% EtOH. Other extracts with high THC content above 30% were numbers 1, 2, 4 and
11 (between 31.08 and 36.18%). In contrast, the lower THC content was 15.52% (extract
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number 6) obtained at 33 MPa, 80°C and 5% EtOH. These results suggest that co-solvent
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levels between 2 and 5% favour the extraction of THC from the cannabis plant. However, an
increase of ethanol, particularly to a higher concentration, can reduce THC recovery. This
effect is evident when comparing extract numbers 9 and 6 (considering no temperature
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effect). Previously, Omar et al. [33] and Rovetto and Aieta [22] reported that the addition of
ethanol as co-solvent in SFE enhanced the cannabinoids extraction efficiency, including
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THC. However, a larger co-solvent concentration could reduce the solvent selectivity [34].
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The Omar et al. [33] study explored the cannabinoids (CBD, CBN and THC) extraction from
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 Cannabis sativa L. strains by SFE with neat SC CO2
and CO2/EtOH. They used a novel extraction procedure based on multistage pressure
increments and pulses of co-solvent. Their results showed THC contents in extracts ranging
between 64.2 and 76.2%. In relation to the present work, THC contents in extracts observed
by Omar et al. are lower whereas those reported by Rovetto and Aieta are higher. It is worth
noting that the cannabis raw material used in the present work and the material used by Omar

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et al. and Rovetto and Aieta studies have different THC content. Therefore the THC content
in extracts can be very different despite using the same extraction method, solvent and similar

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conditions. To evaluate the efficiency of the extraction method for THC recovery from
vegetal material, the THC content in the raw material should be considered. The ratio

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between the THC content in the raw extracts and the total THC in the raw material
(%THCextract / %THCTotal) may be used to evaluate the THC recovery and extraction

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efficiency (see Table 1). In the present work, the total THC content in raw material was

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7.04% (stablished using the procedure described in the section 2.6), and the highest THC
contents in extracts were 37.85% and 36.18% (extract numbers 9 and 11, respectively), then
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the highest THC recoveries were 5.38 and 5.14. These THC recoveries were better compared
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with the Rovetto and Aieta study (76.2% THCextract / 16.6% THCTotal = 4.59), which suggests
a better extraction efficiency.
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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 are the linear
and quadratic coefficient of pressure and the linear coefficient of %EtOH. The second order
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model is therefore adjusted using the Akaike information criterion. Equation (3) represents
the resulting second order model equation adjusted for the THC content in raw extract from
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the Cannabis sativa L. plant. The R2, the R2 adjusted and the ADD (shown in Table 2)
indicate that the adjusted model is acceptable.
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THC content (%) = 72.2 + 3.46EtOH - 3.69P - 0.93EtOH2 + 0.070P2 Eq. (3)

Figure 2 shows the response surface graph of the second order model for the THC content.
This plot allows to visualize the effect of the 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 and 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.
Also, Figure 2 shows that the highest THC contents are obtained at low pressures (15 MPa).

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However, increases of this parameter from 15 to 24 MPa reduce the cannabinoid
concentration in extracts. The effect of pressure observed in the present work agrees with the

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results reported by Rovetto and Aieta [22]. They observed that at constant temperature the
increase in pressure from 17 to 24 MPa decreased the THC content in extracts from 76.23 to

SC
64.17%; however, the change from 24 to 34 MPa enhanced the THC content.

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The results shown in Figure 2 suggest that SFE with CO2/EtOH is an adequate extraction

N
technique for THC recovery from the Cannabis sativa L plant. The ethanol addition as co-
solvent is an important extraction parameter which improves both extraction yields and the
A
THC content in the extracts. The extraction pressure affects the THC content, whereas
M

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),
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however, their extraction pressure levels were different (33 MPa and 15 MPa, respectively).
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Finally, extract numbers 9 and 11 were considered for the isolation and purification of THC
using single step-SPE.
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3.3 THC isolation and purification

The results shown in the previous section indicate that the extract numbers 9 and 11 are
CC

candidates for further purification, these were analyzed by GC-FID and RP-HPLC. Figure 3
shows their respective chromatographic profiles which suggest that the THC in the extract
A

number 11 is purer than in the extract number 9, hence extract number 11 was submitted for
further purification. It is of note that the extract number 11 was obtained using low pressure;
a very important parameter with significant technical implications should the extraction
process is scaled up.
50 mg of the extract number 11 was injected into the solid phase extraction column (ODS, 5
g, 40-60 m) and its components were eluted using a linear water-acetonitrile gradient.
Thirty-one fractions were collected, and each one was analyzed by TLC and RP-HPLC to
detect the presence of THC and purity. The chromatographic analysis allowed the
identification of the highest THC content fractions, i.e. two fractions (number 26 and 27)
were selected for further characterization. The resulting chromatographic profiles of fraction

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numbers 26 and 27 are shown in Figure 4. The fractions selected were combined, and the
organic solvent removed under vacuum, and the water removed by lyophilization. The dried

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final fraction (FF) thus obtained weighed 12.7 mg. and was subjected to NMR analysis to
verify its purity with respect to the THC content.

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The FF was analyzed by NMR: 1H-NMR and 13C-NMR experiments. The 1H-NMR spectra

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of extract number 11 and FF are shown in Figures 5a and 5b, respectively. Table 3 presents

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the NMR signals observed for FF and the THC signals reported in the literature. The 1H-
NMR signals observed for FF are very similar to those reported for THC by Peschel and
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Politi [35]. It is worth noting that the maximum differences observed for the δ values were
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0.02 ppm. Some characteristic 1H-NMR signals for THC were observed in FF spectra (please
see Figure 6): the H-9 and H-10 protons of angular methyl groups at 0.98 ppm and 1.33 ppm,
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respectively; the H-6 proton at 1.62 ppm; the olefinic H-2 proton at 6.37 ppm; the aromatic
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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 provides a distinguishable signal at 9.21 ppm. The ∆-8-
THC and THCA are cannabinoids with similar structures to THC, the analysis of 1H-NMR
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signals observed for FF permitted the elimination of these compounds in the FF. The carboxyl
group on C3’ in THCA changes the stereochemistry of H-1’’ protons, therefore its δ values
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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,
A

the 13C-NMR signals observed for FF were comparable to those assigned previously for THC
by Young et al. [36].

The comparison of the NMR spectra makes it possible to confirm that the extract number 11
consisted mainly of THC. Other cannabinoids such as CBD or CBN could potentially have
been present in the extracts, thus probably reducing the 1H-NMR spectra signals resolution
(e.g., between 0.25 and 2.25 ppm), due to the overlapping of similar NMR signals. The
analysis shown in Figures 5a and 5b indicates that SFE with CO2/EtOH provided a highly
THC enriched extract, which followed the single-SPE step purification of other
cannabinoids. The RP-HPLC and NMR results show that FF corresponds to high purity THC.
Finally, the THC concentration was quantified by GC-FID and 90.1% of purity was observed.

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In general, the results show that SFE coupled with SPE allow to obtain high purity THC from

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the Cannabis sativa L. plant. A global balance of extraction, isolation and purification
process suggests that from 100 g of cannabis material used in this work it is possible to

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recover 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

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a starting cannabis material with a high THC content could increase the THC yield.

Conclusions
N
A
A sequential SFE-SPE process was explored to obtain THC with high purity from Cannabis
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Sativa L plant 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
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response variables, while pressure influenced the THC content. A better extraction yield was
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obtained at higher levels of pressure, temperature and ethanol percentage; however, these
extraction conditions provided a lower THC content. The highest THC recoveries were
achieved using low ethanol percentages (2%), and an additional increase in pressure from 15
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to 33 MPa improved THC extraction, but this reduced the extraction selectivity. SFE
provided two extracts with the highest THC content, numbers 9 and 11 with 37.85 and
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36.18% of THC, respectively. Extract number 11 was selected for the purification-isolation
step due to less complexity. One single SPE step on extract number 11 allowed the extraction
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of THC at 90.1% purity, which was checked by chromatographic and spectroscopic analyses.
On the basis of our results we conclude that from 100 g of Cannabis Sativa L plan material
subjected to a sequential SFE-SPE process it is possible to obtain 13.97 g of THC enriched
extract and 3.55 g of high purity THC. This is a promising candidate for an “in-house
standard”, or to continue the purification until a reference standard is obtained. We have
shown the usefulness of combining SFE with SPE to obtain a raw clean extract. The
foregoing allows us to have possibly a standard material type in-house THC, obtained at low
cost and with fully affordable resources.

Acknowledgements
The authors acknowledge the Universidad Nacional de Colombia (División de Investigación,

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Sede Bogotá) for their financial support and the Fiscalía General de la Nación for their
valuable support in carrying out this work.

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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.

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A
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Fig. 1.
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Fig. 2. Surface response plot for the effect of pressure and co-solvent percentage on THC
content in Cannabis Sativa L. extracts (%THC in dry extract).
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A
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Fig. 2.
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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.
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CC
A
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Fig. 4. RP-HPLC profiles for fractions 26 (a) and 27 (b). The signal at 12.7 corresponds to
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THC.
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2400 VWD: Signal A, 210 nm

Extracto THC run 1 F15
2200 Retention Time
(a)
2000
12.756
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1800
1600
1400
mAU

1200
1000
EP

800
600
400
12.376

200
CC

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Minutes
A
 2400 VWD: Signal A, 210 nm
Extracto THC run 1 F16
2200 Retention Time
(b)
2000
1800
1600

12.716
1400
mAU

1200
1000
800
600
400

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200
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Minutes

Fig. 4.

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Fig. 5. 1H NMR (400 MHz in DMSO-d6) spectra for (a) the extract number 11 and (b) the

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final fraction.

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(a)

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A
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(b)
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CC
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Fig. 5.
Fig. 6. Tetrahydrocannabinol (THC) structure. The numbering presented is based
monoterpenenoid nomenclature.

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Fig. 6.

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A
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Table

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Table 1. Extraction yields and THC contents of SFE extracts from Cannabis sativa L. plant

N
Extraction parameters THC contents
Extraction yields
THC Recovery b

A
Extract Pressure Temperature Co-solvent (% wt d.b.a) %THC in dry %THC in dry
(MPa) (ºC) (% wt) extract cannabis sample

M
1 15 40 0 4.83 32.25 1.56 4.58
2 15 80 0 6.32 31.08 1.96 4.41
3 15 80 5 21.17 24.88 5.27 3.53
4 15 40 5 23.36 30.14 7.04 4.28
ED
5 33 80 0 10.41 24.73 2.57 3.51
6 33 80 5 26.36 15.52 4.09 2.20
7 33 40 5 21.49 20.34 4.37 2.89
8 33 40 0 6.32 25.81 1.63 3.67
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9 33 60 2 16.01 37.85 6.06 5.38

10 24 80 2 17.43 29.54 5.15 4.20
11 15 60 2 13.97 36.18 5.05 5.14
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12 24 40 2 15.57 25.10 3.91 3.57

13 24 60 5 18.27 22.04 4.03 3.13
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14 24 60 0 13.06 28.38 3.71 4.03

15 24 60 2 16.39 24.09 3.95 3.42
16 24 60 2 17.18 26.15 4.49 3.71
17 24 60 2 16.13 23.84 3.85 3.39
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18 24 60 2 18.61 24.53 4.57 3.48

19 24 60 2 15.48 28.28 4.38 4.02
a
d.b. = dry basis
b
THC Recovery values were calculated as the ratio between the THC content in the raw extracts and the total THC in raw material (%THCextract / %THCTotal). The
total THC observed in cannabis sample used was 7.04%, this was extracted followed the procedure described in section 2.6
 PT
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Table 2. Analysis of variance and statistical indicators of second order model for extraction yield and THC content

Extraction yield THC percent in raw extract

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Factor
Estimate p value Adjusted p value Estimate p value Adjusted p value

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Intercept (b0) 4.3923 0.780 8.19 0.0001 *** 51.9855 0.059 * 72.23 0.001 ***
b1 EtOH 5.1500 0.027 ** 4.91 0.0002 *** 4.9502 0.142 3.46 0.081 **

A
b2 MPa 0.4915 0.620 -4.2478 0.020 ** -3.69 0.010 **

M
b3 T -0.1716 0.726 0.8921 0.263
b12 EtOH*MPa -0.0123 0.762 -0.0436 0.498
b13 EtOH*T -0.0071 0.697 -0.0224 0.442
ED
b23 MPa*T 0.0067 0.207 0.0004 0.963
2
b11 EtOH -0.3280 0.230 -0.42 0.0405 ** -0.7464 0.095 * -0.93 0.018 **
2
b22 MPa -0.0155 0.431 0.0832 0.020 ** 0.07 0.016 **
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2
b33 T 0.0007 0.867 -0.0074 0.248
p value 0.01 <0.0001 0.085 0.005
2
R 90.4 83.6 72.3 62.8
E

2
R adjusted 80.7 81.5 44.5 52.1
ADD 9.5 9.1 8.5 7.9
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* = significant
** = highly significant
*** = Very highly significant
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Table 3. 1H-NMR and 13C-NMR spectroscopy data for final fraction and THC

1 13
H-NMR (δ ppm, J Hz) C-NMR (δ ppm)
Position
Final fraction THC Final fraction THC
(400 MHz) (400 MHz)a (100 MHz) (100 MHz)b
1 3.07 (1H, dm) 3.08 33.81 33.6
2 6.37(1H, m) 6.37 125.34 123.7
3 132.45 134.3
4 2.08 (2H, m) 2.08 30.70 31.2
5a 1.25 (1H, m) 1.30

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27.86 25.0
5b 1.86 (1H, m) 1.85
6 1.62 (1H, sw) 1.60 45.90 45.8
7 1.62 (3H, sw) 1.60 23.63 23.4

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8 76.92 76.7
9 0.98 (3H, s) 0.98 24.95 19.3
10 1.33 (3H, s) 1.31 24.95 27.6

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1´ 108.39 110.8
2´ 154.61 154.7
3´ 6.01 (1H, m, J =1.40) 6.01 109.00 107.5
4´ 141.83 142.8

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5´ 6.15 (1H, m, J =1.40) 6.15 107.41 110.1
6´ 154.81 154.4
1´´ 2.35 (2H, m) 2.34 35.54 35.5
2´´
3´´
1.49 (2H, m)
1.25-1.30 (2H, m)
1.48
1.25
N 31.20
31.36
30.6
31.5
A
4´´ 1.25-1.30 (2H, m) 1.25 22.44 22.5
5´´ 0.86 (3H, t) 0.85 14.38 14.0
M

OH 9.24 (1H, s) 9.20

δ (ppm): Chemical shift

J (Hz): Nuclear spin-spin coupling constant
ED

a
Reported data obtained using 400 MHz NMR equipment [35]
b
Reported data obtained using 100 MHz NMR equipment [36]
E PT
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A