Extraction, separation, and purification of
cannabinoids from the Cannabis sativa L. plant
using dry ice and solvents such as hexane and
ethanol to determine the most efficient method
2023 IEEE Seventh Ecuador Technical Chapters Meeting (ECTM) | 979-8-3503-3823-2/23/$31.00 ©2023 IEEE | DOI: 10.1109/ETCM58927.2023.10308993
Lisseth Minchala Espinoza Nicole Alba Naranjo
Universidad Politécnica Salesiana Universidad Politécnica Salesiana
Biotechnology Engineering (GIVABI) Biotechnology Engineering (GIVABI)
Cuenca, Ecuador Cuenca, Ecuador
lminchalae@ieee.org malban1@est.ups.edu.ec
Abstract — This study analyzes the extraction of oil from
Cannabis sativa L, which has a sticky and viscous appearance.
At present it has become popular for its medical potential as an
alternative to the prevention and control of many diseases, a
possible cure for some types of cancer or for the treatment of
neurological diseases. Its preparation using dry ice at -78°C,
96% ethanol and hexane as solvents, following a process of
preheating, decarboxylation, filtration and distillation, have
been an option for the concentrated extraction of cannabinoids.
The time, temperature, amount of raw material used (Black
Dominata) and solvents were mainly studied, in such a way that
the most suitable conditions for the active principles can be
determined and thus propose a method of preparation of
cannabis oil and optimize the results. extraction processes. In
order to obtain the results of this work, an analysis was made
through GC/MS (gas chromatography coupled with mass
spectrometry), where an average of 26.13% CBD (cannabidiol)
was determined; 10.4% yield using 96% ethanol and 12.11%
yield with hexane, and total cannabinoid concentrations in the
range of 75.93%-78.09% between both methods. Unlike other
studies, such as the case of Marchena Pinilla [1], who obtained
<0.0050 cannabinoids in their samples; Ojeda [2] with 7.42%
and the study by Lui et al. [3] talks about the fact that there were
no efficient results. Thus, concluding that the extraction method,
using hexane and ethanol together with dry ice as solvents, is
efficient compared to other studies carried out.
Keywords — Cannabis, oil, extraction, purification, solvents,
CG/MS chromatography
I. INTRODUCTION
Currently the cannabis industry has grown excessively
worldwide in conjunction with productive and technological
advances; being consolidated as a largely profitable,
attractive, sustainable industry allowing the growth of
innovative lines of research, not only scientific but for the
development of products with beneficial properties focused on
health and medicine [4].
The complexity of the products derived from Cannabis
sativa L., has generated a great problem to isolate its bioactive
principles. From this perspective, the search for new methods
of extraction, isolation, purification and quantification that
enhance the final extraction yield for the production of
medicinal cannabis oil [1] has given rise. Currently, in
Ecuador, there are no works specifically related to the
extraction of cannabis compounds since its legalization began
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Myriam Mancheno Cardenas
Universidad Politécnica Salesiana
Biotechnology Engineering (GIVABI)
Cuenca, Ecuador
mmancheno@ups.edu.ec
in 2019. However, it should be noted that innovative research
on the extraction process has been generated internationally.
Therefore, the purpose of this research is to obtain
cannabis oil by means of two different extraction methods, to
determine the adequate yields, considering primarily the
utility of Cannabis sativa L. subsp indica (Black Domina).
In the methodology carried out, the immersion processes
of cannabis in solvent (Hexane and Ethanol 96%) and a
winterization process using dry ice at -78°C were
incorporated.
The results obtained will allow us to contribute innovative
research to obtain cannabis oil, considering the incorporation
of new technologies and methodologies where cannabis oil is
obtained with a high extraction and purification yield.
A. Investigation
In previous investigations, authors have used different
techniques and methods for the extraction of cannabis oil in
order to obtain a product with its active ingredients. As is the
case of Marchena Pinilla [1], who in her research
"Quantitative Determination of CBD present in the cannabis
plant in the Hard Diesel (sativa) and Black Domina (indica)
species from chemical extraction with ethanol" quantitatively
analyzed CBD (cannabidiol), present in the two varieties of
cannabis he studied, using 96% ethyl alcohol. Finally, to
quantify the amount of the active ingredient present, he used
liquid chromatography (HPLC) and estimated a value of
<0.0050 of CBD present in the two varieties.
Murillo & Ojeda [2] in their study "Determination of the
most effective extraction method in obtaining extracts rich in
cannabinoids from three different processes", focused on
implementing three extraction methods where they used
ultrasound/basic solvent, extraction by steam stripping and at
the end used the extraction method by means of dry ice.
Having the following results: 7.42% CBD was obtained by the
steam stripping method and analyzed by final layer
chromatography (TLC), unlike the other methods that did not
show the presence of CBD.
Lui et al. [3] in his article "Bioactive cannabis compounds
and their extraction, separation, purification and identification
technologies: an updated review", he talks about the fact that
the technologies to be able to separate, purify and identify the
 presence of cannabinoids continue to present a problem, since
they need a lot of time and most of them are not ecological.
II. MATERIALS AND METHODS
This section emphasizes the extraction methods and analysis
of the presence of cannabinoids in the samples taken.
A. Phase 0
Subsp indica plant of the Black Domina variety was used
as raw material, where it is previously dry for its use,
prioritizing the use of the buds (flowers), since this part of the
plant contains ten times higher cannabinoids compared to
other parts of it [5].
E. Phase 4: obtaining cannabis sativa L. oil.
The solvent and oil were separated by means of a rotary
evaporator. Through the vacuum distillation process, this
allowed the solvent to evaporate and recover the sample of
interest. It must be considered that cannabinoids do not
denature if it does not pass temperatures above 60°C, that
being said, the time and temperature conditions that were
worked on were: for ethanol 96% at 60°C/90min and for
hexane at 45°C/90min, knowing that its boiling points are
78.37 °C and 69°C respectively.
To estimate the amount of recovered solvent and its yield
(1), the following formula was used [9]:

B. Phase 1: Required pretreatments % = × 100 (1)

I. Shredding of plant material
The parts of the dry plant are broken up, in such a way that
To calculate the yield of the extract (2), the following
the contact area with the solvent can be maximized. It is formula was used [5]:
important to avoid the incorporation of other parts of the plant
such as stems, trunks or other unwanted residues [6]. . . . . .
= = × 100% (2)
. . . .
II. Decarboxylation
F. Phase 5: quantification of cannabinoids
This process refers to activating the active principles of
Cannabis by eliminating the CBDA and THCA carboxyl They were carried out to determine the main cannabinoid
group, which is in its psychoactive form; at a temperature of compounds (D9-THC, D8-THC, CBD and CBN). For the
115°C for 45 minutes, making it its “neutral” CBD and THC analysis, 4 samples of all tests were taken as reference for
analogue [7]. evaluation. Their respective analyses were carried out in the
laboratories of the company HEMP ECUADOR LABS,
C. Phase 2: extraction methods where gas chromatography equipment coupled to mass
Two extraction processes were carried out, differing in the spectrometry was used. As shown in Table 2 the GC/MS
type of solvent used. In the first, ethanol was used with a conditions.
concentration of 96% and in the second, hexane was used. The
TABLE II. CONDITIONS FOR GC/MS
amounts used for each sample varied as indicated in the Table
1: Column 30 m length x 0.25 mm internal diameter x
0.25 µm film thickness.
TABLE I. AMOUNTS USED FOR THE EXTRACTION OF
CANNABIS OIL Split 25:1
Raw Ethanol Hexane Dry ice at injection volume 1.0µL _
material 96% (mL) (mL) -78°C
(g) (kg) injector temperature 250°C
100 1500 1500 2
detector temperature 290°C
110 1650 1650 2
drag gas Helium
120 1800 1800 2
flow per column 1.0 mL/min at constant Flow
130 1950 1950 2
Note: Conditions for GC/MS applied in research [10].
III. RESULTS
When ethanol is used, it is allowed to settle for 24 hours and
for hexane 20 hours, always keeping them at -78°C until their For the final obtaining of cannabis oil, an exhaustive
time is up [8]. review of different bibliographic sources has been made,
including articles, documents, magazines, books, among
D. Phase 3: Filtration others. Where the processes of drying, decarboxylation,
The first filtration used a 25 µm filter to remove larger filtration and distillation by means of rotary steam, they were
plant debris. After this, a winterization is carried out using dry carefully analyzed, thus proposing temperatures, times,
ice again in a time of 20-30 minutes at -78 ° C, this process materials and adequate methods for a profitable extraction.
helps the precipitation of chlorophyll and undesirable A. decarboxylation process
terpenes. After this, a second filtration is done with a 10-13
µm filter together with granulated carbon, to establish a more As stated in the methodology, the plant material must be
efficient purification process. In this way it was possible to in a dry version, since the plant generally contains around 80%
obtain a product with a lighter color and free of impurities. water. In the decarboxylation process the following results
proposed in the Table 3 were obtained.

TABLE III. CALCULATIONS OF DECARBOXYLATION OF

PLANT MATERIAL

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 Number Dry weight Decarboxylated Determination of recovered hexane:
of trials (g) weight (g) loss (g)
In Table 6 can see than in the first extraction phase, when
1 100 88.96 11.04 the cannabis sample is immersed using hexane, an average of
75.68% of the solvent was recovered, the same as 1300 mL in
2 110 99.60 10.40
relation to each test.
3 120 107.02 12.98
TABLE VI. SOLVENT RECOVERED CALCULATIONS IN %: FIRST
4 130 114.45 15.55 EXTRACTION PHASE WITH HEXANE
Sample trial Initial Amount of Percentage
5 100 90.12 9.88 number amount of recovered of solvent
solvent solvent recovered
6 110 97.55 12.45 (mL) (mL) (%)
Black 1 1500 1100 73.33
7 120 104.75 15.25 Domina
(buds) 2 1650 1500 90.91

8 130 113.16 16.84 3 1800 1200 66.67

Total 920 815.61 104.39 4 1950 1400 71.79

Average - - 13.05 Average - 1725 1300 75.68

B. Percentage of ethanol 96% and hexane recovered In Table 7, in the second extraction phase, where the rotary
Determination of 96% recovered ethanol: evaporator equipment was used, 86.43% of the solvent was
In the first extraction phase, when the immersion of the recovered, the same as 1300.75 mL in relation to each test.
cannabis sample is performed with 96% ethanol, an average
TABLE VII. SOLVENT RECOVERED CALCULATIONS IN %: SECOND
of 76.71% of the solvent was recovered, which is equivalent PHASE EXTRACTION WITH HEXANE
to 1325 mL in relation to each test. these results are observed
Sample trial Initial Amount of Percentage
in Table 4: number amount of recovered of solvent
solvent solvent recovered
TABLE IV. CALCULATIONS OF SOLVENT RECOVERED IN %: FIRST (mL) (mL) (%)
PHASE OF EXTRACTION WITH 96% ETHANOL Black 1 1100 1000 90.91
Domina
Sample trial Initial Amount of Percentage (buds) 2 1500 1290 86
number amount of recovered of solvent
solvent solvent recovered 3 1200 1030 85.83
(mL) (mL) (%)
Black 1 1500 1100 73.33 4 1400 1157 82.64
Domina
(buds) 2 1650 1300 78.79
Average - 1300 1119.25 86.35
3 1800 1400 77.78

4 1950 1500 76.92

C. Extraction yield
Average - 1725 1325 76.71 The percentage yields of each sample extracted from
cannabis oil were respectively analyzed by means of four
replicates, as can be seen below in Table 8:
In the second extraction phase, where the rotary
TABLE VIII. YIELD PERCENTAGES OBTAINED IN THE TWO EXTRACTION
evaporator equipment was used, 86.43% of the solvent was PROCESSES
recovered, the same as 1138.75 mL in relation to each test.
Results in Table 5: repetitions Extraction from dry ice Extraction from dry ice
with hexane with 96% ethanol
TABLE V. CALCULATIONS OF SOLVENT RECOVERED IN %: SECOND
PHASE OF EXTRACTION WITH 96% ETHANOL 1 7.71% 11.35%

Sample trial Initial Amount of Percentage 2 11.56% 13.07%

number amount of recovered of solvent
solvent solvent recovered 3 10.79% 11.95%
(mL) (mL) (%)
Black 1 1100 1050 95.45 4 11.61% 12.05%
Domina
(buds) 2 1300 1130 86.92

3 1400 1050 75 The extraction method where dry ice and 96% ethanol
were used was the one with the highest yield percentage, an
4 1500 1325 88.33 average of 13.07%; while in the extraction method where
hexane was used as solvent, a lower percentage of yield was
Average - 1325 1138.75 86.43
obtained, which was 11.61%.

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 D. Cannabis oil characterization
Within the qualitative analysis of cannabinoids, it was
developed and verified by GC/MS chromatography in Percentage of extracted cannabinoids
agreement with the HEMP Ecuador Labs Laboratory, located
in the city of Quito. Through it, 4 types of cannabinoids that 50.00%
were present in the extracted oil samples were identified, as 40.00%
evidenced in Table 9. Thus, highlighting the presence of CBD, 30.00%
CBN, 9 THC and 8THC. Each of them specifically distributed 20.00%
in specific amounts in each sample obtained. 10.00%
0.00%
TABLE IX. CANNABINOIDS PRESENT IN THE SAMPLES CBD CBN Δ9- THC Δ8- THC
cannabinoids Extraction from Extraction from
dry ice using 96% dry ice using 96% Ethanol Hexane
Ethanol Hexane
CBD (Cannabidiol) ✓ ✓ Fig. 1. Percentage of extracted cannabinoids.

CBN (Cannabinol) ✓ ✓
Δ9-THC (Δ9 ✓ ✓
Tetrahydrocannabinol) IV. DISCUSSION
Δ8-THC (Δ8 ✓ ✓
Tetrahydrocannabinol)
Nowadays, the cannabis industry has grown excessively
worldwide in conjunction with productive and technological
On the other hand, in the quantitative analysis through the advances, being consolidated as a largely profitable,
extraction process from dry ice using solvents (96% ethanol attractive, sustainable industry, allowing the growth of
and hexane), the relevant presence of cannabinoids was innovative lines of research, not only scientific but also also
determined. In this way, in contrast to the research by in different areas such as biotechnology for the development
Marchena Pinilla [1], a comparative analysis could be of novel products with beneficial properties focused on health
established in terms of the percentage amounts of and medicine [4].
cannabinoids applying the same extraction method.
According to studies and scientific reports analyzed in
In the research by Marchena Pinilla [1], a percentage recent years, it emphasizes that the active substances that are
<0.0050% and 0.1055% of cannabinoids was determined, in present in cannabis such as cannabidiol [CBD],
contrast to the methods applied in our research, it was higher tetrahydrocannabinol [THC] and cannabinol [CBN] have
with a percentage between 75.9-78.1% of each method. An
potential therapeutic properties acting on CB1 receptors and
important point to consider within the application of these
methods are factors such as temperature, solvent, time, and CB2 thus modulating nervous and physiological functions
amount of plant material used, thus reflecting its influence [11].
during and after the process of obtaining cannabis oil. From this perspective, within the research, the search for
Fig. 1 and the Table 10 show the representative new methods of extraction, isolation, purification, and
percentages of the chromatographic analysis of cannabinoids quantification of its bioactive principles was raised, and with
within each of the established trials, considering the most this, enhance the final extraction yield to produce medical
important within our research to be 8THC, 9THC, CBD and cannabis oil. According to the results obtained, emphasis is
CBN. placed on the application of new techniques for oil extraction,
Cannabinoids Extraction Extraction Thesis of considering its application in new biotechnological processes
from dry ice from dry (Marchena [12].
using 96% ice using Pinilla) [1]
Ethanol Hexane It is important to consider that the method to be carried
CBD 24.18% 28.08% <0.0050% out must be safe and efficient in terms of optimization times
CBN 3.38% 2.61% <0.0050% to maximize the extraction performance [13]. According to
previous studies, the use of extraction methods from solvents
Δ9-THC 47.52% 43.14% 0.1055%
and supercritical CO2 are effective [14]. At the same time,
Δ8-THC 3.04% 2.11% <0.0050%
cannabis crops have a positive impact on the environment,
since there is a high presence of CO2 in the atmosphere and
cannabis absorbs between 20-30 tons of this gas, thus
TABLE X. OVERALL PERCENTAGE OF EXTRACTION SAMPLES
avoiding the greenhouse effect. However, their purification
processes must be taken into account to ensure the
traceability of the samples [15].
The solvents used in this research are organic and non-
toxic, even so, for the person who will handle this material,
complying with the established preventive norms in this
regard. Once the final product has been obtained, it must be
analyzed that all the solvent content has been efficiently
extracted from the cannabis oil samples [16].

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 V. CONCLUSION
According to the study carried out regarding the
extraction methods of essential oil of cannabis sativa L.,
which have been discussed; It is concluded that: the results
when 96% ethanol was used together with dry ice, a yield of
12.10% was obtained in contrast to hexane, which was
10.40%. Thus, emphasizing that the extraction process with
96% ethanol was much more efficient.
However, when performing the gas chromatography
analysis coupled with mass spectrometry, it indicated that
when hexane was used as a solvent, it has a greater amount
of cannabinoids, with 86.96% being the highest percentage.
In this way, it is important to consider the parameters of
temperature, time, type and amount of solvent used, since
they influence when it comes to obtaining positive results in
each oil sample extracted. Therefore, it is important to carry
out research to optimize the results. factors mentioned above,
thus advantageous result to recover bioactive principles of
cannabis.
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