U.S. patent application number 16/552773 was filed with the patent office on 2019-12-19 for cannabis extracts.
The applicant listed for this patent is Canopy Holdings, LLC. Invention is credited to Ryan Beigie, Jacob Black, Alex Mateo.
Application Number | 20190382325 16/552773 |
Document ID | / |
Family ID | 65811711 |
Filed Date | 2019-12-19 |
View All Diagrams
United States Patent
Application |
20190382325 |
Kind Code |
A1 |
Black; Jacob ; et
al. |
December 19, 2019 |
CANNABIS EXTRACTS
Abstract
A method is provided for removing THC from raw cannabis oil.
Additionally, new compositions of cannabis oil are provided.
Further, a new method of obtaining a substantially pure cannabinoid
is provided
Inventors: |
Black; Jacob; (New Haven,
CT) ; Beigie; Ryan; (Littleton, CO) ; Mateo;
Alex; (Lakewood, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Canopy Holdings, LLC |
Littleton |
CO |
US |
|
|
Family ID: |
65811711 |
Appl. No.: |
16/552773 |
Filed: |
August 27, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16266326 |
Feb 4, 2019 |
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16552773 |
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15834826 |
Dec 7, 2017 |
10239808 |
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16266326 |
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62431209 |
Dec 7, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01D 15/322 20130101;
C07C 39/23 20130101; C07C 37/004 20130101; G01N 30/90 20130101;
G01N 30/88 20130101; C07C 37/72 20130101; C07C 37/004 20130101;
C07D 311/78 20130101; G01N 2030/027 20130101; B01D 15/325 20130101;
A61K 2236/00 20130101; A61K 31/352 20130101; G01N 30/02 20130101;
A61K 36/185 20130101; C07C 2601/16 20170501; C07C 39/23 20130101;
C07B 63/00 20130101 |
International
Class: |
C07C 37/72 20060101
C07C037/72; C07D 311/78 20060101 C07D311/78; C07C 39/23 20060101
C07C039/23; C07B 63/00 20060101 C07B063/00; B01D 15/32 20060101
B01D015/32; G01N 30/02 20060101 G01N030/02; C07C 37/00 20060101
C07C037/00; A61K 36/185 20060101 A61K036/185; A61K 31/352 20060101
A61K031/352 |
Claims
1. A method of removing one or more cannabis compounds from a
cannabis oil, the method comprising: obtaining a column packed with
a stationary phase particulate; adding cannabis oil to the packed
column; adding a first eluent to the packed column; adding a second
eluent to the packed column; collecting at least two eluate
fractions comprising one or more compounds; and evaporating solvent
from a subset of the at least two fractions to form a
composition.
2. The method of claim 1, further comprising adding a third eluent
to the packed column.
3. The method of claim 1, wherein the stationary phase particulate
is selected from normal phase or reverse phase stationary
phase.
4. The method of claim 3, wherein the normal phase is silica gel
stationary phase particulate.
5. The method of claim 3, wherein the reverse phase is selected
from the group consisting of C18, C8, C4 and phenyl stationary
phase particulate.
6. The method of claim 4, wherein the first eluent and second
eluent are each solvents selected from one of, or a specific
mixture of two or more of, the group consisting of petroleum ether,
pentane, n-hexane, hexanes, n-heptane, heptanes, diethyl ether,
methyl tert butyl ether, ethyl acetate, and ethanol.
7. The method of claim 6, wherein the first eluent and the second
eluent are each solvents selected from one of, or a specific
mixture of two or more of, the group consisting of petroleum ether,
n-hexane, hexanes, n-heptane, heptanes, diethyl ether, and methyl
tert butyl ether.
8. The method of claim 6, wherein the first eluent and the second
eluent are each a mixture of diethyl ether and petroleum ether.
9. The method of claim 6, wherein the first eluent and the second
eluent are each a mixture of methyl tert butyl ether and petroleum
ether.
10. The method of claim 6, wherein the first eluent and the second
eluent are each a mixture of diethyl ether and n-heptane or a
heptane.
11. The method of claim 6, wherein the first eluent and the second
eluent are each a mixture of methyl tert butyl ether and n-heptane
or a heptane.
12. The method of claim 4, wherein the first eluent added totals
between one and eleven column volumes (CVs), 2 to 7 CVs, or 4 to 6
CVs.
13. The method of claim 4, wherein the second eluent added totals
between one and eleven column volumes (CVs), 2 to 7 CVs, or 4 to 6
CVs.
14. The method of claim 1, the method further comprises pooling the
eluate from 6-7.5 CVs, evaporating the pooled eluate, and
recrystallizing the composition to provide a purified composition
comprising cannabidiol (CBD) in greater than 94% purity, having not
more than 0.3% THC.
15. The method of claim 1, wherein the composition comprises 60-70%
CBD, not more than 0.3% THC, 0.3-5% CBC, and about 10-20% fatty
acids and terpenes.
16. A method for processing raw cannabis oil to provide clean
cannabis oil having less than 0.3 wt % delta9-THC, the method
comprising: obtaining raw cannabis oil; applying the raw cannabis
oil to a normal stationary phase column; eluting the normal
stationary phase column with a binary solvent system wherein the
binary solvent system comprises a first solvent A and a second
solvent B; fractionating the eluate into at least two eluate
fractions; and evaporating solvent from a subset of the at least
two eluate fractions to provide the clean cannabis oil comprising
less than 0.3 wt % THC.
17. The method of claim 16, wherein the first solvent A in the
binary solvent system is a non-polar solvent selected from one or
more of the group consisting of pentane, petroleum ether, hexanes,
n-hexane, n-heptane, heptanes, diisopropyl ether, toluene,
chloroform, and methylene chloride.
18. The method of claim 16, wherein the second solvent B in the
binary solvent system is a polar solvent selected from one or more
of the group consisting of diethyl ether, methyl tert butyl ether,
tetrahydrofuran, ethyl acetate, acetone, acetonitrile, isopropanol,
ethanol, and methanol.
19. The method of claim 16, wherein the column is eluted with a
step gradient using a binary solvent system comprising the first
solvent A and the second solvent B, wherein solvent A is petroleum
ether, a heptane, or n-heptane and solvent B is diethyl ether or
methyl tert butyl ether.
20. A composition comprising about 65-70% CBD, not more than 0.3%
THC, 0.3-5% CBC, 0.3-5% CBG, 0.3-5% CBN, and about 10-20% fatty
acids and terpenes.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application and claims
priority to U.S. patent application Ser. No. 16/266,326, filed Feb.
4, 2019, which is a continuation application of U.S. patent
application Ser. No. 15/834,826, filed Dec. 7, 2017, now issued as
U.S. Pat. No. 10,239,808, which claims priority to U.S. Provisional
Application No. 62/431,209, filed Dec. 7, 2016, which are
incorporated by reference herein in their entirety.
BACKGROUND
[0002] Cannabis plant material contains a variety of potentially
valuable compounds. For example tetrahydrocannabinol (THC),
cannabidiol (CBD), cannabichromene (CBC), cannabigerol (CBG),
cannabinol (CBN), and other compounds are present in varying
amounts in cannabis and hemp plant material.
[0003] As defined by Congress, in order for a cannabis plant to
qualify as industrial hemp, the THC content must be below 0.3% of
overall mass. However, current extraction processes typically
increase THC content of industrial hemp based oil to an average of
3.5% of overall mass. This product is now legally defined by
Congress as "marihuana" and is no longer regulatory compliant. The
most widely employed solution to this problem is the dilution of
product through a myriad of unknown additives, which not only
decreases the THC content, but in decrease turn all medical
cannabinoids by 10 fold. Alternatively CBD isolates are a popular
solution, though these concentrates inevitably render the terpenes,
fatty acids, and full spectrum cannabinoid profile unusable. In
another attempt to solve the problem, fractional distillation is
being explored; however, distillation would be very difficult to
make as a viable solution on a commercial scale, due to costs
associated with industrial refineries and the similar boiling
points of cannabinoids, the need to still add dilutant and the
excessive product loss associated with the process.
[0004] Thus, efficient methods are desirable for processing
cannabis oil to provide a clean cannabis oil comprising not more
than 0.3 wt % THC compared to overall mass, while otherwise
maintaining cannabinoid profile, including other cannabinoids,
terpenes and fatty acids.
[0005] Raw cannabis oil containing these and other compounds may be
extracted from the cannabis flower/plant using techniques such as
CO.sub.2 extraction or liquid-solid solvent extraction. The
concentration of compounds in the raw cannabis oil, however, is
currently not well controlled. Typical control techniques include
diluting the cannabis oil with a foreign substance, such as olive
oil, MCT oil, and hempseed oil. This technique does not change the
relative concentrations of the compounds vis-a-vis other compounds
in the cannabis oil. Other techniques target the removal of
specific compounds, such as THC. This, however, often results in
other compounds being removed or denatured as a result. Thus, it
remains desirous to identify methods to separate raw cannabis oil
into one or more of its constituant compounds.
[0006] It is with respect to these and other considerations that
the technology is disclosed. Also, although relatively specific
problems have been discussed, it should be understood that the
embodiments presented should not be limited to solving the specific
problems identified in the introduction.
SUMMARY
[0007] This Summary is provided to introduce a selection of
concepts in a simplified form that are further described below in
the Detailed Description. This Summary is not intended to identify
key factors or essential features of the claimed subject matter,
nor is it intended to be used to limit the scope of the claimed
subject matter.
[0008] The technology described herein includes systems and methods
to extract various compounds from raw cannabis oil. A liquid
chromatography system may be used to extract specific compounds
from raw cannabis oil.
[0009] In an embodiment, the technology relates to a method for
extracting compounds from raw cannabis oil.
[0010] In some embodiments, a preparatory method is provided for
removing one or more cannabis compounds from a cannabis oil, the
method comprising: obtaining a column packed with a stationary
phase particulate; adding cannabis oil to the packed column; adding
a first eluent to the packed column; adding a second eluent to the
packed column; collecting at least two eluate fractions comprising
one or more compounds; disposing of at least one of the at least
two eluate fractions; and evaporating at least one of the remaining
at least two fractions to form a composition. In some embodiments,
the method comprises adding a third eluent is to the packed
column.
[0011] In embodiments, the stationary phase particulate is selected
from normal phase or reverse phase stationary phase. In a specific
embodiment, the column is a normal phase silica gel stationary
phase column. In other embodiments, a reverse phase stationary
phase is selected from the group consisting of C18, C8, C4 and
phenyl stationary phase particulate.
[0012] In some embodiments, a normal phase column is employed, and
a mobile phase comprising a first eluent and second eluent are used
to elute the column. In some embodiments, the first eluent and
second eluent are different and each is selected from one or more
of petroleum ether, pentane, n-hexane, hexanes, diethyl ether,
ethyl acetate, and ethanol. In some embodiments, the first eluent
and the second eluent are each solvents selected from one or, or a
specific mixture of two or more of, the group consisting of
petroleum ether, pentane, n-hexane, hexanes, diethyl ether, ethyl
acetate, and ethanol. In some embodiments, the first eluent and
second eluent are each solvents selected from one of, or a specific
mixture of two or more of, the group consisting of petroleum ether,
pentane, n-hexane, hexanes, n-heptane, heptanes, diethyl ether,
methyl tert butyl ether, ethyl acetate, and ethanol.
[0013] In embodiments, the first eluent and the second eluent are
each solvents selected from one of, or a specific mixture of two or
more of, the group consisting of petroleum ether, n-hexane,
hexanes, n-heptane, heptanes, diethyl ether, and methyl tert butyl
ether. In embodiments, the first eluent and the second eluent are
each a mixture of diethyl ether and petroleum ether. In
embodiments, the first eluent and the second eluent are each a
mixture of methyl tert butyl ether and petroleum ether. In
embodiments, the first eluent and the second eluent are each a
mixture of diethyl ether and n-heptane or a heptane. In
embodiments, the first eluent and the second eluent are each a
mixture of methyl tert butyl ether and n-heptane or a heptane.
[0014] In some embodiments, the normal phase column is eluted with
a first eluent solvent system selected from 90-97 vol % petroleum
ether with 3-10 vol % of a mixture of from 90-100 vol % diethyl
ether with 0-10 vol % ethanol; 90-97 vol % pentane with 3-10 vol %
of a mixture of a mixture of from 90-100 vol % diethyl ether with
0-10 vol % ethanol; 90-97 vol % n-hexane with 3-10% of a mixture of
from 90-100 vol % diethyl ether with 0-10 vol % ethanol.
[0015] In some specific embodiments, the normal phase column is
eluted with a first eluent solvent system selected from 92 vol %
petroleum ether and 8 vol % of a mixture of 95% diethyl ether/5%
ethanol; 92 vol % pentane and 8 vol % of a mixture of 95% diethyl
ether/5% ethanol; 92 vol % n-hexane and 8 vol % of a mixture of 95%
diethyl ether/5% ethanol; 96 vol % petroleum ether and 4 vol % of a
mixture of 95% diethyl ether/5% ethanol; 96 vol % pentane and 4 vol
% of a mixture of 95% diethyl ether/5% ethanol; or 96 vol %
n-hexane and 4 vol % of a mixture of 95% diethyl ether/5%
ethanol.
[0016] In some embodiments, the normal phase column is eluted with
a second eluent solvent system selected from: 60-80 vol % petroleum
ether with 20-40 vol % of a mixture of from 90-100 vol % diethyl
ether/0-10 vol % ethanol; 60-80 vol % pentane with 20-40 vol % of a
mixture of a mixture of from 90-100 vol % diethyl ether/0-10 vol %
ethanol; or 60-80 vol % n-hexane with 20-40 vol % of a mixture of
from 90-100 vol % diethyl ether/0-10 vol % ethanol.
[0017] In some specific embodiments, the normal phase column is
eluted with a second eluent solvent system selected from: 70 vol %
petroleum ether and 30% of a mixture of 95% diethyl ether/5%
ethanol; 70 vol % pentane and 30 vol % of a mixture of 95% diethyl
ether/5% ethanol; 70 vol % n-hexane and 30 vol % of a mixture of
95% diethyl ether/5% ethanol; 60 vol % petroleum ether and 40 vol %
of a mixture of 95% diethyl ether/5% ethanol; 60 vol % pentane and
40 vol % of a mixture of 95% diethyl ether/5% ethanol; or 60 vol %
n-hexane and 40 vol % of a mixture of 95% diethyl ether/5%
ethanol.
[0018] In embodiments, the second eluent is selected from 30-40 vol
% water with 0.05-1 vol % formic acid and 70-60 vol % ethanol with
0.05-1 vol % formic acid; or 30-40 vol % water with 0.05-1 vol %
formic acid and 70-60 vol % acetonitrile with 0.05-1 vol % formic
acid.
[0019] In embodiments, the second eluent is selected from 20-30 vol
% water with 0.05-1 vol % formic acid and 70-80 vol % ethanol with
0.05-1 vol % formic acid; or 20-30 vol % water with 0.05-1 vol %
formic acid and 70-80 vol % acetonitrile with 0.05-1 vol % formic
acid.
[0020] In some specific embodiments, the normal phase column is
eluted with a first eluent in a volume of between one and eleven
column volumes (CVs), 2 to 7 CVs, or 4 to 6 CVs; and a second
eluent in a volume of between one and eleven column volumes (CVs),
2 to 7 CVs, or 4 to 6 CVs.
[0021] In some embodiments, a reverse phase column is employed and
a mobile phase comprising a first eluent and second eluent are used
to elute the column. In some embodiments, the first eluent and
second eluent are different and each is selected from one or more
of water, acetonitrile, and ethanol, with or without an acidic
modifier. In some embodiments, the reverse phase stationary phase
column is eluted with a first eluent selected from 30-40 vol %
water and 70-60 vol % ethanol optionally containing an organic acid
modifier selected from formic acid or trifluoroacetic acid in from
0.01-0.2 vol %; or 30-40 vol % water and 70-60 vol % acetonitrile;
wherein the first eluent optionally containing an organic acid
modifier selected from formic acid or trifluoroacetic acid in from
0.01-0.2 vol %.
[0022] In some specific embodiments, the reverse phase column is
eluted with a second eluent selected from 20-30 vol % water and
70-80 vol % ethanol; or 20-30 vol % water and 70-80 vol %
acetonitrile, optionally wherein the first and second eluent
contain an organic acid modifier selected from formic acid or
trifluoroacetic acid in from 0.01-0.2 vol %.
[0023] In one embodiment, the method comprises pooling the normal
phase eluate from 6-7.5 column volumes, evaporating the pooled
eluate to form a composition, and recrystallizing the composition
to provide a purified composition comprising cannabidiol (CBD) in
greater than 94% purity, having not more than 0.3% THC. In
embodiments, the purified composition comprises CBD after
recrystallization having at least 94% purity. In embodiments, the
purified composition comprises CBD after recrystallization having
at least 95% purity. In embodiments, the purified composition
comprises CBD after recrystallization having at least 96% purity.
In embodiments, the purified composition comprises CBD after
recrystallization having at least 97% purity. In embodiments, the
purified composition comprises CBD after recrystallization having
at least 98% purity. In embodiments, the purified composition
comprises CBD after recrystallization having at least 99% purity.
In embodiments, the purified composition comprises CBD after
recrystallization having 100% purity. In embodiments, after
evaporation and prior to recrystallization the composition
comprises CBD having a purity of 90-95%. In some embodiments, the
CBD is recrystallized from pentane, n-hexane, petroleum ether, or a
mixture thereof.
[0024] In some embodiments, the method comprises pooling the normal
phase eluate from 1-6 and 9-12 CVs, and evaporating to provide a
composition comprising 60-75% CBD, 0.001-0.5% THC, 0.1-8% CBC, CBG,
CBN, and about 5-25% fatty acids and terpenes. In some embodiments,
the composition comprises 65-70% CBD, not more than 0.3% THC,
0.3-5% CBC, 0.3-5% CBG, 0.3-5% CBN, and about 10-20% fatty acids
and terpenes. In some embodiments, the composition comprises 65-70%
CBD, not more than 0.2% THC, 0.3-5% CBC, and about 15% fatty acids
and terpenes.
[0025] In another embodiment, a method is provided for processing
raw cannabis oil to provide clean cannabis oil, or a CBD isolate,
having less than 0.3 wt % delta9-THC, the method comprising:
obtaining raw cannabis oil; applying the raw cannabis oil to a
normal stationary phase column; eluting the normal stationary phase
column with a binary solvent system wherein the binary solvent
system comprises a first solvent A and a second solvent B;
fractionating the eluate into at least two eluate fractions;
disposing at least one of the at least two eluate fractions; and
evaporating the solvent from the remaining at least two eluate
fractions to provide the clean cannabis oil comprising less than
0.3 wt % THC. In a specific embodiment, the normal stationary phase
column is a silica gel column.
[0026] In some embodiments, the raw cannabis oil is obtained by
supercritical CO.sub.2 extraction of Cannabis spp. plant material,
solvent-solid extraction of Cannabis spp. plant material, or from a
commercial supplier.
[0027] In some embodiments, the first solvent A in the binary
solvent system is a non-polar solvent selected from one or more of
the group consisting of pentane, petroleum ether, hexanes,
n-hexane, heptane, diisopropyl ether, toluene, chloroform, and
methylene chloride; preferably petroleum ether, hexanes, or
n-hexane. In embodiments, the first solvent A in the binary solvent
system is a non-polar solvent selected from one or more of the
group consisting of pentane, petroleum ether, hexanes, n-hexane,
n-heptane, heptanes, diisopropyl ether, toluene, chloroform, and
methylene chloride. In embodiments, the first solvent A in the
binary solvent system is petroleum ether, a heptane, or
n-heptane.
[0028] In some embodiments, the second solvent B in the binary
solvent system is a polar solvent selected from one or more of the
group consisting of diethyl ether, tetrahydrofuran, ethyl acetate,
acetone, acetonitrile, isopropanol, ethanol, and methanol;
preferably diethyl ether, a mixture of diethyl ether and ethanol,
or ethyl acetate. In embodiments, the second solvent B in the
binary solvent system is a polar solvent selected from one or more
of the group consisting of diethyl ether, methyl tert butyl ether,
tetrahydrofuran, ethyl acetate, acetone, acetonitrile, isopropanol,
ethanol, and methanol. In embodiments, the polar solvent is diethyl
ether or methyl tert butyl ether.
[0029] In some embodiments, the normal phase column is eluted with
a step gradient using a binary solvent system comprising the first
solvent A and the second solvent B, wherein solvent A is petroleum
ether, pentane, or n-hexane and the solvent B is a mixture of
90-100:0-10 v/v diethyl ether/ethanol. In a specific embodiment,
the column is eluted with a mixture of solvent A/solvent B at 8%
solvent B for 7 CV, then 30% solvent B for 6 CV. In embodiments,
the column is eluted with a step gradient using a binary solvent
system comprising the first solvent A and the second solvent B,
wherein solvent A is petroleum ether, a heptane, or n-heptane and
solvent B is diethyl ether or methyl tert butyl ether.
[0030] In some embodiments, the normal phase column is eluted and
eluate from 0-6 column volumes and 9-12.5 column volumes are
pooled, and concentrated to provide clean cannabis oil having not
more than 0.3 wt % THC and about 60-70 wt % CBD.
[0031] In specific embodiments, the yield of clean cannabis oil is
not less than 60 wt %, or not less than 65 wt % based on starting
raw cannabis oil.
[0032] In embodiments, the solvent A is petroleum ether, solvent B
is 95% diethyl ether and 5% ethanol, and the eluate fractions from
0-6 column volumes and 9-12 column volumes are pooled, and
concentrated to provide clean cannabis oil having not more than 0.3
wt % THC and about 60-70 wt % CBD.
[0033] In embodiments, applying comprises loading 1 to 20 wt %, 2
to 15 wt %, or 4 to 8 wt % of the raw cannabis oil to the normal
stationary phase column, when compared to the total weight of the
normal stationary phase.
[0034] In a specific embodiment, the disclosure provides a
composition comprising about 65-70% CBD, not more than 0.3% THC,
0.3-5% CBC, 0.3-5% CBG, 0.3-5% CBN, and about 10-20% fatty acids
and terpenes. Also disclosed herein is a composition comprising
about 40-80% CBD, not more than 0.3% THC, 0.3-5% CBC, 0.3-5% CBG,
and 0.3-5% CBN.
[0035] Also disclosed herein are processes that can concentrate
cannabidiol (CBD) from a cannabis oil prior to performing the
chromatography methods disclosed herein. A method of concentrating
CBD from a cannabis oil disclosed herein comprises: mixing
petroleum ether with a heated cannabis oil to provide a mixture;
allowing the mixture to cool to room temperature; cooling the
mixture to 0.degree. C. to provide a cooled mixture; mixing a first
amount of petroleum ether at -10.degree. C. to 0.degree. C. into
the cooled mixture; freezing the cooled mixture for at least two
hours or cooling the cooled mixture at 0.degree. C.; adding a
second amount of petroleum ether at -10.degree. C. to 0.degree. C.
to the mixture; filtering the mixture to provide a filtrate and a
concentrate; and collecting the concentrate, wherein the
concentrate comprises CBD having at least about 94% purity and the
concentrate has not more than 0.3% THC. In embodiments, the
concentrate comprises CBD having 90-95% purity.
[0036] In embodiments, the cooled mixture is frozen for 2-24 hours
or 6-24 hours, for example, for 2-18 hours or for 6-18 hours.
[0037] In embodiments, the concentrate does not include THC. In
embodiments, the concentrate does not include CBG. In embodiments,
the concentrate does not include CBC. In embodiments, the
concentrate does not include CBN. As used herein, in embodiments,
"does not include" means that the component is below a detectable
limit.
[0038] In embodiments, the method further comprises performing a
recrystallization of the concentrate after collecting the
concentrate, wherein the recrystallization provides CBD having
greater than 95% purity. In embodiments, the recrystallization
provides CBD having at least 98% purity. In embodiments, the
recrystallization provides CBD having at least 99% purity. In
embodiments, the recrystallization provides CBD having at least
99.5% purity. In embodiments, the recrystallization provides CBD
having 100% purity. The recrystallization can involve one or more
recrystallization steps. In embodiments, the recrystallization in a
single recrystallization. In embodiments, the recrystallization is
two recrystallizations.
[0039] In embodiments, filting the mixture comprises filtering the
mixture both at atmospheric pressure and under vacuum.
[0040] In embodiments, the method further comprises collecting the
filtrate, wherein the filtrate contains CBD in an amount from about
20 wt % to about 70 wt % less than CBD present in the cannabis oil.
For example, the filtrate can contain CBD in an amount from about
20 wt % to about 50 wt %, from about 40 wt % to about 60 wt %, or
from about 45 wt % to about 60 wt %.
[0041] Further disclosed herein are processes that take a
cannabinoids fraction arising from the chromatography methods
disclosed herein and provide CBG, CBC, or a mixture thereof from
the cannabinoids fraction.
[0042] According to a method, a cannabinoids fraction (e.g.,
provided as a product of the chromatography methods disclosed
herein) is subjected to mixing ethanol at 50-70.degree. C. into the
cannabinoids fraction to provide a mixture; performing reverse
phase chromatography on the mixture providing multiple CVs;
identifying CVs containing CBG; concentrating the CVs containing
CBG; and recrystallizing CBG, wherein the CBG has a purity of at
least 94%. In embodiments, the CBG is a hydrate of CBG having 100%
purity. In embodiments, the CBG is a monohydrate of CBG having 100%
purity. In embodiments, the CBG is not recrystallized. In these
embodiments, concentrating the CVs containing CBG provides an oil
containing CBG having a purity of at least 90%.
[0043] According to a method, a cannabinoids fraction (e.g.,
provided as a product of the chromatography methods disclosed
herein) is subjected to mixing ethanol at 50-70.degree. C. into the
cannabinoids fraction to provide a mixture; performing reverse
phase chromatorgraphy on the mixture providing multiple CVs;
identifying CVs containing CBC; and concentrating the CVs
containing CBC to provide a CBC oil. In embodiments, the CBC oil
has a purity from 94%-96%. In embodiments, the CBC oil has a purity
from 90-96%.
[0044] When the CBG is not recrystallized, the oil containing CBG
and the oil containing CBC can be recombined to provide a mixture
comprising CBC and CBG.
[0045] According to a method, a cannabinioids fraction (e.g.,
provided as a product of the chromatography methods disclosed
herein) is subjected to mixing ethanol at 50-70.degree. C. into the
cannabinoids fraction to provide a mixture; performing reverse
phase chromatography on the mixture providing multiple CVs;
identifying CVs containing CBG and CBC; and concentrating the CVs
containing CBG and CBC to provide an oil comprising CBG and CBC,
wherein the CBG has a purity of at least 90%. In embodiments, the
oil comprises CBC having a purity from 94-96%. In embodiments, the
oil comprises CBC having a purity from 90-96%.
[0046] In embodiments, the CVs containing CBG are identified by
thin layer chromatography. In embodiments, the CVs containing CBC
are identified by thin layer chromatography. In embodiments, the
CVs containing CBG and CBC are identified by thin layer
chromatography.
[0047] In embodiments, performing reverse phase chromatography
comprises: eluting a column with a first solvent for 3 CV; eluting
the column with the first solvent for 4 CV; and eluting the column
with a second solvent for 7 CV, wherein fractions collected from 2
CV to 3.5 CV contain CBG.
[0048] In embodiments, performing reverse phase chromatography
comprises eluting a column with a first solvent for 3 CV; eluting
the column with the first solvent for 4 CV; and eluting the column
with a second solvent for 7 CV, wherein fractions collected from
6-9 CV contain CBC.
[0049] In embodiments, the first solvent is 80% methanol and 20%
distilled water and the second solvent is 85% methanol and 15%
distilled water.
[0050] In embodiments, performing reverse phase chromatography
comprises eluting a column with a step gradient using a binary
solvent system comprising a first solvent A and a second solvent B,
wherein the solvent A is methanol, ethanol, or acetonitrile and the
second solvent B is distilled water. In embodiments, the first
solvent A is methanol and the second solvent B is distilled
water.
[0051] Also disclosed herein is an oil containing CBG produced by
these methods. Further disclosed herein is an oil containing CBC
produced by these methods.
[0052] Additionally disclosed herein is a mixture comprising CBG
and the CBC produced by these methods having less than 0.3% THC. In
embodiments, the mixture comprising CBG and CBC does not include
THC. Again, as used herein, in embodiments, "does not include"
means that the component is below a detectable limit. The mixture
can have a CBG content and a CBC content that is approximately
equal. The mixture can have 50 wt % CBG and 50 wt % CBC.
[0053] Further disclosed herein is a hydrate of CBG. Additionally,
disclosed herein is a monohydrate of CBG.
BRIEF DESCRIPTION OF THE DRAWINGS
[0054] FIG. 1 illustrates a liquid chromatography system;
[0055] FIG. 2 illustrates a liquid chromatographic method for
extracting one or more compounds from raw cannabis oil;
[0056] FIG. 3 illustrates a method for concentrating one or more
fractions.
[0057] FIGS. 4A and 4B illustrate analytical HPLC-MS chromatograms
a combined standard of CBD and THC.
[0058] FIGS. 5A and 5B illustrate analytical HPLC-MS chromatograms
of crude oil.
[0059] FIGS. 6A and 6B illustrate analytical HPLC-MS chromatograms
of diluted raw cannabis oil.
[0060] FIGS. 7A and 7B illustrate analytical HPLC-MS chromatograms
for a combined fraction extracted from sample cannabis oil.
[0061] FIGS. 8A and 8B illustrate analytical HPLC-MS chromatogram
for a combined and diluted sample cannabis oil.
[0062] FIGS. 9A and 9B illustrate analytical HPLC-MS chromatogram
for isolate A.
[0063] FIG. 10 illustrates a preparative chromatogram of a normal
phase separation method monitored at 220 nm and 240 nm where eluate
from 0-6 CVs is pooled with 9-12 CVs to provide a clean cannabis
oil following rotoevaporation, where the clean cannabis oil
exhibits about 68% CBD and not more than 0.3% THC. Fractions 21-30,
or about 6-7.5 CVs are pooled, evaporated, and recrystallized to
provide a CBD isolate with about 95.1% CBD, as shown in Example
1.
[0064] FIG. 11 illustrates a preparative chromatogram of a normal
phase separation method of raw cannabis oil described in Example
2.
[0065] FIG. 12 illustrates a preparative reverse phase separation
method of raw cannabis oil described in Example 3.
DETAILED DESCRIPTION
Definitions
[0066] The terminology used in the disclosure herein is for the
purpose of describing particular embodiments only and is not
intended to be limiting of the disclosure. As used in the
description of the embodiments of the disclosure and the appended
claims, the singular forms "a", "an" and "the" are intended to
include the plural forms as well, unless the context clearly
indicates otherwise. Also, as used herein, "and/or" refers to and
encompasses any and all possible combinations of one or more of the
associated listed items. Furthermore, the term "about," as used
herein when referring to a measurable value such as an amount of a
compound, amount, dose, time, temperature, and the like, is meant
to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of
the specified amount. It will be further understood that the terms
"comprises" and/or "comprising," when used in this specification,
specify the presence of stated features, integers, steps,
operations, elements, and/or components, but do not preclude the
presence or addition of one or more other features, integers,
steps, operations, elements, components, and/or groups thereof.
Unless otherwise defined, all terms, including technical and
scientific terms used in the description, have the same meaning as
commonly understood by one of ordinary skill in the art to which
this disclosure belongs.
[0067] The present technology relates to extracting various
compounds from bulk cannabis oil using liquid chromatography. In
aspects of the technology, an extraction process, such as CO.sub.2
extraction, or liquid-solid solvent extraction, is performed on
cannabis plant material. In some embodiments, the resulting
extract, referred to herein as raw cannabis oil or bulk cannabis
oil, may contain a variety of cannabinoids and other substances.
For example, tetrahydrocannabinol (THC, d9-THC,
1-trans-delta9-tetrahydrocannabinol), cannabidiol (CBD),
Cannabigerol (CBG), Cannabichromene (CBC), Cannabicyclol (CBL),
Cannabinol (CBN), Cannabivarin (CBV), delta-9
Tetrahydrocannabinolic acid (THCA, d9-THCA), Tetrahydrocannabivarin
(THCV), Cannabidiolic acid (CBDA), Cannabigerolic acid (CBGA),
Cannabidivarin (CBDV), Cannabichromevarin (CBCV), Cannabigerovarin
(CBGV), and Cannabigerol Monomethyl Ether (CBGM) may be present in
varying concentrations in the raw cannabis oil.
[0068] In some embodiments, a method is provided to remove or
deplete a THC component from crude cannabis oil, industrial hemp
extracts, or hemp oil. Methods are provided to process crude
cannabis oil, industrial hemp extract, or hemp oil, to provide a
clean cannabis oil. In some embodiments, the clean cannabis oil
comprises from 0.001-3 wt %, 0.01-2 wt %, or 0.1-0.3 wt % THC; or
not more than 1.0 wt %, 0.5 wt %, 0.3 wt %, 0.2 wt %, 0.1 wt %, or
0.05 wt % THC. In other embodiments, a method is provided to remove
or deplete THC from crude cannabis oil, or hemp oil, to provide a
clean cannabis oil having a cannabinoid profile of about 40-70%
CBD, 1-3% CBG, 1-3% CBC, 1-3% CBN, 1-5% CBDA, 1-5% THCA, and 10-30%
fatty acids and terpenes, and not more than 3 wt %, 2 wt %, 0.8 wt
%, 0.5 wt %, 0.4 wt %, 0.3 wt %, 0.2 wt %, 0.1 wt %, or 0.05 wt %
THC.
[0069] In a specific embodiment, a method is provided to process
crude cannabis oil, industrial hemp extracts, or hemp oil to
provide a clean cannabis oil having not more than 70 wt % CBD and
not more than 0.3 wt % THC. In some embodiments, the clean cannabis
oil comprises 40-70 wt %, 50-70 wt %, 55-70 wt %, or 65-70 wt %
CBD, and not more than 0.3 wt % THC. In a specific embodiment, the
clean cannabis oil comprises 65-70% CBD, <0.3% THC, 0.3-5% CBC,
0.3-5% CBG, 0.3-5% CBN, and -15% fatty acids and terpenes.
[0070] In some embodiments, a clean cannabis oil is provided
containing from about 0.1-10 wt %, 0.5-8 wt %, 1-7 wt %, 2-6 wt %,
or about 3-5 wt % terpenes comprising one or more, two or more or
three or more terpenes selected from the group consisting of
myrcene, linolool, limonene, beta-caryophyllene (beta-humulene),
alpha-caryophyllene (alpha-humulene), alpha-pinene, beta-pinene,
alpha-bisabolol, delta-3-carene, trans-gamma-bisabolene, borneol,
terpineol, eucalyptol, trans-ocimene, trans-alpha-farnesene,
cis-beta-farnesene, gamma-curcumene, beta-fenchol, fenchone,
beta-phellandrene, guajol, alpha-guaiene, alpha-eudesmol,
terpinolene, alpha-selinene, camphene, alpha-thujene, and
cineole.
[0071] In some embodiments, a clean cannabis oil is provided
comprising about 5-30 wt %, 6-25 wt %, 7-20 wt %, 8-15 wt %, or
9-12 wt % fatty acids, comprising both unsaturated fatty acids
selected and saturated fatty acids. The clean cannabis oil may
comprise one or more unsaturated fatty acids selected from linoleic
acid, alpha-linolenic acid, oleic acid, gamma-linolenic acid,
stearidonic acid, eicosanoic acid, cis-vaccenic acid, and
isolinolenic acid. The clean cannabis oil may comprise one or more
saturated fatty acids selected from palmitic acid, stearic acid,
arachidonic acid, behenic acid, myristic acid, lignoceric acid,
caproic acid, heptanoic acid, caprylic acid, pelargonic acid,
capric acid, lauric acid, margaric acid, and isoarachidic acid.
[0072] In other embodiments, a method is provided for processing
crude cannabis oil to provide a CBD isolate enriched in CBD,
compared to the starting raw cannabis oil. In some embodiments, the
CBD isolate comprises CBD in from 70 wt %-99.99 wt %, 75-99.9 wt %,
80-99 wt %, 90-98 wt %, or 94-98 wt % CBD, or not less than 90 wt
%, 94 wt %, 95 wt %, 97.5 wt %, 98 wt %, 99 wt %, 99.5 wt %, or not
less than 99.9 wt % CBD, while comprising not more than 0.3 wt % of
THC. In certain aspects, the CBD isolate contains not more than 1
wt %, 0.5 wt %, 0.3 wt % of any of THCV, THCV, CBC, CBN, CBG, THCA,
CBDA, CBGA, and CBDV.
[0073] In some embodiments, the individual components are
identified and/or quantified by any technique known in the art. For
example, comparison to HPLC standards, HPLC, HPLC-MS, GC, GC-MS,
IR, MS, .sup.1H-NMR, .sup.13C-NMR, and/or elemental analysis.
[0074] In other embodiments, because the industrial hemp market is
rapidly expanding and as federal regulations on the medical study
of cannabinoids relax, there will likely be an increased demand for
not only CBD but a myriad of other non-psychoactive
cannabinoids.
[0075] In some embodiments, methods are provided herein for
processing raw cannabis oil to allow for the isolation and or
depletion of individual cannabinoid and custom formulation of over
ten individual medicinally relevant cannabinoids selected from one
or more, two or more, three or more, four or more, five or more,
six or more, seven or more, or eight or more, or nine cannabinoids
selected from the group consisting of CBD, THCV, D9-THC (THC),
D8-THC, CBC, CBN, CBG, THCA, CBDA, CBGA, and CBDV.
[0076] In other embodiments, a method is provided for processing
crude cannabis oil to provide a THCV isolate enriched in THCV,
compared to the starting raw cannabis oil. In some embodiments, the
THCV isolate comprises THCV in from 70 wt %-99.99 wt %, 75-99.9 wt
%, 80-99 wt %, 90-98 wt %, or 95-98 wt % THCV, or not less than 95
wt %, 97.5 wt %, 98 wt %, 99 wt %, 99.5 wt %, or not less than 99.9
wt % THCV, while comprising not more than 0.3 wt % of a cannabinoid
selected from the group consisting of THC, CBD, CBC, CBN, CBG,
THCA, CBDA, CBGA, and CBDV.
[0077] In other embodiments, a method is provided for processing
crude cannabis oil to provide a THC isolate enriched in THC,
compared to the starting raw cannabis oil. In some embodiments, the
THC isolate comprises THC in from 70 wt %-99.99 wt %, 75-99.9 wt %,
80-99 wt %, 90-98 wt %, or 95-98 wt % THC, or not less than 95 wt
%, 97.5 wt %, 98 wt %, 99 wt %, 99.5 wt %, or not less than 99.9 wt
% THC, while comprising not more than 0.3 wt % of a cannabinoid
selected from the group consisting of CBD, THCV, CBC, CBN, CBG,
THCA, CBDA, CBGA, and CBDV.
[0078] In other embodiments, a method is provided for processing
crude cannabis oil to provide a CBC isolate enriched in CBC,
compared to the starting raw cannabis oil. In some embodiments, the
CBC isolate comprises CBC in from 70 wt %-99.99 wt %, 75-99.9 wt %,
80-99 wt %, 90-98 wt %, or 95-98 wt % CBC, or not less than 95 wt
%, 97.5 wt %, 98 wt %, 99 wt %, 99.5 wt %, or not less than 99.9 wt
% CBC, while comprising not more than 0.3 wt % of a cannabinoid
selected from the group consisting of CBD, THC, THCV, CBN, CBG,
THCA, CBDA, CBGA, and CBDV.
[0079] In other embodiments, a method is provided for processing
crude cannabis oil to provide a CBN isolate enriched in CBN,
compared to the starting raw cannabis oil. In some embodiments, the
CBN isolate comprises CBN in from 70 wt %-99.99 wt %, 75-99.9 wt %,
80-99 wt %, 90-98 wt %, or 95-98 wt % CBN, or not less than 95 wt
%, 97.5 wt %, 98 wt %, 99 wt %, 99.5 wt %, or not less than 99.9 wt
% CBN, while comprising not more than 0.3 wt % of a cannabinoid
selected from the group consisting of THC. CBD, THCV, CBC, CBG,
THCA, CBDA, CBGA, and CBDV.
[0080] In other embodiments, a method is provided for processing
crude cannabis oil to provide a CBG isolate enriched in CBG,
compared to the starting raw cannabis oil. In some embodiments, the
CBG isolate comprises CBG in from 70 wt %-99.99 wt %, 75-99.9 wt %,
80-99 wt %, 90-98 wt %, or 95-98 wt % CBG, or not less than 95 wt
%, 97.5 wt %, 98 wt %, 99 wt %, 99.5 wt %, or not less than 99.9 wt
% CBD, while comprising not more than 0.3 wt % of a cannabinoid
selected from the group consisting of THC, CBD, CBC, CBN, THCV,
THCA, CBDA, CBGA, and CBDV.
[0081] In other embodiments, a method is provided for processing
crude cannabis oil to provide a THCA isolate enriched in THCA,
compared to the starting raw cannabis oil. In some embodiments, the
THCA isolate comprises THCA in from 70 wt %-99.99 wt %, 75-99.9 wt
%, 80-99 wt %, 90-98 wt %, or 95-98 wt % THCA, or not less than 95
wt %, 97.5 wt %, 98 wt %, 99 wt %, 99.5 wt %, or not less than 99.9
wt % THCA, while comprising not more than 0.3 wt % of a cannabinoid
selected from the group consisting of THC, CBD, CBC, CBG, CBN,
THCV, CBDA, CBGA, and CBDV.
[0082] In other embodiments, a method is provided for processing
crude cannabis oil to provide a CBDA isolate enriched in CBDA,
compared to the starting raw cannabis oil. In some embodiments, the
CBDA isolate comprises CBDA in from 70 wt %-99.99 wt %, 75-99.9 wt
%, 80-99 wt %, 90-98 wt %, or 95-98 wt % CBDA, or not less than 95
wt %, 97.5 wt %, 98 wt %, 99 wt %, 99.5 wt %, or not less than 99.9
wt % CBDA, while comprising not more than 0.3 wt % of a cannabinoid
selected from the group consisting of THC, CBD, CBC, CBG, CBN,
THCV, THCA, CBGA, and CBDV.
[0083] In other embodiments, a method is provided for processing
crude cannabis oil to provide a CBGA isolate enriched in CBGA,
compared to the starting raw cannabis oil. In some embodiments, the
CBGA isolate comprises CBGA in from 70 wt %-99.99 wt %, 75-99.9 wt
%, 80-99 wt %, 90-98 wt %, or 95-98 wt % CBGA, or not less than 95
wt %, 97.5 wt %, 98 wt %, 99 wt %, 99.5 wt %, or not less than 99.9
wt % CBGA, while comprising not more than 0.3 wt % of a cannabinoid
selected from the group consisting of THC, CBD, CBC, CBG, CBN,
THCV, THCA, CBDA, and CBDV.
[0084] In other embodiments, a method is provided for processing
crude cannabis oil to provide a CBDV isolate enriched in CBDV,
compared to the starting raw cannabis oil. In some embodiments, the
CBDV isolate comprises CBDV in from 70 wt %-99.99 wt %, 75-99.9 wt
%, 80-99 wt %, 90-98 wt %, or 95-98 wt % CBDV, or not less than 95
wt %, 97.5 wt %, 98 wt %, 99 wt %, 99.5 wt %, or not less than 99.9
wt % CBDV, while comprising not more than 0.3 wt % of a cannabinoid
selected from the group consisting of THC, CBD, CBC, CBG, CBN,
THCV, THCA, CBDA, and CBGA.
[0085] In some embodiments, a composition comprising CBD, CBC and
terpenes is provided comprising not more than 0.3 wt % of THC.
[0086] In some embodiments, a composition comprising CBD and fatty
acids is provided comprising not more than 0.3 wt % of THC.
[0087] In some embodiments, a composition comprising CBG and CBD is
provided comprising not more than 0.3 wt % of THC.
[0088] In a specific embodiment, a composition is provided
comprising .about.65-70% CBD, 0.001-0.5% THC, 0.3-5% CBC, 0.3-5%
CBG, 0.3-5% CBN, and .about.10-20% fatty acids and terpenes. In
some aspects, the composition is dependent on the input hemp
extract; however, in the composition the THC content is
significantly reduced compared to starting raw cannabis oil such
that the composition comprises not more than 0.5%, 0.4%, 0.3%, 0.2%
or 0.1% THC, while essentially leaving the profile of the complex
mixture intact. In some embodiments, the individual compounds are
isolated from the composition and quantified to verify their
identity.
[0089] In some embodiments of the technology, raw cannabis oil is
separated using preparative liquid chromatographic methods
described further herein. However, in some embodiments, the eluate
fractionation method is non-traditional in order to reconstitute
most components of the starting raw cannabis oil to provide a clean
cannabis oil, while significantly reducing the amount of THC
compared to the starting raw cannabis oil, such that the
concentration of THC in the clean cannabis oil is not more than
0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.03, 0.01 wt % THC. For example,
raw cannabis oil may be injected into a column and eluted in an
isocratic, gradient, or a step-wise fashion. Fractions may be taken
at (and for) specific column volumes (CVs). These fractions contain
one or more compounds from the raw cannabis oil. Additionally, each
fraction may be combined with at least one other fraction. Further,
each fraction (or combination of fraction) may be concentrated
using a variety of techniques as described further herein.
[0090] FIG. 1 illustrates a system 100 to perform a liquid
chromatographic extraction. As illustrated, FIG. 1 includes a
liquid chromatography column 102, a High Performance Liquid
Chromatography Mass Spectrometry instrument ("HPLC-MS") 104, and
concentration station 108.
[0091] In some embodiments, the crude starting cannabis oil, or one
or more fractions thereof, is isolated and/or purified by
preparative chromatography. Liquid chromatography column 102
includes a column 110. In aspects, the column 110 has a volume of
990 mL, with a height of 291 mm and a diameter of 82 mm. As
illustrated, the column 110 is packed with a material 112, such a
silica gel for normal phase, or phenyl, C4, C8 or C18 for reverse
phase. Additionally, the column may be pre-wet by adding the
pre-wetting solvent (such as water, petroleum ether, diethyl ether
or ethanol) into the column prior to the injection of cannabis oil
sample 114.
[0092] The cannabis oil sample 114 may be raw cannabis oil, or hemp
oil, obtained from bulk extraction of Cannabis spp. plant material.
Extraction of cannabis or hemp plant material may be performed
using super critical CO.sub.2 extraction, liquid-solvent
extraction, or other techniques known in the art to provide bulk
cannabis oil, also known as crude cannabis oil, or raw cannabis
oil. In aspects of the technology, the bulk cannabis oil may
contain approximately 30-60% CBD, 3-5% THC, 1-3% CBG, 1-3% CBC,
1-3% CBN, 1-5% CBDA, 1-5% THCA, and 15-30% fatty acids and
terpenes.
[0093] In some embodiments, raw cannabis oil is first diluted to
form the cannabis oil sample 114. For example, for a process
employing a normal phase silica gel stationary phase column, the
raw cannabis oil may be mixed with a non-polar solvent prior to
loading the column. In specific embodiments, the non-polar solvent
is hexanes, n-hexane, pentane or petroleum ether. For example, the
raw oil can be diluted in a two parts cannabis oil to one part
petroleum ether, pentane, hexanes, or n-hexane to form the cannabis
oil sample 114. In alternative embodiments, when employing a
reverse phase stationary phase column, the raw cannabis oil may be
dissolved in ethanol and diluted with water prior to loading to the
column. mixed in equal parts with ethanol.
[0094] The cannabis oil sample 114 is injected into the column 110
using a pipette or other technique known in the art. In aspects of
the technology, the cannabis oil sample 114 is injected into the
column 110 in an amount selected from 1 to 20 wt %, 2 to 15 wt % or
4 to 8 wt %, of the stationary phase material by weight. After the
cannabis oil sample 114 is injected into the column 110, one or
more eluents 116 may be added to the column 110 to extract one or
more compounds from the cannabis oil sample 114. A first volume of
a first eluent may be added, followed by a second volume of a
second eluent and so on. In various embodiments, the eluents are
selected from one or more solvents, or one or more binary mixtures
of solvents may be used to elute a normal phase column.
[0095] In one embodiment, the normal phase column is a silica gel
column which may be eluted with a non-polar solvent, a polar
solvent, or a a mixture of two or more, three or more, or four or
more solvents. In one embodiment, a mixture of one or more
non-polar solvents and one or more polar solvents is employed to
elute the normal phase column.
[0096] In some embodiments, the non-polar solvent may be selected
from one or more of pentane, petroleum ether, hexanes, n-hexane,
heptane, diisopropyl ether, toluene, chloroform, and methylene
chloride. In specific embodiments, the non-polar solvent is
petroleum ether, pentane, n-hexane, or a hexane. In embodiments,
the non-polar solvent is petroleum ether, a hexane, or n-hexane. In
some embodiments, the polar solvent may be selected from one or
more of diethyl ether, tetrahydrofuran, ethyl acetate, acetone,
acetonitrile, isopropanol, ethanol, and methanol. In specific
embodiments, the polar solvent is selected from one or more of
diethyl ether, ethanol, methanol, or ethyl acetate. In embodiments,
the polar solvent is selected from diethyl ether, a mixture of
diethyl ether and ethanol, or ethyl acetate.
[0097] In some embodiments, the polar solvent is a mix of from
80-99.9 vol % diethyl ether and 20-0.01 vol % ethanol; a mix of
from 90-99 vol % diethyl ether and 10-1 vol % ethanol; or diethyl
ether mix of 95% diethyl ether and 5 vol % ethanol. In some
embodiments, the polar solvent is diethyl ether. In some
embodiments, the polar solvent is ethyl acetate.
[0098] In some embodiments, a binary solvent system is employed to
elute a normal phase silica gel column using a solvent system
selected from petroleum ether/diethyl ether, petroleum
ether/diethyl ether mix, hexane/diethyl ether, hexane/diethyl ether
mix, or hexane/ethyl acetate. For example, a binary mixture of
diethyl ether or a mixture of diethyl ether with ethanol and
petroleum ether may be used to elute the column. As used herein,
the term "diethyl ether mix" refers to a mixture of 95% diethyl
ether and 5% ethanol mixture by volume.
[0099] At high altitude, such as in Denver (5280 ft.), it may be
desirous to compensate for the decreased atmospheric pressure using
low boiling point solvents such as petroleum ether and diethyl
ether. For example, the temperature at which the preparative
chromatography is performed may be controlled, as well as the
pressure, may be controlled to remediate possible discrepancies
between "solvent delivered by pump system" and "solvent actually
present on column".
[0100] In particular, it was observed that, at temperatures above
70 degrees Fahrenheit and high altitude (5280), the pump "sucks"
low boiling solvent into its chamber, and the decreased pressure
causes a large amount of solvent to vaporize. This apparently ends
up filling the pump and line with air bubbles that inhibit reliable
and accurate solvent delivery to the chromatography column. In
order to minimize this effect, the chromatography may be performed
at temperatures below 70 degrees Fahrenheit when in low atmospheric
pressure environments. For example, in some embodiments, the
preparative chromatography may be performed at a temperature from
about 20 to 70 degrees Fahrenheit, 22 to 65 degrees Fahrenheit, 32
to 60 degrees Fahrenheit, or 40 to 55 degrees Fahrenheit. In some
embodiments, the chromatography is performed at a cold room
temperature from about 22-42 degrees Fahrenheit.
[0101] The second specific problem encountered is when diethyl
ether contacts silica, the wetting process is slightly exothermic.
On a large scale the heat released by this exothermic reaction is
enough to vaporize some of the diethyl ether and petroleum ether.
This may lead to unreliable and inaccurate solvent delivery to the
chromatography column. In order to address this second issue, the
solvent gradients must be changed slightly (i.e. from 5% diethyl
ether on a small scale to 7% diethyl ether on the large scale) at
high low pressure environments. Additionally, the column may be
preconditioned for extra column volumes in order to produce
consistent separation over multiple runs on a large scale.
[0102] As illustrated, a stopcock 118 is actuated to begin flow of
the eluent through the packed column. Other means, such as a
mechanical actuator, may be used to control the flow.
[0103] The eluent 116 may be flowed through the column in a
step-wise fashion. For example, the concentration of diethyl either
may be stepped up between the first eluent and the second eluent.
In one aspect, for example, a first eluent is 8% diethyl ether mix
and 92% petroleum ether (by volume) for 7 column volumes, followed
by a second eluent of 30% diethyl ether mix and 70% petroleum ether
(by volume) for four column volumes. In another aspect, a first
eluent is 4% diethyl ether and 96% petroleum ether (by volume) for
6 column volumes, followed by a second eluent of 8% diethyl ether
and 92% petroleum ether (by volume) for four column volumes,
followed by a third eluent of 40% diethyl ether and 60% petroleum
ether (by volume) for four column volumes. In another aspect,
wherein a reverse phase column is employed, the first eluent is 40%
H.sub.2O to 60% ethanol (by volume) for 10 CV and the second eluent
is 10% H.sub.2O and 90% ethanol (by volume). In other embodiments,
a reverse phase colum may be eluted used water and one or more of
acetonitrile, ethanol, methanol. An acidic modifier such as TFA or
formic acid may be added to the water, ethanol, methanol, and/or
acetonitrile. It will be appreciated that the flow rate may vary.
In some aspects, the flowrate varies based on eluent chosen, the
dimensions of the column, and the viscosity of the cannabis oil
sample 114. In aspects, where the column volume is 990 mL and the
solid phase is silica, the flow rate may be between 100 mL/min and
300 mL/min.
[0104] As the extract passes through the column, one through n
fractions 118 may be collected, where n is a number representing
the number of fractions collected. The n-fractions 118 may be
collected at a specific column volume range. For example, one
fraction may be collected between 4 and 7 CVs, another fraction may
be collected between 8.5 and 9 CVs.
[0105] During extraction, liquid chromatography column 102 may be
kept at around atmospheric pressure (1 ATM+/-0.2) and around room
temperature (22.degree. C.+/-5). FIG. 1 also includes an HPLC-MS
104. In aspects, n-fractions 118 collected using the column 102 may
be analyzed. The fractions may first be diluted. For example, a
fraction may be diluted Additionally or alternatively, the
fractions collected may be first concentrated and/or combined with
other fractions prior to being analyzed with the HPLC-MS.
[0106] FIG. 2 illustrates a liquid chromatographic method 200 for
extracting one or more compounds from raw cannabis oil. Method 200
begins with obtain extract operation 202. In operation 202, raw
cannabis oil is extracted from cannabis plant material.
[0107] The raw cannabis oil can be obtained by any method known in
the art for extraction of Cannabis spp. plant material, or the raw
cannabis oil can be purchased from a commercial source. For
example, the raw cannabis oil can be obtained by supercritical (or
subcritical) CO.sub.2 method that uses carbon dioxide under high
pressure and low temperatures to isolate, preserve and maintain the
purity of raw cannabis oil. In one specific embodiment, raw
cannabis oil obtained from a supercritical CO.sub.2 extraction is
used as a starting material for the methods described herein. For
example, supercritical CO.sub.2 extraction may be performed as
described in U.S. Pat. No. 8,895,078, which is incorporated herein
by reference in its entirety. Alternatively, a solvent such as
petroleum ether, ethanol, methanol, butanol, acetone, dry ice, or
olive oil can be used to extract the Cannabis spp. plant material,
at room temperature (ambient temperature) with stirring, by passive
extraction, heated to a temperature above room temperature, or
under reflux, as known in the art to provide the raw cannabis oil.
In another specific embodiment, raw cannabis oil from a butanol
extraction is employed as starting material for methods disclosed
herein. Any Cannabis spp. plant material can be employed. In some
embodiments, the raw cannabis oil is from an extract of Cannabis
sative L. In some embodiments, the cannabis oil is derived from
extraction of of Cannabis spp. plant material parts selected from
one or more of inflorescence of male (staminate) plant, fruiting
female (pistillate) plant, staminate flower, stamen, pollen grains,
pistillate flower with bract, pistillate flower without bract, seed
(archene) with bract, seed without bract, seed without pericarp,
leaves, stalks, and roots.
[0108] Method 200 then proceeds to prepare column operation 204. In
operation 204, a liquid chromatography column is packed. In aspects
of the technology, the column described with reference to FIG. 1 is
used. It will be appreciated that the column will be packed to
compliment the eluent liquid. That is, when a hydrophobic eluent is
chosen, the column will be packed with a hydrophilic material, and
vice-versa. In aspects, the column is packed with a hydrophilic
stationary phase material, such as silica for a normal phase liquid
chromatography extraction. Alternatively, a column may be packed
with a hydrophobic stationary phase material, such as a carbon 18,
phenyl, C4, or C8 reverse phase material.
[0109] The column eluate flow is monitored by any known means in
the art. In some aspects, the eluate flow is monitored by
ultraviolet (UV) absorption, refractive index, thin layer
chromatography (TLC), mass spectrometry (MS) total ion detection,
or MS mass selective detection. In a particular embodiment, the
eluates are monitored by UV and/or mass selective detection. In
specific embodiments, the column eluates are monitored by mass
selective detection for m/z of one or more of delta-9
tetrahydrocannabinol (THC), tetrahydrocannabinol acid (THCA),
cannabidiol (CBD), cannabidiolic acid (CBDA), Cannabigerol (CBG),
Cannabichromene (CBC), Cannabinol (CBN), Cannabicyclol (CBL),
Cannabivarin (CBV), Tetrahydrocannabivarin (TCHV), Cannabidivarin
(CBDV), Cannabichromevarin (CBCV), Cannabigerovarin (CBGV), and
Cannabigerol Monomethyl Ether (CBGM). In a specific aspect, eluate
is monitored at 315.2 m/z and 345.2 m/z. Monitoring at 315.2 (M+1)
will detect CBD and THC, as well as CBC. Monitoring at 345.2 m/z
will detect certain other cannabinoid components, for example
certain carboxylic acid containing cannabinoid compounds. Unless
otherwise specified, percent values refer to weight percent.
[0110] Method 200 then proceeds to insert cannabis oil sample
operation 208. In aspects, the raw cannabis oil extracted at
operation 202 is injected into the column. In other aspects, the
raw cannabis oil is first diluted as described above. In a
particular embodiment, a column volume of 0.05 CVs of cannabis oil
sample may be injected into the column.
[0111] Method 200 then proceeds to stage one elution operation 206.
In operation 206, a first eluent is added to the column. In
aspects, the first eluent is 8% diethyl ether mix and 92% petroleum
ether (by volume), 4% diethyl ether and 96% petroleum ether (by
volume), or 40% H.sub.2O to 60% ethanol (by volume).
[0112] Method 200 then proceeds to collect stage one fractions
operation 207. Where the first eluent is 8% diethyl ether mix and
92% petroleum ether (by volume) the first fraction may be collected
between 0 and 4 CVs. Where the first eluent 4% diethyl ether and
96% petroleum ether (by volume), the first fraction may be
collected between 0 and 2 CVs, and the second fraction may be
collected between 2 and 6 CVs. Further, where the first eluent is
40% H.sub.2O to 60% ethanol (by volume), the first fraction may be
collected between 0 and 4 CVs, and the second fraction may be
collected between 4 and 7 CVs.
[0113] Method 200 then proceeds to stage two elution operation 208.
In operation 208, a second eluent is added to the column. In one
aspect, a second eluent is 30% diethyl ether mix and 70% petroleum
ether (by volume), 8% diethyl ether mix and 92% petroleum ether (by
volume), or second eluent is 10% H.sub.2O and 90% ethanol (by
volume).
[0114] Method 200 then proceeds to collect stage two fractions
operation 209. Where the second eluent is 30% diethyl ether mix and
70% petroleum ether (by volume) the second fraction may be
collected between 4 and 8 CVs, the third fraction may be collected
between 8.5 and 9 CVs, the fourth fraction may be collected between
9 and 9.5 CVs the fourth fraction may be collected between 9.5 and
10 CVs, and the fifth fraction may be collected between 10 and 13
CVs. Where the first eluent 8% diethyl ether and 92% petroleum
ether (by volume), the third fraction may be collected between 7
and 9.5 CVs. Further, where the first eluent is 10% H.sub.2O to 90%
ethanol (by mass), the first fraction may be collected between 7
and 9 CVs the fourth fraction may be collected between 9 and 12
CVs.
[0115] Method 200 optionally proceeds to stage three elution
operation 210. In operation 210, a third eluent is added to the
column. In aspects of the technology, a third eluent of 40% diethyl
ether and 60% petroleum ether (by volume) is added.
[0116] Method 200 then optionally proceeds to collect stage three
fractions operation 211. Where the third eluent of 40% diethyl
ether and 60% petroleum ether (by volume), the fourth fraction may
be collected between 9.5 and 14 CVs.
[0117] FIG. 3 illustrates a method 300 for concentrating one or
more fractions. Method 300 begins with combine fractions operation
302. In some embodiments, the normal phase column first eluent is
8% diethyl ether mix in 92% petroleum ether and is run at a rate of
200 mL/min. After 4 CV (3960 mL where the column volume is 990 mL),
a second eluent of 30% diethyl ether mix and 70% petroleum ether
(by volume) may then be added to the column. In aspects, 120 ml
fractions are collected. In one embodiment, eluate from 1-4 CV and
9-20 CV are collected, pooled, evaporated and combined to provide a
clean cannabis oil having 60-70% CBD and not more than 0.3% THC.
Additionally, fractions 4-8 may be separately combined, evaporated,
and optionally recrystallized to provide a CBD isolate from any
appropriate solvent, e.g., n-hexane, or pentane.
[0118] Operation then proceeds to evaporate eluent operation 304.
In evaporate eluent operation, the eluent is evaporated from the
extract. In some embodiments, the eluent is removed in vacuo. In
aspects, the combined fractions described above with reference to
operation 302 are exposed to an environment of 0.3 ATMs, a
temperature of 45 degreese celsius, and agitated. In other aspects,
each fraction is concentrated prior to combination. Operation 304
may continue until one or more compounds precipitates from the
combined liquid fraction.
[0119] FIGS. 4-9 illustrate various analytical HPLC-MS
chromatograms. To generate each chromatogram, a single-quad MS
detector rather was used. The total ion chromatogram as well as two
specific masses: 315.2 amu and 345.2 amu were monitored.
[0120] FIGS. 4A and 4B illustrate analytical HPLC-MS chromatograms
401 and 403, of standards of a combined CBD and THC monitored at
m/z 315.2 (M+H). The samples of the CBD and THC were obtained as
commercial standards (Sigma-Aldrich). Illustrated in the
chromatogram 401 is CBD peak 402 and THC peak 404. As illustrated
in chromatogram 401, the CBD peak 402 has a retention time of about
1.3 min, and the THC peak 404 has a retention time of about 2.4
minutes. Further, illustrated in chromatogram 403 monitored at m/z
345.2, shows certain very minor cannabinoid components eluting at
peaks 406 (with a retention time of around 0.4 minutes) and 408
(with a retention time of around 1.1 minutes).
[0121] FIGS. 5A and 5B are an HPLC-MS chromatogram 500 of crude
cannabis oil. The crude oil of this sample was prepared using a
supercritical CO.sub.2 extraction method. The sample was prepared
by mixing raw cannabis oil with a diluting solvent. As illustrated,
FIGS. 5A and 5B include two HPLC-MS chromatograms, namely, a first
chromatogram 501 monitored at m/z 315.2 and a second chromatogram
503 monitored at m/z 345.2. The first chromatogram 501 illustrates
a peak 502 at around 1.3 mins retention time. By comparing the peak
502 to the CBD peak 402 illustrated in FIGS. 4A and 4B, it will be
appreciated that peak 502 illustrates the CBD peak. The amount 510
of peak 502 is 64%. Additionally illustrated in chromatogram 501
are other minor peaks 504. Other peaks 504 represent various other
compounds present in the bulk cannabis oil.
[0122] Second chromatogram 503 illustrates peak 506, at m/z 345.2
(M+H). Additionally illustrated in second chromatogram 503 is other
peaks 508, which represents other compounds contained in the raw
cannabis sample.
[0123] FIGS. 6A and 6B illustrate HPLC-MS chromatograms 601 and 603
of raw cannabis oil. The raw cannabis oil is same starting material
as the raw cannabis oil referenced with respect to FIGS. 5A and 5B,
but the raw cannabis oil has been diluted. As illustrated, FIGS. 6A
and 6B include two HPLC-MS Chromatograms, a first chromatogram 601
and a second chromatogram 603. The first chromatogram 601
illustrates a peak 602 at around 1.3 mins retention time. By
comparing the peak 602 to the CBD peak 402 illustrated in FIGS. 4A
and 4B, it will be appreciated that peak 602 illustrates a CBD
peak. Additionally illustrated in chromatogram 601 is other peaks
604. Other peaks 604 represent various other compounds present in
the bulk cannabis oil. The amount 610 of peak 602 is around
65%.
[0124] Second chromatogram 603 illustrates peak 606 at m/z 345.2
(M+H). Additionally illustrated in second chromatogram 603 are
other peaks 608, which represents other compounds contained in the
raw cannabis sample.
[0125] As illustrated, FIGS. 7A and 7B include two HPLC-MS
Chromatograms of the clean cannabis oil obtained in Example 1,
namely, a first chromatogram 701 and a second chromatogram 703. The
first graph 701 illustrates a peak 702 at around 1.3 mins retention
time. By comparing the peak 702 to the CBD peak 402 illustrated in
FIGS. 4A and 4B, it will be appreciated that peak 702 illustrates a
CBD peak. Additionally chromatogram 701 includes other peaks 704.
Other peaks 704 represent various other compounds present in the
clean sample oil. The amount 710 of peak 702 is around 68%.
[0126] Second chromatogram 703 illustrates peak 706 at m/z 345.2
(M+H). Additionally illustrated in second chromatogram 703 is other
peaks 708, which represents other compounds contained in the raw
cannabis sample.
[0127] FIGS. 8A and 8B illustrate HPLC-MS chromatograms for a
combined and diluted sample of clean cannabis oil of Example 1. The
cannabis oil sample was the same as the cannabis oil sample
referenced with respect to FIGS. 7A and 7B, but has been further
diluted. Dilution occurred by addition of a solvent prior to
analytical HPLC. As illustrated, FIGS. 8A and 8B include two
HPLC-MS Chromatograms, namely, a first chromatogram 801 and a
second chromatogram 803. The first chromatogram 801 illustrates a
peak 802 at around 1.3 mins retention time. Comparing the peak 802
to the CBD peak 402 illustrated in FIGS. 4A and 4B, it will be
appreciated that peak 802 illustrates the CBD peak. Additionally,
chromatogram 801 includes other peaks 804. Other peaks 804
represent various other compounds present in the bulk cannabis oil.
The area 810 of peak 802 is around 69%.
[0128] Second chromatogram 803 illustrates peak 806 at m/z 345.2
(M+H). Additionally illustrated in second chromatogram 603 are
other peaks 608, which represents other compounds contained in the
cannabis oil sample.
[0129] As illustrated, FIGS. 9A and 9B include two HPLC-MS
chromatograms, a first chromatogram 901 and a second chromatogram
903. The first chromatogram 901 illustrates a peak 902 at around
1.3 mins retention time. By comparing the peak 902 to the CBD peak
402 illustrated in FIGS. 4A and 4B, it will be appreciated that
peak 902 illustrates the CBD peak. Additionally, chromatogram 901
includes other peaks 904. Other peaks 904 represent various other
compounds present in the cannabis oil sample. The area 910 of peak
902 is around 95.1%.
[0130] Second chromatogram 903 illustrates minor peak 906 at m/z
345.2 (M+H). Additionally illustrated in second chromatogram 903
are other minor peaks 908, which represents other compounds
contained in the cannabis oil sample.
Example 1. Normal Phase Preparative Method 1
[0131] In this example, a Biotage Isolera Flash Chromatography
System was employed to process raw cannabis oil to deplete THC
component to provide clean cannabis oil, and further to provide CBD
isolate. HPLC purity of starting crude cannabis oil used herein was
60.50% CBD and 3.50% THC.
[0132] 45 g hemp oil (injection mass 6 wt %) was dissolved in 22.5
mL petroleum ether and injected to a 750 g normal phase silica gel
column (SNAP KP-Sil 750 g, BIOTAGE) and rinsed with pet ether for a
total injection volume of 67.5 mL. Solvent A was petroleum ether;
Solvent B was 95% diethyl ether and 5% ethanol. Solvents A and B
were employed to elute the column in a step gradient of 8 vol % B
run for 7 column volumes, increased from 8-30 vol % B for 0 column
volume, then 30 vol % B for 6 column volumes at a flow rate of 200
mL/min. The column eluate was monitored by UV-vis at 220 nm, and
240 nm, as shown in FIG. 10. 120 mL fractions were collected.
Following elution, the peak fractions were subjected to analytical
HPLC or TLC analysis. Appropriate fractions were combined and
solvents removed by rotoevaporation. Analytical HPLC by the method
of Example 4 was employed to determine relative amounts of
cannabinoids of interest.
[0133] As shown in FIG. 10, all eluate before 4 CV, fractions 1-20,
(.about.1-6 CV) and all eluate from 9-12 CV were pooled and
concentrated to make isolate 1. Following evaporation of solvents
from isolate 1, clean oil was obtained in about a 70 wt % crude
yield. This is a result of conservative fraction collection and the
loss of THC and minor portions of contaminated CBC. HPLC purity of
clean cannabis oil was 69% CBD and <0.3% THC as shown in FIGS. 7
and 8.
[0134] Fractions 21-30 were collected, pooled and concentrated to
make isolate 2. Isolate 2 was about 25 wt % of the starting
material. Isolate 2 was either sold as is, recrystallized to
further purify, or added back to clean oil in about a 1:2 ratio to
increase the yield of clean oil to 90-95% from starting crude. HPLC
purity of purified isolate is 95% CBD and <0.3% THC, as shown in
FIGS. 9A and 9B.
Example 2. Normal Phase Preparative Method 2
[0135] In this example, a Biotage Isolera Flash Chromatography
System was employed to process raw cannabis oil to deplete THC
component. In this example, a Biotage Isolera Flash Chromatography
System was employed to process raw cannabis oil to deplete THC
component. 45 g hemp oil (injection mass 6 wt %) was dissolved in
22.5 mL petroleum ether and injected to a 750 g normal phase silica
gel column (SNAP KP-Sil 750 g, BIOTAGE) and rinsed with pet ether
for a total injection volume of 67.5 mL. Solvent A was petroleum
ether; Solvent B was 99.9% diethyl ether. Solvents A and B were
employed to elute the column at 200 mL/min in a step gradient of 4
vol % B for 6 column volumes, then 8 vol % B for 4 column volumes,
then 40 vol % B for 4 column volumes. Eluate was monitored at 220
nm and 240 nm. 120 mL fractions were collected. Following elution,
the peak fractions were subjected to analytical HPLC or TLC
analysis. Fractions 1-20 (1-6 CV) and 36-45 (11.5-14 CV) were
combined and solvents removed by rotoevaporation. Analytical HPLC
was employed to determine relative amounts of cannabinoids of
interest.
Example 3. Reverse Phase Preparative Method 3
[0136] In this example, a Biotage Isolera Chromatography System was
employed using a preparative Reverse Phase C18 column to process
raw cannabis oil. 8.0 g hemp oil (injection mass 2 wt %) was
dissolved in 16 mL ethanol and injected to a 400 g RP C18 column
(SNAP C18 400 g) and rinsed with ethanol for a total injection
volume of 24 mL. Solvent A was water; Solvent B was ethanol.
Solvents A and B were employed to elute the column at 100 mL/min in
a gradient of 60-90 vol % B (ethanol/formic acid) over 10 column
volumes. Column eluate was monitored at 220 nm, and 100 mL
fractions were collected. Following elution, the peak fractions
were subjected to analytical HPLC or TLC analysis. Appropriate
fractions were combined and solvents removed by rotoevaporation.
Analytical HPLC was employed to determine relative amounts of
cannabinoids of interest.
Example 4. Reverse Phase Analytical HPLC Method
[0137] Raw cannabis oil and process samples were evaluated by HPLC,
or HPLC-MS by diluting sample at 60 uL/6 mL with an injection
volume of 1 uL. Solvent A is water 0.1% formic acid; Solvent B is
acetonitrile 0.1% formic acid. The C18 analytical column (Agilent
Eclipse Plus C-18/RRHD 1.8 um 21.times.50 mm) is run at 50.degree.
C., at 0.5 mL/mn in a gradient elution of 70% B from 0-3 minutes,
then 70-95% B from 3-5 min. HPLC-MS employed dual ion monitoring
using ES-API, positive ion monitoring at 315.2 amu (Signal 1, MSD1)
and 345.2 amu (Signal 2, MSD2). FIGS. 4-9 illustrate various
analytical HPLC-MS chromatograms. To generate each chromatogram, a
single-quad MS detector rather was used. The total ion chromatogram
as well as two specific masses: 315.2 amu and 345.2 amu.
Calibration curves developed from external reference standards for
Cannabidiol (CBD) (Sigma-Aldrich), and d9-THC (Sigma-Aldrich).
Using this method, HPLC purity of crude cannabis oil used herein
was 60.50% CBD and 3.50% THC. HPLC purity of purified clean
cannabis oil from Method 1 was was 69% CBD and <0.3% THC. HPLC
purity of purified isolate from Method 1 was 95% CBD and <0.3%
THC, as discussed in Example 1.
Example 5. Production Normal Phase CBD Isolate Method
[0138] The following protocol of Example 5 has been used:
[0139] Preparation:
[0140] 1. Dissolve 750 g of crude oil in 400 mL of petro ether. Use
"gentle" heating and cap with foil with a large stir bar. Record
sensory characteristics of starting material description (color,
consistency, smell, etc.) and record actual mass of starting
material (g).
[0141] 2. Once everything is dissolved, let crude oil mixture cool
to room temperature and make sure everything is liquid. There
should be about 1200 mL of total mixture in the syringes. Record
the total volume of mixture prior to injection (mL) and the
description of injection material (color, consistency).
[0142] 3. Prepare 3 gradient tanks:a.G1: 6% ethyl ether in
petroleum ether (3.6 L of ethyl ether in 56.4 L of petroleum
ether); b.G2: 15% ethyl ether in petroleum ether (9 L of ethyl
ether in 51 L of petroleum ether); and c.G3: 80% ethyl ether in
petroleum ether (48 L of ethyl ether in 12 L of petroleum
ether).
[0143] 4. Turn on column compression and set to .about.100 PSI.
[0144] 5. Run G1 for 1.5 CV (13 L) to condition the 5 kg column.
Collect the solvent from this run in a "recycle" solvent container.
Solvent pressure should be between 30-35 PSI and flowing at
.about.1 L per minute.
[0145] 6. Load sample. Increase pressure to .about.80 PSI to load
sample faster. Record Load time (min).
[0146] 7. Decrease solvent pressure back to 30-35 PSI. Begin
running G1 solvent again.
[0147] Collection:
[0148] 8. Initial collection: a.Collect 1 CV (9 L) of solvent in a
10 L carboy labelled A. (If yellow color is observed before 9 L
switch solvent line to a new carboy immediately.) Record volume of
solvent before yellow observed (L). b.Collect an additional 0.5 CV
(4 L) in a 10 L carboy labelled B.
[0149] 9. Fraction collection: Notes: All fractions are 6 L.
Collect all fractions in 10 L carboys.
[0150] a. Collect 1 CV (1 fraction). (6 L).
[0151] b. After Fraction 1 stop solvent flow and switch to G2
ISO.
[0152] c. Continue collecting fractions.
[0153] d. After Fraction 7 stop solvent flow and switch to G3.
[0154] e. Collect Fractions 8, 9, and 10. (If solvent is still
colored yellow after fraction 10 collect additional fractions.
[0155] Analysis:
[0156] 10. Run TLC on fractions to determine CBD containing
fractions for crystallization, disposal, and recombination.
Collected and record each of isolate fractions, raffinate
fractions, THC disposal fractions, collected cannabinoid fractions.
Record description of isolated fractions (approx. volume, color).
Record description of raffinate fractions (approx. volume,
color).
[0157] Raffinate Concentration:
[0158] 11. Collect raffinate fractions in a preweighed 20 L flask.
Concentrate raffinate fractions under reduced pressure. Set vacuum
to 0 mtorr and bath to 55.degree. C. Keep this raffinate for future
use. Record Mass of flask (g). Record Description of concentrated
raffinate oil (color, consistency). Mass of raffinate oil (g).
[0159] Cannabinoid Concentration:
[0160] 12. Collect cannabinoid fractions in a preweighed 20 L
flask. Concentrate cannabinoid fractions under reduced pressure.
Set vacuum to 0 mtorr and bath to 65.degree. C. Keep these
cannabinoids for future use. Record mass of flask (g), description
of concentrated cannabinoids (color, consistency), and mass of
cannabinoid oil (g).
[0161] Crystallization:
[0162] 13. Tare a 5 L round bottom flask. Concentrate isolate
fractions under reduced pressure. Set vacuum to 0 mtorr and bath to
65.degree. C. Record Mass of flask (g), and mass of isolate R1
(g).
[0163] 14. Dissolve isolate R1 in 2.times.(mass of isolate R1) in
mL of hot hexanes. Record volume of hot hexanes used (mL).
[0164] 15. Seed crystallization dish with enough crystals to
sparsely cover the bottom of the container. Cover crystallization
dish with foil and place in freezer at 30.degree. F. for 12 h.
[0165] 16. Decant crystallization dish and rinse crystals with cold
hexanes. The decanted solution and rinse hexanes should be
collected and labelled "mother liquor". Collect and mass rinsed
crystals that remain in the tray. These crystals will be labelled
isolate R2. Record Description of mother liquor/rinse combination
(color). Description of isolate R2 (color, consistency). Mass of
isolate R2 (g).
[0166] 17. Dissolve isolate R2 in .about.2.times.(mass of isolate
R2) in mL of hot hexanes.
[0167] 18. Seed crystallization dish with enough crystals to
sparsely cover the bottom of the container. Cover with foil and let
crystallize for 12 h at room temperature. Record room temperature
(.degree. F.).
[0168] 19. Decant new mother liquor and rinse crystals with cold
petroleum ether. This rinse and decanted material should be
combined with "mother liquor" from Step 16.
[0169] 20. Collect the crystals remaining in tray and crush using
mortar and pestle. Labelled this material isolate F. Mass isolate F
right after grinding. Record Description of final crystals (color,
consistency). Record Mass of isolate F post grinding (g).
[0170] 21. Dry isolate F under reduced pressure or in a vacuum oven
at 50.degree. C. Record Description of isolate F (color,
consistency).Record Mass of isolate F post drying (g).
Example 6. Production THC Removal Method
[0171] The following protocol of Example 6 has been used:
[0172] Preparation:
[0173] 1. Dissolve 500 g of crude oil in 250 mL of petro ether. Use
"gentle" heating and cap with foil with a large stir bar. Record
Starting material description (color, consistency, smell, etc.).
Record Mass of starting material (g).
[0174] 2. Once everything is dissolved, let crude oil mixture cool
to room temperature and make sure everything is liquid. There
should be about 750 mL of total mixture in the syringes. Record
Total volume of mixture prior to injection (mL). Record Description
of injection material (color, consistency).
[0175] 3. Prepare 3 gradient tanks:
[0176] a.G1: 6% ethyl ether in petroleum ether (3.6 L of ethyl
ether in 56.4 L of petroleum ether);
[0177] b.G2: 12% ethyl ether in petroleum ether (7.2 L of ethyl
ether in 52.8 L of petroleum ether); and
[0178] c.G3: 80% ethyl ether in petroleum ether (48 L of ethyl
ether in 12 L of petroleum ether).
[0179] 4. Turn on column compression and set to .about.100 PSI.
[0180] 5. Run G1 for 1.5 CV (13 L) to condition the 5 kg column.
Collect the solvent from this run in a "recycle" solvent container.
Solvent pressure should be between 30-35 PSI and flowing at
.about.1 L per minute.
[0181] 6. Load sample. Increase pressure to .about.80 PSI to load
sample faster. Record Load time (min).
[0182] 7. Decrease solvent pressure back to 30-35 PSI. Begin
running G1 solvent again.
[0183] Collection:
[0184] 8. Initial collection: d.Collect 1 CV (9 L) of solvent in a
10 L carboy labelled A. (If yellow color is observed before 9 L
switch solvent line to a new carboy immediately.) Record volume of
solvent before yellow observed (L).e. Collect an additional 1 CV (9
L) in a 10 L carboy labelled B.
[0185] 9. Fraction collection:
[0186] f. Collect 4 CV (36 L) in 2 L fractions. (18 fractions).
[0187] g. After Fraction 18 stop solvent flow and switch to G2.
[0188] h. Continue collecting Fractions in 2 L portions.
[0189] i. After fraction 31 stop solvent flow and switch to G3.
[0190] 10. Final Fraction Collection:
[0191] j. After Fraction 36 switch collection to 10 L carboys.
[0192] k. Collect two 8 L fractions labelled X and Y. (If solvent
is still colored yellow after fraction Y collect an additional 8 L
fraction labelled Z.)
[0193] Analysis:
[0194] 11. Run TLC on fractions to determine CBD containing
fractions for crystallization, disposal, and recombination. Record
Collected isolate fraction number. Record Collected raffinate
fraction numbers. Record Collected disposal fraction numbers.
Record Description of isolation fractions (approx. volume, color).
Record Description of raffinate fractions (approx. volume,
color).
[0195] Raffinate Concentration:
[0196] 12. Collect raffinate fractions in a preweighed 20 L flask.
Concentrate raffinate fractions under reduced pressure. Set vacuum
to 0 mtorr and bath to 55.degree. C. Record Mass of flask (g).
Record Description of concentrated raffinate oil (color,
consistency). Record Mass of raffinate oil (g).
[0197] Crystallization:
[0198] 13. Tare a 5 L round bottom flask. Concentrate isolate
fractions under reduced pressure. Set vacuum to 0 mtorr and bath to
65.degree. C.Record Mass of flask (g). Record Mass of isolate R1
(g).
[0199] 14. Dissolve isolate R1 in 2.times. (mass of isolate R1) in
mL of hot hexanes. Record Volume of hot hexanes used (mL).
[0200] 15. Seed crystallization dish with enough crystals to
sparsely cover the bottom of the container. Cover crystallization
dish with foil and place in freezer at 30.degree. F. for 12 h.
[0201] 16. Decant crystallization dish and rinse crystals with cold
hexanes. The decanted solution and rinse hexanes should be
collected and labelled "mother liquor". Collect and mass rinsed
crystals that remain in the tray. These crystals will be labelled
isolate R2. Record Description of mother liquor/rinse combination
(color). Record Description of isolate R2 (color, consistency).
Record Mass of isolate R2 (g).
[0202] 17. Dissolve isolate R2 in .about.2.times.(mass of isolate
R2) in mL of hot hexanes.
[0203] 18. Seed crystallization dish with enough crystals to
sparsely cover the bottom of the container. Cover with foil and let
crystallize for 12 h at room temperature. Record Room temperature
(.degree. F.).
[0204] 19. Decant new mother liquor and rinse crystals with cold
petroleum ether. This rinse and decanted material should be
combined with "mother liquor" from Step 16.
[0205] 20. Collect the crystals remaining in tray and crush using
mortar and pestle. Labelled this material isolate F. Mass isolate F
right after grinding. Record Description of final crystals (color,
consistency). Record Mass of isolate F post grinding (g).
[0206] 21. Dry isolate F under reduced pressure or in a vacuum oven
at 50.degree. C. Record Description of isolate F (color,
consistency). Record Mass of isolate F post drying (g).
[0207] Formulation
[0208] 22. Dissolve isolate F in 2.times.(mass of isolate F) in mL
of petroleum ether. Add this mixture to the flask containing
raffinate oil. Stir combined mixture at 55.degree. C. for 20
minutes.
[0209] 23. Concentrate final oil under reduced pressure. Set vacuum
to 0 mtorr and bath to 55.degree. C. Record Mass of flask (g);
(should be the same as Step 12). Record Description of final oil
(color, consistency. Record Mass of final oil (g).
Example 7. Pre-Process. Concentration of CBD
[0210] The following protocol of Example 7 has been used:
[0211] 1. Add 1000 mL of warm crude oil (55.degree. C.) to a 3000
mL beaker. Record mass (g).
[0212] 2. Add 500 mL of petroleum ether to the warm crude oil. Stir
under gentle heat until the crude oil is dissolved to obtain a
homogenous mixture. Use a stir rod and magnetic stirring. Do not
heat mixture over 35.degree. C.
[0213] 3. Let the mixture cool to room temperature over 15
minutes.
[0214] 4. Place the mixture into an ice bath and cool to 0.degree.
C. for 15-60 minutes. Use auto stirrer with a large blade and stir
on the lowest speed setting.
[0215] 5. Add approximately 500 mL of cold petroleum ether to the
solid mixture and use the auto stirrer with the small blade to
re-blend the mixture. Blend until homogeneous. Note: samples can
then be placed into the freezer and left overnight instead of step
6. Placing the samples in the freezer provides slightly better
yield (e.g., 5-10% greater).
[0216] 6. Place the mixture into an ice bath and cool at 0.degree.
C. for 60 minutes. Stir by hand with a large metal spatula every 15
minutes, or with an overhead stirrer constantly to homogenize.
Alternatively, simply let the mixture cool in an ice bath at
0.degree. C. for 60 minutes.
[0217] 7. Add approximately 1 L of cold petroleum ether to the
filter to prime the filter.
[0218] 8. Add 0.5 L of cold petroleum ether to the beaker with the
mixture. Mix gently in the beaker. Pour the mixture into the filter
and stir gently. It should be a light orange/yellow and look like a
smoothie. Break up any clumps at this point by pressing them
against the side of the filter with the spatula.
[0219] 9. Place the filter under vacuum. Mix with a large spatula
until clumpy light yellow solid remains. It should be a cookie
dough like consistency.
[0220] 10. Remove filter from vacuum. Close the vacuum valve. Add
approximately 0.75 L of cold petroleum ether to the filter and stir
with a large spatula until the solid is homogenously mixed. It
should look like a vanilla milkshake.
[0221] 11. Place the filter under vacuum. Mix with a large spatula
until white remains. It should be a powder like consistency.
[0222] 12. Remove filter from vacuum. Close the vacuum valve. Add
approximately 0.75 L of cold petroleum ether to the filter and stir
with large spatula until the solid is homogeneously mixed. It
should look like a vanilla milkshake.
[0223] 13. Place the filter under vacuum. Mix with a large spatula
until white remains. It should be a powder like consistency.
[0224] 14. Using a scoopula or spatula, scrape all the white powder
off of the filter into a tared foil tin. Place into vacuum oven at
50.degree. C. and place under vacuum for one hour.
[0225] 15. Collect the filtrate and concentrate in a 20 L flask for
later use.
Example 8. Production THC Removal Method
[0226] The following protocol of Example 8 has been used:
[0227] Preparation:
[0228] 1. Pour 10.times.20 L bottles of diethyl ether into a 55
gallon drum. Seal the lid on this drum tightly.
[0229] 2. Prepare four gradient tanks:
[0230] a. Prep Tank (PT): 100% petroleum ether (15.9 Gal (60 L) of
petroleum ether)
[0231] b. Gradient 1 (G1): 6% diethyl ether in petroleum ether (1.0
Gal (3.6 L) of diethyl ether in 14.9 Gal (56.4 L) of petroleum
ether)
[0232] c. Gradient 2 (G2): 15% diethyl ether in petroleum ether
(2.4 Gal (9 L) of diethyl ether in 13.5 Gal (51 L) of petroleum
ether)
[0233] d. Gradient 3 (G3): 80% diethyl ether in petroleum ether
(12.7 Gal (48.0 L) of diethyl ether in 3.2 Gal (12.0 L) of
petroleum ether)
[0234] 3. Insert 5 kg Flash 150 silica column into the steel Flash
150 pressure vessel. Gently tap the column with a rubber hammer to
ensure it sits flush with the bottom of the pressure vessel. Close
the lid and tightly seal the pressure vessel lid.
[0235] 4. Ensure all the pressure valves on the entire Flash 150
system are in the closed position.
[0236] 5. Connect the Flash 150 manifold to the house air. Ensure
the house air pressure is no greater than 120 psi using the
regulator.
[0237] 6. Connect the column compression line to the Flash 150
column compression inlet. Turn on column compression and set to 100
psi. Ensure the red pressure indicator is sticking out of the edge
of the Flash 150.
[0238] 7. Connect solvent pressure line A to gradient tank PT via
the quick connect pressure inlet. Connect solvent pressure line B
to the sample loading chamber via the quick connect pressure inlet.
Connect the column inlet line to the solvent outlet valve on the
gradient tank PT.
[0239] 8. Turn on pressure to solvent line A and use the regulator
to set the solvent line pressure at approximately 60 psi. Open the
solvent outlet valve on gradient tank PT.
[0240] 9. Turn column inlet so the valve arrow faces solvent line
PT. This will enable solvent to flow through the column. (Note: If
the column has never been used before, it may take a few minutes
for solvent to start flowing from the column outlet valve.)
[0241] 10. Run gradient tank PT for 17 L (2 CV) to condition the
column. Collect all the solvent from this run in a "recycle"
solvent container. Solvent should flow out of the column at
approximately 2 L per minute. After 17 L of solvent are collected,
turn the solvent inlet to the off position. Turn the solvent outlet
valve on the gradient tank PT to the off position.
[0242] 11. Turn the solvent pressure to line A off. Disconnect line
A from gradient tank PT and connect line A to gradient tank G1 via
the quick connect pressure inlet.
[0243] 12. Turn the column solvent inlet valve so the valve arrow
faces the sample loading vessel line. Increase system pressure to
approximately 80 psi. Turn on pressure to solvent line B. Turn on
the pressure inlet valve of the sample loading vessel.
[0244] 13. The sample will begin loading. Watch the sample travel
through the clear sample loading tube into the column. Immediately
after the entire sample has been loaded onto the column, turn the
solvent inlet valve on the column to the off position.
[0245] 14. Turn off the pressure inlet valve on the sample loading
vessel. Decrease the system pressure to 20-30 psi. Disconnect
solvent pressure line B from the sample loading vessel and connect
solvent pressure line B to gradient tank G2 via the quick connect
pressure inlet.
[0246] Collection:
[0247] 15. Gradient Tank 1 (2.times.10 L fractions, 11.times.2 L
fractions (fractions 1-11)):
[0248] a. Ensure the column outlet tube is placed in a 10 L jug
labelled Pre1.
[0249] b. Turn on solvent pressure line A connected to G1. Ensure
the pressure is at 30 psi. open the solvent outlet valve on
gradient tank G1. Turn column inlet so the valve arrow faces
solvent line G1. Solvent will begin flowing through the column.
[0250] c. Collect 10 L (1 CV) of solvent in a 10 L jug A.
[0251] d. Collect an additional 10 L (1 CV) in a 10 L jug labelled
Pre2. Note: If tallow color is observed before 9 L immediately
switch the column outlet tube to 10 L jug B.
[0252] e. Begin collecting 2 L fractions in fraction vessels (11
total fractions).
[0253] f. After fraction 11, place the column outlet tube in
fraction vessel 12 and immediately turn off the column inlet valve
connected to solvent line G1. Turn off the solvent outlet valve on
gradient tank G1. Turn off solvent pressure line A.
[0254] 16. Gradient Tank 2 (5.times.2 L fractions (fractions
12-16)):
[0255] a. Turn on solvent pressure line B connected to G2. Ensure
the pressure is at 30 psi. Open the solvent outlet valve on
gradient tank G2. Turn column inlet so the valve arrow faces
solvent line G2.
[0256] b. Continue collecting fractions in 2 L portions (5 total
fractions).
[0257] c. While fractions are being collected, disconnect solvent
pressure line A from gradient tank G1 and connect solvent pressure
line A to gradient tank G3 via the quick connect pressure
inlet.
[0258] d. After fraction 16, place the column outlet tube in
fraction vessel 17 and immediately turn off the column inlet valve
connected to solvent line G2. Turn off the solvent outlet valve on
gradient tank G2. Turn off solvent pressure line B.
[0259] 17. Gradient Tank 3 (4.times.2 L fractions (fractions
17-20), 2.times.10 L fractions):
[0260] a. Turn on solvent pressure line A connected to G3. Ensure
the pressure is at 30 psi. Open the solvent outlet valve on
gradient tank G3. Turn column inlet so the valve arrow faces
solvent line G3.
[0261] b. Continue collecting fractions in 2 L portions (4 total
fractions).
[0262] c. After fraction 30 switch collection to 10 L jugs.
[0263] d. Collect one 10 L fraction labelled Post1. (If solvent is
still colored yellow after fraction Post1 collect an additional 8 L
fraction labelled Post2.)
[0264] e. After fraction Post2/3 turn off the column inlet valve
connected to solvent line G3. Turn off the solvent outlet valve on
gradient tank G3. Turn off solvent pressure line A.
[0265] f. Solvent may continue flowing. Continue collecting solvent
in a recycle container until solvent is no longer flowing.
[0266] 18. Ensure all pressure valves on the system are closed.
[0267] 19. Ensure connection to house air is closed.
[0268] 20. Ensure all solvent tanks and the Flash 150 column are
completely vented.
[0269] 21. Clean the sample loading vessel thoroughly with
petroleum ether.
[0270] Analysis:
[0271] 22. Run TLC on fractions to determine CBD containing
fractions for crystallization, disposal, and recombination.
[0272] Raffinate Concentration:
[0273] 23. Collect raffinate fractions in a tared 20 L flask.
Concentrate raffinate fractions under reduced pressure. Set vacuum
to 0 mtorr and bath to 55.degree. C. Analyze final dried raffinate
with LCMS to determine CBD and THC content.
[0274] Crystallization:
[0275] 24. Tare the 20 L round bottom flask. Label with Date/Batch
Number/Initial Tare. Record initial flask tare (g). Record final
flask tare (g). Record mass of isolate oil (g).
[0276] 25. Take the mass of the isolate oil and multiply by 2. The
production of this multiplication is the amount of n-hexane that
should be added to the 20 L round bottom flask. Record volume of
n-hexane (mL).
[0277] 26. Place the round bottom flask on the rotor at 55 rpm in a
hot water bath at 35.degree. C. Do not place under vacuum. Allow to
rotate until all of the isolate oil is dissolved and the mixture is
homogeneous.
[0278] 27. Pour the isolate oil mixture into pyrex trays. Each tray
should be filled appropriately 0.5 to 1 inches high. Immediately
after pouring cover trays with aluminum foil to prevent solvent
evaporation.
[0279] 28. Let the trays cool to room temperature and then lightly
seed with isolate crystals. Recover with aluminum foil.
[0280] 29. Place trays in freezer at -6.degree. C. for
approximately 8-10 hours.
[0281] 30. A layer of crude crystals should have developed. Decant
the orange-yellow hexane layer into mother liquor drum.
[0282] 31. Rinse the crude crystals with approximately 50-100 mL of
cold petroleum ether and decant into mother liquor drum.
[0283] 32. Repeat the rinse.
[0284] 33. Let the crystals dry at room temperature in fume hood
for 1 hour.
[0285] 34. Crush up the crystals and obtain a mass. Record mass of
cyrstals R1 (g).
[0286] Second Crystallization:
[0287] 35. Take the mass of crystals R1 and multiply by 2. The
product of this multiplication is the amount of n-hexane that
should be added to the 20 L Erlenmeyer flask. Cover the flask lid
with a piece of foil to prevent evaporation. Record volume of
n-hexane (mL).
[0288] 36. Stir the n-hexane at 35.degree. C. with a magnetic stir
rod until the solvent comes to a boil.
[0289] 37. Add the crystals from R1 to the hot stirring n-hexane.
Stir at 35.degree. C. with a magnetic stir rod until the solvent
comes to a boil.
[0290] 38. Add the crystals from R1 to the hot stirring n-hexane.
Stir at 35.degree. C. until the solid is dissolved.
[0291] 39. Pour the crystallized mixture into pyrex trays. Each
tray should be filled approximately 0.5 to 1 inches high.
Immediately after pouring cover trays with aluminum foil to prevent
solvent evaporation.
[0292] 40. Let the trays sit at room temperature undisturbed for
8-10 hours.
[0293] 41. A layer of pristine should have developed. Decant the
light-yellow hexane layer into mother liquor drum.
[0294] 42. Rinse the crude crystals with approximately 50-100 mL of
cold petroleum ether and decant into mother liquor drum.
[0295] 43. Repeat the rinse.
[0296] 44. Let the crystals dry at room temperature in fume hood
for 1 hour. Then break up crystals and transfer to grinder.
[0297] 45. Grind crystals into a fine powder and transfer to
aluminum trays. The powder should completely coat the bottom of the
aluminum trays, but the powder layer should be no greater than 0.5
inches.
[0298] 46. Place the aluminum trays with powder into the vacuum
over at 35.degree. C. and place under vacuum for one hour. Record
mass of final crystals (from crystals R1) (g). Record mass of final
crystals (from pre-run isolate) (g).
[0299] Formulation:
[0300] 47. Dissolve specific amount of CBD isolate in 2.times.(mass
of CBD isolate) in mL of petroleum ether. Add this mixture to the
flask containing raffinate oil. Stir combined mixture at 35.degree.
C. for 20 minutes.
[0301] 48. Concentrate final oil under reduced pressure. Set vacuum
to 0 mtorr and bath to 35.degree. C. Record mass of flask (g).
Record description of final oil (color, consistency). Record mass
of final oil (g).
Example 9. Post-Processing. CBG and CBC Isolation
[0302] The following protocol of Example 9 has been used:
[0303] 1. Concentrate the cannabinoids fraction obtained upon
column chromatography of the filtrate oil in Example 8 under
reduced pressure.
[0304] 2. Obtain a mass of the cannabinoids oil. For every gram of
oil, add 1 mL of hot ethanol.
[0305] 3. Stir the hot ethanol and cannabinoids oil until a
homogeneous mixture is obtained (concentration=0.33 g/mL).
[0306] 4. Attach a 400 g reverse phase column to the Biotage
Isolera. Equilabrate for 3 CV with 80% methanol and 20% distilled
water.
[0307] 5. Inject 24 mL of the cannabinoids oil/ethanol mixture (8 g
of cannabinoids oil).
[0308] 6. Run the following step gradient:
[0309] a. flow rate: 100 mL/min
[0310] b. gradient 1: 80% methanol and 20% distilled water for 4
CV
[0311] c. gradient 2: 85% methanol and 15% distilled water for 7
CV
[0312] 7. Collect 120 mL fractions for the duration of the run.
[0313] 8. Run TLC on fractions using 20% diethyl ether and 80%
hexane to confirm the identity of cannabinoid fractions. (Fractions
collected from 2 CV to 3.5 CV are CBG, fractions collected from 6-9
CV are CBC.)
[0314] 9. Concentrate the CBG fractions under reduced pressure.
[0315] 10. Separately concentrate the CBC fractions under reduced
pressure.
[0316] 11. A red oil will remain for the CBG fractions. This can be
recrystallized into a solid using 2:1 heptane:CBG.
[0317] 12. A red oil will remain for CBC fractions. This oil cannot
be recrystallized, but is the pure oil form of CBC.
[0318] By following this protocol, a hydrate of CBG was obtained.
.sup.1H NMR (300 MHz, CDCl3) .delta. 6.26 (s, 2H), 5.50 (s, 2H),
5.27 (t, 1H), 5.06 (t, H), 3.38 (d, 2H), 2.45 (t, 2H), 2.07 (m,
2H), 1.81 (s, 3H), 1.68 (s, 3H), 1.60 (s, 3H), 1.56 (m, 2H) 1.32
(m, 4H), 0.89 (t, 3H) ESMS: 316.24
[0319] Each of the documents identified herein is incorporated in
its entirety by reference. The foregoing description is
illustrative of the disclosure as claimed and is not intended to
limit the scope of the disclosure.
* * * * *