Patent Grant
11084770
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.
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.
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.
Raw cannabis oil containing these and other compounds may be extracted from the cannabis flower/plant using techniques such as CO2 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-à-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.
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.
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.
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.
In an embodiment, the technology relates to a method for extracting compounds from raw cannabis oil.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 %.
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 %.
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.
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.
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 % delta-9-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.
In some embodiments, the raw cannabis oil is obtained by supercritical CO2 extraction of Cannabis spp. plant material, solvent-solid extraction of Cannabis spp. plant material, or from a commercial supplier.
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.
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.
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.
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.
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.
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.
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.
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.
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° C. to provide a cooled mixture; mixing a first amount of petroleum ether at −10° C. to 0° C. into the cooled mixture; freezing the cooled mixture for at least two hours or cooling the cooled mixture at 0° C.; adding a second amount of petroleum ether at −10° C. to 0° 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.
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.
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.
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.
In embodiments, filting the mixture comprises filtering the mixture both at atmospheric pressure and under vacuum.
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 %.
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.
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° 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%.
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° C. into the cannabinoids fraction to provide a mixture; performing reverse phase chromatography 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%.
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.
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° 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%.
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.
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.
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.
In embodiments, the first solvent is 80% methanol and 20% distilled water and the second solvent is 85% methanol and 15% distilled water.
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.
Also disclosed herein is an oil containing CBG produced by these methods. Further disclosed herein is an oil containing CBC produced by these methods.
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.
Further disclosed herein is a hydrate of CBG. Additionally, disclosed herein is a monohydrate of CBG.
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.
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 CO2 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-delta-9-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.
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.
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.
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.
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.
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.
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, 1H-NMR, 13C-NMR, and/or elemental analysis.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In some embodiments, a composition comprising CBD, CBC and terpenes is provided comprising not more than 0.3 wt % of THC.
In some embodiments, a composition comprising CBD and fatty acids is provided comprising not more than 0.3 wt % of THC.
In some embodiments, a composition comprising CBG and CBD is provided comprising not more than 0.3 wt % of THC.
In a specific embodiment, a composition is provided comprising ˜65-70% CBD, 0.001-0.5% THC, 0.3-5% CBC, 0.3-5% CBG, 0.3-5% CBN, and ˜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.
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.
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.
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 CO2 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.
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.
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.
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.
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.
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.
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.
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”.
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.
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.
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.
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% H2O to 60% ethanol (by volume) for 10 CV and the second eluent is 10% H2O and 90% ethanol (by volume). In other embodiments, a reverse phase column 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.
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.
During extraction, liquid chromatography column 102 may be kept at around atmospheric pressure (1 ATM+/−0.2) and around room temperature (22° C.+/−5).
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) CO2 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 CO2 extraction is used as a starting material for the methods described herein. For example, supercritical CO2 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 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.
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
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.
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.
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% H2O to 60% ethanol (by volume).
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% H2O 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.
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% H2O and 90% ethanol (by volume).
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% H2O 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.
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.
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.
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 degrees 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.
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.
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.
As illustrated,
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.
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.
As illustrated,
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.
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.
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
As shown in
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
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.
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.
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 21×50 mm) is run at 50° 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).
The following protocol of Example 5 has been used:
Preparation:
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).
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).
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).
4. Turn on column compression and set to ˜100 PSI.
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 ˜1 L per minute.
6. Load sample. Increase pressure to ˜80 PSI to load sample faster. Record Load time (min).
7. Decrease solvent pressure back to 30-35 PSI. Begin running G1 solvent again.
Collection:
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.
9. Fraction collection: Notes: All fractions are 6 L. Collect all fractions in 10 L carboys.
a. Collect 1 CV (1 fraction). (6 L).
b. After Fraction 1 stop solvent flow and switch to G2 ISO.
c. Continue collecting fractions.
d. After Fraction 7 stop solvent flow and switch to G3.
e. Collect Fractions 8, 9, and 10. (If solvent is still colored yellow after fraction 10 collect additional fractions.
Analysis:
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).
Raffinate Concentration:
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° 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).
Cannabinoid Concentration:
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° C. Keep these cannabinoids for future use. Record mass of flask (g), description of concentrated cannabinoids (color, consistency), and mass of cannabinoid oil (g).
Crystallization:
13. Tare a 5 L round bottom flask. Concentrate isolate fractions under reduced pressure. Set vacuum to 0 mtorr and bath to 65° C. Record Mass of flask (g), and mass of isolate R1 (g).
14. Dissolve isolate R1 in 2×(mass of isolate R1) in mL of hot hexanes. Record volume of hot hexanes used (mL).
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° F. for 12 h.
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).
17. Dissolve isolate R2 in ˜2×(mass of isolate R2) in mL of hot hexanes.
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 (° F.).
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.
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).
21. Dry isolate F under reduced pressure or in a vacuum oven at 50° C. Record Description of isolate F (color, consistency). Record Mass of isolate F post drying (g).
The following protocol of Example 6 has been used:
Preparation:
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).
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).
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: 12% ethyl ether in petroleum ether (7.2 L of ethyl ether in 52.8 L of petroleum ether); and
c. G3: 80% ethyl ether in petroleum ether (48 L of ethyl ether in 12 L of petroleum ether).
4. Turn on column compression and set to ˜100 PSI.
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 ˜1 L per minute.
6. Load sample. Increase pressure to ˜80 PSI to load sample faster. Record Load time (min).
7. Decrease solvent pressure back to 30-35 PSI. Begin running G1 solvent again.
Collection:
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.
9. Fraction collection:
f. Collect 4 CV (36 L) in 2 L fractions. (18 fractions).
g. After Fraction 18 stop solvent flow and switch to G2.
h. Continue collecting Fractions in 2 L portions.
i. After fraction 31 stop solvent flow and switch to G3.
10. Final Fraction Collection:
j. After Fraction 36 switch collection to 10 L carboys.
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.)
Analysis:
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).
Raffinate Concentration:
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° C. Record Mass of flask (g). Record Description of concentrated raffinate oil (color, consistency). Record Mass of raffinate oil (g).
Crystallization:
13. Tare a 5 L round bottom flask. Concentrate isolate fractions under reduced pressure. Set vacuum to 0 mtorr and bath to 65° C. Record Mass of flask (g). Record Mass of isolate R1 (g).
14. Dissolve isolate R1 in 2×(mass of isolate R1) in mL of hot hexanes. Record Volume of hot hexanes used (mL).
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° F. for 12 h.
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).
17. Dissolve isolate R2 in ˜2×(mass of isolate R2) in mL of hot hexanes.
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 (° F.).
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.
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).
21. Dry isolate F under reduced pressure or in a vacuum oven at 50° C. Record Description of isolate F (color, consistency). Record Mass of isolate F post drying (g).
Formulation
22. Dissolve isolate F in 2×(mass of isolate F) in mL of petroleum ether. Add this mixture to the flask containing raffinate oil. Stir combined mixture at 55° C. for 20 minutes.
23. Concentrate final oil under reduced pressure. Set vacuum to 0 mtorr and bath to 55° 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).
The following protocol of Example 7 has been used:
1. Add 1000 mL of warm crude oil (55° C.) to a 3000 mL beaker. Record mass (g).
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° C.
3. Let the mixture cool to room temperature over 15 minutes.
4. Place the mixture into an ice bath and cool to 0° C. for 15-60 minutes. Use auto stirrer with a large blade and stir on the lowest speed setting.
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).
6. Place the mixture into an ice bath and cool at 0° 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° C. for 60 minutes.
7. Add approximately 1 L of cold petroleum ether to the filter to prime the filter.
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.
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.
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.
11. Place the filter under vacuum. Mix with a large spatula until white remains. It should be a powder like consistency.
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.
13. Place the filter under vacuum. Mix with a large spatula until white remains. It should be a powder like consistency.
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° C. and place under vacuum for one hour.
15. Collect the filtrate and concentrate in a 20 L flask for later use.
The following protocol of Example 8 has been used:
Preparation:
1. Pour 10×20 L bottles of diethyl ether into a 55 gallon drum. Seal the lid on this drum tightly.
2. Prepare four gradient tanks:
a. Prep Tank (PT): 100% petroleum ether (15.9 Gal (60 L) of petroleum ether)
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)
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)
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)
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.
4. Ensure all the pressure valves on the entire Flash 150 system are in the closed position.
5. Connect the Flash 150 manifold to the house air. Ensure the house air pressure is no greater than 120 psi using the regulator.
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.
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.
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.
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.)
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.
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.
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.
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.
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.
Collection:
15. Gradient Tank 1 (2×10 L fractions, 11×2 L fractions (fractions 1-11)):
a. Ensure the column outlet tube is placed in a 10 L jug labelled Pre1.
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.
c. Collect 10 L (1 CV) of solvent in a 10 L jug A.
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.
e. Begin collecting 2 L fractions in fraction vessels (11 total fractions).
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.
16. Gradient Tank 2 (5×2 L fractions (fractions 12-16)):
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.
b. Continue collecting fractions in 2 L portions (5 total fractions).
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.
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.
17. Gradient Tank 3 (4×2 L fractions (fractions 17-20), 2×10 L fractions):
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.
b. Continue collecting fractions in 2 L portions (4 total fractions).
c. After fraction 30 switch collection to 10 L jugs.
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.)
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.
f. Solvent may continue flowing. Continue collecting solvent in a recycle container until solvent is no longer flowing.
18. Ensure all pressure valves on the system are closed.
19. Ensure connection to house air is closed.
20. Ensure all solvent tanks and the Flash 150 column are completely vented.
21. Clean the sample loading vessel thoroughly with petroleum ether.
Analysis:
22. Run TLC on fractions to determine CBD containing fractions for crystallization, disposal, and recombination.
Raffinate Concentration:
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° C. Analyze final dried raffinate with LCMS to determine CBD and THC content.
Crystallization:
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).
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).
26. Place the round bottom flask on the rotor at 55 rpm in a hot water bath at 35° C. Do not place under vacuum. Allow to rotate until all of the isolate oil is dissolved and the mixture is homogeneous.
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.
28. Let the trays cool to room temperature and then lightly seed with isolate crystals. Recover with aluminum foil.
29. Place trays in freezer at −6° C. for approximately 8-10 hours.
30. A layer of crude crystals should have developed. Decant the orange-yellow hexane layer into mother liquor drum.
31. Rinse the crude crystals with approximately 50-100 mL of cold petroleum ether and decant into mother liquor drum.
32. Repeat the rinse.
33. Let the crystals dry at room temperature in fume hood for 1 hour.
34. Crush up the crystals and obtain a mass. Record mass of cyrstals R1 (g).
Second Crystallization:
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).
36. Stir the n-hexane at 35° C. with a magnetic stir rod until the solvent comes to a boil.
37. Add the crystals from R1 to the hot stirring n-hexane. Stir at 35° C. with a magnetic stir rod until the solvent comes to a boil.
38. Add the crystals from R1 to the hot stirring n-hexane. Stir at 35° C. until the solid is dissolved.
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.
40. Let the trays sit at room temperature undisturbed for 8-10 hours.
41. A layer of pristine should have developed. Decant the light-yellow hexane layer into mother liquor drum.
42. Rinse the crude crystals with approximately 50-100 mL of cold petroleum ether and decant into mother liquor drum.
43. Repeat the rinse.
44. Let the crystals dry at room temperature in fume hood for 1 hour. Then break up crystals and transfer to grinder.
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.
46. Place the aluminum trays with powder into the vacuum over at 35° 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).
Formulation:
47. Dissolve specific amount of CBD isolate in 2×(mass of CBD isolate) in mL of petroleum ether. Add this mixture to the flask containing raffinate oil. Stir combined mixture at 35° C. for 20 minutes.
48. Concentrate final oil under reduced pressure. Set vacuum to 0 mtorr and bath to 35° C. Record mass of flask (g). Record description of final oil (color, consistency). Record mass of final oil (g).
The following protocol of Example 9 has been used:
1. Concentrate the cannabinoids fraction obtained upon column chromatography of the filtrate oil in Example 8 under reduced pressure.
2. Obtain a mass of the cannabinoids oil. For every gram of oil, add 1 mL of hot ethanol.
3. Stir the hot ethanol and cannabinoids oil until a homogeneous mixture is obtained (concentration=0.33 g/mL).
4. Attach a 400 g reverse phase column to the Biotage Isolera. Equilabrate for 3 CV with 80% methanol and 20% distilled water.
5. Inject 24 mL of the cannabinoids oil/ethanol mixture (8 g of cannabinoids oil).
6. Run the following step gradient:
a. flow rate: 100 mL/min
b. gradient 1: 80% methanol and 20% distilled water for 4 CV
c. gradient 2: 85% methanol and 15% distilled water for 7 CV
7. Collect 120 mL fractions for the duration of the run.
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.)
9. Concentrate the CBG fractions under reduced pressure.
10. Separately concentrate the CBC fractions under reduced pressure.
11. A red oil will remain for the CBG fractions. This can be recrystallized into a solid using 2:1 heptane:CBG.
12. A red oil will remain for CBC fractions. This oil cannot be recrystallized, but is the pure oil form of CBC.
By following this protocol, a hydrate of CBG was obtained. 1H NMR (300 MHz, CDCl3) δ 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
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.
This application is a continuation application and claims priority to U.S. patent application Ser. No. 15/834,826, filed Dec. 7, 2017, 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.
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| 2146731 | Jan 2010 | EP |
| 2332533 | Jun 2011 | EP |
| 2444081 | Apr 2012 | EP |
| 2968259 | Jan 2016 | EP |
| 3028697 | Jun 2016 | EP |
| 3041815 | Jul 2016 | EP |
| 3067058 | Sep 2016 | EP |
| 3119391 | Jan 2017 | EP |
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| 3247402 | Nov 2017 | EP |
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| 3439759 | Feb 2019 | EP |
| 3442515 | Feb 2019 | EP |
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| 3445838 | Feb 2019 | EP |
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| Number | Date | Country | |
|---|---|---|---|
| 20190270691 A1 | Sep 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62431209 | Dec 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15834826 | Dec 2017 | US |
| Child | 16266326 | US |
