Methods for Removing Pesticides from Cannabinoid Extract Oils Using Countercurrent Partition Chromatography
The disclosure provides methods and reagents for removing pesticides or pesticide residues from plant matter such as cannabis plant matter. The method uses Countercurrent Partition Chromatography (CPC).
This application claims the benefit of, and priority to, U.S. Ser. No. 62/567,581 filed Oct. 3, 2017, the content of which is incorporated herein by reference herein in its entirety.
FIELD OF THE DISCLOSUREThe disclosure relates to systems and methods for removing pesticides from plant matter.
BACKGROUND OF THE PRESENT DISCLOSURECannabis is used for recreational purposes, for example, when consuming cannabis flowers or extracts. Also, cannabis finds use in medicine, for example, for relief of pain, nausea, and epilepsy (see. Clinical Decisions (2013) New Engl. J. Med. 368:866-868; Kilmer (2017) New Engl. J. Med. 376:705-707; Berkovic (2017) New Engl. J. Med. 376:2075-2076).
Plant matter, including cannabis plant matter, may contain contaminants such as pesticides, microbes, and heavy metal (see, Dryburgh, Bolan, Grof (2018) Br. J. Clin. Phamracol. DOI: 10.1111/bcp.13695; Moulins, Blais, Montsion (2018) J. AOAC. DOI: 10.5740/jaoacint.17-0495). The concern for adverse health effects of pesticides in foods is evident by the fact that pesticide content is measured by a Hazard Index (HI) (see, Jensen, Petersen, Nielsen (2015) Food Chem. Toxicol. 83:300-307; Evans, Scholze (2015) Food. Chem. Toxicol. 84:260-269). Pesticides are not effectively removed by ethanol extraction alone, or using butane extraction alone, or using carbon dioxide extraction alone. The present disclosure addresses the unmet need for removing pesticides from plant matter, such as cannabis plant matter, by novel methods that use Countercurrent Partition Chromatography (CPC).
SUMMARY OF THE DISCLOSUREWhat is provided is a method for removing pesticides from an oil extract of plant matter, wherein the plant matter contains one or more different kinds of pesticides, the method comprising: (1) The step of washing, soaking, or washing and soaking the plant matter with ethanol, thereby extracting cannabinoids from the plant matter, and resulting in an oil extract that is dissolved or dispersed in the ethanol, (2) The step of removing the ethanol from the oil extract, wherein the removing is optionally with a rotary evaporator, resulting in an ethanol-free oil extract, (3) Dissolving the ethanol-free oil extract in hexane, resulting in a hexane solution that is optionally at a ratio of 1:5 ethanol-free oil extract/hexane (vol./vol.), (4) The step of preparing the stationary phase that comprises of 3 parts ethanol and 1 part deionized water, (5) The step of adjusting the pH of the stationary phase to effect acid-induced ionization or alkali-inducible ionization of an ionizable group on at least one of the one or more different pesticides to produce a pH-adjusted liquid composition, (6) The step of running the pH-adjusted liquid composition in a Countercurrent Partition Chromatography (CPC) cartridge, wherein the cartridge is rotated, and wherein the CPC is run in an ascending mode that produces an upper phase of a binary solvent mixture, and (7) The step of collecting the upper phase of the binary solvent mixture.
Ratio embodiments. In embodiments, ratio of ethanol-free oil extract/hexane can be any of the following values, or a range consisting of any pair of any of these values: 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:14, 1:16, 1:18, or 1:20. In “about” embodiments, the ratio can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:14, about 1:16, about 1:18, or about 1:20.
Ethanol/water embodiments. In embodiments, ratio of ethanol/deionized water can be any of the following values, or an “about” version of any one of the following values, or a range consisting of a pair of any of these values, 0.5 parts ethanol/1 part water, 0.6 parts ethanol/1 part water, 0.7 parts ethanol/1 part water, 0.8 parts ethanol/1 part water, 0.9 parts ethanol/1 part water, 1 part ethanol/1 part water, 1.2 parts ethanol/1 part water, 1.4 parts ethanol/1 part water, 1.6 parts ethanol/1 part water, 1.8 parts ethanol/1 part water, 2 parts ethanol/1 part water, 2.2 parts ethanol/1 part water, 2.4 parts ethanol/1 part water, 2.6 parts ethanol/1 part water, 2.8 parts ethanol/1 part water, 3 parts ethanol/1 part water, 3.5 parts ethanol/1 part water, 4 parts ethanol/1 part water, 4.5 parts ethanol/1 part water, 5 parts ethanol/1 part water, 5.5 parts ethanol/1 part water, 6 parts ethanol/1 part water, 6.5 parts ethanol/1 part water, 7 parts ethanol/1 part water, 7.5 parts ethanol/1 part water, 8 parts ethanol/1 part water, and the like.
Details of the stationary phase and the mobile phase. Step (4) comprises preparing the stationary phase for the CPC run, which is a composition of 3 parts ethanol, 1 part deionized water at 2.0 using phosphoric acid. Water:ethanol solution is the stationary phase, and it is prepared separately from oil hexane and hexane mobile phase.
Further details of Step (6) and other steps. The first step oft should be preparing the stationary phase by homogenizing 3 part of ethanol and 1 part water—once the stationary phase is homogenized you will have to modify the pH to 2.0 using phosphoric acid—this is then run in the CPC as the bed. You have to run the system to complete a 2 volume equivalence of the cartridge (example if the cartridge volume is 1 L then you will have to run 2 liter of the stationary phase-once the stationary phase if in place you can now equilibrate the system with mobile phase (hexane). It should be noted that at this point the equilibration of the system depends on the rotation of the cartridge and the flow of the liquid, (the more rotation more stationary will bleedout)—once the mobile and stationary phase are equilibrated, you can inject the sample the parameters of the ran should be the same as the equilibration state (RPM and flow)—determine the fraction to be collected via HPLCs—perform the collection. Also, note that, the CPC can be run with collection in an ascending mode or with collection in a descending mode, wherein with the ascending mode the hexane phase is collected, and wherein with the descending mode the ethanol/water phase is collected. Step (6), which is disclosed above, depends on the mode of collection ascending mode or descending mode. Ascending mode means that you will be collecting the hexane phase and descending mode means that you will be collecting the ethanol/water phase.
Also provided is the above method, wherein the ionizable group comprises a nitrile group, a pyrrole group, or both a nitrile group and a pyrrole group.
Moreover, what is encompassed is the above method, wherein the ethanol is removed using a rotary evaporator. In another aspect, what is provided is the above method, wherein at least one or the one or more different kinds of pesticides comprises an ionizable group as set forth in Table 1. Furthermore, what is provided is the above method, wherein the one or more different kinds of pesticides is selected from Table 3, Table 4, and Table 5.
What is additionally embraced, is the above method, wherein the step of collecting the upper phase of the binary solvent mixture comprises collecting fractions or collecting one or more runtime cuts from the CPC cartridge.
Moreover, what is contemplated is the above method, wherein the plant matter comprises cannabinoid acids, and wherein the method comprises the step of decarboxylating the cannabinoid acids prior to introducing a plant extract into the CPC cartridge. Additionally, what is contemplated is the above method, wherein the plant matter is derived from Cannabis sativa.
In yet another aspect, what is embraced is the above method, wherein the plant matter is derived from Cannabis sativa, and wherein the derived comprises derived by one or more of chopping, drying, drying and powdering, of removing stems and seeds. Additionally, what is provided is the above method, wherein the plant matter comprises cannabinoid acids, and wherein the method comprises the step of decarboxylating the cannabinoid acids.
What is provided is the above method, wherein the plant matter comprises plant sugars, the method further comprising the step of removing the plant sugars prior to introducing to the CPC cartridge and running the CPC. Additionally, what is embraced is the above method, wherein the step of adjusting the pH to effect acid-induced ionization or alkali-inducible ionization of an ionizable group on at least one of the one or more different pesticides to produce a pH-adjusted liquid composition, consists of adjusting the pH to effect acid-induced ionization.
Distillation and removing plant sugars. Plant sugars can be removed by way of distillation, where distillation fits into The overall scheme of the process. This overall scheme is as follows:
Extraction of cannabinoid using ethanol.
Removal of ethanol to acquire crude oil devoid of ethanol.
Activation of the crude oil to convert THF-acid to THC.
Distillation of the activated crude to acquire 90% cannabinoid content.
CPC run of the distilled oil.
This is the most efficient way of cleaning the oil. Since your starting material is already at 90% potency.
Moreover, what is provided is the above method, wherein the step of adjusting the pH to effect acid-induced ionization or alkali-inducible ionization of an ionizable group on at least one of the one or more different pesticides to produce a pH-adjusted liquid composition, consists of adjusting the pH to effect alkali-induced ionization.
Exclusionary embodiments for alkali modification are as follows. But note that, regarding alkali modification, the inventor prefers to not include an alkali modification in order to avoid soap formation in the CPC cartridge. In an exclusionary embodiment, the present disclosure can exclude any method or reagents that are used for an alkali modification of ionizable pesticides, when using CPC method.
Furthermore, what is provided is the above method, wherein the one or more different kinds of pesticides is selected from Table 3, Table 4, and Table 5, wherein the method results in partial or substantial depletion of at least one of said one or more different kinds of pesticides, and wherein said partial or substantial depletion is measurable by comparing the total milligrams of pesticide in an aliquot of fraction or runtime cut of the CPC run, where the comparing is with the total milligrams of pesticide in the plant matter that was used in the CPC run.
In composition embodiments, what is provided is a composition that comprises cannabinoids, wherein the composition is prepared by the method or methods detailed above, wherein the composition is partially or substantially depleted in pesticides, and wherein the partially or substantially depleted is measurable by comparing the total milligrams of pesticide in an aliquot of said composition with the corresponding quantity of plant matter from which said aliquot was derived.
Working definition of substantially depleted. The term “substantially depleted” can refer to a relationship between plant matter from which an extract is prepared and then processed to give a product. Where “substantially depleted” refers to a pesticide, or perhaps to a contaminant, it can mean that the product contains less than 50% that in the starting material, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 2% that of that contained in the starting material. In a preferred calculation, what is compared is the amount in the entire starting material, and the amount in the product, where the product represents all of the available product. In an acceptable calculation, what can be compared is the amount in the entire starting material and the amount of product, correcting for intermediate compositions that were removed from the process. For example, removal can be for archiving and storage of a fraction of material from an intermediate step. Also, removal can be for accidental spilling and loss of a fraction of material from an intermediate step.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENTAs used herein, including the appended claims, the singular forms of words such as “an,” and “the” include their corresponding plural references unless the context clearly dictates otherwise. All patent references cited herein are incorporated by reference to the same extent as if each individual patent, and published patent application, as well as figures, drawings, compact discs, and the like, was specifically and individually indicated to be incorporated by reference.
CannabinoidsOne of more of the following cannabinoids can be included in the compositions of the present disclosure. Alternatively, one of more of the following cannabinoids can be excluded (omitted) from the compositions and methods of the present disclosure. Cannabinoids and related compounds include, for example, cannabigerol; carmabichromene; carmabitriol; cannabidiol; cannabicyclolol; cannabinodiol; cannabinol; delta-8-tetrabydrocannabinol; delta-9-tetrahydrocannabinol; cannabichromanone; carmabicoumaronone; cannabicitran; 10-oxo-delta-6a10a-tetralaydrocannabinol; cannabiglendol; delta-7-isotetrahydrocannabinol; CBLVA; CBV; CBEVA-B; CBCVA; delta-9-THCVA; CBDVA; CBGVA; divarinolic acid; quercetin; kaemferol; dihydrokaempferol; dihydroquercetin; cannflavin B; isovitexin; apigenin; naringenin; eriodictyol; luteolin; orientin; cytisoside; vitexin; canniprene; 3,4′-dihydroxy-5 -methoxy bibenzyl; dihydroresveratrol; 3,4′-dihydroxy-5,3′-dimethoxy-5′-isoprenyl; cannabistilbene 1; cannabistilbene 11a; cannabistilbene 11b; cannithrene 1; cannithrene 2; cannabispirone; iso-cannabispirone; cannabispirenon-A; cannabispirenone-B; cannabispiradienone; alpha-cannabispiranol; beta-canniabispiranol; acetyl-cannabispirol; 7-hydroxy-5-methoxyindan-1-spiro-cyclohexane; 5-hydroxy-7-methoxyindan-1-spiro cyclohexane; myristic acid, palmitic acid, oleic acid, stearic acid, linoleic acid, linolenic acid, arachidic acid, eicosenoic acid, behenic acid, lignoceric acid, 5,7-dihydroxyindan-1-cyclohexane; cannabispinidienone; 3,4′-dihydroxy-5-methoxybibenzyl; canniprene; cannabispirone; cannithrene 1; cannithrene 2; alpha-cannabispiranol; acetyl-cannabispirol; vomifoliol; dihydrovomifoliol; beta-ionone; dihydroactinidiolide; palustrine; palustridine; plus-cannabisativine; anhydrocannabsativine; dihydroperiphylline; cannabisin-A; cannabisin-B; cannabisin-C; cannabisin-D; grossamide; cannabisin-E; cannabisin-F; cannabisin-G; and so on (see, e.g., Flores-Sanchez and Verpoorte (2008) Secondary metabolism in cannabis in Phytochem. Rev. DOI 10.1007/s11101-008-9094-4).
Regarding different numbering systems for the same compound, AVIV (US 2004/0110827) states that: “It should be noted that for historical reasons, these cannabinoid analogs are still named following the previous nomenclature, where the terpenic ring was the base for the numbering system. Then the chiral centers of THC type cannabinoids were at carbon atoms 3 and 4. The accepted nomenclature is now based on the phenolic ring as the starting point for numbering. Thus, THC that was previously described as delta-1-THC was later renamed delta-9-THC, similarly delta-6-THC was renamed delta-8-THC, and the chiral centers are at carbons 6a and 10a.” AVIV also has this comment about enantiomers: “delta-9-THC was established by Mechoulam R. et at in 1967 and found to be of (−)-(3R,4R) stereochemistry. It was later found that the psychotropic activity of cannabinoids resides in the natural (3R,4R) OH series, while the opposite enantiomeric synthetic series (3S,4S) was free of these undesirable effects.”
According to Chulgin, the numbering system most broadly used recognizes both the terpene nature and the aromatic nature of the two different parts of the cannabinoid. Here, the terpene is numbered from the ring carbon that carries that branched methyl group, and this is numbered 7, and the remaining three carbons of the isopropyl group are then numbered sequentially. The advantage to this numbering system is that this numbering system is applicable whether the center ring is closed or open. Other numbering systems are the biphenyl numbering system, the Chemical Abstracts system (substituted dibenzoman numbering), and the Todd numbering system (pyran numbering) (see, Chulgin A T (1969) Recent developments in cannabis chemistry. J. Psychedelic Drugs. pp. 397-415.
Thermal decarboxylation. Thermal decarboxylation of cannabinoids, for example, delta-9-tetrahydrocannabinolic acid (THCA-A), cannabidiolic acid (DBDA), and cannabigerolic acid (CBGA) is described (see, Wang, Aula, Khan (2016) Cannabis and Cannabinoid Res. 1.1:262-271).
Removing sugar. In a sugar-removing embodiment, what is provided is a method for removing sugar from an oil matrix, the method comprising providing an oil matrix that contains at least sugar, followed by the steps of: (a) Dissolving an oil matrix to homogeneity, wherein the dissolving is in an organic solvent, (b) Cooling the oil matrix to about minus 50 degrees C., (c) Filtering and collecting the filtered material which is herein called a supernatant, (d) Alkalizing the supernatant and allowing phases to develop where the developed phases include an organic phase, (e) Separating the phases, (f) A phase collection step that collects the organic hexane phase, (g) Repeating at least once the phase separation step, (h) Adding bentonite to the organic phase followed by collecting the hexane layer, (i) The step of separating the hexane from the distillate oil, (j) The step of distillation to remove any residual hexane.
Sources of Oil Extract. Without implying any limitation, an oil extract of the present disclosure is a starting material, and where the starting material is the oil that is collected during extraction. An oil extract acquired from plant matter can include one or more of the following: (1) Chlorophyll; (2) Pigments and tannins; (3) Sugars and fats; (4) Phytocannabinoids; (5) Traces of extracting solvent; (6) Pesticides.
Cannabinoids, terpenes, and a combination of cannabinoids and terpenes, can be extracted from plant matter. One method for extraction is to press it to get what is called a live resin. Other methods called liquid-solid extraction use an extracting solvent such as hexane, heptane, ethanol, butane, or carbon dioxide (CO2). The use in the methods of the present disclosure, the inventor received various types of crude oil that were produced by extraction using ethanol, butane, or carbon dioxide. Also, crude oil of the present disclosure can be produced by extraction using acetone and hexane.
Partitioning of each member of a collection of solutes into sure of two different solvent layers. Liquids that do not dissolve into each other are called immiscible. When combined with each other, two immiscible liquids arrange themselves into two layers. If one of the solvents is water and the other is hexane (an organic solvent), solutes that are hydrophilic will accumulate more in the water layer. But if the solute has low polarity and is hydrophobic, this solute will accumulate more in the hexane layer. Partitioning coefficient (Kp) equals [solute concentration in organic layer]/[solute concentration in water layer]. The following concerns the situation where two immiscible solvents are competing, with each other to dissolve a given solute. The pH of the solvent can influence which solvent (water versus hexane) is chosen by the solute. In other words, the pH of the solvent can influence the partitioning coefficient. A solute of high polarity will preferably dissolve in the water layer (also known as, water phase). Using the example of phenol, the partitioning coefficient is 1.25, where the system has an upper benzene layer and a lower water layer. If some sodium hydroxide is added to the system, resulting in alkalinization, this encourages protons to dissociate from the phenol, where the result is phenol with a greater negative charge (it takes the form of a phenolate anion). The greater charge on the phenol causes it to avoid the upper benzene layer and instead to migrate into the lower water layer. See, Ronald B. Davis. Partitioning and Liquid-Liquid Extraction. ChemSurvival. Georgetown University, Wash. D.C.
The following concerns a mixture of desired chemicals that you want to keep and undesired chemicals that you want to get rid of. An example is a mixture of cannabinoids (need to keep) and pesticides (need to get rid of). Please imagine that all of these chemicals partition equally between an upper benzene phase and a lower water phase. However, in the event that at a pH extreme, what materializes is a difference in partitioning coefficients between the desired and undesired chemicals. If this happens, this difference that materializes can be used to exploit the phenomenon of partitioning to purify the desired cannabinoids.
Countercurrent Partition Chromatography (CPC). According to one source, “Counter-current chromatography is a . . . versatile separation technique which does not require a solid stationary phase. It relies simply on the partition of a sample between the two phases of an immiscible solvent system . . . [c]rude plant extracts and semi-pure fractions can be chrotnatographed, with sample loads ranging from milligrams to grams. Aqueous and non-aqueous solvent systems are used and the separation of compounds with a wide range of polarities is possible” (see, Marston and Hostettman (2006) J. Chromatography A. 1112:181-194).
In the following commentary, the abbreviation “SS” means “solvent systems” and the abbreviation “CCS” means “countercurrent separation.” The use of multiphasic SSs to effect separations is the unifying feature of liquid-liquid separation techniques. Many formulations are available for producing biphasic SSs. Two or more solvents may be mixed in a great number of proportions. The biphasic SS is optionally pre-equilibrated in a separatory funnel before the two phases are separated and equilibrated in the CCS column. In addition, modifying solutes may be added to the mixture either before or after pre-equilibration. Describing SS formulation gives the solvent combinations followed by their volume ratios. Generally, solvents are reported in the order of least polar to most polar. In many cases, the volume ratios are given in whole number ratios, but decimals are sometimes used (see, Friesen, McAlpine, Pauli (2015) J. Nat. Prod, 78:1765-1796). Solvents suitable for countercurrent separation include, petroleum ether (Pet), hexane (H), cyclohexane (Cy), toluene (Tol), methyl tent-butyl ether (ter), tetrahydrofuran (Tet), diethyl ether (De), ethyl acetate (E), chloroform (Ch), acetone (At), DMSO (So), acetonitrile (Ac), isopropyl alcohol (Iso), n-propanol (Pro), n-butanol (Bu), acetic acid (Aa), ethanol (Et), methanol (M), and water (Wat). While most two-phase SSs are mixtures of three, four, or even more solvents, there are several combinations of just two liquids that are immiscible and suitable for CCS. Examples are HepM, EtWat, BuWat, ChWat, and finally OctWat (see, Friesen, McAlpine, Pauli (2015) J. Nat. Prod. 78:1765-1796). “Oct” may refer to octane or octanol.
Countercurrent Partition Chromatography may be further defined by comparing it with conventional column chromatography, such as chromatography where the column is situated vertically and flow of solvent that separates and elutes various compounds is driven by gravity. This comparison has been described as, “Liquid-liquid partition chromatographic techniques have advantages over chromatographic techniques such as . . . column chromatography and HPLC . . . [b]ecause irreversible adsorption does not occur, simple rinsing of the instrument allows full recovery of non-eluted compounds. Moreover, pretreatment of samples is unnecessary and purification can be accomplished in a short time. Significantly, liquid-liquid partition chromatographic techniques do not require expensive solid support” (see, Kato and Hatanaka (2008) J. Lipid Res. 49:2474-2478).
Suppliers of Reagents and EquipmentFor Countercurrent Partition Chromatography (CPC) embodiments of the present disclosure, inventor used equipment from Kromaton (Kromaton, Groupe Rousselet-Robatel, 07110 Annonay, France). Kromaton supplies Fast Centrifugal. Partition Chromatography (FCPC®) equipment, including FCPCA, FCPCB, FCPCC, and FCPCD. Additionally, Kromaton supplies peripheral equipment, such as a pump (isocratic mode, gradient mode), detection (UV-visible, ELSD detector (evaporative light scattering detector), mass spec), Fraction collector, and the like.
Chromatography media and columns, pumps, thermometers, mixers, chemical reagents, and the like, are available from Bio-Rad Laboratories, Hercules, Calif., Cole-Panner, Vernon Hills, Ill., Grainger, Lake Forest, Ill., Sigma-Aldrich, St. Louis, Mo., Fisher Scientific, Pittsburgh Pa., VWR International, Radnor, Pa. Useful labels include 33P, 35S, 14C, 3H, stable isotopes, fluorescent dyes, or fluorettes (see, e.g., Rozinov and Nolan (1998) Chem. Biol. 5:713-728).
This concerns the “6 liter to 25 liter cartridge” that is used in the present disclosure. The cartridge is Gilson, Inc., Madison, Wis. Gilson manufactures the CPC cartridge from Stainless Steel-316 (58316) (this includes all microfluidic lines). For all seals for the connectors, mainly composed of polyvinylidene fluoride for a highly inert material and thermoplastic fluoro polymer. Methods, equipment, and solvents for preparing extracts of cannabis are available (see, Romano and Hazekamp (2013) Cannabinoids, 1:1-11; Pure Extraction (2017) Super/Subcritical. Pure Extraction, Vancouver, BC, Green Mill Supercritical, Pittsburgh, Pa.; Hazekamp (accessed July 2018) Cannabis Oil: What is the Best and Healthies way to Produce Cannabis Oil. IAM Bulletin, Int. Association for Cannabis Medicines). Extracts can be made using naphtha, petroleum ether, ethanol, butane, olive oil, isopropyl alcohol, propane, vegetable oil, butter, carbon dioxide, ethanol combined with carbon dioxide, acetone, and so on. In exclusionary embodiments, the present disclosure can exclude any method, crude extract, and system that uses or that was prepared with one or more of the above solvents. The present disclosure can include, or exclude, and method that uses or any extract prepared by supercritical fluid extraction or subcritical fluid extraction.
Dilated phosphoric acid embodiments. System, compositions, reagents, an methods of the present disclosure can use pure, reagent grade phosphoric acid, as well as pure, reagent grade phosphoric acid that occurs at various dilutions in water, preferably distilled water, deionized water, and the like. Deionized water can be prepared with EV ID Millipore® Milli-DI™ Water Purification System. The present disclosure provides diluted phosphoric acid at dilutions of 90% phosphoric acid/10% water (vol./vol.), 85% phosphoric acid/15% water (vol./vol.), 80% phosphoric acid/20% water (vol./vol.), 75% phosphoric acid/25% water (vol./vol.), 70% phosphoric acid/30% water (vol./vol.), 65% phosphoric acid/35% water (vol./vol.), 60% phosphoric acid/40% water (vol./vol.), 50% phosphoric acid/50% water (vol./vol.), 40% phosphoric acid/60% water (vol./vol.), 30% phosphoric acid/70% water (vol./vol.), 20% phosphoric acid/80% water (vol./vol.), 15% phosphoric acid/85% water (vol./vol.), 10% phosphoric acid/90% water (vol./vol.), and the like. “About” embodiments of the above values are also provided by the present disclosure.
In “range” embodiments, the present disclosure provides diluted phosphoric acid, with phosphoric acid at the indicated percent range (with the remaining percentage being water), and where the phosphoric acid percent ranges that are encompassed by the present disclosure include, 98-100%, 95-100%, 90-100%, 90-95%, 85-90%, 85-95%, 80-85%, 80-90%, 75-80%, 75-85%, 70-75%, 70-80%, 65-70%, 65-75% 60-65%, 60-70%, 55-60%, 55-65%, 50-55%, 50-60%, 45-50%, 45-55%, 40-45%, 40-50%, 35-40%, 35-45%, 30-35%, 30-40%, 25-30%, 25-35%, 20-25%, 20-30%, 15-20%, 15-25%, 10-15%, 10-20%, 5-10%, 5-15%, and the like.
Less-preferred alkalinized embodiments. The present disclosure provides plant extracts and oil extracts that are alkalinized, for example, by adding sodium hydroxide solution, and where the sodium hydroxide solution is added to effect partial or substantial ionization of one or more pesticides. For example, alkalinization can encourage dissociation of a proton from an alcohol group, resulting in a negatively charged ion, and where the negative charge influences partitioning of the pesticide, where the end-result is separation and removal of that pesticide (undesired) from cannabinoids (desired). Sodium hydroxide solution (e.g., NaOH dissolved in water) can be added, where the result is adjusting the pH of any solution, or any suspension, or any oil, extract, to a pH of about pH 7.5, about pH 8.0, about pH 8.5, about pH 9.0, about pH 9.5, about pH 10.0. about pH 10.5, about pH 11.0, about pH 11.5, about pH 12.0, about pH 12.5, or about 13.0, and so on.
Less-preferred alkalinized embodiments (continued). Moreover, sodium hydroxide solution can be added, where the result is a solution, suspension or oil extract, that has been adjusted to a pH in the range of pH 7.5-8.0, in the range of pH 8.0-8.5, in the range of pH 8.5-9.0, in the range of pH 9.0-9.5, in the range of pH 9.5-10, in the range of pH 10-10.5, in the range of pH 10.5-pH 11, in the range of pH 11-pH 11.5, in the range of pH 11.5-pH 12, in the range of pH 12-pH 12.5, in the range of pH 12.5-13, and so on.
In an exclusionary embodiment the present disclosure can exclude alkali modification of ionizable pesticides, in order to avoid soap formation in the CPC cartridge.
Fractions and runtime cuts. The system, apparatus, instrumentation, and methods of the present disclosure can separate eluant from Countercurrent Partition Chromatography (CPC) cartridge into a plurality of fractions or into “runtime cuts.” The entire eluate, or substantially the entire eluate, from the cartridge can be collected in fractions. For example, each fraction can be collected in a test tube, where each fraction has a volume of about 1 mL, about 2 mL, about 5 mL, about 10 mL, about 20 ml, about 50 mL, about 75 mL, about 100 mL, about 150 mL, about 200 mL, about 500 mL, about 750 mL, about 1,000 mL (one liter), and so on. Following collection of the fractions, each fraction can be subjected to an assay method that is sensitive to a cannabinoid, where fractions that are enriched in one or more cannabinoids can be combined.
Alternatively, the CPC cartridge can be optimized, so that the elution throes of the desired cannabinoids are known. Where the elution times have been pre-determined, fluid eluting from the CPC cartridge can be selectively collected, with fluid occurring before and fluid occurring after the selectively collected fluid being diverted (or discarded). For example, a “runtime cut” can take the form of all fluid eluting from the CPC cartridge between 2.5 minutes and 3.0 minutes.
Ionization of pesticides versus pH of solution. The following discloses, for a number of chemical groups, if the group is ionized in a solution of extremely low pH or if the group is ionized at extremely high pH. These chemical groups are found in some of the pesticides that are removable by the methods, systems, and reagents of the present disclosure (see, Table 1). For use in the present compositions and methods for removing pesticides, the pH value of the solution or buffer can be adjusted in order to ensure ionization of the pesticide that needs to be removed.
The Countercurrent Partition Chromatography (CPC) method takes advantage of how pesticide and the oil can be isolated from each other via liquid-liquid separation. CPC uses an approach that is similar to that used with HPLC (high pressure liquid chromatography) where analytes that you feed in the column have various retention times and mobilities based on the chemistry of the analyte and the mobile phase in addition to the flow and type of solid phase bed. However, the main differences that is used for the present disclosure, is that in CPC there is no solid phase media that facilitate the separation. Instead, in the CPC method of the present disclosure, the analytes of interest are separated based on their behavior in a solution composing of two phases (such as heptane and ethanol) by changing the polarity of the biphasic solvent you can change the migration of the analyte and possibly separating them. The CPC method of the present disclosure allows one to change the pH of the solution, where the result is acceleration of the separation and acquisition of greater resolution. In this process the inventor used low pH module in heptane-ethanol-water system to remove residual pesticide from the oil mixture.
Extracting Cannabinoids with One or More Solvents. Various methods and solvents are available for extracting cannabinoids from plants, cannabis plants, cannabis flowers, dried plants, dried cannabis flowers, and the like to produce a crude extract. The sample is activated into its decarboxylated form and run on the distillation unit once to raise the total cannabinoid potency From 70% to 89% which then can be feed in the CPC method. For CPC, there is no need for distillation but the material has to be activated. For both the butane and CO2 extracts, the crude oil must be winterized, activated and ran on the distillation unit to raise it total cannabinoid concentration from 60% to 89%. For CPC method, the activated oil can be run directly into it. For any other form of extraction, the crude oil must be filtered via charcoal column to remove excessive chlorophyll, tannins, protein matter, and certain carbohydrates. This is then run into a still where the total cannabinoids concentration is raised from 13-50 to 65-70%. Further step for this crude oil is to remove the sugar and water soluble alcohol prior to remediation as it will interfere with the polarity of the solvent system for CPC method, which can cause deleterious effect to the outcome of the process.
Stationary Phase (Ethanol, Water, Phosphoric Acid) for Use in CPCThe following provides a step-by-step description now all of the following things are used, and fit in together, in a preferred method and system of the present disclosure. The things are: (1) Cannabis plant matter. This is the source of cannabinoids, more specifically, of phytocannabinoids, (2) Oil extract (this is the starting material for the process), (3) (this is the dissolving media for the process), (4) Water (to allow the cationic/anionic exchange), and (5) Acid water (pH modifier).
Step 1. Plant material that has been depleted of cannabinoids by way of extraction using ethanol as the extraction solvent. The result is an oil extract.
Step 2. Oil extract is collected once the plant material is washed with ethanol and that ethanol is rotary evaporator.
Step 3. Once the oil extract is devoid of ethanol it will be disssolved in hexane, hexane will be added in the ratio of 1:5 oil:hexane.
Step 4. Acid water (phosphoric acid in water) will be added in the hexane mixture to protonate amino groups of the pesticides. 50 ml of phosphoric acid is added water then added to the solution, then a pH will be used to determine the pH (the pH, has to be between steps 1-2).
Adjusting pH to ionize nitrite groups and pyrrole groups (use in CPC). The present inventor discovered how to adjust the pH of the system in a way that allows us to slightly ionize the nitrite and pyrrole groups. Table 2 discloses pesticides, which can be removed by the methods of the present disclosure, where the pesticides contain a nitrite group, a pyrrole group, an azole group, an amino group, an alcohol group, or a nitrate group (Table 2).
Nitriles can be protonated and acquire a positive charge. The protonation reaction involves conversion of R—C≡N: to positively charged R—C≡N+—H (see, www dot chemistry score dot com).
The pyrrole group can be protonated at the C2-carbon of the pyrrole, as a result of the pyrrole donating the ring-nitrogen's lone pair to the ring, resulting in the materialization of a net negative charge at the C2-carbon. The end-result is a stable positive charge on the pyrrole's ring-nitrogen atom (see, www dot chegg dot com).
By inducing the partial charges on these molecules (Table 2) with the aid of pH manipulation, pesticide exhibit as an ion exchangable compound. Furthermore, there are analytes that in extreme pH can naturally degrade, and these are the pesticides that contains esters such as bifenazate, spinosad, cypermetrin, permethrin, triofloxystrobin, cyftutrin, spirotetramat and pipetonyl butoxide. Degradation by-product contains an alcohol and a carboxylic acid derivative which can be trapped in silica alumina column. Furthermore, the alcoholic derivatives can be easily distilled to remove all remnants in the oil mixture solution. See, it is the concerted effect in each stages of the cleaning provides an additionally mechanism for removal to increase the efficiency of the remediation process. Collectively in a step-wise process, the degree of removal depends on the following hierarchy:
Esters<alcohols<carboxylic acids<nitrile<pyrrole<azole<nitrate<amino
For heterocyclic pyrroles and azoles the degree of activation depends on the degree of its ionizability which are directly correlated to the eleetronegativity of the atoms included in the heterocyclic ring. This refers to the electron withdrawing and electron donating capacity of those atoms which are describe in accordance to the Huckel rule.
This provides a detail regarding the above-disclosed degradation by-products. Pesticides with esters, when exposed to an extreme pH, can degrade to an alcohol derivative or to a carboxylic acid derivative. This is documented below for the example of bifenthrin (Fecko A (1999) Environmental Fate of Bifenthrin. Environmental Monitoring and Pest Management Branch, Dept. of Pesticide Regulation, Sacramento, Calif.). Bifenthrin is, (2-methyl-1,1-biphenyl-3-yl)-methyl-3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethyl cyclopropanecarboxylate. The Fecko publication discloses that bifentrin can be degraded to produce, bifentrin alcohol, bifentrin acid, TFP acid, 4′-hydroxy bifentrin (hydrolysis product), and bifentrin aldehyde.
Equipment:
Counter Current Partition Chromatography (CCP): 6.0 liter to 25 liter cartridge, with SSI pumps to deliver 200 mL/minute. Fraction collector. (Waters Corp., Milford, Mass.). Column Filter: 200/300 column chromatography. Homogenizer: Overhead mixer with variable control. 5 liter beaker with borosilicate insert. Rotary evaporator (5.0 liter capacity). Distillation unit: 2 inch still (Pope Scientific, Milwaukee, Wis.). Solid-Liquid Separator: Cole-Parmer particle separator. Pump system: 30 gallons per minute (gpm) with inline value for solvent velocity control.
Pesticides that can be Removed. Table 3 pro a list of pesticides that can be removed by process of the present disclosure.
There are other pesticides that can be removed with this process, but where the process requires high dosage and usually two passes depending on the amount of pesticide in the mixture. These residues are listed in Table 4.
Countercurrent Partition Chromatography (CPC) for Removing Pesticides
The present disclosure provides a CPC process of remediation, where this process is unique in a way that the pH modification enhances the separation of pesticide from the oil mixture, allowing them to dissolve more in the stationary phase (the converse of the statement is also true, since the ionic strength of the alcohol is high, this decreases the solubility of THC (thin layer chromatography) and effectively affecting its partition coefficient increasing the resolution of separation). A solvent system of the present disclosure, heptane/ethanol/water, are used for purification purposes but its limitations are in the sense of removing pesticide as pesticides are very lipophilic where if not derivatized, they will be interact in the solvent system as THC would. The following explains the statement about “limitations” of the solvent system. The solvent system on the present disclosure, heptane/ethanol/water as described, is used for the purification purposes of phytocannabinoids but there may be limitations in the removing pesticides. Pesticides are very lipophilic which favors their partition coefficient closely to those in phytocannabinoids.
How the cartridge used in CPC processes the upper phase and the lower phase. CPC works by taking advantage of the partition coefficient of the compound. Meaning that if one was to create an azeotropic system of hexane and ethanol (biphasic) a given compound would have a relative distribution on both phases. This equation is illustrated as Kd=[Corganic]/[Caqueous]. This separation is done inside the cartridge at about 1000 times consecutively increasing the resolution of separation each time a wash is done inside the cartridge. With that being said, the azeotropic solution inside is said to be immiscible and it should have two layer (heptane on the top and ethanol water on the bottom).
Further illustration is that the separation can be compared to that of an HPLC (where the stationary phase is solid-silica) here the stationary phase is the aqueous part (ethanol/water) the ethanol water will stay inside the cartridge throughout the run (this should be equilibrated), and once the run has started the solution that come our will be the heptane (or the upper phase) this process is said to be ascending mode since you are collecting the upper phase. If you ran for example in descending mode, this means that the stationary phase will be the upper phase and you will be collecting the ethanol/water phase.
The time frame for these process depends on the parameter of the method. In the experiments of the present inventor, the upper phase start to collect at fifth fraction (at 32 ml per fraction at 26 ml/min flow if one does the math it should come out at 5.7 min) this will continue till you get the traction in which your analyte elute (on our experiment the active was in fraction 28-25—conservative collection). Furthermore, the stationary phase had to be replaced depending on the method injection volume which well be determined during the method validation. For the experiments of the present inventor, experiment the inventor did 3 different composition for stationary phase, that is, only with water, at pH 4 then at pH 2.
Description of the ascending mode. In “4.1. Part one: Fraction method 1” it refers to an “ascending mode.” The ascending mode refers to the differentiation of which solvent is used as the mobile phase and as the stationary phase. In ascending more, you are using the upper phase (heptane) as the mobile phase that carries the eluted active, and in descending more you are using heptane as the stationary phase. Whatever solvent is used as the stationary phase, it will stay in the column until it is saturated and you will have to replace it with a new one.
Description of the upper phase. The term “upper phase” does not mean the chemical constituents that move more slowly, and does not mean the chemical constituents that move more quickly during CPC. There is no vector differentiation here, upper phase simply means organic solvent since most organic solvent are less denser than water (unless of course you are using chlorinated solvent and water in that case organic phase will be in the bottom). Think of this system as if you were separating compound in a separator funnel. If the researcher was to use hexane and water, the upper phase would be hexane, and if the researcher was to use DCM and water in this case my upper phase would be water. DCM stands for Dichloromethane.
Activating the secondary amine of a pesticide by adjusting the pH of the CPC chromatography medium. This provides further details on controlling and adjusting the pH of the medium, in the Countercurrent Partition Chromatography (CPC) method of the present disclosure. The statement, “This process can be aided by fully activating the amino to their salt form to get an efficient absorption,” refers to the following. This concerns pesticides that have a moiety that takes the form of a secondary amine. At neutral pH, the secondary amine can take the form that is not protonated and not ionized, such as the following: R1—NH—R2. The present CPC method activates this non-protonated secondary amine so that it takes a form, R1—NH—R2 where the lone pair of electrons on the nitrogen binds a proton (H+). Alternatively, the present CPC method activates this non-protonated secondary amine so that the lone pair of electrons on the nitrogen binds a sodium atom (Na+).
Some pesticides are ionized when the medium has a high pH and some are ionized when the medium has a low pH. This process is dictated by the pKa of the pesticide, meaning that one can generate the proper pH strength to force an ionizable condition (an ionizable condition taking the form of an anion or cation).
(J) Example Ten. Steps in CPC Method(1) Procedure: Countercurrent Partition Chromatography (CPC)
(1.1) Part One: Fraction Method 1
(1.1.1) Dissolve the oil mixture at 5 to 1 ratio aliphatic hydrocarbon to oil acid allow it to homogenize.
(1.1.2) Prepare a mobile phase of 3 parts alcohol and 1 part water (deionized), and feed it to the CCP at 32 ml/min and allow it to equilibrate. The rotation of the cartridge should be 900 rpm
(1.1.3) Once the column is equilibrated inject the sample in the 28 ml/min and feed the mobile phase
(1.1.4) Run the system on ascending mode to collet the upper phase of the binary solvent mixture.
(1.1.5) Collect the eluate at 30 ml per fraction and the sample should come out at 28 fractions. To 36 fraction.
(1.1.6) Calculated elution time for collection is 30 min to start and ends at 38.5 min
(1.1.7) Collect the fraction at the prescribe tubes or at the prescribe runtime and separate the aliphatic hydrocarbon from the oil mixture via rotary evaporation.
(1.1.8) Place the oil mixture in the vacuum oven heated at 100 C to dried over hours for every 5000 g in a container with bottom surface area of 1.600 cm̂2 at a height of 2-4 centimeter.
(1.2) Part Two: Fraction Method 2
(1.2.1) Dissolve oil mixture sample in aliphatic hydrocarbon at 5:1 ratio aliphatic hydrocarbon:oil mixture and allow it to homogenized
(1.2.2) Prepare a stationary phase composing of 3 part alcohol and 1 part water and adjust the pH to 2.0 using an inorganic acid, once ready, feed it in the column to repeat step 4.1.2. and allow it to equilibrate, once equilibrated follow step 4.1.3 to 4.1.8.
(1.2.3) Dried the oil mixture in vacuum at 105 C for 5 hours with intermittent mixing
(1.3) Part Three: Fraction Method 3
(1.3.1) Dissolve oil mixture sample in aliphatic hydrocarbon at 5:1 aliphatic hydrocarbon:oil mixture ratio
(1.3.2) Prepare a mobile phase composing of 2 part alcohol and 2 part water decrease the pH to 2.0 using an acid and repeat step 4.1.2 to 4.1.8 to run the material and collect the sample.
(1.3.3) Dried the oil mixture at 105 degrees C. for 5 hours with intermittent mixing
The table below is relevant to the Countercurrent Partition Chromatography (CPC) procedure.
Table 4: Pesticide profile that are found to be present in the oil mixture. Note that blank boxes indicate that the pesticide was not detected. Red coding (in this table, as originally drafted) indicate a pesticide that is present in a method and that its commonly found in the oil mixture of our common samples in the laboratory. Such as trifloxystrobin, pyrethrins, hyxythiazox, fenpyroximate, cyflutrin, acequinocyl and abamectin. The red coded pesticides were bifenazate (Method 1); Chlorfenapyr (Method 2; Method 3); Cypermetrin (Method 1); Etaxazole (Method 1, Method 3); Pyrethrin (Method 1); and Trifloxystrobin (Method 3). Red indicated that pesticide residue failed the state requirement and its commonly found in our common oil mixture samples.
Green (in this table, as originally drafted) indicate a passable value or non-existence of a particular pesticide in the oil sample collected. The green-colored cells in the table are those that are not colored red, with the exceptions of the following cells, which did not have any color. The cells in the table that did not have any color were: acequinoxyl (Method 1, Method 2, Method 3); Abamectin (Method 1); Cyflutrin (Method 1; Method 2); Fenpyroximate (Method 3); Hexythiazox (Method 1).
The result also should be interpreted in a way that all samples used in each CPC experiment runs are all coming from the same bulk starting material that is contaminated quantitatively to 100 ppm of pesticide residues to illustrate the effectiveness of each experimental independently from each other. By combining three methods the inventor can eliminate all state requirement pesticide in one run. However, method 1 and 2 combine do remove commonly found pesticide in the oil mixture With the exception of cyflutrin which is not commonly found in our processing samples.
Exclusionary Embodiments. In embodiments relating to solvents, the present disclosure can exclude any reagent, solution, composition, method, or system that comprises one or more of petroleum ether, hexane, cyclohexane, toluene, methyl tert-butyl ether, tetrahydrofuran, diethyl ether, ethyl acetate, chloroform, acetone, DMSO, acetonitrile, isopropyl alcohol, n-propanol, n-butanol, acetic acid, ethanol, methanol, water, octane, octanol, a biphasic solvent, a triphasic solvent, or a tetraphasic solvent. Also, the present disclosure can exclude any solution (such as a mixture of two or more solvents) that comprises greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95% of any of the above chemicals, by volume. In other exclusionary embodiments, the preset disclosure can exclude any solution that comprises less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95% of any of the above chemicals, by volume.
In embodiments relating to Countercurrent Partition Chromatography (CPC) methods, the present disclosure can exclude any system or method that uses one or more of spiral disk, column rotation, hydrodynamic bobbin design, hydrodynamic I-type centrifuge, hydrodynamic vortex countercurrent separation, centrifugal partition chromatography, biphasic solvent, centrifugal partition extraction, and pulsed countercurrent separation.
Example OnePrepare Cannabinoids. This describes how to prepare the cannabinoids for injection into the cartridge, for the goal of separating cannabinoids from pesticides. The starting material can take the form of cannabis leaves and stems, but usually with buds removed. One kilogram (1 kg) (wet weight) of plant material is washed with ethanol (99.9% ethanol), using an extracting machine. The extracting machine is gentle and does not damage the plant. The ethanol extract is kept, and the plant material is discarded. Naturally-occurring (wet) cannabis plant material can be used. Alternatively, dried cannabis plant material can be used. If wet cannabis plant material is used, the ethanol extract will contain water from the plant, because water and ethanol are soluble (miscible) in each other. The ethanol extract is then concentrated in a rotary evaporator at 60 degrees C., resulting in an oil. The oil is then heated at 200 degrees C. for 50 minutes. During this heating, bubbling occurs. The bubbling results from the removal of carbon dioxide. The source of the carbon dioxide is decarboxylation of cannabinoid acids, where the decarboxylation was induced by the heating. Also, during the 50 minutes of heating, terpenes and other volatile materials were removed. Finally, distillation is used, where the result of the distillation is a starting material that is about 78 percent cannabinoids to a distilled product that is about 92 percent cannabinoids. Finally, the decarboxylated/distilled cannabinoid oil is ready to dissolve in hexane.
Stationary Phase. The stationary phase is an aqueous liquid. The stationary phase is made of ethanol, water, and enough phosphoric acid to bring the pH to about pH 2.0. The proportions of ethanol and water are three volumes of ethanol and one volume of water. When the stationary phase is run into the cartridge, it is called, “charging with the stationary phase.” The stationary phase is injected into the cartridge until it fills up the entire cartridge. The “cartridge” may be called a “drum.” The volume of the stationary phase that is injected into the cartridge is 810 mL.
Mobile Phase. After charging the cartridge with the stationary phase, it is time to plug in the mobile phase. The stationary phase must be inside the cartridge before you start to inject the mobile phase into the cartridge. The mobile phase is hexane. The volume of the mobile phase that is injected into the cartridge is 190 mL. The operator knows that equilibrium has been reached when liquid that is being eluted out of the cartridge takes the form of only the mobile phase (the mobile phase is hexane). When equilibrium is reached at a point when about 190 mL of aqueous phase has been pushed out of the cartridge. When the mobile phase is being injected into the cartridge, the motor must be running in order to make the CPC device rotate. What occurs is rotational mixing.
Inject Cannabinoids. After injecting the stationary phase, followed by injecting, the mobile phase, it is time to inject the cannabinoids. About 50 mL of cannabinoids are injected over twenty minutes. This 50 mL preparation takes the form of cannabinoids dissolved in hexane. The proportion is 40% cannabinoids and 60% hexane, by volume. After separation in the cartridge, the cannabinoids have an elution position, as measured by elution time, of 32-42 minutes. The entire CPC run takes about 60 minutes. The flow rate is 28 mL per minute. The elution results are as follows. Cannabinoids elute in the time frame of 35-42 minutes. Bifentrin elutes after 60 minutes. Myclobutanil elutes early, that is, at 8 minutes. Trifloxystrobin elutes at 52-56 minutes, that is, about ten minutes after the cannabinoids have eluted. Paclobutrazol elutes very early, that is, at 3-5 minutes. Etoxazole elutes at 73 minutes.
Additional Embodiments. Solvent system composition 6 part aliphatic alkane, 3 part alcohol and 1 part water at 2.0 pH.
System composition 6 part aliphatic alkane, part alcohol and 2 part water at pH 2.0.
Solvent system composition 6 part aliphatic alkane, 3 part alcohol, 1 part water.
Stationary pH was adjusted to 2.0 using inorganic acid and bases such as hydrochloric acid, phosphoric acid, nitric acid and citric acid, Sodium hydroxide and potassium hydroxide depending on the saponification and acid value of the material.
Alcohol composition used are Propanol, ethanol, methanol, isopropanol butanol depending on the viscosity, peroxide value and APHA value of the starting material.
Aliphatic hydrocarbon used in the process are pentane, hexane and heptane depending on the sugar and phenol content of the starting material.
Additives such as ethyl acetate and acetonitrile in the presence of selected pyrethrin.
The present invention is not to limited by compositions, reagents, methods, diagnostics, laboratory data, and the like, of the present disclosure. Also, the present invention is not be limited by any preferred embodiments that are disclosed herein.
Claims
1. A method for removing pesticides from an oil extract of plant matter, wherein the plant matter contains one or more different kinds of pesticides, the method comprising:
- (1) The step of washing, soaking, or washing and soaking the plant matter with ethanol, thereby extracting cannabinoids from the plant matter, and resulting in an oil extract that is dissolved or dispersed in the ethanol,
- (2) The step of removing the ethanol from the oil extract, wherein the removing is optionally with a rotary evaporator, resulting in an ethanol-free crude oil extract,
- (3) Dissolving the crude ethanol-free oil extract in hexane, resulting in a hexane solution that is optionally at a ratio of 1:5 ethanol-free oil extract/hexane (vol./vol.),
- (4) The step of preparing the stationary phase that comprises 3 parts ethanol and 1 part deionized water,
- (5) The step of adjusting the pH of the stationary phase to effect acid-induced ionization of an ionizable group on at least one of the one or more different pesticides to produce a pH-adjusted liquid composition,
- (6) The step running the stationary phase into the system to equilibrium running the pH-adjusted liquid composition in a Countercurrent Partition Chromatography (CPC) cartridge, wherein once the stationary phase is in place, the mobile phase is run into the system to equilibrium wherein the cartridge is rotated, and wherein the CPC is run in an ascending mode that, produces an upper phase of a binary solvent mixture, and
- (7) The step of collecting the upper phase of the binary solvent mixture.
2. The method of claim 1, wherein the ionizable group comprises a nitrile group, a pyrrole group, or both a nitrite group and a pyrrole group.
3. The method of claim 1, wherein the ethanol is removed using a rotary evaporator.
4. The method of claim 1, wherein at least one or the one or more different kinds of pesticides comprises an ionizable group as set forth in Table 1.
5. The method of claim 1, wherein the one or more different kinds of pesticides is selected from Table 3, Table 4, and Table 5.
6. The method of claim 1, wherein the step of collecting the upper phase of the binary solvent mixture comprises collecting fractions or collecting one or more runtime cuts from the CPC cartridge.
7. The method of claim 1, wherein the plant matter comprises cannabinoid acids, and wherein the method comprises the step or decarboxylating the cannabinoid acids prior to introducing a plant extract into the CPC cartridge.
8. The method of claim 1, wherein the plant matter is derived from Cannabis saliva.
9. The method of claim 1, wherein matter is derived from Cannabis sativa, and wherein the derived comprises derived by one or more of chopping, drying, drying and powdering, of removing stems and seeds.
10. The method of claim 1, wherein the plant matter comprises cannabinoid acids, and wherein the method comprises the step of decarboxylating the cannabinoid acids.
11. The method of claim 1, wherein the plant matter comprises plant sugars, the method further comprising the step of removing the plant sugars prior to introducing to the CPC cartridge and running the CPC.
12. The method of claim 1, wherein the step of adjusting the pH to effect acid-induced ionization of an ionizable group on at least one of the one or more different pesticides to produce a pH adjusted liquid composition, consists of adjusting the to effect acid-induced ionization.
13. The method of claim 1, further including the step of alkali-inducible ionization of an ionizable group on at least one of the one or more different pesticides to produce a pH-adjusted liquid composition, consists of adjusting the pH to effect alkali-induced ionization.
14. The method of claim 1, wherein the one or more different kinds of pesticides selected from Table 3, Table 4, and Table 5, wherein the method results in partial or substantial depletion of at least one of said one or more different kinds of pesticides, and wherein said partial or substantial depletion is measurable by comparing the total milligrams of pesticide in an aliquot of fraction or runtime cut of the CPC run, where the comparing is with the total milligrams of pesticide in the plant matter that was used in the CPC run.
15. A composition that comprises cannabinoids, wherein the composition is prepared by the method of claim 1, wherein the composition is partially or substantially depleted in pesticides, and wherein the partially or substantially depleted is measurable by comparing the total milligrams of pesticide in an aliquot of said composition with the corresponding quantity of plant matter from which said aliquot was derived.
Type: Application
Filed: Oct 3, 2018
Publication Date: Apr 4, 2019
Inventor: Erwin Sibal (North Hollywood, CA)
Application Number: 16/151,288
