METHOD, SYSTEM AND/OR APPARATUS FOR USE OF LIQUID OR FLUID CARBON DIOXIDE IN EXTRACTION AND/OR SOLUBILISING SOURCE MATERIAL AND BINDING AND/OR ELUTION WITH A MOLECULARLY IMPRINTED POLYMER
20240058726 ยท 2024-02-22
Assignee
Inventors
Cpc classification
B01D15/3852
PERFORMING OPERATIONS; TRANSPORTING
B01J20/268
PERFORMING OPERATIONS; TRANSPORTING
B01D15/40
PERFORMING OPERATIONS; TRANSPORTING
G01N30/88
PHYSICS
International classification
B01D15/42
PERFORMING OPERATIONS; TRANSPORTING
B01D15/38
PERFORMING OPERATIONS; TRANSPORTING
Abstract
This disclosure relates to a method/system for associating at least one target molecule with a molecularly imprinted polymer and/or a method/system of obtaining at least one target molecule from a source material. More particularly, this disclosure relates to use of liquid or fluid carbon dioxide in various steps related to use of molecularly imprinted polymers.
Claims
1. A method of obtaining at least one target molecule from a source material, the method comprising: a) bringing a source material into contact with a molecularly imprinted polymer (MIP) to associate at least one target molecule present in the source material with the molecularly imprinted polymer, b) eluting the at least one target molecule off the MIP using liquid or fluid carbon dioxide as an elution solvent, wherein the liquid or fluid carbon dioxide used as an elution solvent is a pressure of above about 7 MPa and a temperature of above about 31 C., or wherein the liquid or fluid carbon dioxide used as an elution solvent is a pressure of about 0.5 to 41 MPa and a temperature of about 50 to 30 C.
2. The method of claim 1 wherein the liquid or fluid carbon dioxide used as an elution solvent is a pressure of about 7 to 100 MPa and a temperature of about 31 to 70 C.
3. The method of claim 1 wherein the liquid or fluid carbon dioxide is at a pressure of about 5 to 30 MPa and temperature of about 10 to 25 C., or the liquid or fluid carbon dioxide is at a pressure of about 7 to 15 MPa and temperature of about 10 to 25 C.
4. The method of claims 1 or 2 wherein the liquid or fluid carbon dioxide used as an elution solvent is supercritical carbon dioxide.
5. The method of claim 4 wherein the liquid or fluid carbon dioxide used as an elution solvent has a density of about 0.4 to 1.0 g/cm.sup.3, or about 0.6 to 1.0 g/cm.sup.3 or about 0.8 to 1.0 g/cm.sup.3.
6. The method of claims 1 or 3 wherein the liquid or fluid carbon dioxide used as an elution solvent is subcritical carbon dioxide.
7. The method of claim 6 wherein the liquid or fluid carbon dioxide used as an elution solvent has a density of about 0.1 to 1.1 g/cm.sup.3 or about 0.2 to 1.1 g/cm.sup.3, or about 0.2 to 1.0 g/cm.sup.3 or about 0.5 to 1.0 g/cm.sup.3 or about 0.7 to 1.0 g/cm.sup.3.
8. The method of any one of claims 1 to 7 wherein the elution solvent further comprises at least one co-solvent.
9. The method of claim 8 wherein the co-solvent is selected from one or more of water, ethanol, methanol, ethyl acetate, isopropyl alcohol, acetonitrile, acetone, THF.
10. The method of claim 8 or 9 wherein the co-solvent is greater than 0% to about 75%, or greater than 0% to about 70%, or greater than 0% to about 60%, or greater than 0% to about 50%, or greater than 0% to about 50%, or greater than 0% to about 40%, or greater than 0% to about 30% volume of the liquid or fluid carbon dioxide elution solvent or greater than 0% to about 20% of mass flow of the liquid or fluid carbon dioxide elution solvent.
11. The method of any one of claims 1 to 10 wherein the source material is any one or more of a liquid, a wax, an oil, a solid and/or an extract of an animal, plant or synthetic source material.
12. The method of any one of claims 1 to 11 wherein the source material is in a binding solvent when it is brought into contact with the MIP and the binding solvent is selected from any one or more of liquid or fluid carbon dioxide, water, ethanol, methanol, ethyl acetate, isopropyl alcohol, acetonitrile, acetone, THF.
13. The method of claim 12 wherein the binding solvent comprises supercritical carbon dioxide.
14. The method of claim 12 wherein the binding solvent comprises subcritical carbon dioxide.
15. The method of any one of claims 12 to 14 wherein the binding solvent comprises liquid or fluid carbon dioxide at a density that is lower than the density of the liquid or fluid carbon dioxide used as the elution solvent.
16. The method of any one of claims 12 to 15 wherein the binding solvent comprises liquid or fluid carbon dioxide and a co-solvent.
17. The method of claim 16 wherein the co-solvent is selected from one or more of water, ethanol, methanol, ethyl acetate, isopropyl alcohol, acetonitrile, acetone, THF.
18. The method of claim 16 or 17 wherein the co-solvent is greater than 0% to about 75%, or greater than 0% to about 70%, or greater than 0% to about 60%, or greater than 0% to about 50% of mass flow of the liquid or fluid carbon dioxide.
19. The method of any one of claims 1 to 18 wherein the source material is solubilised in at least one solvent prior to being brought into contact with the MIP.
20. The method of claim 19 wherein the source material is solubilised in one or more of water, ethanol, methanol, ethyl acetate, isopropyl alcohol, acetonitrile, acetone, tetrahydrofuran (THF), liquid or fluid carbon dioxide.
21. The method of claim 20 or 21 wherein the source material is solubilised in supercritical carbon dioxide.
22. The method of claim 20 or 21 wherein the source material is solubilised in supercritical carbon dioxide.
23. A method for associating at least one target molecule with a molecularly imprinted polymer, the method comprising: i) solubilising a source material in liquid or fluid carbon dioxide and ii) bringing the solubilised source material in the liquid or fluid carbon dioxide into contact with a molecularly imprinted polymer (MIP) to associate at least one target molecule present in the source material with the molecularly imprinted polymer, wherein the liquid or fluid carbon dioxide is at a first pressure and temperature combination when solubilising the source material and at a second pressure and temperature combination when bringing the solubilised source material into contact with the molecularly imprinted polymer (MIP).
24. The method of claim 23 wherein the first pressure and temperature combination is about 7.4 MPa to 35 MPa and about 31 C. to 80 C. or about 0.5 MPa to 41 MPa and about 50 C. to 30 C.
25. The method of claim 23 or 24 wherein the liquid or fluid carbon dioxide at the first pressure and temperature combination is supercritical carbon dioxide.
26. The method of claim 23 or 24 wherein the liquid or fluid carbon dioxide at the first pressure and temperature combination is subcritical carbon dioxide.
27. The method of any one of claims 23 to 26 wherein the liquid or fluid carbon dioxide at the second pressure and temperature combination is supercritical carbon dioxide.
28. The method of any one of claims 23 to 26 wherein the liquid or fluid carbon dioxide at the second pressure and temperature combination is subcritical carbon dioxide.
29. The method of any one of claims 23 to 28 wherein the liquid or fluid carbon dioxide at the first pressure and temperature has a higher density than the liquid or fluid carbon dioxide at the second pressure and temperature combination.
30. The method of any one of claims 23 to 29 wherein the source material is solubilised in the carbon dioxide by extracting the source material from a solid material using supercritical carbon dioxide.
31. The method of any one of claims 23 to 30 wherein the source material is solubilised in the carbon dioxide by extracting the source material from a solid material using supercritical carbon dioxide at pressure of about 7.4 MPa to 35 MPa and about 31 C. to 80 C.
32. The method of any one of claims 23 to 31 wherein the source material is solubilised in the carbon dioxide by extracting the source material from a solid material using supercritical carbon dioxide at density of about 0.4 to 1.0 g/cm.sup.3.
33. The method of any one of claims 23 to 29 wherein the source material is solubilised in the carbon dioxide by extracting the source material from a solid material using subcritical carbon dioxide.
34. The method of claim 33 wherein the source material is solubilised in the carbon dioxide by extracting the source material from a solid material using subcritical carbon dioxide at pressure of about 0.5 MPa to 41 MPa and temperature of about 50 C. to 30 C.
35. The method of claim 33 or 34 wherein the source material is solubilised in the carbon dioxide by extracting the source material from a solid material using subcritical carbon dioxide at density of about 0.01 to 1.3 g/cm.sup.3.
36. A system for obtaining at least one target molecule from a source material, the system comprising: a molecularly imprinted polymer, a device for providing a flow of liquid or fluid carbon dioxide, wherein the system is adapted to bring a source material into contact with a molecularly imprinted polymer to associate at least one target molecule present in the source material with the molecularly imprinted polymer, and wherein the system is adapted to elute the target molecule that is associated with the molecularly imprinted polymer using the flow of liquid or fluid carbon dioxide as an elution solvent, and wherein the carbon dioxide is at a pressure of above about 7 MPa and temperature of above about 31 C., or wherein the system is adapted to elute the target molecule that is associated with the molecularly imprinted polymer using the flow of liquid or fluid carbon dioxide as an elution solvent, and wherein the carbon dioxide is at a pressure of about 0.5 to 41 MPa and a temperature of about 50 to 30 C.
37. A system for associating at least one target molecule with a molecularly imprinted polymer, the system comprising: a molecularly imprinted polymer, at least one device for providing a flow of liquid or fluid carbon dioxide at a first pressure and temperature combination and a second pressure and temperature combination, wherein the system is adapted to solubilise a source material in the liquid or fluid carbon dioxide, and wherein the system is adapted to bring the solubilised source material into contact with a molecularly imprinted polymer to associate at least one target molecule present in the source material with the molecularly imprinted polymer, wherein the liquid or fluid carbon dioxide is at the first pressure and temperature combination when solubilising the source material and at the second pressure and temperature combination when bringing the solubilised source material into contact with the molecularly imprinted polymer (MIP).
Description
BRIEF DESCRIPTION OF THE FIGURES
[0274] Preferred embodiments of the disclosure will be described by way of example only and with reference to the following drawings.
[0275]
[0276]
[0277]
[0278]
[0279]
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[0281]
[0282]
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[0284]
DETAILED DESCRIPTION
[0285] The present disclosure broadly relates to a method/system for associating at least one target molecule with a molecularly imprinted polymer.
[0286] The present disclosure relates a method for associating at least one target molecule with a molecularly imprinted polymer, the method comprising: i) solubilising a source material in liquid or fluid carbon dioxide, and ii) bringing the solubilised source material in the liquid or fluid carbon dioxide into contact with a molecularly imprinted polymer (MIP) to associate at least one target molecule present in the source material with the molecularly imprinted polymer. The liquid or fluid carbon dioxide is at a first pressure and temperature combination when solubilising the source material and at a second pressure and temperature combination when bringing the solubilised source material into contact with the molecularly imprinted polymer (MIP).
[0287] The present disclosure further or alternatively relates to a system for associating at least one target molecule with a molecularly imprinted polymer, the system comprising: a molecularly imprinted polymer, at least one device for providing a flow of liquid or fluid carbon dioxide at a first pressure and temperature combination and a second pressure and temperature combination. The system is adapted to solubilise a source material in the liquid or fluid carbon dioxide and bring the solubilised source material into contact with a molecularly imprinted polymer to associate at least one target molecule present in the source material with the molecularly imprinted polymer. The liquid or fluid carbon dioxide is at the first pressure and temperature combination when solubilising the source material and at the second pressure and temperature combination when bringing the solubilised source material into contact with the molecularly imprinted polymer (MIP).
[0288] The present disclosure further or alternatively relates to a method for associating at least one target molecule with a molecularly imprinted polymer, the method comprising: i) solubilising a source material in liquid or fluid carbon dioxide and ii) bringing the solubilised source material in liquid or fluid carbon dioxide into contact with a molecularly imprinted polymer (MIP) to associate at least one target molecule present in the source material with the molecularly imprinted polymer. The liquid or fluid carbon dioxide is at a first density when solubilising the source material and at a second density when bringing the solubilised source material into contact with a molecularly imprinted polymer (MIP). The first density is different to the second density.
[0289] The present disclosure further or alternatively relates to a system for associating at least one target molecule with a molecularly imprinted polymer, the system comprising: a molecularly imprinted polymer (MIP), at least one device for providing a flow of liquid or fluid carbon dioxide at a first density and a second density, wherein the system is adapted to solubilise a source material in the liquid or fluid carbon dioxide, and bring the solubilised source material into contact with a molecularly imprinted polymer to associate at least one target molecule present in the source material with the molecularly imprinted polymer. The liquid or fluid carbon dioxide is at the first density when solubilising the source material and at the second density when bringing the solubilised source material into contact with the molecularly imprinted polymer (MIP). The first density is different to the second density.
[0290] The present disclosure further or alternatively relates to a method for associating at least one target molecule with a molecularly imprinted polymer, the method comprising: i) solubilising a source material in liquid or fluid carbon dioxide and ii) bringing the solubilised source material in liquid or fluid carbon dioxide into contact with a molecularly imprinted polymer (MIP) to associate at least one target molecule present in the source material with the molecularly imprinted polymer. The liquid or fluid carbon dioxide is at a first density when solubilising the source material and at a second density when bringing the solubilised source material into contact with a molecularly imprinted polymer (MIP). The first density is higher than the second density.
[0291] The present disclosure further or alternatively relates to a system for associating at least one target molecule with a molecularly imprinted polymer, the system comprising: a molecularly imprinted polymer (MIP), at least one device for providing a flow of liquid or fluid carbon dioxide at a first density and a second density. The system is adapted to solubilise a source material in the liquid or fluid carbon dioxide and bring the solubilised source material into contact with a molecularly imprinted polymer to associate at least one target molecule present in the source material with the molecularly imprinted polymer. The liquid or fluid carbon dioxide is at the first density when solubilising the source material and at the second density when bringing the solubilised source material into contact with the molecularly imprinted polymer (MIP). The first density is higher than the second density.
[0292] The present disclosure further or alternatively relates to a method of obtaining at least one target molecule from a source material, the method comprising: a) bringing a source material into contact with a molecularly imprinted polymer (MIP) to associate at least one target molecule present in the source material with the molecularly imprinted polymer, b) eluting the at least one target molecule off the MIP using liquid or fluid carbon dioxide as an elution solvent. The liquid or fluid carbon dioxide used as an elution solvent is at a pressure of above about 7 MPa and a temperature of above about 31 C., or the liquid or fluid carbon dioxide used as an elution solvent is a pressure of about 0.5 to 41 MPa and a temperature of about 50 to 30 C.
[0293] The present disclosure further or alternatively relates to a system for obtaining at least one target molecule from a source material, the system comprising: a molecularly imprinted polymer, a device for providing a flow of liquid or fluid carbon dioxide. The system is adapted to bring a source material into contact with a molecularly imprinted polymer to associate at least one target molecule present in the source material with the molecularly imprinted polymer, and the system is adapted to elute the target molecule that is associated with the molecularly imprinted polymer using the flow of liquid or fluid carbon dioxide as an elution solvent. The carbon dioxide is at a pressure of above about 7 MPa and temperature of above about 31 C., or the carbon dioxide is at a pressure of about 0.5 to 41 MPa and a temperature of about 50 to 30 C.
[0294] The present disclosure further or alternatively relates to a method of obtaining a at least one target molecule from a source material, the method comprising: a) bringing a source material into contact with a molecularly imprinted polymer (MIP) to associate at least one target molecule present in the source material with the molecularly imprinted polymer, b) eluting the at least one target molecule off the MIP using liquid or fluid carbon dioxide as an elution solvent. The liquid or fluid carbon dioxide used as an elution solvent is a pressure of above about 7 MPa and temperature above about 30 C., preferably a pressure of about 20 to 35 MPa and a temperature of about 40 to 70 C.
[0295] The present disclosure further or alternatively relates to a system for obtaining at least one target molecule from a source material, the system comprising: a molecularly imprinted polymer and a device for providing a flow of liquid or fluid carbon dioxide. The system is adapted to bring a source material into contact with a molecularly imprinted polymer to associate at least one target molecule present in the source material with the molecularly imprinted polymer. The system is adapted to elute the target molecule that is associated with the molecularly imprinted polymer using the flow of liquid or fluid carbon dioxide, and wherein the carbon dioxide is at a pressure of above about 7 MPa and temperature above about 30 C., preferably a pressure of about 20 to 35 MPa and temperature of about 40 to 70 C.
[0296] The present disclosure further or alternatively relates to a method of obtaining at least one target molecule from a source material, the method comprising: a) bringing a source material into contact with a molecularly imprinted polymer (MIP) to associate at least one target molecule present in the source material with the molecularly imprinted polymer, b) eluting the at least one target molecule off the MIP using liquid or fluid carbon dioxide as an elution solvent. The liquid or fluid carbon dioxide used as an elution solvent is a pressure of about 0.5 to 41 MPa and a temperature of about 50 to 30 C.
[0297] The present disclosure further or alternatively relates to a system for obtaining at least one target molecule from a source material, the system comprising: a molecularly imprinted polymer, a device for providing a flow of liquid or fluid carbon dioxide. The system is adapted to bring a source material into contact with a molecularly imprinted polymer to associate at least one target molecule present in the source material with the molecularly imprinted polymer. The system is adapted to elute the target molecule that is associated with the molecularly imprinted polymer using the flow of liquid or fluid carbon dioxide as an elution solvent. The carbon dioxide is at a pressure of about 0.5 to 41 MPa and a temperature of about 50 to 30 C.
[0298] The present disclosure further or alternatively relates to a method of obtaining at least one target molecule from a source material, the method comprising: a) bringing a source material into contact with a molecularly imprinted polymer (MIP) to associate at least one target molecule present in the source material with the molecularly imprinted polymer, and b) eluting the at least one target molecule off the MIP using liquid or fluid carbon dioxide as an elution solvent. The liquid or fluid carbon dioxide used as an elution solvent has a density of about 0.2 to 1.0 g/cm.sup.3, preferably a density of about 0.6 to 1.0 g/cm.sup.3.
[0299] The present disclosure further or alternatively relates to a system for obtaining at least one target molecule from a source material, the system comprising: a molecularly imprinted polymer, a device for providing a flow of liquid or fluid carbon dioxide. The system is adapted to bring a source material into contact with a molecularly imprinted polymer to associate at least one target molecule present in the source material with the molecularly imprinted polymer. The system is adapted to elute the target molecule that is associated with the molecularly imprinted polymer using the flow of liquid or fluid carbon dioxide, wherein the carbon dioxide has a density of about 0.2 to 1.0 g/cm.sup.3, preferably a density of about 0.6 to 1.0 g/cm.sup.3.
[0300] The present disclosure further or alternatively relates to a method of obtaining at least one target molecule from a source material, the method comprising: a) bringing a source material into contact with a molecularly imprinted polymer (MIP) to associate at least one target molecule present in the source material with the molecularly imprinted polymer, b) eluting the at least one target molecule off the MIP using liquid or fluid carbon dioxide as an elution solvent. The liquid or fluid carbon dioxide used as an elution solvent has a density of about 0.01 to 1.3 g/cm.sup.3.
[0301] The present disclosure further or alternatively relates to a system for obtaining at least one target molecule from a source material, the system comprising: a molecularly imprinted polymer, a device for providing a flow of liquid or fluid carbon dioxide. The system is adapted to bring a source material into contact with a molecularly imprinted polymer to associate at least one target molecule present in the source material with the molecularly imprinted polymer. The system is adapted to elute the target molecule that is associated with the molecularly imprinted polymer using the flow of liquid or fluid carbon dioxide as an elution solvent, wherein the carbon dioxide has a density of about 0.01 to 1.3 g/cm.sup.3.
[0302] The methods and/or systems described herein are generally concerned with separating or purifying one or more target molecules form a source material using a molecularly imprinted polymer (MIP).
[0303] The source material may be solubilizing in order to get it into form/solution or suspension to be introduced to the MIP. The source material may be solubilized in a solvent/liquid, for example in one or more of water, ethanol, methanol, ethyl acetate, isopropyl alcohol, acetonitrile, acetone, tetrahydrofuran (THF), liquid or fluid carbon dioxide.
[0304] Where liquid or fluid carbon dioxide is used to solubilize, it may be super or sub-critical carbon dioxide, for example supercritical carbon dioxide at a pressure of about 7.4 MPa to 35 MPa and temperature of about 31 C. to 80 C. or subcritical carbon dioxide at a pressure of about 0.5 MPa to 41 MPa and temperature of about 50 C. to 30 C. The density of the liquid or fluid carbon dioxide may be about 0.4 to 1.0 g/cm.sup.3 or about 0.01 to 1.3 g/cm.sup.3. Where the liquid or fluid carbon dioxide is supercritical carbon dioxide the density may be about 0.4 to 1.0 g/cm.sup.3 or about 0.6 to 1.0 g/cm.sup.3 or about 0.7 to 1.0 g/cm.sup.3 or about 0.8 to 1.0 g/cm.sup.3 or about 0.8 to 0.9 g/cm.sup.3. Where the liquid or fluid carbon dioxide is subcritical carbon dioxide the density may be about 0.01 to 1.3 g/cm.sup.3, or about 0.3 to 1.0 g/cm.sup.3, or about 0.7 to 1.0 g/cm.sup.3.
[0305] Where liquid or fluid carbon dioxide is used to solubilize a co-solvent may also be used (for example one or more of water, ethanol, methanol, ethyl acetate, isopropyl alcohol, acetonitrile, acetone, THF).
[0306] In some cases, solubilizing encompasses extracting the target molecule from animal or plant material, for example, liquid, a wax, an oil, or a solid. Examples of solid material are a fungus, plant seed, plant fruit, plant root, plant stem and/or leaf, algae, fish. Examples of source material include coffee beans, kava plant, cannabis plant, mushroom, kiwi fruit, avocado, hops, tobacco, tea, grapes, blackcurrant, any one of which being whole or parts of.
[0307] In other cases, the source material to be solubilized may be partially processed, for example a crude extract, such as crude cannabis extract, which is winterized or non-winterized.
[0308] Winterization is a process used to reduce fats/waxes/other lipids in an extract. Winterization involves dissolving the extract in solvent, then chilling for around 24-48 hours to solidify the fats/waxes/other lipids, which are then filtered off. The solvent is then usually removed from the remaining extract which is depleted in the fats/waxes/other lipids. The extract/solubilised material used may be a winterised extract. However it may not be necessary to remove the solvent from the extract after the solution is winterised. The source material may be solubilised in at least one solvent prior to being brought into contact with the MIP, for example the solvent may be one or more of water, ethanol, methanol, ethyl acetate, isopropyl alcohol, acetonitrile, acetone, THF, preferably ethanol or methanol. The solubilised source material may be filtered, warmed, cooled (for example partial winterized), winterized, sonicated (for example ultrasonicated) and/or mixed (for example high shear mixing) prior to being brought into contact with the MIP. For example, the solubilised source material may be cooled and filtered (i.e. winterised) to remove solidified fats/waxes/other lipids. The solubilised source material may then be brought into contact with a MIP to associate at least one target molecule present in the source material with the MIP. The solvent may not be removed from the source material or only partially removed from the source material. This may avoid processing steps usually associated with the winterisation process or other filtering steps and/or reduce use of solvents.
[0309] The source material (which may be solubilized first) is brought into contact with a molecularly imprinted polymer (MIP) to associate at least one target molecule present in the source material with the molecularly imprinted polymer. This may be alternatively referred to as binding or the binding step. The source material may be in a binding solvent when it is brought into contact with the MIP. The binding solvent may be selected from any one or more of liquid or fluid carbon dioxide, water, ethanol, methanol, ethyl acetate, isopropyl alcohol, acetonitrile, acetone, THF. Where a liquid or fluid carbon dioxide is used it may be supercritical carbon dioxide or subcritical carbon dioxide. The liquid or fluid carbon dioxide may be at pressure and temperature combination of about 7.4 MPa to 100 MPa and about 31 C. to 120 C. or about 0.5 MPa to 41 MPa and about 50 C. to 30 C. The density of the liquid or fluid carbon dioxide used for binding may be about 0.1 to 1.2 g/cm.sup.3 or about 0.01 to 1.3 g/cm.sup.3.
[0310] Where the liquid or fluid carbon dioxide used for binding is supercritical carbon dioxide the density may be about 0.1 to 1.2 g/cm.sup.3, or about 0.1 to 1.0 g/cm.sup.3 or about 0.2 to 0.7 g/cm.sup.3 and/or at pressure and temperature combination of about 7.4 MPa to 100 MPa and about 31 C. to 120 C., about 8 to 13 MPa and about 37 to 63 C., or about 9 to 12 MPa and about 37 to 55 C.
[0311] Where the liquid or fluid carbon dioxide used for binding is subcritical carbon dioxide, the density may be about 0.01 to 1.3 g/cm.sup.3 or about 0.4 to 1.0 g/cm.sup.3 or about 0.8 to 1.0 g/cm.sup.3 and/or at a pressure and temperature combination of about 0.5 MPa to 41 MPa and about 50 C. to 30 C., or about 3 MPa to 20 MPa and about 25 C. to 20 C., or about 5 MPa to 10 MPa and temperature of about 0 C. to 13 C.
[0312] The binding solvent may comprise liquid or fluid carbon dioxide and a co-solvent, for example one or more of water, ethanol, methanol, ethyl acetate, isopropyl alcohol, acetonitrile, acetone, THF.
[0313] The co-solvent may have a flow rate of greater than 0% (for example 0.001%) to about 75% of mass flow rate of the liquid or fluid carbon dioxide, or greater than 0% to about 70%, or greater than 0% to about 60% or greater than 0% to about 50% of mass flow rate of the liquid or fluid carbon dioxide.
[0314] Where liquid or fluid carbon dioxide is used for both solubilizing the source material and binding to the MIP, the pressure and temperature combination of the carbon dioxide for the two steps may be different, and/or the density may be different, and/or the density of the liquid or fluid carbon dioxide is higher for solubilizing than binding.
[0315] The binding solvent containing the target molecule may be cycled over/through the MIP multiple times to allow more of the target molecule to bind to the MIP. i.e. the binding solvent may be collected after it has passed over/thought the MIP and reintroduced to the MIP to contact the MIP again.
[0316] The MIP may non-covalently associate with the target molecule, while the non-target, waste, or impurities wash over or through the MIP, either with the binding solvent, or with an optionally additional rinse or wash step. The target molecule is eluted off the MIP using liquid or fluid carbon dioxide as an elution solvent.
[0317] The liquid or fluid carbon dioxide used as an elution solvent may be at a pressure of above about 7 MPa and a temperature of above about 31 C., or a pressure of about 0.5 to 41 MPa and a temperature of about 50 to 30 C. Alternatively, the liquid or fluid carbon dioxide used as an elution solvent may have a density of about 0.2 to 1.0 g/cm.sup.3, or about 0.01 to 1.3 g/cm.sup.3. The liquid or fluid carbon dioxide used as an elution solvent may be supercritical carbon dioxide or subcritical carbon dioxide.
[0318] Where the liquid or fluid carbon dioxide used for elution is supercritical carbon dioxide the carbon dioxide may be at a pressure of above about 7 MPa and a temperature of above about 31 C., or pressure about 7 to 100 MPa and a temperature of about 31 to 70 C., or pressure of about 7 to 55 MPa and a temperature of about 31 to 70 C., or pressure of about 7 to 35 MPa and a temperature of about 31 to 70 C., or pressure of about 10 to 35 MPa and a temperature of about 31 to 70 C., or pressure of about 10 to 35 MPa and a temperature of about 40 to 70 C., or pressure of about 20 to 35 MPa and temperature of about 40 to 70 C., or pressure of about 25 MPa to 35 MPa and temperature of about 35 C. to 50 C. The density of the carbon dioxide may be 0.4 to 1.0 g/cm.sup.3 or about 0.6 to 1.0 g/cm.sup.3. or about 0.8 to 1.0 g/cm.sup.3.
[0319] Where the liquid or fluid carbon dioxide used for elution is subcritical carbon dioxide the carbon dioxide may be at a pressure of about 0.5 to 41 MPa and temperature of about 50 to 30 C. or pressure of about 5 to 30 MPa and temperature of about 10 to 25 C. or pressure of about 7 to 15 MPa and temperature of about 10 to 25 C. The density of the carbon dioxide may be about 0.1 to 1.1 g/cm.sup.3 or about 0.2 to 1.1 g/cm.sup.3 or about 0.2 to 1.0 g/cm.sup.3 or about 0.5 to 1.0 g/cm.sup.3 or about 0.7 to 1.0 g/cm.sup.3.
[0320] The elution solvent may include at least one co-solvent, for example one or more of water, ethanol, methanol, ethyl acetate, isopropyl alcohol, acetonitrile, acetone, THF. The co-solvent may have a flow rate of greater than 0% (for example 0.001%) to about 75% of mass flow rate of the liquid or fluid carbon dioxide elution solvent, or greater than 0% to about 70%, or greater than 0% to about 60%, or greater than 0% to about 50%, or greater than 0% to about 50%, or greater than 0% to about 40%, or greater than 0% to about 30%, or greater than 0% to about 20% of mass flow rate of the liquid or fluid carbon dioxide elution solvent. The use of less co-solvent, while still achieving good elution is particularly beneficial, as elution can use large quantities of classic (for example organic) solvents.
[0321] Various embodiments are described with reference to the Figures. Throughout the Figures and specification, the same reference numerals may be used to designate the same or similar components, and redundant descriptions thereof may be omitted.
[0322]
[0323] Solid material (for example plant material) may be packed in a vessel 4a for extraction, for example a jacketed extraction chamber. The supercritical or subcritical CO.sub.2 may be passed through the vessel 4a at set/selected pressure and temperature.
[0324] Following extraction, the CO.sub.2 containing the extract (i.e. the solubilized source material) may be passed to pressure and/or temperature adjustment device 5a, 5b, where the pressure adjustment causes the source material to lose solubility in the CO.sub.2, for example pressure reduction causes CO.sub.2 to change into gas phase and the source material remains in the liquid phase.
[0325] The source material may then be physically separated at separator 6 and collected in collection vessel 7a, 7b, if wishing to collect the source material. Physical separation in separator 6a, 6b may be by various means, including gravity and/or cyclone.
[0326] Pressure reduction/adjustment physical separation may be done in multiple stages, for example two sequential stages are shown in
[0327] Following pressure adjustment/reduction/separation the CO.sub.2 may be recycled by cooling and/or increasing the pressure to convert back to the liquid state at converter 8. The liquid CO.sub.2 may be reused in the method/system, for example by being returned to the storage tank 1a.
[0328]
[0329] Plant material may be packed in vessel 4a for extraction, for example a jacketed extraction chamber. The supercritical or subcritical CO.sub.2 and co-solvent may be passed through the vessel 4a at set pressure and temperature.
[0330] Following extraction, the CO.sub.2 and co-solvent containing the source material is optionally passed to pressure and/or temperature adjustment device 5a, 5b, where the pressure adjustment causes the source material to lose solubility in the CO.sub.2 and co-solvent, for example pressure reduction causes CO.sub.2 to change into gas phase and the source material remains in the liquid phase.
[0331] The source material may be then physically separated at separator 6a, 6b and the source material and co-solvent collected in collection vessel 7. Physical separation in separator 6a, 6b may be by various means, including gravity and/or cyclone.
[0332] Pressure reduction/adjustment physical separation may be done in multiple stages, for example two sequential stages are shown in
[0333] The co-solvent and source material may optionally be separated at co-solvent separation unit 12, for example the source material may be collected at collection unit 15 and co-solvent may be evaporated and condensed at condensation unit 13 and may be passed to co-solvent tank 14 for recycling back to co-solvent storage 9a.
[0334] Following pressure adjustment/reduction/separation the CO.sub.2 may also be recycled by cooling and/or increasing the pressure to convert back to the liquid state at converter 8. The liquid CO.sub.2 may be reused in the method/system, for example by being returned to the storage tank 1a.
[0335]
[0336] Solid material (for example plant material comprising source material, e.g. plant extract) may be packed in vessel 4a for extraction, for example a jacketed extraction chamber. CO.sub.2 may be passed through the vessel 4a at set pressure and temperature.
[0337] The CO.sub.2 containing the source material may be passed to the MIP. In this example the MIP beads are contained in a separate vessel 4b. The source material may be brought into contact with the MIP (for example MIP beads), for the target molecule(s) to associate with the MIP while the remainder of the extract (that does not associate with the MIP) may be flushed through with the CO.sub.2. The remainder of the extract that is flushed through may be collected and optionally may be passed to the MIP again or to another MIP (not shown), for further binding and elution.
[0338] Further CO.sub.2 at a different selected pressure and temperature or density (which does not contain source material) may be passed though/over the MIP in separate vessel 4b via a bypass valve (bypassing vessel 4a) to elute the target molecule(s) from the MIP. The pressure and temperature or density may be changed sharply or gradually from the temperature/pressure/density used to bring the source material into contact with the MIP.
[0339] The target molecule(s) may be collected, for example the CO.sub.2 containing the target molecule(s) may be passed to pressure and/or temperature adjustment device 5a, 5b, where the pressure adjustment causes the target molecule(s) to lose solubility in the CO.sub.2. Pressure reduction causes CO.sub.2 to change into gas phase and the target molecule(s) remains in the liquid phase, the target molecule(s) may be then physically separated at separator 6a, 6b and collected in collection vessel 7a, 7b.
[0340] Physical separation in separator 6a, 6b may be by various means, including gravity and/or cyclone. Pressure reduction/adjustment physical separation may be done in multiple stages, for example two sequential stages are shown in
[0341] Following pressure adjustment/reduction/separation the CO.sub.2 may be recycled by cooling and/or increasing the pressure to convert back to the liquid state at converter 8. The liquid CO.sub.2 may be reused in the method/system, for example by being returned to the storage tank.
[0342]
[0343] Solid material (for example plant material comprising source material, e.g. plant extract) may be packed in vessel 4a for extraction, for example a jacketed extraction chamber.
[0344] CO.sub.2 may be passed through the vessel 4a at set pressure and temperature. The pressure and temperature (and therefore density) of the CO.sub.2 containing the source material may be adjusted using the pressure regulating device 31 and/or temperature regulating device 32. This may cause some of the solubility of some of the components extracted from the solid material to drop out of the CO.sub.2 solution. These may be removed (for example by extract separator 33 and/or extract collection vessel 34) to avoid fouling of the MIP in the second vessel/column 4b.
[0345] The extract pressure regulating device 31 and/or extract temperature regulating device 32 optionally allow the pressure/temperature/density of the CO.sub.2 to be adjusted prior to the CO.sub.2 containing the source material being contacted with the MIP. The CO.sub.2 containing the source material may be passed to the MIP.
[0346] In this example (see
[0347] CO.sub.2 at a different selected pressure and temperature or density (which does not contain source material/extract) may be passed though/over the MIP in separate vessel 4b via a bypass valve (bypassing vessel 4a) to elute the target molecule(s) from the MIP. The pressure and temperature or density may be changed sharply or gradually from the temperature/pressure/density used to bring the source material into contact with the MIP.
[0348] The target molecule(s) may be collected, for example the CO.sub.2 containing the target molecule(s) may be passed to pressure and/or temperature adjustment device 5a, 5b, where the pressure adjustment causes the target molecule(s) to lose solubility in the CO.sub.2. Pressure reduction may cause CO.sub.2 to change into gas phase and the target molecule(s) remains in the liquid phase. The target molecule(s) may be then physically separated at separator 6a, 6b and collected in collection vessel 7a, 7b. Physical separation in separator 6a, 6b may be by various means, including gravity and/or cyclone.
[0349] Pressure reduction/adjustment physical separation may be done in multiple stages, for example two sequential stages are shown in
[0350] Following pressure adjustment/reduction/separation the CO.sub.2 may be recycled by cooling and/or increasing the pressure to convert back to the liquid state at converter 8. The liquid CO.sub.2 may be reused in the method/system, for example by being returned to the storage tank 1a.
[0351]
[0352] The system/method allows for co-solvent to be added to the CO.sub.2 stream at any one or more of (1) solubilising the source material or extraction of source material from a solid material in vessel 4a (2) when the source material in the CO.sub.2 is contacted with the MIP in vessel 4b (e.g. binding) (3) when the CO.sub.2 steam elutes at least one target molecule off the MIP in vessel 4b.
[0353] The system/method may include a CO.sub.2 storage or top up tank(s) 1a, 1b, CO.sub.2 pressure adjustment device 2, CO.sub.2 temperature adjustment device 3a and/or pre-cooling device 3b as previously described in relation to
[0354] Solid material (for example plant material comprising source material, e.g. plant extract) may be packed in extraction vessel 4a, for example a jacketed extraction chamber.
[0355] CO.sub.2 and optionally co-solvent may be passed through the extraction vessel 4a at set/selected pressure and temperature.
[0356] The pressure and temperature (and therefore density) of the CO.sub.2 containing the source material may be adjusted using the pressure regulating and/or temperature regulating device(s) 31, 32. This may cause some of the solubility of some of the components extracted from the solid material to drop out of the CO.sub.2 solution. These may be removed to avoid fouling of the MIP in the second vessel/column 4b (for example by extract separator 33 and/or extract collection vessel 34).
[0357] The extract pressure regulating and/or temperature regulating device(s) 31, 32 may further allow the pressure/temperature/density of the CO.sub.2 to be adjusted prior to the CO.sub.2 (and optionally co-solvent) containing the source material being contacted with the MIP.
[0358] The CO.sub.2 (and optionally co-solvent) containing the source material may be passed to the MIP. In this example the MIP may be contained in a separate vessel 4b. The source material may be brought into contact with the MIP (for example MIP beads), for the target molecule(s) to associate with the MIP while the remainder of the extract (that does not associate with the MIP) may be flushed through with the CO.sub.2 (and optionally co-solvent). The extract/CO.sub.2/co-solvent that is flushed through may be collected and optionally may be passed to the MIP again or to another MIP (not shown), for further binding and elution.
[0359] Further CO.sub.2 at a different selected pressure and temperature or density (which does not contain source material/extract) and optionally co-solvent may be passed though/over the MIP in separate vessel 4b via a bypass valve (bypassing vessel 4a) to elute the target molecule(s) from the MIP. The pressure and temperature or density may be changed sharply or gradually from the temperature/pressure/density used to bring the source material into contact with the MIP.
[0360] The target molecule(s) may be collected, for example the CO.sub.2 (and optionally co-solvent) containing the target molecule(s) may be passed to pressure and/or temperature adjustment device 5a, 5b, where the pressure adjustment causes the target molecule(s) to lose solubility in the CO.sub.2. Pressure reduction may cause CO.sub.2 to change into gas phase and the target molecule(s) remains in the liquid phase, the target molecule(s) may be then physically separated at separator 6a, 6b and collected in collection vessel(s) 7 (7a-7f).
[0361] Physical separation in separator 6a, 6b may be by various means, including gravity and/or cyclone. Pressure reduction/adjustment physical separation may be done in multiple stages, for example two sequential stages are shown in
[0362] Following pressure adjustment/reduction separation the CO.sub.2 may be recycled by cooling and/or increasing the pressure to convert back to the liquid state at converter 8. The liquid CO.sub.2 may be reused in the method/system, for example by being returned to the storage tank 1a.
[0363]
[0364] Solid material (for example plant material comprising source material, e.g. plant extract) may be packed in the first portion of vessel 4, for example a jacketed extraction chamber.
[0365] CO.sub.2 may be passed through the first portion of vessel 4 at set pressure and temperature. The CO.sub.2 containing the source material may then flow to the MIP in the second portion of vessel 4. The source material may be brought into contact with the MIP (for example MIP beads), for the target molecule(s) to associate with the MIP while the remainder of the extract (that does not associate with the MIP) may be flushed through with the CO.sub.2. The extract that may be flushed through may be collected and may be passed to the MIP again or to another MIP (not shown), for further binding and elution.
[0366] Further CO.sub.2 at a different selected pressure and temperature or density (which does not contain source material/extract) may be provided via a bypass valve (shown between temperature adjustment device 3a and vessel 4) and enters vessel 4 by an alternative entry and may be passed though/over the MIP in vessel 4 to elute the target molecule(s) from the MIP.
[0367] The pressure and temperature or density may be changed sharply or gradually from the temperature/pressure/density used to bring the source material into contact with the MIP.
[0368] The target molecule(s) may be collected, for example the CO.sub.2 containing the CO.sub.2 containing the target molecule(s) may be passed to pressure and/or temperature adjustment device 5a, 5b, where the pressure adjustment causes the target molecule(s) to lose solubility in the CO.sub.2. Pressure reduction may cause CO.sub.2 to change into gas phase and the target molecule(s) remains in the liquid phase, the target molecule(s) may be then physically separated at separator 6a, 6b and collected in collection vessel 7a, 7b.
[0369] Physical separation in separator 6a, 6b may be by various means, including gravity and/or cyclone. Pressure reduction/adjustment physical separation may be done in multiple stages, for example two sequential stages are shown in
[0370] Following pressure adjustment/reduction/separation the CO.sub.2 may be recycled by cooling and/or increasing the pressure to convert back to the liquid state at converter 8. The liquid CO.sub.2 may be reused in the method/system, for example by being returned to the storage tank 1a.
[0371]
[0372] Solubilised source material may be fed from tank 24 through vessel/column 4 containing the MIP to associate the target molecule(s) with the MIP. The remaining extract may pass over the MIP to recovery tank 27. Recovery tank 27 collects the extract so that it may be passed to the MIP again or to another MIP (not shown), for further association/binding and elution.
[0373] Rinse tank 25 optionally provides rinse solvent (for example water/ethanol mix) which may be pumped through vessel 4 (over the MIP). Having passed through the MIP the rinse solvent may go to recovery tank 28 for disposal or reprocessing. A rinse may be used to wash off or reduce fouling of the MIP from the source material.
[0374] A purge (for example low pressure compressed CO.sub.2) is optionally provided from purge source 29, to the vessel 4, to remove residue rinse solvent from the MIP/vessel. Excess rinse solution may pass to gas-liquid separator 30, to separate CO.sub.2 and rinse solvent, the rinse solvent may be collected (for example at 31) for disposal or reprocessing.
[0375] A vacuum pump 32 is optionally provided to further remove residual rinse solution from the vessel/MIP. The CO.sub.2 gas collected may be vented or collected at 33.
[0376] Once the target molecule(s) are associated (for example bound) the MIP in vessel 4, (and optionally the vessel/MIP rinsed and/or purged and/or vacuum applied and released), a flow of fluid or liquid CO.sub.2 may be provided, for example from CO.sub.2 tank 1a, and selected pressure and temperature (for example via pre-cooler device 3b, pump, or high pressure pump 2 and/or temperature adjustment 3a).
[0377] An elution co-solvent (for example ethanol) is optionally provided from co-solvent tank 9a. The co-solvent may be injected into the CO.sub.2 flow prior to entering vessel 4. The amount/ratio of co-solvent is optionally controlled at co-solvent dosing pump 10 and/or the temperature of the co-solvent adjusted at co-solvent temperature adjuster 11.
[0378] The CO.sub.2 flow pressure and/or temperature may be adjusted during elution to elute different target molecules from the MIP, thereby at least partially separating the target molecules. For example, the CO.sub.2 flow may start at lower pressure and lower temperature to remove weakly associated target extract or target molecule(s). Such conditions may also or alternatively be used as an alternative to a rinse (as described above). The pressure and temperature may be increased over time and/or the amount of co-solvent changed (for example increasing ratio of ethanol). The extract and/or target molecules eluted under different conditions may be collected as fractions as they exit the vessel 4.
[0379] The fractions may be collected, for example the CO.sub.2/co-solvent containing target molecule(s) may be passed to pressure and/or temperature adjustment device(s) 5a-5e, where the pressure adjustment may cause the target molecule(s) to lose solubility in the CO.sub.2/co-solvent.
[0380] Pressure reduction may cause CO.sub.2 to change into gas phase and the target molecule(s) remains in the liquid phase, the target molecule(s) may be then physically separated at separator(s) 6a-6e and collected in collection vessel(s) 7a-7e.
[0381] Physical separation in separator 6a-6e may be by various means, including gravity and/or cyclone. Following pressure adjustment/reduction separation the CO.sub.2 may be recycled by condensing at condenser 8 to convert back to the liquid state. The liquid CO.sub.2 may be reused in the method/system, for example by being returned to the storage tank 1a.
[0382] Where, in the foregoing description reference has been made to integers or components having known equivalents thereof, those integers are herein incorporated as if individually set forth.
[0383] Although the present disclosure has been described in terms of certain embodiments, other embodiments apparent to those of ordinary skill in the art also are within the scope of this disclosure. Thus, various changes and modifications may be made without departing from the spirit and scope of the disclosure. For instance, various components may be repositioned as desired. Moreover, not all of the features, aspects and advantages are necessarily required to practice the present disclosure. Accordingly, the scope of the present disclosure is intended to be defined only by the claims that follow.
EXAMPLES
1. Extraction and Binding of Cannabinoids with Carbon Dioxide
1.1 Experimental
[0384] The experiment was carried out using crude cannabis extract as the input material, with the intent of binding cannabinoids.
[0385] Cannabis plant material was dried and then ground into a fine powder, picking out large stems and sticks.
[0386] A column was packed with dried ground plant material, then the remaining space was filled with MIP to ensure the CO.sub.2 flowed into the plant material then through the MIPs (carrying the cannabinoids).
[0387] The Supercritical CO.sub.2 machine was run at 40 C. at 4000 psi (27.58 MPa) for 15 mins (allowing for a heat soak to allow the vessel/column and the material inside to get to the same temperature).
[0388] The initial extraction process was 15 mins with CO.sub.2 running.
[0389] For the binding process the pressure was reduced to 1500 psi (10.34 MPa) and held for 10 minutes.
[0390] The experimental parameters are given in Table 1.
TABLE-US-00001 TABLE 1 Parameter Details MIP MIP beads (templated to non-covalently bind cannabinoids as a class) Mass: 12.581 grams Input Material Cannabis leaf, no flower/bud Mass: 18.779 grams Extraction from Plant 40 C. and 4000 psi (27.6 MPa) for total of 15 mins Binding to MIP 40 C. and 1500 psi (10.3 MPa), closed system held for 10 mins
[0391] The following time schedule was used for the experiment: [0392] Heating as turned ON at 1.10 pm at 40 C. and was left to heat soak through the column and material. [0393] Supercritical CO.sub.2 cylinders were turned ON at 1.35 pm, the pressure got to 4000 psi at 1.40 pm [0394] Extracted with flowing CO.sub.2 at 4000 psi for 15 mins (until 1.55 pm)Collected the material that passed over the beads and exited the cylinder. [0395] At 1.55 pm the Super Critical CO.sub.2 cylinders were switched off, and the pressure reduced to 1500 psi (while still capturing any expelled material). The bleed-off valve was closed to hold at this pressure for 10 min (until 2.05 pm). [0396] At 2.05 pm the bleed-off valve was opened, and all remaining pressure was slowly released (while still capturing any expelled material).
1.2 Results
[0397] The results are shown in Table 2.
[0398] Each dilution was done in duplicate (for example A and E, B and F, C and G, D and H). Cells A-H give what was bound to beads within supercritical conditions Cells I-P give what flowed past the beads, and into a collection vial outside the CO.sub.2 cylinder. Bold cells in the last two columns are the sample dilutions that fit within the calibration curve from the LCMS cannabinoid standards.
1.3 Discussion of Results
[0399] The approach for this experiment was to exploit the solubility of cannabinoids in supercritical CO.sub.2, which increases as the density of the fluid increases.
[0400] Higher pressure, (4000 psi) was used initially to extract from the plant material and to ensure the supercritical fluid had higher amounts of cannabinoids solubilised in solution. Then during the binding process, the pressure was decreased (1500 psi) as the density decreases, the aim is for the cannabinoids to drop out of solution and find preferential binding in the MIPs.
[0401] The results in the bold cells in the last two columns, suggest an average of 186.0 mg of CBD (cannabidiol) and 78.0 mg of THC (tetrahydrocannabinol) was bound to the MIP beads. The bold cells have been chosen as they are dilutions that fit in the calibration curve (whereas the other cells fall outside).
[0402] This experiment has provided initial evidence to support binding target molecules from MIPs using supercritical CO.sub.2.
TABLE-US-00002 TABLE 2 RAW LCMS data to ppb conversion Sample (using standard Correction for ppb to ppm Volume ppm to mg RAW LCMS data curve) dilution series conversion (L) conversion Dilution Factor CBD THC CBD THC CBD THC CBD THC CBD THC Dilution Factor CBD THC CBD THC CBD THC CBD THC CBD THC A Bound 10 54855264 51673977 53282 34365 532822 343652 533 344 0.13 69.3 44.7 B to 100 11381913 8266748 11055 5517 1105460 551745 1105 552 0.13 143.7 71.7 C Beads 1000 1482931 930094 1439 642 1439271 641627 1439 642 0.13 187.1 83.4 D 10000 149270 101230 144 91 1438254 907769 1438 908 0.13 187.0 118.0 E 10 55055893 51895499 53477 34512 534771 345124 535 345 0.13 69.5 44.9 F 100 11339558 8190221 11013 5467 1101346 546659 1101 547 0.13 143.2 71.1 G 1000 1489327 968771 1445 667 1445483 667331 1445 667 0.13 187.9 86.8 H 10000 145573 93791 140 86 1402343 858331 1402 858 0.13 182.3 111.6 I Flowed 10 15227827 19491681 14790 12977 147903 129774 148 130 0.005 0.7 0.6 J Past 100 2376471 2704107 2307 1821 230721 182061 231 182 0.005 1.2 0.9 K Beads. 1000 285430 301318 276 224 276084 223752 276 224 0.005 1.4 1.1 L 10000 35056 26018 33 41 328841 407922 329 408 0.005 1.6 2.0 M 10 14855672 19371233 14429 12897 144288 128973 144 129 0.005 0.7 0.6 N 100 2326755 2590838 2259 1745 225892 174533 226 175 0.005 1.1 0.9 O 1000 245673 286500 237 214 237466 213904 237 214 0.005 1.2 1.1 P 10000 30984 34617 29 47 289288 465069 289 465 0.005 1.4 2.3
2. Extraction and Binding of Cannabinoids with Carbon Dioxide
2.1 Experimental
[0403] The experiment was carried out using crude cannabis extract as the input material, with the intent of binding cannabinoids.
[0404] The crude cannabis extract was loaded into a small mesh bag, taking weights to calculate exactly how much extract was loaded.
[0405] A column was placed vertically, and glass beads poured in to make a 1-2 cm bed at the bottom of the column.
[0406] The mesh bag containing the extract was dropped in on top of the glass beads.
[0407] More glass beads were poured in to surround and cover the mesh bag by 1-2 cm.
[0408] A mesh frit was dropped in to keep glass beads separate from MIP.
[0409] MIP were loaded in to fill the rest of the column.
[0410] The setup of the column is shown in
[0411] The super critical CO.sub.2 was started 40 C. and 4000 psi for extraction.
[0412] For the binding to the MIP the CO.sub.2 was reduced to 1500 psi and leave at 40 C.
[0413] The parameters of the experiment are given in Table 3.
TABLE-US-00003 TABLE 3 Parameter Details Input Crude, non-winterised cannabis extract material Potency of 462.7 mg of crude 9.2% CBD | 1.1% THC 350 ppm CBD | 35 ppm THC Equipment Small non-commercial/benchtop scale supercritical CO.sub.2 extraction machine. Range of 20 C.-65 C. and 4500 psi max., extraction chamber is ~110 cm.sup.3. Direct attachment to CO.sub.2 cylinder, no flowrate control, vents directly to atmosphere. MIP Cannabinoid MIP beads (templated to non- covalently bind cannabinoids as a class) Mass: ~35 grams (est. only) Processing conditions: Extraction 40 C. and 4000 psi (27.6 MPa) Conditions Binding 40 C. and 1500 psi (10.3 MPa) Conditions
[0414] The following time schedule was used for the experiment: [0415] Heating was set at 40 C. and turned on at 2.15 pm, left to heat soak for 30 mins. [0416] The CO.sub.2 was turned on, and cylinders turned on at 2.45 pm to increase the pressure to 4000 psi. [0417] The pressure got to 4000 psi by 2.47 pm and continued to hold at this pressure with CO.sub.2 flowing until 3.17 pm. [0418] The CO.sub.2 was turned off at 3.17 pm, and the pressure was manually decreased to 1500 psi to hold for 15 mins. [0419] At 3.32 pm the cylinder valve was fully opened to allow the chamber to completely depressurise.
2.2 Results
[0420] The results are shown in Table 4.
[0421] The initial extract potency was 9.2% CBD (cannabidiol) and 1.1% THC (tetrahydrocannabinol).
[0422] The captured extract potency (in the MIPs) was 12.6% CBD and 1.3% THC.
TABLE-US-00004 TABLE 4 CO.sub.2 flow direction .fwdarw. Extract that passed Extract Glass beads Mesh holder MIPS through MIPS 350 ppm CBD 2 ppm CBD 2 ppm CBD 623 ppm CBD 164 ppm CBD 35 ppm THC 0 ppm THC 0 ppm THC 63 PPM 4 ppm THC (initial weight (522.8 mg oil before recovered with extraction and rotovap) binding 462.7 mg)
2.3 Discussion of Results
[0423] The data shows is 99.9% of the extract is solubilised and carried out from the mesh holder. It appears that the glass beads slow down/separate and hold onto the fats and waxes as the glass beads were covered in a green sticky residue, while the MIP's that bound the cannabinoids turned a more yellow colour.
[0424] The data further shows the MIP's ability to bind both CBD and THC within supercritical conditions.
[0425] Greater mass of oil/extract appeared to be recovered from the MIP's than was initially loaded into the mesh holder. This is believed to be due to added mass from a small amount of dust from the MIPs being washed off during the soxhlet wash into the ethanol that was later reduced in the rotovap and/or the margin of error with relatively small weights.
[0426] This experiment provided proof-of-concept evidence to support the ability of MIPs to bind target molecules in a supercritical CO.sub.2 medium.
3. Elution with Supercritical Carbon Dioxide
3.1 Experimental
[0427] The experiment was carried out using crude cannabis extract as the input material, with the intent of eluting cannabinoids. 5 g of non-winterised crude cannabis extract was dissolved into a 50% EtOH/H.sub.2O solution. MIPs were added to the solution to bind the cannabinoids, then dried in dehydrator. The super critical CO.sub.2 was run at three different conditions, with varying temperature and pressure. The parameters are given in Table 5.
TABLE-US-00005 TABLE 5 Parameter Details Input material Crude, Non-winterised cannabis extract Potency of 4987 mg of crude - 6.4% CBD and 2.6% THC Equipment Small non-commercial/benchtop scale supercritical CO.sub.2 extraction machine. Range of 20 C.-65 C. and 4500 psi (31.0 MPa) max., extraction chamber is ~110 cm.sup.3, Direct attachment to CO.sub.2 cylinder, no flowrate control, vents directly to atmosphere. MIP Cannabinoid MIP beads (templated to non- covalently bind cannabinoids as a class) Mass: ~41 grams (est. from column size) Beads bound: 99.8% THC | 99.6% CBD Processing Conditions 40 C. & 2000 psi First hour condition Potency of 336.1 mg of extracted oil 8.7% CBD and 2.1% THC 65 C. & 1400 psi Second hour condition Potency of 24 mg of extracted oil 18.8% CBD and 2.1% THC 67 C. & 3400 psi Third hour condition Potency of 29.5 mg of extracted oil 23.4% CBD and 2.4% THC Post-processing Steps: 200 mL neat EtOH Soxhlet wash contained 85% CBD and 40% THC
3.2 Results
[0428] The results are shown in Table 6.
TABLE-US-00006 TABLE 6 Physically Calculated Amount Total Weighed Column Stock of Cannabinoids Calculated Amount of Equivalent Volume Solution ppm (mg) Cannabinoids Extracted Oil Condition (mL) CBD THC CBD THC (mg) (mg) Extract Solution - Matrix 200 1597 638 319.3 127.7 447 5 g in 200 mL of 50% eth. Extract Solution - Pass Through 200 7 1 1.5 0.3 1.8 After Beads had soaked for 24 h 1st Hour - 40 C. CBD elution 5 5840 1447 29.2 7.2 36.4 336.1 @ 2000 psi scCO.sub.2 condition elution 2nd Hour - 65 C. THC elution 5 910 100 4.5 0.5 5 24 @ 1400 psi scCO.sub.2 condition elution 3rd Hour - 67 C. Total CANA 5 1370 137 6.9 0.7 7.6 29.5 @ 3400 psi scCO.sub.2 elution elution condition After scCO.sub.2 Total extract 200 1351 258 270.2 51.5 321.7 200 mL wash with recovery 100% EtOH
3.3 Discussion of Results
[0429] The initial 24-hour soak of MIP in 50% EtOH+Extract, bound 99.6% of both the CBD and THC present.
[0430] From the three elution methods, a total of 13.6% CBD (cannabidiol) and 6.6% THC (tetrahydrocannabinol) was eluted from the bound cannabinoids using supercritical CO.sub.2.
[0431] The total amount of recovered cannabinoids (including all three CO.sub.2 elutions, the washed beaker and the 200 mL pure ethanol wash) was: 98.6% CBD and 47.8% THC, suggesting the THC has a higher affinity to the beads and is not as easily eluted off.
[0432] This experiment provided initial evidence of the potential to apply this invention to selectively separate CBD and THC (due to the difference in affinities of CBD and THC to the MIP beads and the affinities to the supercritical CO.sub.2 at the different temperatures and pressures/conditions).
4. Elution with Supercritical Carbon Dioxide
4.1 Experimental
[0433] The experiment was carried out using crude cannabis extract as the input material, with the intent of determining whether supercritical CO.sub.2 elution has the potential to selectively separate bound targets (cannabinoids). 5 g of non-winterised crude cannabis extract was dissolved into 200 mL of 50% EtOH solution. MIPs were added to the solution to bind the cannabinoids, then dried. The super critical CO.sub.2 was run at one condition (55 C. and 2200 psi) for 1 hour. Samples of extracted oils were taken at different time periods
[0434] The parameters are given in Table 7.
TABLE-US-00007 TABLE 7 Parameter Details Input Crude, Non-winterised cannabis extract material Potency of 4970 mg crude 4% THC | 1% CBC | 34% CBD | 1% CBG | 0% CBN 35% CBDA | 1% THCA and 76% total cannabinoid content Equipment Small non-commercial/benchtop scale supercritical CO.sub.2 extraction machine. Range of 20 C.-65 C. and 4500 psi max., extraction chamber is ~110 cm.sup.3, Direct attachment to CO.sub.2 cylinder, no flowrate control, vents directly to atmosphere. MIP Cannabinoid MIP beads (templated to bind non-covalently cannabinoids as a class) Mass: ~41 grams (est. from column size) Beads bound: 100% THC | 100% CBC | 96% CBD | 100% CBG 100% CBN | 88% CBDA | 100% THCA from 50% EtOH Processing 55 C. and 2200 psi for a total of 1 hour condition Bound Target (input): Extract Physically calculated mass: About 3925 mg bound LCMS data on Cannabinoids shows: 3527 mg to MIP Potency 5% THC | 41% CBD | 39% CBDA Eluted Target (output): Extract 0-20 min Fraction #1 Potency of 64.3 mg extracted oil 2% THC | 35% CBD | 3% CBDA Extract 20-35 min Fraction #2 Potency of 8.2 mg of extracted oil 0% THC | 146% CBD | 12% CBDA Extract 35-48 min Fraction #3 Potency of 15.9 mg extracted oil 6% THC | 107% CBD | 6% CBDA Post-processing Steps: Soxhlet 3370.0 mg was still bound on MIP after CO.sub.2 elution Recovery (weight obtained via rotavap and tared glassware) of extract Potency 5% THC | 2% CBC | 50% CBD | 2% CBG | 1% CBDA
4.2 Results
[0435] The results are shown in Table 8.
[0436] The total elution from all three fractions was 88.4 mg. The total cannabinoids on MIP 3527.0 mg=2.51% total cannabinoid elution.
TABLE-US-00008 TABLE 8 Calculated Amount Total Physically Column Stock of Cannabinoids Calculated Weighed Equivalent Volume Solution ppm (mg) CANA Amounts Condition (mL) CBD THC CBD THC (mg) (mg) Extract Solution - 5 g in Matrix 200 8359 1066 1672 213 1885 4970 200 ml of 50% eth. Extract Solution -After Beads Pass 200 400 0 43 0 43 713.9 (after rotavap) had soaked for 24 h Through 20% Ethanol wash Rinse step 200 81 0 19 0 19 331.6 (after rotavap) with 20% EtOH scCO2 Elution #1 5 2229 121 22 1 23 64.3 (tared vial) scCO2 Elution #2 5 1187 41 12 0 12 8.2 (tared vial) scCO2 Elution #3 5 1740 71 17 1 18 15.9 (tared vial) Recovered extract from used 276.5 6118 610 1692 169 1861 3370 (after rotavap) MIP beads (Soxhlet recovery)
4.3 Discussion of Results
[0437] The results appear to show CO.sub.2 elution favours CBD extraction over THC.
[0438] This experiment has provided initial evidence to support elution of bound target molecules from MIPs using supercritical CO.sub.2.
[0439] This experiment further has provided initial evidence of the potential to apply this supercritical CO.sub.2 extraction to selectively separate CBD and THC (due to the difference in affinities of CBD and THC to the MIP beads and the affinities to supercritical CO.sub.2 at the different temperatures and pressures/conditions).
[0440] The results appear to imply that a greater mass of the cannabinoids were eluted than what was physically weighed in the sample vials. This is believed to be merely due to non-evaporated solvent, dust from the MIPs and/or difficulty of accuracy with small weights.
5. Solubilising, Binding, and Elution of Cannabis Extract
5.1 Experimental
[0441] The experiment was carried out using crude cannabis extract as the input material, with the intent of eluting cannabinoids. The following steps were followed: [0442] 100 g of extract was dissolved in 2 L of 50% EtOH, 50% water to make a solution. [0443] MIPs were added to the solution and left to soak for 24 hours to bind all the cannabinoids, then dried ready for the supercritical CO.sub.2. [0444] 45 g samples of bound MIP were weighed out for each supercritical extraction. [0445] The supercritical CO.sub.2 was loaded with a 45 g sample of dried bound MIP, and a range of pressures and temperatures were run, each as a 40 min extraction.
[0446] The parameters are given in Table 9.
TABLE-US-00009 TABLE 9 Parameter Details Input material Cannabis Extract, Winterised Potency 59.6% CBD Equipment Small non-commercial/benchtop scale supercritical CO.sub.2 extraction machine. Range of 20 C.-65 C. and 4500 psi max. Extraction chamber is ~110 cm.sup.3. Direct attachment to CO.sub.2 cylinder, no flowrate control, vents directly to atmosphere. MIP Cannabinoid MIP beads (templated to non-covalently bind cannabinoids as a class) Mass: 45 g samples for each run Beads bound: 98.8% CBD Processing Conditions: Initial 45 g Sample - Soxhlet 5.36 g total extract weight (after rotovap) 3.68 g CBD was calculated (LCMS Data) Total potency is 68.7% 2500 psi (17.3 MPa) @ 40 C. Percent elution = 31.39% 2500 psi (17.3 MPa) @ 50 C. Percent elution = 23.59% 2500 psi (17.3 MPa) @ 60 C. Percent elution = 13.04% 3000 psi (20.7 MPa) @ 40 C. Percent elution = 32.34% 3000 psi (20.7 MPa) @ 50 C. Percent elution = 21.85% 3000 psi (20.7 MPa) @ 60 C. Percent elution = 22.45% 3500 psi (24.1 MPa) @ 40 C. Percent elution = 32.83% 3500 psi (24.1 MPa) @ 50 C. Percent elution = 31.82% 3500 psi (24.1 MPa) @ 60 C. Percent elution = 29.97% 4000 psi (27.6 MPa) @ 40 C. Percent elution = 26.30% 4000 psi (27.6 MPa) @ 50 C. Percent elution = 18.40% 4000 psi (27.6 MPa) @ 60 C. Percent elution = 30.19% Post-processing Steps: 45 g sample after scCO.sub.2 - 2.622 g of CBD still remained after CO.sub.2 elution Soxhlet recovery of extract Potency = 68.3%
5.2 Discussion of Results
[0447] The initial 24-hour soak of MIP in 50% EtOH/50% water+extract bound 98.8% of the total CBD. The binding of the extract to the MIP increased the potency by 10%
[0448] The percentages eluted (% of the total amount of CBD from the initial 45 g soxhlet sample, i.e. % of the 3.68 g calculated from the LCMS data) compared to the density of the CO.sub.2 are shown in
[0449] Most of the higher temperatures of each pressure range showed a decrease in extraction, one exception being when the pressure got to 4000 psi (27.6 MPa). At 60 C. it showed the highest elution in this pressure range, even though the density is less than the other two conditions set at 4000 psi (27.6 MPa).
[0450] The post-processing step shows that the CO.sub.2 extraction does not elute all possible CBD, as there was still 2.622 g of CBD after a CO.sub.2 extraction (done at 2500 psi (17.2 MPa) at 40 C.)
[0451] It was noted that the binding of the extract to the MIP increased the potency of the crude material from the 59.6% CBD to 68.7%.
6. Binding of Caffeine with Sub-Critical Carbon Dioxide and Elution with Super-Critical Carbon Dioxide and a Co-Solvent
6.1 Experimental
[0452] The experiment was carried out to demonstrate sequential binding and elution of MIPs, using caffeine as the target molecule.
Binding
[0453] A 5 L column was packed with clean virgin MIP (see Table 10 for details).
[0454] Sub-critical CO.sub.2 (986 psi & 10 C.) was pumped through the column to precool the system (MIP and column).
[0455] Using the co-solvent pump, ethanol solution containing caffeine isolate was circulated through the column with the CO.sub.2. The ethanol was circulated at about 15% of the CO.sub.2 flow.
[0456] Binding solution (Sub-critical CO.sub.2 with 15% ethanol with caffeine) was recirculated to allow the caffeine time to bind to the MIP.
[0457] The binding solution was sampled for final concentration verification. The change in solute concentration allowed first quantitative determination of MIP absorption in CO.sub.2-ethanol system at saturation.
[0458] MIP was unloaded and air/freeze dried then weighed. The difference in weight allowed a second quantitative determination of MIP absorption in CO.sub.2-ethanol system at saturation. The dried MIP was ready for the elution step.
Elution
[0459] A 5 L column was packed with caffeine-bound MIPs (from the above sub-critical binding).
[0460] Supercritical CO.sub.2 (3002 psi and 40 C.) was pumped through the column of MIP.
[0461] The eluted target in supercritical CO.sub.2 was collected in a collection chamber.
[0462] When CO.sub.2 exited the column and entered the collection chamber the CO.sub.2 turned from liquid to gas and was separated from the caffeine. The CO.sub.2 evaporated and was recycled back to the CO.sub.2 reservoir. The extract/caffeine remained in the collection chamber.
[0463] The collection chamber was rinsed with ethanol to ensure that all target was collected.
[0464] As an additional step (elution step 2), the supercritical CO.sub.2 (4351 psi and 40 C.) with ethanol as a co-solvent (5.2 litres or 4.1 kg) was pumped through the column.
[0465] The eluted target was collected in a collection chamber. The collection chamber was rinsed with ethanol to ensure that all target was collected.
[0466] The general parameters of the binding and elution are given in Table 10.
TABLE-US-00010 TABLE 10 Details Parameter MIP MIP beads templated to non-covalently bind caffeine Mass: 2.0 kg Equipment Natex Super Critical Extractor 5 L Vessel Research and Development Unit. Flow rate of 30 kg/hr of CO.sub.2. Input material 60 grams of caffeine isolate mixed with 11,940 grams (Binding solution) EtOH 11,940 grams = 15,133 mL (15.133 L) 60,000 mg caffeine per 15.133 L = 3965 ppm Processing Conditions: Binding CO.sub.2 - 10 C. & 986 psi (68 bar, 6.8 MPa). Approx. 85 L Sub-critical CO.sub.2 + Co-solvent of CO.sub.2 was used (67.66 kg). EtOH Ethanol - 15 litres (11.940 kg), approx. 15% co-solvent (EtOH) flow by mass of the CO.sub.2 flow. Elution Step 1: 40 C. & 3002 psi (207 bar, 20.70 MPa) Supercritical Elution Elution Step 2: 40 C. & 4351 psi (300 bar, 30 MPa) Supercritical + Co-solvent Ethanol: 4095 g (5.2 L) Elution Co-Solvent/MIP g/g: 2.25 Co-Solvent to MIP ratio based on ethanol to stripped MIP. 3.70% co-solvent to CO.sub.2 flow by mass. e.g. in total 110 kg of CO.sub.2 was used and 5.2 litres or 4.1 kg of EtOH.
6.2 Results
[0467] The samples were tested using HPLC. The results are shown in Table 11.
TABLE-US-00011 TABLE 11 Mass Result Input material (Caffeine isolate) 60.0 g Binding: Target bound by MIP 6.1 g 10.2% binding Elution Step 1: Target eluted from 6 g 98.3% elution MIP from step 1 Elution Step 2: Target eluted from 0 g 0% elution MIP from step 2
6.3 Discussion of Results
[0468] MIP was used to bind caffeine from a CO.sub.2/ethanol solution then the bound caffeine was eluted from the MIP using CO.sub.2 as the eluent.
[0469] At 10 C. and 986 psi (68 bar) binding conditions, 2000 g of MIP bound 6.1 g of caffeine. This is equivalent to a binding capacity of 3.05 mg of caffeine per g of MIP or 10% binding efficiency based on the total initial caffeine available. It is expected that this capacity and efficiency can be further altered by adjusting the binding conditions.
[0470] At 40 C. & 3002 psi (207 bar) eluting conditions using CO.sub.2, approximately 98% elution of bound caffeine was achieved. For comparison
[0471] No comparison between elution with CO.sub.2 only and CO.sub.2 plus co-solvent (elution step 2) was able to be made due to the majority of caffeine being eluted prior to introduction of the co-solvent.
7. Binding of Phenols with Subcritical Carbon Dioxide and Elution with Supercritical Carbon Dioxide and a Co-Solvent
7.1 Experimental
[0472] This experiment demonstrates sequential binding and elution of two phenols 4-ethylphenol and guaiacol on/from a MIP.
Binding
[0473] A 5 L column was packed with clean virgin MIP.
[0474] Sub-critical CO.sub.2 (986 psi & 10 C.) was pumped through the packed column to pre-cool the system (MIP and column).
[0475] Ethanol solution containing 4-ethylphenol and guaiacol was circulated with the CO.sub.2 through the column at about 15% (CO.sub.2 flow) using the co-solvent pump.
[0476] Binding solution (Subcritical CO.sub.2 with ethanol with phenols) was recirculated to allow the phenols time to bind to the MIP.
[0477] The ethanol was sampled for final concentration verification (CO.sub.2 was allowed to evaporate off). This allowed comparison to the initial sample to calculate how much of the phenols had bound to the MIP. The change in solute concentration allows first quantitative determination of MIP absorption in CO.sub.2-ethanol system at saturation.
[0478] The MIP was unloaded and air/freeze dried then weighed. The difference in weight allows a second quantitative determination of MIP absorption in CO.sub.2-ethanol system at saturation.
[0479] The MIP was then dry and ready for elution.
Elution
[0480] A 5 L column was packed with 4-ethylphenol and guaiacol-bound MIPs.
[0481] Supercritical CO.sub.2 (3002 psi & 40 C.) was pumped through the column.
[0482] The eluted target in CO.sub.2 was collected in a collection chamber. When the CO.sub.2 exited the column and entered the collection chamber the CO.sub.2 turned from liquid to gas, so was separated from the phenols. The collection chamber was rinsed with ethanol to ensure that all target was collected.
[0483] As an additional step (elution step 2), supercritical CO.sub.2 (4351 psi & 40 C.) with ethanol as a co-solvent was pumped through the column.
[0484] The eluted target was collected in a collection chamber. When the CO.sub.2 exited the column and entered the collection chamber the CO.sub.2 turned from liquid to gas, so was separated from the phenols. The collection chamber was rinsed with ethanol to ensure that all target is collected.
[0485] The general parameters of the binding and elution are given in Table 12.
TABLE-US-00012 TABLE 12 Details Parameter MIP MIP beads, templated to non-covalently bind small phenols Mass: 1.822 kg Equipment Natex Super Critical Extractor 5 L Vessel Research and Development Unit. Flow rate 30 kg/hr of CO.sub.2. Input material 89.716 grams of solid 4-ethylphenol (4-EP) (Binding solution) 89.715 grams of liquid Guaiacol Mixed with 11,821 grams EtOH (14.982 L) 89,716 mg of 4-EP per 14.982 L 4-EP = 5988 ppm 89,715 mg of Guaiacol per 14.982 L Guaiacol = 5988 ppm Processing Conditions: Binding CO.sub.2 at 10 C. & 986 psi (68 bar, 6.8 MPa) Sub-critical CO.sub.2 + EtOH Ethanol 11,821 grams = 14,982 mL (14.982 L) EtOH at about 15% of the CO.sub.2 flow Elution Step 1: CO.sub.2 at 40 C. & 3002 psi (207 bar, 20.70 MPs) Supercritical CO.sub.2 Elution Elution Step 2: CO.sub.2 at 40 C. & 4351 psi (300 bar, 30 MPa), approx. Supercritical CO.sub.2 + Co- 100 kg. solvent (ethanol) Ethanol: 4.474 kg (5.7 L) Co-Solvent/MIP g/g: 2.46 Co-Solvent to MIP ratio based on ethanol to stripped MIP (approx. 4.5% flow rate of the CO.sub.2 by mass)
7.2 Results
[0486] The samples were tested using HPLC. The results are shown in Table 13.
TABLE-US-00013 TABLE 13 4-ethylphenol Total Mass Guaiacol Phenols Total Result Input material 89.716 g 89.715 g 179.431 g Binding: 25.8 g 35.7 g 61.5 g 34.3% Target bound by binding MIP Elution Step 1: 5.73 g 7.89 g 13.62 g 22.1% elution Target eluted from from step 1 MIP Elution Step 2: 1.27 g 0.26 g 1.53 g 3.2% elution Target eluted from from step 2 MIP
7.3 Discussion of Results
[0487] MIP was used to bind two types of phenol (4-ethylphenol and guaiacol) from a sub-critical CO.sub.2 plus ethanol solution then the bound phenols were eluted from the MIP using supercritical CO.sub.2 as the eluent. This demonstrated the use of CO.sub.2 as a binding solvent (or co-solvent) and elution solvent for MIP processes which capture phenols.
[0488] At the 10 C. and 986 psi (68 bar) binding conditions, 1822 g of MIP bound 25.8 g of 4EP and 35.7 g of guaiacol. This is equivalent to a binding capacity of 13.71 mg of 4EP per g of MIP and 19.59 mg of guaiacol per g of MIP. This is equal to 29% binding for 4EP and 40% binding for guaiacol based on the total initial 4EP and guaiacol available. It is likely that this capacity and efficiency can be further altered by adjusting the binding conditions.
[0489] At the 40 C. & 3002 psi (207 bar) CO.sub.2 elution conditions (elution step 1), approximately 5.73 g of the bound 4EP and 7.89 g of the bound guaiacol was eluted. This is equal to about 22% elution for both 4EP and guaiacol. It is of interest that under these conditions, the elution percentage was identical for both phenols whereas under the binding conditions the two phenols had different binding characteristics.
[0490] Using the 40 C. & 4351 psi (300 bar) CO.sub.2 plus ethanol as a cosolvent elution conditions (elution step 2), 1.27 g of 4EP and 0.26 g of guaiacol was eluted. This is equal to about 3.2% of the remaining phenols that are believed to be bound to the MIP after elution step 1. The lower amount of recovered phenols in this step would suggest the addition of the co-solvent reduced elution efficiency. It is possible that due to the volatility of the phenols they could have flashed off and have been lost to atmosphere and not recovered.
[0491] No residual phenols were found in the samples used for HPLC several weeks later. This is believed to confirm that the phenols were eluted effectively, but that the volatile nature of the phenols means they are lost to atmosphere or possibly entrained in the CO.sub.2. It is believed the mass recovery could therefore be improved by careful capture of the volatile phenols.
8. Elution of Cannabinoids from Pre-Loaded MIP Using Supercritical CO.SUB.2 .Only and then Supercritical CO.SUB.2 .with Co-Solvent
8.1 Experimental
[0492] This experiment demonstrated elution of CBD from MIP.
Binding
[0493] 4224 g of MIP (templated to non-covalently bind cannabinoids) were pre-loaded with 150 g of CBD isolate combined with 100 grams cannabinoid extract by suspending the mixture in 10 liters of 60% ethanol and 40% water matrix solution.
[0494] The MIP was separated from the matrix solution and dried on metal trays to remove as much water and ethanol as possible.
Elution
[0495] The dry MIP (4224 g) were then placed in a 10 L basket with sintered filter discs at both ends, and the basket was then loaded into a pressure vessel.
[0496] The vessel was pressurized to the elution pressure and CO.sub.2 was circulated through the vessel in an up flow direction.
[0497] After passing through the MIP, the CO.sub.2 containing the dissolved extract was depressurized to approximately 50 bar and 45 C. into a first separation vessel where the extract was accumulated. Gas phase CO.sub.2 was then condensed and recirculated.
[0498] Extract accumulating in the separation vessel was manually recovered through a valve periodically during the run to determine the progress of the extraction.
[0499] After a solvent:MIP ratio of 19:1 had been circulated (i.e. CO.sub.2 19 times the mass of the MIP was circulated through the MIP), the ethanol co-solvent pump was started (elution step 2) and extract collection was moved to a second separation vessel.
[0500] The CO.sub.2 flow rate was halved during the co-solvent stage, and the ratio of CO.sub.2:ethanol flow rate was set to approximately 2:1.
[0501] When a total ratio of 2:1 ethanol:MIP had been pumped in, the ethanol pump was stopped and neat CO.sub.2 was briefly circulated (10 minutes) to remove some of the excess ethanol from the extraction basket, before slowly depressurizing the plant.
[0502] Samples were rotary evaporated to remove ethanol and the mass was recorded.
[0503] The general parameters of the binding and elution are given in Table 14.
TABLE-US-00014 TABLE 14 Details Parameter MIP MIP beads, templated to non-covalently bind cannabinoids Mass: 4.224 kg Equipment Super Critical Extractor approx. 10 L Vessel Research and Development Unit. Input material 150 grams of CBD isolate 100 grams cannabinoid extract. Containing in total approximately 230 g of CBD. Processing Conditions: Binding 10 litres of 60% ethanol and 40% water matrix solution. Elution Step 1: CO.sub.2: 40 C. & 3989 psi (275 bar, 27.50 MPa) Supercritical Elution: Flow rate 26.4 kg/hr, 3.1 column volumes/hr Elution Step 2: CO.sub.2: 40 C. & 3989 psi (275 bar, 27.50 MPa), Supercritical + flow rate 12.6 kg/hr, 1.4 column volumes/hr, Co-solvent Elution Ethanol: 8448 g (10.7 L), flow rate 5.6 kg/hr, 0.7 column volumes/hr (approx. 44% flow rate of the CO.sub.2 by mass)
8.2 Results
[0504] The samples were tested using HPLC. The results are shown in Table 15.
TABLE-US-00015 TABLE 15 CBD Mass Total Result Input material 230 g Binding: 195 g 85% binding Target bound by MIP Elution Step 1: 59 g 30% elution from Target eluted from step 1 MIP Elution Step 2: 74 g 54.4% elution from Target eluted from step 2 MIP
8.3 Discussion of Results
[0505] This experiment demonstrated the ability to integrate a non-CO.sub.2 binding step (classic solvent) and an CO.sub.2 elution step together. MIP was used to bind CBD from a 60% ethanol, 40% water matrix solution using a liquid solubilising and binding procedure. Traditionally the CBD would then be eluted from the MIP using large volumes of ethanol. This traditional process produces an eluent which is CBD suspended in ethanol. The ethanol then would need to be removed using solvent recovery equipment.
[0506] This experiment provides evidence that the CBD can be eluted from the MIP using CO.sub.2, this significantly reduces the traditional solvent volume utilised in this process.
[0507] At 40 C. and 3989 psi (275 bar) CO.sub.2 eluting conditions (elution step 1), approximately 59 g of the bound CBD was eluted. This is equal to 30% elution of CBD.
[0508] At 40 C. and 3989 psi (275 bar) eluting conditions using CO.sub.2+ethanol as a cosolvent (elution step 2), a further 74 g of CBD was eluted. This is equal to about 54% of the CBD believed to remain on the MIP after the first elution step, (or 37% of the total CBD) and indicated the improvement in CBD elution that the addition of co-solvent provided.
[0509] For comparison, when eluting with ethanol only in a similar system, the average CBD elution efficiency is 69% of total bound CBD. The use of CO.sub.2+ethanol as a cosolvent (elution step 2) gave comparable results without the large quantities of ethanol.
9. Binding of CBD with Subcritical Carbon Dioxide and Elution with Supercritical Carbon Dioxide with Co-Solvent
9.1 Experimental
[0510] This experiment demonstrates sequential binding and elution of CBD from a MIP.
Binding
[0511] A vessel was loaded with 4224 g of MIP.
[0512] Cannabinoid solution containing a total 250 g of CBD extract mixed with CBD isolate in ethanol (2 litres, 1.6 kg) and was injected into the vessel using subcritical CO.sub.2.
[0513] In the first pass, 1835 g of solution was injected, and 366 g was collected in a separation vessel.
[0514] The extract collected after the first pass was then fed back into the liquid tank and recirculated into the MIP packed vessel. This process was repeated six times.
[0515] A total of 2880 g of cannabinoid solution was injected into the vessel, and 169 g of carried over extract was collected from the separation vessel after the sixth pass. Small samples of each pass were taken for analysis.
[0516] Once depressurized, the MIP was mostly visibly wet with ethanol and allowed to dry off in a warm room to remove ethanol so that it would not act as a cosolvent during the CO.sub.2 only phase of elution.
Elution
[0517] The dry MIPs (4224 g) were then placed in a 10 L basket with sintered filter discs at both ends, and the basket was then loaded into a pressure vessel.
[0518] The vessel was pressurized to the elution pressure and CO.sub.2 was circulated through the vessel in up flow direction.
[0519] After passing through the bed, the CO.sub.2 containing the dissolved extract was depressurized to approx. 50 bar and 45 C. into a first separation vessel where the extract was accumulated. Gas phase CO.sub.2 was then condensed and recirculated.
[0520] Extract accumulating in the separation vessel was manually recovered through a valve periodically during the run to determine the progress of the extraction.
[0521] After a solvent:MIP ratio of 19:1 had been circulated (i.e. CO.sub.2 19 times the mass of the MIP was circulated through the MIP), the ethanol co-solvent pump was started (elution step 2) and extract collection was moved to a second separation vessel.
[0522] The CO.sub.2 flow rate was halved during the co-solvent stage, and the ratio of CO.sub.2:ethanol flow rate was set to approximately 2:1.
[0523] When a total ratio of 2:1 ethanol:MIP had been pumped in (i.e. 8.448 kg ethanol to 4.224 kg of MIP), the ethanol pump was stopped and neat CO.sub.2 was briefly circulated (10 minutes) to remove some of the excess ethanol from the extraction basket, before slowly depressurizing the plant.
[0524] Samples were rotary evaporated to remove ethanol and the mass was recorded.
[0525] The general parameters of the binding and elution are given in Table 16.
TABLE-US-00016 TABLE 16 Details Parameter MIP MIP beads, templated to non-covalently bind cannabinoids Mass: 4.224 kg Contained approximately 30 g of residual CBD already on the MIP. Equipment Super Critical Extractor approx. 10 L Vessel Research and Development Unit. Input material 150 grams of CBD isolate 100 grams cannabinoid extract. Containing in total 197 g of CBD. (Solubilised in 2 L of 96% ethanol solution) Processing Conditions: Binding CO.sub.2: 40 C. & 1015 psi (70 bar, 7 MPa) Ethanol 2 litres (1.6 kg) Ethanol flow rate 50% of the CO.sub.2 flow rate by mass. Elution Step 1: CO.sub.2: 40 C. & 3989 psi (275 bar, 27.50 MPa) Supercritical Elution: Flow rate 24.6 kg/hr, 2.8 column volumes/hr Elution Step 2: CO.sub.2: 40 C. & 3989 psi (275 bar, 27.50 MPa) Supercritical CO.sub.2 + Co- Flow rate 11.4 kg/hr, 1.3 column volumes/hr solvent Ethanol: 8448 g (10.7 L) Flow rate 5.7 kg/hr, 0.7 column volumes/hr (approx. 50% flow rate of the CO.sub.2 by mass)
9.2 Results
[0526] The results are shown in Table 17.
TABLE-US-00017 TABLE 17 CBD Mass Total Result Residual CBD on MIP 30 g Input CBD material in binding 197 g solution Binding: 180 g 91% binding Target (CBD) bound by MIP Total CBD on MIP after binding 210 g Elution Step 1: 73 g 35% elution from Target eluted from MIP step 1 Elution Step 2: 103 g 75.2% elution from Target eluted from MIP step 2
9.3 Discussion of Results
[0527] This experiment demonstrated the ability to integrate a subcritical CO.sub.2 solubilising and binding and supercritical CO.sub.2 elution together into a cannabinoid refinement process.
[0528] MIP was used to bind CBD from a subcritical CO.sub.2 with ethanol cosolvent matrix containing CBD. This suggests CO.sub.2 (optionally with cosolvent) could be used to extract CBD or cannabinoids from plant material (see examples 1 and 2), then the liquid fed to the MIP for binding. This could potentially allow in line winterisation or refinement. Once cannabinoids are bound to the MIP, supercritical CO.sub.2 with or without cosolvent could be used to elute them from the MIP into a refined form. Utilising this approach also removes the need for a water/ethanol matrix for solubilising and is an alternative low liquid matrix option to the traditional matrix binding methods.
[0529] At 40 C. & 1015 psi (70 bar), approximately 91% CBD (180 g) was bound to the MIP. This is equal to a binding capacity of 43 mg/g. It is likely that this could be optimised by altering the CO.sub.2 temperature and pressure. For comparison, when binding using water and ethanol, the average CBD binding is 78%. Without wishing to be bound by theory, the high binding may be due to CO.sub.2 being a relatively poor solvent for cannabinoids but being a good penetrative solvent. This double effect may provide the CBD good access to the complete internal structure of the MIP where it binds to the MIP due to its favourable interaction with the MIP instead of the CO.sub.2.
[0530] At 40 C. and 3989 psi (275 bar) CO.sub.2 eluting conditions (elution step 1), approximately 73 g of the bound CBD was eluted. This is equal to 35% elution of CBD.
[0531] At 40 C. and 3989 psi (275 bar) CO.sub.2+ethanol cosolvent eluting conditions (elution step 2), 103 g of CBD was eluted. This is equal to 49% elution of the total CBD (or about 75% of the CBD believed to remain on the MIP after elution step 1) and indicated the improvement in CBD elution that the addition of co-solvent provided.
[0532] For comparison, when eluting with ethanol only in a similar system, the average CBD elution efficiency is 69%.
[0533] For comparison, the elution efficiency of CBD from MIP in a similar system using only ethanol ranges from 2-8 g of CBD per litre of ethanol. When ethanol was used in this experiment in the presence of CO.sub.2, the efficiency was 9.6 g of CBD per litre of ethanol. This was likely to be higher, if a significant portion of CBD had not already been removed in the first phase of the experiment where CO.sub.2 only was used at the eluent.
[0534] In this example the MIP used had 30 g (0.7%) of residual CBD already bound to the MIP. When a MIP purification process is used in an industrial process it is likely the MIP will contain residual bound target molecule, as removal of all the target molecule between cycles takes time and is likely unnecessary. Use of the MIP with 0.7% residual CBD content therefore may simulate binding to a MIP in a commercial setting where economic or time constraints prevent returning the MIP back to zero residual target content.