The Use of Preparative Supercritical Fluid Chromatography for the Isolation of Cannabidiolic Acid (CBDA) From Hemp Extract

Transcription

1 The Use of Preparative Supercritical Fluid Chromatography for the Isolation of Cannabidiolic Acid (CBDA) From Hemp Extract Andrew Aubin and John Flynn Waters Corporation, Milford MA, USA #emeraldconference 2/22/ Emerald Conference 2018 Waters Corporation 1

2 Cannabidiolic Acid (CBD-A) CAS Number Molecular Formula - C 22 H 30 O 4 Formula Weight ,4-dihydroxy-3-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-6-pentyl-benzoic acid Unfortunately, substantial research on CBDA is lacking. Preliminary research in the lab suggests that CBDA may be helpful in four distinct therapeutic areas. These include: Inflammation, Nausea, Anti-cancer, Psychosis 2018 Waters Corporation 2

3 General Purification Workflow 2018 Waters Corporation 3

4 Sample and Sample Extraction Cannabis Sativa (20 lb) grown in Vermont, buds and leaves ground and homogenized. Sample was extracted using a Waters BBES with 100% CO 2 Total cannabinoid content was ~ 5% (wt/wt). Total yield was ~ 670 g raw extract SFE Conditions: Flow Rate Extraction Pressure Extraction Temperature Time 170 g/min 344 bar 50 C 210 min Collection Conditions: CS1 CS2 CS3 158 bar/45 C 75 bar/40c 53 bar/35 C Three extract fractions were collected from each extraction, one from each cyclone separator (CS 1-3). Four of the resulting CS1 extracts were combined and homogenized in cannabinoid containing ethanol washes performed post extraction (~85g raw extract in 1.2L of ethanol wash) and stored at -20C until analysis and purification. Sample provided by, and extractions performed at ProVerde Labs, Milford MA 2018 Waters Corporation 4

5 Chlorophyll Removal AU Minutes 44.5 mg/ml CBD-Acid Pre-Removal Post-Removal 2018 Waters Corporation 5

6 SFC Method Development All of the method development activities were performed using a Waters UPC 2 system with a SDQ2 mass spectrometer. Generic Column Screening Separation Method Development on Selected Column Loading Study 2018 Waters Corporation 6

7 Columns and Mobile Phases Mobile Phase Carbon Dioxide was the primary mobile phase Co-Solvent 190 Proof Food Grade Ethanol 9 Separate Columns Screened (3X50 mm 5 µm) 2018 Waters Corporation 7

8 Screening Procedure Generic Isocratic Conditions 15% 190 Proof EtOH as co-solvent, 2 ml/min, ambient temp, 2000 psi BPR 1. Ethanol Blank 2. 6 Compound Mix (CBD, THC, CBN, THCA, CBDA, CBGA) 3. Filtered extract 1. Evaluate 2. Re-Run at different co-solvent if required 2018 Waters Corporation 8

9 Standard Mix BEH DIOL 2EP 2PIC CBD, THC, CBN, THCA, CBDA, CBGA 2018 Waters Corporation 9

10 Diluted Filtered Extract CBDA BEH CBDA DIOL CBDA 2EP CBDA 2PIC 2018 Waters Corporation 10

11 UPC 2 Analytical Column 2018 Waters Corporation 11

12 Separation Method Development Parameters Separation Technique Isocratic or Gradient Column Bonded Phase 2-PIC based on column screening Column Length Based on the Screening 50 mm not enough Co-Solvent Food Grade 190 proof Ethanol Co-Solvent % Screenings were at 15% Additives Water, peak shapes indicated no other additional additive needed Flow Rate Screening was at 2 ml/min Pressure Screening was at 138 bar (2000 psi) Temperature Ambient 2018 Waters Corporation 12

13 Separation Method Development 10% CS 15% CS 2018 Waters Corporation 13

14 Final Analytical Method Column 2-PIC 3 X 150 mm 5 µm Separation Mode Isocratic Mobil Phase 10% 190 proof Ethanol: 90% Carbon Dioxide Flow Rate 3 ml/minute Temperature Ambient Pressure 138 bar (2000 psi) THC/CBD/CBN THC-A CBD-A CBG-A CBC-A 2018 Waters Corporation 14

15 Loading Study A loading study should be performed to determine how much sample can be loaded while maintaining a resolution adequate for producing a highly pure isolate. Column loading is dependent upon the sample volume and concentration, which both affect resolution. These studies are performed at small scale and are executed by one of the following techniques; a sample is prepared at a high concentration and the injection volume incrementally increased, or a sample is prepared at multiple concentrations and the injection volume held constant Waters Corporation 15

16 Injection Mechanisms 2018 Waters Corporation 16

17 Mixed vs Modifier 2018 Waters Corporation 17

18 Converting UPC 2 to MSI To Column Aux Valve 2 1 Simple Tee 3 6 Plumbing for modifier stream injection Co-Solvent CO Inject Valve 2 1 Co-Solvent CO 2 Replace Mixer with 2 unions 2018 Waters Corporation 18

19 Loading Study 2018 Waters Corporation 19

20 Loading Study 15 ul Injection 10 ul Injection 5 ul Injection 2018 Waters Corporation 20

21 Scaling to Prep 3.0 X 150 mm to 30.0 X 150 mm Average Pressure 2018 Waters Corporation 21

22 Prep 350 SFC 2018 Waters Corporation 22

23 Scaled Test Injection 30.0 X 150 mm 2-PIC 5 um 10% 190 Proof EtOH 300 g/ml total Flow 132 Bar 2018 Waters Corporation 23

24 Stacked Injections Injections are made while an already injected sample is on (or eluting from) the column. To utilize stacked injections, isocratic methods are required. In order to stack injections successfully, the correct cycle time (or time between injections) needs to be determined. Volts Time (min) Time (min) Time (min) Sequential injections Volts uv uv uv Time (min) Time (min) Time (min) Stacked injections 2018 Waters Corporation 24

25 Prep System Final Steps 1. Scaled separation method 2. Make collection Method 3. Make Stacked Injection Method 4. Run the sample list 2018 Waters Corporation 25

26 Stacked Injection Run One injection every 1.7 min 2018 Waters Corporation 26

27 Collection Results Total Volume Collected = 370 ml of Ethanol Assay of Collected Fraction = mg/ml Mass of collected CBD-A = mg Total Injected amount of CBD-A = mg Recovery = 94% Calculated Total Throughput mg per 1.73 min = mg/hr 5.60 g/ 8 hr day 2018 Waters Corporation 27

28 Potential Throughput ~ Every 1.7 minutes, collected ~ 20.2 mg CBD-A Calculated Total Throughput mg/2 min = mg/hr = 5.60 g/8 hr day Potential Total Throughput 56 mg/1.73 min = 1942 mg/hr = 15.5 g/8 hr day 2018 Waters Corporation 28

29 Post Purification 0.90 ~370 ml of Ethanol RotoVap/Freeze Dry CBDA Assay of recovered powder ~96.3% CBDA UV Purity = 80.7% to 97.0 % AU Minutes No indication of THC-A or THC UV@228 nm or LC/MS Major impurity was CBG-A 2018 Waters Corporation 29

30 Conclusions A SFC method for purification was developed using food grade ethanol at an analytical scale Separation method was scale to prep (30X150 mm column) From a SFE hemp extract, ~300 mg of CBD-A was produced with a purity of ~96.3% Inject to inject cycle time was ~ 1.7 minutes A calculated throughput of ~700 mg/hr was achieved using the 30X150 mm 2-PIC column 2018 Waters Corporation 30

31 Acknowledgments Shawn Helmueller Waters Chris Hudalla ProVerde Labs Jaci Runco Waters Catharine Layton Waters 2018 Waters Corporation 31

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