Humulone has been isolated in many ways. To isolate humulone we used the process described above by Sharp and Ormrod. An o-Phenylene Diamine/ Humulone Complex was made by combining 30.236g of a humulone rich hop oil that was extracted with supercritical CO2. The oil was dissolved in 50 mL benzene and 12.028g of o-phenylene diamine was added into the erlenmeyer flask, then mixed and heated on a hot plate. Once dissolved, decolorizing charcoal was added after boiling, and the mixture was immediately vacuum filtered using a buchner funnel. The filtrate was then cooled to crystallize and the crystals collected again by vacuum filtration. We repeated the recrystallization two times and were left with an o-phenylene diamine complex which was a yellow color. To extract the humulone from the o-phenylene diamine complex we added 6M HCl, a portion of the complex, and diethyl ether into a separatory funnel. This was shaken with frequent venting, and after separation the aqueous layer was drained. The organic layer was collected and the aqueous layer was re-extracted twice with ether. The collected organic layers were then washed four times with 6M HCl, NaCl, and then two aliquots of DI water. The ether was then evaporated off, to do this we used a rotovap. The remaining solid was humulone.
H NMR’s were taken after the extraction of humulone to verify that it was the correct substance. H NMR’s were also taken of the o-phenylene diamine complex.
Once humulone was obtained, we added drops of NaOH to 10 mL of DI water to give it a pH of 10. 0.05 g of humulone was then added to the water and dissolved. The water and dissolved humulone were poured into a covered polarimeter cell and polarimeter readings were taken every 15 minutes.
We tested the effects of another solvent on polarimeter readings related to isomerization. 8 mL of ethanol and 2 mL of pH 10 Ammonium Hydroxide Buffer were combined and 0.05 g of humulone was dissolved in it. Another set of polarimeter readings were taken.
We followed patent 3,354,219 on a Method for Preparation of Isohumulone Concentrates in an attempt to obtain isohumulone in order to take and H NMR for future reference and comparison. We first refluxed 300 mL DI water, 8.001 g 15% hops, and 10 mL 3N NaOH for 10 minutes in a light shielded round bottom. The mixture was then filtered into a flask and 7.5 mL of 6M HCl was added so that the organics would crash out. The isohumulone attaches to the organics and therefore crashes out as well. The organics were then vacuum filtered out with a buchner funnel. The compound was then combined with 15 mL methanol, 15 mL hexanes, and 15 mL 2% H2SO4 in a separatory funnel. After shaking and venting the compound did not dissolve and the funnel became clogged. Since we only wanted the hexanes layer from the top we pipetted it off and filtered it. The hexanes were then rotovapped off of the isohumulone.
H NMR’s were taken after the extraction of humulone to verify that it was the correct substance. H NMR’s were also taken of the o-phenylene diamine complex.
Once humulone was obtained, we added drops of NaOH to 10 mL of DI water to give it a pH of 10. 0.05 g of humulone was then added to the water and dissolved. The water and dissolved humulone were poured into a covered polarimeter cell and polarimeter readings were taken every 15 minutes.
We tested the effects of another solvent on polarimeter readings related to isomerization. 8 mL of ethanol and 2 mL of pH 10 Ammonium Hydroxide Buffer were combined and 0.05 g of humulone was dissolved in it. Another set of polarimeter readings were taken.
We followed patent 3,354,219 on a Method for Preparation of Isohumulone Concentrates in an attempt to obtain isohumulone in order to take and H NMR for future reference and comparison. We first refluxed 300 mL DI water, 8.001 g 15% hops, and 10 mL 3N NaOH for 10 minutes in a light shielded round bottom. The mixture was then filtered into a flask and 7.5 mL of 6M HCl was added so that the organics would crash out. The isohumulone attaches to the organics and therefore crashes out as well. The organics were then vacuum filtered out with a buchner funnel. The compound was then combined with 15 mL methanol, 15 mL hexanes, and 15 mL 2% H2SO4 in a separatory funnel. After shaking and venting the compound did not dissolve and the funnel became clogged. Since we only wanted the hexanes layer from the top we pipetted it off and filtered it. The hexanes were then rotovapped off of the isohumulone.