1. The Isomerization of Humulone into Isohumulone Dylan Walker 1, Bixby Daugherty 1, and Sean Johnson 2 1 Frontiers of Science Institute, 2 Department of Chemistry and Biochemistry, University of Northern Colorado, Greeley, CO Introduction Results Conclusions Abstract Methods and Materials Beer has been brewed for thousands of years. Modern beer is brewed with hops for a preservation agent and for flavor. The compound in hops, humulone, isomerizes into isohumulone in the brewing process when wort is boiled. Isohumulone is the main compound that causes the bitter flavor of beer. If a factor such as pH is found and used to manipulate the isomerization rates of humulone, it could have a significant effect due to the large world market of brewing. The quality control of isomerization will help maintain reproducible flavors, and breweries want to have a consistent flavor of their product. To isolate humulone from hops we used the process developed by Sharpe and Ormrod. We started with a humulone rich hop oil extracted with supercritical CO 2 from HopSteiner, Inc.. The hop oil was heated to boil with benzene and o-phenylenediamine (OPD) until dissolved, and then vacuum filtered. The filtrate was cooled to crystallize and again vacuum filtered to collect the crystals leaving us with an OPD humulone complex. Recrystalization was preformed 2-3 times. To extract the humulone from the OPD complex, we combined the complex with 6M HCl and diethyl ether in a separatory funnel. The aqueous layer was drained and the organics collected. The ether was then evaporated off. The remaining solid was humulone. A polarimeter measures the degree to which a substance rotates plane polarized light. Since humulone and isohumulone rotate light differently, -212° per g/mL for humulone and 46.7° and -7.8° per g/mL for cis- and trans-isohumulone respectively, a polarimeter can be used to gauge when humulone isomerizes into isohumulone. We isolated isohumulone following US patent 3,354,219. Hops were combined and refluxed with DI water and NaOH at a pH of 10. It was then filtered and HCl was added so that the organics crashed out. Isohumulone adheres to the filtrate. This was vacuum filtered. Hexanes were added and then pipetted off. The isohumulone was extracted with the hexanes. The hexanes were mostly removed leaving isohumulone that needs to undergo further purification. The 1 H NMRs of the two processes were compared to better understand the isomerization. Acknowledgements References Clark, J. (2000). Plane Polarised Light. Retrieved June 25, 2015, from http://www.chemguide.co.uk/basicorg/isomerism/polarised.html Haseleu, G., Intelmann, D., & Hofmann, T. (2009). Structure determination and sensory evaluation of novel bitter compounds formed from β-acids of hop (Humulus lupulus L.) upon wort boiling. Food Chemistry, 71-81. doi:10.1016/j.foodchem.2009.02.008 Johnson, S. (2014). A Study of the Isomerization Humulone into Isohumulone, Using Synthetic and Spectroscopic Studies. University of Northern Colorado Department of Chemistry and Biochemistry, 1-23. Meussdoerffer, F. (2009). A Comprehensive History of Beer Brewing. Handbook of Brewing, 1-42. Retrieved June 24, 2015, from http://www.wiley- vch.de/books/sample/3527316744_c01.pdf Rigby, Francis L. Method for Preparation of Isohumulone Concentrates. Haas Inc John I, assignee. Patent 3354219. 21 Nov. 1967. Print. Sharpe, F., & Ormrod, I. (1991). Fast Isomerisation Of Humulone By Photo-Reaction: Preparation Of An Hplc Standard. Journal of the Institute of Brewing, 97, 33-37. Unknown. (2014). Chirality. Retrieved June 25, 2015, from http://0-search.credoreference.com.source.unco.edu/content/entry/heliconhe/chirality/0# Unknown. (2015). Isomerization, chemical reaction. Retrieved June 25, 2015. Urban, J., Dahlberg, C., Carroll, B., & Kaminsky, W. (2012). Absolute Configuration of Beer′s Bitter Compounds. Angew. Chem. Angewandte Chemie, 1593-1595. doi:10.1002/anie.201208450 Advisor: Sean Hoskins Mentors: Dr. Mosher Sean E. Johnson Research Sponsors: Kinder Morgan Foundation (Dylan) Union Pacific Foundation (Bixby) Scholar Sponsors: Adolph Coors Foundation (Bixby) Suncor Energy (Dylan) University of Northern Colorado Chemistry and Biochemistry Department Table 1: Polarimeter readings for 0.05g of humulone in 10 mL pH 10 buffer and ethanol. Figure 1: An 1 H NMR of our OPD and humulone complex. Figure 2: An 1 H NMR of our humulone. The peaks in our humulone match the peaks in the known humulone, except for the ones at 7 and 7.3 which are benzene and our solvent deuterated chloroform. (Figure 2 and 5). Figure 3: An 1 H NMR of our compound after combining with pH 10 buffer and ethanol. The peaks from this do not match the known humulone peaks, so we believe it may have formed a salt complex with NH 4 OH. Figure 4: An 1 H NMR of isohumulone. This sample need further purification to remove the hexanes. This was following the patent. In the brewing process, the compound humulone in hops isomerizes into isohumulone when the wort is boiled. Isohumulone is the main compound that causes beer to have a bitter flavor. Humulone was isolated in order to test isomerization. Isohumulone was also isolated in order to obtain an 1 H NMR. The 1 H NMR spectra were compared to unknown compounds to see if they isomerized. Figure 5: An annotated humulone compound and its respective 1 H NMR peaks. Future Research Isohumulone needs to be purified further. Cis- and trans-isohumulone need to be separated using chromatography to determine which peaks correlate to which compound on the 1 H NMR. Humulone can be isolated using the process described by Sharpe and Ormrod (1991). Isohumulone can be isolated by the methods described in US patent 3,354,219. Dissolving humulone in basic ethanol changes the compound into a possible isohumulone. Further purification of the isohumulone from the hexanes is needed to accurately compare the two 1 H NMRs.