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Isomerization of Humulone into IsohumuloneDylan Walker2015 Frontiers of Science InstituteUniversity of Northern Colorado, Greeley, COMentored by Sean E. Johnson and Dr. MosherIntroductionHops are a plant used in the process of brewing beer. Beer has been brewed with hops for hundreds of years as a preservation agent. The compounds in hops responsible for the bitter flavor many people of age experience are isohumulones. Humulone is photosensitive and can change to an isohumulone through a process known as isomerization. The goal of the research is to isolate humulone and see how different conditions will affect the isomerization. Light is a known factor, but there are other factors that can be explored. Temperature can affect isomerization rates along with pH levels of water. Figure 1. Humulone and its isomers.If a factor 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 isohumulones contribute to the bitter taste of beer and breweries want to have a consistent flavor of their product. Humulone is not very soluble in acidic water, which is the type of water used in brewing, but its isomers are more soluble, which allows the taste of the beer to change. UV light treatment is not ideal for brewing conditions, so other ways of manipulating the isomerization rates are extremely helpful.Humulone and its isomers are optically active meaning they can rotate polarized light emitted by a polarimeter. The polarimeter will give a measurement of the rotation of the polarized light and from the measurement, it can be determined from a control of previously defined specific rotations what is present in the sample. After extraction of the humulone from hops oil, the sample can be placed in a polarimeter cell and then inside the instrument chamber and tested to record the degree to which the light is rotated. Figure 2. Use of a polarimeter cell inside of a polarimeter. The variable α is used as the degree of rotation the instrument detects.Literature ReviewHumulone, being light sensitive, degrades in light. This degradation is minimized by keeping reactions out of light and bottling the beverage in colored glass bottles. This adds more cost to the manufacture process as colored glass is more costly than clear glass bottles. If the isomerization of humulone can be manipulated, the price of manufacturing beer will drop, in turn allowing consumers a price drop as well (Keukeleire, D. D., 2000).Humulone rotates polarized light at -212° per g/mL. This is humulone’s optical rotation. Cis- and trans- isohumulone have an optical rotation of 46.7° and -7.8° respectively. Knowing this, we can calculate the amounts of cis- and trans- isohumulone using a polarimeter during and after isomerization. Sharpe and Ormrod used a preparation of an o-phenylenediamine/ humulone complex from 150 g of an alpha-acids-rich hop extract and 600 mL of benzene. The mixture they used was stirred on a hotplate with 12 g of decolorizing charcoal and 55 g o-phenylenediamine added prior to the mixture boiling. Sharpe and Ormrod refluxed the mixture for two minutes and then added another 2 g of decolorizing charcoal before immediately filtering under vacuum through a pre-heated sintered glass funnel. The filtrate was then cooled to 5 degrees C and the precipitate was removed by the vacuum filtration. This was done another four times with decolorizing charcoal added to the first two times. This method of re-crystallization produced 90 g of complex.Ensuing the re-crystallization came separation of humulone from the complex. This was achieved through a separatory funnel. 60 ml of 6 M hydrochloric acid was poured into a250 ml glass separating funnel, and aluminum foil wrapped round the outer surface to exclude light. 5 g of the o-phenylenediamine/ humulone complex was added to the separatingfunnel, followed by 30 ml of ether. The funnel was stoppered, shaken vigorously, and vented frequently. The lower aqueous layer was slowly drained, with the organic layer being collected. The aqueous layer was re-extracted twice with two 30 ml aliquots of ether, and the organic layers collected and combined with the initial organic fraction in a second, light-shielded 250 ml separating funnel.The combined ether extracts were then washed with 6 M HCl (50 ml), saturated NaCl (50 ml), and two 100 ml aliquots of distilled water. The mixture was allowed to separate, the aqueous layer discarded, and the ether evaporated by rotary evaporation. The remaining white solid was humulone. Using this procedure, 5 g of o-phenylenediamine complex yielded 3.8 g of humulone (Sharpe and Ormrod, 1991).Francis L. Rigby found a way to get a concentrate of isohumulones. 3000 mL of hot water was added with 90 mL of 3.0 N NaOH and 80 g of hops and refluxed and boiled for 10 minutes. After boiling, the mixture was filtered to get the spent hops separated from the liquid. Then 74 mL of 6 N HCl was added and the acid formed a precipitate. The isohumulones adhere to this precipitate for extraction.Following the patent is useful because getting an H-NMR spectrum of isohumulone will determine if using ammonium hydroxide is a faster way to get humulone to isomerize. H-NMR spectra will be used to compare the two humulone samples. If the ammonium hydroxide buffer works, it would be much easier to get humulone to isomerize after extraction from hops oil.Materials and MethodsHops oil is the starting substance to extract the humulone from. o-phenylenediamine is also used alongside benzene. A hot plate, magnetic stir rod, pipette, syringe, Erlenmeyer flask, foil, rubber stop, activated charcoal, Buchner funnel, vacuum filter, filter paper, filter flask, vial, balance, balance paper, rotary evaporator (rotovap), polarimeter, polarimeter cell, round bottom flasks of multiple volumes, bump bulb, magnesium sulfate, hydrochloric acid (6M and 12M), ether, deionized water, pH buffer, deuterated chloroform, NMR tube, separatory funnel, graduated cylinder, and vacuum pump were all used in different quantities throughout the research.Humulone can be extracted from hops oil. According to F.R. Sharpe and I. H. L. Ormrod, humulone can be extracted and isolated by preparing an o-phenylenediamine/ humulone complex (OPD complex). This is useful to the experiment because humulone needs to be isolated to be able to explore its isomerization rates. With the o-phenylenediamine/ humulone complex, the complex is able to crystallize after being added with benzene as a solvent. This allows for the complex to be more pure after every crystallization. Figure 3. UV absorbance spectra by Sharpe and Ormrod. Spectrum of OPD complex (left) and purified humulone (right).The complex is crystallized by bringing the complex and benzene to a boil while mixing. The complex is then treated with decolorizing charcoal. Vacuum filtration is next while keeping the complex heated. Once cooling, the complex re-crystallizes with the precipitate left behind. In the lab, this was done two times to be able to extract the humulone.To re-crystallize, 30.236 g of hops oil, 12.028 g OPD, and 50 mL of benzene were used. The hops oil was heated along with o-phenylenediamine and benzene until the mixture was dissolved and boiling. A Buchner funnel was prepared by placing it in the oven to keep it hot in preparation for filtering. Once the mixture came to a boil, heat was turned off while a pea sized amount of decolorizing charcoal was added and stirred. The mixture was then placed back on the hotplate while the now hot Buchner funnel was placed in the vacuum filtration setup. The mixture was quickly transferred to the vacuum filter and immediately filtered. After filtration, the filter flask was placed in the refrigerator to crystallize for fifteen minutes. After the fifteen minutes, the crystals were removed and vacuum filtered a second time by using benzene. The mass of the remaining yellow crystals was 34.472 g.Sharpe and Ormrod also describe how to separate the humulone from the OPD complex. This is the essential part in the research because a supply of humulone is needed to conduct research with. The complex has other compounds, so the purification process will help to get just humulone. Figure 3 shows the difference between purified and non-extracted humulone.The separation process requires a separatory funnel, the complex, deionized water, hydrochloric acid, ether, magnesium sulfate as a drying agent. 30 mL of 6 M HCl was obtained by mixing 15 mL of DI water and 15 mL of 12 M HCl. 1.05 g of the complex was added with 20 mL ether in a separatory funnel, then shaken and vented. Then 6 mL of 6 M HCl was added to the funnel and shaken and vented. The bottom aqueous layer was separated. The entire process was repeated and then the organic layer was removed. The aqueous layer was re-added to the separatory funnel with ether to collect the aqueous layer again. The organic layer was added back and washed with 6 mL 6M HCl, and then again with about 6 mL brine, and twice more with DI water. The organic layer remaining was put in an Erlenmeyer flask with magnesium sulfate as a drying agent to remove water. The magnesium sulfate was gravity filtered out and the organic layer was put inside of a round bottom flask. The ether was rotovapped out and an orange substance was left over A proton nuclear magnetic resonance (H-NMR) spectrum was collected from this sample to determine what exactly is in the crystallized humulone. The separation process was repeated with Sean Johnson’s humulone crystals and an H-NMR was also taken of his sample for comparison.Following US patent 3,354,219, the next goal was to isolate isohumulone. 4 g of hop oil and 25 mL of the pH 10 buffer were refluxed for 10 minutes at a boil. Water was filtered into an Erlenmeyer flask by wetting the filter paper with DI water to separate the water and oil. 4 mL of 6 M HCl was added to the water to crash out the organic material that according to the patent, contains isohumulone. There was no crash out of organic material so the compound was placed in a separatory funnel with ether, shook and vented, and collected the aqueous and ether layers. The ether layer was dried with magnesium sulfate and then filtered into a vial and rotovapped. An H-NMR was taken and the results were inconclusive.Due to the inconclusive results, the patent was replicated using hops instead of hops oil. 300 mL of DI water, 8.001 g of Australian Galaxy 15% hops, and 10 mL of 3 N NaOH were refluxed in a light-shielded 500 mL round bottom flask for 10 minutes. The mixture was filtered into a 500 mL Erlenmeyer. 7.5 mL of 6 M HCl was added to the water and hops mixture and mixed. The organics with attached isohumulone crashed out. The white organics containing isohumulone was then filtered out of the water under a vacuum filter and Buchner funnel. The organic material and isohumulone left weighed 8.366 g.The organic material with isohumulone was combined with 15 mL of methanol in attempt to dissolve it. Although not completely dissolved, the solution was placed in a separation funnel with 15 mL of hexanes and 2% sulfuric acid. The funnel was shaken and vented in attempt to dissolve the compound. It dissolved some, so the aqueous layer was going to be drained, but it clogged the separatory funnel. The hexanes layer was needed from the top, so it was pipetted off and filtered. It was then put in a vial and rotovapped. The final compound weight was 0.171 g.Results and DiscussionAfter making the o-phenylenediamine/ humulone complex, an H-NMR was taken to verify that the complex obtained was the complex desired, shown in Figure 4. The H-NMR does show that the desired complex was obtained. This is great because 34.472 g of the crystals were obtained. Figure 4. H-NMR spectrum of o-phenylenediamine/ humulone complex.After the isolation of humulone, H-NMR spectra were obtained in two different deuterated solvents. The two solvents were used since each solvent has its own peak that is seen in the spectrum and the peak may overlap a peak present in the compound. Figure 5 shows humulone in deuterated chloroform and Figure 6 shows humulone in deuterated acetone. Figure 5 shows chloroform and benzene remnants in the sample. The chloroform and benzene peaks are the peaks to the far left in the spectrum. The benzene had just not evaporated fully and the chloroform is present because not all of the chloroform in the solvent was deuterated. The peak is not deuterated chloroform, it is just chloroform.Figure 5. H-NMR spectrum of humulone in deuterated chloroform (CDCl3).Figure 6. H-NMR spectrum of humulone in deuterated acetone (Acetone-D6).Possible isohumulone was used for an H-NMR spectrum. Figure 7 shows the spectrum of humulone after being dissolved in a basic solution of sodium hydroxide (NaOH). The humulone did not fully dissolve in the NaOH. This likely gave us skewed polarimeter results that are inconclusive.Figure 7. H-NMR spectrum of possible isohumulone in deuterated chloroform.A polarimeter reading was taken for humulone that was dissolved in DI water with a pH of 10.Table 1. Polarimeter data of humulone dissolved in pH 10 DI water and NaOH.Time3:103:253:403:554:104:254:404:55Optical Rotation-0.159-0.183-0.189-0.189-0.186-0.184-0.177-0.179Standard Deviation0.00180.00050.00060.00030.00030.00050.00050.0000n200199201204201208201201A polarimeter reading was taken of the humulone dissolved in 8 mL of ethanol and 2 mL of pH 10 ammonium hydroxide buffer. The ethanol dissolved the humulone and then adding the ammonium hydroxide immediately changed the color of the sample inside the polarimeter cell. There is little change in the optical rotation degrees.Table 2. Polarimeter data of humulone dissolved in pH 10 buffer and ethanol.Time2:182:332:483:033:18Optical Rotation0.1960.1970.1970.1970.197Standard Deviation0.00010.00000.00050.00000.0000n201201202209205 ConclusionAlthough the isohumulone was isolated, more time was needed to study the isomerization rates. There were time limitations to the research. With more time, the isohumulone could have been further purified and used.Humulone can be isolated using the process found by Sharpe and Ormrod (1991). The humulone isolated is significant and their method is viable in getting humulone from hops. The H-NMR spectrum shows that we did isolate humulone with only slight impurities such as left over benzene from crystallization.NaOH buffer did not dissolve humulone so using ether and ammonium hydroxide allowed tests using the polarimeter. The ammonium hydroxide changed the humulone to what was first believed to be isohumulone. More likely, the ammonium hydroxide changed the humulone and turned it into a salt complex.Following US Patent 3,354,219, isohumulone can be isolated. There were impurities in the spectrum obtained of the product, but further purification will allow a better spectrum with definite findings. There were still hexanes in the product and the hexanes made the spectrum unclear with the peaks.Once an H-NMR spectrum is taken of the isohumulone from the patent, it can be compared to the spectrum of the humulone with ammonium hydroxide and ether. Comparing the peaks will tell if what the ammonium hydroxide created was an isohumulone or a salt complex. ReferencesChirality and Optical Activity. Retrieved July 10, 2015, from , B. J., & Hildebrand, R. P. (1965). The Isomerization Of Humulone. Journal Of the Institute of Brewing, 71(1), 26–36. , D. D. (2000). Fundamentals of beer and hop chemistry. Quím. Nova Química Nova, 23(1), 108–112. , Francis L. Method for Preparation of Isohumulone Concentrates. Haas Inc. John I, assignee. Patent 3354219. 21 Nov. 1967. Print.Sharpe, F. R., & Ormrod, I. H. L. (1991). Fast Isomerisation Of Humulone By Photo-Reaction: Preparation Of An Hplc Standard. Journal Of the Institute of Brewing, 97(1), 33–37. ................
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