Running head: ISOMERIZATION OF HUMULONE INTO ISOHUMULONE

Isomerization of Humulone into Isohumulone

Dylan Walker
2015 Frontiers of Science Institute
University of Northern Colorado, Greeley, CO

Mentored by Sean E. Johnson and Dr. Mosher

Introduction

Hops 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 is used as the degree of rotation the
instrument detects.

Literature Review
Humulone, 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 humulones 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 a
250 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 separating

funnel, 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 Methods

Hops 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 Johnsons 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 Discussion

After 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.

Time

3:10

3:25

3:40

3:55

4:10

4:25

4:40

4:55

Optical
Rotation

-0.159

-0.183

-0.189

-0.189

-0.186

-0.184

-0.177

-0.179

Standard
Deviation

0.0018

0.0005

0.0006

0.0003

0.0003

0.0005

0.0005

0.0000

200

199

201

204

201

208

201

201

A 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.

Time

2:18

2:33

2:48

3:03

3:18

Optical
Rotation

0.196

0.197

0.197

0.197

0.197

Standard
Deviation

0.0001

0.0000

0.0005

0.0000

0.0000

201

201

202

209

205

Conclusion
Although 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.

References

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