Isabella Pinque Chem219 Page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018
Ultraviolet – Visible Spectroscopy For Determination of α- and β- acids in beer hops
Isabella Pinque
Lab Partner: Lucas Paquin
TA: Kevin Fischer
Date lab performed: 02/06/2018
Date report submitted: 03/14/2018
Isabella Pinque Chem 219 page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018
ABSTRACT
A spectrophotometric analysis at three wavelengths was used to determine the concentration of
α-acids, β-acids, and a third component that is associated with the degradation of hops. Two different samples of a simple extraction of hops were analyzed using a Shimadzu UV2450 to determine the concentration of each component. While doing a three component analysis, the third break down component had the highest concentration within the hops sample (0.05391 g/L
± 0.00493) , followed by the α-acids (0.01507 g/L ± 0.001291) and finally β-acids (0.005062 ±
0.0004816). When doing a two component analysis, the α-acid had a higher concentration than the β-acids, 0.01783 ± 0.001539 and 0.004354 ± 0.0004520, respectively. When taking the percentage of all three components, the overall percent did not add up to 1 due to the fact that there are more than just three components in the samples. In conclusion, the α-acids are more prevalent in a sample of hops which can be taken into consideration when home brewing to achieve the bitterness and the flavor desired.
INTRODUCTION
Hops are an essential aspect of the brewing process that provide flavor and aromas due to the oils and resins that reside in the lupulin glands of a hops cone (1). A simple extraction from ground up hops pellets were spectrophotometrically analyzed to determine the quantities of α- and β-acids in a sample. While the α- and β-acids are the two major components extracted, there is a third component that appears over time as degradation occurs. The third component is not purified and presumed an alternative breakdown component of the hops.
The bitterness of a beer is derived from the amount of α- acid and β-acids present in a brew (2). Knowing the concentration is critical for a brewer as they have significant impacts on Isabella Pinque Chem 219 page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018 the flavor of beer. The α-acids isomerize to form iso-α-acids, which is the main chemical source of the bitterness. With known concentrations, it is possible to balance the flavors in a specific brew by regulating the amount of hops, and thus the α- and β acids in the brewing process.
A spectrophotometric analysis at three wavelengths of the hops sample was used to quantify the concentration of the α- and β-acids, as well as the third component. The third component yields a strong absorption at the 275, 325, and 355 nm wavelength. At the 325 and
355 nm wavelength, a significant absorption is yielded as it increases the apparent absorption of the α- and β-acids. The data points collected were then used to determine the concentration of each absorber, using excel and the inverse matrix function. Both a two component, and three component analysis were utilized, and the percentage of the components in each sample was determined.
EXPERIMENTAL
Theory. A UV-Visible Spectrometer allows the measurement of the absorbance versus wavelength of a specific compounds within the wavelength range of 190-750 nm. The ultraviolet
(UV) region typically scans from 200-400 nm while the visible region typically scans from 400-
800 nm (3). The absorbance’s vary for different compounds; and so that light energy can reach the detector, solutions need to be diluted prior to examination. An optical spectrometer measured the wavelengths at which absorptions occur, and a spectrum is plotted comparing absorbance (A) versus wavelength (λ).
Interaction with infrared light can lead to molecules undergoing electronic transitions.
The energy from the UV or visible light is absorbed by the molecule and the energy jumps from a low energy molecular orbital to a higher energy molecular orbital. There are four types of Isabella Pinque Chem 219 page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018 molecular electronic transitions: σ-σ*, π-π*, n-π*, and n-σ*. A π-π* transition are analytically the most useful as the energy required is only moderate and falls within the range measurable by the instrument. The other three types of transitions while sometimes can be used require a large amount of energy and energetic photons below 190 nm for σ-σ* transitions and n-π*, and n-σ* transitions are molecules with lone pair(s) of electrons that do not can absorb energy and move to excited states, yet do not participate in chemical bonds.
An absorption spectrometer passes series of wavelengths through a solution in a sample cell and a blank in the reference cell. The concentration of a sample has an effect on the absorbance recorded. This is due to the fact that the light absorbed is proportional to the amount of molecules it interacts with. Therefore, the higher the concentration, the larger the absorbance and vice versa (4). The molar absorptivity measures how a chemical species absorbs at a given wavelength. To calculate the concentration (c), the absorbance (A), the path length (l) and the molar absorptivity coefficient (ε) are needed. Beer’s-Lambert law relates the four components with the relationship shown below in equation 1.
$ " = (Eq. 1) %&
Materials. The materials provided to us for this lab was commercial hops that are used for home brewing in a dried, pellet form, spectrophotometric grade methanol and reagent grade sodium hydroxide (NaOH). The NaOH and methanol were also utilized to prepare a solution of methanolic NaOH. Methanol is a hazardous reagent as it is highly flammable, and toxic upon ingestion, skin absorption or inhalation. NaOH causes severe burns as it is corrosive, thus protective equipment was imperative. A labeled non-halogenated waste container was provided for proper disposal of waste. Isabella Pinque Chem 219 page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018
Instrumentation. The spectrophotometer used was a Shimadzu UV2450 double beam instrument. It is a monochromatic meaning each wavelength is analyzed sequentially. The instrument contained a tungsten and a deuterium lamp which analyzed the visible region and the
UV region, respectively. The absorbance at a range of wavelengths (210-510 nm) was measured with a resolution of 1 nm yielded the data points.
Procedures. Using a hand-held coffee grinder, approximately 25 g of hops pellets were ground up. Into three 100 mL beakers, exactly 50.0 mL of methanol was pipetted and approximately 2.5 g of hops was weighed out by difference and added to the beakers. The beakers were placed on a magnetic stir plate, stir bars were added. To minimize the evaporative loss of methanol while stirred for 30 minutes at room temperature, each beaker was covered with parafilm. The beakers where then stood for 10 minutes without stirring or any form or irritation to allow for the settling of the particulate mass. Into a clean, dry 125 mL Erlenmeyer flask that had been rinsed with methanol, the solutions were suction filtered using a Buchner funnel.
A stock solution of methanolic NaOH was prepared by pipetting 0.5 mL NaOH into a
250 mL volumetric flask and bringing it up to volume with spectrophotometric grade methanol.
A 50 µL aliquot of the three filtrate samples were pipetted into three separate 25 mL volumetric flasks, and brought up to volume using the methanolic NaOH. A blank solution was prepared by pipetting a 50 µL aliquot of spectrophotometric grade methanol into a separate 25 mL volumetric flask and brought up to volume with the previously prepared methanolic NaOH.
A 1-cm quartz cuvette was filled with the blank solution so that it was approximately ¾ full, and placed into the reference cell holder. A separate cuvette was prepared by rinsing it several time using the hops extract, and then the cuvette was filled so that it was ¾ full. The Isabella Pinque Chem 219 page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018 cuvette was then placed into the sample cell holder for analysis. An absorbance sample was taken for each of the the extract solution samples from 210-510 nm.
The same procedure as aforementioned for the first three samples was replicated in triplicate but used a stoppered 200 mL Erlenmeyer flask rather than a beaker. After being stirred and let settled, the samples were filtered using gravity filtration rather than suction filtration. The extract solutions were analyzed in the same way using UV-Vis Spectroscopy.
Data Analysis. The two major components being analyzed in the hop extract were the α- acids and the β-acids. For an ideal system, these could be analyzed using a two-component analysis as only the molar absorptivity coefficients at two different wavelengths are needed.
However, as neither the α-acids and the β-acids are single compounds, there is also a third component present in the solution that appears over time as the hops are degraded. Figure 1 shows the components that α- and β-acids are comprised of as they are not singular compounds.
Figure 1: The structures of the major α- and β-acids that are found in hops
α-acids R β-acids humulone CH2CH(CH3)2 lupulone cohumulone CH(CH3)2 colupulone adhumulone CH(CH2)CH2CH3 adlupulone Figure 1. Structures of major α – and β – acids found in hops The third component is not purified and it interferes with a two component analysis. It is more rational to use specific absorptivity rather and molar absorptivity as there are no single Isabella Pinque Chem 219 page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018 absorbing compounds. The specific absorptivity relates absorbance with the total concentration of 1 g/L at a given path length to the absorbance of a mixture of compounds.
Table 1: Reported specific absorptivity’s (L mg-1) for pure α- acids, β-acids and the third degradation component as reported in the lab manual.
355 nm 325 nm 275 nm α-acids 31.8 38.1 9.0 β-acids 46.0 33.1 3.7 Component 3 1.0 1.5 3.1 The sum of the absorbance’s of each component is the total absorbance at any wavelength. For a three component system, and using the specific absorptivity values listed in table one, three equations can be formed to describe the three component system at the three wavelengths being examined, 355, 325, and 275 nm.
'()) = 31.8./ + 46.0.4 + 1.0.56789 (Eq 2) '(:) = 38.1./ + 33.1.4 + 1.5.56789 (Eq 3) ':<) = 9.0./ + 3.7.4 + 3.1.56789 (Eq 4)
Using the absorption equations, it was possible to determine the concentrations of the α- acids (./), the β-acids (.4), and the break down component (.56789). The inverse matrix function as well as a system of linear equations in excel were utilized so that the concentration of each component could be determined.
To determine the percentage of each component within a hops sample, the concentration of the α- and β-acids, and the third component, for each of the six samples was multiplied by
0.025 L to account for the methanolic NaOH, thus determining the grams in each sample. To calculate the percentage, the grams were then divided by the total amount of hops in each respective sample and multiplied by 100. The same calculation was used following both the two component and three component analysis. Isabella Pinque Chem 219 page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018
RESULTS Six samples were prepared for hops extraction and were analyzed using a Shimadzu 2450 to obtain absorbance spectra comparing the wavelengths versus the absorbance of each specific sample. The masses of the hops in grams in each sample are shown in Table 2.
Three key wavelengths of the α- acids, β-acids, and the third degradation component were further analyzed. Table 3 shows the data collected at each specific wavelength for each sample.
Using the coefficients from Equations 2, 3, and the absorbance values at the wavelengths of 355 and 325 nm from Table 3, it was possible to calculate the concentrations of the α- acids
(./), the β-acids (.4) in each sample of hops extract, using the inverse matrix function in excel for a two component analysis. Table 4 shows the concentration of the α- acids (./), the β-acids
(.4) component for each sample.
After the concentrations had been solved for with the two-component analysis, the percentage of the α- and β-acids present in each sample was determined. These percentages are shown in Table 5.
Using the coefficients from Equations 2, 3, 4 and the absorbance values at the wavelengths of 355 and 325 nm from Table 3, the concentrations of the α- acids (./), the β- acids (.4), and the break down component (.56789) in each sample of hops extract could be determined. The inverse matrix function in excel was used again, but for a three-component analysis rather than for two different components. Table 6 shows the concentration of all three components in each sample. Isabella Pinque Chem 219 page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018
The percentage of the α-acids, β-acids and the break down component present in each
sample utilizing the concentrations that had been solved for with the two-component analysis.
These percentages are shown in Table 7.
Replicate measures were made, so the average and standard deviation for the
concentrations and percentages of the components following the calculations were taken. These
statistics are reported in Table 8 and Table 9.
Chart 1 shows the absorbance spectrum for the first three samples that had been prepared
following the first procedure. Chart 2 shows the absorbance spectrum for the last three samples
that were prepared following the second procedure. Chart 3 is the spectrum for the blank versus
blank sample which was run following the first six samples. These “blank” samples ideally
contain no hops, thus none of the three components of interest. Chart 4 shows all of the
absorbance spectrum generated throughout the experiment together. Thus, Chart 4 provides a
visual representation of the variation among all the samples that had been analyzed.
Table 2: Mass of the hops in each sample being analyzed.
Sample 1A Sample 2A Sample 3A Sample 1B Sample 2B Sample 3B
Mass of hops (g) 2.449 2.451 2.4884 2.5309 2.4715 2.5125
Table 3: Absorbance of each sample recorded at three specified wavelengths: 355, 325, and 275 nm Sample Sample Sample Sample Sample Sample 1A 2A 3A 1B 2B 3B 355 nm 0.32364 0.33203 0.37083 0.29829 0.31207 0.29182 325 nm 0.83537 0.86023 0.93916 0.76161 0.79741 0.74101 275 nm 0.77837 0.86022 0.87474 0.70847 0.74295 0.68854
Isabella Pinque Chem 219 page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018
Table 4: Two component analysis of the concentrations (g/L) of the sample for α- acids (./), the β-acids (.4) Sample Sample Sample Sample Sample Sample 1A 2A 3A 1B 2B 3B α-acids 0.018089 0.018595 0.020353 0.016548 0.01727 0.016136 β-acids 0.004416 0.004585 0.004946 0.003962 0.004212 0.003813
Table 5: Percentage of α- acids and β-acids in hops extract samples, two component analysis Sample 1A Sample 2A Sample 3A Sample 1B Sample 2B Sample 3B α-acids (%) 0.01847 0.01897 0.02045 0.01635 0.01747 0.01606 β-acids (%) 0.004508 0.004676 0.004969 0.003914 0.004261 0.003794
Table 6: Three component analysis of the concentrations (g/L) of the α- acids (./), the β-acids (.4), and the break down component (.56789) in each sample of hops extract Sample Sample Sample Sample Sample Sample 1A 2A 3A 1B 2B 3B α-acids 0.01533 0.01578 0.01712 0.01398 0.01458 0.01361 β-acids 0.005153 0.005338 0.005810 0.004649 0.004932 0.004489 Third Component 0.05373 0.05493 0.06298 0.05009 0.05245 0.04927
Table 7: Percentage of α- acids, β-acids , and the break down component in hops extract samples, three component analysis Sample 1A Sample 2A Sample 3A Sample 1B Sample 2B Sample 3B α-acids 0.01565 0.01609 0.01720 0.01381 0.01475 0.01354 β-acids 0.005260 0.005445 0.005837 0.004592 0.004989 0.004466 Third 0.05485 0.05603 0.06327 0.04948 0.05306 0.04902 Component
Table 8: Statistics performed following the two component analysis to determine the average and standard deviation for each component as replicate measures were taken Average Standard Deviation Concentration: α-acids 0.01783 0.001539 Concentration: β-acids 0.004322 0.0004167 Percentage: α-acids 0.01796 0.001669 Percentage: β-acids 0.004354 0.0004520
Table 9: Statistics performed following the three component analysis to determine the average and standard deviation for each component as replicate measures were taken Average Standard Deviation Concentration: α-acids 0.01507 0.001291 Concentration: β-acids 0.005062 0.0004816 Concentration: Third Component 0.05391 0.004931 Percentage: α-acids 0.01517 0.001406 Percentage: β-acids 0.005098 0.0005216 Percentage: Third Component 0.05429 0.005223 Isabella Pinque Chem219 Page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018
Chart 1: Wavelength Versus Absorbance for Samples 1A, 2A, 3A following procedure
1.8
1.6
1.4
1.2
1
0.8 Absorbance
0.6
0.4
0.2
0 200 250 300 350 400 450 500 Wavelength (nm)
Sample 1A Sample 2A Sample 3A
Isabella Pinque Chem 219 page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018
Chart 2: Wavelength versus Absorbance for Samples 1B, 2B, 3B following procedure 2
1.6
1.4
1.2
1
0.8
0.6 Absorbance
0.4
0.2
0 200 250 300 350 400 450 500 Wavelength (nm)
Sample 1B Sample 2B Sample 3B
Isabella Pinque Chem 219 page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018
Chart 3: Wavelength Versus Absorbance for a Blank versus Blank sample
0.08
0.07
0.06
0.05
0.04
0.03 Absorbance 0.02
0.01
0 200 250 300 350 400 450 500
-0.01 Wavelength (nm)
Isabella Pinque Chem 219 page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018
Chart 4: Wavelength versus Absorbance for all six samples of hops extract as well as a blank versus blank sample.
1.6
1.4
1.2
1
0.8
0.6 Absorbance 0.4
0.2
0 200 250 300 350 400 450 500
-0.2 Wavelength (nm)
Sample 1A Sample 2A Sample 3A Sample1B Sample 2B Series6 Blank vs. Blank Isabella Pinque Chem219 Page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018
DISCUSSION
The quantities of α- and β-acids, as well as a third component linked to hop degradation in the sample were analyzed from a simple extraction from a hops sample followed by a spectrophotometric analysis at three wavelengths (275, 325, and 355 nm). The amount of α- and
β-acids are what provide bitter flavor to a brew, so knowing the concentrations of each in a sample of hops is important when home brewing.
From Chart 4 and the relative concentration of each component that had been determined throughout this experiment, it is possible to make the assumption that there are more α-acids than β-acids in the six samples analyzed. All of the spectrum fall relatively within the same range and the peaks and troughs of the graph occur at the same wavelength. Using equation 1, and knowing that the relationship between concentration and absorbance are directly proportional, it is possible to conclude that the higher the absorbance, the higher the concentration. It had been calculated that the third component had the highest concentration, followed by the α-acids and finally the β-acids. Therefore, it can be concluded that the α-acids will yield higher peaks than those for β-acids, and it is evident that there are more α-acids than
β-acids in the samples investigated.
Using Excel, it was possible to complete a two component analysis by utilizing the inverse matrix function of and α- and β-acids. The two component analysis allows for calculations to be made without accounting for the third component related to the breakdown of hops. To determine the percentage of the two components within a hops sample, the concentration of the α- and β-acids were used to determine the grams in each sample along with the the total amount of hops in each respective sample. As evident in Table 5, the α-acids yielded larger gram values than the β-acids. Isabella Pinque Chem 219 page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018
The same procedure was done in excel, but for a three component analysis of the α- and
β-acids, as well as the third breakdown component. The mass and weight percent for all of the components are shown above in Table 6 and Table 7 respectively. From these calculations, it could be determined that the component with the largest mass and weight component was the third breakdown component, followed by the α-acids and lastly the β-acids.
Comparing the results from the two component analysis and the three component analysis, the α-acids were more concentrated than the β-acids in both. However, the weigh percent for the α- and β-acids were greater in the two component analysis. This result is logical as there is one less component being analyzed and in the three component analysis, the third component accounted for the majority of the concentration. This also makes sense because in the two component analysis, the third degradation component hadn’t yet formed or influenced the breakdown in any way.
Throughout the experimental procedure there were many circumstances that could be classified as sources of error. The first source of error is the noise that comes from the instrument. Putting all 25 g of hops into the coffee grinder took much more time than anticipated; and as a result, it left the hops open to gain moisture which could have altered the mass of the hops added to each sample. To minimize evaporation during the extraction and settling processes, it was imperative to cover the sample. However, if the parafilm or stopper used during the stirring process had not been fully secured it is possible that a lot of methanol evaporated out yielding results with values due to error. This could be corrected for by using appropriately sized watch glasses, or stoppers that ensure the flasks are properly sealed. If there was no openings/breaks in a seal, it is likely there would be a minimal amount of evaporation of methanol during the stirring process. Isabella Pinque Chem 219 page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018
Three blank solutions were accidentally made instead of just one which limited the amount of stock solution of methanolic NaOH available. It was imperative to only use one stock sample of methanolic NaOH. If an error has been made in the preparation of the solution, the error would have consistently been carried through all six of the samples analyzed. Thus, to keep the samples consistent, all methanolic NaOH used throughout the experiment came from that one stock solution. However, while creating sample 2B, a glass pipette broke into the 25 mL volumetric flask being utilized. The stock solution was nearly empty so a different sample 2B was not made and there is a possibility that the glass from the pipette altered the results obtained.
If some of the glass had gotten into the cuvette, the absorbance yielded in the analysis could have been obstructed and incorrect due to the presence of the glass. If a new sample had been prepared it would have ensured that there was no possibility of broken glass from the pipette in the cuvette. However, rather than introduce a new error from using a different stock solution, the decision was made to carefully pipette sample 2B into the cuvette, avoiding the glass shards in the flask.
It would be interesting to investigate the concentration of α- and β-acids in varying forms of hops as each can provide a different flavor, aroma, and makeup of a brew. While the dried pellet, as used in this experiment is most common, other forms such as hops extract, liquid, whole leaf, or powder can also be used. If more trials had been run, the likelihood of error could decrease as there is a larger sample size.
In conclusion, spectrophotometric analysis at three absorbing wavelengths was utilized to determine the quantities of three components: α-acids, β-acids, and a third component, present in a sample of hops.
Isabella Pinque Chem 219 page 1 UV-Vis Spectrophotometric Analysis of Beer Hops date: 3/14/2018
LITERATURE CITED 1.! Hops http://www.aperfectpint.net/Hops.pdf (accessed Feb 14, 2018).
2.! Growing Hops is Thirsty Work https://www.usahops.org/enthusiasts/brewing.html
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A_Organic_Chemistry_with_a_Biological_Emphasis_(Soderberg)/Chapter_04%3A_Stru
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2018).
4.! Libretexts. The Beer-Lambert Law
https://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/Spectroscopy/Elec
tronic_Spectroscopy/Electronic_Spectroscopy_Basics/The_Beer-Lambert_Law (accessed
Mar 3, 2018).