Ultraviolet – Visible Spectroscopy for Determination of Α- and Β- Acids in Beer Hops

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.

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