Behind the Bar: Simplifying Headspace Analysis Samantha Reiter, Gabrielle Rigden, Claudia Tourville, Lindsey Coonen, Anna Meier, and Dr. Scott Bailey-Hartsel  Chemistry Department  University of Wisconsin-Eau Claire Abstract Results: Profiling and Comparing Beer Analyses As the popularity of microbreweries grows in Wisconsin, young such as Modicum Octanoic acid, ethyl ester ethyl acid, Octanoic acidOctanoic found in the Eau Claire area are encountering obstacles not faced by larger, more AlcoholPhenylethyl Figure 1: Floating versus Headspace Collection Figure 2: Carbon versus C18-Silica Collection Pellet established facilities. Chemical analysis of volatile flavor compounds greatly improves a brewer’s Floating (red) Carbon (red) Phenylethyl AlcoholPhenylethyl ability to characterize and fine-tune their product, but the instrumentation required for this task Headspace (green) C18-Silica (black)

tends to be highly specific, expensive, and requiring of expertise in both assembly and Linalool methodology. In our research, we have developed a novel approach to solid phase microextraction (SPME) as a method to prepare beer samples for analysis using standard gas Octanoic acidOctanoic 4-vinylguaiacol ester ethyl acid, α-Decanoic α-Terpineol Acetic acid, 2-phenylethyl ester 2-phenylethyl acid, Acetic chromatography and mass spectrometry techniques while removing the need for additional ester ethyl acid, Octanoic attachments traditionally used in beer analysis. To collect volatile compounds contributing to n-Decanoic acidn-Decanoic flavor, our method simply floats a Monotrap ® monolithic pellet in the sample that collects Citronellol Geraniol compounds found both in the headspace as well as less volatile compounds in solution that also α-Terpineol Citronellol largely contribute to flavor. This collection pellet is then extracted into a standard GC/MS solvent Geraniol and run through the instrument to be semi-quantitatively analyzed for target compounds in the interest of the brewer. As an added benefit, this method requires a smaller sample volume than conventional headspace methods, making multiple analyses with varying parameters possible with a single sample. Through our collaboration with Modicum Brewery, this method has produced promising results that can someday be employed by microbrewers with similar Modicum Original () Figure 5: versus Ale standardization goals. 2-Methyl-1,4-butanediol 1-Butanol, 3-methyl- 1-Butanol, 2-Pentanol, 4-methyl- 2-Pentanol, 1-Butanol, 2-methyl- 1-Butanol,

Introduction: Volatile Compound Extraction 4-Penten-1-ol linalool If utilizing conventional methods, surveying the chemical profile of flavor-contributing acetate Ethyl compounds in beer may be quite an expensive process. Multiple techniques exist to acetate 2-methyl-, 1-Butanol, provide brewers with options, but each method has its own instrumentation and consumables that put conventional analysis outside the realm of possibility for small brewers like Modicum Brewery. Headspace analysis followed by gas chromatography – mass spectrometry (GC/MS) is often most meaningful in order to emulate the chemical profile that is tasted by the consumer. This requires specialized apparatus and preparations to accurately and reliably withdraw and concentrate gas from the WWHG (lager) headspace of a sample in an amount significant enough to be detected via GC/MS. We have developed a method using a solid phase microextraction method (SPME) with MonoTrap® monolithic collection pellets which is much more cost effective and simpler. These pellets were either floated directly in the beer sample (Figure A) or held above the Beer Name Beer Type Hops Microbes beer in the headspace (Figure B). AB Peel WheatAle with Citrus Nugget Yeast

We also explored the use of the two coating options for Monotrap’s ® collection pellets: Moniker Bierede Garde Mt. Hood House Lager Yeast an octadecylsilane (ODS) – coated pellet (DSC18) and a pellet coated with both ODS and activated carbon (DCC18). The ODS pellets are preferable for less hydrophobic Divine Dramedy Sterling House Ale Yeast compounds with low to medium boiling points while the ODS-activated carbon pellets better collect compounds with greater hydrophobicity and medium to high boiling points. Idiom American Amber Lager Mackinac House Ale Yeast These coatings were compared by the peak intensity and reproducibility of compounds of interest during GC/MS analysis. Several other parameters were tested to extract more volatile compounds, such as incubation time. Using the best methods once found, TheHeavens Golden Lager Nugget House Ale Yeast different types of beer were profiled and compared. Along with this an analysis of the Table 1: Names and ingredients for five beer samples in Figure 6 volatile compounds throughout the fermentation process was done. This analysis shows Figure 6 (Left): Terpene profiles in five beer samples the success of this novel approach to beer quality control. Conclusion Future Directions

Based on our initial development of the MonoTrap ® extraction method, we • Evaluate the repitchability (reusability) of yeast cultures between find it to be very promising for cost-efficient analysis of the flavor- batches • Quantitatively determine the ideal resting period post- for contributing compounds in beer. Based on our data, we conclude that the elimination of additional off flavors ODS-activated carbon pellets provide the most efficient binding of relevant • Semi-quantitatively profile major flavor-contributing compounds volatile compounds while also providing sufficient surface area to detect • Empirically compare the products of yeasts, hops, malts, and other compounds existing at relatively low concentrations in beer. key ingredients in the brewing process Materials & Methods Furthermore, we conclude the floating method to be superior to the headspace method for our purposes. Initially, our goals were more focused 10mL beer samples were prepared by adding 15% sodium chloride and incubating at on the headspace method to emulate the process of retronasal olfaction, References 60°C at 200rpm for 90 minutes with the floating MonoTrap ® monolithic pellets. These but this method of collection was found to be inferior in detecting larger, TM pellets were coated with ODS alone (MonoTrap ® DSC18) or ODS – activated carbon -- (2014) MonoTrap Guide to Proper Use, In Monolithic Material Sorptive Extraction (Inc., G. less volatile molecules that do significantly contribute to flavor (e.g. …S., Ed.), pp 1-36, Tokyo, Japan. (MonoTrap ® DCC18). Following this incubation period, the collection pellets were octanoic acid and phenylethyl ). These were much more detectable -- Barnes, T. (2011-13) The Complete Beer Fault Guide v. 1.4, 1-42. transferred to 1mL of dichloromethane solvent for sonification for 5 minutes at room -- Charry-Parra, G., DeJesus-Echevarria, M., and Perez, F. J. (2011) Beer Volatile Analysis: temperature. The solvent, now containing compounds targeted for profiling, was with the floating method while still capturing other molecules found in the …Optimization of HS/SPME Coupled to GC/MS/FID, Journal of Food Science 76 , C205-C211. -- Marotta, L., and Tipler, A. (2011) Monitoring Volatile Organic Compounds in Beer Production analyzed using GC/MS under the following conditions: headspace method. Along with this the additional parameters as …Using the Clarus SQ 8 GC/MS and TurboMatrix Headspace Trap Systems, In Gas mentioned in the materials and methods such as incubation time were …Chromatography/ Mass Spectrometry(PerkinElmer, Ed.), p 6, Shelton, CT. . Table 1: GC Conditions Table 2: MS Conditions proven to be the most effective mean of volatile compound extraction. GC/MS Agilent Technologies 7820A with quad MSD Scan Range 35.0 to 450.0 Da J&W DB-624 GC Column, 60 m, 0.32 mm, 1.80 Scan Speed 1,562u/s Acknowledgements Column µm, 7 inch cage This project provided a undergraduate-centered way to utilize university Frequency 3.5 scans/s --Eric Rykal, Modicum Brewery Oven 40⁰C for 5 min., then 6⁰C/min. to 250⁰C instrumentation in the best interest of the Eau Claire community. We hope Interscan Delay 9 minutes --Dr. James Boulter, UWEC Chemistry Department 1µL injection volume, Programmable Split- that our efforts working with Modicum Brewery can be extended to other --University of Wisconsin – Eau Claire Office of Research and Sponsored Programs Injector Splitless, 250⁰C and 13psi, Splitless Source Temp 230⁰C microbreweries for the production of better products and standardization ( (ORSP) Carrier Gas Helium at 20mL/min after 3 minutes Inlet Line Temp 250⁰C of brewing methods. --University of Wisconsin – Eau Claire Chemistry Department --University of Wisconsin – Eau Claire Learning and Technology Services