1. Quantitative Analysis of Humulene in Beer Joseph Frederick CHEM 4101, Fall 2010 December 10, 2010

2. What is Humulene?  Humulene is an essential oil found in hops used for brewing.  Has an “elegant” flavor.  Higher levels found in aroma hops.

3. Hypothesis  There is a certain concentration of humulene that the majority of people will prefer in beer. Importance of solving hypothesis  If this concentration was to be determined then the beer that would appeal to the largest amount of people could be produced.

4. Background  Normal levels in Japanese beers (usually have lower levels of essential oils than Western beers): 0.23 – 0.73 ppb  These low levels are suitable for this method implying that it would be acceptable for American and European beers which will have higher concentrations of humulene.

5. Procedure  Survey needed in order to determine which level of humulene in beer is preferred by the most people.  Stir bar-sorptive extraction using PDMS coated stir bar added to 30 mL beer diluted to 150 mL with distilled water, stir for 2 hours at 40 °C  Use Twister desorption unit to get sample to GC/MS

6. Stir Bar-Sorptive Extraction  Stir Bar-Sorptive Extraction preferable to liquid extraction methods.  No solvent used.  Lower loss of product due to analysis.  Far more efficient extraction of humulene.  Need to use Twister desorption unit to desorb compounds from PDMS.

7. Method of Choice: GC/MS  Allows for separation and then identification simultaneously  Good limit of detection: 0.035 ppb

8. Agilent 6890  Gas Chromatograph  Carrier Gas: Helium  DB-WAX GC Column with polyethylene glycol as the stationary phase (60 m length X 0.25mm i.d,; film thickness = 0.25 μm)

9. MSD5973N  Mass spectrometer  Quadrupole  Electron Impact Mode at 70 eV  Resolution: 0.1 amu  Sensitivity: 1 pg

10. Other Analytical methods MethodProsCons IRFast, low sample preparation Qualitative, not quantitative UV-visSimple sample Preparation, inexpensive Interference from other hydrocarbons, insufficient LOD AES/AASSimple sample preparation Complex matrix would interfere

11. Conclusion  Humulene levels at a certain level will make beer more appealing to the most people.  Stir bar-sorptive extraction and GC/MS allows for relatively cheap determination of humulene levels.  Since low amounts of beer are required for this method (30 mL) and no solvent is used, constant monitoring of humulene (and other essential oils) may be implemented.

12. References  1. Kishimoto, Toru, et al., "Analysis of Hop-Derived Terpenoids in Beer and Evaluation of their Behavior Using the Stir Bar-Sorptive Extraction Method with GC-MS". Journal of Agricultural and Food Chemistry, 2005, 53, 4701-4707.  2. Fix, George. Principles of Brewing Science. Boulder, Colorado: Brewers Publications, 1989  3. Dev, S. “Studies in Sesquiterpenes-XVIII: The Proton Magnetic Resonance Spectra of Some Sesquiterpenes and the Structure of Humulene”. Tetrahedron. 1960, 9, 1-9  4. Musenga, A., et al, "analysis of aromatic and terpenic constituents of pepper extracts by capillary electrochromatography". J. Sep. Sci., 2007, 30, 612-619