Characterization of Varietal Differences in Essential Oil Components of Hops (Humulus lupulus) by SFC FTIR Spectroscopy

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1 AUERBACH ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 3, FOOD COMPOSITION AND ADDITIVES Characterization of Varietal Differences in Essential Oil Components of Hops (Humulus lupulus) by SFC FTIR Spectroscopy RITA H. AUERBACH,KENAN DOST, and GEORGE DAVIDSON 1 University of Nottingham, Department of Chemistry, University Park, Nottingham, NG7 2RD, UK A supercritical fluid chromatographic method combined with Fourier-transform infrared spectroscopy detection (SFC FTIR) was developed for determination of varietal differences in essential oil constituents in hops (Humulus lupulus). Infrared spectra (IR) of the major constituents of essential oil of hops were taken as films deposited on AgCl discs and compared with those obtained after chromatographic separation in the IR flow-cell with supercritical carbon dioxide (scco 2 ). Spectra from AgCl discs were comparable to those in scco 2, but in scco 2 most of the bands appeared approximately 8 10 cm 1 to higher wave numbers. Open-tubular SFC FTIR analysis of the essential oil of 4 different hop varieties was performed. The SFC FTIR chromatograms showed differences in the location and relative intensity of the peaks depending on the variety, which was further confirmed by consideration of their FTIR spectra. (Figure 1). Together they represent 80 90% of the total essential oil (2). The increasing number of hop varieties complicates the brewing process with respect to the specific taste and flavor in a particular beer. In addition, brewers wish to improve efficiency and consistency in their brewing procedure by using processed hop products of constant quality. Therefore, it is important to characterize hop varieties by chemical analysis. Fingerprinting the hop essential oil is the best way to do this, and many applications, mainly gas chromatographic (GC) methods, have been published (3 15). Using a supercritical fluid chromatographic Fourier-transform infrared (SFC FTIR) method provides another promising alternative. Combination of capillary SFC with an FTIR detector allows chromatographic separation with high resolution plus structural information to be achieved simultaneously (16). In contrast to GC, decomposition of thermolabile substances in essential oils is unlikely in SFC. We investigated the potential of this method for the analysis of the essential oil components of hops. The female inflorescence of hops is known as the hop cone or strobile. Hop cones have lupulin glands which occur on the outer lower surface of the bracteoles and the entire surface of the perianth. The constituents of these glands include bitter acids and essential oil components. The bitter taste of beer is mainly determined by iso- -acids, which are formed in the brewing process from the -acids naturally occurring in hops. Hoppy flavors in beer are derived from the essential oil of hops (1). The chemistry of hop aroma in beer is not clearly understood, but complex mixtures of substances primarily derived from hop oil terpenes, especially humulene, may be responsible. As with most essential oils, hop oil consists mainly of terpene hydrocarbons and their oxidation products such as carbonyl compounds, esters, acids, oxygen heterocyclics, and sulfur derivatives. The most important constituents of essential oils are the terpene hydrocarbons myrcene, humulene, and caryophyllene Received August 10, Accepted by JL December 23, Author to whom correspondence should be addressed. Experimental Supercritical Fluid Chromatography (SFC) Open-tubular SFC with supercritical carbon dioxide was performed with a system linked to an FTIR spectrometer and a flame ionization detector (FID) (17). The chromatographic conditions were as follows: columns (Dionex Corp. Salt Lake City, UT) 10 m 50 µm id SB-Phenyl-5 column, 0.25 µm film; 10 m 50 µm id SB-Cyano-50 column, 0.25 µm film; 10 m 50 µm idsb-carbowax column, 0.25 µm film; injection, 200 nl, flow-split 1:10; flow rate, 3 ml/min using a 50 µm frit restrictor which ended in the FID; oven temperature, 80 C; FID temperature, 350 C; pressure program, 80 bar for 10 min isobaric, then up to 350 bar with 3 bar/min; FTIR, Perkin-Elmer 1760 x; resolution 8 cm 1, scan range cm 1, mercury cadmium telluride (MCT) detector. (Note: Dionex Corp. no longer furnishes this equipment. The only equivalent is now furnished by Selerity in North Salt Lake, UT).

2 622 AUERBACH ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 3, 2000 Figure 1. The most important hop oil terpenes. Samples The sample oils (English Hop Products Ltd., Hop Pocket Lane, Paddock Wood, Tonbridge, Kent, UK) were liquid CO 2 extracts from the varieties Styrian Golding, Challenger, Cascade, and Northdown. Solutions of these oils were made at a concentration of 20 mg/ml in methylene chloride. Standards Standard solutions of trans-caryophyllene, -humulene, and myrcene (Sigma-Aldrich Company Ltd., Fancy Rd, Poole, Dorset, UK) were made at a concentration of 2 mg/ml in methylene chloride. Figure 3. FTIR chromatograms of Styrian Golding and Northdown hop oils. Results and Discussion Chromatographic Column Performance Figure 2. FTIR chromatograms of Challenger and Cascade hop oils. The performance of 3 chromatographic columns was tested for a standard mixture commonly used to characterize essential oil constituents of hops. It contained a mixture of the even carbon numbered n-alkanes for C8, to C24, limonene, linalool, linalyl acetate, cinnamyl alcohol, acetophenone, and naphthalene. The columns used were SB-Cyano- 50, SB-Phenyl-5, and Carbowax. To optimize the separation conditions, a number of experiments were made by systematically altering the SFCoven temperature (80, 100, 120, 140 C) and applying 2 different pressure programs: (a) 80 bar for 10 min isobaric, proceeding to 350 bar with 3 bar/min; and (b) 120 bar for 10 min isobaric, proceeding to 400 bar with 5 bar/min. The best separation was achieved with the SB-Phenyl-5 column at an oven temperature of 80 C and using pressure program (a). All components of the standard mixture eluted within 60 min, from which 9 components were separated, with subsequent identification. For the 2 other columns, separation was unsatisfactory at all conditions; therefore, they were not useful for separating hop samples.

3 AUERBACH ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 3, Figure 4. FTIR spectra of (a) the Challenger peak at 37.7 min and (b) a standard of -humulene. Chromatographic Separation of Essential Oil of 4 Hop Varieties by SFC FTIR Differences in the chromatograms of different hop varieties were identified with the aid of their FTIR spectra. The FTIR spectrometer can be used for recording infrared spectra and for recording chromatographic data. Two basic types of infrared chromatogram were used in this work: Gram-Schmidt chromatograms, which measure the total infrared absorption as a function of time; and window chromatograms, which are generated by plotting integrated infrared absorbance for predefined spectral windows as a function of time. Figure 2 shows the window chromatograms ( cm 1 ) of the hop varieties Challenger and Cascade; Figure 3 shows the window chromatograms of Styrian Golding and Northdown. The first peak at min appears in all chromatograms, and is due to the solvent and not taken into account. The chromatogram of Challenger shows 8 labeled peaks. The 2 most intense peaks eluted at 37.7 and 39.0 min with an intensity ratio of 1:1, and were not well resolved. These peaks are flanked by 2 medium intensity peaks, with the first eluting at 27.2 min and the other at 40.4 min. The third intense peak had a retention time of 27.3 min. A small peak before this peak was apparent at 24.9 min and another small peak at 33.0 min. The chromatogram of Cascade is very similar to that of Challenger, but crucial differences are necessary to assign varietal differences. For instance, in the chromatogram of Challenger the 2 most intense peaks are at 37.7 and 39.0 min, with similar intensities, whereas in the chromatogram of Cascade the peak at 37.9 min is much more intense than that at 39.0 min, which is only of medium intensity and not well resolved. Another characteristic of Cascade is that the peak eluting at 26.9 min shows almost the same intensity as the most intense peak at 37.9 min. In the chromatogram of Challenger this peak is significantly weaker and has a retention time of 27.3 min. The chromatograms of Styrian Golding and Northdown are also similar to each other, but are different from those of Challenger and Cascade. In both chromatograms of Styrian Golding and Northdown, the most intense peak has a retention time of min. This peak is part of a series of 4

4 624 AUERBACH ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 3, 2000 peaks which are almost identical to those in the chromatogram of Cascade. A difference in the chromatogram of Northdown compared with that of Cascade is that the peak at 26.9 min is less intense for Northdown. In the chromatogram of Styrian Golding this peak is not observed. Unlike all other chromatograms, Styrian Golding shows no peaks at 39.0 and 40.4 min but shows 2 late eluting peaks at 45.9 min and 48.2 min. Thus, the SFC FTIR chromatograms showed significant and characteristic differences between the varieties of hop studied. Identification of Peaks in the Chromatograms Using the SFC FTIR Spectra To further characterize the differences, the species responsible for the chromatographic peaks must be identified. In the chromatogram of Challenger, useful IR spectra were obtained for the peaks with the retention time of 27.3, 33.0, and 37.7 min. The strongest peak, eluting at 37.7 min, was identified as -humulene. Figure 4(a) shows the FTIR spectrum of this peak and Figure 4(b) shows the spectrum of an -humulene standard passed through the same column under the same chromatographic conditions. Figure 5(a) shows the FTIR spectrum of the peak with a retention time of 27.3 min. The very strong absorption at 1272 cm 1 probably indicates an epoxide rather than an aryl ether, which is also consistent with the presence of the medium strong absorptions at 901 and 808 cm 1 (18 ). The C H stretches of the CH 3 and CH 2 groups are very weak. The absorption which occurs at 3094 cm 1 indicates either an unsaturated or an aromatic compound. The FTIR spectrum of a very weak peak at 33.2 min is shown in Figure 5 (b). It has an absorption at 1725 cm 1 typical of a carboxyl group in one of the following types of compound: 6-membered ring ketones,, -unsaturated aldehydes, or, -unsaturated aryl esters. The bands at 801 cm 1 and at 3017 cm 1 also indicate an unsaturated compound. As for Challenger, the strongest peak in the chromatogram of Cascade with a retention time of 37.9 min could be identified as -humulene. The spectrum for the peak at 26.9 min is identical to that at 27.3 min in the chromatogram of Challenger. For all other peaks, the FTIR spectra were too weak to provide useful information. In the chromatogram of Styrian Golding the peaks with the retention times of 33.2, 38.1, and 48.2 min could be assigned. The most intense peak at 38.1 min was identified as Figure 5. FTIR spectra of Challenger peaks at (a) 27.3 and (b) 33.2 min.

5 AUERBACH ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 3, Figure 6. FTIR spectra of (a) the Styrian Golding peak at 37.4 min and (b) the Northdown peak at 39.0 min. -humulene. The spectrum of the peak at 33.2 min is identical to that of Challenger at 33.0 min, indicating either 6-membered ring ketones,, -unsaturated aldehydes, or, -unsaturated aryl esters. The spectrum of the peak in the chromatogram of Styrian Golding with a retention time of 48.2 min is due to a mixture of humulones and lupolones, the -, and -acids of hops, shown in Figure 6(a) (19). For Northdown, the peaks with retention times of 33.2, 37.9, and 39.0 min gave useful spectra data. As for all varieties analyzed, the most intense peak at 37.9 min was identified as -humulene. The spectrum of the peak at 26.9 min is Figure 7. FTIR spectrum of a standard of trans-caryophyllene.

6 626 AUERBACH ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 3, 2000 identical to that at 27.3 min in Challenger; the spectrum of the peak at 39.0 min [Figure 6 (b)], may be due to transcaryophyllene. Figure 7 shows the FTIR spectrum of a transcaryophyllene standard passed through the same column under the same chromatographic conditions. Conclusions The SFC FTIR chromatograms of several hop varieties showed differences in the location and intensity ratios of the peaks depending on the variety. This was further confirmed by the IR spectra. These differences can, therefore, be used to characterize the sources of hop oils in terms of hop varieties used in their production. The chromatograms of Challenger and Cascade were very similar, as were those of Styrian Golding and Northdown. -Humulene was found in all varieties as a major component. It eluted with a retention time of about 38 min. Challenger and Cascade showed a very strong early eluting peak at about 27.1 min, which is due to an epoxide. Styrian Golding showed no such peak, and Northdown showed only a very weak intensity in this region. A very weak peak at 33.2 min was apparent in chromatograms of all 4 varieties. The SFC FTIR spectra indicated either of the following compounds: 6-membered ring ketones,, -unsaturated aldehydes, or, -unsaturated aryl esters. The variety Northdown showed a peak that could be due to trans-caryophyllene. The FTIR spectra of the very late eluting peak at 48.2 min in the chromatogram of Styrian Golding revealed traces of a mixture of the, -acids of hops. This information is useful with respect to the quality of the essential oil. Although the FTIR spectra gave useful information on the chemical nature of the species present in the hop oils, further work will be necessary to obtain more specific identifications. Such information may be obtained by mass spectrometry. Acknowledgments We thank our colleagues in the Chemistry Department at the University of Nottingham, especially M. Poliakoff for fruitful discussions and advice. We also thank English Hops Ltd. for providing us with all extracts. We are also grateful to Celal Bayar University, Manisa, Turkey, and Nottingham University for Studentships (K.D and R.H.A). References (1) Moir, M. (1994) Hop Aromatic Compounds, European Brewery Convention, Monograph XXII, Zocterwoude, p 165 (2) Verzele, M. (1986) J. Inst. Brew. 92,32 (3) Maier, J. (1996) Brauwissenschaft 19, 425 (4) Buttery, G., & Ling, L. (1967) J. Agric. Food Chem. 15, 531 (5) Tressl, R., Engel, K.H., Kossa, M., & Koeppler, H. (1983) J. Agric. Food Chem. 31, 892 (6) Stenroos, L.E., & Siebert, K.J. (1984) J. Am. Soc. Brew. Chem. 42,54 (7) Seeleitner, G., Seif, P., & Püspök, J. (1985) Brew. Conv. Proc. Congr. 20, 571 (8) Narziss, L., Miedaner, H., & Gresser, A. (1986) Brauwelt 1, 17 (9) Freundorfer, J. (1988) Monogr. Eur. Brew. Conv. 13,15 (10) Katsiotis, S.T., Langezaal, C.R., Scheffer, J.J.C., & Verpoorte, R. (1989) Flav. Fragr. J. 4, 187 (11) Kenny, S.T. (1990) J. Am. Soc. Brew. Chem. 48,3 (12) Freundorfer, J., Maier, J., & Reiner, L. Monatsschr. Brauwiss. 44, 416 (13) Kralj, D., Zupanec, J., Vasilj, D., Kralj, S., & Psenicnik, J. (1991) J. Inst. Brew. 97, 197 (14) Peacock, V.E., & McCarty, P. (1992) Techn. Quart. Master Brew. Assoc. Am. 29,81 (15) De Keukeleire, D., De Cooman, L., & Everaert, E. (1998) Phytochem. Anal. 9, 145 (16) Jenkins, T.J., Kaplan, M., & Davidson, G. (1994) J. High Resolut. Chromatogr. 17,1 (17) Jenkins, T.J., Kaplan, M., Davidson, G., Healy, M.A., & Poliakoff, M. (1992) J. Chromatog. 626,53 (18) Bellamy, L.J. (1975) The Infra-red Spectra of Complex Molecules, Chapman and Hall, London, UK (19) Auerbach, R.H. (1997) Ph.D. Thesis, University of Nottingham, UK

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