Journal of Chromatographic Science, Vol. 30, October 1992
Fractionation by SFE and Microcolumn Analysis of the Essential Oil and the Bitter Principles of Hops M. Verschuere and P. Sandra Laboratory of Organic Chemistry, University of Gent, Krijgslaan 2 8 1 , S4, B-9000 Gent, Belgium
F. David Research Institute for Chromatography, Kennedypark 20, B-8500 Kortrijk, Belgium
Abstract Supercritical fluid extraction (SFE) is evaluated and optimized for the enrichment and fractionation of the essential oil and the bitter principles of hops (Hamulus lupulus), both of which contribute to the flavor of beer. Profiles of the essential oil of different hop varieties are compared. The bitter principles, the humulones and lupulones, are analyzed by miniaturized liquid chromatography (micro-LC) and by micellar electrokinetic chromatography (MEKC).
lizing power can be adjusted, which provides the possibility of selective extraction. The possibilities of SFE for hop extraction have already been illustrated by David et al. (2). This paper de scribes the optimization of the SFE conditions for the selective extraction of the essential oil fraction and the a- and β-acid frac tion. Some limitations of analytical SFE are addressed. The bitter acids are analyzed by miniaturized LC (micro-LC) (3) on packed fused-silica columns and by micellar electrokinetic chro matography (MEKC) (4). The essential oil fraction is analyzed by capillary GC. Essential oils from different hop cultivars are compared.
Introduction Hops (Hwnulus lupulus) is a climbing perennial plant, flowers of which are indispensable in the production of beer. The essen tial oil of hops, primarily composed (80%) of the hydrocarbons myrcene, humulene, and β-caryophyllene, contributes to the aroma of beer. The most important constituents of hops, however, are the a- and β-acids, the precursors of beer bitterness. The αacids, or humulones, and the β-acids, or lupulones, each consist of three main homologues: the normal-, ad-, and co-compounds. During brewing, the α-acids are converted into the cis- and trans-iso-α-acids, or isohumulones, which are responsible for the typical bitter taste of beer (Figure 1). The analysis of the essential oil and of the a- and β-acids in hops is therefore an important aspect of quality control in the brewery. It is not the aim of this contribution to present an overview of the analytical methods nowadays applied in the brewery, but rather to discuss the possibilities of new analytical techniques, i.e., supercritical fluid extraction (SFE) and micro column analysis. For knowing more about hop chemistry and analysis, we refer the reader to the excellent book of Verzele and De Keukeleire (1). SFE has been used for many years as a preparative extraction method for the isolation of flavors and fragrances from plant material, i.e., the bitter principles of hops, but SFE is a relatively new technique in the field of analytical chemistry, having evolved in the past five years as an alternative method of preparing samples prior to chromatographic analysis. Besides the high extraction speed and the low extraction temperature, su percritical fluids have the unique property that their solubi-
388
Figure 1. Structures of α- and β-hop acids and iso-α-beer acids..
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
Journal of Chromatographic Science, Vol. 30, October 1992
Experimental
Results and Discussion
Plant material. Hop flowers of the cultivars Saaz, Northern Brewer, Hallertau, and Target, harvested in 1991, were dried and milled. Fifty-milligram quantities were used for SFE.
The total SFE of the essential oil, the bitter principles, and the lipid fraction of hops is routinely performed with pure C O at a density of 0.83 g/mL (temperature 50°C, pressure 250 bar) during 60 min and a supercritical fluid flow rate of 1 mL/min. The addition of modifiers on the quantitative recovery of the humulones and the lupulones has been studied. Hexane, dichloromethane, methanol, and acetone (20 μL each) were added on top of the hop sample in the extraction cartridge. The recovery was followed by comparing the peak areas of co-humulone and co-lupulone, representing respectively the humu lones and the lupulones versus the peak area of the p-nitroanilide derivative of myristic acid as internal standard. A typical chromatogram by micro-LC is represented in Figure 2.
Supercritical fluid extraction. SFE extractions were performed on the HP7680 SFE (Hewlett-Packard). This fully automated ex traction system includes a self-sealing extraction cell and a vari able restrictor that allows independent control of flow and pres sure. This restrictor reduces the risk of plugging when large quantities are extracted and provides an instantaneous pressure drop as the supercritical fluid (mostly C O ) exits the nozzle. The extracts are collected on a solid-phase trap that is flushed by sol vent after completion of the extraction. The extract is collected in a 2-mL vial. The system also allows multiple extractions of one sample. The solubilizing power of the extracting fluid can be adjusted for each step via the density, temperature, or the addition of modifiers. By sequential extractions, fractionation can be eval uated. Milled hops (50 mg) were introduced in the cartridge having a volume of 7 mL. Pure C O was applied with sequential steps of increased density by 0.05-0.1 g/mL. This was carried out using extraction times of 15 and 30 min at each density. The ex traction temperature was 50°C and a supercritical C O flow of 1 mL/min was applied. The collection trap was filled with 30-μm octadecylsilica (ODS) particles. The trap temperature was 15°C. After extraction, the trap was rinsed with 1 mL acetonitrile at a rate of 1 mL/min. The extract was analyzed as such by micro-LC for the determination of the bitter principles. As an alternative, the extract was evaporated to dryness and dissolved in the electrolyte for MEKC analysis. For the GC analysis of the essential oil, the extract was diluted 1:1 in ethylacetate. 2
2
2
Chromatographic analysis Micro-LC. A Varian 5000 LC pump operated in the split flow mode was used as the solvent delivery system. Injections were made with a 60-nL internal sample loop valve (Valco C14 W ) . A Wescan variable wavelength UV detector, equipped with a 320-μm i.d. fused-silica microcell, was used. The FSOT column (46 cm × 320 μm i.d.) was packed at 500 bar with a 10% CCI4 slurry of cyanopropyl PMSC1 (3). The humulones and lupulones were an alyzed using a gradient from 40% acetonitrile-60% water-0.05% phosphoric acid to 100% acetonitrile-0.05% phosphoric acid in 1 h. The flow through the microcolumn was 3 μL/min. Micellar electrokinetic chromatography. MEKC was performed on a P/ACE System 2000 (Beckman Instruments). The column was 75-μm i.d. × 43 cm in length, operated at 20°C with an applied voltage of 20 kV. The electrolyte consisted of 40 mM SDS in 25 mM tris (hydroxymethyl)amino methane-HCl, pH 8.55. Detection was at 214 nm (5). An HP 5890 Series II GC Capillary gas chromatography. equipped with an HP 3396 integrator (Hewlett-Packard) was used for the analysis of the essential oils. The extracts were intro duced via cool on-column injection on a 30-cm x 0.32-mm i.d. capillary column coated with 0.25-μm methyl silicone. A retention gap of 1.5 m x 320 μm i.d. was coupled to the analytical column to avoid the peak splitting that normally occurs when polar sol vents such as the acetonitrile-ethylacetate mixture are injected. The column was temperature programmed from 50° to 260°C at 5°/min. Helium was the carrier gas at 2.0 mL/min. The flame ionization detector (FID) was operated at a temperature of 280°C. For identification of the most important constituents in the essential oil fraction, the column was installed in an HP 5988A mass spectrometer.
2
One hundred percent recovery is obtained with acetone and methanol as modifier in 15 min at the density of 0.83 g/mL for both co-humulone and co-lupulone. Under the same conditions, 92% recovery for co-lupulone is obtained with dichloromethane and hexane and for co-humulone, there is a 70% recovery with hexane and an 82% recovery with dichloromethane. The reason for this difference is the more apolar nature of the lupulones, compared to the humulones. Addition of modifiers increases extraction speed. This, however, also corresponds to less selec tive extraction. Indeed, the methanol and acetone extracts are more colored because of the co-extraction of chlorophylls. There fore, for routine determination of bitter principles, pure C O is 2
Figure 2. Micro-LC analysis of the a- and β-acids. Peaks: (1) co-humulone, (2) humulone, (3) ad-humulone, (4) co-lupulone, (5) lupulone, (6) ad-lupulone, and (7) p-nitroanilide of myristic acid.
389
Journal of Chromatographic Science, Vol. 30, October 1992
normally applied. Compared to conventional methods for the de terminations of the a- and β-acids, relative standard deviations (RSDs) are higher with SFE. Typical values are on the order of 10% for six experiments on the same hop sample. This is because of the small sample size requirements if contamination problems have to be avoided. With the extractor used, and taking into consideration that the concentration of bitter principles in hops ranges as high as 10%, a 250-mg sample size is the maximum that can be extracted without breakthrough or contamination of the analyte trap. Such small samples may not be representative of the overall sample, biasing the analytical result. According to the Official Methods of Analysis 1990 (6), at least 5 g should be extracted to compensate for inhomogeneities in the sample. This is also the main reason why off-line SFE is preferred over on-line SFE for samples in which the analytes are present in high con centration. Another advantage of off-line methods is that the extract can be analyzed by any appropriate technique, and is available for multiple analyses. Nowadays, high-performance liquid chromatographic (HPLC) analysis of the a- and β-acids in hops, and of the iso-α-acids in beer, has almost completely re placed older, less specific, and less selective analytical methods (polarimetry, conductimetry, spectrophotometry, etc.). However, routine LC analysis is frequently characterized by coelutions, e.g., of normal and ad-homologues, or cis- and trans-iso-αacids, and by poor quantitation. The main problem in HPLC is the propensity of hop and beer iso-α-acids to form complexes with metal ions, causing peak tailing and irreversible adsorption. All metal components in conventional LC (pump, frits, injector, column, etc.) contribute to metal ion occurrence and require the addition of high concentrations of phosphoric acid to the mobile phase to compete with possible adsorbing sources. Micro-LC in fused-silica capillaries alleviates most of the metal ion problem, but requires the use of special demineralized silica materials. Electrophoretic techniques, where all possible sources of metal ions are excluded, can be an attractive alternative for the analysis of a- and β-acids in hops (5), and the analysis of iso-α-acids in beer (7). The analysis of a- and β-acids of an SFE extract by MEKC is presented in Figure 3.
of the homologues has been performed by combining both microLC and MEKC with diode array detection and by the analysis of reference compounds isolated by counter current distribution (8). In a second series of experiments, the possibility of selective SFE of the essential oil from the bitter compounds was investi gated. The recovery of myrcene, humulene, and β-caryophyllene, as main compounds of the essential oil, and of humulone, rep resenting the α-acids, and lupulone, representing the β-acids, was followed in function of time and C O density. The terpenes were monitored by capillary GC with n-nonadecane as internal stan dard and humulone and lupulone were monitored by micro-LC with the p-nitro anilide of myristic acid as internal standard. Figure 4A shows the graph at 15 min extraction time. For clarity, only the humulene line is drawn. At a density of 0.2 g/mL, 91% humulene, 98% myrcene, and 95% β-caryophyllene were ex tracted in a selective way from hops during the first 15 min. No bitter acids were found in the extract. A second extraction of the same sample during 15 min, but at a density of 0.25 g/mL gives 0.4% n-humulone and 8% n-lupulone. This also illustrates that the lupulones are better solubilized in the supercritical medium than the humulones, which are more polar. Extraction of the bitter compounds is completed at a density of 0.9 g/mL, which corresponds very well with the standard extraction conditions of 0.83 g/mL. The different extraction times and densities have to be taken into account, e.g., nine extractions of 15 min at in creasing densities versus 60 min of extraction at 0.83 g/mL. The graph also shows that the highest amount of both humulone and 2
Compared to the micro-LC analysis in Figure 2, analysis time is reduced by a factor of 6 for a better resolution. The elucidation
Figure 3. MEKC analysis of the α- and β-acids. Peaks: (1) formamide (t marker), (2) co-lupulone, (3) lupulone, (4) ad-lupulone, (5) co-humulone, (6) adhumulone, and (7) humulone. 0
390
Figure 4. SFE recovery as a function of density. Extraction time at (A) 15 min and (B) 30 min.
Journal of Chromatographic Science, Vol. 30, October 1992
lupulone is found at a density of 0.4 g/mL after 1 h extraction time (49% humulone and 50% lupulone). Lupulone is extracted completely at a density of 0.7 g/mL, whereas humulone requires a density of 0.9 g/mL. In order to determine if complete frac tionation of the essential oil from the bitter acids was possible,
the density program of Figure 4A was repeated but with extrac tion times of 30 min. The resulting graph is represented in Figure 4B. Ninety-nine percent humulene, 100% myrcene, and 100% βcaryophylene were extracted at a density of 0.2 g/mL during the first 30 min. At these conditions, n-humulone and n-lupulone were not extracted. Extraction at 0.2 g/mL for 60 min resulted in co-extraction of 1% humulones and 4.5% lupulones. From these data it is clear that sharp fractionation is possible if 100% re covery of the essential oil, without traces of the bitter acids, is at tempted. For further experiments on essential oils, an extraction time of 30 min and a density of 0.2 g/mL was used. In our studies to classify different hop cultivars by chemotaxomy, SFE of the cultivars Saaz, Northern Brewer, Hallertau, and Target was performed. The essential oil profiles of Northern Brewer, Hallertau, and Target are very similar and differ only by some minor quantitative differences which have to be evaluated as a function of harvest year in order to be of any value for chemotaxonomic characterization. However, the profile of Saaz, the top hops quality based on organoleptic evaluation of beers ac cording to brewers, differs qualitatively. Farnesene is one of the main components, and this sesquiterpene is absent in other hop varieties. Figures 5A and 5B show the capillary GC-FID trace of the SFE extracts of Northern Brewer and Saaz. The compounds marked were identified by capillary GC-MS.
Conclusion SFE can be applied to fractionate the essential oil and bitter principles of hops. Miniaturized separation systems, e.g. capillary GC, micro-LC, MEKC, offer several advantages over conven tional chromatographic techniques. The described methodologies are applied for chemotaxonomic studies and elucidation of vari ations as a function of geography and climatological conditions.
Acknowledgment We thank the Belgian National Fund for Scientific Research and the National Lottery for financial support to our laboratory. Μ . V. thanks the IWONL for their grant.
References
Figure 5. Capillary GC analysis of essential oils. (A) Northern Brewer and (B) Saaz. Peaks: (1) myrcene, (2) β-caryophyllene, (3) humulene, (4)
1. M. Verzele and D. D e Keukelelre. Chemistry and Analysis of Hop and Beer Bitter Acids, Elsevier, Amsterdam, 1 9 9 1 . 2. F. David, P. Sandra, and W.S. Pipkin. Supercritical Fluid Extraction of Hops, Application Note 228-115, Hewlett-Packard, Avondale, PA 1990. 3. P. Sandra, G. Steenbeke, M. Ghijs, and G. Schomburg. Micro liquid chromatography-diode array detection of hop bitter acids. J. High Res. Chromatogr. 13: 5 2 7 - 2 9 (1990). 4. J. Vindevogel and P. Sandra. Introduction to Micellar Electrokinetic Chro matography, Huethig Verlag, Heidelberg, Germany, 1992. 5. J. Vindevogel, P. Sandra, and L.C. Verhagen. Separation of hop bitter acids by capillary zone electrophoresis and micellar electrokinetic chromatog raphy with UV-diode array detection. J. High Res. Chromatogr. 1 3 : 2 9 5 - 3 0 0 (1990). 6. K. Helrich, Ed., Official Method of Analysis, Acids (Alpha and Beta) in Hops, 15th ed., AOAC, Arlington, VA, 1990, p. 732. 7. J. Vindevogel, R. Szücs, P. Sandra, and L.C. Verhagen. Analysis of beer isoα-acids by micellar electrokinetic chromatography and multi-wavelength UV de tection. J. High Res. Chromatogr. 1 4 : 5 8 4 - 8 8 (1991). 8. P. Sandra, unpublished results.
farnesene, and (5) humuladienone. Manuscript received July 3 1 , 1992.
391
Fractionation by SFE and Microcolumn Analysis of the Essential Oil and the Bitter Principles of Hops M. Verschuere and P. Sandra Laboratory of Organic Chemistry, University of Gent, Krijgslaan 2 8 1 , S4, B-9000 Gent, Belgium
F. David Research Institute for Chromatography, Kennedypark 20, B-8500 Kortrijk, Belgium
Abstract Supercritical fluid extraction (SFE) is evaluated and optimized for the enrichment and fractionation of the essential oil and the bitter principles of hops (Hamulus lupulus), both of which contribute to the flavor of beer. Profiles of the essential oil of different hop varieties are compared. The bitter principles, the humulones and lupulones, are analyzed by miniaturized liquid chromatography (micro-LC) and by micellar electrokinetic chromatography (MEKC).
lizing power can be adjusted, which provides the possibility of selective extraction. The possibilities of SFE for hop extraction have already been illustrated by David et al. (2). This paper de scribes the optimization of the SFE conditions for the selective extraction of the essential oil fraction and the a- and β-acid frac tion. Some limitations of analytical SFE are addressed. The bitter acids are analyzed by miniaturized LC (micro-LC) (3) on packed fused-silica columns and by micellar electrokinetic chro matography (MEKC) (4). The essential oil fraction is analyzed by capillary GC. Essential oils from different hop cultivars are compared.
Introduction Hops (Hwnulus lupulus) is a climbing perennial plant, flowers of which are indispensable in the production of beer. The essen tial oil of hops, primarily composed (80%) of the hydrocarbons myrcene, humulene, and β-caryophyllene, contributes to the aroma of beer. The most important constituents of hops, however, are the a- and β-acids, the precursors of beer bitterness. The αacids, or humulones, and the β-acids, or lupulones, each consist of three main homologues: the normal-, ad-, and co-compounds. During brewing, the α-acids are converted into the cis- and trans-iso-α-acids, or isohumulones, which are responsible for the typical bitter taste of beer (Figure 1). The analysis of the essential oil and of the a- and β-acids in hops is therefore an important aspect of quality control in the brewery. It is not the aim of this contribution to present an overview of the analytical methods nowadays applied in the brewery, but rather to discuss the possibilities of new analytical techniques, i.e., supercritical fluid extraction (SFE) and micro column analysis. For knowing more about hop chemistry and analysis, we refer the reader to the excellent book of Verzele and De Keukeleire (1). SFE has been used for many years as a preparative extraction method for the isolation of flavors and fragrances from plant material, i.e., the bitter principles of hops, but SFE is a relatively new technique in the field of analytical chemistry, having evolved in the past five years as an alternative method of preparing samples prior to chromatographic analysis. Besides the high extraction speed and the low extraction temperature, su percritical fluids have the unique property that their solubi-
388
Figure 1. Structures of α- and β-hop acids and iso-α-beer acids..
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
Journal of Chromatographic Science, Vol. 30, October 1992
Experimental
Results and Discussion
Plant material. Hop flowers of the cultivars Saaz, Northern Brewer, Hallertau, and Target, harvested in 1991, were dried and milled. Fifty-milligram quantities were used for SFE.
The total SFE of the essential oil, the bitter principles, and the lipid fraction of hops is routinely performed with pure C O at a density of 0.83 g/mL (temperature 50°C, pressure 250 bar) during 60 min and a supercritical fluid flow rate of 1 mL/min. The addition of modifiers on the quantitative recovery of the humulones and the lupulones has been studied. Hexane, dichloromethane, methanol, and acetone (20 μL each) were added on top of the hop sample in the extraction cartridge. The recovery was followed by comparing the peak areas of co-humulone and co-lupulone, representing respectively the humu lones and the lupulones versus the peak area of the p-nitroanilide derivative of myristic acid as internal standard. A typical chromatogram by micro-LC is represented in Figure 2.
Supercritical fluid extraction. SFE extractions were performed on the HP7680 SFE (Hewlett-Packard). This fully automated ex traction system includes a self-sealing extraction cell and a vari able restrictor that allows independent control of flow and pres sure. This restrictor reduces the risk of plugging when large quantities are extracted and provides an instantaneous pressure drop as the supercritical fluid (mostly C O ) exits the nozzle. The extracts are collected on a solid-phase trap that is flushed by sol vent after completion of the extraction. The extract is collected in a 2-mL vial. The system also allows multiple extractions of one sample. The solubilizing power of the extracting fluid can be adjusted for each step via the density, temperature, or the addition of modifiers. By sequential extractions, fractionation can be eval uated. Milled hops (50 mg) were introduced in the cartridge having a volume of 7 mL. Pure C O was applied with sequential steps of increased density by 0.05-0.1 g/mL. This was carried out using extraction times of 15 and 30 min at each density. The ex traction temperature was 50°C and a supercritical C O flow of 1 mL/min was applied. The collection trap was filled with 30-μm octadecylsilica (ODS) particles. The trap temperature was 15°C. After extraction, the trap was rinsed with 1 mL acetonitrile at a rate of 1 mL/min. The extract was analyzed as such by micro-LC for the determination of the bitter principles. As an alternative, the extract was evaporated to dryness and dissolved in the electrolyte for MEKC analysis. For the GC analysis of the essential oil, the extract was diluted 1:1 in ethylacetate. 2
2
2
Chromatographic analysis Micro-LC. A Varian 5000 LC pump operated in the split flow mode was used as the solvent delivery system. Injections were made with a 60-nL internal sample loop valve (Valco C14 W ) . A Wescan variable wavelength UV detector, equipped with a 320-μm i.d. fused-silica microcell, was used. The FSOT column (46 cm × 320 μm i.d.) was packed at 500 bar with a 10% CCI4 slurry of cyanopropyl PMSC1 (3). The humulones and lupulones were an alyzed using a gradient from 40% acetonitrile-60% water-0.05% phosphoric acid to 100% acetonitrile-0.05% phosphoric acid in 1 h. The flow through the microcolumn was 3 μL/min. Micellar electrokinetic chromatography. MEKC was performed on a P/ACE System 2000 (Beckman Instruments). The column was 75-μm i.d. × 43 cm in length, operated at 20°C with an applied voltage of 20 kV. The electrolyte consisted of 40 mM SDS in 25 mM tris (hydroxymethyl)amino methane-HCl, pH 8.55. Detection was at 214 nm (5). An HP 5890 Series II GC Capillary gas chromatography. equipped with an HP 3396 integrator (Hewlett-Packard) was used for the analysis of the essential oils. The extracts were intro duced via cool on-column injection on a 30-cm x 0.32-mm i.d. capillary column coated with 0.25-μm methyl silicone. A retention gap of 1.5 m x 320 μm i.d. was coupled to the analytical column to avoid the peak splitting that normally occurs when polar sol vents such as the acetonitrile-ethylacetate mixture are injected. The column was temperature programmed from 50° to 260°C at 5°/min. Helium was the carrier gas at 2.0 mL/min. The flame ionization detector (FID) was operated at a temperature of 280°C. For identification of the most important constituents in the essential oil fraction, the column was installed in an HP 5988A mass spectrometer.
2
One hundred percent recovery is obtained with acetone and methanol as modifier in 15 min at the density of 0.83 g/mL for both co-humulone and co-lupulone. Under the same conditions, 92% recovery for co-lupulone is obtained with dichloromethane and hexane and for co-humulone, there is a 70% recovery with hexane and an 82% recovery with dichloromethane. The reason for this difference is the more apolar nature of the lupulones, compared to the humulones. Addition of modifiers increases extraction speed. This, however, also corresponds to less selec tive extraction. Indeed, the methanol and acetone extracts are more colored because of the co-extraction of chlorophylls. There fore, for routine determination of bitter principles, pure C O is 2
Figure 2. Micro-LC analysis of the a- and β-acids. Peaks: (1) co-humulone, (2) humulone, (3) ad-humulone, (4) co-lupulone, (5) lupulone, (6) ad-lupulone, and (7) p-nitroanilide of myristic acid.
389
Journal of Chromatographic Science, Vol. 30, October 1992
normally applied. Compared to conventional methods for the de terminations of the a- and β-acids, relative standard deviations (RSDs) are higher with SFE. Typical values are on the order of 10% for six experiments on the same hop sample. This is because of the small sample size requirements if contamination problems have to be avoided. With the extractor used, and taking into consideration that the concentration of bitter principles in hops ranges as high as 10%, a 250-mg sample size is the maximum that can be extracted without breakthrough or contamination of the analyte trap. Such small samples may not be representative of the overall sample, biasing the analytical result. According to the Official Methods of Analysis 1990 (6), at least 5 g should be extracted to compensate for inhomogeneities in the sample. This is also the main reason why off-line SFE is preferred over on-line SFE for samples in which the analytes are present in high con centration. Another advantage of off-line methods is that the extract can be analyzed by any appropriate technique, and is available for multiple analyses. Nowadays, high-performance liquid chromatographic (HPLC) analysis of the a- and β-acids in hops, and of the iso-α-acids in beer, has almost completely re placed older, less specific, and less selective analytical methods (polarimetry, conductimetry, spectrophotometry, etc.). However, routine LC analysis is frequently characterized by coelutions, e.g., of normal and ad-homologues, or cis- and trans-iso-αacids, and by poor quantitation. The main problem in HPLC is the propensity of hop and beer iso-α-acids to form complexes with metal ions, causing peak tailing and irreversible adsorption. All metal components in conventional LC (pump, frits, injector, column, etc.) contribute to metal ion occurrence and require the addition of high concentrations of phosphoric acid to the mobile phase to compete with possible adsorbing sources. Micro-LC in fused-silica capillaries alleviates most of the metal ion problem, but requires the use of special demineralized silica materials. Electrophoretic techniques, where all possible sources of metal ions are excluded, can be an attractive alternative for the analysis of a- and β-acids in hops (5), and the analysis of iso-α-acids in beer (7). The analysis of a- and β-acids of an SFE extract by MEKC is presented in Figure 3.
of the homologues has been performed by combining both microLC and MEKC with diode array detection and by the analysis of reference compounds isolated by counter current distribution (8). In a second series of experiments, the possibility of selective SFE of the essential oil from the bitter compounds was investi gated. The recovery of myrcene, humulene, and β-caryophyllene, as main compounds of the essential oil, and of humulone, rep resenting the α-acids, and lupulone, representing the β-acids, was followed in function of time and C O density. The terpenes were monitored by capillary GC with n-nonadecane as internal stan dard and humulone and lupulone were monitored by micro-LC with the p-nitro anilide of myristic acid as internal standard. Figure 4A shows the graph at 15 min extraction time. For clarity, only the humulene line is drawn. At a density of 0.2 g/mL, 91% humulene, 98% myrcene, and 95% β-caryophyllene were ex tracted in a selective way from hops during the first 15 min. No bitter acids were found in the extract. A second extraction of the same sample during 15 min, but at a density of 0.25 g/mL gives 0.4% n-humulone and 8% n-lupulone. This also illustrates that the lupulones are better solubilized in the supercritical medium than the humulones, which are more polar. Extraction of the bitter compounds is completed at a density of 0.9 g/mL, which corresponds very well with the standard extraction conditions of 0.83 g/mL. The different extraction times and densities have to be taken into account, e.g., nine extractions of 15 min at in creasing densities versus 60 min of extraction at 0.83 g/mL. The graph also shows that the highest amount of both humulone and 2
Compared to the micro-LC analysis in Figure 2, analysis time is reduced by a factor of 6 for a better resolution. The elucidation
Figure 3. MEKC analysis of the α- and β-acids. Peaks: (1) formamide (t marker), (2) co-lupulone, (3) lupulone, (4) ad-lupulone, (5) co-humulone, (6) adhumulone, and (7) humulone. 0
390
Figure 4. SFE recovery as a function of density. Extraction time at (A) 15 min and (B) 30 min.
Journal of Chromatographic Science, Vol. 30, October 1992
lupulone is found at a density of 0.4 g/mL after 1 h extraction time (49% humulone and 50% lupulone). Lupulone is extracted completely at a density of 0.7 g/mL, whereas humulone requires a density of 0.9 g/mL. In order to determine if complete frac tionation of the essential oil from the bitter acids was possible,
the density program of Figure 4A was repeated but with extrac tion times of 30 min. The resulting graph is represented in Figure 4B. Ninety-nine percent humulene, 100% myrcene, and 100% βcaryophylene were extracted at a density of 0.2 g/mL during the first 30 min. At these conditions, n-humulone and n-lupulone were not extracted. Extraction at 0.2 g/mL for 60 min resulted in co-extraction of 1% humulones and 4.5% lupulones. From these data it is clear that sharp fractionation is possible if 100% re covery of the essential oil, without traces of the bitter acids, is at tempted. For further experiments on essential oils, an extraction time of 30 min and a density of 0.2 g/mL was used. In our studies to classify different hop cultivars by chemotaxomy, SFE of the cultivars Saaz, Northern Brewer, Hallertau, and Target was performed. The essential oil profiles of Northern Brewer, Hallertau, and Target are very similar and differ only by some minor quantitative differences which have to be evaluated as a function of harvest year in order to be of any value for chemotaxonomic characterization. However, the profile of Saaz, the top hops quality based on organoleptic evaluation of beers ac cording to brewers, differs qualitatively. Farnesene is one of the main components, and this sesquiterpene is absent in other hop varieties. Figures 5A and 5B show the capillary GC-FID trace of the SFE extracts of Northern Brewer and Saaz. The compounds marked were identified by capillary GC-MS.
Conclusion SFE can be applied to fractionate the essential oil and bitter principles of hops. Miniaturized separation systems, e.g. capillary GC, micro-LC, MEKC, offer several advantages over conven tional chromatographic techniques. The described methodologies are applied for chemotaxonomic studies and elucidation of vari ations as a function of geography and climatological conditions.
Acknowledgment We thank the Belgian National Fund for Scientific Research and the National Lottery for financial support to our laboratory. Μ . V. thanks the IWONL for their grant.
References
Figure 5. Capillary GC analysis of essential oils. (A) Northern Brewer and (B) Saaz. Peaks: (1) myrcene, (2) β-caryophyllene, (3) humulene, (4)
1. M. Verzele and D. D e Keukelelre. Chemistry and Analysis of Hop and Beer Bitter Acids, Elsevier, Amsterdam, 1 9 9 1 . 2. F. David, P. Sandra, and W.S. Pipkin. Supercritical Fluid Extraction of Hops, Application Note 228-115, Hewlett-Packard, Avondale, PA 1990. 3. P. Sandra, G. Steenbeke, M. Ghijs, and G. Schomburg. Micro liquid chromatography-diode array detection of hop bitter acids. J. High Res. Chromatogr. 13: 5 2 7 - 2 9 (1990). 4. J. Vindevogel and P. Sandra. Introduction to Micellar Electrokinetic Chro matography, Huethig Verlag, Heidelberg, Germany, 1992. 5. J. Vindevogel, P. Sandra, and L.C. Verhagen. Separation of hop bitter acids by capillary zone electrophoresis and micellar electrokinetic chromatog raphy with UV-diode array detection. J. High Res. Chromatogr. 1 3 : 2 9 5 - 3 0 0 (1990). 6. K. Helrich, Ed., Official Method of Analysis, Acids (Alpha and Beta) in Hops, 15th ed., AOAC, Arlington, VA, 1990, p. 732. 7. J. Vindevogel, R. Szücs, P. Sandra, and L.C. Verhagen. Analysis of beer isoα-acids by micellar electrokinetic chromatography and multi-wavelength UV de tection. J. High Res. Chromatogr. 1 4 : 5 8 4 - 8 8 (1991). 8. P. Sandra, unpublished results.
farnesene, and (5) humuladienone. Manuscript received July 3 1 , 1992.
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