Rapid Analysis of Hop Acids in Beer using Strata -X Solid Phase Extraction and Kinetex.6 µm Core-Shell Technology Column Philip J. Koerner, Matthew Trass, and Jeff Layne Phenomenex, Inc., Madrid Ave., Torrance, CA 95 USA An improved HPLC analysis for iso-alpha hop acids in beer is demonstrated using the Kinetex.6 µm core-shell column, resulting in improved peak shape, easier quantitation, and reduced analysis times. Sample cleanup using Strata-X can also remove many potential interferences in the beer matrix, resulting in cleaner samples for HPLC analysis. Introduction Iso-alpha acids, derived from hops, are the compounds responsible for beer s bitter taste. During the beer brewing process compounds known as alpha acids are extracted from the hops and isomerized to form iso-alpha acids (Figure ). The conversion of alpha acids into iso-alpha acids takes place when the hops are added to the wort (unfinished beer) and boiled. The amount and type of isoalpha acids formed is dependent on a number of factors including the boiling time, the variety and age of the hops, and the ph of the wort. The bitterness derived from iso-alpha hop acids is a primary flavor attribute of beer and accurate determination of beer bitterness is of great importance to the brewer. Therefore, to maintain a consistent product, brewers must carefully monitor the levels of iso-alpha acids throughout the manufacturing process and in the final beer product. Iso-alpha acid levels are typically monitored using reversed phase HPLC. Until recently, these analyses have been performed using columns packed with fully porous silica particles, with run times of 5- minutes or longer. By switching to HPLC columns packed with Kinetex core-shell particles, these analyses can be significantly improved and performed in a fraction of the currently accepted analysis time. Materials and Methods Reagents and Chemicals All reagents and solvents were HPLC or analytical grade. HPLC Grade methanol and acetonitrile were purchased from Honeywell, Burdick & Jackson (Muskegon, MI), Milli-Q water was used for solid-phase extraction and sample-preparation. HPLC Grade water was purchased from Honeywell, Burdick & Jackson and used to prepare the LC mobile phase. Standards The hop standards were purchased from the American Society of Brewing Chemists. The standards came in two mixtures: one contained the normal iso-alpha acids (Isocohumulone, Isohumulone, and Isoadhumulone) and the other contained the reduced tetrahydroiso-alpha acids (Tetrahydroisocohumulone, Tetraisohumulone, and Tetrahydroisoadhumulone). The tetrahydroiso-alpha acid standard mix contains both the cis and trans isomers of each compound. Equipment and Materials Agilent Series HPLC (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with quaternary pump, autosampler, column oven, and variable wavelength detector. Solid Phase Extraction Each beer sample was degassed by stirring for approximately min at room temperature. Cartridge: Strata-X mg/6 ml Figure. Structures of Iso-Alpha Acids R = a-acids trans-iso-a-acids cis-iso-a-acids Part No.: Condition: Equilibrate: Load: Wash: Dry: Elute: 8B-S-FCH ml acidified methanol (- ml/min) ml water (- ml/min) Note: Do not let sorbent run dry 5 ml of beer degassed ( ml/min) ml of % methanol in water > Hg for 5 minutes to remove residual water ml of acidified methanol ( ml/min) CH(CH ) cohumulone trans-isocohumulone cis-isocohumulone CH CH(CH ) humulone trans-isohumulone cis-isohumulone CH(CH )C H 5 adhumulone trans-isoadhumulone cis-isoadhumulone Drydown: Nitrogen gas at 55 ºC Reconstitute: 5 µl of mobile phase Page of 8
HPLC Conditions Column: Dimensions: Mobile Phase: Kinetex.6 μm C8 Å as noted Methanol / Water / Phosphoric acid (75::, v/v/v) Figure. Iso-Alpha Acid Standards a) Kinetex.6 µm C8 5 x.6 mm Flow Rate: Injection Volume: Temperature:. ml/min as noted as noted 5 6 7 App ID 98 Detection: Sample: UV @ 7 nm (ambient) as noted 6 min Results and Discussion Figure is a chromatogram of hop acid standards (obtained from the American Society of Brewing Chemists) run on a Kinetex.6 μm C8 column. All six of the iso-alpha acids that are most commonly tested for by brewers, were resolved isocratically in less than 7 min. The first three hop acids isomers (isocohumulone, isohumulone, and isoadhumulone) are derived from the naturally occurring alphaacids in hops. The last three hop acids isomers (tetrahydroisocohumulone, tetrahydroisohumulone, and tetrahydroisoadhumulone) are specially modified reduced forms of the corresponding iso-alpha acids (tetrahydro-iso-alpha acids) and resistant to photo degradation. Both the naturally derived and the tetrahydro-iso-alpha acids have cis and trans isomers, therefore partial or even full resolution of some of the cis and trans iso-alpha acids is to be expected. Both the 5 mm and mm Kinetex columns completely separate all 6 iso-alpha acids (Figures a and b). The mm column provides near baseline separation for cis- and trans-tetrahydroisocohumulone (peaks & 5), while the 5 mm column only partially separates these compounds. Cis- and trans-tetrahydroisohumulone coelute on both Kinetex column dimensions (peak 6), but because of the better separation of the tetrahydroisocohumulone isomers, more accurate quantitation is achieved with the longer mm Kinetex column. It is easy to see that the separation with the Kinetex column is substantially improved versus the fully porous C8 column; providing faster separation and improved resolution and peak shape for all compounds. The chromatographic resolution for all of the hop acids, in particular for isohumulone and isoadhumulone, is much better on the Kinetex column. This increases the ability to more accurately monitor the brewing process and the final beer product. Column: Kinetex.6 μm C8 Å Dimensions: 5 x.6 mm Part No.: B-6-E Mobile Phase: Methanol / Water / Phosphoric acid (75::, v/v/v) Flow Rate:. ml/min Injection Volume: µl Temperature: 5 C Detection: UV @ 7 nm (ambient) Sample:. Isocohumulone. Isohumulone. Isoadhumulone. trans-tetrahydroisocohumulone 5. cis-tetrahydroisocohumulone 6. Tetrahydroisohumulone 7. Tetrahydroisoadhumulone b) Kinetex.6 µm C8 x.6 mm 6 8 min 5 6 Column: Kinetex.6 μm C8 Å Dimensions: x.6 mm Part No.: D-6-E Mobile Phase: Methanol / Water / Phosphoric acid (75::, v/v/v) Flow Rate:. ml/min Injection Volume: µl Temperature: 5 C Detection: UV @ 7 nm (ambient) Sample:. Isocohumulone. Isohumulone. Isoadhumulone. trans-tetrahydroisocohumulone 5. cis-tetrahydroisocohumulone 6. Tetrahydroisohumulone 7. Tetrahydroisoadhumulone 7 App ID 98 Page of 8
Figure a is a customer supplied chromatogram of an analysis of a beer product using a conventional, fully porous packing. As evidenced in the chromatogram, the peak shapes for these acids are poor and isohumulone and isoadhumulone are not well resolved. When a similar analysis is performed on a beer sample (Red Stripe Jamaican Lager) containing the same iso alpha-acids using Kinetex.6 μm C8 (Figure b), all of the hop acids are well resolved with excellent peak shape. In addition, the run time has been reduced from ~ to less than minutes. Figure. Analysis of Iso-Alpha Acids from commercial beer sample a) Fully porous 5 µm C8 5 x.6 mm Figures a and b are chromatograms obtained from the analysis of beer samples that contain tetrahydro-iso-alpha acids. The customer supplied chromatogram (Figure a) obtained using an HPLC column packed with fully porous particles shows that the tetrahydro-iso-alpha hop acids exhibit significant peak tailing and poor resolution, and the total analysis time is about minutes. However, when a similar sample (Miller Genuine Draft; Figure b) is analyzed using the Kinetex.6 μm C8 column, the tetra acids are eluted in less than 7 minutes. Note that the cis and trans isomers are partially resolved under these conditions, but quantified as a single unit. Figure. Analysis of Miller Genuine Draft beer samples containing Tetrahydro-iso-alpha Acids.5.5 -.5 - -.5 - -.5 6 8 min a) Fully porous 5 µm C8 5 x.6 mm 8 6 b) Kinetex.6 µm C8 x.6 mm 6 8 min 8 6 App ID 98 6 min Column: Kinetex.6 μm C8 Å Dimensions: x.6 mm Part No.: D-6-E Mobile Phase: Methanol / Water / Phosphoric acid (75::, v/v/v) Flow Rate:. ml/min Injection Volume: 5 µl Temperature: C Detection: UV @ 7 nm (ambient) Sample:. Isocohumulone. Isohumulone. Isoadhumulone Page of 8
b) Kinetex.6 µm C8 x.6 mm 98 Figure 5. Iso-Alpha Acids from Red Stripe beer following SPE a) 6.. 6 8 min App ID 98 6 8 min App ID 985 Column: Kinetex.6 μm C8 Å Dimensions: x.6 mm Part No.: D-6-E Mobile Phase: Methanol / Water / Phosphoric acid (75::, v/v/v) Flow Rate:. ml/min Injection Volume: 5 µl Temperature: C Detection: UV @ 7 nm (ambient) Sample:. trans-tetrahydroisocohumulone. cis-tetrahydroisocohumulone. Tetrahydroisohumulone. Tetrahydroisoadhumulone Column: Kinetex.6 μm C8 Å Dimensions: x.6 mm Part No.: D-6-E Mobile Phase: Methanol / Water / Phosphoric acid (75::, v/v/v) Flow Rate:. ml/min Injection Volume: 5 µl Temperature: C Detection: UV @ 7 nm (ambient) Sample:. Isocohumulone. Isohumulone. Isoadhumulone The iso-alpha acids are present in sufficient concentration to be able to be detected by simply injecting 5 µl of degassed beer straight onto the LC column without any sample preparation (other than degassing). All analytes are completely separated from any obvious matrix interferences. However, the complex nature of the beer matrix makes sample preparation worth investigating. The isoalpha acids were successfully extracted from packaged beer using Strata -X SPE products. A % methanol wash was found to remove some sample matrix components without adversely affecting analyte recovery. Two ml of acidified methanol was sufficient for eluting the analytes from the SPE cartridge. Recoveries from two commercial beer samples are shown in Tables and and the corresponding chromatograms are shown in Figure 5. Tetrahydro-iso-alpha acids from Miller Genuine Draft beer following SPE b) 6 8 min App ID 986 Column: Kinetex.6 μm C8 Å Dimensions: x.6 mm Part No.: D-6-E Mobile Phase: Methanol / Water / Phosphoric acid (75::, v/v/v) Flow Rate:. ml/min Injection Volume: 5 µl Temperature: C Detection: UV @ 7 nm (ambient) Sample:. trans-tetrahydroisocohumulone. cis-tetrahydroisocohumulone. Tetrahydroisohumulone. Tetrahydroisoadhumulone Page of 8
Table. SPE Recoveries of Iso-alpha Acids from Red Stripe Jamaican Lager Beer using Strata -X Iso-Alpha Acids % Recovery Isocohumulone Isohumulone Isoadhumulone 8 Table. SPE Recoveries of Tetrahydro-Iso-alpha Acids from Miller Genuine Draft Beer using Strata-X Tetra Iso-alpha Acids % Recovery Tetrahydroisocohumulone 8 Tetrahydroisohumulone 8 Tetrahydroisoadhumulone 69 Conclusions Typical HPLC methods for measuring iso-alpha acids throughout the brewing process have relied upon HPLC columns packed with fully porous particles. Converting these methods over to columns packed with high efficiency Kinetex.6 μm core-shell particles significantly improves chromatographic resolution while drastically reducing analysis times. While the iso-alpha acids are typically present in sufficient concentration to allow for direction analysis of the beer sample, a simple SPE cleanup was demonstrated to remove potential matrix interferences with good recovery. Page 5 of 8
Ordering Information Sample Preparation Vacuum Manifolds Part No. Description Unit AH-6 SPE -Position Vacuum Manifold Set, complete assembly ea AH-6 SPE -Position Vacuum Manifold Set, complete assembly ea AH-75 SPE -Position Tall-Boy Vacuum Manifold Set, complete assembly ea AH-78 96-Well Plate Manifold, Acrylic ea Vacuum Manifold Accessories Part No. Description Unit AH-79 Adapter Caps for, and 6 ml SPE tubes, polyethylene, with Luer tip 5/pk AH-778 Adapter Caps for and ml SPE tubes, polyethylene, with Luer tip 5/pk AH-779 Adapter Caps for 6 ml SPE tubes, polyethylene, with Luer tip 5/pk AH-878 Strata Syringe and Adapter Kit ea AH-6 SPE Manifold Needles, polypropylene /pk AH-65 SPE Manifold Needles, stainless steel /pk AH-65 SPE Drying Attachment for -position manifold ea Strata -X Sorbent Mass Part No. Unit Tube mg 8B-S-TAK ml (/box) mg 8B-S-TBJ ml (5/box) 6 mg 8B-S-UBJ ml (5/box) mg 8B-S-EBJ ml (5/box) mg 8B-S-ECH 6 ml (/box) mg 8B-S-FBJ ml (5/box) mg 8B-S-FCH 6 ml (/box) 5 mg 8B-S-HBJ ml (5/box) 5 mg 8B-S-HCH 6 ml (/box) Giga Tube 5 mg 8B-S-HDG ml (/box) g 8B-S-JDG ml (/box) g 8B-S-JEG ml (/box) g 8B-S-KEG ml (/box) 5 g 8B-S-LFF 6 ml (6/box) 96-Well Plate mg 8E-S-AGB Plates/Box mg 8E-S-TGB Plates/Box 6 mg 8E-S-UGB Plates/Box Additional sizes available. Contact your Phenomenex Sample Preparation Specialist for additional information. AH-65 SPE Drying Attachment for -position manifold ea AH-65 Female Luer Fittings /pk AH-65 Male Luer Fittings /pk AH-657 Vacuum Gauge and Valve Assembly ea AH-66 Control Valve, Teflon 5/pk AH-66 Teflon Needles /pk AH-665 Teflon Needles 5/pk Page 6 of 8
Kinetex Ordering Information KrudKatcher.6 μm Analytical Columns (mm) Ultra In-Line Filter* x.6 5 x.6 75 x.6 x.6 5 x.6 /pk XB-C8 B-96-E C-96-E D-96-E F-96-E AF-897 C8 A-6-E B-6-E C-6-E D-6-E F-6-E AF-897 C8 B-97-E C-97-E D-97-E F-97-E AF-897 PFP A-77-E B-77-E C-77-E D-77-E F-77-E AF-897 HILIC B-6-E C-6-E D-6-E F-6-E AF-897 KrudKatcher.6 μm MidBore Columns (mm) Ultra In-Line Filter* x. 5 x. 75 x. x. 5 x. /pk XB-C8 B-96-Y C-96-Y D-96-Y AF-897 C8 A-6-Y B-6-Y C-6-Y D-6-Y F-6-Y AF-897 C8 B-97-Y C-97-Y D-97-Y AF-897 PFP A-77-Y B-77-Y C-77-Y D-77-Y F-77-Y AF-897 HILIC A-6-Y F-6-Y AF-897 KrudKatcher.6 μm Minibore Columns (mm) Ultra In-Line Filter* x. 5 x. x. 5 x. /pk XB-C8 A-96-AN B-96-AN D-96-AN F-96-AN AF-897 C8 A-6-AN B-6-AN D-6-AN F-6-AN AF-897 C8 A-97-AN B-97-AN D-97-AN F-97-AN AF-897 PFP A-77-AN B-77-AN D-77-AN F-77-AN AF-897 HILIC B-6-AN D-6-AN F-6-AN AF-897 KrudKatcher.7 μm Minibore Columns (mm) Ultra In-Line Filter* 5 x. x. 5 x. /pk XB-C8 B-98-AN D-98-AN AF-897 C8 B-75-AN D-75-AN F-75-AN AF-897 C8 B-99-AN D-99-AN AF-897 PFP B-76-AN D-76-AN F-76-AN AF-897 HILIC B-7-AN AF-897 *KrudKatcher Ultra requires 5 /6 in. wrench. Wrench not provided. UHPLC / HPLC Sure-Lok High Pressure PEEK Male Nut Fittings Part No. Description Unit AQ-85 Sure-Lok High Pressure PEEK -Pc Nut -, /pk for / 6 in. Tubing,, psi (87 bar) AQ-85 Sure-Lok Fitting Tightening Tool, Aluminum ea Page 7 of 8
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