Analysis of Iso-α-Acids, α-Acids, and Humulinonesunder EBC 9.47

Significance of the Topic

Beer bitterness is a critical quality attribute that influences consumer perception and product consistency in brewing. Bittering compounds such as α-acids, iso–α-acids, and humulinones determine flavor balance and stability. Reliable analytical methods are essential for recipe optimization, quality control, and regulatory compliance in the brewing industry.

Objectives and Overview of the Study

This application note evaluates a reversed-phase liquid chromatography method following EBC 9.47 for simultaneous separation and quantification of humulinones, iso–α-acids, and α-acids. The aim is to demonstrate performance of a Shimadzu Nexera XR system equipped with a Shim-pack GIST C8 column under defined mobile phase conditions and gradient program.

Methodology and Instrumentation

The analysis was performed under the following conditions:

• System: Nexera XR liquid chromatograph
• Column: Shim-pack GIST C8, 250 mm × 4.6 mm I.D., 5 µm particle size
• Mobile phase A: Acetonitrile/1 % citric acid buffer (pH 7.0) = 30 : 70
• Mobile phase B: Methanol
• Gradient: B conc. 15 % (0–5 min) → 80 % (30–33 min) → 15 % (35–45 min)
• Flow rate: 1.0 mL/min
• Column temperature: 35 °C
• Injection volume: 50 µL
• Detection: UV at 270 nm

Main Results and Discussion

The method achieved baseline resolution of humulinones, iso–α-acids, and α-acids within a 33 min gradient run. Retention times were consistent with EBC 9.47 guidelines. Chromatograms exhibited sharp peaks and minimal tailing, reflecting high column efficiency. Method repeatability was confirmed with relative standard deviations below 2 % for retention time and area.

The gradient profile enabled clear separation of multiple isomeric forms of iso–α-acids and α-acids. Humulinones eluted earlier due to higher polarity, followed by iso–α-acids and more hydrophobic α-acids.

Practical Benefits and Applications

This LC method provides breweries and analytical laboratories with a robust tool for routine bitterness profiling. Key advantages include:

• Compliance with EBC standard procedure
• High resolution of closely related hop compounds
• Reproducible quantitation for quality control
• Compatibility with standard UV detection

Future Trends and Applications

Advancements in column technology and detection systems can further enhance throughput and sensitivity. Potential developments include:

• Use of sub-2 µm UHPLC columns to reduce analysis time
• Integration of mass spectrometry for compound identification and lower detection limits
• Automated sample preparation and online monitoring of brewing process
• Digital data analysis for predictive quality management

Conclusion

The described reversed-phase LC method employing a Shim-pack GIST C8 column on a Nexera XR system effectively separates and quantifies key bittering compounds in beer. It meets EBC 9.47 requirements, offering reliable performance for breweries and research laboratories focused on flavor quality and process control.

References

Application News L590 (JP, ENG), Shimadzu Corporation, First Edition: December 2021