Determination of iso-α-acids, α-acids and β-acids in beer, beer-based beverages and wort
Suitable for all beer, beer-based beverages and wort
iso-α-Acids (isohumulones), α-acids (humulones) and β-acids (lupulones) are chromatographically separated using this method.
The bitter substances are chromatographically separated by means of RHPLC. The humulones and lupulones are detected at 314 nm, whereas the iso-α-acids are detected at 270 nm.
Equipment
Analytical balance, ± 0.0001 g
Water bath, 20 °C
Ultrasound bath
Centrifuge, 750 g
Vials, crimp caps, crimping tool
Disposable syringes, 20 ml
Syringe filter, 0.45 µm, PTFE
High performance liquid chromatography set up for gradient operations, column oven 35 °C, UV-VIS detector (270 nm and 314 nm)
High pressure liquid chromatograph and UV-VIS detector, equipped for gradient operation
HPLC column, Machery & Nagel CC 125 / 4 Nucleodur 100 5 C18 EC or a comparable column
Graduated cylinder, 1000 ml, 50 ml
Beaker, 50 ml
Volumetric flasks, 100 ml, 50 ml
Volumetric pipettes, 30 ml, 10 ml
Erlenmeyer flask, 100 ml
Simultaneous determination of iso-a-acids and reduced iso-a-acids (rho, tetra, hexa) in beer and beer-based beverages using HPLC
This method is not suitable for beer and beer-based beverages containing a mix of hexa- and iso-products (co-elution).
This method separates iso-α-acids and reduced iso-α-acids (rho, tetra, hexa) chromatographically [2].
The separated compounds are detected spectrophotometrically at 270 nm. Quantification is performed using International Calibration Standards (ICS).
These standards do not contain all of the isomers of a certain type of iso-α-acids. Currently, ICS-I (DCHA-Iso) contains only the trans-isomeric complexes with dicyclohexylamine (DCHA). ICS-R (DCHA-rho) only has the cis-isomers in a complex with DCHA. ICS-T (tetra) comprises both cis- and trans-isomer complexes with DCHA, and ICS-H (DCHA-hexa) includes only cis-isomers with DCHA.
Commercially available hop products contain a greater number of isomers than the standards.
Thus, there are more peaks in the chromatogram than in the corresponding international calibration standards, in particular, with the cis-iso-cohumulone as well as the cis-iso-n-humulone and cis-iso-ad-humulone. The application of this method to these kinds of beers should be conducted with this in mind, since it is not entirely known whether these additional peaks are actually isomers of iso-α-acids.
Determination of xanthohumol and isoxanthohumol
All beers, beer-based beverages, wort, ethanol extracts, CO2 spent hops and xanthohumol products
Xanthohumol and isoxanthohumol are dissolved with acetonitrile from the sample and following separation, are determined using a Nucleodur C18 column and UV detection.