Hop acids

Fig. 2. Representative structures of d- and P-hop acids. R groups are defined in Table 6.

There are many methods available in the scientific literature describing the separation and quantification of the hop acids. They may be classified according the mobile phase employed, for most if not all the reverse-phase methods are based on octyl (C8) and octadecyl (C18) columns (Table 3). 

Verzele and de Keukeleire have reviewed the difficulties associated with the standardization of hop acid analyses (33). In particular, at the time of writing, standardization for isomerized and chemically modified components is highly topical. As mentioned earlier, the trans-iso-a-acids specifically precipitate when a mixture of iso-a-acids is treated with dicyclohexylamine, and it has recently been shown that this behavior extends to the hexahydro-, p-, and (with difficulty) tetrahydroiso-a-acids (55). An international subcommittee has recently been convened to establish standards for the full range of chemically modified iso-a-acids to encourage international standardization for ensuring beer quality control and to provide an agreed basis on which these products can be traded. 

P Ting, H Goldstein. Preparation and purification of hop acids and their derivatives. J Am Soc Brew Chem 54 103-109, 1996. 

AJP Hofte, RAM van der Hoeven. Characterization of hop acids by liquid chromatography with negative ion electrospray ionization mass spectrometry. J Am Soc Brew Chem 56 118-122, 1998. 

R Schwarzenbach. HPLC of hop acids on buffered silica gel systems. J Am Soc Brew Chem 37 ISO-184, 1979. 

RK Krueger, AJ Rehberger, PD Payne. The trimethylsilyl derivatives of hop acids and their transformation products. Proc Am Soc Brew Chem, 1967, pp 84-97. 

Chromatography methods combined with NMR have been used for the analysis of hop acids and beer bitter acids.378 379 Supercritical fluid chromatography has been used in the analysis of ascorbigens in Brassicas, but in this case the NMR was not on line with the chromatography system.380... 

In the normal isomerization of humulone (70) the bond between C-1 and C-6 is broken and a new bond is formed between C-1 and the carbonyl group at C-5 (Fig. 14.6). The reversed isomerization of humulone (Fig. 14.8), in which the bond between C-5 and C-6 is broken and the new bond formed between C-5 and the carbonyl on C-1, has now been found to occur and the unti-products account for about 10% of the isomerization mixture [93]. By boiling humulone in a buffer solution at pH 11-0 the cis- and tmn.r-isomers of both acetylhumulinic acid (88) are formed. The bitter tasting hop acids known [94]. In the mixture of isomerization mixture are deacylated anti-derivatives deacyl-ated anti-isohumulone (90), deacylated anti-acetylhumulinic acid (91), and deacylated onti-humulinic acid (92) [93]. Deacylated humulone (89) is readily isomerized to these products whereas the deacylation of anti-isohumulone only occurs to a limited extent (< 2 %) so it has been proposed [95] that (90), (91) and (92) are formed via deacylated humulone (89). However, the relative ease of deacylation of humulone and Deacylated derivatives of isohumulone have not been characterized in isomerization mixtures. 

The trend toward beer-mix beverages, low-alcohol and alcohol-free beers is a worldwide driver of product innovations in the brewery sector. The low contents of natural preservatives such as ethanol or hop acids and the increased amounts of (fermentable) sugars have led to a higher microbial susceptibility of these products. If tunnel pasteurization is avoided or is not possible (with plastic packaging), the filling process should occur under highly controlled hygiene conditions. 

The acyl and alkenyl side chains in 163 are separated by four carbon atoms, which is not the case for the six-membered ring hop acids. The three-carbon connection, which is found in the five-membered ring hop derivatives next to the bridge of four carbon atoms, has clearly been cleaved and replaced by a C-O-C chain. An... 

Comparison of the development of the hop acids in fertilized and unfertilized hops shows that about 20% more alpha acids and 100% more essential oil are produced in the unfertilized hops. A corresponding increase in the fraction of beta acids was not observed and the synthesis of the hop acids was completed simultaneously. The production of the essential oil continued for one more week in unfertilized hops (45). 

Fig.115. Chromatogram of an ethanol hop extract obtained on a HP 1090 LC instrument with DAD and with two coupled conventional columns of 250 x 4.6 mm, packed with 5 pm RoSiL-C18 (for "Hop Acids ). Gradient elution at 1 ml.min with solvent A CH3CN H2O H3PO4 60 40 0.5 (+ 100 ppm EDTA) to solvent B CH3CN ...

We do not know how the Nucleosil material is processed to become a phase for "Hop Acids . The RSL material is boiled out twice with acid (information from the producer). This is sufficient for alpha acids analysis, but is not enough for iso-alpha acids analysis. That the acid treatment has a negative influence on the packing particles is shown by the increased pressure drop for the "Hop Acid" columns (Table 15). 

Apparently (see Table 16) the addition of EDTA leads to correct results on both commercial columns specially designed for hop acids analysis. It must be noted that EDTA has to be added to the water used for preparing the eluent. In the eluent itself it is very difficult to dissolve the indicated amount of EDTA. This is the reason why EDTA addition failed many years ago, when we first tried this. According to the Merck Index 1 litre of water can dissolve 500 mg of EDTA. Still, to dissolve 100 mg of EDTA in 300 ml of water may take considerable time. The disodium salt of EDTA is more soluble, but although results with this chemical were reasonable, EDTA seems to be better. With the disodium salt of EDTA, larger amounts were tried, but this did not further improve the results. It is noteworthy that the standard deviation (s%) in the tables becomes smaller with every improvement of the analytical results due to changes in eluent composition. 

Data showing the difference in metal sensitivity for different hop and beer bitter acids are presented in Table 18. The increasing metal sensitivity in the series deoxyhumulone, colupulone, humulone, trans isohumulone is revealed. These results agree with the observation that RoSil-C18 for "Hop acids" and Nucleosil-C18 for "Hop acids" are satisfactory for LC analysis of alpha acids, but not for iso-alpha acids. Other commercial columns, newer or older columns may of course give figures more or less different from those shown in Table 18. Calibration and the use of alpha acids of known composition as ES may get around the trace metal problem, but clearly this is not the way to go. Trace metal activity of columns will vary by production variation, with column age and with duration of the chromatographic run. Special columns must be used and EDTA must be added to the eluent as shown in this section. 

A 25 X 0.46 cm column packed with either 5 pm R0SN-CI8 "Hop Alpha Acids" (Bio-Rad-RSL) or with 5 pm Nucleosil-C18 "Hop Acids" (Macherey Nagel) has to be used in a convenient chromatograph, fitted with a 10 pi sample loop injector. The variable wavelength detector set at 314 nm must be calibrated against a naphthalene solution in hexane or in iso-octane (sharp absorption peak at 311 nm). 

Instead of using an IS, calibration can also be obtained by using a hop extract with known composition. The peak area of the reference extract is then directly compared with that of the sample to be analysed. The unknown and reference extract should be analysed consecutively, which means double analysis time. The choice of internal or external reference was discussed in more detail earlier in this chapter. In some cases oxidation during the sample preparation and during the chromatography may be feared or is a reality. Therefore the addition of 1 % BHT to all solvents is sometimes needed. Whether such oxidation occurs or not can be evaluated by running the analysis with and without BHT addition. If the results are similar, the system is not oxidizing the hop acids and BHT can be ommitted. 

Fig.119. Chromatograms for the magnesium salt mentioned in Table 24 on the two discussed columns. BHT was added to the sample solution. The first doublet is for the trans and cis isocohumulones, the next triplet is for the trans and cis isohumulones and the isoadhumulones. Varian 5040 LC with Varian detector at 270 nm. 1 ml solvent per minute. 5 pm packing particles. Left (A) RoSiL-C18 for "Hop Acids Analysis", right (B) Nucleosil-CI 8 for "Hop Analysis". The IS is 2,6-di-t.butylphenol.

Fig. 120. Micro-LC of an iso-octane extract of lager beer. Column 210 mm x 0.32 mm i.d., 5 pm RoSiL-C18 (for "Hop Acids Analysis"). Mobile phase as described before, except for 5 % Cetavlon hydroxide added. Flowrate 6.4 pl.min. Pressure 104 kg.cm 2. Detection at 270 nm with Varian 2050 UV detector provided with a miniaturized detection cell (320 pm capillary). Sample loop injection of 200 pi of the iso-octane extract. Peak 1 trans and cis isocohumulones. Peak 2 trans and cis isohumulones + isoadhumulones Peak 3 2.6-di-t.butylphenol (IS).

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