QUANTITATIVE ANALYSIS OF HOPS AND HOP EXTRACTS BY HIGH PRESSURE ION EXCHANGE CHROMATOGRAPHY

A new analysis for hop acids in hops and hop extracts is described. It is based on recent developments in high pressure liquid chromatography using pellicular anion exchange column filling material. Quantitative evaluation of α-acids, β-acids and oxidation products in hops and hop extracts is carried out by standard addition of pure humulone. The α-acids are completely separated from other hop substances before quantitation. The results of α-acids determinations must therefore be more accurate than was formerly possible and they are compared with conductometric titration results which are systematically higher. This is to be expected as it becomes more and more obvious that paper strip and conductometric analysis are not selective enough and determine fractions as “α-acids” which are in fact oxidation products of the hop acids.     

GAS CHROMATOGRAPHY AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY OF SOFT RESINS AND RELATED COMPOUNDS

The α-acids, β-acids, iso-α-acids, hulupones and humulinic acid have been separated by gas chromatography utilising the cool on-column injection technique on various bonded phase capillary columns. The α-acids were separated into cohumulone and humulone; the β-acids into colupulone and lupulone; and the hulupones into cohulupone and hulupone. Iso-α-acids were separated into four peaks; cis-isocohumulone, trans-isocohumulone, cis-isohumulone and trans-isohumulone. Flame ionization detection (FID) and chemical ionization mass spectrometry (CIMS) were used for chromatographic detection. Best results were achieved by employing a DB-1 fused silica capillary column with isooctane as the sample solvent .     

PREPARATION OF HOP EXTRACTS RICH IN PARTICULAR CONSTITUENTS*

When a column containing powdered hops is extracted with liquid carbon dioxide, chromatographic separation of hop components occurs. They are extracted in the order essential oils, β-acids, α-acids; and the separation is enhanced when finely milled hops are extracted. Early fractions (～0·5 hour) contain a high proportion of the available essential oils when hops are extracted at ?20°C and such extracts are suitable as a replacement for dry hops. Fractions can be obtained from extractions at ～7°C which are rich in α-acids and contain low levels of β-acids. Small amounts of fats and waxes are normally present in fractions collected towards the end of a run when seeded hops are extracted.     

NON-IMPLICATION OF HOP α- AND β- ACIDS AS SOURCES OF SULPHUR COMPOUNDS IN BEER

Whereas hop oil terpenoids can give rise to organoleptically undesirable sulphur compounds in beer brewed using hops dressed on the bine with sulphur, the hop resin α- and β-acids and their transformation products appear incapable of reactions with sulphur under analogous conditions. The evidence indicates that the hop resins are not potential sources of sulphur compounds in beer     

ESTIMATION OF α- AND β-ACIDS IN HOP EXTRACTS BY HPLC

A procedure relying on high performance liquid chromatography for the estimation of α-acids and β-acids in hop extracts has been collaboratively tested by members of a Sub-Committee of the Institute of Brewing Analysis Committee and is recommended for use. No significant differences were found between precision values obtained using peak height and peak area measurements. For α-acids, the mean repeatability (r₉₅) and reproducibility (R₉₅) values were 1-3 and 2-4% m/m respectively over the range 41–62% m/m. For β-acids they were 0-9 and 2-0% m/m respectively over the range 11 to 35% m/m. The precision values were judged to be independent of concentration.     

SOLUBILITY OF HOP α- AND β-ACIDS IN LIQUID CARBON DIOXIDE*: ADDENDUM: SOLUBILITY OF PESTICIDES IN LIQUID CARBON DIOXIDE

A procedure is described for determining the solubility of hop α- and β-acids in liquid carbon dioxide. Results have shown that the optimum temperature range for the extraction of hops with liquid carbon dioxide is +5 to +10°C. A number of pesticides used by hop growers are appreciably soluble in liquid carbon dioxide.     

ENZYMIC DEGRADATION OF α-ACIDS IN HOPS

Evidence is presented for the biological oxidation of α-acids in hop cones by an enzyme having molecular weight 17,000 daltons, K_m 6–0 and Mn as the active metal ion. The enzyme is located in bracts and bracteoles and not in lupulin glands. The presence of an endogenous inhibitor was demonstrated. The specific activity of protein in hop cones increased with maturity and this was probably associated with release of α-acids from lupulin glands.     

THE DETERMINATION OF α- AND β-ACIDS IN HOPS AND HOP PRODUCTS USING HPLC

A rapid reversed phase HPLC method for the analysis for α- and β-acids in hops and hop products is described and has been evaluated. The method uses citric acid in the eluent as a complexing agent to overcome the irreversible adsorption effects shown by some columns, thus allowing optimum eluent pH to be selected. The precision of the method for analysis of hop extract has been determined giving relative standard deviations of 1·0% and 2·1% for α- and β-acids respectively. General agreement with results obtained using a polarimetric α-acids analysis method for hop extracts and hops has been demonstrated.     

RAPID SPECTROPHOTOMETRIC METHOD FOR DETERMINATION OF THE BITTERING VALUE OF ISOMERIZED HOP EXTRACTS

A method is described which allows the rapid determination of the bittering value of isomerized hop extracts. The method is also applicable in the case of isomerized hop extracts containing impurities such as α-acids and β-acids.     

HOP RESIN ANALYSIS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ON MICROPARTICULATE SILICA

A high-performance liquid chromatographic separation of hop resins on a commercially available microparticulate silica has been developed. The chromatography involves a new mechanism in hop resin separation. A solvent containing di-n-butylammonium acetate is used. The method gives good separation of α-acid, iso-α-acid, β-acid and humulinic acid, and has been used for the quantitative analysis of α-acid and iso-α-acid in hop extracts and isomerised hop extracts.     

STABILITY OF RESINS IN PELLETISED HOPS

The α-acids content and Lead Conductance Value of hop pellets, manufactured from seedless Pride of Ringwood hops grown in Australia and stored at 5°C, 20°C or 30°C, were monitored over a twelve month storage period. No decrease in α-acids content occurred during storage at 5°C, whereas at 20°C the decrease (ca. 7%) was approximately one-third that experienced for baled hops of the same variety. At both 5°C and 20°C the Lead Conductance Value decreased at approximately one-third the rate of that in baled hops. During twelve months storage at 30°C the α-acids content of the hop pellets decreased by approximately 40% and the Lead Conductance Value by 30%. The hop pellets stored at 20°C and 30°C developed rancid odours during the twelve months storage period.     

KINETICS OF HOP STORAGE

After an initial ‘delay period’ the storage losses of α- and β-acids follow a first-order rate equation and not a zero-order equation as has been suggested by others.     

PREPARATION,PURIFICATION AND SEPARATION BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF HUMULINIC ACIDS,DEHYDROHUMULINIC ACIDS,AND HULUPONES

Modification of published procedures are described for the preparation of humulinic acids, dehydrohumulinic acids and hulupone, using iso-α-acids as starting material. These procedures provide facile routes for preparation of these compounds in higher yields than was hitherto reported, and also in sufficient purity for their use as standards in quantitative High Performance Liquid Chromatography. An alternative and more feasible route to hulupones, using β-acids as starting material, is also outlined.     

PROPOSALS FOR THE EFFICIENT AND ECONOMICAL USE OF HOPS IN BREWING

The hop lupulin glands, which contain the essential oils and resin acids, can be separated effectively from the bulky and worthless bracts and residues in a mechanical process which avoids solvent contamination. The separation is accomplished in an air stream through a centrifugal sifter after the lupulin has been rubbed off in a disc pin mill. The separated lupulin can be broken open by grinding in a colloid mill and used to obtain a dry hop aroma in beers by an ‘instant’ process. The lupulin can be used to bitter beers either directly in the copper or in a pre-boiling process. It can also be used in an aqueous extraction process to yield, nearly quantatively, pure and separate extracts of the α- and β-acids. The α-acids can then be isomerized almost quantitatively and the β-acids can be oxidized to give a 50% yield of hulupones. The hop residues, when boiled with wort, give an approximately 50% utilization of the remaining α-acids. These isohumulones can then be enhanced by those obtained from the extracted α-acids and the bitterness can be supplemented by the hulupones obtained from the β-acids. Overall, this process is calculated to give a bitterness utilization equivalent to 87% calculated on the original α-acids.     

啤酒花活性成分与抗氧化活性的相关性

以3 个品种的萨斯特啤酒花为原料,比较了其石油醚、氯仿、乙酸乙酯、正丁醇和水相等不同极性溶剂提取物中α-酸、β-酸、总黄酮和总多酚的含量,采用?OH、1,1-二苯基-2-三硝基苯肼自由基、β-胡萝卜素-亚油酸3 种不同的抗氧化活性评价体系对各极性溶剂提取物的抗氧化活性进行了研究,并与其活性成分的含量进行了相关性分析。结果表明,啤酒花各极性部位均具有不同程度的抗氧化活性。α-酸、β-酸在石油醚相中含量较高,在3 个啤酒花样品中的含量范围分别为α-酸444.29～583.81 mg/g、β-酸131.83～293.19 mg/g,在氯仿相中含量相对较低,其余提取物中均未检出;相关性研究表明α-酸、β-酸是石油醚和氯仿相抗氧化活性的主要物质基础。中等极性和极性较大溶剂提取物中啤酒花总黄酮、总多酚含量较高,相关性研究也表明黄酮、多酚类物质是这类提取物抗氧化活性的主要物质基础;其中,3 个啤酒花样品中总黄酮、总多酚在乙酸乙酯相中含量最高,分别为20.43～24.13 mg/g和34.97～40.24 mg/g,正丁醇次之,分别为9.45～11.68 mg/g和12.12～19.14 mg/g。     

ON THE ANALYSIS OF HOP BITTER ACID

A High Performance Liquid Chromatography (HPLC) method for automated routine analysis of the hop bitter acids is described. It allows the simultaneous determination of α-acids, β-acids, cohumulone and colupulone. This analysis based on two chromatographic runs takes less than 1/2 h. With this new method as reference, the defects of existing conductometric titration techniques were studied and a new improved conductometric method based on toluene-buffer extraction was developed. The new methods are applicable to all forms of hop products containing α- and β-acids. Analysis for α-acids with ether-acid aqueous methanol extraction gives results which are too high because of several basic defects.     

ANALYSIS OF HOPS AND HOP PRODUCTS FOR α-ACIDS OR ISO-α-ACIDS

Two methods based on the resolution of mixtures of hop compounds by chromatography on Sephadex columns have been adopted by E.B.C. and A.S.B.C. as ‘International Methods’.     

SEPARATION AND QUANTIFICATION OF ALL MAIN HOP ACIDS IN DIFFERENT HOP CULTIVARS BY MICROEMULSION ELECTROKINETIC CHROMATOGRAPHY

Microemulsion electrokinetic chromatography has been applied to the analysis of hop acids in extracts of different hop cultivars. With this new analytical method, the 6 main hop bitter acids are completely separated within 10 minutes. Quantitative analyses of extracts from Saaz, Nugget and Wye Target cultivars have been compared to those obtained with reversed-phase high-performance liquid chromatography. The cohumulone and colupulone ratios are in good agreement. Microemulsion electrokinetic chromatography however is much faster and provides, in addition, data for the adhumulone and adlupulone ratios.     

DIRECT ANALYSIS OF INDIVIDUAL ANALOGUES OF ALPHA-ACIDS,BETA-ACIDS AND 4-DESOXYHUMULONES IN HOPS USING GAS-LIQUID CHROMATOGRAPHY AND NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

The techniques of gas-liquid chromatography and nuclear magnetic resonance allow simultaneous analysis of the individual α-acids and β-acids and the corresponding 4-desoxyhumulones. The rates of appearance in the growing hop-cone of humulone, lupulone, 4-desoxyhumulone and their co-homologues and ad-isomers are reported. The results indicate a relatively enhanced initial formation of the 4-desoxy compounds and β-acids. The α-acids reach their maximum concentration later than the desoxyhumulones and β-acids but eventually become the predominant group. It appears that formation of the α-acids is associated with a decline in the relative proportion of both β-acids and 4-desoxyhumulones. For each class of compounds, the co-homologues increase their relative concentrations during the growth of the hop; adhumulone and adlupulone remain in almost constant proportions during this period.     

ANALYSIS OF HOPS OF THE 1987 CROP

Analytical values for α-acid content, as measured by lead conductance value, of twelve varieties of hops from the 1987 crop are presented in relation to the district of production. Satisfactory variabilities have been achieved by Fuggle, Progress, Bramling Cross, Northern Brewer, Zenith, Wye Challenger and Omega. Wye Northdown has increased in variability, reaching the upper limit of acceptability despite an increase in sampling rate. Goldings, W.G.V. and Wye Target have continued to show higher than normal variabilities. This year Yeoman has moved in variability above the acceptable limit. The sampling rates have been considered and in view of the unusual growing conditions experienced with the 1987 crop, with hop yields considerably reduced and α-acids increased to very high levels, no changes have been recommended.