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.     

ESTIMATION OF α-, β- AND ISO-α-ACIDS IN HOPS,HOP PRODUCTS AND BEER USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Methods are described in which high performance liquid chromatography (HPLC) is used to estimate α-, β- and iso-α-acids in hops, hop products and beer. The chromatography relies on an isocratic elution of components from a polythene ‘cartridge’ column, and the method is calibrated with the pure substances as primary standards. Using such a column over 1000 analyses have been carried out without any significant loss in resolution or precision. The procedures are sufficiently rapid for use in commercial transactions and for quality control purposes. For hops and hop extracts coefficients of variation (%) of 2·5 and 0·8 were obtained respectively for α-acids. Values of 0·9 and 0·3 were obtained for iso-α-acids in isomerised extracts and beers respectively. For some isomerised extracts it has been observed that peaks in addition to those given by the iso-α-acids are present on the chromatogram. The current method recommended by the EBC over estimates the iso-α-acid content since these other constituents are included in the analysis.     

异构酒花浸膏中异α-酸氢化工艺优化

采用单因素筛选试验考察了氢化温度、pH值、氢气压力、氢化时间、催化剂用量、浸膏中异α-酸浓度对异α-酸氢化的影响。再利用Plackett-Burman设计研究了各因素对异α-酸氢化的影响。结果表明,底物异α-酸浓度、氢气压力和pH对异α-酸酒花浸膏氢化效果影响显著;在此基础上,采用L9(34)正交设计法对影响异α-酸酒花浸膏氢化的3个主要因素异α-酸浓度、氢气压力、pH进行了参数优化试验。结果表明,影响异α-酸酒花浸膏氢化的主次因素顺序为异α-酸浓度>氢气压力>pH值,异α-酸氢化的最佳工艺参数为:异α-酸的浓度55mg/mL,氢气压力0.2 MPa,pH值10,催化剂Pb/C用量3%,氢化时间4h。浸膏中四氢异α-酸的浓度达到58.89%,异α-酸氢化转化率达到89.73%。     

AUTOMATED REPORTING ON THE QUALITY OF HOPS AND HOP PRODUCTS

The quality of a hop variety or a hop product can readily be assessed by a fully automated sequence of selective extraction, fractionation and quantitative analysis. To illustrate the elegance of the method, nine hop varieties and three hop extracts were compared with respect to the content of important marker compounds in the hop oils and of the hop acids. Supercritical fluid extraction at different densities of carbon dioxide was applied to extract selectively, the hop oils and the hop acids, respectively. The hop oils were further fractionated into an apolar and a polar fraction by solid phase extraction and consecutive elution with n-hexane and ethyl acetate. Separation and identification were achieved by capillary gas chromatography coupled to mass spectrometry. Myrcene, β;-caryophyllene, α;-humulene and β;-farnesene in the apolar fraction, linalool, undecan-2-one, tridecan-2-one and humuladienone in the polar fraction were selected for quantitative evaluation of the respective hop oils. Sulphur-containing compounds were revealed by capillary gas chromatography using sulphur-selective atomic emission detection. Complete separation and quantification of all hop α;-acids and β;-acids was effected by microemulsion electrokinetic chromatography coupled to diode array detection .     

THE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF HOP RESINS

Hop resins are eluted from a specially developed weak anion exchange bonded phase silica using citric acid, methanol, water, mixtures as eluent. Fresh and deteriorated hops and isomerized extracts have been examined. The ratio of the humulone congeners in different hop varieties and the conversion of α-acid to iso-α-acid during the hop boil have been measured.     

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 .     

酒花制品中异α-酸浓度的测定方法

采用分光光度法探索测定异α 酸、四氢异α 酸异构体、六氢异α 酸异构体等酒花制品中异α 酸的浓度 ,利用该方法对已知标准样品中异α 酸浓度进行检测 ,其重复性与精密度的结果表明 ,该方法的变异系数≤ 1 %,回收率在 99 8%～ 10 0 5 %之间 ;方法操作简单 ,可作为啤酒企业衡量评价异α 酸、四氢异α 酸异构体、六氢异α 酸异构体等酒花制品中异α 酸浓度的检测方法     

STUDIES ON THE EFFECT OF POLLINATION ON THE YIELD AND RESIN CONTENT OF HOPS (HUMULUS LUPULUS L.)

Pollination trials in otherwise male-free hop gardens are described. The results showed that a reduction in cone yield of up to 30% may be expected from seedless hops, but an increase in α-acid content of the cones more or less compensates for this yield reduction and the yield of α-acid per acre can be almost as high from seedless as seeded hops. Northern Brewer, in particular, performed better than other varieties when grown seedless but most of the high α-acid varieties gave reasonable results. It is suggested that the variation in response may depend on the number of resin glands present on the seeds of the different varieties, and differences in the distribution of resin glands on other parts of the cone.     

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.     

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.     

ISO-α-ACIDS CONTENT OF ISOMERIZED HOP EXTRACTS

Two chromatographic procedures for the analysis of iso-α-acids in isomerized extracts have been tested. Although one of the methods—a variant of the Hansen, Hetzel & Miller procedure—performed better than the other, it still falls short of the demands of commercial transactions. The methods available for the assay of iso-α-acids are reviewed.     

HOP INDUSTRY ADVISORY COMMITTEE: ANALYSIS OF HOPS OF THE 1975 CROP

Analytical values for α-acid content of eleven varieties of hops from the 1975 crop are presented in relation to the district of production. For most of the varieties the variation within growths was lower than in the previous year. With amended sampling rates, the tolerance limits of ±0·5 % were achieved for all hop varieties and the results are acceptable for commercial purposes. Some α-acid values for seedless hops from the Seedless Hop Trials are included in the Report and comparisons with seeded hops are shown over a three year period.     

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.     

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.     

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.     

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.     

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.     

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     

AUTOMATIC ANALYSIS OF HOP RESINS BY ION EXCHANGE CHROMATOGRAPHY

Ion exchange chromatography has been used to provide a quantitative automatic analysis of lupolunes, humulones, humulinic acid, iso-α-acid and hulupones. The resins are extracted and chromatographed on a column of Q.A.E. Sephadex in the acetate form, using an acetic acid gradient in methanol as eluent. The absorbance of the effluent is recorded and the peaks are integrated. A comparison of peak areas with known standards gives a quantitative analysis of the hop components. The analysis takes I h if only the α and β resins are required, 2 h for an analysis including iso-α-acid and h if hulupone is included. The method is applicable to hops, hop extracts, wort and beer.     

Analysis of hop acids by capillary electrophoresis

Micellar electrokinetic chromatography (MEKC) was used to separate components of hop extracts. The separation of a sample of iso-α-acids by MEKC was better and faster than by an established HPLC method, giving <0.8% RSD on migration times and 5–10% RSD on peak areas. MEKC was also successfully used to separate the oxidation products of the α- and β-acids and thus to monitor the stability of hop products containing them. Furthermore, MEKC distinguished among samples of reduced iso-α-acids (rho-, tetrahydro- and hexahydro- derivatives).