their chemical specificity and, therefore, may well provide simple keywords for setting up reference files of low voltage Py-MS spectra of different nonvolatile organic or biological samples.
Finally we want to report an attractive procedure for data reduction of low voltage Py-MS spectra. Formally, this procedure is analogous t o that described by Crawford et al. (13). Its application to low voltage Py-MS, however, is based on the observation that complex pyrolyzates often contain typical series of homologous compounds (6, 9 ) , e.g., alkenes, ketones, amines, nitriles, and acids. The members of these series differ only by 14 mass units or a multiple of this. If the spectrum is now reduced to the summed ion intensities a t multiples of 14 mass units, the resulting condensed mass spectrum (see Figure 5) is perhaps best called “mixed series of homologous ions.” In our experience such condensed spectra still retain much of
ACKNOWLEDGMENT The authors gratefully acknowledge the valuable support and advice of J . F. K. Huber and A. J. H. Boerboom. Received for review October 18, 1972. Accepted January 2, 1973. Part of this work has been presented a t the Second International Symposium on Pyrolysis Gas Chromatography held on September 28 and 29, 1972, in Paris. This research is sponsored by the organization for Fundamental Research on Matter (F. 0. M.) and the Dutch Ministry of Health.
(13) L R Crawford and J D Morrison, Anal Chem , 40, 1469 (1968)
Gas Chromatography of Beer Bitter Acids Maurice Verzele, Eric Vanluchene, and Joseph Van Dyck Department of Organic Chemistry. State University of Ghent, Belgium
Gas chromatography (GC) of the trimethylsilyl derivatives of beer bitter acids was first introduced by Dalgliesh (2). Further developments are rather scarce (2, 3) and applications of the technique have not been reported. Informal discussions a t congress meetings revealed that several laboratories had trouble in duplicating the published results because of instability problems of the trimethylsilyl derivatives of the beer bitter acids. Indeed, packed column GC of these substances is only partly successful and this only in extremely optimized conditions. Even more important is the fact that the available resolution with packed columns is insufficient considering the complexity of the mixture involved. We therefore adapted open-tubular columns for this analysis. Most problems were overcome by using wide bore glass capillaries with on column injection in a gold capillary interphase and by adopting hydrogen as carrier gas a t rather high gas rate. There is a general tendency toward such solutions for capillary GC of labile compounds 14) and this approach to the methodology involved was published recently (5). Hops and Beer Bitter Acids. The bitter acids of hops are the so-called a and @-acids (I and I1 in Figure 1). These substances do not occur in beer; they are oxygen sensicive and are gradually oxidized to very complex mixtures which are even today not fully known. In the beer brewing process, hops is boiled with wort and the a acids are in this way isomerized into beer soluble cis and trans iso-a-acids (111).Since there are three major a acids (humulone, cohumulone,and adhumulone), there are six major iso-a-acids representing normally about 70 to 80% of the beer bitter substances. The other beer bitter substances are further transformation products of the iso-a(1) C. Dalgilesh, A . Mills. and S. Shaw, Biochem. d.. 101, 792 (1966). (2) E. Segel and R. Molyneux. Amer. SOC. Brew Chem.. Proc.. 280 (19711. (3) S. Shaw. Tefrahedron Left.. 2 6 , 3033 (1968). ( 4 ) R. Teranishi, T. Mon. A . Robinson P . Cary. and L. Pauling. Advafl. Chromafogr.. 135 (1971) ( 5 ) M . Verzele. M . Verstappe. P. Sandra, E. Vanluchene, and A . Vuye. J Chromatogr. S o . . 668 (1972).
acids (IV to VII) and oxidation products of the 6-acids. Important oxidation products of the 6 acids, because they occur in beer, are the hulupones (VIII). Transformation of a-acids into iso-a-acids and further breakdown to humulinic acids (VII) is catalyzed by alkali. In the normal brewing process at p H 5.4, the degradation of iso-a-acids is negligible and practically stops a t the alloiso-a-acids (IV) stage, but the utilization yield of a-acids is only around 25%. Industrial (alkaline) isomerization can increase this yield markedly, but there is the possibility that nonbitter acetylhumulinic acids (VI) or even humulinic acids (VII) are then formed. I t is therefore important to be able to analyze industrial isomerized hop extracts for these further transformation products. GC Derivatives of Bitter Acids. Hop bitter acids contain vinylogous acid and enolic and alcoholic hydroxyl groups which should all be derivatized to produce thermostable derivatives with sufficient GC volatility. Dalgliesh ( I ) reports unsuccessful attempts with diazomethane, methoxylamine, and trifluoroacetic acid anhydride and this we can indeed confirm. Better results are claimed with hexamethyldisilazane (HMDS) in pyridine or in dimethylformamide. Humulone on packed columns indeed produces apparently only a single derivative after trimethylsilylation in pyridine (not in dimethylformamide), but the five-membered ring compounds all give complex mixtures. Capillary column GC reveals that the humulone derivative is particularly labile and gives unreproducible and complex results. Butylboronic ester derivatives as described by Shaw (3) were also tried by us. Butylboronic acid and phenylboronic acid were both synthetized and obtained from Applied Science Laboratories but derivatization of the hop bitter acids, some of which have the suitable 1,3-dihydroxylic function, gave no useful results. We therefore studied derivatization of the hops bitter acids with different trimethylsilyl donors and in a wide range of possible solvents. While bistrimethylsilylacetamide (BSA) probably enolizes the bitter acids carbonyl functions and so produces a complex, unuseable mixture, trimethylA N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 8, J U L Y 1973
1549
Table I. Kovats Indices of Important Hop and Beer Bitter Compounds (For Structural Formulas see Figure 1) 200
Cohulupone Hulupone -I- adhulupone cis-Cohumulinicacid trans-Cohumulinic acid cis-Humulinic acid trans-Humulinic acid trans-Dehydrohumulinicacid trans-Acetylhumulinicacid cis- Acetylhumulinic acid
230"
Cohulupone Hulupone adhulupone cis-Cohumulinicacid trans-Cohumulinicacid cis-Hurnulinic acid trans-Humulinicacid Cohumulone Humulone trans-Dehydrohumulinicacid trans-Acetylhurnulinic acid cis-Acetylhumulinic acid trans- I socohurnulone cis- lsocohumulone trans- lsoadhumulone Colupulone trans- I sohumulone cis- lsoadhumulone Lupulone
2053 21 24 2142 2152 2208 221 6 2293 2353 2376
silylimidazole (TSIM) apparently does not react. Good results were then obtained for all bitter acids except humulone, with HMDS in dimethoxypropane (DMP) as a solvent. Reaction with the iso-a-acids is faster than with, for example, hulupones or humulinic acids and it is for the latter that heating is required.
+
I
2105 2166 2183 2184 2240 2242 2253* 231 8* 2330 2374 2396 2572 2597 2607 2626 2629 2652 2682
I
EXPERIMENTAL The developed standard procedure for derivatization of beer bitter compounds is as follows: 0.2-2 mg of the acid, in a well closed small test tube, is treated with 150 ~1 of DMP and 100 p1 of HMDS, and the mixture is heated at 50" in a water bath for 3 hr. A sample (1~ 1e . g . ) of the solution is injected as such. GC Analysis of Beer Bitter Acids. Beer (250 ml) is acidified weighed in a 30-ml test tube. Methanol ( 5 ml) and isooctane (10 ml) are added. Hydrochloric acid 10 ml 0.2N is added stepwise with intermittent shaking. One milliliter of the isooctane is pipetted in a derivatization tube and the solvent is removed by blowing nitrogen over it; this takes about 10 min. After solvent removal, the standard derivatization procedure is carried out on the residue. Gc Analysis of Beer Bitter Acids. Beer (250 ml) is acidified with hydrochloric acid ( p H 2) and is extracted with isooctane (1 1.). The isooctane extract, 750 ml, is evaporated in cucuum to about 10-20 ml. The residue is transferred to a pear shaped distillation flask of about 20-30 ml and evaporated in vacuum without shaking. This procedure concentrates the residue in the pear tip and derivatization is carried out in the flask. Gas Chromatographic Specifications. Packed columns as used by Dalgliesh ( 1 ) do not produce sufficient resolution. Extensive deterioration of the silyl derivatives also occurs. Therefore a glass capillary column procedure was needed. Gas chromatographic specifications are then: glass capillary column about 0.8 t o 1 m m i.d. and 60-85 m long; OV-1 is the preferred phase with a coating solution of 4 mg/ml for the static coating technique previously described by our laboratory 16. 71; injection is on column in a "gold capillary interphase" adapted to a Varian 2100 gas chromatograph with FID; the hydrogen flow rate is usually around 10 to 20 ml/min.
RESULTS AND DISCUSSION Some results shown in Figures 2 and 3 are chromatograms at 230 "C of an unpurified and purified isomerized hop extract, respectively. Figure 4 compares chromatograms of beer bitter hop acids with an isomerized extract (at 200 "C; part before ( 2 2 4 ) . (61 J. Bouche and M . Verzele, J . Chrornatogr. Sci.. 501 ( 1 9 6 8 ) . ( 7 ) T. Boogaerts, M . Verstappe. and M . Verzele, J . Chrornatogr S c i , 217 ( 1 9 7 2 ) .
1550
ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, JULY 1973
m
rn
PT
Figure 1 . Important hop bitter acids: ( I A ) humulone; ( i B ) cohumulone; ( I C ) adhurnulone; ( I I A ) lupulone; ( I le) coiupulone: i l l c ) adlupulone; ( I l l ) cis and trans refers to the 4 - O H and 5alkenyl side chain. For the other compounds see the text
Kovats Indices of Hop Bitter Compounds. Kovats index values were deduced using eicosane, tetracosane, and octacosane standards. The indices of the compounds giving suitable peaks are shown in Table I. More volatile derivatives are better analyzed at 200 "C for correct identification. In these conditions, the iso-aacids are not eluted. For the iso-a-acids a temperature of 230 "C is needed, but then the precision of the identification of the more volatile substances is inadequate. As can be seen in the table, the retention index is rather strongly temperature dependent. Humulone and cohumulone are not eluted a t 200 " C . A t 230 "C they give irregular results and their indices of the table (starred) are for the major peak on a broad hump. Relatively large amounts of humulone and cohumulone are needed to give a detectable peak. Undoubtedly decomposition is quite extensive with these derivatives. Humulinone does not produce any GC peaks at all. Hydrated iso-a-acids give very broad humps in the iso-a-acids region. Alloiso-a-acids give broadened
~~
Figure 2. Chromatogram of unpurified isomerized hop extract at 230 "C. For other conditions, see text. The last major peak before C28 is lupulone
Figure 3. Chromatogram of purified isomerized hop extract at 230 "C. For other conditions see text (about 2-3 times too broad for index determination) skewed peaks just before the isohumulones (index k2620). For the peaks between eicosane and tetracosane (20002400), the precision of the index determination is best a t 200 "C and for the peaks obtained a t 230 "C the precision is best for the peaks with index above 2400. The standard deviation for the same column and instrument and for a consecutive series of determinations (10 analyses in 3 days) is around 0.05%. For determinations on two different instruments, four columns, and spread in time over 3 months, the standard deviation is around 0.1%. Considering the small difference in some of the Kovats index values, absolute identification of peaks can be difficult and requires repeated analyses. Indeed standard deviation of 0.1% means 2 to 3 index units absolute in this case and peak widths are also about this size. The analysis a t 230 "C can be run several hundred times on one column without apparent column deterioration. In the specified conditions, the C28 peak produces about 40.000 plates for an 85-m column. Some Applications of the GC Analysis of Hop Bitter Acids. The ideal of complete quantitative analysis of all hop bitter acids and their transformation products is not possible with GC. Most derivatives are slowly decomposed on the column as can be deduced from the unstable base line, tailing, and the well known phenomenon of the higher base line after a peak, dropping after some time quite suddenly to the original position. The FID response is also unstable and suffers from nonlinearity because of silica deposits a t the flame tip. A large excess of silicium-con-
-t
N -¶
u
I
Figure 4. Chromatogram at 200 "C of isomerized extract (lower) compared with beer bitter substances (upper).The extra peaks before C20 in the latter indicated by an arrow come from the beer matrix ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, J U L Y 1973
1551
taining HMDS is indeed introduced with each injection. Ways to improve this appear to be obvious, but so far experiments in this direction were unsuccessful. Another negative point is that from the very complex oxidation mixtures of humulone and lupulone, only two or three substances produce derivatives which can be chromatographed. It is known that those oxidation products occur in beer. The analysis discussed in the present paper can therefore only give qualitative results for part of the hop bitter acids; it happens, however, it is the most important and most bitter part. Furthermore, unhopped beer, prepared with yeast which has never been in contact with bitter acids, analyzed by the procedure described above, produces a small number of not very important peaks. Their Kovats index a t 200 “C in the conditions of Figure 2 is 1824, 1864, 1931, 1963, and 2155, respectively. These peaks are indicated with an arrow in the chromatogram of Figure 4. These substances obviously come from the malt or other carbohydrate material used in brewing. Against these drawbacks, the new GC procedure allows the study of a number of problems about the bitter acids of beer and isomerized hop extracts. Examples of this are as follows. A. According to a recent theory (8), cohumulone could be a negative hop quality factor. This question is far from settled, but analysis of the beer bitter compounds allows a t least an evaluation of the relative contribution of cohumulone (relative peak heights of the peaks for cis-isohumulone and cis-isocohumulone in Figure 3). B. Another important question is whether older hops or nonisomerized hop extracts (with little or no a-acids) contain iso-a-acids or if their remaining bittering power is due to other substances. This point is also important because the conductometric routine a-acids determination is known to give high results for older (oxidized) hops. A slightly different way to put the above question is therefore: Is the conductometric brewing value of older hops partly due to iso-a-acids or not? (8) F. Rigby. Amer SOC. Brew C h e m . , P r o c , in press
1552
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 8, J U L Y 1973
To answer this question, older hops and an extract showing clear symptoms of oxidation were extracted, dissolved, and the possible iso-a-acids first separated from the bulk material by 300 transfer counter current distribution in the usual phase system (9). The cells which should have contained the iso-a-acids band were collected and worked-up to produce the material for GC analysis. Only traces (less than 0.1%) of iso-a-acids could be detected. The main bittering substances in these hops seemed to be the hulupones. C. Commercial isomerized hop extracts are prepared by alkaline treatment of hop extracts and, as indicated in Figure 1, this can easily lead to humulinic acids. These nonbitter reaction products easily escape routine photometric analysis and it is therefore obvious that the presence of humulinic acids in an isomerized hop extract is a negative quality factor. Humulinic acids can be detected by counter current distribution and thin layer chromatography but for small concentrations this is rather difficult if not impossible. Adding either pure cis- or trans-humulinic acid in the concentration range 1 to 15% to isomerized extracts produces appropriate GC peaks for which the heights relative to the tetracosane standard give a straight line in a chart: relative height us. added humulinic acid amount. This GC procedure was applied to the six commercial isomerized hop extracts now available and astonishingly none of these contains humulinic acids, but “normal” beer, prepared from hops, does contain traces of humulinic acids. D. Other obvious applications of the technique are: purity and identity check for individual bitter acids in the research laboratory, and finger printing of isomerized hop extracts.
Received for review November 29, 1972. Accepted January 30, 1973. Heineken Breweries Rotterdam are thanked for financial assistance to our hop research program. (9) M. Verzele, H. Claus, and J Van Dyck, J Inst. Brew.. /London), 73, 39 (1 967).
