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JOURNAL OF FOOD COMPOSITION AND ANAL YSIS 4, 304-3 14 (199 1)
Analysis of Anisated Fermented Grape Distillates
by Gas-Liquid Chromatography
A. GEAHCHAN,*” C. KHALIFE,~ P. CHAMBON,* AND R. CHAMBON*
*Laboratoire de Toxicologic, Faculte’ de Pharm acie, 8, avenue Rocke feller, 69373, Lyon, Franc e; and
YLaboratoire de Toxicologic, Faculte’ de Pharma cie, UniversitP Saint-Joseph, Bert& Lebanon
Received February 26, 199 1, and in revised form June 14, 199 1
Tw o gas-liquid chromatographic metho ds have been developed for the identification andquantitative determination of methanol and other volatile compounds in anisated fermented
grape distillates. Methano l, acetaldehyde, ethanol, propanol, isopropanol, ethyl acetate, butanol,isobutanol, and amy l + isoam yl alcohols were all resolved on a Porapak Q column, using tert-
butanol as internal standard. A column containing 15% EGA on 80- 100 mes h Supelcoport wasused for rapid quantitative estimation of anethole in the presence of anisaldehyde as an internalstandard. Confirmation of the presence of different constitu ents in anisated spirits was investigated
using a head-space injector coupled to a capillary gas liquid chromatograph. Analysis o f 30 com-mercial and 19 artisanal sam ples respectively obtained for a survey of anisated grape distillates
constituen ts in Lebanon revealed the absence of ethyl acetate , isobutanol, butanol, am yl, andisoamyl alcohols in 10 comm ercial samp les produced by manu facturers of high proof industrial
alcohol. Ten other comm ercial samp les showed methanol levels above the max imum allowableconcentration (100 mg/liter) defined by the European Econom ic C omm unity. o 1991 Academic
Press. Inc.
INTRODUCTION
Alcoholic beverages obtained from fermented grape juice and flavored by adding
aniseed and distilling again are largely consumed in Lebanon and to a limited extent
in the neighboring countries of the middle east such as Israel, Turkey, and Greece
where they are known as arak, raki, and ouzo respectively. Arak is the national alcoholic
drink in Lebanon. It is exported to different European and American countries. With
the continuing and rising popularity of anise-flavored grape disti llates, there has been
a corresponding increase in the brands available with wide price differentials. This
type of situation increases the possibil ities of the less expensive brands being illegally
substituted for the more expensive brands by some retail alcoholic beverage dealers.
Arak is subject to different falsifications, such as adding extract to industrial alcohol
obtained from fermented sugar cane or sugar beet juice. Imitations include any type
of distilled spirit to which aniseed extract has been added. Artisanal manufacturers
have also been using methanol instead of alcohol resulting in severe intoxications and
death (unpublished data).
Quantitation of volatile alcoholic congeners by gas-liquid chromatography was sug-
gested for the characterization of distilled alcoholic beverages. The distribution of
volatile alcoholic congeners was used by Schoeneman et al. ( 197 1) and Schoeneman
and Dyer (1973) to establish analytical profiles of classes of distilled spirits such as
bourbon, scotch, vodka, gin, and tequila. Few and incomplete data are available on
’ To w hom reprint requests should be addressed.
0889-1575/91 $3.00Copyright 0 1991 by Academic Press, Inc.
All rights of reproduction in any form reserved.
304
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ANALYSIS OF GRAPE DISTILLATES BY GLC 305
TABLE 1
LINEAR REGRESSION OF DIFFERENT COMPONENTS: INTERNAL STANDARD RESPON SE RATIO
vs COMPO NENTS CONCENTRA TIONS (n = 3)
component Concentration Peak ht ratio Correlation coefficient(g/L) I-
Methan ol 1-8 0.676 - 5.546 0.999
Acetaldehyde 1-8 0.508 - 3.953 0.993
Ethyl acetate 1-8 0.218 - 1.700 0.998
Propanol 1-8 0.528 - 3.873 0.999
Isopropanol l-8 0.510 - 3.998 0.999
Isobutanol 1-8 0.260 - 2.110 0.998
Butano l l-8 0.246 - 1.782 0.998
Amy1 + Isoamyl
alcohols 1-8 0.110 - 1.001 0.998
Anethole 1-6 0.840 - 4.928 0.999
Note . Peak height ratio = compon ent peak height/internal standard peak height. Internal standard, an-
isaldehyde for Anethole measu reme nt, terbutanole for all other comp onents.
the composition of anisated grape disti llates (Dagher and Ruhayem, 1975). Grape
brandy disti llates which are manufactured in a similar way to arak except for the
manner in which the flavor is added were reported to contain 70-300 ppm (w/v) ofpropyl alcohol, lo-250 ppm (w/v) of isobutyl alcohol, and 220-880 ppm (w/v) of
amyl alcohol (Guymon, 1970). A number of brandy samples from various parts of
the world have been analyzed by Askew and Lisle (197 1) who found propyl alcohol
to be present in the range of 20-l 3,320 ppm, isobutyl alcohol 10-1080 ppm, amyl
alcohol 40-3000 ppm, and methanol in the range of 20-8750 ppm (w/v). Bamett and
Einsmann (1977) reported the presence of isobutanol, butanol, amylol, and 1-hexanol
in grape brandy disti llates in the range of 3-36, 3-12, 10-40, and 7-57 ppm (w/v),
respectively. Other distilled beverages have also been analyzed for their fuse1 oil content
(Kahn and Blessinger, 1972; Martin and Caress, 197 1, Onishi et al., 1978).
No data are available on the anethole content of anise-flavored grape distil lates.Anethole, a primary constituent of anise oil, contributes largely as fuse1 oil constituents
to the consistency in flavor and quality of the final product which requires a suitable
and constant level of these constituents.
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306 GEAHCHAN ET AL.
0 4 8 12 16MI N
20 24
FIG. 1. Gas chromatog ram of ethanolic standard solution on Porapak Q containing 2 mg /ml of the
following constitue nts: (1) methanol, (2) acetaldehyde, (3) ethanol, (4) isopropanol, (5) propanol, (6) internalstandard let?-butanol, (7) ethyl acetate, (8) isobutanol, (9) butanol, and (10) amyl + isoamyl a lcohols.
Various methods have been reported for analysis and separation of fuse1 oils in
brandy disti llates (Kahn and Blessinger, 1972; Martin and Caress, 197 1; Schoeneman
and Dyer, 1973) and volatile compounds in wines (Reglero et al., 1986; Loyola et al.,
1988; Herraiz et al., 1989), and anethole in anisated liqueurs (Parker, 1974), using
gas-liquid chromatography and different stationary phases. This paper reports the
development of a new and rapid method for determining methanol, acetaldehyde,
ethyl acetate, fuse1 oils, and anethole in anise-flavored brandy distil lates. The method
was used to analyze a large number of samples and resulted in a full range of data
concerning commercial, and to a lessextent artisanal manufactured, anisated disti llates.
Apparatus
METHOD
Equipment specified is not restrictive; other suitable equipment may be used.
Gas chromatographs. (1) Pet-kin-Elmer Model Sigma 2B equipped with flame ion-ization detector. GC operating conditions: injection port, 19O’C; detector, 250°C.
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ANALYSIS OF GRAPE DISTILLATES BY GLC 307
0 4 8M,N12 16 20 24
FIG . 2. Gas chromatog ram of an&ted distilled spirit produced from fermented grape juice on PorapakQ. (1) methan ol, (2) acetaldehyde, (3) ethanol, (5) propanol, (6) internal standard terf-butano l, (7) ethylacetate , (8) isobutanol, and (10) amyl + isoamyl a lcohols.
Columns: (a) stainless steel, 1.8 X 2-mm i.d. packed with Porapak Q; carrier gas flow,
nitrogen, 30 ml/min, oven temperature, 165°C. (b) Glass column, 2 X 2-mm i.d.
packed with 15% ethylene glycol adipate (EGA) coated on 80- 100 mesh Supelcoport;
carrier gas flow, nitrogen, 25 ml/mm, oven temperature, 180°C. Computing integrator,
Perkin-Elmer Model LCI 100. Operating conditions: chart speed, 10 mm/min, atten-
uation, 1.
(2) Perkin-Elmer Model Sigma 2000 capillary gas chromatograph. GC column: fused
silica 50 X 0.32-mm i.d. CP Sil8B with 1.2~pm Iilm thickness. GC operating conditions:
temperature programmed as follows: initial, 45°C; initial hold time, 3 min; program-
ming rate, S”C/min (45-250°C); hold 2 min at 250°C; carrier gas flow, helium, 1 ml/
min. Detector: flame ionization, temperature, 300°C. Head-space sampler: Perkin-Elmer Model HS- 100 with the following operating conditions: sample temperature,
55°C; transfer temperature, 60°C; thermostating time, 60 min, pressurization time,
2 min; injection time, 8 min; withdrawal time, 0.2 min; ventilation and split mode
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308 GEAHCHAN ET AL.
O 2 4 ii
MI N
FIG . 3. Gas chromatog ram of anisated distilled spirit on glass column packed with EGA 15% coated onSupelcoport. (1) anethole, (2) anisaldehyde.
injection. Computing integrator Hitachi-Merck D 2000. Operating conditions: chart
speed, 10 mm/min; attenuation, 2.
Reagents
Unless otherwise noted, all the chemicals used in this work were analytical grade,
commercially available reagents (from Merck). Anethole and anisaldehyde were ob-
tained from Fluka Chemical Corp. They are supplied as pure grade chemicals with a
purity > 98% GC. Water was distilled and deionized. Ethanol + water solution (50
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ANALYSIS OF GRAPE DISTILLATES BY GLC 309
TABLE 2
ACETALDE HYDE, ETHYL ACETATE, AND VARIOUS ALCOHOLS CONTENT OF 30 ARAK SAMPLES
MANLJFAC~URED BY COMMERCIAL DISTILLERS“
20
ND
ND
Note. N D = not detected.
y The limits of detection were as follows: 5 mg/liter for methanol, acetaldehyde, isopropanol, and propanol;11, 8, 10, and 22 mg/liter for ethyl acetate, isobutanol, butanol, and amyl + isoamyl alcohols, respectively.
+ 50 v/v) was used for the preparation of internal and standard solutions. Tert-butanol
and anisaldehyde internal standard solutions were prepared at 400 and 800 mg/l,
respectively. Alcohols, aldehyde, and ester standard mixture were prepared and adjusted
to give a final solution containing 2 mg of each component per millili ter. The final
reference solution was stored in refrigerator and was stable for 15 days. Anethole
standard solution was prepared at 2 mg/ml. The solution was kept in the dark and
made fresh every 24 h.
Standardand SampleAnalysis
(a) Methanol,acetaldehyde,thyl acetate, nd use1oils determination.Quantita-tively transfer 1 ml aliquot of standard solution to a 5-ml glass stoppered test tube.
Add 5 ~1 of tert-butanol internal standard solution. Vortex and inject 2 ~1 into gas
chromatograph using Porapak Q as the stationary phase. To a l-ml an&ted distil late
sample in a 5-ml glass stoppered test tube add 5 ~1 of tert-butanol internal standard
solution and continue as above.
(b) Anetholedetermination.Quantitatively transfer a l-ml aliquot of standard so-
lution to a 5-ml glass stoppered test tube and add 5 ~1 of anisaldehyde internal standard
solution. Vortex and inject 1 ~1 nto the gas chromatograph using EGA as the stationary
phase and relative operating conditions described above. Repeat the same procedure
for sample analysis substituting standard solution with an an&ted brandy distillate
sample.
(c) Calculation.Determine methanol, acetaldehyde, ethyl acetate, and fuse1 oils
concentrations by measuring the area ratio of each analyte to tert-butanol of sample
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310 GEAHCHAN ET AL.
TABLE 3
ACETALD EHYDE, ETHYLACETATE,ANDVAR IOUSALCOHOLS CONTENT OF 19 AUK SAM PLE S
MANUFACTURED BY ARTISANAL DISTILLERS a
Note. ND = not detected.
(1The limits of detection were as follows: 5 mgJliter for methanol, acetaldehyde, isopropanol, and propanol;
1 I, 8, 10, and 22 mg/liter for ethyl acetate, isobutanol, butanol, and amyl + isoamyl alcohols, respectively.
and standard peaks. Determine anethole concentration by measurement of the area
ratio of anethole to anisaldehyde of sample and standard peaks.
Proof and Total Acidity Determination
Proof was determined by the AOAC hydrometer method (957.03), and total acidity
by the AOAC titrimetric method (945.08) (Dyer, 1990).
RESULTS AND DISCUSSION
Linearity
In order to study the linearity of the above method, concentrations of different
analytes in anisated fermented grape disti llates were assayed in concentrations from
1 to 8 mg/ml. Anethole levels were varied from 0 to 6 mg/ml. For each component,
response ratios were plotted vs analyte concentrations. Results of the least-squares
linear regression analysis (Table 1) indicated good linearity. Nonzero intercepts were
negligible.
Reproducibility
Within-day precision of the method was assessed by analyzing standard solution
samples containing all different constituents (including anethole) at 2 mg/ml of each.
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ANALYSIS OF GRAPE DISTILLATES BY GLC
TABLE 4
PROOF, ACIDITY, AND ANETHOLE CONTENT OF 30 ARA K S AMP LES
MANUFACTURED BY COMMERCIAL DISTILLERS
Commercial name Proof Acidity Anetho le( mg/lOO mL acetic acid ) ( g/L 1
311
Abi - chakerAbi - hai la
Abou - akl
Al - arz
AyoubBalaban
Chabat
DakachDawal i
Excel lent
FakraGergi Abi-raad
Ghantous & Abou-raadJeitaKef raya
KE3PSJXl
Khazen
KortbawiKsarak
LebrunLobos
Marechal
Mechaa lan i
Musar
NakadRayes Abou-kal i l
R if
S ibhe l i
S tephanTOLUI~
10 1 60 110 2 71 5
140 1080
96 2210
113 190790 476
11 010 810 3
10 0
12 296
10 188
10 211 0
12 2
10 010 3
10 6
10 497
90
10 7
10 196
10 7
98
8392
1669
13111320
1072 2.4
1192 3.51490 2. 2
476 2.4541 1.2
1549 2.41503 3.8
1847
2861
47 6
202636 0
26
66 1
333883 4
1625
25032980
83 0
59
2. 62. 6
2. 8
2. 4
2. 4
1. 82. 5
2. 7
3. 0
3. 0
2. 5
2. 1
3. 02. 3
1. 6
2. 0
2. 72. 3
2. 1
2. 9
2. 3
1. 31. 9
At this concentration, the method yielded for methanol, acetaldehyde, propanol, ethyl
acetate, isobutanol, butanol, amyl + isoamyl alcohols, isopropanol, and anethole coef-
ficients of variation (n = 6) of 6.7,5.7, 1.9,3.6, 1.3,0.93, 1.2, 1.5, and 3.0%, respectively.
Day-to-day precision was determined by six repeated analyses of the same samples
in three different days, The coefficients of variation (n = 18) were 9.2, 8.1, 3.1, 6.4,
1.3, 3.0, 3.0, 3.1, and 6.1%, respectively.
Limits of Detection
The limit of detection was calculated by assuming arbitrarily that a detector response
equivalent to 5 times the baseline noise was the minimum peak height that could be
appreciated with a reasonable confidence. Limits of detection were 5 mg/liter for
methanol, acetaldehyde, isopropanol, and propanol, 11, 8, 10, and 22 mg/liter for
ethyl acetate, isobutanol, butanol, and amyl + isoamyl alcohols, respectively.
Resolution of D@erent Constituents on Column Chromatography
Different column packings and stationary phases for the separation and quantitative
estimation of different fuse1 oil constituents of various brands of disti lled alcoholic
beverages were reported in the literature (Guymon, 1970; Barnett and Einsmann,
1977; Onishi et al., 1978). The use of these stationary phases resulted in an incomplete
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312 GEAHCHAN ET AL.
TABLE 5
ANETHOLE CONTENT OF 11 ARAK SAMPLES MANUFACTURED BY ARTISANAL DISTILLERS’
Samp le number Anethole ( g/L )
1 4.2
2 3.9
3 1.8
4 3. 1
5 1.9
6 3. 1
7 2.1
8 0.5
9 0. 9
10 1.8
11 3. 5
a Anethole level was not measured in al l ar t isanal samples. Rema ining samples have been destroyed by
artillery shellin g of Beirut.
separation of methanol and acetaldehyde in the presence of excess of ethanol. Better
results were obtained with Porapak Q for which high resolution of all constituents was
observed (see Figs. 1 and 2). Identity of compounds was confirmed using head-space
capillary gas-liquid chromatography. Ten millil iter standard and sample solutions
were run, respectively, and different peaks retention times were compared. Anethole
was rapidly separated and estimated using a glass column packed with 15% ethylene
glycol adipate coated on Supelcoport. No interferences were observed from volatile
constituents occurring in brandy distil lates purchased from artisanal distillers before
the final anisation step (Fig. 3).
Methanol, Acetaldehyde, Ethyl Acetate, and Fuse1 Oil Components in Commercial
and Artisanal Arak Samples
The samples analyzed in this survey were randomly drawn from anisated brands
which were commercially available in Lebanon from 1986 to 1989. One sample of
each brand was analyzed. Nineteen other samples were purchased directly from local
artisanal distillers producing anisated distilled spirits for their own use. The results of
the quantitative determination of the various alcohols, ethyl acetate, and acetaldehyde
in arak are presented in Tables 2 and 3. Methanol, acetaldehyde, ethyl acetate, and
fuse1 oil constituents are absent in 10 commercial samples produced by manufacturers
suspected of having used high proof alcohol for the anisation step.
Methanol, acetaldehyde, and propanol are present in all remaining commercial and
artisanal samples. Their levels vary from 9 to 540 mg/liter, 6 to 220 mg/liter, and 17
to 132 mg/liter, respectively, in commercial samples, and 141 to 1754 mg/liter, 34 to4 14 mg/liter, and 16 to 2 14 mg/liter, respectively, in artisanal distil lates. Ten com-
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ANALYSIS OF GRAPE DISTILLATES BY GLC 313
mercial as well as nineteen artisanal samples showed methanol levels above the max-
imum allowable concentration (100 mg/liter) defined by the European Economic
Community. Methanol and acetaldehyde levels are much higher in artisanal samples.
Highly efficient rectifying columns used by commercial distillers reduce their levels
appreciably as they are the most volatile components. Propanol is almost always presentin commercial samples. One commercial sample showed a high propanol level (760
mg/liter) and absence of all other constituents, due probably to the use of highly
contaminated industrial alcohol for the anisation step.
Ethyl acetate was present in 15 commercial samples and was almost always accom-
panied by fuse1 oil components of grape disti llates. Ethyl acetate, isobutanol, and amyl
alcohols levels varied considerably between samples analyzed ranging from 18 to 870
mg/liter, 13 to 523 mg/liter, and 7 1 to 1692 mg/liter, respectively, in commercial
samples, and 85 to 62 1 mg/liter, 40 to 1687 mg/liter, and 204 to 363 1 mg/liter, re-
spectively, in artisanal samples. The differences in the fermentation conditions such
as temperature and yeast strain, the disti llation technique, and the grape varieties arethe most important factors that control the levels of these alcohols. Artisanal and
commercial samples obtained from fermented grape juice show the same analytical
profile. One aspect of these useful parameters is their consistent presence in anisated
distilled spir its obtained from fermented grape juice. The applications of these param-
eters are obvious in quality control and forensic situations. Tables 2 and 3 show that
butanol has little application as a discriminating parameter. It is significant that it is
found in only two commercial and one artisanal beverage at 111, 400, and 412 mg/
liter, respectively.
The results of the quantitative determination of anethole in 30 commercial and 11
artisanal samples are presented in Tables 4 and 5. Anethole levels vary from 1.2 to3.8 g/liter and 0.5 to 4.2 g/liter, respectively. Higher variation levels are observed in
artisanal samples. It has been demonstrated in an earlier study that level variations of
anethole are in direct relation with the amounts of aniseed used in the anisation step
(unpublished data).
Total Acidity
Determination of total acidity was performed on commercial samples. Total acidity
varied from 26 to 3400 mg/lOO ml expressed as acetic acid. No significant variations
were observed between commercial samples produced from fermented grape juice
and high proof industrial alcohol. No relationship was observed between proof andindividual volatile components levels in beverage distillates.
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