analysis of grape distillates

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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



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.



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.




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



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




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



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.



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



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-




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.

REFERENCES

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DAG HER, S. M., AN D RUHA YEM , I. G. (1975). F use1 oi l and methanol content of Lebanese arak. J. FoodSci. 40, 9 17-9 18.

DYE R, R. H. (1990). Beverages, distilled liquors. In Oficial Metho ds ofAn alysis (K. Hehich, Ed.), 15th ed.pp. 690-707. AOAC , Arlington, VA.

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HERRAIZ, T., MARTIN-ALVAREZ, P., PEDRO, J., REGLERO, G., HERRAIZ, M., AND CABEZUDO, M. D.

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