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Gas-Liquid Chromatographic Determination of Ethanol in "Alcohol-Free"

Beverages and Fruit Juices

A. M. Morad* / A. H. Hikal / R. Buchanin


Department of Pharmaceutics, College of Pharmacy, Riyad University, Riyad, Saudi Arabia

Key Words
Alcohol-free beverages
Gas-liquid chromatography

Summary
h rapid, sensitive and direct method is described for the
determination of ethanol in "Alcohol-Free" beverages
and fruit juices. A known amount of internal standard
is added to the sample and a 0.1 mm 3 aliquot is injected
directly into a gas chromatograph. Ethanol concentrations as low as 0.005 % v/v can be detected and quantitatively determined. Five brands of beer, seven brands
of grape juice and four brands of apple juice; all labelled
"Alcohol-Free" were analyzed. Ethanol in concentrations ranging from 0.009 % v/v to 0.385 % v/v was found.
These findings indicate that these products are not
actually "Alcohol-Free" but alcohol-low or alcoholpoor.

tains ethanol in concentrations of 0.5-2.5% v/v. Another


procedure is described for the preparation of non-alcoholic
beer that involves heating beer at 55 ~ under a pressure of
2 kg/cm 2, followed by spraying under a reduced pressure
of 1 mmHg [4]. This procedure results in a beer containing
0 . 2 - 1 % v/v ethanol.
Several methods are reported for the quantitative determination of ethanol in liquor and pharmaceutical preparations. A procedure involving the distillation of ethanol and
measurement of the specific gravity or refractive index has
been described [5, 6]. Another procedure involving the
redox-titration of the distillate is also described [7]. A gasliquid chromatographic procedure for ethanol determination in liquid preparations containing less than 10 % v/v is
reported [6, 8-10]. The aim of the present study is twofold: (a) development of a highly sensitive gas-liquid chromatographic procedure for the direct determination of
ethanol in beverages and fruit juices (in concentrations as
low as 0.005 % v/v); and (b) assessment of the validity of
the labelled claim that these products are actually "AlcohoIFree".

Materials and Method


Introduction
In recent years, beverages and fruit juices carrying the label
"Alcohol-Free" have appeared on world markets. Because
of this labelling, it is understood, or assumed, by the consumer that these products are absolutely free from alcohol.
A literature survey of the manufacturing procedure used in
making these products, indicated that they are actually
alcohol-low or alcohol-poor beers and juices. Alcohol-free
beer is made either by brewing a mixture of water, malt and
hops, a process in which fermentation is prematurely stopped', or by reducing the alcohol content of normally produced beer. In the latter case, reduction in alcohol content
is achieved by reverse osmosis [1], distillation of alcohol
under reduced pressure [2], or by either process coupled
with appropriate dilution with low-alcohol, strongly hopped
beer [3]. The beer produced by any of these processes con-

0009-5893/80/03 0161-03 ~ 02.00/0

All samples were stored at 4 ~


temperature prior to use.

but brought to room

Gas Chromatography
A Pye Unicam series 204 gas chromatograph with a flame
ionization detector was used. The conditions were as
follows: column, 1.5 m 4 mm glass column packed with
10 % polyethylene glycol 20 M on 100-120 mesh diatomite
MAW; oven, 150 ~ (isothermal); injector, 175 ~ detector,
200~
carrier gas, nitrogen 3 0 c m 3 rain -1, hydrogen
33 cm 3 rain -1, air 3 3 0 c m 3 rain -1 ; chart speed 1 0 m m
rain -~ . Retention times; ethanol 42 s amyl alcohol 90 s.
* Riedel-De Haen A G , Sleeze-Hannover, FRG
** E. Merck, Darmstadt, FRG

* To whom enquiries should be addressed.

Chromatographia Vot. 13 No. 3, March 1980

The "non-alcoholic" beverages and fruit juices were obtained


locally. The amyl alcohol* and absolute ethanol** B.P.
were of chromatographic grade.

Originals
(9 1980 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH

161

Procedure
3.5

Two standard curves using absolute ethanol were prepared


viz., 0.01-0.1 (%v/v), 0 . 1 - 1 . 0 ( % v / v ) each with its appropriate concentration of amyl alcohol as internal standard, 0.05, 0.5, respectively, as this would provide more
accuracy in measuring traces of ethanol. An 0.1 mm 3 sample
was injected and the most suitable attenuation selected.
This attenuation was compared with those used for the
standard curves, and the concentration of internal standard
to be used was determined. The appropriate amount of
internal standard was pipetted into a 100 cm 3 volumetric
flask and diluted to volume with the sample. The flask
was inverted 3 - 4 times and allowed to stand for 10 rain;
care was taken to prevent excessive frothing; four or five
injections of 0.1 mm 3 were injected. Eight samples of each
brand were analyzed.

13

3.0

-2

~: 2.0

-g

1.0

Results and Discussion


In the case of ethanol, the best internal standard is another
alcohol. Amyl alcohol was chosen after examining methanol,
isopropanol, and n-butanol, as its retention time is sufficiently long to give good peak separation and yet a short
analysis time. Fig. 1 shows the chromatogram and indicates
the retention times for ethanol and amyl alcohol.

Fig. 1
9 Chromatogram showing detector
responsel Column temperature:

150 ~ isothermal; injector: 175 ~


detector: 200 ~ The indicated
peaks are: a ethanol; b water;
c amyl alcohol,

o
rime (min)

Sharp, symmetrical peaks were obtained enabling peak


height measurements to be used with as much accuracy as
peak areas (Wesselman, 1960).
The standard curves were obtained by accurately measuring
the peak heights of ethanol and internal standard, calculating the ratios and plotting against percentage ethanol. Fig. 2
shows the straight line relationship between peak height
ratios and ethanol concentration. The slopes of the least
squares lines for both concentration ranges viz., 0 . 0 1 0 . 1 % v / v and 0 . 1 - 1 % v/v; were calculated using a programmable calculator* and values of 29.62 and 3.29 were
* Texas Instruments, Programmable Calculator, ModellT159,
Texas USA.

162

/
i

0.01 0.02

Fig. 2

0.04

0.06
0.08
% Ethanol

0.10

0.12

9 Calibration curve for ethanol determination.

obtained, respectively (correlation coefficient 0.998 for


both concentration ranges). Two standard solutions from
each standard curve were analyzed daily as a check on
instrument performance. From the standard curves, the
concentration of ethanol in the samples was determined
using the equation:
Ethanol % (v/v) = peak height ratio X slope -1
The described procedure has the advantages of speed and
accuracy since samples are directly analyzed without any
prior treatment. Furthermore, ethanol concentrations as
low as 0.005 % v/v are detected and quantitatively deter.
mined.
Tables I and II show the results obtained from the analysis
of "Alcohol-Free" beer and fruit juices. Ethanol was found
in all the beverages analyzed in concentrations ranging from
0.009-0.385 % v/v. Though these concentrations are relatively low compared to regular beer or other alcoholic
beverages, this is contrary to the claim that these beverages
are "Alcohol-Free". The presence of ethanol in these be.
verages can possibly be attributed to the inefficiency of
the alcohol removing technique. This is evidenced by the
data in Table I, where two brands showed ethanol concen.
trations as high as 0.4 % v/v while others showed concentrations aslow as 0.009 % v/v. It is also possible that ethanol
might be formed after packaging and during storage if
pasteurization is inadequate in destroying microorganisms.
Data in Table II showed that fruit juices contain ethanol in
concentrations ranging from 0.02 to 0.146 %v/v. Since
fruit juices are not subjected to fermentation, the presence
Chromatographia Vol. 13 No. 3, March 1980

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Table I. Ethyl alcohol content of "Alcohol-Free" beer

Brand*

A
B
C
D
E

Container
volume
ca 3

Mean alcohol
content**
(+ S.D.)

0.368
0,059
0.009
0.009
0.385

(+ 0.056)
(+ 0,0007)
(-+-0.0024)
(+ 0.0002)
(+ 0.026)

Volume of
alcohol per
container
cm 3

330
330
330
330
355

of ethanol indicates that pasteurization was inadequate.


However, further work is needed to study the effect of
storage conditions on ethanol content.
The amount of ethanol per container could be substantial
if ethanol concentration reaches 0.4 % v/v or higher, particularly in "Alcohol-Free" beer.
In conclusion, the present investigation revealed the presence of ethanol in beverages labelled "Alcohol-Free".

1.22
0.20
0.03
0.03
1.37

References
Brand names have been omitted for obvious reasons.
** % v/v EtOH,
*

Table II. Alcohol-content

of "Alcohol-Free" grape and

apple juices
Brand*

Mean alcohol
content**
(+ S.D.)

Container
volume
cm 3

Volume of
alcohol per
container
cm 3

I. Grape juice
A
B
C
D
E
F
G

(+ 0.005)
(+ 0.004)
(-+ 0,003)
(+ 0.001 )
(+ 0,0003)
(-+ 0.002)
(-+ 0.006)

730
1000
750
700
1000
730
750

0.68

0.019 (_+0,001)
0.013 (-+ 0.0006)
0.03 (-+ 0.004)
0.02 (+- 0.008)

1000
1000
210
700

0.19
0.13
0.06
0.14

0.093
0.146
0.093
0.054
0.022
0.031
0.083

1.46

0.70
0.38
0.22
0.23
0.62

II. Apple juice


A
B
C
D

111 P. K, Wucher and S. Neubert, Brauwelt 43, 1422 (1976).


121 S. Masio, T. Kuchciak and J. SurminskL Przem. Ferment.
Rolny. 20, 2 (1976).
131 tr Kleber and iV. Hunes, Chem. Abst. 83, 204823 u (1975).
14] Sociedale Central de Cervejas S.a.r.1. Spain, through Chem.
Abst. 86, 187670j (1977).
I51 Official Methods of the Association of Official Analytical
Chemists, Association of Official Analytical Chemists,
Washington DC., 1975, pp. 153.
[61 United States Pharmacopeia, 19th Rev., Mach Publishing Co.,
Easton, Pa., 1975, pp. 634-635.
[71 Official Methods of the Association of Official Analytical
Chemists, Association of Official Analytical Chemists,
Washington DC., 1975, pp. 194.
181 1-1. P. Warrington and J. M. Lennon, J. Pharm. Sci., 56, 404
(1967).
191 H.J. Wasselman, J. Pharm. Sci. 49, 320 (1960).
I101 Official Methods of the Association of Official Analytical
Chemists, Association of Official Analytical Chemists,
Washington DC, 1975, pp. 328.
Received: Aug. 23, 1979
Accepted: Sept. 14, 1979
C

* Brand names have been omitted for obvious reasons.


** % v/v EtOH.

Chromatographia Vol. 13 No. 3, March 1980

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