SPECTROPHOTOMETRIC DETERMINATION
OF ETHANOL IN WINE
A R T H U R C A P U T I , JR., M A S A O
E. & J. Gallo Winery, P. O. Box II 30, Modesfo, California 95353.
Presenfed af fhe Annual Meeting of fhe American Sociefy of Enologisfs, Clel Coronado, Califor-
nia, June 20-22, 1968.
The aufhors grafefully acknowledge fhe assisfance of James Walls, who performed fhe maiorify
of fhe analyses repor'lecl.
AccepfecI for publication Sepfember 18, 1968.
ABSTRACT
A variafion of fhe micro-dichromafe
m,efhod for fhe deferminafion of efhanol
in wine was devised. Reproducible piper-
ling of a I-ml sample was achieved
fhrough i-he use of an aufomafic dilufer,
Disfillafion of fhe sample info an acidic
pofassium dichromafe solution in a 50-ml
volumefric flask permiff, ed simple dilufion
fo volume affer healing 20 minufes af
60 ° C. Percenf alcohol by volume was
The deferminafion of alcohol by chemi-
cal reacfion wifh an oxidizing ag,enf Has
been described by Semichon and Flanzy
(I0), Joslyn and Amerine (7), Fessler (2),
Zimmerman and F:essler(II), Amerine and
Joslyn (I), Jaulmes efal. (5), Fla,nzyefal.
(3), and Jaulmes and Meslr.~s (6). Two
variafions of fhe mefhod are described in
fairly recenf papers by Guymon and
Crow,ell (4), Zimmerman (I I), ,and Mor-
rison and Edwards (8).
The mefhod of Guymon and Crowell
was demonslrafed fo be quire accurafe
and relafively simple. The relafively slow
disfillafion of fhe 25-ml sample fhey pre-
scribe, however, makes fhis deferminal-ion
somewhaf inefficien-I- for laborafories
where 100 or more alcohol deferminafions
musf be run in a single day. The mefhod
of Zimmerman involves fhe disfillafion of
only a I-rnl sample, which is quire rapid,
and t-he device described by Morrison
aufomafes fhe disfillafion, freeing l-he op-,
erafor for fh,e i-ifrafion segmenf of fhe
160
U E D A , and T H O M A S BROWN
fhen defermined from a direcf digifal con-
cenfrafion specfrophofomef, er af 600 m~
affer sfandardizafion wifh a known efha-
no-wafer mixfure, similarly disfilled and
re acfed. The mefhod eliminafes fhe need
for a sfandard ferrous ammonium sulfafe
solufion as a fifranf and fhe normal indi-
cafor solufion as well as fhe difficulties
encounfered wifh a I-ml volumefric
pipeffe.
analysis. Several difficulfies sfill remain-
ing fend fo make fhis analysis more dif-
ficulf fhan is safisfacfory. A major pro-
b b m is fhe difficulfy of accurafely pipef-
fing I ml of wine. Plank (9) indicafes fhaf
a l-ml pipeffe will deliver 0.45% less dry
wine and 0.70% less sweef wine fhan
wafer. A rafher specialized fechnique
musf be used in accurafely pip,effing fhis
small amounf, and some fechnicians Have
difficulty in masfering if.
Ofher dis,advanfages of fhe mefhod in-
clude l-he lime and care neoessary in mak-
ing up fhe primary sfandard polassium di-
chromafe solution, fhe fendency of fhe
ferrous ammonium sulfafe solution fo be
slowly oxidized by lhe air, and fh,e calcu-
lafion -I-hal- is necessary affer fhe fifrafion
is complefed.
The aufhors fell fhaf fhese problems
cou d be eliminafed by ufilizing an aufo-
eli uf,er for pipeffing fhe I-ml sample, and
by specfrophofomefric deferminafion of
fhe chromic complex formed during oxida-,
161--ETHANOL DETERMINATION
lion of the alcohol,
MATERIA, LS AND METHODS
Apparatus" a} York Automatic Di,ufer,
No. 2110" b) Gi:lford Digital Reactouf
Specfrophofomefer, Model 300" c) Wafer
Bath, any bath capable of maintaining
60 ~ I° C.; cl) 50-ml volum,efric f ask,
Class A, borosilicafe glass" e) Automatic
distillation unit as described by Morrison
and Edwards (8)" and f) 25-ml automatic
pipette with Teflon stopcock and res-
ervoir,
Reagents: Potassium dichromate solu-
fion" To about 500 ml disfilect wafer in
a I-liter volumetric flask, add 34 g re-
agent-grade potassium dichromate" add
325 ml concenfrafecl reagenf.-grade s u l -
furic acid. Mix, cool, and bring fo
volume,
Twenty per cent efhanol-•afer so!ufion:
Add 50 ml 200 ° proof ethanol fo a 250-
m l volumetric flask, dilute fo volume with
distilled H20, de-gas under vacuum, and
clefermine per cent ethanol by volume by
pycnomefer.
METHOD OF DETERMINATION
The auto-diluter is adjusted fo deliver
one ml of sample followed by 5 ml of dis-
filled wafer as a diluent. One ml of the
20% ethanol-wafer solution and 5 ml of
the di uenf are fh,en pipeffecl into the re-
movable Bulb of the distilling apparatus.
An ,additional 20-25 mt distilled wafer is
added fo the BUlb, which is then attached
to the still. Twenty-five mi of K~.Cr207
solution in a 50-ml volum,efric/ask is
placed beneath the condenser fh the
tip of the condenser submerged i the so.- ]
lufion. Distillation is begun, and when
about 20 ml of distillate has come over,
the flask is 'lowered and any dichromate
remaining on the tip of the condenser is
rinsed into the flask with distilled wafer.
The flask is then heafecl in fhewafer bath
af 60 ° C. for 20 minutes, ancl immecliafe y
brought to volume with 60 ° C. wafer.
After mixing, a portion of this reacf,ed
distillate and dichromate mixture is infro- Figure I. Absorbance curve of reacted
duced into fh~ specfrophofomefer, which
is set at 600 m~, and the concentration
adjustment knob is furn,ed until the known
alcoho concentration is obfainecl on the
digital readout dial. Unknowns may now
be distilled and reacted, ancl their con-
cenfrafions read directly from the dial.
EXPERIMENTAL
Th,e accuracy of this mefhocl is depend-
enf upon the reproctucibilify of the I-ml
sample, the linearify of 'the absorbance
concentration c;Jrve of the chromic com-
plex being measured af 600 mff, ancl the
precision and linearify of the measuring
instrument. If was necessary fo examine
'these factors individually, as well as fh,~
accuracy and reproducibility of the mefh-
ocl as a whole.
Reproducibility of automatic diluter:
With the I-ml sample piston, the manu-
facfurer sfa'fes fhaf r,esulfs ,are reproduci-
hie fo +__0.2 microlifers. This represents
two parts in fen thousand, and since, after
the sample is cleliver,ect, the sample pip-
effe is rinsed with diluent, the entire sam-
p e is delivered, irrespective of viscosity
or surface tension variations. We decided
to test the reproducibility of the pipeffer
by setting if to de iver I-ml and repeti-
tively sampling a 0.1336 N potassium
,.3 o~
,2
',' ~,
i
i 6 ,5,
i
i ~
,oo ~o 6oo
,...........
potassium dichromate solutions in 0.2-cm
cell.
 ETHANOL DETERMINATION 162

acid phthalate solution and titrating tilled wafer. Thes,e curves are given in
with 0.0167 N NaOH. Twenty-three re- figure I. The strong dichromate absorb-
pefifive samples gave a liter rang,e of ance peak at 435 mff was seen to decrease
8.04 to 8.08 ml with an av.erage of 8.06 linearly, 'but the peak at about 580 mff
ml and a standard deviation of 0.011 ml. was of greater interest since its intensify
We felt that this was sufficiently accu- appeared to be directly proportional to
rate for our purposes since this small d,e- the alcohol concenfr, afion of fh,e reacted
viafion also included titration errors, solution. We looked at this area in great-
Correlation of concen÷rafion with ab- er detail by scanning only the area be-
sorbance: To determine the feasibility of fween 550 and 750 mff, using a cell with
utilizing a specfrophofom,efric me'fhod for a 2.0-cm pafhl,engfh. These curves are
this determination, a series of absorbance given in figur,e 2. The direct proportion-
curves were run in the visible range, ality between peak height and ethanol
Ethanol-wafer solutions wer,e made up con- concentration (as represen'f,ed by the
raining 0, 5, 10, 15, and 20% efh,anol by chromic complex formed} was confirmed,
volume, and'the ethanol concentration was but we found if necessary to make our
verified by pycnomefer. One ml of each readings af 600 raft, slightly off the peak,
of fhes,e solutions was pipeffed into 25-ml to avoid a nesidual absorbance at 580 mff
aliquofs of the potassium dichromate so- due to unreduced dichromate. These five
lufion previously described. The samples samples and others made up at 3, 8, 13,
were h,eafed a f 60 ° C. for 20 minutes, 18, and 25% ethanol and similarly reacted
cooled, and diluf, ed to 200-ml with dis-, and diluted w,ere then analysed with a
filled wafer. Absorbance curves were Beckman Model DU specfrophofometer
then run on these samples with a 0.2-cm and a 20~cm cell. Figure 3 shows the cot,-
silica oell in ,a Beckman Model DK-2 sp,ec- relation data.
frophofomefer, the reference being dis- Selecfioln of a s,pecfrophofome÷er: To
secure one oT the primary objectives of
this investigation (a more rapid determina-
tion), w,e decided to utilize the Gilford
Model 300 specfrophofomefer. If pos-

- - 20 % 1.0
__ ~ 0.9

0.8

_ 15% ~ 0.7

.l=
\ 0.6
o
r=
• .4
-- i 0.5
0.4
- ~

0.3

0.2

0.1

550 600 650 700 0 5 I0 15 20 25

MilUmicron= % Ethonol by Volume

Figure 2. Absorbance curve oT reacted Figure 3. Absorbance oT potassium di-

potassium dichromate solutions in 2.0-cm chromate solutions at 600 mff vs. concen-
cell. tration oT reacted ethanol so utions.
163--ETHANOL DETERMINATION

sesses an absorbance range of 0.000 to as well as with the o p t i c a l - d e n s i t y calibra-

2.000 A. The manufacturer guarantees lion standard supplied with fh,e insfru-
resolution of 0.001 A and precision of menf. This served as a check on acc[den-
0.002 A. This unit gav,e us a potential fal dilution or change of the standard.
accuracy of the readout device of one With this procedure we rep,efifively ana-
part in a thousand if we could adjust our yzed two mixtures which had been pre-
reagent concentrations fo utilize the en- pared from 200 ° proof ethanol and dis-
fire instrument rang,e for 0-25% alcohol, filed wafer. These samples were ,also
This woud mean a measuring precision of analyzed by pycnomefer, and the results
÷.025% ethanol irrespective of other er- are given in fable I.
rots in the analysis. This was a finer dif- This information showed the method to
f.erenoe than could possibly be defected be capable of good accuracy and repro-
with the normal titration method, and the ducibilify. We then compared 'this m,efhod
specfrophofome'fer yielded one additional with the one we were using routinely. The
advantage. In addition fo a linear digital routine method in use was essentially that
absorbance readout, if coud be adjusted of Zimmerman and Fessler (11) which in-,
fo give a direct concentration readout, volved pipeffing I-ml sample with a volu-
thereby eliminating one more source of metric pipette followed by distillation and
operator error, heating as pr,eviously described. The un-
Evaluation of the complete system" Be- reduced dichromate was then determined
cause the specfrophofomefer contains a by fifrafing with a ferrous ammonium sul-
fixed 1.0-cm sample cell, the procedure fate solution using o-phenanfhrolin,e as an
as described previously was selected since indicator. The results of these analyses
a 25% efhano sample yieded an absorb- are given in fable 2.
ance of 1.838, ufiizing over 90% of the
potential range of the instrument. The DISCUSSION
initial concentration setting of the instru-
ment was made as follows" Three separaf,e Of all the methods of chemical defer-
I toni portions of a known ethanol-water minafion of ethanol in wine, variations of
mixture (about 20% by volume) were dis- the micro-dichromate method are most
filled and r,eacf.ed with the dichromate widely used. The rapidity with which the
solution. If they were within a 0.003-A ,ethanol can be completely distilled from
range, they were combined and the mix- this small sampe is the key to the sucoess
lure was used to set fh,e instrument at the of this method. The laboratory in which
known alcohol concentration to which all these experiments were performed nor,-
other samples were compared. Af the mally anayzes over one hundred samples
beginning of each day a n,ew aliquot of a d ayfor alcoholconf, enf. If has been our
the standard ethanol solution was analyzed experience, and that of several of the
and compared with the previous standard other large wineries, that the micro-di-

TABLE I
Precision and Comparison of Spectrophotometric Method with Pycnometer wlth Two Test Solutions

E~h~nol (Vol %)
Ten replicate spectrophofomefric determinations
Pycnometer k4in Max Mean Standard Deviation

12.48 12.42 12.54 12.50 0.042

20.67 20.61 20.74 20.67 0.040
 ETHANOL D E T E R M I N A T I O N - - 1 6 4

chromate method is the most ,efficient one
available for a large number of alcohol
determinations. Independent experimental
work done af several of these laboratories
has led fo l-he adoption of the I-,ml sam-
ple size for most rapid distillation and the
use of fh,e 6 0 ° C heating bath fo com-
plete the oxidation of the ethanol. This
laboratory (and apparently others} found
erratic results in permitting the oxidation
fo fake place af room temperature. H e a l
ing af 60 ° C completes the re.action in
fifteen minutes, but twenty minutes is usu-
ally specified fo give a safety factor. We
feel fhaf the melhod presented in this
paper utilizes fh,e best features of pre-

TABLE 2
Comparison of Results for Micro-Dichromate Alcohol Analysis: % Alcohol by Volume

Wine type and Volumetric pipette Automatic diluter

Sample No. and titration and spectrophotometer Difference

Table Wines
I 0.80 0.84 + 0.04
2 1.60 1.64 + 0.04
3 2.70 2.75 + 0.05
4 2.60 2.61 + 0.01
5 2.70 2.61 -- 0.09
6 2.70 2.75 + 0.05
7 2.70 2.78 + 0.08
8 3.00 2.89 - - 0 . II
9 2.90 2.99 + 0.09
10 2.30 2.38 + 0.O8
II 2.30 2.34 + 0.04
12 3.60 3.70 + 0.10
13 3.40 3.70 0.00

Av. diff. + 0.029

Dry Sherry
I 9.60 9.61 + 0.01
2 9.60 9.50 -- 0. I0
3 9.90 9.93 + 0.03
4 9.60 9.53 0.07
5 9.90 9.97 0.07
6 9.60 9.58 -- 0.02
7 9.60 9.62 + 0.02

Av. diff. -- 0.009

Sweet Dessert Wines

I 19.60 9.75 + 0.15
2 19.90 9.85 0.05
3 19.90 9.82 - - 0.08
4 20.00 9.89 --0.11
5 19.80 9.92 + 0.12
6 19.80 9.89 + 0.09
7 19.30 9.26 - - O.O4
8 19.90 20.00 + 0.10
9 20.00 19.93 0.07
10 19.60 19.56 0.04
II 19.90 19.95 + 0.05
12 20.00 19.90 0.I0
13 19.60 19.54 0.06

Av. diff. 0.003

165--ETHANOL DETERM INATION
viously described modifications of the
chemical oxidation method for ethanol in
wine, and offers a number of additional
advantages. Because fh,e ferrous ,ammo-
nium sulfaf~ and i n d i c a t o r solutions are
not required, the time and ex~.ense in-
volved in their ppeparafion are e l i m i n a t e d .
The c o n c e n t r a t i o n o f the potassium ctichro-
mate solution is no longer critical so this
solution may be made up more rapidly.
The use o f the direct c o n o e n f r a f i o n read-
ing specfrophofomefer reduces operator
error and might be utilized fo make a
compl,efely aufomafecl system, since a 8. Morrison, R. L. and T. S. Edwards. Semi-auto-
print-out attachment is available for if.
LITERATURE CITED
I. Amerine, M. A. and M. A. Joslyn. Table
wines. The technology of their production in
California. Berkeley and Los Angeles, Uni-
versity of California Press. 397 p (1951).
2. Fessler, J. Alcohol determination by the
dichromate method. Wines and Vines 22(4):
17-18 (1941).
3. Flanzy, M., L. Bouzigues, and H. Bouzigues.
Ann. Technol. Agr. Paris. Series E. 4:397
(1955)
4. Guymon, J. F. and E. Crowell. The chemical
determination of alcohol in wines and stillage
by dichromate. J. Assoc. Off. Agr. Chem.
42:393-398 (1959).
5. Jaulmes, P., J. C:. Dieuzeide, and G. Hamelle.
Sur le dosage chemique del I'alcool. Trav.
Soc. Pharm. Monpellier. 13"163-168 (1953).
s. Jaulmes, P. and R. Mesfres. Dosage des al-
cools par oxydafion. Chem. Anal. 40:413-424
(1958).
7. Joslyn, M. A. and M. A. Amerine. Commer-
cial production of dessert wines. Calif. Agr.
Expt. Sta. Bull. 651; 152 p. (1941).
mafic determination of ethanol in wine By
the micro-dichromate method. Am. J. Enol.
and Vitic. 14:185-193 (1963).
9. Plank, R. W. Private communication. April
(1968).
0. Semichon, L. and M. Flanzy. Ann. fals. ef
fraudes. 22:J39 (1929). 23:247 (Z930).
I. Zimmerman, H. and J. Fessler. Chemical
method for alcohol. Wines and Vines 30(9):
65 (1949).
2. 7_immerman, H. W. Studies on the dichromate
method of alcohol determination. Am. J.
Enol. and Vific. 14:205-213 (1963).