September, 1938] 425
A COLORIMETRIC METHOD FOR THE DETERMINATION OF
DISSOLVED OXYGEN
Bv Hakold Rothchild and Irwin M. Stonk
In recent literature dealing with the influence
of air in beer, DeClerck {Modern Brewer,
1937, 18, No. 6, 38; 1938, 19, No. 1, 49;)
M. Wallerstein, Commun. Sci. Practice Brew
ing, 1937, No. 1, 5; this Jmirn., 1938, 111),
there has been a growing realisation that,
at best, the rH concept and measurements,
even if applicable to beer, can prove only of
limited assistance in studying the problems
connected with the loss of stability and
other changes through oxidation in beer.
This has served to emphasise the need for
a simple, accurate, analytical method capable
of permitting the determination of the
concentration of dissolved oxygen in beer.
It was visualised that if a method, which
would not require special skill and com
plicated equipment, and, at the same time
would yield fairly accurate results as to
oxygen concentration were available, it
' could be of considerable value both as a
research tool and as a control measure.
The practical effeots of oxygen (air) on
beer have been amply demonstrated. Methods
for studying the rate at which such oxidation
takes place for different sets of experimental
conditions are required as a basis for further
light on the problem.
With a method available for the determina
tion of oxygen, the rate at which oxygen
disappears can be measured and in this way
data can be accumulated as to the influence
of time, temperature, pa value, composition,
etc., on the oxidation reactions involved.
For control purposes, such a method could
be utilised to estimate the oxygen content
of beer in different tanks at different lovels
and under varying conditions as a basis for
establishing uniformly the most satisfactory
conditions possible.
It is the purpose of this paper to describe
a procedure as worked out in our laboratories
which is particularly adapted for doter-
mining dissolved oxygen content of beer as
drawn from tanks. At present, the only
methods available employ a gasometric
technique in which all the gas is evolved
from the beer and then resolved into its
components by the usual gasometric pro
cedures (c/. Murray, this Journ., 1925, 137;
IN BEER
Helm and Richardt, ibid., 1930, 191;
Siegfried, Schweiz. Brau.-Bund., 1937, 48, 1;
this Journ., 1937, 128; DeClerok, Bull, de
VAssoc. Anc. Elud. de I'Ecole Sup. de Brass,
de I'Univ. de Louvain, 1937, 53, 175; this
Journ., 1937, 350; 1938, 59).
These methods require rather complicated
apparatus, they are time-consuming and
contain inherent errors which may affect
their accuracy for measuring small quantities
of dissolved oxygen. There may also be
cited the paper by Gray and Stone (J. Ind.
and Eng. Chem.* Analyt. Edn., 1938, 10,
15; this Journ., 1938, 171), from our labora
tories, in which apparatus and methods for
transferring evolved gas from beer and
subsequently analysing the mixture of gases
so evolved are referred to.
Considerable work has been done on the
determination of dissolved oxygen in water.
Various procedures have been employed,
such as the titrimetric alkaline MnSO4
method, (Winklor Ber., 1888, 21, 2843);
the cuprous ammonium chloride method,
(Frankforter, Walker and Wilhoit J. Amer.
Chem. Soc, 1909, 31, 35); the colorimetric
methods utilising the photographic developers;
Adurol (Winkler, Z. angew. Chem., 1911,
24, 341); and Amidol, (McCrumb and Kenny,
(J. Amer. Water Works Asm., 1929, 21, 400);
and Linossier's phenosafranine and alkaline
tartrate method, Miller (J. Soc. Chem. Ind.,
1914, 33, 185). All of the above methods
are inapplicable to beer because of the
presence of large quantities of organic matter.
Pasteur {Etudes sur la Biere, 1876, p. 277)
refers to the method employed by Schiitzen-
berger and Gerardin {Compt. rend., 1872,
75, 879) in which the oxygen was titrated
with a solution of sodium hydrosulphite,
using aniline blue as an indicator and taking
precautions to prevent interference by
atmospheric air. Tho hydrosulphite was sub
sequently standardised against ammoniacal
CuSO4. Pasteur modified this method and
used it for determining dissolved oxygen in
beer and wort. He substituted indigo
carmine for the aniline blue as an indicator
and standardised the hydrosulphito against
distilled water saturated with air. The
426 kothciiild and stone : dissolved oxyqen in beer [September, 1938

dissolved oxygen content of the water at of bottle used, no further calculations are
the temperature employed was calculated necessary.

from Bunsen's tables. Pasteur found it
necessary to neutralise the wort or boor
with ammonia before titration. The entire
titration was carried out in an atmosphere of
hydrogen. Apart from the cumbersome
apparatus and unwieldy technique employed,
this method has been criticised by DeClerck
(toe. cit. 1937) because of the likelihood that
the hydrosulphito, especially at the higher
pn, reacts with substances other than oxygen
in the beer.
Under the conditions of this test, it has
been found empirically that 1 molecule of
oxygen is required to oxidise one molecule
of the leuco indigo disulphonate. With an
excess of the leuco base present, therefore,
32 grms. of oxygen would yield 466 grms.
of oxidised indigo disulphonate. This is borne
out by the results in Table I.
Tabu: I
Production of Dye by Oxygen

Efimov (Biochem. ZeiL, 1925, 155, 371),

while studying the physico-chemical con Mgrm. 0, in r> t-
Ratio Dve
pf—
stants of cultures of protozoa, used a method sample of H,0. Mgrms.
indigo
for determining the oxygen content of the
(Determined by disulphonate
culture in which indigo carmine was con Winkler method). produced. Mgrms. Mofe.
verted to its leuco base by reduction with
glucose and potassium carbonate for 24 hours. 0-251 3-78 16-1 103
0149 2-08 14-0 0-97
On adding the leuco base to the solution to
0-166 2-27 13-7 0-94
be tested, the indicator was oxidised by the 0-247 3-59 14-5 1-00
dissolved oxygen. The colour produced 0-577 8-31 14-4 0-99
was compared with standards which were 0-459 6-61 14-4 0-99
standardised against the Winkler method.
The chief drawback of Efimov's method is
the character and amount of the reducing Method for the Determination of
agent used in convert the dye into its leuco Dissolved Oxygen in Beer
form. The action of his reducing agent is
much too slow for practical purposes, and Reagents—(1) Indigo Disulphonate.—The
it is present to excess in his reagent. Further, purified assayed dye may be readily obtained.
his method required that the dye solution Ifit is found necessary to standardise the in
be standardised against the Winklor method digo disulphonate, anyofthe standard methods
before use. may be employed (see Methods of Analysis,
Briefly, the method as developed by us 4th edn., Association of Official Agricultural
consists in bringing an indigo carmine solution Chemists, 1935, 255-6; Allen's Commercial
exactly to its leuco form by a strong reducing Organic Analysis, 5th edn., 1927, Vol. 5,
agent such as sodium hydrosulphite. The leuco 368-71). The concentration of the dye
indicator is then added in excess to the sample solution should be adjusted so that 1 ml. in
which has been collected in a suitable bottle. the particular volume used is equivalent to
Simple anaerobic conditions are observed the colour produced by 1 part oxygen per
throughout the test. Reaction then takes million. The formula for calculating the
place between the dissolved oxygen and requisite dye concentration is: Grms. per
the leuco base, the amount of dye regenerated 100 ml. indigo disulphonate
being dependent upon the amount of oxygen 466 net volume (ml.)*
originally present in the sample. The colour
3200 per cent, assay of dye.
produced ranges from a light green through
intermediate shades of blue to a deep violet,
Example.—If net volume of bottle = 130
depending on the oxygen concentration.
ml. and assay of dye is 91 per cent., then
Comparison is then made with standards
prepared by adding varying quantities of an i^X *g « 0-208 grm. per 100 ml.
indigo carmine dye solution to distilled
water in bottles similar to those used in the
* Net volume represents the total volume of the
determination. By adjusting the concen
bottlo minus the volume occupied by the rubber
tration of the dye so that 1 ml. equals 1 part stopper and tho volume of the boor displaced by
oxygen per million for the particular size the indicator solution (5 ml.).
September, 1938] bothchild and stone: dissolved oxygen in beer
Triturate in a mortar the requisite amount
of dye, as calculated above, with distilled
water and dilute to 100 ml. It is advisable
to prepare a fresh solution every few days.
(2) 5 per cent. Sodium Hydrosulphite.—
Dissolve approximately 2-5 grms. of sodium
hydrosulphite in 50 ml. ofwater. This solution
is unstable and should be prepared imme
diately before using.
(3) Reduced Indigo Disulphonate.—Cover a
portion of the above indigo disulphonate
solution contained in a small flask or bottle
with a one-inch layer of paraffin oil. Fill a
Pro. 1.
pipette with the sodium hydrosulphite solu
tion, insert the tip below the oil and titrate to
a yellow tint. Fifty ml. dye requires approxi
mately 0-8 to 1*0 ml. of the hydrosulphite
solution. Titrate the reduced dye solution
to a faint pale green colour with the un
reduced dye. The adjustment of the colour
to the pale green tint with the unreduced
dye should always be made immediately
before using.
Apparatus.—The bottle as shown in
Figure 1 is rectangular-shaped with dimen
sions of 2 in. X If in. X 4 in. (to neck).
427
Contents with stopper total 135 ± 1 ml.
The bottle described, while desirable, is not
essential. Any other glass bottle or large
test tube may be used. The receptacles,
however, should be of uniform size. A two-
holed rubber stopper, containing two glass
tubes, is wired on to the bottle. One of the
glass tubes (B) extends to the bottom of
the bottle and is bent at a right angle, the
other tube (A), a short straight length, is
flush with the under surface of the rubber
stopper and extends about 1 cm. above the
stopper.
Rubber tubes are wired on to the glass
tubing, and screw clamps on both rubber
tubes complete the apparatus.
Collection of Sample.—Flush the test
bottle well with CO2 and clamp shut under
slightly higher pressure than that of the
sample to be collected. Allow the beer to
flow freely from the vessel in which it is
contained, through a rubber tube having a
glass tube at the end, until a steady stream
with no evolution of gas in the tube is secured.
Without stopping the flow of beer, connect
the bottle by tube "B" and carefully open
clamp "B." Open clamp "A" slightly and
allow the beer to rise in the bottle slowly
in a quiescent state, until the bottle is com
pletely filled and all bubbles have been forced
out. Clamp "A" is then opened wide, and
beer permitted to pass through the apparatus
for some time until a representative sample
is obtained. Clamp "A" is first shut and
then clamp "B." The sample should be
kept cold until ready to be tested.
Determination.—Into a 5 ml. transfer
pipette, draw up a portion of the paraffin
oil covering the reduced indigo disulphonate
(to prevent surface oxidation of the leuco
dye in the pipette) and then 5 ml. of the
reduced dye itself. (This is sufficient for
beer containing less than 5 parts per million
of oxygen.) Open clamp "A" of the bottle
containing the sample, allow a drop of dye
to fall from the pipette, and immediately
insert the pipette through the rubber tubing.
Open clamp "B" and force the reduced dye
into the beer. Care must be taken that no
air is blown into the beer, the flow from the
pipette being stopped before the paraffin oil
reaches the tip. It is not necessary to add
exactly 5 ml. as the reduced dye is present
in excess. Close the clamps and withdraw
the pipette. Invert the bottle several times
428 rothchild and stomb: dissolved oxyoen in beer
to ensure mixing and place in a 25° 0. water
bath for one hour. Release the pressure
after 10 minutes, to prevent possible breakage
of the bottles, by opening and closing
clamp "B."
Estimate the dissolved oxygen by com
parison with standards. Prepare the stan
dards by adding varying quantities of the
indigo disulphonate solution to distilled water
in bottles similar to those used for sampling,
Fio. 2.
but without the fittings. The bottles should
be marked at the level similar to the volume
of beer tested (130 ml. in the above example)
■and made up to that volume with water
after the addition of the dye.
The standards are readily prepared when
needed. A little experience enables one to
-estimate the approximate oxygen content of
the sample. Standards at 10 per cent,
intervals (i.e., 0-20 part per million, 0-22 part
per million, etc.; or 2-0 parts per million,
2-2 parts per million, etc.) are then prepared.
[September, 1938
Thus, the 0-50 part per million standard
would be prepared by adding 0-50 ml. of
the unreduced dye to the bottle, and diluting
to the mark with distilled water. Com
parison with the standards is best made with
the bottles in a comparator as shown in
Figure 2.
Daylight is necessary as a light source,
artificial sources generally yielding off-shades.
A yellow filter between the source of light
*•***"
and the comparator aids the comparison.
Yellow-tinted cellophane has been found
satisfactory for this purpose.
Comparison with the WinkUr Method on
Water.—Several large samples of water
containing varying amounts of dissolved
oxygen were prepared by bubbling nitrogen
through and allowing to come to equilibrium
under several pounds pressure. The bottles
in which the samples were collected were
fitted with inlet and outlet tubes and con
nected in series with the sample. The
September, 1938] rothchild and stone: dissolved oxygen in beer 429

dissolved oxygen content was determined so-called redox range of beer. Experiments
by the usual Winkler method (loc. cit., p. 511) were conducted to show that the indigo
and by the colorimetric procedure described carmine is neither reduced nor oxidised by
above. The yellow filter was dispensed with the beer within the limits of the test. To show
in comparing with the standards. The that no reducing action takes place, graded
results are tabulated below in Table II. amounts of the oxidised indicator, ranging
from 0-5 ml. to 4-0 ml., were added to beer
Table II
in bottles similar to those used for the
Comparison of Winkleb and Leuco Dye
Methods ok Distilled Water
determination of oxygen. Comparison with
standards after two hours showed that the
beer had had no reducing (decolorising)
Winkler method Leuco dye method
effect upon the oxidised indicator.
Sample. (parts oxygen (parts oxygon
per million). per million). In order to determine whether the beer
constituents had any oxidising action on the
A 1-9 2-0 leuco indigo carmine, the oxygen was
B 0-58 0-68
removed from a sample of beer by heating
C 1-2 M
D 1-3 1-2 to 75° C. for 30 minutes. Samples were
E 1-9 1-9 collected in the oxygen bottles, precautions
F 4-4 4-4 being taken to exclude air. Five ml. of
G 3-5 3-5
the leuco indigo carmine were then added
to the beer. After two hours, no regenera
tion of colour had taken place.
Comparison with the Evolution Method of
Effect of pB.—pa variations apparently
Beer.—Comparative tests on samples of beer
have no effect upon the oxidation of the
were made by the described colorimetric
leuco dye by oxygen within the pu range of
method and an evolution method. In the
beer. Small additions of dilute acid and
evolution method, the samples were collected
alkali were made to the test bottles before
in quart bottles fitted with two-holed rubber
collection of the samples. The test bottles
stoppers containing inlet and outlet tubes.
were then filled with beer from one large
The "air" was determined according to the
sample and the dissolved oxygen content of
evolution method of Gray and Stone (loc. cit.)
each determined. The pu values of the
in which the CO2-air mixture is first collected
mixtures ranged from 3*4 to 5*6. While the
over acid-sodium sulphate solution and the
regeneration of colour proceeded at a more
C02 then absorbed by alkali. The oxygen
rapid rate in the more alkaline samples, the
content of this evolved "air" was sub
final colours produced at the end of one hour
sequently determined by adsorption with
were all alike.
alkaline pyrogallol in the usual manner.
Effect of SO2—The possibility that sul
The results are given in Table HI.
phites in beer might interfere in the deter
Table HI mination was also studied. Potassium meta-
Comparison of Evolution akd Leuco Dye
bisulphite was added to the bottles before
Methods on Beeb collection of the sample and the tests carried
out as described above. The greatest amount
Evolution method Leuco dye method
of the bisulphite used, equivalent to 100
Sample. (parts oxygen (parts oxygen parts SO2 per million, showed no interference
per million). per million). in determining the dissolved oxygen content
of the beer.
A 0-31 0-30
B
Effect of Yeast.—Since yeast is known to
2-9 3-0
C 2-4 2-4 possess reducing properties, it was necessary
D 1-6 1-5 to investigate (A) the possibility of such
E 0-88 0-85 action upon indigo carmine, and (B) whether
F 1-6 1-65
G
or not the yeast inhibited the oxidation of
1-6 1-7
the leuco dye by dissolved oxygen.
(A) A sample of fermenting beer was
The dye used in this method, indigo collected in a sealed bottle and known
carmine, was selected because, as an oxida amounts of the oxidised indigo carmine
tion-reduction indicator, it is near the added. Comparison with standards after
430 bothchild and stone: dissolved oxygen in beer [September, 1938

two hours showed that none of the dye had
been reduced.
(B) Fermenting beer, containing con
siderable yeast in suspension, was collected
in four stoppered 50 ml. centrifuge tubes,
connected in series. Two of the tubes were
centrifuged to settle out the yeast. Then,
to each of the four tubes was added 2 ml.
of the reduced dye solution. The dye was
mixed with the contents of the tubes, care
being taken not to disturb the sedimented
yeast in the centrifuged tubes. All the tubes
were then allowed to stand for 1 hour at
25° C. In other words, the reduced dye was
no inhibitory action on the regeneration of
colour from the leuco dye by dissolved
oxygen.
The above described method was applied
in a number of breweries in order to deter
mine quantitatively the concentration of
dissolved oxygen through the various brewery
operations. Several brews were followed
from beginning to end. The results were
obtained in three large, modern, well
equipped American breweries and are tabu
lated in Table IV. The values are here
presented primarily to illustrate the fact
that definite values can be obtained, rather

Table IV

Dissolved oxygon (parts per million).

Browory operation. Brewery Brewery Brewerj'

"A." "B." "C."

Aerated wort* 40 50 4-5

Fermenting wort—
After 1 day 0-26 0-43 0-28
End of fermentation. 0-18 019 0-20
Storage beer—
Start 0-25 (Kraeusenod) 0-32 0-31 (Kraeusened)
Middle 0-20 0-20
End 0-13 0-18 0-27
At the filter (sampled from line)—
Mixed with filter medium before filtration 0-48 0-55
After filtration 0-33 0-50
Filtered beer in tank—
Just filled 0-60 0-08
After 1 day 000 0-40 0-67
After 3 days 0-42
After 7 days 0-30
Second filtration (sampled from line)—
Before 0-48
After 0-43
In bottling tank 0-68 058

* Due to high turbidity of the wort at this stage, these figures are only approximate.

added, in one case, to beer containing con
siderable yeast in suspension, and, in the
other case, to the same beer in which the
yeast had been centrifugod out of the sphere
of reaction. After the 1-hour period, the
tubes containing yeast in suspension were
centrifuged in order to permit comparison
of the intensity of the regenerated colour.
Upon comparison, the colouration in the
four tubes was found to be identical.
As fermenting beer contains very little
oxygen, the above test was repeated on
another sample of beer which was given a
preliminary aeration immediately before
testing. Results similar to those described
above were obtained, showing that yeast has
than to establish any correlations in regard
to oxygen content.
The dissolved oxygen content of beer may
vary considerably at different levels of an
individual tank. In transferring beer from
one tank to another against air counter-
pressure, the first portion of beer entering
the empty tank mixes intimately with air
and dissolves some. The beer following
enters in a more quiescent state and has no
direct contact with the atmospheric oxygen.
Normally, diffusion, though slow, tends to
diminish these differences.
Greater differences were observed when
prolonged contact of the beer with air in
the head space of the tank had occurred.
September, 1938] bothckeld and stoke: dissolved oxygen in beer
Table V
Dissolved oxygen (parts per million).
Four hours after
Sampled from top of tank
« •< middle of tank
» - bottom of tank ..
In Table V are the results obtained from
testing a tank of beer at different levels
four hours after kraeusening and then three
weeks later. Due to faulty bunging off, an
air pocket had formed. An analysis of the
head space gas showed an air content of
25 per cent.
The described method has been shown to
be applicable to water as well as beer
(see Table II). It is particularly suitable
where the dissolved oxygen content of the
water is low as the errors of the usual
titrimetric procedures are then significant.
This leuco dye method may also find
application in determining the dissolved
oxygen content of liquids other than beer
or water in which the presence of considerable
organic matter or other interfering substances
prohibit the use of other methods.
Summary
(1) A simple, accurate method for deter
mining dissolved oxygen in tank beer is
described.
(2) The method has been compared with
the Winkler method on water and with an
431
Three weeks after
kraeusoning. kraeusening.
0-48 1-8
0-35 0-25
0-35 0-25
evolution method on beer, and found to give-
concordant results.
(3) The dye employed has been shown to-
be neither reduced nor oxidised by beer
within the limits of the test.
(4) The effects of pa, S02 and yeast, within
the limits likely to be encountered, are
shown to be without action on the method.
(5) Typical results found in several brew
eries are tabulated.
(6) Variations possible in oxygen content
of beer at different levels of a tank are
shown.
(7) It is suggested that the method may
be applicable to waters of low oxygen content.
The authors wish to express their gratitude
to Mr. Leo Wallerstein and Mr. Philip P.
Gray for their continued encouragement,
and advice in the course of this work.
Wallerstein Laboratories,
180, Madison Avenue, New York.
June, 1938.