A METHOD FOR THE QUANTITATIVE DETERMINATION

OF PHENOLS*
BY ROGER W. STOUGHTON
(From the Department of Pharmacology, Vanderbilt University School of
Medicine, Nashville)

(Received for publication, June 1, 1936)

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In studying the anthelmintic properties of certain alkylphenols
it became necessary to determine the degree of absorption and
excretion of these substances. Although satisfactory quantitative
tests for many types of phenols are known and have been reviewed
by Gibbs (l), an exhaustive search of the literature failed to
reveal any method that was applicable to those p-substituted
phenols which are only slightly soluble in water. Hence, it
became necessary to devise a method for the estimat.ion of these
compounds.
Nitric or nitrous acid, alone or in the presence of metallic salts,
has been used as a qualitative color reagent for phenols, but has
had quantitative application only in the case of certain water-
soluble phenols. The new calorimetric procedure described here
is based upon the action of nitric acid in acetic acid solution and
subsequent neutralization with alkali. Although this method was
originally devised for the quantitative determination of palkyl-
phenols, it may be employed equally as well with most other types
of phenolic substances.
Method
The standard is prepared by pipetting 1 cc. of a 1: 1000 glacial
acetic acid solution of the phenol under investigation into a 50 cc.
volumetric flask, diluting with 5 cc. of acetic acid, and then adding
6 drops of sulfuric acid and 2 drops of nitric acid. The mixture
is thoroughly shaken and warmed on a steam bath until the pale
* The funds for carrying out this work were given by the International
Health Division of the Rockefeller Foundation.
293
294 Determination of Phenols
yellow color that develops has reached its maximum intensity,
which takes approximately 2 minutes. This solution is diluted
with 5 to 10 cc. of water, cooled, neutralized carefully with 15 cc.
of concentrated ammonia, and made up to the mark with distilled
water. On neutralization the color deepens to a clear yellow.
This standard solution can be kept for several days without change
in either the shade or intensity of the color.
In the quantitative determinations of phenols in biological
matter, such as feces or urine, the phenol is first separated from
this extraneous matter by acidifying with phosphoric acid and

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distilling with steam until a few drops of the distillate give a
negative test for phenol. Distillates containing more than 1 part
of phenol in 10,000 parts of water may be used directly for making
the solution for comparison with the standard. However, very
much more dilute solutions are almost invariably encountered and
they must be extracted three times with ether, in order to con-
centrate the phenol. An aliquot of the combined ether extracts
is then used.
To prepare the unknown for calorimetric comparison, an aliquot
of the ether extracts, containing approximately 1 mg. of the
phenol, is evaporated just to dryness on a steam bath, and the
residue taken up with 5 cc. of glacial acetic acid and transferred
to a 50 cc. volumetric flask. If a concentrated aqueous solution
is used directly instead of its ether extract, 5 cc. are diluted with
an equal volume of acetic acid.
Either of these acetic acid solutions of the phenol is then treated
exactly as in preparing the standard-namely, 6 drops of sulfuric
acid and 2 drops of nitric acid are added, the flask is shaken,
warmed on the steam bath, diluted with 5 to 10 cc. of water,
cooled, and neutralized carefully with 15 cc. of ammonia. After
it has been made up to the mark with distilled water, it is com-
pared with the standard. When the aqueous phenol solution is
used, rather than the ether extract, heating must be continued
from 2 to 5 minutes longer in order to bring out the color com-
pletely.

DISCUSSION

The reaction upon which this determination is based is believed

to be the formation of a nitrosophenol, the alkali salt of which is
 R. IV. Stoughton
colored. Gibbs (2), in studying the action of nitric acid in the
detection of phenols, found that dilute solutions act as nitrous
acid with the formation of nitroso compounds. We have found
that if nitrous acid was used in the method described above, a
color was obtained which compared exactly with that formed by
nitric acid. The reaction was, however, so very rapid that if the
mixture was warmed for more than a few seconds, side reactions
occurred and produced a change in the shade of color formed. On
this account, the use of nitric acid is preferable. It is very unlikely
that such a small amount of acid could have caused nitration, and

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the fact that nitric and nitrous acids gave the same colors virtually
eliminates this as a possibility. In spite of the fact that there

TABLE I
Effect of the Concentration of Acetic Acid
1 mg. of o-phenylphenol in 100 per cent acetic acid compared with 1 mg.
in varying concentrations of acetic acid. Standard set at 20.0.
Acetic acid Time of color Colorimetor reading Calculated
development concentration

per cent min. mm. mg.

100 1 20.1 0.996
75 1 20.2 0.990
50 2.5 20.1 0.996
25 5 20.1 0.996
10 5 Reddish tint

must be sulfuric acid present, there was no evidence of a Lieber-

mann condensation (3) as orange or yellow colors were always
obtained. If such a condensation had taken place, the indophenol
produced would have imparted a green or blue color to the solution.
The success of this method depends upon the use of acetic acid
as a solvent. This not only makes the method applicable to
slightly soluble phenols, but it appears to be the factor responsible
for the constancy of the colors obtained. Water solutions of
phenols, when treated with nitric and sulfuric acids, gave varying
shades and intensities of color. The importance of the strength of
the acetic acid solution used may be seen in Table I. The time
required to bring out the color was found to be slightly longer
when the more dilute solutions were used. Although satisfactory
296 Determination of Phenols

comparisons were obtained with 25 per cent solutions, it seems

desirable to use as strong an acetic acid solution as possible.
Some phenols required a slightly longer period of heating than
others in order to complete the reaction. Prolonged warming for
more than 10 minutes produced side reactions that caused a change
in the shade of the color ultimately obtained and consequently
must be avoided. A safe rule is to heat just twice as long as is
necessary to bring out the first visible sign of a color.
The amount of sulfuric or nitric acid used could be varied by as
much as 100 per cent without affecting the reaction other than to

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change slightly the time of color development. The more reagent
used, the sooner the color appeared.
TABLE II
Comparison of Known Solutions with 1 Mg. of p-Tertiary Amylphenol or
o-Phenylphenol
-
Calorimeter reading
%lculated
ElT0r
amount
Standard Unknown
.-
mg. mm. mm. ml. per cent
p-Tertiary amyl- 0.5 20.0 39.9 0.504 1
phenol 1.0 20.0 20.0 1.000 0
1.5 20.0 13.6 1.470 2
o-Phenylphenol 0.5 15.0 29.7 0.505 1
0.8 20.0 24.9 0.802 0.5
1.0 20.0 20.1 0.996 0.5
1.2 20.0 16.1 1.242 3.5
1.5 20.0 13.5 1.481 1
1.8 20.0 11.4 1.754 2.5
2.0 20.0 9.8 2.041 2

Although sodium or potassium hydroxide gave a slightly deeper

color than ammonia, very concentrated solutions must be used
in order to keep the total volume below 50 cc. On this account,
it was found more convenient to use ammonia. A large excess of
alkali was without noticeable effect.
The accuracy of this method has been found to be as great as
the precision with which the individual observer can read the
calorimeter. In Table II figures are given for the results obtained
in the comparison, in a Klett calorimeter, of known solutions of
p-tertiary amylphenol and o-phenylphenol. In no case was a
greater error observed than when the same solution was used in
 R. W. Stoughton
both cups of the calorimeter. In other experiments from 95 to
103 per cent of the p-tertiary amylphenol added to samples of
urine was determined by the procedure described above.
This method gave a qualitative test with as little as 0.1 mg.
of p-tertiary amylphenol, but for the most accurate calorimetric
comparisons 0.5 to 1.0 mg. should be present.

TABLE III
General Application
b&f-

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Compound color
sensitivity

WI.
Phenolic com- Phenol Yellow 0.1
pounds o-Hexylphenol ‘I 0.1
o-Phenylphenol ‘I 0.1
o-Nonylphenol Cloudy
p-Hexylphenol Yellow 0.1
Thymol ‘I 0.1
2,4,6-Dimethylphenol Pale yellow 0.1
a-Nsphthol Orange-yellow 0.05
&Naphthol ‘I 0.05
Catechol Reddish orange 0.1
Hydroquinone Rose 0.5
Pyrogallol Yellow 0.1
Phloroglucinol Rose 0.5
Hexylresorcinol Yellow 0.1
p-Chlorophenol ‘( 0.1
2,4,6-Trichlorophenol Pink 0.2
p-Aminophenol Pale yellow 5
Salicylic acid I‘ ‘6 10
p-Hydroxyacetophenom ‘I I, 10
Non-phenolic Aniline ,I I, 10
compounds Toluene ‘I ‘I 10
Benzoic acid ‘I ‘1 20
Benryl alcohol Opalescent 10

As indicated in Table III, certain other aromatic compounds

gave faint colors in high concentrations and special care should be
taken to be sure they are not present in concentrations great
enough to interfere with the test. It was found by running blanks
on urine, feces, etc., that these substances are not present in
normal biological fluids in amounts great enough to cause inter-
ference, but they might be troublesome in the analysis of other
298 Determination of Phenols
materials, such as some pharmaceutical preparations, unless
special precautions are taken.
General Application
In order to determine the extent of the application of this
reaction, investigations were carried out on different types of
phenolic substances. Table III lists representative examples of the
types studied, together with the color produced and the minimum
amount of each compound that will give a readily distinguishable
color. Most simple alkylphenols or halogenated phenols, irre-

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spective of the number or position of the substituents, gave very
good colors. Some of the di- and trisubstituted compounds gave
a red or reddish orange color instead of the yellow most commonly
observed.
Phenols with a carbonyl group attached directly to the benzene
ring, such as the salicylic acid derivatives or hydroxyphenyl
ketones, gave such very faint colors, even at higher concentrations,
that they cannot be determined by this method. Compounds
which are themselves colored, as nitro- or nitrosophenols, are
obviously unsuited for such a calorimetric determination.
It was observed that alkylphenols with side chains containing
more than 7 or 8 carbon atoms gave cloudy solutions. This was
due to the fact that, as the molecular weight of the alkylphenol
increases, the solubility, even in alkali, decreases and those phenols
which are very insoluble cannot be determined by this method.
In a few border line cases, the use of aqueous or alcoholic sodium
hydroxide in place of the weaker ammonia gave better results.
SUMMARY

The nitrosophenol test has been so modified by the use of acetic

acid as a solvent, that it can be used for the quantitative deter-
mination for a wide variety of phenols. This is the first method
to be reported that is generally applicable for the quantitative
determination of psubstituted alkylphenols which are only slightly
soluble in water.
BIBLIOGRAPHY

I. Gibbs, H. D., Chem. Rev., 3, 291 (1926).

2. Gibbs, H. D., J. Biol. Chem., 71,445 (1926-27).
3. Liebermann, C., Ber. them. Ges., 7,247 (1874).
A METHOD FOR THE QUANTITATIVE
DETERMINATION OF PHENOLS
Roger W. Stoughton
J. Biol. Chem. 1936, 115:293-298.

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