A VOLUMETRIC METHOD FOR THE ESTIMATION OF

TOTAL SULPHUR IN URINE.

BY G. W. RAIZISS AND H. DUBIN.

(From the Department of Dermatological Research, Jay F. Schamberg,
Director. Philadelphia Polyclinic and College for Graduates in
Medicine, Philadelphia, Pa.)

(Received for publication, June 1, 1914.)

During the last ten years, it has been shown that the quantitative

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determination of sulphur could be accomplished not only by weigh-
ing it as barium sulphate, but also, with the same degree of accu-
racy, by precipitating it as benzidine sulphate, and titrating the
sulphuric acid with standard alkali. It is known that when a
soluble salt of benzidine, e.g., benzidine hydrochloride, is mixed
with a solution containing sulphuric acid or soluble sulphates, a
precipitate is formed consisting of benzidine sulphate. This reac-
tion, carried out under certain conditions which have been de-
termined by Miiller,l Raschig, Friedheim and Nydegger, and
others, is quantitative. The compound, benzidine sulphate, be-
haves, in water, like a mixture of benzidine and sulphuric acid,
and this work2 was begun with the object of ascertaining whether
in the estimation of total sulphur in urine according to Benedict,$
it would be possible to substitute the titration of the sulphuric acid
in the benzidine sulphate precipitate by standard alkali for the
gravimetric determination of barium sulphate, now universally
practiced.

i W. Miiller: Ber. d. deutsch. Chem. Gesellsch., xxxv, p. 1587, 1902; Miil-

ler and Durkes: Zeitschr. f. anal. Chem., xlii, p. 477, 1903; F. Raschig: Zeit-
schr. f. angew. Chem., 1903; Friedheim and Nydegger: ibid, 1907.
2 After this work was completed our attention was called to the some-
what similar work of 0. Rosenheim and J. C. Drummond: Biochem. Journ.,
viii, April, 1914. These authors applied the benzidine method for the de-
termination of inorganic and ethereal sulphates in urine, with good results.
3 This Journal, vi, p. 363, 1909.
298 Volumetric Estimation of Sulphur in Urine
Two distinct disadvantages were immediately encountered :
First, the amount of sulphuric acid derivable from 10 cc. of urine
is so small as to make its titrimetric estimation uncertain. The
use of larger amounts of urine makes the procedure less simple
than the original method of Benedict. Second, the end point in
the titration of the sulphuric acid in a bensidine sulphate precip-
itate is not reached at once, being obtained only after repeated
boilings and additions of alkali.
To overcome these difficulties, we have devised a methodfor esti-
mating the benzidine in the precipitate of beneidine sulphate,

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rather than the sulphuric acid. We have found that benzidine,
in acid medium, reduces potassium permanganate quantitatively,
with the instant production of a canary-yellow soluble product
which, on further addition of permanganate, is converted into a
colorless substance. Further, a small. amount of benzidine reduces
a relatively large volume of 6 potassium permanganate.
In applying this reaction to the quantitative estimation of the
benzidine in benzidine sulphate, certain conditions must be
observed. Towards the end of the titration, the pink color of the
permanganate disappears more and more slowly, and this enables
us to obtain a definite end point, which, in our work, is a distinct
pink lasting for twenty seconds. Variations in temperature
influence the titration: it is therefore necessary to titrate at a
certain definite temperature. If the benzidine sulphate is dis-
solved in 200 cc. of water at room temperature, and to it are added
20 cc. of sulphuric acid (cont.), then the resulting temperature is
taken as a standard for all titrations. It was noticed that benzi-
dine sulphate, in suspension, was oxidized irregularly, and therefore
it was necessary to have the benzidine sulphate in solution before
titrating. This is possible, since a small amount of benzidine sul-
phate is soluble in boiling water. Once brought in solution, it
remains so both after cooling and after addition of sulphuric
acid (cont.). For the sake of rapid filtration and thorough wash-
ing of the benzidine sulphate, we use strong suction, without fear
of compressing the precipitate. The addition of a small amount of
sodium hydroxide greatly facilitates the process of dissolving the
benzidine sulphate, even if the latter is suspended in the form of
compressed lumps.
It was found that the precipitation of benzidine sulphate was
 G. W. Raiziss and H. Dubin 299

markedly interfered with by the presence of free hydrochloric acid;

a series of experiments showed that the more free hydrochloric
acid there was present, the less benzidine sulphate was formed.
Inasmuch as we were dealing with small amounts of benzidine
sulphate, it was essential to reduce the amount of free hydro-
chloric acid to a minimum. This was accomplished by neutraliz-
ing the clear hydrochloric acid solution with sodium hydroxide
(10 per cent), until one drop produced a precipitate of copper
hydroxide, which was then redissolved with one drop of hydro-
chloric acid (1: 4). If the solution is neutralized and acidified

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carefully, the benzidine sulphate precipitates quantitatively.
The speed and accuracy of the work are increased by the use of
asbestos filters, a scheme devised by us for vacuum filtration of
precipitates, which are subsequently analyzed by volumetric
methods. The preparation of an asbestos filter for small precipi-
tates is as follows:-Fit a 4 cm. funnel to a 250 cc. filtering flask;
fill the funnel two-thirds full with absorbent cotton, moisten it
with water, and press the cotton down, forming a flat surface.
Pour a suspension of asbestos on the cotton, sufficient to form a
thin layer of asbestos, which is easily separated from the cotton
when the filtration is finished. With such tilters, a strong suction
may be safely used.4
The $ potassium permanganate was standardized by Sorensen’s
sodium oxalate method. A weak sulphuric acid solution was
analyzed gravimetrically, four results agreeing closely; 1 cc. of
this sulphuric acid contained 0.000166 gram of sulphur. In
order to establish the value of 1 cc. of & potassium permanganate
in terms of sulphur, 5 and 10 cc. respectively, of the sulphuric acid
were treated with benzidine hydrochloride, the precipitate filtered,
washed, and titrated. Below are the results of sixteen such analy-
ses, and their average value, we think, may safely be taken as a
basis for the proposed method of analysis.

4 We have used a larger form of filter to advantage in the filtration of

ammonium urate obtained in the Folin-Shaffer method for uric acid, and
in the filtration of ammonium phosphomolybdate in Neumann’s method
for the determination of phosphorus. In each case, a 6 cm. funnel was used,
and the filtering and washing was accomplished with great ease and thor-
oughness; good duplicates were obtained, the asbestos present having no
influence on the end point in the titration.
300 Volumetric Estimation of Sulphur in Urine

For 5 cc. H&S04 were required 8.35, 8.48, 8.39, 8.39, 8.44, 8.44, 8.25, 8.49;
average, 8.40 cc. $ KMnOd. For 10 cc. HGSOb were required 16.83, 16.80,
16.82, 16.60, 16.64, 16.73, 16.94, 16.83; average 16.77 cc. KMnOd.
Taking the average of these analyses we find that 1 cc. of $
potassium permanganate corresponds to 0.000099 gram of sulphur.
The method, which we propose for the determination of total
sulphur in urine, is as follows: To 2 cc. of urine, in an 8 cm. por-
celain dish, add 0.5 cc. of Benedict’s reagent, and evaporate to
dryness on the water bath. Heat the dish carefully over a small
flame, till the contents are black, and then heat to rednessfor only

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two minutes. Cool, add 2 cc. of hydrochloric acid (1: 4), and
warm. The clear solution is neutralized with sodium hydroxide
(10 per cent), and again acidified with one drop of hydrochloric acid
(1: 4) ; 25 cc. of a solution of benzidine hydrochloride6 are added,
with stirring, the resultant precipitate allowed to stand fifteen
minutes and then filtered off on an asbestosfilter. The precipitate
of benzidine sulphate is quantitatively transferred to the filter by
means of the filtrate, and washed with 5 cc. of cold water, drop by
drop. The filter is put into a 500-cc. Erlenmeyer flask, 1 cc. of
sodium hydroxide (10 per cent) and 200 cc. of water are added, the
suspensionboiled for five minutes, and then cooled to room tem-
perature; 20 cc. of sulphuric acid (cone.) are now added, and the
warm solution immediately titrated with 3 potassium permangan-
ate, till a distinct pink coloration is obtained, which should last for
twenty seconds. In titrating, at first add only about 0.5 cc. at
a time, and towards the end, only 2 drops at a time, waiting till the
color disappears before further addition of potassium perman-
ganate. As the titration progresses, it will be noticed that the
yellow color gradually disappears, the solution turning practically
colorless. It is at this stage of the titration, that care should be
taken in adding only 2 drops of permanganate at a time.
If the urine is concentrated, and contains a large amount of
sulphur, 1 cc. may be taken for analysis; if very diluted urines, or
those of small animals are analyzed, 5 cc. or more may be taken.
In Benedict’s method, we have a comparatively quick and accur-
ate procedure for the determination of total sulphur in the urine.
6 6.7 gm. of benzidine (Merck reagent) are put into a l-liter flask, 29 cc.
of hydrochloric acid (sp. gr., 1.12) added, and the solution diluted up to
the mark.
 G. W. Raiziss and H. Dubin 301

We had the opportunity, in our laboratory, of making hundreds of

these determinations according to the original process. We also
compared Benedict’s method with Folin’@ peroxide method, and
with Denis’ modification of Benedict’s method. It was found
that all three methods gave concordant results.
The first part of the method we propose is similar to Benedict ‘s.
The gravimetric determination of barium sulphate, however, is
substituted by a volumetric oxidation procedure. Since it was
found possible to estimate amounts of sulphur smaller than have
ever before been determined, and since 2 cc. of urine instead of 10

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cc. may be analyzed Benedict’s method, with our modification,
becomes extremely convenient and rapid.
Below are the results of analyses by the new method, compared
to those obtained with Benedict’s procedure.
Urine A. By Benedict’s method two analyses, each of 10 cc., showed
0.0368 grams and 0.0368 gram of barium sulphate; in a 24-hour sample, this
is equal to 1.0120 grams of sulphur.
By the volumetric method, the following results were obtained:
5 cc. urine analyzed.
6 KMnOd s S in 24 hrs.
cc. gm. gm.
25.30 0.002505 1.0020
25.10 0.002488 0.9952
25.20 0.002497 0.9988
__~___
2 cc. urine analyzed.

10.08 0.000998 0.9980

10.08 0 .ooo998 0.9980
9.88 0.000979 0.9790
9.97 0,000987 0.9870
10.08 0.000988 0.9980
10.08 0.000988 0.9980

Urine B. By Benedict’s method two analyses, each of 10 cc., showed

0.0395 gram and 0.0400 gram of barium sulphate; in a 24-hour sample, this
is equal to 1.0920 grams of sulphur.

6 Folin: Journ. Amer. Chem. Sot., xxi, 1908.

7 Denis: This Journal, viii, 1910.
30’ Volumetric Estimation of Sulphur in Urine

The volumetric method yielded the following results:

- 2 cc. urine analyzed.

$ KMn04 S S in 24 hrs.
cc. Qm. !Jm.
10.78 0.001067 I. 0670
10.85 0.001074 1.0740
10.94 0.001082 1.0820
10.92 0.001081 1.0810
10.82 0.001071 1.0710
10.80 0.001069 1.0690

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10.70 0.001059 1.0590

Urine C. By Benedict’s method three analyses, each of 10 cc., showed

0.0195 grams, 0.0195 grams and 0.0197 gram of barium sulphate; in a 24-hour
sample, this is equal to 0.6725 gram of sulphur.
The volumetric method yielded the following results:
5 cc. urine analyzed.

2 cc. urine analyzed.

5.48 0.0005425 0.6780

5.44 0.0005380 0.6730
5.44 0.0005380 0.6730

A glance at the above tables will show that our method gives
results similar to Benedict’s. An analogous procedure for the
estimation of different sulphur fractions in the urine, and also
other applications of the new method are being worked out.

SUMMARY.

1. A volumetric method is proposed for the determination of

total sulphur in urine.
 G. W. Raiziss and H. Dubin 303
2. This method has for its basis, the estimation of benzidine in
the precipitate of benzidine sulphate, rather than the sulphuric
acid.
3. The procedure occupies only one-fifth of the time required by
the Benedict method.
4. Very small amounts of sulphur may be accurately determined
by the proposed method.

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 A VOLUMETRIC METHOD FOR THE
ESTIMATION OF TOTAL SULPHUR IN
URINE
G. W. Raiziss and H. Dubin
J. Biol. Chem. 1914, 18:297-303.

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