THE DETERMINATION OF INORGANIC SULFATE, TOTAL,

SULFATE, AND TOTAL SULFUR IN URINE

BY THE BENZIDINE METHOD.
BY CYRUS H. FISKE.
(From the Biochemical Laboratory, Haructrd Medical School, Boston.)

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(Received for publication, April 28, 1921.)

As far as it’s use for the analysis of urine is concerned, the

benzidine method for the determination of sulfur is subject to
two sources of error that may under certain conditions be serious.
One of these, the contamination of the benzidine sulfate pre-
cipitate by phosphate, has been recognized before, and can be
eliminated by adding acid before precipitating with benzidine.
For this purpose Rosenheim and Drummondl recommend the
addition of hydrochloric acid to the urine until Congo red paper
shows an acid reaction. Gauvin and Skarzynski,2 on the other
hand, add the same quantity of hydrochloric acid in every in-
stance, without the use of an indicator, and this scheme has
also been adopted by Drummond3 in a more, recent modification
of the Rosenheim and Drummond method on a smaller scale.
In the-methods for inorganic sulfate proposed by all these writers,
the concentration of benzidine during the precipitation is about
the same ( 0.017 to 0.02 N ), but the concentration of hydro-
chloric acid is much less uniform. In Table I are collected data
showing the concentrations of sulfuric acid, benzidine, and hydro-
chloric acid existing during the precipitation from sulfate solu-
tions by these three methods. The figure for hydrochloric acid
given in the table for Rosenheim and Drummond’s method holds
also with urine, since in this method the urine is acidified before
adding the benzidine reagent. In the other two methods, in-

1 Rosenheim, O., and Dr;lmmond, J. C., Biochem. J., 1914-15, viii, 143.
* Gauvin, R., and Skarzynski, V., Bull. Sot. chim., 1913, 4th series,
xiii, 1121.
a Drummond, J. C., Biochem. J., 1915, ix, 492.

59
 Determination of Sulfur in Urine

asmuch as this step is omitted, the concentration of hydrochloric

acid during the precipitation of benzidine sulfate from urine is
less than stated by an amount Ohat varies with, the character
of the urine.
In spite of these differences, all three methods have given re-
sults that agree fairly well with the gravimetric method, but
they have done so only because the comparisons have been con-
fined to urines that are very much alike with respect to the factors
that affect the accuracy of the determination. As as their iong
use is restricted to ordinary 24 hour urines, any one of a great
many possible modifications would suffice to avoid trouble due
to the presence of phosphate, simply because in such urines there

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is never a very large amount of phosphate in proportion t.o the
sulfate content. But the much more exacting requirements of
TABLE I.

Hydro-
Method. Sulfuric acid. Benxidine. chloric
I I acid.

Rosenheim and Drummond ............. 0.0044.007

Drummond .................. ............ o.oo34.010 /
Gauvin and Skarzynski ................. 0.0024.003

the less uniform urines obtained in short period metabolism ex-

periments are satisfactorily met neither by any modification of
the benzidine method that has been suggested so far, nor by any
that I have been able to find that’does not involve the pre-
liminary remova,l of the phosphate.
If phosphate were the only source of trouble, it would be easy
to devise a method that would do for all circumstances that are
likely to arise, although such a method would call for a more
precise adjustment of the acidity than has heretofore been thought
necessary. But unfortunately there is a second cause of error,
in some respects even more troublesome. This is the increase
in solubility of benzidine sulfate due to the presence of chloride,
which often exists in urine in sufficient concentration to be in-
jurious in this way. That this may rarely or never be the case
in 24 hour urines presumably accounts for the fact that the
effect of chloride has previously escaped notice in connecbion with
urine analysis.
 C. H, Fiske 61

In 1 hour urines, on the other hand, for each mg. of sulfur in

the form of inorganic sulfate, there may be as much as 5 mg.
of inorganic phosphorus and 30 mg. of chlorine, and even more
under conditions that can hardly be called unusual, although
they may not be especially frequent. The influence of the addi-
tion of sodium chloride and disodium phosphate on the anal-
ysis of sulfate solutions by Drummond’s method will be seen
from the figures recorded in Table II. That the same factors
TABLE II.

Analysis of Sodium Sulfate Solution by Drummond’s Method.

Composition of solution.
Sulfur found.

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Sulfur. Phosphorus. Chlorine.

WI. mg. mg. mll.

1 0 0 1.00
1 5 0 1.04
1 0 30 0.97
1 0 60 0.91

TABLE III.

Analysis of Urine by Drummond’s Method.

T
-
Expe?-i?t Urine. Inorganic
phosphorus. Chlorine.

cc. VW. m7. m7. mg. per 100 ce.

1 0.66 14.8 0.674 67.4

1 3.36 14.8 0.678 67.8
1 0.66 34.8 0.642 64.2
2 1.32 29.6 1.39 69.5
2 6.72 29.6 1.44 , 72.0
-
are not without considerable effect on sulfate determinations
in urine is evident from the results in Table III, all obtained with
one sample of urine, which was analyzed alone (Experiments
1 and 4) and after the addition of disodium phosphate (Experi-
ments 2 and 5) and of sodium chloride (Experiment 3). De-
pending upon the amount of phosphate and chloride present
(and the conditions in this respect are not extreme for 1 hour
periods) the figures vary by more than 10 per cent.
A considerable experience with short period metabolism ex-
periments has led me to the conclusion that any sulfur method,
62 Determination of Sulfur in Urine
to be safe for such work, must give accurate results in the pres-
ence of 10 mg. of phosphorus or 60 mg. of chlorine for each mg.
of sulfur in the form of inorganic sulfate. Many different ben-
zidine reagents have been tried, under all sorts of conditions,
but none has been found equal to these requirements, and it is
apparent.ly necessary to accept the fact that both these difficul-
ties cannot be successfully contended with at the same time.
Once the phosphate has been removed, the situation is much
simplified, as it is then possible to avoid trouble from the pres-
ence of chloride by precipitating the benzidine sulfate at a much
lower acidit,y than would otherwise be permissible.

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Removal of Phosphate.

For the removal of phosphate from urine as a preliminary to

the precipitation of sulfate with benzidine, nothing has been
found equal in effectiveness to magnesia mixture in some form,
and it is fortunately possible to precipitate the phosphate nearly
quant,itatively as magnesium ammonium phosphate without the
introduction of injurious quantities of electrolyt.es, which, like
sodium chloride, would prevent the complete precipitat,ion of ben-
zidine sulfate. This is accomplished by shaking the urine (pre-
viously neutralized with ammonia) with basic magnesium car-
bonate in the presence of a small amount of ammonium chloride.
The whole process of precipitation and filtration requires only
a very few minutes. If the first 15 or 20 cc. of filtrate are
poured back on the paper and filtered again, the solution will
percolate through a layer of magnesium carbonate mixed with
triple phosphate crystals, and this is a particularly effective
way of removing phosphate. The final filtrate should t,hen con-
tain less then 0.1 mg. of inorganic phosphorus in 5 cc.
The urine must be fairly dilute before the phosphate is removed,
for magnesium ammonium phosphate crystallizes with 8 mol-
ecules of water, and the removal in this way of more than 0.2
per cent of phosphorus would appreciably alter the concentration
of sulfate in the filtrate.
In the following directions for preparing the essentially phos-
phate-free filtrate, the quantities prescribed are sufficient for
duplicate determinations of all three forms of sulfur (inorganic
sulfate, total sulfate, and total sulfur).
 C. H. Fiske
Transfer to a 50 cc. volumetric flask sufficient urine to contain
between 5 and 10 mg. of sulfur in the form of inorganic sulfat’e,
and dilute to about 25 cc. with mater. Add 1 drop of phenol-
phthalein solution and 1 drop of concentrated ammonium hy-
droxide (or as much as is necessary to make the solution faintly
pink), followed by 5 cc. of a 5 per cent solution of ammonium
chloride. Make up to the mark, mix, and pour the solution
into a dry Erlenmeyer flask containing about 0.65 gm. of finely
powdered basic magnesium carbonate.4 Shake for 1 minute, and
transfer to a 9 cm. filter paper enough of the suspension to fill
the paper nearly to the t’op. Allow this first filtrate to drain
back into the Erlenmeyer flask, and then filter the ent,ire sus-

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pension through the same paper into a dry container.
In case the urine is already extremely dilute, the phosphate
can be precipitated without appreciably altering the concen-
tration by using solid ammonium chloride (0.25 gm.) instead
of a solution. Urines obt.ained in short period experiments are
sometimes so dilute as to make this modification necessary.
The filtrate, prepared as described above, is now used for all
three sulfur determinations.

Determination of Xulfur in the Phosphate-Free Filtrate.

Inorganic Sulfate.--Pipette 5 cc. of the filtrate into a 100 cc.
bea.ker. Adcl 2 drops of a 0.04 per cent alcoholic solution of
brom-phenol blues and 5 cc. of water. Then add approximately
N HCI, drop by drop, until the solution is yellow without a
trace of blue. Run in, from a pipette, 2 cc. of benzidine reagent,
and let stand for 2 minutes. Finally, add 4 cc. of 95 per cent
acetone, and let stand for 10 minutes more. Filter through a
mat of paper pulp in a special filtration tube (described below).
Wash the beaker and the filter, first with three 1 cc. portions
of 95 per cent acetone, and then once with 5 cc. Transfer about
2 cc. of water to the filtration tube, and poke the precipitate
4 This reagent must obviously be free from sulfate. Baker’s analyzed
magnesium carbonate has proved satisfactory.
6 Clark, W. M., The determination of hydrogen ions, Baltimore, 1920,63.
6 Suspend 4 gm. of benzidine in about 150 cc. of water in a 250 cc. volu-
metric flask. Add 50 cc. of N HCl (standardized). Shake until dissolved,
and make up to volume. Filter if necessary.
64 Determination of Sulfur in Urine
and mat through the hole in the lower end into a large Pyrex
test-tube (200 by 20 mm.), using a sharpened nichrome wire.
Rinse off the wire with a few drops of water, and heat the contents
of the test-tube just to boiling, leaving the filtration tube sus-
pended in the mouth of the test-tube. Add 2 drops of a 0.05
per cent aqueous solution. of phenol red (monosodium salt),6
and run in from a micro-burette,” * through the filtration tube,
about 1 cc. of 0.02 N NaOH. Rinse down the wall of the filtra-
tion tube with 2 or 3 cc. of water from a wash bottle, heat
again to boiling until steam escapes actively from the test-tube,
and rinse a second time with sufficient water to bring the total
volume up to about 10 cc. This treatment should suffice to

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remove all traces of precipitate from the filtration tube, which
may now be removed, and the titration with 0.02 N NaQH
continued. When the color begins to change from yellow to
red, again heat to boiling, and pour the hot solution into the
beaker (in which the precipit.ation took place) and back.g This
will decompose any trace of precipitate that may have adhered
to the wall of the beaker. From t’his point on the standard
alkali should be added, not more than 0.02 cc. at a time, until
the solution acquires a definite pink color, which further boiling
does not discharge.
Total Sulfate.-To 5 cc. of the filtrate in a 100 cc. beaker add
1 cc. of 3 N HCI (approximate). Heat on the water bath
until the solution has evaporated to dryness, and for 10 minutes
longer. Immediately add 10 cc. of wat,er, and break up the
residue by rotating the beaker. Add 2 cc. of the benzidine
reagent and (2 minutes later) 4 cc. of acetone, exactly as in the
method for inorganic sulfate, and complete the determination
as described above.
Total #u&-.-Transfer 0.25 cc. of Benedict’s total sulfur
reagentlO to a 6 cm. evaporating dish, and add 5 cc. of the urine
’ Folin, O., and Peck, E. C., J. Bid. Chem., 1919, xxxviii, 289.
8 Fiske, C. H., J. Biol. Chem., 1921, xlvi, 285.
9 This step may be avoided by conducting both precipitation and titra-
tion in a large lipped test-tube, but except for the inorganic sulfate deter-
mination a beaker is on the whole more convenient.
10 Benedict, S. R., J. Biol. Chem., 1909, vi, 363. The reagent contains
20 gm. of copper nitrate crystals and 5 gm. of potassium chlorate per 100
cc. A blank (gravimetric) must, of course, be run on the reagent unless
the copper nitrate is free from sulfate.
 C. H. Fiske

filtrate. Evaporate to dryness, preferably on an electric hot

plate at low heat. When the mixture has become dry, increase
the heat by steps to the maximum, and finish the ignition with
a microburner, allowing 2 minutes at red heat after the contents
of the dish have become thoroughly black. Cool for 5 minutes.
Add 1 cc. of 3 N HCl, and evaporate to dryness on the hot plate
(low heat). When the residue is thoroughly dry, dissolve and
wash into a 100 cc. beaker with five 2 cc. portions of water.
Add 1 drop of N HCl, and precipitate with t’he benaidine reagent
and acetone as in the other two methods. The rest of the deter-
mination is likewise the same as before, with the single excep-
tion that 2 cc. of 50 per cent acetone should be used in place

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of the first of the three 1 cc. portions of 95 per cent aceDone,
otherwise it will be impossible to wash the filter free from copper.
The amount of sulfur in the 5 cc. of filtrate analyzed is in each
case obtained (in mg.) by multiplying the titration figure by
0.32.

DISCUSSION.

Use of Acetone.--Xt a time when there was still some hope

of avoiding the necessity of removing the phosphate, the addi-
tion of acetone during the precipitation with benzidine was
introduced for the purpose of diminishing the solubility of the
precipitate. Although this may not be altogether necessary under
the conditions finally adopted, the modification has been retained,
and the same liquid used for washing the precipitate, for various
reasons. Washing with a saturated solution of the precipitate
is a thing to be avoided whenever possible, and acetone is to
be preferred on that account. Since acetone wets glass more
readily than does water, it does not collect in drops on the wall
of the beaker, a matter of some consequence when a fairly large
surface must be washed with a small volume of liquid. But
the most important consideration of all is that the use of acetone
altogether prevents t!he benzidine sulfate from assuming the form
of large flakes, which can be decomposed only by prolonged boil-
ing at the end of the titration, and on account of which Rosenheim
and Drummond,’ who wash the precipitate with a saturated
solution of benzidine sulfate, have been obliged to recommend
that the fiber never be allowed to be sucked dry.
66 Determination of Sulfur in Urine

Indicator.-Boiling aqueous solutions of purified benzidine, at

concentrations corresponding with the conditions at the end-
point of the titration described above, give a brownish color
with phenol red, inOermediat,e between yellow and red. The
addition of less than 1 per cent of one equivalent of sodium
hydroxide is sufficient to change t,he color to a definite pink,
whereas phenolphthalein under these con&ions is still color-
less. Since phenol red is besides a much more brilliant indicat’or,
it is to be preferred on all counts.
Filtration Tube.-This is, in principle, the same as the tube
recently described in connection with a method for the determi-
nation of inorganic phosphate in urine.8 The narrow t.ube recom-

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mended for the filtration of magnesium ammonium phosphate

FIG. 1. Filtration tube (one-half natural size).

is readily clogged by the much more compact benzidine sulfate

precipitate, and for sulfate determinations it should be replaced
by one considerably larger in diameter. The tubes that have
been found most convenient are made from glass tubing” 15
mm. in internal diameter, shrunken at one end so as to leave a
hole 3 mm. in diameter, cut to a length of 70 mm., and flanged
at the cut end. The somewhat elongated tip shown in Fig. 1
gives the best results.
In using this tube, only enough paper pulp should be intro-
duced to form a thin cup-shaped mat lining the constricted tip.
It is neither necessary nor desirable to fill the tip with pulp.
The mat having been prepared, the tube should be filled with the
11 Pyrex tubing is preferable. Since the tube is subjected to the action
of steam during the titration, soft glass should not be used.
 C. H. Fiske 67

solution to be filtered before starting the suction, and the suction,

when it is started, should be very gentle. Any attempt to has-
ten the filtration by applying strong suction is almost certain
to produce the opposite result by packing down the precipitate.
Adjustment of Acidity.-The purpose of the preliminary acid-
ification ( to brom-phenol blue ) in the determination of inorganic
sulfate is to neutralize any basic substances that may be present
and to liberate weak acids from their salts. No appreciable
TABLE IV.

Sulfur per hr.

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Urine Total sulfate. Total sulfur.
No.
i -. _--
Zlavi- Titra- , Zravi- l!itrs- Gravi- Titra-
metric tion. 1 metric tion. metric tion.

1
mo. mg. mg. mg. nzg. mg.

1 1.8 14 15.0 15.4 17.4 17.3 21.1 21.1

10.0 14 15.4 17.3 20.9
1.8 60 15.3 17.3 21.1

2 9.5 0.9 13 22.7 22.6 25.4 25.6 32.6 32.5

10.0 13 22.8 25.6
0.9 60 22.4 25.3 32.3

3 8.5 1.0 18 14.3 14.8 17.7 17.8 22.1 22.4

10.0 18 14.7 17.9 22.3
1.0 60 14.7 18.0 22.3

4 20.5 0.8 9 34.5 34.7 37.5 37.7 42.0 41.7

10.0 9 34.8 37.7 41.8
0.8 60 34.3 37.7 42.2
- - -
amount of the hydrochloric acid in the benzidine reagent will
then be neutralized by constituents of the urine filtrate. In
the method for total sulfate, this adjustment is made automat-
ically by evaporating the urine with hydrochloric acid.
Hydrolysis of Ethereal Sulfate.- Evaporation with hydrochloric
acid is substituted for the usual boiling mainly because the
additional acid required for hydrolysis would be enough to
cause low results in the presence of large amounts of chloride.
68 Determination of Sulfur in Urine

Total Sulfur Method.-The only special modification introduced

in this determination is the removal of the excess hydrochloric
acid, after dissolving the residue of copper oxide, by evaporation
to dryness instead of by neutralization with alkali. Neutral-
ization would introduce more sodium chloride, which is unde-
sirable for reasons that have been mentioned. After the excess
acid has been removed by evaporation, the residue is at times
so nearly neutral that a drop of dilute acid should be added
(as stated) before running in the benzidine reagent.

Results.
The method as described gives satisfactory results in the pres-

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ence of 10 mg. of inorganic phosphorus or 60 mg. of chlorine
for each mg. of sulfur in the form of inorganic sulfate. A series
of analyses is given in Table IV. Each of the urines was ana-
lyzed first in the manner described above (a), secondly aft.er add-
ing sufficient disodium phosphate to bring the inorganic phos-
phate content up to the stated figure (b), and finally after the
addition of enough sodium chloride to make the ratio of chlorine
to inorganic sulfate S equal to 60 (c). For comparison the same
urines were analyzed gravimetrically (Folin’s*2 method for inor-
ganic and total sulfate; Benedict’slO method for total sulfur).
Differences greater than about 1 per cent sometimes occur
in the determination of inorganic sulfate, especially when the
sulfur excretion is small, however closely duplicates by each
method may agree. In such cases there is apparently no way
of deciding which method is the more accurate. Discrepancies
of this nature have not, been observed in the determination of
total sulfate or total sulfur.
12 Folin, O., J. Bid. Chem., 1905, i, 131.
THE DETERMINATION OF INORGANIC
SULFATE, TOTAL SULFATE, AND
TOTAL SULFUR IN URINE BY THE
BENZIDINE METHOD
Cyrus H. Fiske
J. Biol. Chem. 1921, 47:59-68.

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