A Colorimetric Method for Determination

of Serum Chloride

R. G. Schoenfeld and C. J. Lewellen

A procedure is outlined for the determination of body fluid chloride levels from as
little as 0.2-mi. of fluid. The results are highly reproducible and compare very
favorably with the widely used method of Schales and Schales. The color produced
is quite stable, and the interference of extraneous compounds has been found to
have no effect on the accuracy of the method.

HAVE INSTITUTED a colorimetric chloride procedure which has

been in use for 3 years in our laboratory and has met with great suc-
cess. This colorimetric method is based on a procedure used in auto-
mated syStems* (2, 3). It is extremely simple, quite accurate, and
gives highly reproducible results even in the hands of student tech-
nicians. Those laboratories not having access to a chloridometer or
automated systems, or even those not wanting to burden their auto-
mated systems with more work, may find this method suitable for their
needs.
Basically, the method consists of the addition of chloride ions to a
solution of mercuric thiocyanate and ferric nitrate. The chloride ions
upset an equilibrium established between the latter two salts, thereby
allowing the formation of a brown ferric thiocyanate complex which
is quantitatively proportional to the amount of chloride added.

Method
Reagents

1. Saturated solution of Hg(SCN)5 Add 2.0 gIn, of Hg(SCN).,

to 1 L. of distilled water. Leave the solution at room temperature for

From the Department of Pathology, Chemistry Section, Veterans Administration Hos-

pital, Albuquerque, N. M.
Received for publication Nov. 13, 1962.
AutoAnalyzer, Technicon Instruments Corp., Chauncey, N. Y.

533
534 SCHOENFELD & LEWELLEN Clinical Chemistry

48 hr. or longer, with occasional shaking. Decant and filter the super-
natant solution before using. If Hg(SCN)2 is not readily available, it
can easily be prepared by dissolving 33 gm. llg(N03)2H20 in 100 ml.
of distilled 1120 containing 5.0 ml. concentrated 11N03. In a separate
1000-mi. Erlenmeyer flask, dissolve 20 gm. of KCNS in 100 ml. of dis-
tilled 1120. With constant stirring, mix the two solutions together.
Allow the precipitated Hg(SCN)2 to settle to the bottom of the flask
and decant the supernatant solution. Wash the precipitate 4 times
with 900-1000 ml. of distilled water, decanting the supernatant each
time. (The llg(SCN)., is of sufficiently low solubility that any excess
Hg(N03)2 .1120 or KCNS will be washed out while losing very little
Hg(SCN)2.) At this point the wet Hg(SCN)9 can be used to prepare
a saturated solution of the salt.
2. a% (w/t’) Mercuric nitrate solution Place 6.0 gIn, of Hg(N03)
#{149}
1120 in a 100-mi. volumetric flask. Add 80.0 ml. distilled water and
1.0 ml. of concentrated HNOa. Mix until dissolved, dilute to volume.
3. Standard chloride solution (0.1 N) Dry reagent grade sodium
chloride overnight in a 1150 oven and allow to cool to room tempera-
ture in a desiccator. To prepare 0.1000N NaCl, weigh exactly 5.845
gm. of the salt and transfer it quantitatively to a 1000-mi. volumetric
flask. Dissolve and dilute to the mark with distilled water. Under the
conditions of this test, 0.5 ml. of 0.1N NaC1 is equivalent to 100 mEq.
of chloride per liter of serum.
4. Reagent blank solution Place 13.0 gm. of Fe(N03)3 . 911..0 in a
l-L. volumetric flask. Add approximately 500-600 ml. of distilled wa-
ter and 1.5 ml. of concentrated nitric acid. Shake until the salt is dis-
solved and dilute to volume.
Prepare the color reagent by dissolving 13.0 gm. of Fe(NOa)a’9H20
in approximately 400 ml. of distilled water in a 1-L. volumetric flask.
Add 1.5 ml. of concentrated nitric acid and 500 ml. of saturated mer-
curic thiocyanate, and dilute to volume. Next, add 6% mercuric nitrate
(approximately 5.0 to 6.0 ml.) until the absorbance of an 80.0 mEq./L.
standard (0.4 ml. of the standard chloride solution plus 15.0 ml. of this
color reagent) is between 0.07 and 0.10. If desired, 4 or 5 L. of reagent
may be prepared at one time to save time in standardization.

Standardization Procedure

Either a precalibrated card or a standard curve should be prepared

for this procedure. The absorption-concentration curve does not pass
through zero absorbance for a zero chloride concentration, and there-
Vol. 10, No. 6, 1964 SERUM CHLORIDE 535

fore calculations of chloride values, using absorbance values of the

standard and the unknown, are not possible.
A standard curve can be constructed by transferring 0.40, 0.45, 0.50,
0.55, and 0.60 ml. of the 0.1N NaC1 standard chloride solution to each
of five separate spectrophotometer tubes. These tubes then contain
the equivalent of 80, 90, 100, 110, and 120 mEq. chloride per liter of
serum, respectively. Bring the volume of each tube to 0.60 ml. and add
15.0 ml. of color reagent to each tube. Prepare a reagent blank by
transferring 0.60 ml. of 0.1N NaC1 to a spectrophotometer tube and
adding 15.0 ml. of reagent blank solution. Using this blank, set the
spectrophotometer to zero absorbance at 480 mjz and obtain the ab-
sorbance values for each of the standards. A plot of the absorbance
against milliequivalents Cl per liter should yield a straight line on
regular graph paper. The range of absorbance for the 80- to 120-
mEq./L. standards will usually fall from 0.07 to 0.70, respectively,
when using spectrophotometer tubes of 19 mm. diameter. It will be
noticed that there is a discrepancy of 0.1 ml. in the total volume be-
tween the standards and the unkiiowii, but this amount is negligible in
relation to the total volume and contributes no detectable error in the
procedure.

Procedure
This entire procedure is best carried out directly in the spectro-
photometer tubes.
Transfer 0.5 ml. of serum to each of two spectrophotometer tubes.
To one tube add 15.0 ml. of color reagent; to the second tube add 15.0
ml. of reagent blank. Shake the tubes as the reagents are added. A
slight precipitation will form, but this will disappear on standing.
Allow 10 mm. for the color to develop and also for the precipitate to
dissolve. Set the spectrophotometer for zero absorhance at 480 m
with the blank and then obtain the absorbance of the unknown. The
concentration of the unknown can be obtained from the precalibrated
card or standard curve, whichever is preferred.

Discussion

The absorption curve of the ferric thiocyanate complex formed is

shown in Fig. 1. Since the curve has a rather broad peak, the color
can be read over a wide spectral range. A wavelength of 480 m is rec-
ommended for this procedure; however, any wavelength chosen be-
536 SCHOENFELD & LEWELLEN Clinical Chemistry

tween 450mg and 480 m will give satisfactory results. This is an ad-
vantage for laboratories using photometers with fixed filter systems.
Although this procedure suggests the use of 0.5 ml. of serum and
15.0 ml. of color reagent, these quantities can be varied to suit the re-

040

0.35

0.30

Cl,
C
a)
a
0.25
C
U
0,
0
0.20

0.15

0.10
400 420 440 460 480 600 520 540

Wavelength in m
Fig. 1. Absorbanee curve of ferric-thiocyanate complex formed in chloride determination.

quirements of the laboratory. Any volume of serum may be used, as

long as a 1:30 ratio of serum to reagent is maintained.
This method can also be easily used for urine and cerebrospinal
fluid chlorides. Usually 0.2 ml. of cerebrospinal fluid and 6.0 ml. of
color reagent are used, while for urine, 0.5 ml. of urine and 15.0 ml. of
color reagent are sometimes more conveniently used.
As with most other chloride methods, this procedure is not specific
for chloride ions. Other halogens will react the same as chlorides, but
since inorganic fluorides, bromides, and iodides normally contribute a
total of less than 1.0 mEq./L. of serum, their presence can be neglected.
Patients who have been receiving large amounts of bromides, aspirin,
PASA, or any salicylic acid derivates, will have an apparently ele-
vated chloride level unless the reagent blank is used with the serum
Vol. 10, No. 6, 1964 SERUM CHLORIDE 537

from such patients. At high serum levels these drugs can also alter the
chloride values obtained by the method of Schales and Schales (1).
It must be emphasized that a separate reagent blank should be pre-
pared for each serum tested. Even though no drugs may be present

0.70

0.60

0.50

Fig. 2. Influence of nitric ‘

“ 0.40
acid concentration on color de-
velopment. a
C
U
0.
0
0.20

0.10

0 0.05 0.10 0.15 0.20 0.25

HNO3 Concentration (Normality)

to produce abnormal colors with the color reagent, there are normal
constituents in serum which react with ferric salts to produce a variety
of colors. For instance, serum from jaundiced patients produces a
color with the color reagent distinctly different from that produced
by chlorides. Since the concentrations of these constituents vary from
serum to serum, a single serum blank should not be used for a series of
chloride determinations.
The concentration of nitric acid in the color reagent has a marked
effect on color development, as shown in Fig. 2. When HNO3 was
added to give a concentration above 0.25N, proteins in the specimen
were precipitated. A nitric acid concentration of approximately 0.03N
gives optimum conditions for the determination of physiologic serum
chloride levels.
Results of a study on the recovery of chloride added to pooled hu-
man serum are presented in Table 1. Pooled serum was diluted ap-
proximately two-fold with distilled water. To 0.5-ml. aliquots of the
538 SCHOENFELD & LEWELLEN Clinical Chemistry

Table 1. RECOVERY o’ ADDED CHLORIDE FROM POOLED SERUM

Chloride (mEqIL.) *

In pooled Total I)iflerenee

serlhrn* Added Total content* determined* (mEq./L.) I.ecorery (%)

55.0 40.0 95.0 95.5 0.5 100.5 ± S.D.

55.0 40.0 95.0 96.0 1.0 101.0
52.0 40.0 92.0 94.0 2.0 102.1
75.0 40.0 115.0 116.0 1.0 100.9
55.0 45.0 100.0 100.0 - 100.0
55.0 45.0 100.0 98.0 2.0 98.0
52.0 45.0 97.0 99.5 2.5 102.5
75.0 45.0 120.0 119.0 1.0 100.9
55.0 50.0 105.0 103.0 2.0 98.1
55.0 50.0 105.0 104.0 1.0 99.1
52.0 50.0 102.0 103.0 1.0 101.0
75.0 50.0 125.0 122.0 3.0 97.6
55.0 55.0 110.0 107.0 3.0 97.2
55.0 55.0 110.0 107.0 3.0 97.2
52.0 55.0 107.0 108.0 1.0 100.9
55.0 55.0 110.0 110.0 - 100.0
55.0 60.0 115.0 112.0 3.0 97.4
55.0 60.0 115.0 113.0 2.0 98.2
52.0 60.0 112.0 114.0 2.0 101.7
55.0 60.0 115.0 116.0 1.0 100.9

*s.D., ± 1.74; 99% confidence limits; 94.78-104.74% recovery.

S.D. = / - where d is the difference between the actual and the determined
N-i
chloride content.

diluted serum, various amounts of 0.1N NaC1 were added to bring the
total chloride concentration within the normal physiologic range.
(Correction for the varying total volume was made in the blank in
each case.) Both a Coleman Jr. spectrophotometer and a Bausch &
Lomb Spectronic 20 instrument were routinely used in the authors’
laboratory- for the colorimetric determinations. Recovery values of
added chloride ranged from 102.5 to 97.4%, with an average recovery
of 99.76%.
This method was checked against that of Schales and Schales and
found to agree, as shown in Table 2. The mean difference between the
chloride content found by the two methods was ± 1.6 mEq./L. over a
Vol. 10, No. 6, 1964 SERUM CHLORIDE 539

Table 2. COMPARISON OF RESULTS WITH VARIoUs CHLORIDE METHODS

Method (mEq. Cl-L.

Difference
Specimen Mercuric-thiocyanate Ech ales & Schales (rnEq. (‘I-IL.)

1 98 97 1
2 112 110 2
3 107 104 3
4 98 98 0
5 100 102 2
6 102 102 0
7 97 98 1
8 116 113 3
9 82 84 2
10 104 104 0
11 101 100 1
12 86 89 3
13 88 92 4
14 102 100 2
15 107 107 0
16 89 90 1
17 97 96 1
18 110 112 2
19 96 98 2
20 103 100 3

S.D. = -- where d is the difference between the two methods. S.D., ± 2.06.
N-i

range from 86 to 116 mEq./L., with a standard deviation of ± 2.1.

mEq./L. The normal range values for serum chlorides by this colon-
metric method thus remain at 98-110 mEq./L.

References
1. Schales, 0., and Sehales, 5. S., J. Biol. Chem. 140, 879 (1941).
2. ZaJI, D. M., Fisher, D., and Garner, M. Q., Ann. Chem. 28, 1665 (1956).
3. Skeggs, L. T., Jr., Am. J. Clin. Path. 28, 311 (1957).