Chlorine
in Distilled Water
of Laboratory
Wendell T.
SYSTEMATIC
Error
Caraway
of low and
INVESTIGATION
as a Source
erratic
results
obtained
in
one laboratory
on standard solutions of uric acid revealed the presence of considerable
free residual chlorine in the supply of distilled
water. A further study was made of the effect of free chlorine on
the determination
of uric acid and other substances
susceptible
to
oxidation-reduction
reactions.
Collier and Stuart
(1) previously
reported on the interference
of chlorine in distilled water with bacterial viability and with micro-iodometric
titrations.
EXPERIMENTAL
“Free
chlorine”
in water
was
determined
colorimetrically
by the
o-tolidine method (2). As a confirmatory
test, solutions
of potassium
iodide and starch were added, respectively,
to an aliquot of water
and the color compared with a standard
solution of iodine treated
similarly.
Three lots of water were used for comparative
tests:
1.
Chlorine-free
distilled
2. Chlorine-contaminated
mately 1.0 part per million
water.
distilled
water,
free chlorine.
containing
approxi-
3. Chlorine
water prepared
by bubbling
chlorine
gas into distilled
water, then adjusted
by dilution
to approximately
100 p.p.m. of free
chlorine.
This was used to accentuate
any slight effect of chlorine.
These three lots of water were used in the preparation
of dilute
working
standards
or for the initial
dilution
of specimens.
Unless
Prom the Laboratories
Flint Medical Laboratory,
Received
for publication
of the MeLaren
Flint, Mich.
May 27, 1958.
General
513
Hospital,
St.
Joseph
Hospital,
and
the
514
CARAWAY
otherwise
specified,
chlorine-free
distilled
reagents
water.
were
Clinical Chsinhfry
prepared
for
all
methods
from
RESULTS AND DISCUSSION
The following
equilibrium
is established
in chlorinated
water:
Cl2 +H2OHCl+HClO
On distillation,
chlorine
chiorous
acid could appear
The chlorine-contaminated
is volatilized
and both chlorine
and hypo-
in the distillate
in appreciable
quantities.
water used in this study was found to
contain
1.0 p.p.m. of free chlorine
definite
chlorine
and had a
in the
area at this time contained free chlorine in amounts ranging from a
trace to 1.0 p.p.m.
The values appeared
to vary with the rate of
operation of the still; rapid distillations
were associated with higher
free chlorine contents in the distillate.
When an aliquot of the
odor.
Distilled
by the o-tolidine
water
from
four
method
different
stills
chlorine-contaminated
water was boiled down to three-fourths
of its
original
volume, both tests for chlorine
became negative.
It was also
possible
to remove all but a trace of free chlorine
by treatment
with
activated
resin
carbon or by passage
of the water
through
an ion-exchange
(Deeminac).
The raw tap water supplied to the still at this time contained approximately
1.0 p.p.m. of free chlorine, a figure in agreement
with
that reported by the laboratory
of the city water supply department.
The tap water had no appreciable
odor of free chlorine.
The high
alkalinity of the raw water (pH 10.2) would result in a shift of the
equilibrium
to form hypochlorite
ion since the pK of hypochlorous
acid is 7.4. Chlorination
of this water supply was
ment with chlorine
gas, lime, and sodium carbonate.
effected
by treat-
URIC ACID
Dilute standards
correspond
were prepared
to 5 mg.
of uric
acid
with the respective
per
100 ml.
lots of water
of serum
and
to
were
analyzed without delay by four different methods. Results are shown
in Table 1. Free chlorine had a marked inhibitory
effect on color
development
in all methods.
cyanide method of Brown
(3);
The least effect was noted with
however,
this is largely
a reflection
the
of
the smaller volume of standard used in this method relative to the
final volume of solution.
In all instances addition of strong chlorine
water to the solutions after full color development
resulted in a
further decrease in optical density.
Vol. 4, No. 6, 1958
Table
TypicAl.
1.
op Fanz Cntoanr
RF’PZCTS
ON
Relative
Determination
Uric
Uric
Uric
Uric
Bilirubin
Phosphatase
(phenol
reaction step)
Hemoglobin
Methemoglobin
Cyanide
(3)
Silicate (4)
Carbonate (5)
Carbonate
(6)
Mafloy-Evelyn
(7)
5.0 mg./100
5.0 mg./100
5.0 mg./100
5.0 mg./100
7.3 mg./100
(8)
King-Armstrong
Oxyhemoglobin
Evelyn-Malloy
(9)
30.0
KA
Paocznunas
ANALYTICAL
Values
Obtained
Chlorine
water
1 pp.m.
Chlorine-free
water
Method
Acid
Acid
Acid
Acid
515
CHLORINE IN DISTILLED WATER
nil.
ml.
ml.
ml.
nil.
units
12.6 Gm./100
0.0 Gm.f100
ml.
ml.
Vaing:
Okiorins
100
water
p.p.w.
4.7
0.8
3.9
3.6
4.0
6.4
0.0
0.0
0.0
1.6
29.3
8.1
12.6
0.5
10.9
1.9
BILIRUBIN
Aliquots of jaundiced
serum were diluted with the three lots of
water and analyzed without delay by the method of Malloy and
Evelyn
(7). Results
are shown in Table 1. Use of chlorine-contaminated water resulted
in a 12 per cent decrease
in optical density
compared with chlorine-free
water.
When strong chlorine water was used
for the diluent, very little color developed and the control tube turned
green, presumably
by the oxidation
of bilirubin
to biliverdin.
Addition of chlorine water to the fully developed azobilirubin
had only a
slight effect on the optical density other than that calculated
for
simple
dilution.
PHOSPHATASE
(Phenol
reaction
step) Dilute standard
solutions
of phenol
equiva-
lent to 30 units of alkaline
phosphatase
were prepared
with the three
lots of water and analyzed
by the method of King and Armstrong
(8).
Results
are shown in Table 1. A negligible
reduction
in color development was obtained
with the chlorine-contaminated
water;
with strong
chlorine
strong
sulted
water
the color was decreased
by 73 per cent.
chlorine
water
to the fully developed
in a further
decrease
in optical density.
reaction
Addition
mixture
of
re-
HEMOGLOBIN AND METHEMOGLOBIN
For hemoglobin determinations
0.06 M ammonium hydroxide was
prepared
with the different lots of water.
Aliquots of blood were
diluted 1:200 with the ammonia water and measured
spectrophotometrically.
No effect on optical density was observed with one p.p.m.
516
CARAWAY
Clinkel Chemistry
free chlorine;
with 100 p.p.m. the optical density
decreased
by 13 pei
cent and the solution
turned brown.
Methemoglobin
determinations
on whole blood were performed
by
the method
of Evelyn
and Malloy
(9).
The different
lots of water
were used for preparation
of M/60 phosphate buffer for the initial
1 :100 dilution of blood. As expected, appreciable
concentrations
of
methemoglobin
were formed by the oxidizing action of free chlorine.
At high concentrations
of chlorine,
the solution
assumed
a brown
color, which did not completely disappear
on addition of neutralized
cyanide
pletely
solution,
converted
indicating
that the pigments
to cyanmethemoglobin.
formed
were
not com-
PROTEIN-BOUND IODINE
Presence
of free chlorine
in distilled
water decreases
the stability
of dilute standard
solutions
of sodium iodide, presumably
by oxidation of iodide to volatile
iodine.
A solution
containing
0.04 jg per
ml. of iodide,
used as a working
standard
in the determination
of
protein-bound
iodine
(10), was found to have excellent
stability
as
normally
prepared.
During
one dry summer
season when the water
supply
was heavily
chlorinated
it was observed
that this standard
would decrease
in strength
as much as 20 per cent over a three-day
period.
Test of the distilled
water at this time revealed
the presence
of appreciable
amounts
of free chlorine.
The water was freed from
chlorine by boiling in an open beaker, after which the dilute standards
of sodium iodide showed their usual good stability.
OTHER DETERMINATIONS
Chloride
determinations
by direct titration
with mercuric
nitrate
(11) were not affected
by the small amount
of chloride
present
in the
chlorine-contaminated
water.
As expected,
strong
chlorine
water
contained
appreciable
amounts
of “available”
chloride.
No significant
effects were observed
on the final color development
of standards
when chlorine
water was substituted
for chlorine-free
water in the following
determinations:
inorganic
phosphate
by the
Fiske-SubbaRow
method
(12); glucose by the Benedict
method
(13);
protein
by the biuret method
(14); ammonia
nitrogen
by nessleriza-
tion. (15).
TESTSFOR FREE CHLORINE IN WATER
Distilled
water
supplies
presence
of free chlorine.
should
be checked
occasionally
for the
The test reagent
is prepared
by dissolving
Vol. 4. N.
6, 1959
CHLORINEIN DISTILLEDWATER
0.1 Gm. of o-Lolidine dihydrochioride
517
in 100 ml. of 1 N hydrochloric
acid. This solution
is stable and should be colorless.
To test a water
supply,
1.0 ml. of reagent
is added to 100 ml. of water,
thoroughly
mixed, and the color noted at the end of 5 minutes.
A yellow color
is presumptive
evidence for the presence
of “free chlorine.”
Actually,
the test measures
total available
chlorine,
regardless
of the form in
which it is present in the water.
A set of permanent
visual standards
may be prepared
from appropriate
mixtures
of potassium
dichromate
and cupric sulfate
(16). Reagents
and standards
are also available
commercially
(Hach Chemical
Co., Ames, Iowa).
Manganic,
ferric,
and nitrite
ions will produce
some color with
the o-tolidine
reagent
(16). When color is obtained,
a sample of the
original
water is boiled down to three-fourths
of its original
volume
and retested.
If the contaminant
is chlorine
only, no color should be
obtained
in the boiled sample.
The test is sensitive
to approximately
0.01 p.p.m. of free chlorine.
Raw tap water frequently
contains
0.25
to 0.50 p.p.m. or more of free chlorine
and may be tested for comparison.
An alternate
test for free chlorine
is to add a crystal
of potassium
iodide, 1 ml. of concentrated
hydrochloric
acid, and 1 ml. of 1 per cent
soluble
starch,
respectively,
to 200 ml. of water.
A blue color is
presumptive
evidence
of free chlorine.
SUMMARY
Some distillation
processes
may result in the inadvertent
contamination of distilled
water with free chlorine.
One part per million of
free chlorine
in distilled
water has been shown to inhibit
markedly
the color development
in the usual determinations
of uric acid and
bilirubin.
The effect of free chlorine
on other clinical
chemistry
determinations
is discussed
and simple tests for the detection
of free
chlorine
in water are reviewed.
REFERENCES
1.
2.
Collier, H. B., and Stuart, R. D., Ca’nad. M. A. J. 69, 321 (1953).
Standard
Method8
for the Examination
of Water,
Sewage,
and Indu8trial
York, American
Public Health Association,
Inc., 1955, 10th ed.
3. Brown, H., J. Biol. Cliem. 158, 601 (1945).
4. Archibald, R. M., GUn. Chern. 3, 102 (1957).
5. Caraway, W. T., Am. J. GUn. Path. 25, 840 (1955).
6.
7.
8.
9.
Henry,
Malloy,
R. J., Sobel, C., and Kim, J., Am. J. Clin. Path. 28, 152 (1957).
H. T., and Evelyn,
K. A., J. Biol. Chem. 119, 481 (1937).
King, E. J., and Armstrong, A. R., Canad. M. A. J. 31, 376 (1934).
Evelyn, K. A., and Malloy, H. T., J. Biol. Chein. 126, 655 (1938).
Wa8te8.
New
518
10.
11.
12.
13.
CARAWAY
Barker, S. B., Humphrey,
M. J., and Soley, M. H., J. CUn. Inve8t.
Seliales,0., and Sehales,
S. S., J. Biol. Chem. 140, 879 (1941).
Fiske, C. H., and SubbaRow,
Y., J. Biol. Chem. 66, 375 (1925).
Benethct, S. R., J. BioL Chem. 92, 141 (1931).
Gornail, A. G.. Bardawill,
C. 3., and David, M. M., J. BiOZ. Chem.
Clinical
CJemistry
30, 55 (1951).
177, 751 (1949).
15. Connerty, H. V., Brigga, A. R., and Eaton, E. H., Am. J. Gun. Path. 25, 1321 (1955).
16. Sneil, F. D., and Snell, C. T., Coiorinnetrw
Methods
of Anauyais Princeton,
N. 3., D.
Van Nostrand, 1949, 3rd. ad., Vol. II, p. 707.
14.