454
NOTES
AND
cision of the Winkler analysis including
sampling errors. Thus, at low concentration levels, the Winkler method can be in
error by 10 pg-atoms/liter
and has ten
times poorer precision than the colorimetric method.
WILLIAM W. BROENKOW
JOEL D. CLINE
Department of Oceanography,
University of Washington,
Seattle 98105.
REFERENCES
ALSTERBERG, G. 1925.
Methoden
zur Bestimmung von in Wasser gelosten elementaren
Sauerstoff
bei gegenwart
von salpetriger
Saure.
Biochem. Z., 159: 3647.
1969.
BROENKOW, W. W.
An interface
sampler using spring-actuated
syringes.
Limnol.
Oceanog., 14: 288-291.
CARPENTER, J. H.
1965. The accuracy of the
COMMENT
Winkler
method for dissolved oxygen analysis. Limnol. Oceanog., 10: 135-140.
1966.
CARRITT, D. E., AND J. H. CARPENTER.
Comparison
and evaluation
of currently
employed modifications
of the Winkler
method
for determining
dissolved oxygen in seawater;
J. Marine Res., 24: 286a NASCO report.
318.
1968. Kinetics of the sulfide-oxyCLINE, J. D.
gen reaction in seawater; An investigation
at
constant temperature
and salinity.
M.S. Thesis, Univ. Washington,
Seattle. 68 p.
CUSTER, J. J., AND S. NATELSO?\T. 1949. Spectrophotometric
determination
of microquantities
of iodine.
Anal. Chem., 21: 1005-1009.
1939.
THOMPSON, T. G., AND R. J. ROBINSON.
Notes on the determination
of dissolved oxygen in sea water.
J. Marine Res., 2: 1-8.
1955.
WHEATLAND, A. B., AND L. J. SMITH.
Gasometric determination
of dissolved oxygen
in pure and saline water as a check of titrimetric methods.
J. Appl. Chem. (London),
5: 144-148.
WINKLER, L. W.
1888. Die Bestimmung des im
Wasser gelosten Sauerstoffes.
Chem. Ber.,
21: 2843-2855.
SPECTROPHOTOMETRIC
DETERMINATION OF HYDROGEN SULFIDE IPI:
NATURAL
The determination
of small concentrations of hydrogen sulfide by the methylene
blue method has been widely used since
its introduction
by Fischer ( 1883). The
method is sensitive and specific for sulfidesulfur and is readily adaptable for rouof the
tine analyses. Many modifications
method have been made for specific purposes (Almy 1925; Sheppard and Hudson
1930; Sands et al. 1949; Fogo and Popowsky 1949; Budd and Bewick 1952). The
procedure has also been used in determining sulfide-sulfur
in sewage (Pomeroy
1936) and seawater (Fonselius 1962).
In our experience, the methylene blue
method was somewhat erratic, and studies
were undertaken to define the optimum
conditions for determining sulfide-sulfur in
natural waters. The study evolved during
1 Contribution
No. 493 from the Department
of
Oceanography,
University
of Washington,
Seattle.
This research was supported by National Science
Foundation
Grant GA-644 to Dr. F. A. Richards.
WATERS~
investigations of anoxic marine basins in
which hydrogen sulfide occurs as a respiration product of sulfate reduction.
A procedure using unsubstituted p-phenylenediamine to produce Lauth’s violet in
the determination of sulfide-sulfur
in seawater was described recently (Strickland
and Parsons 1968). The method described
here has several advantages over the
Lauth’s violet procedure, including 1) the
use of a single reagent, containing N,iV-dimethyl-p-phenylenediamine
sulfate, which
need not be recrystallized before use; 2)
superior color and reagent stability at low
sulfide concentrations; 3) applicability to a
wide range of sulfide concentrations; 4)
approximately a 10% increase in sensitivity,
and 5) a simplified procedure for standardization.
The method is applicable to
natural waters containing l-1,000 pg-atems/liter sulfide-sulfur ( 0.03-32 ppm ) and
is free of salt effects and temperature
dependence.
NOTES
AND
TABLE
1.
Suggested reagent concentrations
and
dilution factors to be used in the determination
of
sulfide-sulfur
in the stated concentration
ranges
Sulfide
concn
(,umoles/
liter)
Diamine
concn
1-3
3-40
40-250
250-1,000
0.5
2.0
8.0
20.0
Ferric
concn
(g/500
cgLYo
ml)
0.75
3.0
12.0
30.0
Dil.
factor
(ml : ml)
‘~~~t~
1:l
1:l
2:25
1:50
10
1
1
1
Path
I am indebted to Dr. F. A. Richards who
critically reviewed the manuscript and to
Mr. W. Broenkow for his pertinent and
valuable suggestions. I also thank Mr. D.
Kester who, as a co-worker, carried out
much of the initial
experimental
background of this work.
EXPERIMENTAL
To determine dissolved hydrogen sulfide
( H& HS-, S2-) in the concentration range
1.0 to 1,000 pug-atoms/liter, it is necessary
to use reagents of various concentrations
and dilutions (Table 1).
Reagents
Mixed diumine reagent. Dissolve the
amounts of NJV-dimethyl-p-phenylenediamine sulfate (Eastman Kodak No. 1333)
and ferric chloride ( FeC& *6H2O ) shown
in Table 1 in 500 ml of cool 50% ( v/v)
reagent grade hydrochloric
acid. All but
the most dilute reagents are stable for several months, particularly
if kept in dark
bottles under refrigeration.
The reagent
concentrations for the lower two ranges
are in large excess to enhance reagent
stability.
Instrumentation
Beckman
photometer.
DU
or comparable
spectro-
Sampling
Because hydrogen sulfide is volatile and
is oxidized by dissolved oxygen, exposure
to air and manipulation
of the samples
must be kept to a minimum.
We use a
50-ml syringe fitted with a 16-gauge stainless steel cannula coated with Siliclad
455
COMMENT
(Clay-Adams, New York) to prevent corrosion The syringe is mounted rigidly in
either a wooden or plastic frame with
calibrated stop positions for convenient
sample volumes.
The drain cock of most sampling bottles
can be fitted with an appropriate size rubber septum to permit uncontaminated samples to be drawn into the syringe. The
inclusion of air bubbles during sampling
should be avoided.
If enough mounted syringes are available, the calorimetry can be carried out
directly in each syringe, eliminating
the
need to transfer samples. If the number
of syringes is insufficient,
the procedure
below may be followed.
Procedure
A 50-ml sample is transferred from the
syringe to a 50-ml serum bottle, to which
4 ml of the appropriate mixed diamine
reagent is added. A 5-ml syringe with a
4-ml stop position is satisfactory for reagent delivery. The serum cap is replaced
promptly to reduce volatilization
of the
hydrogen sulfide, and the solution is mixed
gently. After 20 min the absorbance is
determined spectrophotometrically
at 670
rnp in the appropriate cuvette. All necessary dilutions should be made after the
color development time and in volumetric
glassware.
The concentration of sulfide in the sample is calculated from the expression
Czs = F(A-Ah),
where Cxs is the concentration of sulfide
in the units given to the factor F, A is the
absorbance of the sample, and Ab is the
blank absorbance. The factor F is evaluated by standardization with known concentrations of sulfide. The value of A,
will depend on reagent strength and purity,
sample turbidity, and cell matching.
Standardization
It is convenient to prepare oxygen-free
water by purging distilled water with tank
nitrogen. The nitrogen is bubbled through
1 liter of distilled water in an aspirator
456
NOTES
AND
COMMENT
0
I
0.4
I
I.2
I
0.8
I
1.6
I
2.0
PH
FIG.
2. The effect of pH on pure methylene
blue solutions in the pH range of zero to 2.0.
on the
(--a-).
Th e e ff ec t o f sample dilution
apparent molar extinction
coefficient
(normalized)
is shown for comparative
purposes:
1, Undiluted;
2, dilution 2 : 25; 3, dilution 1: 50.
I
IO
I
0
I
40
I
200
I
80
I
400
I
20
I
120
I
600
6
I
30
I
40
I
50
c
I
160
I
200
I
240
I
I
I-J
I
800
1000
SULFIDE (,&g-atoms/liter)
FIG.
tion of
ranges.
1. Calibration
sulfide-sulfur
curves for the determinain the four concentration
reagent, Nusbaum (1965) suggested the
use of oxalate in place of sulfate.
The calibration factor F should be determined at each concentration level. The
final absorbance (after dilution) should be
less than 1.0 (and preferably less than OS),
because aqueous methylene blue solutions
do not conform strictly to Beer’s law at
higher concentrations.
RESULTS
bottle fitted with a two-way stopcock.
After approximately 20 min, sodium sulfide
( Na$ . 9Hz0 ) , washed free of oxidation
products, wiped dry with a cellulose tissue, and weighed, is added to the water
and a slight nitrogen overpressure is applied. The concentration of the standard
can be calculated from the amount of sulfide added or, if improved accuracy is
needed, the solution can be standardized
iodometrically
( Budd and Bewick 1952).
Subsamples of this standard can be drawn
from the lower vent fitted with a rubber
tube. Although standards prepared in this
way have not become oxidized after several days when kept under a nitrogen
is recatmosphere, daily standardization
ommended. The diamine reagent yields
( within a few per cent) the same calibration factor from lot to lot, although some
lots are more oxidized than others. To
improve the reproducibility
of the amine
AND
DISCUSSION
The effects of reagent strength, pH,
temperature, salt effect, and interfering
substances on the reaction have been investigated. In general, these findings confirm the observations of others (Pomeroy
1936; Sands et al. 1949; Budd and Bewick
1952) and will not be discussed in detail.
Range of concentrations
The usable concentration range was investigated in four intervals to obtain the
maximum
sensitivity
and adherence to
Beer’s law. The ranges are 0 to 3, 3 to 40,
40 to 250, and 250 to 1,000 pug-atoms/liter,
the last two requiring dilution after the
color is developed. All studies were conducted at the optimum pH of 0.35, which
had been determined previously.
Except at concentrations of less than 1
pg-atom/liter,
Beer’s law is followed (Fig.
1). Departures from Beer’s law were ob-
NOTES
TABLE 2.
Expected
Molar
(liter
l-4
4-40
40-250
25&1,000
* The
mean
of the methylene
sensitivities
blue method
Sulfide
concn
(fimoles/liter)
absorptivity
mole-l
cm-l)
30.2
29.5*
32.9
33.4
calculated
from
12
AND
calibration
x
x
x
x
lo3
lo3
lo3
lo3
curves.
served when the molar diamine concentration was less than seven times the total
sulfide molar concentration and when the
final absorbance was greater than 1.0. The
latter effect was in agreement with the
findings of Sheppard and Geddes ( 1944))
who showed that more concentrated methylene blue solutions do not conform to
Beer’s law.
Sensitivity,
precision,
and accuracy
The sensitivity of the method, defined
in terms of the apparent molar absorptivity
(E’), has been optimized with respect to
reagent strength and ;pH. A comparison of
the apparent molar absorptivity, calculated
from Fig. 1 and listed in Table 2, shows
an increase in sensitivity at higher concentrations. This arises, in part, from the
increase in “pH accompanying sample dilution and its concomitant effect on the ;pHdependent molar absorptivity of methylene
blue ( Fig. 2). The average value of E’ for
this method at pH 0.35 (undiluted)
is
approximately 29.5 X 103 liter mole-l cm-l,
a value comparable to the sensitivities reported by Sands et al. ( 1949), Johnson and
Nishita ( 1952 ) , and Fogo and Popowsky
( 1949). It should be noted that the experiments of Sands and his co-workers were
carried out at pH values of less than zero.
At these values, the primary absorption
band of methylene blue occurs at 750 rnp
instead of 670 mp; however, our sensitivities were comparable.
A comparison of the apparent molar absorptivity
with that of pure methylene
blue solutions under comparable conditions
(Fig. 2) suggests that the reaction is approximately 62% complete.
457
COMMENT
TABLE
Sulfide
(w-de/
liter)
3
10
20
125
276
525
817
3.
Precision
at selected
Dil. factor
(ml : ml)
1:l
1:l
1:l
2:25
2:25
1: 50
1:50
* S is an estimate
t 7t is the number
of the methylene
concentration
Optical
path
(cm)
10
1
1
1
1
1
1
of the standard
of samples.
blue method
levels
2s*
(%)
n-t
3.0
2.0
3.0
2.1
1.7
2.0
2.3
6
25
25
10
12
10
10
deviation.
The precision of the method was investigated by determining the absorbance of n
replicate sulfide standards at various concentration levels. The results (Table 3)
indicate that the overall method, not including any sampling errors, will give a
precision of +2% at the 95% confidence
level.
The accuracy of weighed standards was
compared with those determined iodometrically. Twenty standard sulfide solutions
were prepared, and their concentrations
were determined by iodine titration using
a pair of polarized platinum electrodes to
detect the end point (Potter and White
1957). Although
the amperometric
end
point is more time consuming than the
conventional starch end point, it is more
accurate, allowing iodine concentrations to
be determined within *0.5% (95% confidence level) of the true value. Potassium
biiodate,
KH ( 103)2, was used as the
primary standard.
The concentrations of the sulfide solutions estimated gravimetrically
were always within 22% of the titrated value at
the 95% confidence level.
Temperature
effect
The effect of temperature on the sensitivity was investigated by two methods. In
the first, the diamine and ferric ion were
added to the sulfide solutions separately
as is the usual practice; in the second case
they were added mixed. Care was taken
458
NOTES
- 32,
IO
‘0
x 30
-i
[
E
0
-i
al
z
E
&
t
z
3
I
I
I
I
AND
the interferences
ganic thiols.
I
by mercaptans
JOEL
B
and orD.
CLINE
Department of Oceanography,
University of Washington,
Seattle 98105.
/
28’
REFERENCES
1
26’
i
241
0
COMMENT
5
I
IO
1
15
I
20
I
25
30
T PC)
FIG. 3. The effect of temperature
on the apparent molar extinction
coefficient
of methylene
blue. Curve A shows the results of separate addition of the amine and ferric reagents and curve B
shows the results of the mixed diamine reagent.
to ensure the same final pH in both cases.
The decrease in the apparent molar absorptivity noted in curve A, Fig. 3, presumably
arose from the effect of temperature on
the volatilization
of hydrogen sulfide from
the solution after the addition of the acidic
diamine reagent. When the reagents were
mixed, the volatilization
of hydrogen sulfide was appreciably reduced and the sensitivity correspondingly increased.
Interferences
The method is without salt effect over
the salinity range of 0 to 40%0. Thiosulfate
and sulfite (0 to 100 pmoles/liter)
did not
prevent the full development of the color,
but thiosulfate inhibited the reaction, the
inhibition
time depending on the concentration of thiosulfate. This inhibition is the
basis of a clock-reaction method for the
determination
of thiosulfate
(Risk and
Strickland 1957) and serves as the qualitative indication of the presence of thiosulfate.
No attempt was made to investigate the
effects of organo-sulfur compounds on the
methylene blue method because of its intended application
to natural waters in
which the nature and concentrations of
these compounds are largely unknown.
Others (Almy 1925; Sands et al. 1949;
Siegel 1965) have completed some work on
ALMY, L. H. 1925. A method for the estimation of hydrogen sulfide in proteinaceous
food
products.
Am. Chem. Sot., 47: 1381-1390.
BUDD, M. S., AND H. A. BEWICK. 1952. Photometric determination
of sulfide and reducible
sulfur in alkalies.
Anal. Chem., 24: 15361540.
FISCHER, E . 1883. Bildung
von Methylenblau
als Reaction auf Schwefelwasserstoff.
Chem.
Ber., 26: 2234-2236.
FOGO, J. K., AND M. POPOWSKY. 1949. Spectrophotometric
determination
of hydrogen
sulfide.
Anal. Chem., 21: 732-734.
FONSELIUS, S. H. 1962. Hydrography
of the
Baltic deep basins.
Fish. Bd. Swed. Ser.
Hydrol.,
Rep. 13, p. 31-32.
JOHNSON, C. M., AND H. NISHITA.
1952. Microestimation of sulfur.
Anal. Chem., 24: 736-
742.
NUSBAUM, I. 1965. Determining
sulfides in water and wasterwater
[sic].
Water Sewage
Works, 112: 113, 150.
POMEROY, R. 1936. The determination
of sulphides in sewage.
Sewage Works J., 8:
572-591.
POTTER, E. C., AND J. F. WHITE.
1957.
The
microdetermination
of dissolved
oxygen in
water.
III.
Titrimetric
determination
of iodine in submicrogramme
amounts.
J. APP~.
Chem. ( London),
7: 309-317.
RISK, J. B., AND J. D. H. STRICKLAND. 1957. Determination
of milligram
quantities
of thiosulfate by a clock reaction.
Anal. Chem.,
29: 434-437.
SANDS, A. E., M. A. GRAFIUS, H. W. WAINWRIGHT, AND M. W. WILSON.
1949. The
determination
of low concentrations
of hydrogen sulfide in gas by the methylene
blue
method.
U.S. Bur.
Mines
Rept.
Invest.
4547, p. 1-18.
SHEPPARD, S. E., AND A. L. GEDDES. 1944. Effect of solvents upon the absorption
spectra
of dyes. V. Water as solvent:
Quantitative
examination
of the dimerization
hypothesis.
J. Am. Chem. Sot., 66: 2003-2009.
AND J. H. HUDSON. 1930. Determination of labile sulfur in gelatin and proteins.
Ind. Eng. Chem., Anal. Ed., 2: 73-75.
SIEGEL, L. M.
1965. A direct microdetermination for sulfide.
Anal. Biochem., 11: 126132.
STRICKLAND, J. D. H., AND T. R. PARSONS. 1968.
A manual of seawater analysis.
Bull. Fisheries Res. Board Can. 167. 311 p.