Colorimetric Determination of Dissolved Oxygen at Low Concentrations

Author(s): William W. Broenkow and Joel D. Cline

Source: Limnology and Oceanography, Vol. 14, No. 3 (May, 1969), pp. 450-454
Published by: American Society of Limnology and Oceanography
Stable URL: http://www.jstor.org/stable/2833812 .
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450 NOTES
COLORIMETRIC DETERMINATION
Low
The Winkler (1888) method for the de-
terminationof dissolved oxygen is used
almost exclusively in limnological and
oceanographic work. Divalent manganese
is oxidized by molecular oxygen in basic
solution and, upon subsequent acidifica-
tion, iodide is oxidized to free iodine by
the oxidized manganese; the free iodine
concentrationis then determinedby titra-
tionwiththiosulfate.Althoughthe method
has been subject to some criticismand
modificationsince its inception(Thompson
and Robinson 1939; Wheatland and Smith
1955; Carpenter1965; Carrittand Carpen-
ter 1966), it is probablythe most-usedana-
lytical method in the chemistryof natural
waters.
When oxygen concentrationsare less
than 10% of the saturation value the
Winklermethod is imprecise,atmospheric
contaminationcan be serious, and special
analytical and sampling procedures must
be used. Our work in oxygen-deficient en-
vironmentshas led us to devise a method
to measure preciselyoxygenconcentrations
between zero and 50 ,ug-atoms/liter. The
method is not intended to replace the
Winklermethod,and it is useful only with
low oxygen concentrations.
MATERIALS AND METHODS
Oxygen concentrationscan be deter-
mined colorimetricallyby measuring the
absorption of ultravioletlight by the tri-
iodide ion that is produced when Winkler
reagents are added to the sample (Custer
and Natelson 1949). Unless the sampling
procedure is precise, atmosphericoxygen
contaminationwill invalidate the results.
Samples are thereforedrawn into syringes
to eliminate contact with the atmosphere
(Broenkow 1969). Reagents similar to
those used in the Winkler method are
injected into the syringes,and iodine is
1 Contribution
No. 494 fromthe Departmentof
Oceanography,University of Washington,Seattle.
This researchwas supportedby NationalScience
FoundationGrantGA-644 to Dr. F. A. Richards.
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AND COMMENT
OF DISSOLVED OXYGEN AT
CONCENTRATIONS'
released in proportion to the dissolved
oxygen originallyin the sample.
REAGENTS
1. Manganous sulfatesolution. Dissolve
25 g of reagent grade MnSO4 4H2O or 20
g of MnSO4*H20 in 500 ml of distilled
water. This reagent is stable indefinitely.
2. Alkaline iodide solution. Dissolve 14
g of reagent grade KOH and 60 g of
reagent grade KI in 200 ml of distilled
water. This reagenttends to discolor after
a time because of the oxidation of iodide
to iodine, and only small quantitiesshould
be prepared. Afterthe calibration factor
has been determined,add 0.1 g of reagent
grade sodium azide (NaN3) to the alka-
line iodide solution. Sodium azide is used
to eliminatenitriteinterference(Alsterberg
1925); however,it also interfereswith the
oxidationof iodide by iodate.
3. Phosphoricacid solution. Dilute 100
ml of 85% phosphoricacid with 400 ml of
distilled water. This reagent is stable in-
definitely.If the pH of the treatedsample
is less than about 2, the rate of air oxida-
tion of the iodide is increased, and high
apparentoxygenconcentrations result.The
use of phosphoricacid permitsbetter pH
control, and phosphoric acid is less cor-
rosive than the sulfuricacid normallyused
in the Winklermethod.
4. Primarybiiodate solution. Dissolve
exactly 0.1300 g of dry, reagent grade
KH(IO3)2 in distilledwater and dilute to
exactly1 liter (0.3333x 10-3 M). It is
stable indefinitely.
5. Secondary biiodate solution. Dilute
25.00 ml of the primarybiiodate solution
to exactly 1 liter. This potassium biiodate
solution is 0.8333 x 10-5 M and is equiva-
lent to a solutionwith an oxygen concen-
trationof 50 pg-atoms/liter.
6. Iodine solution. Dissolve 25 g of
reagent grade KI and 0.1 g (one pellet)
of KOH in about 200 ml of distilledwater.
Dissolve 0.32 g of reagent grade I2 in this
solutionand dilute to 1 liter. This solution
 NOTES AND COMMENT 451
should be weakly basic to retardair oxida- fromlightto preventphotochemicalreduc-
tion of the iodide ion. It should be stored tion of the iodine.
in an amber bottle and protected from Measure the absorbance of the triiodide
intenselight. The solutionis about 1.25 x ion at 352 mjt in a 1-cm glass cell. A
10-3 M in I2, and the concentrationin- spectrophotometer having either a tung-
creases slowly as the iodide oxidizes. sten or a hydrogenlamp can be used.
The oxygen concentrationsin the man- In some oxygen-deficient environments,
ganous sulfate and alkaline iodide nor- compoundsare presentthatcan produce or
mally used in the Winkler determination remove iodine. If this is the case, a sepa-
are about 30% of the saturationvalue for rate analysis must be made to determine
distilled water. The errorassociated with the iodine equivalence of the interfering
the introductionof oxygen with the re- substances. To do this, add to 25 ml of
agents in the Winkler method is about 1 the sample 0.5 ml of the phosphoricacid,
jtg-atom/liter when 1 ml of each reagent shake well, then add 0.5 ml of the alkaline
is added to 300-ml samples. Oxygen con- iodide solution and shake. Add 0.500 ml
taminationfromthe reagentsin the method of the iodine solution and shake. The
described here would be about 20 ug- iodine solutioncan be added accuratelyto
atoms/literif the oxygenwere not removed the sample syringewith a micrometersy-
from the manganous sulfate and alkaline ringe fittedwith a small-borecannula. If
iodide. To minimize this contamination, no interferingsubstances are present,the
the manganous sulfateand alkaline iodide resultingsolutionshould contain25 umoles
are stored in aspiratorbottles fittedwith I2/liter. If the absorbance of the sample
rubber septums, and nitrogenis bubbled treatedfor interfering substancesindicates
throughthe reagentsfor30 min beforeand more or less than this concentration,a
duringthe analyses. Because the reagents correctionmust be applied.
are added to the samples with syringes,
the reagent viscosity must be kept low, STANDARDIZATIONS
so the reagents are about one-tenththe Standardizations preferably should be
concentrationof those used in the Winkler carried out in the syringesso that dilution
method. effects (about 4%) are duplicated in the
PROCEDURE standardsand in the samples; standardiza-
Samples (25 ml) are drawn into glass tions can be done in suitable glassware
syringes(Broenkow 1969) fromsampling and a correctionapplied to account for
bottles fitted with rubber septums using dilution. As in the Winkler analysis, the
large-bore (16 gauge) cannulas. Air con- iodate ion is the primary standard, and
taminationmust be avoided, and the sy- oxygen contaminationduring standardiza-
ringes must be flushed with the sample tion is not a factor. For standardization,
water. draw a syringefulof secondary biiodate
Immediatelyaftersampling,add 0.5 ml solution. To this add 0.5 ml of phosphoric
of manganous sulfate and 0.5 ml of alka- acid, shake well, and add 0.5 ml of alkaline
line iodide solutionsthroughthe tip of the iodide. Mix well and add 0.5 ml of man-
sample syringeusing calibrated1-mltuber- ganous sulfate solution. The iodine lib-
culin syringesfitted with small-bore (20 erated is equivalent to an oxygen concen-
gauge) cannulas. After these reagents trationof 50 jtg-atoms/liter.
have been added, cover the tip of the The reagentblank is determinedby add-
sample syringeand shake. After 30 sec, ing 0.5 ml of phosphoricacid and 0.5 ml
add 0.5 ml of phosphoricacid solutionand of alkaline iodide to a syringefulof dis-
shake. The reactionis sufficientlyfastthat tilled water. Shake well and add 0.5 ml
it is unnecessaryto allow the precipitate of manganous sulfate solution. A small
to settle before acidification. absorbance results from the presence of
The treatedsamples should be protected reducing compounds in the reagents and

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452 NOTES AND COMMENT

TABLE 1. Reproducibility
studyof the colorimet- TABLE 2. Effectof sodiumazide in eliminating
ric oxygenmethod nitriteinterference

Oxygenconcn Iodine concn

Sample Absorbance (ALg-atoms/liter) (ALmoles/liter)
(oxygenequiv-
1 0.484 20.1 Absorb- alence gg-
ance atoms/liter)
2 0.486 20.2
3 0.497 20.7 Withoutsodiumazide added to alkaline-iodide
4 0.484 20.1 0.0 Ag-atoms/liter
NO2- 0.458 19.0
5 0.492 20.4 21.4
5.0 ug-atoms/liter
NO2- 0.515
6 0.492 20.4
7 0.494 20.5 Withsodiumazide added to alkaline-iodide
8 0.481 20.0 0.0 Ag-atoms/liter
NO2- 0.460 19.1
9 0.481 20.0
5.0 Ag-atoms/liter
NO2- 0.462 19.1
10 0.489 20.3
Avg 0.488 20.3
SD ?+0.23 AR is the absorbance of the test solution
treated for reducing compounds.

DISCUSSION
fromsmall amounts of I2 in the KOH-KI
An estimate of the overall precision of
solution.
the colorimetricoxygenanalysiswas made
The calibrationfactor,F, is determined
fromthe expression by determiningthe oxygen content of 10
replicate samples of low oxygen water.
F = 5O/(As-Ab), The sample water was withdrawnwith the
sample syringesfrom an airtight,piston-
whereAs is the average absorbance of three fittedchamber(Cline 1968) thatprevented
standards,and Ab is the average absorb- atmospheric contamination. The average
ance of two reagent blanks. The value of oxygen concentrationwas 20.3 ,ug-atoms/
F should be about 43. Oxygen concentra- liter with +0.5 ,ug-atoms/liter (2 SD) pre-
tions are computed fromthe expression cision (Table 1).
02 (,ug-atoms/liter)= F ( As - Ab), The efficiencyof sodium azide in elim-
where An,is the absorbanceof the unknown inating nitriteinterferencewas shown by
sample. adding free iodine to samples containing
The iodine solutionmustbe standardized nitrite.When azide was not present,nitrite
daily by adding 0.5 ml of the alkaline oxidized iodide to iodine giving higher
iodide solution and 0.5 ml of phosphoric apparent oxygen values. When azide was
acid solutionto 25 ml of distilledwater in added to the alkaline-iodidesolution,how-
a sample syringe. Shake well, and add ever, the nitrite interferencewas com-
0.500 ml of iodine solutionwith a microm- pletely eliminated (Table 2).
eter syringeand shake again. The absorb- Certain reducingsubstancessuch as sul-
ance of the triiodideion is determinedas fite, thiosulfate,or sulfide are sometimes
before. presentin polluted waters or reducing en-
The concentration of reducing sub-
stances is computed fromthe relation:
TABLE 3. Effectof thiosulfate
interference
reducing compounds
(Ftmoles 12/liter) = F (AI -AR,), Iodine concn
(,umoles/liter)
(oxygenequiv-
where the concentrationof reducing sub- Absorb- alence ,ug-
ance atoms/liter)
stances is reported in iodine equivalents,
F is the calibrationfactordeterminedfrom 0.0 umoles/liter S2032- 0.458 19.0
the biiodate standardization,A, is the ab- 10.7 umoles/liter
S2032- 0.325 13.3
sorbance of the iodine standardization,and 21.4 umoles/liter
S2032- 0.203 8.2

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 NOTES AND COMMENT 453

DISSOLVED OXYGEN(/4g-atoms/liter) COLORIMETRIC

OXYGEN
(ml/liter)
O 10 20 30 40 50 60 0 0., 0.2 0.3 0.4 0.5
0 70 I

0.6
a) 60 *-g,? /

E 50 - * * X
200 -

4 ~~~~~~0.3
g30 *u;7K/ x
400 -
o o
20* 0: ':

zL o
0/
lo 20* Q050 6-0.
CL

600
0 10 20 30 40 50 60
OXYGEN
COLORIMETRIC (/.g-atoms/liter)
set
FIG.1: 2. orlton ahdln
Comparisonbetween rpeet
colorimetrich
and
Winkleranalysesof dissolvedoxygenduringRV
800 _ ThomasG. Thompsoncruise28. Solid line repre-
sents 1: 1 correlation.Dashed line represents
t-he
least squares regressionwith a regressioncoeffi-
cient, b - 1.06, with standarddeviationon the
slope, Sb = +0.13.
1000 , I x,
0 0.1 0.2 0.3 0.4 0.5
DISSOLVED OXYGEN (mI/liter)
1968 (University of Washington,unpub-
lished data), oxygen concentrationsin the
FIG. 1. Verticaldistribution of oxygendeter- low oxygenwaterwere determinedby both
minedcolorimetrically in the easternPacificOcean the Winkler and colorimetric methods.
(RV Thomas G. Thompsoncruise26, station67,
8? 50' N lat, 900 21' W long,21 February1968).
Colorimetric samples were drawn from
conventionalsamplingbottlesthroughrub-
ber septums. A comparisonof the Winkler
vironments. These substances reduce io- and colorimetric values (Fig. 2) shows that
dine and give lower apparent oxygen Winkler values are about 10 ytg-atoms/
values, and their concentrationsmust be liter higher than the colorimetricvalues,
determined by the described procedure. and the data suggest that oxygen contam-
The addition of 19 )umoles/literof free ination in the reagents and atmospheric
iodine to a sample 21.4 )uM in thiosulfate contaminationof the samples are impor-
decreased the iodine concentrationby 10.8 tantsourcesof errorin the Winklermethod
ytmoles/liter, in agreementwith the iodine- at these low concentrationlevels. The
thiosulfatestoichiometry(Table 3). modificationof the Winkler method used
An extensive body of oxygen-deficient here employsreagentsas recommendedby
water occurs in the eastern Pacific Ocean Carrittand Carpenter (1966), but differs
along the coasts of Mexico and Central by titrating100-ml subsamples. The re-
America between about 100 and 200 N gression coefficientbetween the Winkler
latitude. In much of this region, oxygen and colorimetricvalues is 1.06 and is not
concentrations below 10% of the saturation significantly differentfrom1.00. The stan-
value occur between depths of 100 and dard deviationof the Winklervalues about
1,000m (Fig. 1). DuringRV ThomasG. the regressionline is Sz,I,= ?7 )ug-atoms/
Thompson cruise 26, January to March literand representsan estimateof the pre-

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454 NOTES AND COMMENT

cision of the Winkler analysis including Winklermethodfor dissolvedoxygenanaly-

sampling errors. Thus, at low concentra- sis. Limnol. Oceanog., 10: 135-140.
CARRITT, D. E., AND J. H. CARPENTER. 1966.
tion levels, the Winklermethod can be in Comparisonand evaluationof currently em-
error by 10 ug-atoms/literand has ten ployed modifications of the Winklermethod
times poorer precision than the colori- fordetermining dissolvedoxygenin seawater;
metricmethod. a NASCO report. J. MarineRes., 24: 286-
318.
WILLIAM W. BROENKOW CLINE, J. D. 1968. Kineticsof the sulfide-oxy-
gen reactionin seawater;An investigation at
JOEL D. CLINE
constanttemperature and salinity. M.S. The-
sis, Univ. Washington,Seattle. 68 p.
Department of Oceanography, CUSTER, J. J., AND S. NATELSON. 1949. Spectro-
Universityof Washington, photometric determination of microquantities
Seattle 98105. of iodine. Anal. Chem., 21: 1005-1009.
THOMPSON, T. G., AND R. J. ROBINSON. 1939.
REFERENCES
Notes on the determination of dissolvedoxy-
gen in sea water. J. MarineRes., 2: 1-8.
ALSTERBERG, G. 1925. Methoden zur Bestim- WHEATLAND, A. B., AND L. J. SMITH. 1955.
mung von in Wasser gelosten elementaren Gasometricdetermination of dissolvedoxygen
Sauerstoff bei gegenwart von salpetriger in pure and saline wateras a check of titri-
Saure. Biochem. Z., 159: 36-47. metricmethods. J. Appl. Chem. (London),
BROENKOW, W. W. 1969. An interface sam- 5: 144-148.
pler using spring-actuated syringes. Limnol. WINKLER, L. W. 1888. Die Bestimmung des im
Oceanog., 14: 288-291. Wasser gelostenSauerstoffes. Chem. Ber.,
CARPENTER, J. H. 1965. The accuracy of the 21: 2843-2855.

SPECTROPHOTOMETRIC DETERMINATION OF HYDROGEN SULFIDE IN

NATURAL WATERS'

The determinationof small concentra- investigationsof anoxic marine basins in

tions of hydrogensulfideby the methylene
blue method has been widely used since
its introductionby Fischer (1883). The
methodis sensitiveand specificforsulfide-
sulfur and is readily adaptable for rou-
tine analyses. Many modificationsof the
method have been made for specific pur-
poses (Almy 1925; Sheppard and Hudson
1930; Sands et al. 1949; Fogo and Popow-
sky 1949; Budd and Bewick 1952). The
procedurehas also been used in determin-
ing sulfide-sulfurin sewage (Pomeroy
1936) and seawater (Fonselius 1962).
In our experience, the methyleneblue
method was somewhaterratic,and studies
were undertaken to define the optimum
conditionsfor determiningsulfide-sulfur
in
naturalwaters. The studyevolved during
1ContributionNo. 493 fromthe Departmentof
Oceanography,University of Washington,Seattle.
This researchwas supportedby National Science
FoundationGrantGA-644 to Dr. F. A. Richards.
which hydrogensulfide occurs as a respi-
ration product of sulfate reduction.
A procedureusing unsubstitutedp-phen-
ylenediamineto produce Lauth's violet in
the determinationof sulfide-sulfur in sea-
water 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,containingN,N-di-
methyl-p-phenylenediamine sulfate, which
need not be recrystallizedbefore use; 2)
superiorcolor and reagent stabilityat low
sulfideconcentrations;3) applicabilityto a
wide range of sulfide concentrations;4)
approximatelya 10% increase in sensitivity,
and 5) a simplifiedprocedure for stan-
dardization. The method is applicable to
natural waters containing 1-1,000 ug-at-
oms/litersulfide-sulfur(0.03-32 ppm) and
is free of salt effects and temperature
dependence.

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